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Formulation and Stability Testing of Eye Drop Preparations Containing Phenylephrine Hydrochloride
Formulation and Stability Testing of Eye Drop Preparations Containing Phenylephrine Hydrochloride
Formulation and Stability Testing of Eye Drop Preparations Containing Phenylephrine Hydrochloride
METROPOLITAN UNIVERSITY
DECEMBER 2012
ii
DECLARATION
I, Chinedum Oluchukwu Okafor, 205010351, hereby declare that the dissertation for
Magister Scientiae is my own work and that it has not previously been submitted for
assessment or completion of any postgraduate qualifcaton to another University or
for another qualification.
iii
ACKNOWLEDGEMENTS
I wish to thank the following people and institutions for their assistance during the
compiling of this dissertation:
My exceptional families Okafor and Makamure for their steadfast support and
love;
Dr. M Worthington, as without sponsorship and guidance there could be no
research;
My supervisors, Mrs. M. Keele, and Prof. G. Kilian for guidance and support;
Prof. Milne, for his unwavering support, no words in the dictionary can
describe his help;
Michael (Aspen), Jean, Charne and Arista (NMMU) for her input, support and
exceptional skills at sourcing materials for me;
Aspen Pharmacare, for financial assistance and the use of equipment and
materials needed to perform my experiments;
All my friends all around the world, every moment with you was a blessing.
The Pharmacy, Biochemistry and Microbiology and Chemistry Departments of
the Nelson Mandela Metropolitan University for the use of laboratory facilities
and technical assistance;
Above all, God Almighty, only through His Grace can I achieve all things.
iv
SUMMARY
v
Changes in the appearance and colour of products containing glycerol under
accelerated storage conditions were observed. The sodium carboxy methylcellulose
(SCMC) containing formulation was found to be physically and chemically stable in
two conditions, namely 30 °C/65%RH and 25 °C/60%RH. The formulations
containing hydroxypropyl methylcellulose along with an antioxidant showed to be
most stable as it remained aesthetically pleasing did not change colour and did not
have a reduction in phenylephrine hydrochloride concentrations. This meant that
phenylephrine hydrochloride did not degrade while the viscosity modifying agents
remained stable.
The preservatives in the formulations were active against the microbial organisms
use to challenged them.
vi
TABLE OF CONTENTS
DECLARATION ...................................................................................................................... iii
ACKNOWLEDGEMENTS ....................................................................................................... iv
SUMMARY ...............................................................................................................................v
TABLE OF CONTENTS ......................................................................................................... vii
LIST OF ABBREVIATIONS .....................................................................................................x
LIST OF FIGURES ............................................................................................................... xiv
LIST OF TABLES ...............................................................................................................xxviii
1. INTRODUCTION ............................................................................................................. 1
1.1 Background and motivation ...................................................................................... 1
1.2 Aim and objectives ................................................................................................... 2
1.3 Plan of work .............................................................................................................. 3
2. LITERATURE REVIEW ................................................................................................... 4
2.1 Anatomy and physiology of the eye ......................................................................... 4
2.2 Pathophysiology of the eye ......................................................................................... 10
2.3 Phenylephrine hydrochloride and its ocular uses ........................................................ 14
2.3.1 Phenylephrine hydrochloride .................................................................................... 14
2.3.2 Pharmacological actions and uses ........................................................................... 15
2.3.3 Mechanism of action ................................................................................................ 15
2.3.4 Pharmacokinetics ..................................................................................................... 16
2.3.5 Adverse effects ......................................................................................................... 16
2.3.6 Drug interactions ...................................................................................................... 17
2.3.7 Bioavailability ............................................................................................................ 17
2.3.7.1 Reasons for poor ocular bioavailability .................................................................. 18
2.3.7.2 Strategies for improving drug availability in ocular adminstration ......................... 19
2.3.7.2.1 Increasing ocular residence time ........................................................................ 19
2.3.7.2.2 Increasing ocular absorption .............................................................................. 19
2.3.7.2.3 Altering drug structure ........................................................................................ 20
2.3.8 Polymorphism and pseudomorphism of phenylephrine hydrochloride ..................... 21
2.4 Ophthalmic formulations .............................................................................................. 21
2.4.1 Eye drops as an ophthalmic dosage form ................................................................ 22
2.4.2 Eye drop formulation characteristics ........................................................................ 24
2.4.2.1 Clarity .................................................................................................................... 24
2.4.2.2 Stability, pH and buffer systems ............................................................................ 25
2.4.2.3 Tonicity .................................................................................................................. 26
vii
2.4.2.4 Viscosity ................................................................................................................ 27
2.4.2.5 Additives ................................................................................................................ 30
2.5 Sterilization .................................................................................................................. 34
2.5.1 Steam under pressure as a method of sterilization .................................................. 35
2.5.2 Filtration as a method of sterilization ........................................................................ 35
2.5.3 Laminar-flow principles ............................................................................................. 35
2.5.4 Preservatives used in eye drop formulations ........................................................... 36
2.5.4.1 Quaternary ammonium compounds ...................................................................... 38
2.5.4.2 Parahydroxybenzoic acid esters ........................................................................... 40
2.6 Efficacy of antimicrobial preservation .......................................................................... 42
2.7 Packaging .................................................................................................................... 43
2.9 Formulation development ............................................................................................ 44
2.9.1 Validation of HPLC analytical methods .................................................................... 46
2.9.1.1 Stability indicating HPLC analysis ......................................................................... 47
2.9.1.2 Choice of analytical column and conditions ........................................................... 48
2.9.1.3 Steps for HPLC method validation ........................................................................ 50
2.9.1.4 Linearity ................................................................................................................. 50
2.9.1.5 Accuracy and precision ......................................................................................... 51
2.9.1.6 Limit of detection and limit of quantification ........................................................... 51
2.9.1.7 Range .................................................................................................................... 51
2.9.1.8 Specificity .............................................................................................................. 51
2.9.2 Active-excipient compatibility studies ....................................................................... 53
2.10 Determining formulation stability study ...................................................................... 54
3. METHODOLOGY .......................................................................................................... 57
3.1 HPLC method validation .............................................................................................. 57
3.1.1 Equipment ................................................................................................................ 57
3.1.2 Materials and reagents ............................................................................................. 57
3.1.3 Mobile phase preparation and standard curve construction ..................................... 57
3.1.4 Chromatographic conditions ..................................................................................... 58
3.1.5 Linearity .................................................................................................................... 58
3.1.6 Accuracy and precision ............................................................................................ 59
3.1.7 Limit of detection and limit of quantification .............................................................. 59
3.1.8 Range and system suitability .................................................................................... 60
3.1.9 Specificity ................................................................................................................. 60
3.2 Determination of active–excipient compatibility .......................................................... 62
3.3 Manufacture of products .............................................................................................. 62
viii
3.3.1 Materials ............................................................................................................... 63
3.3.2 Product manufacture ................................................................................................ 63
3.3.2.1 Sterilization for heat sensitive API and exipients ................................................... 64
3.3.3 Manufacturing methods for products I–V ................................................................. 64
3.4 Stability Tests .............................................................................................................. 69
3.5 Qualitative and quantitative analysis of the formulations ............................................ 70
3.5.1 Appearance and pH ................................................................................................. 70
3.5.2 Phenyleprine hydrochloride concentration ............................................................... 70
3.6 Test for preservative efficacy ...................................................................................... 70
3.6.1 Procedure for standard plate count .......................................................................... 71
3.6.2 Procedure for plating the bacteria and fungi ............................................................ 71
3.6.3 Standardization of cultures using turbidimetry method ............................................ 72
3.6.4 Preservative efficacy ................................................................................................ 73
3.7 Determination of viscosity ........................................................................................... 73
3.8 Statistical analysis ....................................................................................................... 74
4. RESULTS AND DISCUSSION ...................................................................................... 75
4.1 Validation of the stability indicating assay ................................................................... 75
4.1.1 Linearity .............................................................................................................. 75
4.1.2 Accuracy .......................................................................................................... 76
4.1.3 Precision ......................................................................................................... 76
4.1.4 Limit of detection (LOD) and quantification (LOQ) .......................................... 77
4.1.5 Specificity and system suitability ..................................................................... 77
4.2 Active and excipient study ......................................................................................... 113
4.3 Stability study ............................................................................................................ 123
4.4 Determination of yield point and viscosity of products .............................................. 137
4.5 Effectiveness of the ophthalmic solution preservatives ............................................. 144
5. CONCLUSION AND RECOMMENDATIONS .............................................................. 147
REFERENCES ................................................................................................................... 151
APPENDIX A ...................................................................................................................... 176
CONCEPT ARTICLE ....................................................................................................... 176
APPENDIX B ...................................................................................................................... 192
LIST OF EQUIPMENT .................................................................................................... 192
APPENDIX C ...................................................................................................................... 193
LIST OF SOLUTIONS ..................................................................................................... 193
ix
LIST OF ABBREVIATIONS
Å angstrom
BP British Pharmacopeia
COMT catechol–O–methyltransferases
R2 Correlation coefficient
Da Dalton
ET Eustachian tube
x
> Greater than
kg kilogram
log logarithmic
µg microgram
µL microliter
µm micrometer
mg milligram
xi
mm millimeter
min minute
OTC Over–The–Counter
% Percentage
RH Relative humidity
~ roughly similar
s seconds
SA South Africa
SD Standard Deviation
xii
Tf Tailing factor
T Temperature
UV Ultraviolet
UK United Kingdom
λ Wavelength
xiii
LIST OF FIGURES
Figure 1: Anatomy of the eye (Del Amo & Urtti, 2008). ............................................. 4
Figure 2: Structure of Phenylephrine, its base and salts (Trommer et al., 2010). .... 15
Figure 3: Diagram of Ocular Absorption (Nanjawade et al., 2007). ......................... 17
Figure 4: Diagram of a typical HPLC-UV absorbance peak and plots of noise (or
threshold) and purity angles (Krull & Swartz, 2001). ................................................ 52
Figure 5: Laboratory scale 1000 ml manufacturing process of product I ................. 65
Figure 6: Laboratory scale 1000 ml manufacturing process of product II ................ 66
Figure 7: Laboratory scale 1000 ml manufacturing process of product III ............... 67
Figure 8: Laboratory scale 1000 ml manufacturing process of product IV............... 68
Figure 9: Laboratory scale 1000 ml manufacturing process of product V................ 69
Figure 10: Graph showing a mean peak area versus concentration of replicate
samples of phenylephrine hydrochloride standards. Linear regression equation: y =
8541.1x + 438.55, R2 = 0.9999. ............................................................................... 75
Figure 11: HPLC Chromatogram for mobile phase alone........................................ 78
Figure 12: HPLC Chromatogram for phenylephrine hydrochloride dissolved in
mobile phase with a retention time of 7.80 minutes. ................................................ 79
Figure 13: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and
purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999999.
................................................................................................................................. 79
Figure 14: HPLC Chromatogram for product I dissolved in mobile phase with a
retention time of 7.87 minutes. ................................................................................. 79
Figure 15: Peak purity profile calculated using PDA data (from 190–800 nm) for
Product I prepared in mobile phase. Peak shown in pink and purity curve in black.
Peak purity index = 1.00000; Single point threshold = 0.999054 ............................. 80
Figure 16: HPLC Chromatogram for product II dissolved in mobile phase with a
retention time of 7.82 minutes. ................................................................................. 80
Figure 17: Peak purity profile calculated using PDA data (from 190–800 nm) for
Product II prepared in mobile phase. Peak shown in pink and purity curve in black.
Peak purity index = 1.00000; Single point threshold = 0.996482. ............................ 80
xiv
Figure 18: HPLC Chromatogram for product III dissolved in mobile phase with a
retention time of 7.83 minutes. ................................................................................. 81
Figure 19: Peak purity profile calculated using PDA data (from 190–800 nm) for
Product III prepared in mobile phase. Peak shown in pink and purity curve in black.
Peak purity index = 1.00000; Single point threshold = 0.998178. ............................ 81
Figure 20: HPLC Chromatogram for product IV dissolved in mobile phase with a
retention time of 7.89 minutes. ................................................................................. 81
Figure 21: Peak purity profile calculated using PDA data (from 190–800 nm) for
Product IV prepared in mobile phase. Peak shown in pink and purity curve in black.
Peak purity index = 1.00000; Single point threshold = 0.996947. ............................ 82
Figure 22: HPLC Chromatogram for product V dissolved in mobile phase with a
retention time of 7.84 minutes. ................................................................................. 82
Figure 23: Peak purity profile calculated using PDA data (from 190–800 nm) for
product V prepared in mobile phase. Peak shown in pink and purity curve in black.
Peak purity index = 1.00000; Single point threshold = 0.999587. ............................ 82
Figure 24: HPLC Chromatogram for phenylephrine hydrochloride stressed under UV
light dissolved in mobile phase with a retention time of 7.81 minutes. ..................... 83
Figure 25: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride prepared in mobile phase. Peak shown in pink and
purity curve in black. Peak purity index = 1.00000; Single point threshold = 0.999999.
................................................................................................................................. 83
Figure 26: HPLC Chromatogram for product I stressed under UV light dissolved in
mobile phase with a retention time of 7.86 minutes. ................................................ 84
Figure 27: Peak purity profile calculated using PDA data (from 190–800 nm) for
product I prepared in mobile phase. Peak shown in pink and purity curve in black.
Peak purity index = 1.00000; Single point threshold = 0.999054. ............................ 84
Figure 28: HPLC Chromatogram for product II stressed under UV light dissolved in
mobile phase with a retention time of 7.82 minutes. ................................................ 84
Figure 29: Peak purity profile calculated using PDA data (from 190–800 nm) for
product II in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 1.00000; Single point threshold = 0.999116. ............................................... 85
Figure 30: HPLC Chromatogram for product III stressed under UV light dissolved in
mobile phase with a retention time of 7.85 minutes. ................................................ 85
xv
Figure 31: Peak purity profile calculated using PDA data (from 190–800 nm) for
product III in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 0.999999; Single point threshold = 0.997116. ............................................. 85
Figure 32: HPLC Chromatogram for product IV stressed under UV light dissolved in
mobile phase with a retention time of 7.81 minutes. ................................................ 86
Figure 33: Peak purity profile calculated using PDA data (from 190–800 nm) for
product IV in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 1.000000; Single point threshold = 0.997916..................................... 86
Figure 34: HPLC chromatogram for product V stressed under UV light dissolved in
mobile phase with a retention time of 7.87 minutes. ................................................ 86
Figure 35: Peak purity profile calculated using PDA data (from 190–800 nm) for
product V in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 1.000000; Single point threshold = 0.997475. ............................................. 87
Figure 36: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M
HCl dissolved in mobile phase with a retention time of 7.86 minutes. ...................... 88
Figure 37: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in
black. Peak purity index = 1.000000; Single point threshold = 0.999574. ................ 88
Figure 38: HPLC chromatogram of Product I stressed with 0.2 M HCl dissolved in
mobile phase with a retention time of 7.82 minutes. ................................................ 88
Figure 39: Peak purity profile calculated using PDA data (from 190–800 nm) for
product I stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 0.999999; Single point threshold = 0.997116. ... 89
Figure 40: HPLC chromatogram for product II stressed with 0.2 M HCl dissolved in
mobile phase with a retention time of 7.83 minutes. ................................................ 89
Figure 41: Peak purity profile calculated using PDA data (from 190–800 nm) for
product II stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 1.000000; Single point threshold = 0.996296. ... 89
Figure 42: HPLC chromatogram for product III stressed with 0.2 M HCl dissolved in
mobile phase with a retention time of 7.91 minutes. ................................................ 90
Figure 43: Peak purity profile calculated using PDA data (from 190–800 nm) for
product III stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index =0.999999; Single point threshold = 0.995179. .... 90
xvi
Figure 44: HPLC chromatogram for product IV stressed with 0.2 M HCl dissolved in
mobile phase with a retention time of 7.86 minutes. ................................................ 90
Figure 45: Peak purity profile calculated using PDA data (from 190–800 nm) for
product IV stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index =1.000000; Single point threshold = 0.997097. .... 91
Figure 46: HPLC chromatogram of product V stressed with 0.2 M HCl dissolved in
mobile phase with a retention time of 7.80 minutes. ................................................ 91
Figure 47: Peak purity profile calculated using PDA data (from 190–800 nm) for
product V stressed with 0.2 M HCl in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index =1.000000; Single point threshold = 0.999578. .... 91
Figure 48: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M
NaOH dissolved in mobile phase with a retention time of 7.82 minutes ................... 92
Figure 49: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride stressed with 0.2 M NaOH in mobile phase. Peak
shown in pink and purity curve in black. Peak purity index =0.999999; Single point
threshold = 0.996847. .............................................................................................. 92
Figure 50: HPLC chromatogram of product I stressed with 0.2 M NaOH dissolved in
mobile phase with a retention time of 7.89 minutes. ................................................ 93
Figure 51: Peak purity profile calculated using PDA data (from 190–800 nm) for
product I stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index =1.000000; Single point threshold = 0.995815. .... 93
Figure 52: HPLC chromatogram of product II stressed with 0.2 M NaOH dissolved in
mobile phase with a retention time of 7.9 minutes. .................................................. 93
Figure 53: Peak purity profile calculated using PDA data (from 190–800 nm) for
product II stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 0.999999; Single point threshold = 0.996370. ... 94
Figure 54: HPLC chromatogram for product III stressed with 0.2 M NaOH dissolved
in mobile phase with a retention time of 7.79 minutes. ............................................. 94
Figure 55: Peak purity profile calculated using PDA data (from 190–800 nm) for
product III stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 0.999999; Single point threshold = 0.998771. ... 94
Figure 56: HPLC chromatogram of product IV stressed with 0.2 M NaOH dissolved
in mobile phase with a retention time of 7.91 minutes. ............................................. 95
xvii
Figure 57: Peak purity profile calculated using PDA data (from 190–800 nm) for
product IV stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and
purity curve in black. Peak purity index = 0.999999; Single point threshold =
0.996930. ................................................................................................................. 95
Figure 58: HPLC chromatogram for product V stressed with 0.2 M NaOH dissolved
in mobile phase with a retention time of 7.85 minutes. ............................................. 95
Figure 59: Peak purity profile calculated using PDA data (from 190–800 nm) for
product V stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 0.999999; Single point threshold = 0.999587. ... 96
Figure 60: HPLC chromatogram for phenylephrine hydrochloride stressed with 0.2
M H2O2 dissolved in mobile phase with a retention time of 7.82 minutes. ................ 96
Figure 61: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride stressed with 0.2 H2O2 in mobile phase. Peak shown in
pink and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.999987. ................................................................................................................. 97
Figure 62: HPLC chromatogram for product I stressed with 0.2 M H2O2 dissolved in
mobile phase with a retention time of 7.81 minutes. ................................................ 97
Figure 63: Peak purity profile calculated using PDA data (from 190–800 nm) for
product I stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 1.000000; Single point threshold = 0.999522. ... 97
Figure 64: HPLC chromatogram for product II stressed with 0.2 M H2O2 dissolved in
mobile phase with a retention time of 7.9 minutes. .................................................. 98
Figure 65: Peak purity profile calculated using PDA data (from 190–800 nm) for
product II stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 0.999999; Single point threshold = 0.999722. ... 98
Figure 66: HPLC chromatogram of product III stressed with 0.2 M H2O2 dissolved in
mobile phase with a retention time 7.94 minutes. .................................................... 98
Figure 67: Peak purity profile calculated using PDA data (from 190–800 nm) for
product III stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 0.999999; Single point threshold = 0.999706. ... 99
Figure 68: HPLC chromatogram for product IV with 0.2 M H2O2 dissolved in mobile
phase with a retention time of 7.81 minutes. ............................................................ 99
xviii
Figure 69: Peak purity profile calculated using PDA data (from 190–800 nm) for
product IV stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 1.000000; Single point threshold = 0.999896. ... 99
Figure 70: HPLC chromatogram for product V stressed with 0.2 M H2O2 dissolved in
mobile phase with a retention time of 7.88 minutes. .............................................. 100
Figure 71: Peak purity profile calculated using PDA data (from 190–800 nm) for
product V stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 0.999999; Single point threshold = 0.999536. . 100
Figure 72: HPLC chromatogram for phenylephrine hydrochloride stored at 100 °C
for 24 hours dissolved in mobile phase with a retention time of 7.8 minutes. ......... 102
Figure 73: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride stored at 100 °C for 24 hours in mobile phase. Peak
shown in pink and purity curve in black. Peak purity index = 1.000000; Single point
threshold = 0.999999. ............................................................................................ 102
Figure 74: HPLC chromatogram for phenylephrine hydrochloride stored at 65 °C for
1 month dissolved in mobile phase with a retention time of 7.83 minutes. ............. 103
Figure 75: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride stored at 65 °C for 1 month in mobile phase. Peak
shown in pink and purity curve in black. Peak purity index = 1.000000; Single point
threshold = 0.999989. ............................................................................................ 103
Figure 76: HPLC chromatogram for product I stored at 65 °C for 1 month dissolved
in mobile phase with a retention time of 7.92 minutes. ........................................... 103
Figure 77: Peak purity profile calculated using PDA data (from 190–800 nm) for
product I stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 0.999998; Single point threshold = 0.999956. . 104
Figure 78: HPLC chromatogram for product II stored at 65 °C for 1 month dissolved
in mobile phase with a retention time of 7.84 minutes. ........................................... 104
Figure 79: Peak purity profile calculated using PDA data (from 190–800 nm) for
product II stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 1.000000; Single point threshold = 0.999056. . 104
Figure 80: HPLC chromatogram for Product III stored at 65 °C for 1 month dissolved
in mobile phase with a retention time of 7.89 minutes. ........................................... 105
Figure 81: Peak purity profile calculated using PDA data (from 190–800 nm) for
product III stored at 65 °C for 1 month in mobile phase. Peak shown in pink and
xix
purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.999720. ............................................................................................................... 105
Figure 82: HPLC chromatogram for Product IV stored at 65 °C for 1 month dissolved
in mobile phase with a retention time of 7.87 minutes. ........................................... 105
Figure 83: Peak purity profile calculated using PDA data (from 190–800 nm) for
product IV stored at 65 °C for 1 month in mobile phase. Peak shown in pink and
purity curve in black. Peak purity index =0.999999; Single point threshold =
0.999803. ............................................................................................................... 106
Figure 84: HPLC chromatogram for Product V stored at 65 °C for 1 month dissolved
in mobile phase with a retention time of 7.90 minutes. ........................................... 106
Figure 85: Peak purity profile calculated using PDA data (from 190–800 nm) for
product V stored at 65 °C for 1 month in mobile phase. Peak shown in pink and
purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.999752. ............................................................................................................... 106
Figure 86: HPLC chromatogram for phenylephrine hydrochloride stored at 40
°C/75%RH for 1 month dissolved in mobile phase with a retention time of 7.86
minutes................................................................................................................... 107
Figure 87: Peak purity profile calculated using PDA date (from 190–800 nm) for
phenylephrine hydrochloride stored at 40 °C/75%RH for 1 month in mobile phase.
Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single
point threshold = 0.999056. .................................................................................... 107
Figure 88: HPLC chromatogram for Product I stored at 40 °C/75%RH for 1 month
dissolved in mobile phase with a retention time of 7.87 minutes. ........................... 107
Figure 89: Peak purity profile calculated using PDA data (from 190–800 nm) for
product I stored at 40 °C/75% RH for 1 month in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999999; Single point threshold =
0.995566. ............................................................................................................... 108
Figure 90: HPLC chromatogram for Product II stored at 40 °C/75%RH for 1 month
dissolved in mobile phase with a retention time of 7.84 minutes. ........................... 108
Figure 91: Peak purity profile calculated using PDA data (from 190–800 nm) for
product II stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.999579. ............................................................................................................... 108
xx
Figure 92: HPLC chromatogram for product III stored at 40 °C/75%RH for 1 month
dissolved in mobile phase with a retention time of 7.81 minutes. ........................... 109
Figure 93: Peak purity profile calculated using PDA data (from 190–800 nm) for
product III stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.994609. ............................................................................................................... 109
Figure 94: HPLC chromatogram for Product IV stored at 40 °C/75%RH for 1 month
dissolved in mobile phase with a retention time of 7.85 minutes. ........................... 109
Figure 95: Peak purity profile calculated using PDA data (from 190–800 nm) for
product IV stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.994609. ............................................................................................................... 110
Figure 96: HPLC chromatogram for Product V stored at 40 °C/75%RH for 1 month
dissolved in mobile phase with a retention time of 7.83 minutes. ........................... 110
Figure 97: Peak purity profile calculated using PDA data (from 190–800 nm) for
product V stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999999; Single point threshold =
0.995332. ............................................................................................................... 110
Figure 98: HPLC chromatogram for phenylephrine hydrochloride alone dissolved in
mobile phase with retention of 7.82 minutes. ......................................................... 115
Figure 99: Peak purity profile calculated using PDA date (from 190–800 nm) for
phenylephrine hydrochloride in mobile phase. Peak shown in pink and purity curve in
black. Peak purity index = 1.000000; Single point threshold = 0.999999. .............. 115
Figure 100: HPLC chromatogram for phenylephrine hydrochloride with sodium
citrate dihydrate (1:1) dissolved in mobile phase with a retention time of 7.92
minutes................................................................................................................... 115
Figure 101: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride with sodium citrate dihydrate (1:1) in mobile phase.
Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single
point threshold = 0.999896. .................................................................................... 116
Figure 102: HPLC chromatogram for phenylephrine hydrochloride with
carboxymethycellulose sodium (1:1) dissolved in mobile phase with a retention time
of 7.82 minutes. ...................................................................................................... 116
xxi
Figure 103: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride with carboxymethycellulose sodium (1:1) in mobile
phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000;
Single point threshold = 0.999999. ......................................................................... 116
Figure 104: HPLC chromatogram for phenylephrine hydrochloride with hypromellose
(1:1) dissolved in mobile phase with a retention time of 7.81 minutes. .................. 117
Figure 105: Peak purity profile calculated using PDA date (from 190–800 nm) for
phenylephrine hydrochloride with hypromellose (1:1) in mobile phase. Peak shown in
pink and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.999999. ............................................................................................................... 117
Figure 106: HPLC chromatogram for phenylephrine hydrochloride with glycerol (1:1)
dissolved in mobile phase with a retention time of 7.85 minutes. ........................... 117
Figure 107: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride with glycerol (1:1) in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.999989. ............................................................................................................... 118
Figure 108: HPLC chromatogram for phenylephrine hydrochloride with
benzalkonium chloride (1:1) dissolved in mobile phase with a retention time of 7.88
minutes................................................................................................................... 118
Figure 109: Peak purity profile calculated using PDA date (from 190–800 nm) for
phenylephrine hydrochloride with benzalkonium chloride (1:1) in mobile phase. Peak
shown in pink and purity curve in black. Peak purity index = 0.999999; Single point
threshold = 0.999918. ............................................................................................ 118
Figure 110: HPLC chromatogram for phenylephrine hydrochloride with EDTA (1:1)
dissolved in mobile phase with a retention time of 7.86 minutes. ........................... 119
Figure 111: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride with EDTA (1:1) in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999999; Single point threshold =
0.999991. ............................................................................................................... 119
Figure 112: HPLC chromatogram for phenylephrine hydrochloride with boric acid
(1:1) dissolved in mobile phase with a retention time of 7.92 minutes. .................. 119
Figure 113: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride with boric acid (1:1) in mobile phase. Peak shown in
xxii
pink and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.999999. ............................................................................................................... 120
Figure 114: HPLC chromatogram for phenylephrine hydrochloride with sodium
metabisulfite (1:1) dissolved in mobile phase with a retention time of 7.82 minutes.
............................................................................................................................... 120
Figure 115: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride with sodium metabisulfite (1:1) in mobile phase. Peak
shown in pink and purity curve in black. Peak purity index = 1.000000; Single point
threshold = 0.999870. ............................................................................................ 120
Figure 116: HPLC chromatogram for phenylephrine hydrochloride with disodium
edetate (1:1) dissolved in mobile phase with a retention time of 7.82 minutes....... 121
Figure 117: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride with disodium edetate (1:1) in mobile phase. Peak
shown in pink and purity curve in black. Peak purity index = 1.000000; Single point
threshold = 0.998891. ............................................................................................ 121
Figure 118: HPLC chromatogram for phenylephrine hydrochloride with propyl
paraben (1:1) dissolved in mobile phase with a retention time of 7.83 minutes. .... 121
Figure 119: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride with propyl paraben (1:1) in mobile phase. Peak shown
in pink and purity curve in black. Peak purity index = 1.000000; Single point
threshold = 0.999948. ............................................................................................ 122
Figure 120: HPLC chromatogram for phenylephrine hydrochloride with methyl
paraben (1:1) dissolved in mobile phase with a retention time of 7.82 minutes. .... 122
Figure 121: Peak purity profile calculated using PDA data (from 190–800 nm) for
phenylephrine hydrochloride with methyl paraben (1:1) in mobile phase. Peak
shown in pink and purity curve in black. Peak purity index = 1.000000; Single point
threshold = 0.999982. ............................................................................................ 122
Figure 122: HPLC chromatogram for product I stored at 30 °C/65%RH for 3 months
dissolved in mobile phase with a retention time of 7.87 minutes. ........................... 123
Figure 123: Peak purity profile calculated using PDA date (from 190–800 nm) for
product I stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999999; Single point threshold =
0.999950. ............................................................................................................... 124
xxiii
Figure 124: HPLC chromatogram for product II stored at 30 °C/65%RH for 3 months
dissolved in mobile phase with a retention time of 7.85 minutes. ........................... 124
Figure 125: Peak purity profile calculated using PDA date (from 190–800 nm) for
product II stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.996473. ............................................................................................................... 124
Figure 126: HPLC chromatogram for product III stored at 30 °C/65%RH for 3
months dissolved in mobile phase with a retention time of 7.87 minutes. .............. 125
Figure 127: Peak purity profile calculated using PDA date (from 190–800 nm) for
product III stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999999; Single point threshold =
0.996955. ............................................................................................................... 125
Figure 128: HPLC chromatogram for product IV stored at 30 °C/65% RH for 3
months dissolved in mobile phase with a retention time of 7.88 minutes. .............. 125
Figure 129: Peak purity profile calculated using PDA date (from 190–800 nm) for
product IV stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999998; Single point threshold =
0.998694. ............................................................................................................... 126
Figure 130: HPLC chromatogram for product V stored at 30 °C/65%RH for 3 months
dissolved in mobile phase with a retention time of 7.87 minutes. ........................... 126
Figure 131: Peak purity profile calculated using PDA date (from 190–800 nm) for
product V stored at 30 °C/65%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999998; Single point threshold =
0.994533. ............................................................................................................... 126
Figure 132: HPLC chromatogram for product I stored at 25 °C/60%RH for 3 months
dissolved in mobile phase with a retention time of 7.91 minutes. ........................... 127
Figure 133: Peak purity profile calculated using PDA date (from 190–800 nm) for
product I stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.995873. ............................................................................................................... 127
Figure 134: HPLC chromatogram for product II stored at 25 °C/60%RH for 3 months
dissolved in mobile phase with a retention time of 7.89 minutes. ........................... 128
Figure 135: Peak purity profile calculated using PDA date (from 190–800 nm) for
product II stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink
xxiv
and purity curve in black. Peak purity index = 0.999998; Single point threshold =
0.999950. ............................................................................................................... 128
Figure 136: HPLC chromatogram for product III stored at 25 °C/60%RH for 3
months dissolved in mobile phase with a retention time of 7.92 minutes. .............. 128
Figure 137: Peak purity profile calculated using PDA date (from 190–800 nm) for
product III stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999999; Single point threshold =
0.998742. ............................................................................................................... 129
Figure 138: HPLC chromatogram for product IV stored at 25 °C/60%RH for 3
months dissolved in mobile phase with a retention time of 7.92 minutes. .............. 129
Figure 139: Peak purity profile calculated using PDA date (from 190–800 nm) for
product IV stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999999; Single point threshold =
0.996396. ............................................................................................................... 129
Figure 140: HPLC chromatogram for product V stored at 25 °C/60%RH for 3 months
dissolved in mobile phase with a retention time of 7.84 minutes. ........................... 130
Figure 141: Peak purity profile calculated using PDA date (from 190–800 nm) for
product V stored at 25 °C/60%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.998093. ............................................................................................................... 130
Figure 142: HPLC chromatogram for product I stored at 40 °C/75%RH for 3 months
dissolved in mobile phase with a retention time of 7.84 minutes. ........................... 131
Figure 143: Peak purity profile calculated using PDA date (from 190 – 800 nm) for
product I stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.998093. ............................................................................................................... 131
Figure 144: HPLC chromatogram for product II stored at 40 °C/75%RH for 3 months
dissolved in mobile phase with a retention time of 7.84 minutes. ........................... 131
Figure 145: Peak purity profile calculated using PDA date (from 190 – 800 nm) for
product II stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.998093. ............................................................................................................... 132
Figure 146: HPLC chromatogram for product III stored at 40 °C/75%RH for 3
months dissolved in mobile phase with a retention time of 7.84 minutes. .............. 132
xxv
Figure 147: Peak purity profile calculated using PDA date (from 190–800 nm) for
product III stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.998093. ............................................................................................................... 132
Figure 148: HPLC chromatogram for product IV stored at 40 °C/75%RH for 3
months dissolved in mobile phase with a retention time of 7.84 minutes. .............. 133
Figure 149: Peak purity profile calculated using PDA date (from 190–800 nm) for
product IV stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 1.000000; Single point threshold =
0.998093. ............................................................................................................... 133
Figure 150: HPLC chromatogram for product V stored at 40 °C/75%RH for 3 months
dissolved in mobile phase with a retention time of 7.91 minutes. ........................... 133
Figure 151: Peak purity profile calculated using PDA date (from 190–800 nm) for
product V stored at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink
and purity curve in black. Peak purity index = 0.999999; Single point threshold =
0.998093. ............................................................................................................... 134
Figure 152: A graph showing standard error and phenylephrine hydrochloride left in
product I–V after 12 weeks at 30 °C/65%RH, 40 °C/75%RH, 25 °C/60%RH. ........ 134
Figure 153: Flow and viscosity curve of Prefrin® and products I–V at time zero for
storage condition 30 °C/65% RH. ........................................................................... 138
Figure 154: Flow and viscosity curve of Prefrin® and products I–V after 3 months for
storage condition 30 °C/65% RH. ........................................................................... 138
Figure 155: Flow and viscosity curve of Prefrin® and products I–V at time zero for
storage condition 40 °C/75% RH. ........................................................................... 139
Figure 156: Flow and viscosity curve of Prefrin® and products I–V after 3 months for
storage condition 40 °C/75% RH. ........................................................................... 139
Figure 157: Flow and viscosity curve of Prefrin® and products I–V at time zero for
storage condition 25 °C/60%RH. ............................................................................ 140
Figure 158: Flow and viscosity curve of Prefrin® and products I–V after 3 months for
storage condition 25 °C/60% RH. ........................................................................... 140
Figure 159: Graph showing viscosity of products I–V stored in a stability chamber of
40 °C/75%RH tested at time zero (T0), three months later (T1) and compared to an
original marketed product Prefrin®. ........................................................................ 141
xxvi
Figure 160: Graph showing viscosity of products I–V stored in a stability chamber of
25 °C/60%RH tested at time zero (T0), 3 months later (T1) and compared to an
original marketed product Prefrin®. ........................................................................ 141
Figure 161: Graph showing viscosity of products I–V stored in a stability chamber of
30 °C/65%RH tested at time zero (T0), 3 months later (T1) and compared to an
original marketed product Prefrin®. ........................................................................ 142
xxvii
LIST OF TABLES
xxviii
Table 18: Physical appearance of products I–V before and after varying storage
conditions for 3 months. ......................................................................................... 137
Table 19: Results of one-way ANOVA analysis for viscosity of products I–V stored at
25 °C/60%RH, 30 °C/65%RH and 40 °C/75%RH for 3 months. The values shown
indicate differences in p-values and significance in differences of mean was defined
as p < 0.05. ............................................................................................................ 143
Table 20: Antimicrobial preservative efficacy of the eye-drop products I–V
challenged with E. coli, S.aureus, P. aeruginosa, C.albicans. ................................ 145
xxix
1. INTRODUCTION
The conjunctival sac holds a limited capacity of fluids which poses another problem,
as most of the eye drop solution is drained into the nasal cavity thereby reducing the
portion of drug that reaches its site of action. Systemic absorption of phenylephrine
hydrochloride into the nasal mucosa may produce unwanted side effects such as
headache, hypertension and tachycardia (Bartlett & Jaanus, 2008). Moreover,
phenylephrine hydrochloride solutions may irritate the eye due to the presence of
1
preservatives such as benzalkonium chloride and methyl paraben amongst others
(Giaconi et al., 2009).
The contact time of eye drops is considered as being the most important factor in
ophthalmic drug delivery (Agarwal et al., 2002). This research study will focus on
formulating an eye drop solution which improves contact time. Various formulations
of phenylephrine hydrochloride eye drops have been made in the quest to improve
contact time. These include viscous solutions (Saettone et al., 1984), rods (Alani,
1978), gels (Durrani et al., 1996), ointments (Saettone et al., 1980; Gurjar et al.,
1998), and a polyvinyl alcohol flag (O’Donnell & Gillibrand, 1995; Maitani et al.,
1997).
However, there is still scope for an eye drop that can be administered easily in less
frequent doses which produces consistent, rapid results and minimizes the risk of
adverse effects to the patient.
2
Validate a high performance liquid chromatographic (HPLC) method for the
quantitative determination of phenylephrine hydrochloride in the finished
product.
Propose formulations of phenylephrine hydrochloride eye drops and
manufacture laboratory scale batches of these.
Characterize the physicochemical properties of the formulations by assessing
appearance, rheology, pH and degradation.
Conduct real time and accelerated stability studies on the formulations; in
accordance with the International Conference for Harmonization (ICH) and
Medicines Control Council guidelines.
Determine the efficacy of antimicrobial preservation.
In order to achieve the above objectives, a well laid out plan was to be followed.
Literature reviews, on the theory relating to phenylephrine and its prodrugs were
undertaken in an effort to understand the API, eye drops, solutions and product
formulation. Active–excipients compatibility studies were conducted using HPLC and
various eye drops were formulated and manufactured on a laboratory scale. These
underwent stability studies in accordance with International Conference on
Harmonization and Medicine Control Council guidelines. Rheological tests and
efficacy of antimicrobial preservation were demonstrated.
3
2. LITERATURE REVIEW
The human eye provides a challenge to formulators who seek to produce dosage
forms where the API is administered ocularly. This is due to (a) the permeability of
the cornea and (b) the protective operation of the eyelids and lacrimal system. The
operation of the eyelids and lacrimal system rapidly removes materials instilled into
the eye; however, this clearance does not apply to materials that are small in volume
and which are chemically and physiologically compatible with surface tissues
(Hughes, 2004).
The eyes are highly specialized organs of photoreception and are protected by
eyelids and the orbit in which they are placed (Rathore & Nema, 2009). The eye can
be divided into two segments, namely the anterior and posterior segments. The
anterior segment comprises of the cornea, iris, the ciliary body, the anterior chamber
and the posterior chamber while the posterior segment comprises of retina and the
vitreous body as seen in Figure 1 (Ghosh & Jasti, 2005).
The unique anatomy, physiology and biochemistry of the eye make it resistant to
foreign substances (Del Amo & Urtti, 2008) this protective mechanism poses a
4
challenge to the formulator who is required to bypass the barriers without causing
damage to the eye (Meqi & Deshpande, 2002). The corneal barrier poses
physiological constraints due to its poor permeability which reduces the absorption of
ophthalmic drugs. The cornea is made up of three membranes; the epithelium, the
endothelium and inner stroma (Chien et al., 1990). The epithelium has tight
junctions that serve as a selective barrier to ion transport, thereby limiting the
diffusion of macromolecules via the paracellular route. The stroma is a highly
lipophilic layer that lies beneath the epithelium, and the more lipophilic a drug is, the
less resistance it will have crossing the stroma (Patel et al., 2010).
The eyelids, conjunctiva, lacrimal systems, cornea–precorneal film and its absorption
are discussed below as they play a major role in the absorption metabolism of eye
drops.
Eyelids: The eyelids have two functions: mechanical protection of the globe (eye)
and creation of an optimum environment for the cornea. The eyelids are lubricated
and kept moist by secretions of the lacrimal glands and specialized cells found in the
bulbar conjunctiva. The antechamber is shaped in a narrow cleft manner directly
over the front of the eyeball, with pocket-like extensions upward and downward. The
pockets are called the superior and inferior fornices (vaults), and the entire space is
called the cul-de-sac. The oval opening between the eyelids is called the palpebral
fissure (Zide, 2006).
Lacrimal system: The conjunctival and lacrimal glands secrete a film of fluid which
covers and lubricates the conjunctival and corneal surfaces. The lacrimal glands
produce tears which are delivered through a number of fine ducts into the
5
conjunctival fornix. A tear is a clear, watery fluid containing salts, glucose, other
organic compounds, approximately 0.7% protein, and the enzyme lysozyme. Small
accessory lacrimal glands are situated in the conjunctival fornices. Their secretion
provides lubrication and cleansing during ordinary conditions and also maintains a
thin fluid film covering the cornea and conjunctiva (the precorneal film). The stability
of the film is maintained through the mucin–protein layer. The sebaceous glands of
the eyelids secrete an oily fluid that prevents overflowing of tears at the lid margin
and reduces evaporation from the exposed surfaces of the eye by spreading over
the tear film (Hughes, 2004).
Blinking helps in replenishing the fluid film by pushing a thin layer of fluid ahead of
the lid margins as they come together. The excess fluid is directed into the lacrimal
lake which is a small, triangular area lying in the angle bound by the innermost
portions of the lids. The skin of the eyelids is the thinnest in the body and folds
easily which permits rapid opening and closing of the palpebral fissures. The eyelids
provide controlled movement such as narrowing of the palpebral fissures in a zipper-
like action from the lateral canthus toward the medial canthus. The transport or
movement of fluid toward the lacrimal lake is aided by the eyelids (Del Amo & Urtti,
2008).
Tears are drained from the lacrimal lake by two small tubes–the lacrimal canaliculi–
which go into the upper part of the nasolacrimal duct, called the lacrimal sac. The
drainage of tears into the nose does not depend only on gravity. Fluid moves along
the lacrimal canaliculi by capillary attraction supported by aspiration caused by
contraction of muscle found in the eyelids. The blinking action causes contraction of
the muscles inducing dilation of the upper part of the lacrimal sac and compression
of its lower portion (Cohen et al., 2006). Tears are aspirated into the sac, which is
collected in its lower part by forcing down the tears through the nasolacrimal duct
toward its opening into the nose. As the muscle relaxes, the lids open. Owing to
muscle relaxation, the upper part of the sac forces fluid into the lower part where it is
simultaneously released from compression. The act of blinking therefore exerts a
suction force-pump action in removing tears from the lacrimal lake as well as
emptying them into the nasal cavity (Cohen et al., 2006). Lacrimation is induced by
reflex action through the stimulation of nerve endings of the cornea or conjunctiva.
6
The reflex could be abolished by anaesthetization of the surface of the eye and by
disorders affecting its nerve components (Cohen et al., 2006).
The cul-de-sac is free of pathogenic organisms and is sterile. The sterility is due to
the action of lysozyme, in the tears, which destroys saprophytic organisms but has
little action against pathogens. Certain diseases cause the lacrimal gland, to undergo
involution, resulting in scanty lacrimal fluid production and a change in the
conjunctival glands also leads to changes in the amount of the secretions. This may
lead to symptoms of dryness, burning and general discomfort and may interfere with
visual acuity (Kaur & Kanwar, 2002).
Precorneal film: The precorneal film is composed of a thin outer lipid layer, a thicker
middle aqueous layer, and a thin inner mucoid layer. It is renewed during each blink,
and when blinking is suppressed, either by drugs or by mechanical means, it dries in
patches. The precorneal film is unaffected by the addition of concentrations of up to
2% sodium chloride into the conjunctiva. The precorneal film, which is part of the
tear fluid, maintains the cornea’s moist surfaces and its functionality depends on the
condition of the corneal epithelium. A pH below four or above nine causes
derangement of the film (Grosvenor, 2007).
Cornea: The cornea has a thickness of 0.5 to 1 mm and consists mainly of the
following structures (from the front backward): Corneal epithelium, subtantia propia
(stroma), corneal endothelium. The cornea is transparent to visible light due to the
special laminar arrangement of the cells and fibres and because of the absence of
blood vessels (Chien & Schoenwald, 1990). Cloudiness of the cornea may be due to
several factors including excess pressure in the eyeball as in glaucoma, and scar
tissue due to injury, infection, deficiency of oxygen or excess hydration such as may
occur during the wearing of improperly–fitted contact lenses. Trauma to the cornea
usually heals as an opaque patch that can permanently impair vision unless it is
located in the periphery of the cornea (Kaur et al., 2003).
The outer surface of the cornea is most responsible for the refraction of light
whereby the index of refraction changes from that of air (1.00) to that of precorneal
substance (1.38). Alteration in its shape or transparency interferes with the formation
7
of a clear image; therefore any pathological process, however little, may seriously
interfere with the resolving power or visual acuity of the eye (Lang et al., 2005).
The normal cornea possesses no blood vessels except at the corneoscleral junction.
The cornea derives its nutrition from the aqueous humour by diffusion and has
certain permeable characteristics. It also receives nourishment from the fluid
circulating through the chambers of the eye and from the air. The corneal epithelium
provides an efficient barrier against bacterial invasions due to its poor blood supply.
Foreign bodies that either scratch the cornea or lodge or become embedded in the
cornea are of serious concern, as the presence of any of these could play a role in
permitting pathogenic bacteria to gain access to the cornea. Trauma plays an
important part in most of the infectious diseases of the cornea that occurs
exogenously (Ghosh & Jasti, 2005).
Eye drops penetrate the eye primarily through the cornea. Typical drug penetraton
occurs as follows:
a) The drug molecule as a free base and the salt will be in an equilibrium
depending on the drugs physicochemical characteristics and the pH.
8
b) If the formulation is slightly acidic at the moment of instillation, the acidity is
neutralised by the lacrimal fluid which converts it rapidly to the physiological
pH range ( pH 7.4).
c) There will be sufficient free base to begin penetration of the corneal
epithelium at this pH. An undissociated drug molecule will penetrate the
stroma, epithelium, and endothelium because it is water–soluble.
d) The dissociated drug leaves the endothelium for the aqueous humour, as it
can readily diffuse to the iris, the ciliary body, and the site of the
pharmacological actions (Nanjawade et al., 2007).
Drugs penetrate the corneal epithelium with the use of transcellular or paracellular
pathways which are both types of transcorneal penetration. Transcellular and
paracellular pathways are used by both lipophilic and hydrophilic drugs; these
pathways involve passive diffusion or modified diffusion through intercellular spaces
and most topically–applied drugs diffuse passively along their concentration gradient.
However, the stereospecific carrier–mediated transport system is used by certain
drugs as their mode of transport such as l-lysine which makes use of the Na+ –K+ –
ATPase pump as medium of transport in the cornea (Urtti, 2006). Physicochemical
properties of drugs have properties such as lipophilicity, solubility, molecular size
and shape, charge and degree of ionization affects the route and rate of permeation
in the cornea (Järvinen et al., 1995). The hydrophilic drugs are rate–limited by the
9
lipophilic corneal epithelium, while lipophilic drugs partitioning from the epithelium to
the hydrophilic stroma are rate limiting (Järvinen et al., 1995).
Non-corneal absorption pathways involve the penetration of drug across the bulbar
conjunctiva and sclera into the uveal tract and vitreous humour. The conjunctiva and
sclera provide a route for large hydrophilic molecules for example inulin and p-
aminoclonidine (Nanjawade et al., 2007; Lang et al., 2005).
10
Seasonal allergic conjunctivitis (SAC) is the most common form of allergic eye
disease that constitutes of 90% of the allergy cases with perennial allergic
conjunctivitis at 5% (Bielory, 2008). SAC is most frequently caused by grass, tree
and weed pollens and outdoor moulds which peak at different times of the year
(Bielory, 2008; Chigbu, 2009), often as part of seasonal rhinoconjunctivitis (hay
fever). However, PAC occurs year round due to house dust mites, animal dander,
insects and indoor moulds (Chigbu, 2009). The signs and symptoms of SAC develop
gradually but sometimes it can develop suddenly in contact with an offending
allergen (Bielory, 2000 & Bielory 2008). Some of the signs and symptoms of SAC
includes itching, tearing, eyelid oedema and conjunctival hyperaemia, chemosis and
papillary reaction in which the severity often varies with pollen counts (Cox, 2007).
PAC and SAC have similar signs and symptoms but differ as PAC is milder and
chronic in nature and may have seasonal exacerbations (Bielory, 2008 & Buckley,
1998). The impact of allergic eye disease on the quality of life can be profound even
though the signs of symptoms of SAC and PAC are relatively mild (Bielory, 2006). It
affects daily activities, productivity at work, school performance and the
economy (Pitt et al., 2004; Smith et al., 2005; Palmares et al., 2010). The severities
of presentation limits the complications of SAC and are linked to steroid use in cases
refractory to conventional treatment (Hingorani and Lightman, 1995; Joss & Craig,
1998; Bielory et al., 2010). The treatments provided for SAC are mainly for
preventing and alleviating symptoms rather than cure due to the fact that no cure has
been found or produced for SAC or any allergy. However, immunotherapy shows
promise (Bielory, 2008).
11
mediators as well as the secretion of chemokines and cytokines (Tkaczyk et al.,
2002).
Both SAC and PAC are IgE-mast cell mediated hypersensitivity reactions, divided
into two phases with the mast cell playing a central role (Bacon et al., 2000; Choi &
Bielory 2008). The reaction involves a very complex series of immunological events
coordinated by various mediators initiated by an allergen (Ono & Abelson, 2005 ;
Hodges & Kean–Myers, 2007) An allergen such as pollen reacts with specific IgE
antibodies bound to a sensitised mast cell, triggering cross linkage of the IgE
molecules and an influx of calcium ions into the mast cell. This causes the mast cell
to degranulate and release preformed inflammatory mediators such as histamine
which cause the signs and symptoms associated with the early phase response in
sensitised individuals. The early phase response is immediate and lasts clinically for
20–30 minutes (Leonardi et al., 2008).
Mast cell degranulaion also initiates a series of cellular and extracellular events
which lead to the late phase response, including production of prostaglandins,
thromboxanes and leukotrienes derived from arachidonic acid. Mast cells also
release cytokines and chemotactic factors which induce the production of IgE from
B-cells, enhance production of Th2-lymphocytes, attract eosinophils and activate
12
vascular endothelial corneal and conjunctival cells to release chemokines and
adhesion molecules. The chemokines and adhesion molecules mediate the
infiltration of eosinophils, basophils, neutrophils and Th2-lymphocytes to the site of
inflammation and coupled with the newly formed mediators and sustained mast cell
activation they result in the late phase response (Stahl et al., 2002). This may occur
3–12 hours after the initial reaction and symptoms can continue up to 24 hours. The
year round symptoms associated with PAC are the result of chronic mast cell
activation and Th2-lymphocyte infiltration (Bonini et al., 1989).
The most important and most effective step in treating allergic eye disease is
avoiding the offending allergen to prevent the hypersensitivity reaction from being
triggered. This necessitates the identification of the offending allergen and complete
avoidance is not always possible (Friedlaender, 2001). In SAC a detailed history is
essential as knowledge of the period of time of year symptoms occur can allow
identification using a pollen calendar to some extent but peak levels of common
causative pollens often overlap. However, effective measures for allergen avoidance
in SAC and PAC are based upon control of the environment. Given that pollens are
the main cause of SAC, preventative measures include limiting outdoor activity
during the symptomatic period, closing windows and using air conditioning when in a
car or indoors, avoid touching/rubbing eyes after being outdoors, wash hands after
being outdoors and wearing close fitting or wrap around style sunglasses when
outdoors (Veys, 2004). As PAC can affect the patient all year round, more thorough
avoidance measures are necessary. Dust mite levels in the home can be reduced by
using and regularly replacing protective pillow, mattress and duvet covers; washing
bedding regularly at least at 60 °C; vacuum and damp dust entire house on weekly
basis; reduce humidity to between 35 and 50% and remove or regularly clean
carpets, upholstery, curtains and any other areas that gather dust (Gotzche &
Johansen, 2008; Scheikh et al., 2010). Animal dander can be reduced by eliminating
all pets/animals from the home or keep them outdoors; regular vacuuming;
minimising exposure to areas that gather animal dander; avoid touching animals;
washing hands and avoid eye touching/rubbing after contact with animals; and
washing all clothes that have come into contact with animals (Eggleston & Wood,
1992; Bush, 2008).
13
Despite implementing these avoidance measures, complete avoidance is not always
possible so use of anti–allergic medication may become necessary to prevent and
alleviate symptoms. With increased knowledge of the pathophysiology of the
hypersensitivity reaction in SAC and PAC over the years, there has been a rapid
increase in the number of anti-allergic medications that target the immunological
cells and inflammatory mediators involved in the allergic expression. Ophthalmic
anti-allergic medications include topical mast cell stabilisers, antihistamines,
vasoconstrictors (phenylephrine hydrochloride), antihistamine–vasoconstrictor
combinations and dual action agents with mast cell stabilising and antihistaminic
properties (Schultz, 2006).
Studies on the ocular use of phenylephrine hydrochloride started in 1933 and were
first reported in 1936 (Greaves et al., 1992). The present research or study is based
on the insight into phenylephrine hydrochloride and its properties.
14
2.3.2 Pharmacological actions and uses
Phenylephrine, or 3-(1–hydroxyl–2–methylamino-ethyl) phenol (Trommer et al.,
2010) is a synthetic, imidazole-derivative, completely used in the optically active form
(Pandey et al., 2003). Figure 2 shows the structure and available salts of
phenylephrine in the pharmaceutical industry.
Figure 2: Structure of Phenylephrine, its base and salts (Trommer et al., 2010).
Ornato and Peberdy (2005) showed that phenylephrine hydrochloride solution could
be used in place of epinephrine to increase regional cerebral blood flow following a
prolonged cardiac arrest during cardiopulmonary resuscitation.
15
2.3.4 Pharmacokinetics
The protective mechanisms that are present in the eye to shield the visual pathway
from foreign chemicals make it hard for drug to be delivered to the eye. These
mechanisms have led to the development of optimized ophthalmic drug delivery
systems that are based on the understanding of the drug disposition pathways in the
eye and the ophthalmic pharmacokinetic profile (Worakul & Robinson, 1997).
Phenylephrine hydrochloride is administered topically to avoid extensive first pass
metabolism and to reduce systemic side effects (Aschenbrenner & Venable, 2008).
Peak ocular concentrations of 1.2% to 5% are achieved after a single dose between
20 to 60 minutes. Maximal dilation of the pupil occurs within 60–90 minutes and the
effect lasts 5–7 hours (Rossiter, 2010).
Phenylephrine has a half-life of a few minutes when circulating in the blood. It can be
degraded either through methylation by COMT or by deamination by MAO in the
blood stream (Flancbaum et al., 1997). The dehydrated form of phenylephrine
hydrochloride is a major degradation derivative during pharmaceutical processing
and stability determination (Trommer et al., 2010).
Eye drops containing 10% of API may have profound effects on the cardiovascular
system, and the risk is smaller with 2.5% products, which are said to be equally
effective as mydriatics (Rossiter, 2010). The hypertensive effects of phenylephrine
may be treated with an alpha–adrenoceptor blocking agent such as phentolamine
mesylate, 5 to 10 mg intravenously which should be repeated as necessary (Beyene
& Maren, 2004).
16
2.3.6 Drug interactions
2.3.7 Bioavailability
The average human being has a tear volume of 7 µL while the cul-de-sac has a
maximum capacity of about 30 µL. Many ophthalmic formulations range from 50 to
75 µL in volume; however, volumes in excess of 50 µL are probably unable to enter
the cul-de-sac (Greaves et al., 1992). Systemic absorption occurs through solution
drainage into the nose, which causes the loss of an instilled drug as shown in Figure
3 (Jarvinen et al., 1995).
Ocular tissues
The equation describing the above processes in terms of drug concentration is:
17
Where F is the fraction of dose absorbed, D is the dose, k and K are absorption and
elimination rate constants, respectively, and V, is the apparent volume of distribution
(Lee & Robinson, 1979; Worakul &Robinson, 1997; Lee & Robinson, 1986).
The most common method for the administration of therapeutic treatment for ocular
diseases is the topical delivery of eye drops. The rapid elimination of drugs from the
precorneal lachrymal fluid by solution drainage, lachrymation, and poor absorption
by the conjunctiva creates problems for topical delivery (Bartlett & Jaanus, 2008).
The eye has the ability to permanently maintain its residence volume at 7–10 µL due
to high drainage rate. The volumes instilled with eye drops range from 20–50 µL,
therefore the residence time of solutions is limited to a few minutes, causing the
overall absorption of a topically applied drug to be limited to 1–10% (Felt et al.,
1999).
18
2.3.7.2 Strategies for improving drug availability in ocular adminstration
There are three main ways to maximise the systemic bioavailability of drugs
administered ocularly, these are:
Lacrimal secretions and drainage systems act to remove foreign bodies and
substances from the corneal epithelium as quickly as possible. In order to increase
residence time, or delay clearance, drops should be instilled in the anterior segment
of the ocular cavity. Products can also be formulated with polymers such as
methylcellulose, hydroxypropyl methylcellulose or polyacrylic acid (Carbopol), which
increases the viscosity of the formulation and act as bio–adhesives with the ocular
tissue (Patel et al., 2010).
Enhancers act by increasing the rate at which drugs penetrate the epithelium. They
alter the structure of the epithelial cells to increase absorption; this is achieved
without destruction of the cells. Some examples are dimethyl sulfoxide,
decamethonium, EDTA and glycocholate. Ideal absorption enhancers should
possess the following qualities:
19
Should be stable (Saettone et al., 1996; Patel et al., 2010).
The effect of pH and the partition coefficient: The partition coefficients of drugs
are dependent upon environmental pH which affects the ionization of drugs (local pH
can be modified by ocular formulation). The rate of absorption is increased when the
drug or substance is un–ionized. The movement of ionizable drugs through
membranes depends on the chemical equilibrium between ionized and unionized
drug in the eye drop and the lacrimal fluid (Geroski & Edelhauser, 2000). The ionized
form does not easily penetrate the lipid membrane compared to the unionized
molecule. The partitioning of ionized molecule does not only depend on the degree
of ionization but also on the charge of the molecules (Liaw et al., 1992). The corneal
epithelium is negatively charged (isoelectric point is pI 3.2), and as a result,
hydrophilic–charged cationic substances permeate easily through the cornea than
anionic compounds (Liaw et al., 1992). Compounds below pI 3.2 are negatively-
charged and pass through the cornea making the pH too acidic, and irritating for
clinical use. Consequently, in practice, charge–discriminating effects of the corneal
20
epithelium decrease the absorption of negatively charged compounds (Rojanasakul
& Robinson, 1989; Conroy & Maren, 1995; Taylor, 2002; Lee & Robinson, 2009;
Hecht, 2000).
A polymorph is a solid substance with the ability to crystallize in at least two different
crystal structures that produce distinct crystal species. Polymorphism is important in
the drug development process as it can affect drug dissolution, chemical stability, as
well as drug bioavailability (Yoshihashi et al., 2002). Polymorphs are the collection of
different crystal structures that can exist for a single chemical entity and its hydrates.
The difference between a solvate and polymorph is that a solvate contains different
quantities of solvent trapped within the crystal structure (as is the case for
MgSO4.7H2O and MgSO4.10H2O) and polymorphism is when there is no solvent
trapped in the crystal structure and the same chemical species are found in various
crystal structures. Solvates or false polymorphs are called pseudopolymorphs.
Pseudopolymorphs can be obtained by changing the recrystallizing solvent.
Common solvents used to induce polymorphic change are water, methanol, ethanol,
and acetone and chloroform (Lee et al., 2006). Phenylephrine hydrochloride does
not exhibit polymorph or solvate forms, its chemical stability will therefore not be
hampered when dissolved in solvents like water and glycerol (Yoshihashi et al.,
2002).
Eye drops are sterile products, free from foreign particles, compounded and
packaged for ocular drug delivery. Eye drops can be prepared as single or multidose
products (Van Santvliet & Ludwig, 2004).
Compared to the buccal, nasal, rectal, vaginal or dermal routes, eye drops are easier
to use, non-invasive, accurate and less expensive for systemic delivery of drugs
(Pillion et al., 1994). The ocular route helps eliminate painful parenteral injections or
gastro-intestinal degradation (Chiou et al., 1991 and Pillion et al., 1994).
21
Absorption of the eye drops across the mucosa in the nasal cavity that is near to the
conjunctival sac helps eye drops with active pharmaceutical ingredients reach the
bloodstream (Winfield et al., 2009). Physicochemical drug properties, such as
lipophilicity, solubility, molecular size and shape (Huang et al., 1989; Liaw &
Robinson, 1992), charge (Rojanasakul et al., 1992; Liaw et al., 1992) and degree of
ionization (Brechu & Maren, 1993) affect the route and rate of permeation in the
cornea.
Drugs can be absorbed through the naso-lacrymal system and reach systemic
circulation without passing metabolism by the liver only if there is (a) drainage loss of
topically-applied solutions,(b) increase in bioavailability of the instilled drug (Saettone
et al., 1984), and (c) an increase in viscosity (Flach, 2002). Mild to severe side
effects may therefore be observed. The phenomenon has led to the reduction in the
size of eye drops (Saettone et al., 1984; Ludwig & Van Ooteghem, 1989; Salminen,
1990; Flach, 2002).
Liquid formulations (eye drops and eye lotions), semisolids (creams, ointment and
gels), solids (biodegradable implants) are various ophthalmic dosage forms used to
deliver therapeutic agents topically (Kaur & Kanwar, 2002). Eye drops are
conventional dosage forms that account for 90% of the currently accessible
ophthalmic formulations. Eye drop solutions are common dosage form as they offer
advantages like administrations, easy preparation and low costs. Eye drops also
have some disadvantages like short contact time with ophthalmic surface and
nasolacrimal drainage which causes poor bioavailability of the drug (Ali &
Lehmussaari, 2006). Ophthalmic delivery systems are being investigated in order to
increase the corneal permeability and prolong the contact time with the ocular
surface through the addition of viscosity modifying agents such as hydroxypropyl
methylcellulose, carboxy methylcellulose sodium and other cellulose derivatives
(Gad, 2008).
Suspensions are defined as the dispersion of finely divided, relatively insoluble drug
substances in aqueous vehicle containing suitable suspending and dispersing
agents (Hecht, 2000). Due to the tendency of particles being retained in the cul-de-
22
sac, the contact time and duration of action of a suspension could theoretically
exceed that of a solution. There are difficulties associated with suspension dosage
forms such as dosage uniformity, crystal growth and polymorphism. Dosage
uniformity requires a brisk “shake well” approach to distribute the suspended drug.
This can contribute towards poor patient compliance as significant numbers of
patients do not adhere to instructions (shake well before use). Polymorphism can
occur during storage resulting in an increased or decreased solubility whereby
changes are reflected in increased or decreased bioavailability (Hecht, 2000).
Ointments offer longer contact time, greater total drug bioavailability albeit with
slower onset and time to peak absorption. Ointments interfere with vision and are
usually restricted to bedtime use. They remain a popular paediatric dosage form and
for postoperative use (Gad, 2008).
Solutions are the preferred ophthalmic dosage forms for treatment of conditions such
as uveitis (Ali & Lehmussaari, 2006). These forms are preferred because:
Limited bioavailability due to short contact time between the eye and the active
pharmaceutical ingredients is one shortcoming of solutions. The short contact time
between the active pharmaceutical ingredient and the eye surface can be increased
by the inclusion of a viscosity-modifying agent such as methylcellulose. The optimum
level of drug retention and visual comfort is within the viscosity range of 0.15 to 0.25
Pa·s (Lang et al., 2005).
There are factors to be considered in the preparation of eye drop solutions. These
include sterility, clarity, buffer, buffer capacity and pH, tonicity, viscosity, stability,
comfort, additives, particle size, packaging and preservatives. These factors are
23
interrelated and assessed collectively in the preparation of an eye drop product. The
buffer system is considered along with tonicity and comfort. Stability of the eye drop
product depends on pH, buffer system, and packaging. Viscosity-modifying agents
which may or may not be present should not affect the therapeutic effectiveness of
the active ingredient (Lang et al., 2005).
The pH and buffer capacity is a compromise between stability of the drug and
comfort in the eye. Optimum patient comfort is found at the pH of the tear fluid, or at
7.4 pH, while optimum stability for many drugs is lower than 5 pH. Buffers are
therefore needed to maintain the desired pH for drug stability and allow it to be
altered to 7.4 immediately after instillation in the eye (Aulton, 2002).
Formulation Number %
Gels 2 0.7
Injectables 11 3.8
Inserts 11 3.8
Ointments 50 17.4
Orals 9 3.1
Solutions 179 62.4
Suspensions 25 8.7
2.4.2.1 Clarity
24
particles to the solution during prolonged contact such as shelf-life storage (Michael
& Richards, 2009).
Stability is not only the chemical stability of a single product component but the total
product. A well-planned stability programme will consider and evaluate the chemical
stability of the active ingredient’s preservative efficacy against selected test
organisms, and test the adequacy of the package over time (McDonnell, 2007).
Ideally, the prepared eye drop should be formulated at a pH equivalent to the tear
fluid value of pH 7.4. The majorities of active ingredients used in eye drops are salts
of weak bases and are stable at acidic pH. The acidic pH selected should therefore
be maintained throughout the product’s shelf life, however, if the buffer capacity is
sufficient to resist adjustment by tear fluid, and the overall eye pH remains acidic for
a considerable period, stinging and discomfort may result (Florence & Attwood,
2006). The buffer capacity should thus be adequate for stability but minimized, as far
as possible, to allow the overall pH of the tear fluid to be disrupted only momentarily
(Florence & Attwood, 2006). Common buffering agents found in eye drop
formulations are sodium citrate dihydrate and boric acid (Florence & Attwood, 2006).
25
Boric acid is odourless, colourless and greasy to the touch. It has weak bacteriostatic
and fungistatic properties. It is commonly used as a buffer and antimicrobial in eye
drops at concentrations of 1.22%. Boric acid volatilizes in steam (Stewart, 2004).
2.4.2.3 Tonicity
Tonicity refers to the osmotic pressure exerted by salts in aqueous solution. An eye
drop solution is isotonic with another solution when the magnitudes of the colligative
properties of the solutions are equal. An eye drop solution is considered isotonic
when its tonicity is equal to that of a 0.9% sodium chloride solution. The calculation
of tonicity at one time was stressed rather heavily to the detriment of other factors
such as sterility and stability. In actuality the eye is much more tolerant of tonicity
variations than it was formerly thought. The eye tolerates solutions equivalent to a
range of 0.5 to 1.8% sodium chloride (Ansel et al., 2011).
26
2.4.2.4 Viscosity
Medicated eye drops usually have poor bioavailability due to the barrier created by
the precorneal area. The site of absorption is cleared rapidly by protective
mechanisms such as blinking and nasolacrimal drainage (Ali & Lehmussaari, 2006).
This necessitates frequent instillation, increasing side effects associated with the
active pharmaceutical ingredient (Di Colo et al., 2009). There is need therefore to
increase API residence in the eye by increasing or adding the polymer that prolongs
drug contact time with the ocular surface (Wilson, 2004).
27
commonly used for antimicrobial protection. Aqueous solutions may be sterilized by
autoclaving; the coagulated polymer must be redispersed on cooling by shaking
(Harwood, 2006).
28
(hydroxypropyl methylcellulose, hydroxyl propylcellulose, hydroxyl ethylcellulose,
methylcellulose and carboxy methylcellulose). Cellulose esters are
celluloseacetophtalate, microbial polysaccharides (dextran, xanthan gum, gellan,
gum and scleroglucan), algal polysaccharides (sodium alginate and carrageenan)
and animal polysaccharides (sodium hyaluronate, chondroitin sulphate and
chitosan).
In this study the following excipients were used as the viscosity modifying agent’s
carboxy methylcellulose sodium, glycerol and hydroxylpropyl methylcellulose (Rowe
et al., 2003). They were used to thicken and stabilize the formulation (Swarbrick et
al., 2000). They also lack reactivity with other excipients and PE and do not irritate
the eye (Ranucci & Silverstein, 1998).
29
2.4.2.5 Additives
Very few additives in eye drop solutions are permissible because they either reduce
the efficacy of the active pharmaceutical ingredient or are toxic for use in the ocular
region. Antioxidants like sodium bisulfite or sodium metabisulfite are permitted in
concentrations of up to 0.3%, particularly in solutions containing phenylephrine and
epinephrine salts. The antioxidant acts as a stabilizer to minimize oxidation of PE
and epinephrine. Other types of antioxidants are ascorbic acid and acetylcysteine
(Järvinen et al., 1995).
Non-ionic surfactants which are least toxic to the ophthalmic tissues are permitted at
concentrations of approximately 0.005%–0.2%. Surfactants are used as co-solvents
to increase solubility and to improve topical bioavailability of the API and to improve
the therapeutic response of an ophthalmic drug. Some surfactants can prolong
ocular residence time of the drug enhancing ocular permeation of the drug molecules
(Urtti & Salminen, 1993; Järvinen et al., 1995).
A common penetration enhancer is EDTA which loosens the tight junctions between
the superficial epithelial cells, facilitating paracellular transport (Saettone et al., 1996;
Hochman & Artursson, 1994). It is used in concentration of 0.1%.
30
Sodium metabisulfite has antioxidant, antimicrobial (in acidic preparations), and
antibrowning agent properties. In acidic preparations, sodium metabisulfite is used at
concentrations of 0.01–1% and less than 0.5% in alkaline preparations is slowly
oxidized to sodium sulfate on exposure to air and moisture. Sodium metabisulfite is
immediately converted to sodium ions (Na+) and bisulfite ions (HSO3+) when mixed
with water. The crystals also disintegrate when exposed to air and moisture.
Decomposition of aqueous sodium metabisulfite occurs as it exposed to air. The bulk
material should be stored in a well-closed container protected from light, in a cool,
dry, place (Stewart, 2006).
Edetic acid and edetate salts are used in ophthalmic pharmaceutical formulations as
chelating agents. Edetic acid and edetates are primarily used as antioxidant
synergists by sequestering trace amounts of metal ions, particularly copper, iron, and
manganese, which might otherwise catalyze autoxidation reactions. Edetic acid and
edetates may be used alone or in combination with true antioxidants, the usual
concentration employed being in the range 0.005–0.1% w/v. Edetic acid possesses
antimicrobial activity against Gram-negative microorganisms, Pseudonomas
aeruginosa, some yeasts, and fungi, although this activity is insufficient for edetic
acid to be used effectively as an antimicrobial preservative on its own. Many
solutions used for the cleaning, storage and wetting of contact lenses contain
disodium edentate. Edetic acid occurs as a white crystalline powder (Weller, 2006).
Aqueous solutions of edetic acid or edetate salt may be sterilized by autoclaving,
and should be stored in an alkali-free container. Edetic acid and edentates should be
stored in well-closed containers in a cool, dry, place (Weller, 2006).
The most widely used vehicle for pharmaceutical products which is also a solvent is
water. This is due to its physiological compatibility with many compounds and
absence of toxicity. It has a high dielectric constant which is necessary for ensuring
the dissolution of a wide range of ionisable materials (Mido et al., 2003).
There are two types of water used in the production of pharmaceutical products:
purified water and water for injections. Purified water must be free of ionic and
organic chemicals and microbial increase. It is produced by mains water going
through a unit that deionizes and distills the water, has an ion-exchange, provides for
31
reverse osmosis and finally filters. All purified water systems must be validated.
Purified water should be supplied constantly at ≥ 80 °C (Potdar, 2007).
Water for injection is used in the production of injections and other pharmaceutical
applications like cleaning of critical equipment and preparation of pharmaceutical
products. The water for injection gets its feed from purified water, which is subjected
to further distillation and reverse osmosis. The requirements for bacterial endotoxin
tests are much higher values as it must be free of microbial contamination and
endotoxins. Water for injection must show no reaction with the limulus amebocyte
lysate (LAL) reagent which is used to test for microbes (Potdar, 2007).
An important factor for eye drops is that they should be sterile when dispensed in a
multiple-application container (Reddy & Ganesan, 1996). Aseptic techniques during
the manufacture of injections have to be adopted when manufacturing sterile eye
drops (Turco & King, 2005).
32
any deficiencies in the manufacturing procedures, and the second is that the
preservative should be an integral part of the formulation, chosen to afford protection
in that particular environment (Parker, 2002).
Serious ocular infection can result from the use of contaminated eye drop solutions.
Such solutions are the cause of corneal ulcers and loss of eyesight. Contaminated
solutions have been found in use and dispensed on prescription in community and
hospital pharmacies (Hodges, 2002).
33
organisms penetrate through an abrasion into the corneal stroma, within the corneal
stroma, traces of antimicrobial agents are neutralized by tissue components and the
organisms finds an excellent culture medium for rapid growth and dissemination
through the cornea and the anterior segment of the eye (Fassihi, 2001).
2.5 Sterilization
34
2.5.1 Steam under pressure as a method of sterilization
35
be found in different shapes and sizes and in two categories, horizontal and vertical
laminar-flow (Turco & King, 2005). Laminar-flow is not a guarantee of sterility and
correct procedures and sterile techniques are necessary (McHugh, 2010).
Laminar flow areas are supplied with air passed through high effiecincy particulate
filter (HEPA) filters which are high density mats composed of randomly arranged
fibres and are used as air filters to remove much smaller particles such as dust
pollutants and microbes. The fibres, made of fibreglass, have a diameter-range of
0.5 and 2.0 µm.
Factors that affect function of HEPA filters are fibre diameter, filter thickness, and
face velocity (Abdel-Magid & Caron, 2006).
The HEPA filters remove finer particles in air passed through them, and virtually free
it from foreign matter. Ultraviolet light (UV) is installed in the air ducts on the
downstream side of the filter to kill microbes that may have escaped through or
around the filter (Brooks et al., 2007). The hood air flow should have an air velocity
of 100 ft. / minute (Potdar, 2007).
Preservative substances must be evaluated as part of the total eye drop preparation
in the proposed package. The growth or presence of microorganisms in the eye drop
can lead to destruction of the product or transmission of disease to the consumer.
Destruction can be in the form of chemical degradation of drugs or excipients by the
enzymes produced by the microorganism or a breakdown of the physical attributes
such as tactile change, visible change in colour, or smell (Amin et al., 2010).
The USP (2004) states that eye drop solutions may be packaged in multiple-dose
containers and must contain a substance or mixture of substances to prevent the
growth of, or to destroy, microorganisms introduced accidentally when the container
is opened during use. Eye drop solutions prepared and packaged for a single
application, need not contain a preservative because they are not intended for reuse.
36
Preservatives are not to be used in solutions intended for intraocular use because of
the risk of irritation.
37
storage conditions. Common compounds used as preservatives are discussed
below.
Benzalkonium chloride solutions are active against a wide range of bacteria, yeasts,
and fungi. Activity is more marked against Gram-positive than Gram-negative
bacteria and minimal against bacterial endospores and acid-fast bacteria. The
antimicrobial activity of benzalkonium chloride is significantly dependent upon the
alkyl composition of the homolog mixture. Benzalkonium chloride is ineffective
against some Pseudomonas aeruginosa strains, Mycobacterium tuberculosis,
38
Trichophyton interdigitale, and Trichophyton rubrum. However, combined with
disodium edetate (0.01–0.1%w/v), benzyl alcohol, phenylethanol, or phenylpropanol,
the activity against Pseudonomas aeruginosa is increased. Antimicrobial activity
may also be enhanced by the addition of phenylmercuric acetate, phenylmercuric
borate, chlorhexidine, cetrimide, or m-cresol. In the presence of citrate and
phosphate buffers (but not borate), activity against Pseudonomas can be reduced.
Benzalkonium chloride is relatively inactive against spores and moulds, but is active
against some viruses including human immunodeficiency virus (HIV). Inhibitory
activity increases with pH although antimicrobial activity occurs between pH 4–10
and organisms inhibited at specific concentrations can be seen below in Table 2
(Kibbe, 2006).
Benzalkonium chloride is hygroscopic and may be affected by light, air, and metals.
Solutions are stable over a wide pH and temperature range and may be sterilized by
autoclaving without loss of effectiveness. Solutions may be stored for prolonged
periods at room temperature. It is incompatible with aluminium, anionic surfactants,
citrates, flourescein, hydrogen peroxide, iodides, kaolin, lanolin, nitrates, and non-
ionic surfactants in high concentration (Kibbe, 2006).
39
2.5.4.2 Parahydroxybenzoic acid esters
Table 3: Minimum inhibitory concentration for propylparaben in aqueous solution (Rieger, 2006b)
40
room temperature while solutions at pH 8 or above are subject to rapid hydrolysis
(Rieger, 2006b).
41
2.6 Efficacy of antimicrobial preservation
The preservative properties of the formulation are adequate if, in the conditions of
test, there is a significant fall or no increase, as appropriate, in the number of micro-
organisms in the inoculated product after the times and at the temperatures
prescribed (B.P, 2011).
42
other sources. Strains derived from contaminated medicines will be more resistant to
preservatives. This is because the characteristics of the organism (including its
resistance to antimicrobial chemicals) over a period of time changes as a result of
mutation and natural selection (Curtin and Cormican, 2003). To get results that are
reproducible by a variety of laboratories it is necessary to specify the strain of the
organism used for the experiment. The common strains used are cultures of
Escherichia coli, Candida albicans, Pseudomonas aeruginosa, Staphylococcus
aureus. Methods used to assess the activity of antimicrobial preservatives involve an
inoculum of the test organisms which are added to a solution of the product in
question. Samples are then removed over a period of time, the preservative is
inactivated and the surviving cells calculated (Brooks et al., 2007).
2.7 Packaging
According to the American Society for Testing and Materials, a plastic is a material
that contains, as an essential ingredient, one or more polymeric organic substances
of large molecular weight, is solid in its finished state and at some stage of
manufacture or processing into finished articles it can be shaped by flow (Rabinow
& Roseman, 2005). Important mechanical properties required in plastic packaging
materials are: tensile strength, impact strength, stiffness, flex resistance, tear
strength, coefficient of friction, blocking, fatigue resistance and creep failure
(Massey, 2004).
43
Ophthalmic glass containers with glass droppers have largely been replaced by
plastic bottles, only in rare cases are glass containers still in use (Van Santvliet &
Ludwig, 2004). Plastic containers are used more often due to their increased
resistance to shock, they are lightweight, and they present more options with regard
to design opportunities (Jenkins & Osborn, 1993).
Plastic containers used to store ophthalmic products should have the following
characteristics:
Most plastic containers are shaped with round or oval bases containing substances
of 3 to 15 ml (Santvliet & Ludwig, 2004). There are disadvantages in plastic
containers, these are:
Plastic packaging is not interchangeable with glass (Shah et al., 2008). Plastic
packages may contain a variety of extraneous substances such as mould-release
agents, antioxidants, and reaction quenchers amongst others which can readily
leach out of the plastic and into the contained solution (Tokiwa & Calabia, 2004) and
label glues, inks and dyes also have been reported to penetrate (Van Santvliet &
Ludwig, 2004). Commercially-produced plastic eye drop bottles are made of
polyethylene or polypropylene. They are common containers for various drugs
including phenylephrine hydrochloride solutions (Winfield et al., 2009).
Each formulation and API is unique. Formulation experiments begin with a well-
structured formulation plan. The formulation strategy is a result of thorough analysis
44
of the preformulation data report and manufacturing process. The following criteria
are to be met in order to begin formulation development:
The assessment of the final formulation can be achieved by scaling to require pilot
batches, and packaged into all possible configurations intended for future
commercialization. The batch is placed into accelerated (40 °C/75%RH) storage
conditions for a period of three months. The batches are tested using validated
analytical methods, should the batch prove to be stable over the 3 months period of
exposure, it would have a high degree of probability that the product is stable and
formulae succeeded. It is pivotal to ensure that all desirable characteristics are
achieved. The generic drug product must demonstrate the minimum 3 months
satisfactory stability, before the following are achieved:
45
Development of specifications for both API and the dosage form.
Ordering of the API and excipients for batch manufacturing.
Ordering of all relevant tooling, change parts.
Completion of development report (Ansel et al., 2011).
46
Gupta & Parasrampuria, 1987; Bastos & de Oliveira, 2009). Most reversed phase
HPLC separations are done in the isocratic mode, where the composition of the
mobile phase is held constant during the analysis. The external standard method is
the most general method for determination of the concentration of a compound in an
unknown sample. It involves the construction of a calibration plot using external
standards of the analyte. A fixed volume of each standard solution of known
concentration is injected onto the column and the peak response of each injection is
plotted versus the concentration of the standard solution. The standards are called
‘external standards’ because they are prepared and analyzed separately from the
unknown samples. After constructing the calibration plot, the unknown sample is
prepared, injected and analyzed in exactly the same manner. The concentration of
the analyte in the unknown sample is then determined from the calibration plot
(Wong, 2006).
Other types of standards are the internal standard, mass balance and area
normalization. The internal standard procedure allows the analyst to identify a region
of the chromatogram that is devoid of peaks (quiet region). The analyst then
attempts to identify a compound of known purity, that is structurally related to the
analyte, and which elutes in the quiet region of the chromatogram. The internal
standard should have a relative response factor that is about the same as the
analyte (Cecil & Sheinin, 2005).
Reversed phase HPLC has been extensively used for the pharmaceutical analysis of
phenylephrine hydrochloride in pharmaceutical dosage forms (Sprieck, 1974;
Muhammad and Bodnar, 1980; Chien & Schoenwald, 1985; Zeise et al., 1996;
Muszalska et al., 2000; Goicoechea & Olivieri, 2001; Savic et al., 2008; Samadi–
Maybodi & Nejad–Darzi, 2009; & Al-Shaalan, 2010).
Analytical procedures used for the assay of the API alone or in the final product
during stability studies should be stability indicating. A stability indicating assay is
one that accurately quantifies the API without interference from degradation
products, process impurities, excipients, or other potential impurities. Samples are
47
obtained by placing the pure API under stress intentionally, for example, by
subjecting the API to acid, base, heat, light or oxidation. This process is often called
forceful degradation or purposeful degradation. Usually, the goal is to degrade the
parent API by 10–20%. Degradation much greater than 10–20% could result in
secondary degradants that will complicate the development process (ICH
Harmonised Tripartite Guideline Q2A, 1994).
In the mass balance approach, all impurities are quantified and subtracted from the
absolute API value of 100%. This approach will result in a purity value that, if all
impurities are accounted for, is more accurate than the external or internal standard
methods. However, the ability to identify all impurities in a given drug substance may
require the use of hyphenated detection techniques and could be extremely costly to
complete on a regular basis. A related approach is called area normalization, and is
often used where the majority of the impurities can be identified and quantified in a
single chromatogram. All of the impurities would be assumed to have the same
relative response factor as the parent drug. The quantification of the individual
impurities would be reported as a percentage of the parent drug rather than an
absolute value in milligrams (Cecil & Sheinin, 2005).
48
which is five times higher than that of a monofunctional bonded phase (Saettone et
al., 1980; O’Donnell & Gillibrand, 1995; Palabiyik & Onur, 2007).
Buffered to pH 7.5.
Form ion pairs with strong and weak acids.
Suppresses weak base ions.
Buffered to pH 3.5.
Form ion–pairs with strong and weak bases.
Buffering suppresses weak acid ions.
The longer the alkyl chain, the greater is the retention time (Florence and
Attwood, 2006).
Ion–pairing agents and pH increase the retention time with increasing concentration
of ion–pairing agent. The true origins of the ions which pair with drug ions is not
clear, but there is evidence that ion–pair formation aids absorption. Ion–pairing
provides the interaction between a drug ion and an organic ion of opposite charges
to form an absorbable neutral species. The formation of ion pairs will depend on
solvent–ion interactions: hydrophobic ions might be encouraged to form ion pairs by
the mechanism of water-structure enforced ion–pairing in which water attempts to
minimize the disturbance on its structuring, and achieves this end by reducing the
polarity of the species in solution by ion–pair formation (Florence and Attwood,
2006).
49
2.9.1.3 Steps for HPLC method validation
Specificity.
System suitability.
Linearity.
Accuracy.
Precision.
Limit of detection.
Limit of quantification.
Range (ICH Harmonized Tripartite Guideline Q2A, 1994).
2.9.1.4 Linearity
Linearity of an analytical procedure is its ability, within a given range, to obtain test
results that are directly proportional to the concentration of analyte in the sample
(ICH Harmonized Tripartite Guideline Q2A, 1994). The requirements for linearity are
that the correlation coefficient of the regression line must be greater than or equal to
0.9999 and that the y-intercept must not be significantly different from zero (ICH
Harmonized Tripartite Guideline Q2A, 1994).
50
2.9.1.5 Accuracy and precision
The limit of detection (LOD) is the lowest analyte concentration that produces a
response detectable above the noise level of the system and the limit of
quantification (LOQ) is the lowest level of analyte that can be accurately and
precisely measured (Kupiec et al., 2004). The LOD is thus the lowest concentration
for which the relative standard deviation of multiple injections is less than 5.0%. By
convention, the LOD value is taken as 0.3 times the LOQ (Thomsen et al., 2003).
2.9.1.7 Range
Range of an analytical procedure is the interval between the upper and lower
concentrations of analyte in the sample (including these concentrations) for which it
has been demonstrated that the analytical procedure has a suitable level of linearity,
accuracy and precision (ICH Harmonized Tripartite Guideline Q2A, 1994).
2.9.1.8 Specificity
51
analyte peak is indeed the desired analyte structure and is 100% pure (Krull &
Swartz, 2001). The peak purity results from the photodiode-array analysis must show
that the phenylephrine hydrochloride peak is pure. In other words the purity angle
must be less than the threshold angle. Depending on the wavelength, a tungsten
lamp and a deuterium lamp are used as light sources. The polychromatic light beam
is focused on a flow-cell and subsequently dispersed by a holographic grating or
quartz prism. The spectral light then reaches a chip that contains 100 to 1000 light-
sensitive diodes arranged side by side. Each diode only registers a well-defined
fraction of the information and in this way all wavelengths are measured at the same
time. At the end of the run, a three-dimensional spectrochromatogram (absorbance
as a function of wavelength and time) is stored on the computer and can be
evaluated qualitatively and quantitatively. Peak identity and peak homogeneity (peak
purity) can be investigated by analyzing spectrum collected during chromatographic
seperations and detection, a pure compound will produce a peak with spectra that
have the same shape across the peak. In contrast any interefence from coeluting
analytes will produce composite spectra with varying degrees of spectra
dissimilarities across the peak. This is the basis of peak purity index. Diode array
detection offers the advantage that knowledge of the spectra of compounds of
interest enables interfering peaks to be eliminated such that an accurate
quantification of peaks of interest can be achieved despite less than optimal
resolution. Peak purity is of the utmost importance in the quality control of
pharmaceutical products (Kupiec et al., 2004).
Figure 4: Diagram of a typical HPLC-UV absorbance peak and plots of noise (or threshold) and purity
angles (Krull & Swartz, 2001).
Figure 4 above shows a typical HPLC-UV absorbance peak with plots of noise and
purity angles. Stress testing is undertaken to demonstrate specificity when
52
developing stability indicating methods (Reynolds et al., 2002). The API and finished
products will be subjected to stress studies in order to force phenylephrine
hydrochloride degradation and thereby verify or exclude the presence of co-eluting
impurities or degradation products in the mobile phase, solvent or unstressed /
stressed solutions.
53
DSC is a technique that is used extensively in the field of pharmaceutics. The main
benefit of DSC, compared to stress storage methods, is its ability to quickly screen
potential excipients for incompatibilities derived from the appearance, shifts or
disappearances of peaks and/or variations in the corresponding peak. DSC is mostly
used to show instability resulting from solid-solid interactions. Another feature of
DSC possesses is low sample consumption making it an attractive method. Although
DSC is unquestionably a valuable technique, interpretation of the data may not be
straightforward. In this method, the sample is exposed to high temperatures (up to
300 °C or more), which in reality is not experienced by the dosage form. Thus, DSC
results should be interpreted carefully, as the conclusions based on DSC results
alone can be often misleading and inconclusive. The results obtained with DSC
should therefore always be confirmed with isothermal stress testing, IST (Giron,
1998).
Ideally, both techniques, DSC and IST should be used in combination for the
selection of excipients. That was not the case in this research study and only IST
was used as it could satisfactorily indicate the results.
Stability, with respect to drug dosage form, refers to the chemical and physical
integrity of the dosage unit and the ability of the dosage unit to maintain protection
against microbiological contamination. The shelf life of the dosage form is the time
lapse from initial preparation to the specified expiration date. The product within its
shelf life must fulfil the monograph specification for identity, strength, quality, and
54
purity. Stability parameters of a drug dosage form can be influenced by
environmental conditions of storage (temperature, light, air, and humidity) as well as
packaging (USP, 2004).
Selection of batches.
Test procedure and criteria.
Specification storage test conditions.
Testing frequency.
Packaging material.
Evaluation statements and labelling (Miller–Meeks et al., 1991).
Stability study is one of the most important areas, in relation to the registration of
pharmaceutical products, as it predicts shelf life and storage instructions for batches.
It also determines degradation of products, mechanism of breakdown and conditions
under which the breakdown occurs. With the help of stability studies, any parameter
subject to change within the eye drop during storage can be measured, such as
appearance, pH, viscosity and density, (where relevant), solubility time
(reconstitution and appearance thereof) sterility, preserving ability and preservative
content (where relevant). Tests are also performed to ensure compatibility between
the container-closure system and the product. Stability testing is the cornerstone of
drug development or formulation (Jeffs, 2009).
During storage, one or more of the following changes may take place:
55
c) Mould or bacterial growth commonly referred to as biological change.
Chemical change involves the drug itself because this is normally the most reactive
component of the system. Occasionally, it doesn’t involve the drug but is limited to
the excipient and/or the container for example the rusting of cans or flaking of glass,
this thus gives plastics added advantage. A hazardous occurrence within plastic is
the extraction of substances from the walls of plastic containers. Other chemical
changes, such as discoloration, may be harmless and quantitatively negligible but
could have serious effect on the acceptability of the product. Chemical change
occurs when stimulated by heat light, moisture and aeration. Thus antioxidants,
buffers and chelating agents are commonly added to offer protection against light
and moisture. Physical change may make a product inconvenient or impossible to
use and occasionally can lead to danger to patient. The growth of micro-organisms
can cause spoilage either by their appearance or because they have induced
significant chemical change (Huynh-Ba, 2009).
56
3. METHODOLOGY
3.1.1 Equipment
The HPLC system consisted of a complete FPLC Shimadzu® HPLC system which
has a SPD-M20A Prominence diode array detector, SIL-20A Prominence auto-
sampler, DGU-20A5 Prominence degasser, LC-20AB Prominence liquid
chromatography and CTO-10AS vp Prominence column oven ( Shimadzu, Tokyo,
Japan). The stationary phase consisted of a reverse phase Phenomenex® Luna C18
(2) column 250 mm × 4.60 mm, 5 μm particle size (Separations, Johannesburg, SA).
The flow rate was set at one millimetre per minute. The column temperature was set
at its temperature of 40 °C and column back-pressure varied between 2800 to 3000
psi. A PDA was used, therefore all runs were analysed at wave length, 280 nm. The
injection volume was 20 μl (USP, 2004). The assay was performed utilizing a reverse
phase Phenomenex® Luna C18 (2) column (250 mm × 4.60 mm, 5 μm particle size).
To identify the phenylephrine hydrochloride, the retention times of the peaks were
noted and to quantitate the amount of the API, values found of AUC (peak area)
which is computer-stored and generated was used. A graph of the concentrations vs.
peak area was plotted. Equation of the line was calculated using Microsoft Excel®
program. With the above, the following were determined: specificity, system
suitability, linearity, accuracy, precision, limit of detection/limit of quantification and
range.
3.1.5 Linearity
58
by plotting the peak areas of phenylephrine hydrochloride versus the respective
phenylephrine hydrochloride concentrations and a linear regression trend line was
fitted to the plot on Microsoft Excel® 2007, (Microsoft Corporation).
Sensitivity of the method was determined by means of the detection limit (LOD) and
quantification limit (LOQ). Calculations for LOD were based on the standard
deviation of the calibration curve (σ) and the slope of curve (S), using the equation
LOD = 3.3 × σ divided by S. LOQ was calculated using the equation LOQ = 3.33
multiplied by LOD (ICH Harmonized Tripartite Guideline Q2 (R1), 1994). Standard
solutions of decreasing concentration were produced by successive dilution of the
59
lowest calibration standard and the resulting solutions were injected in triplicate. A
volume or concentration of 0.01 ml, the least calibration standard concentration was
diluted 10 times.
The range was determined by observing the interval between the upper and lower
concentration of phenylephrine hydrochloride for which the HPLC analytical assay
has suitable level of linearity, accuracy and precision (ICH Harmonized Tripartite
Guideline Q2 (R1), 1994).
3.1.9 Specificity
60
stressed products. The products are referred to as product I, II, III, IV, V and were
formulated as indicated in Table 5 (section 3.3.2). The nature of the specificity
samples and the relevant stress conditions are listed below:
All five products and the phenylephrine hydrochloride were subjected to the following
stress conditions after which they were analysed:
61
UV energy of not less than 200 watt hours/m2 all according to ICH Harmonized
Tripartite Guideline Q1B, (2005).
All impurities and degradation products should be between 10 – 20 % of the API.
This includes samples stored under relevant stress conditions: light, heat, humidity,
acid/base hydrolysis and oxidation (ICH Harmonized Tripartite Guideline Q2 (R1),
2005). Degradants and impurities will be labeled as unknown (Ω) and if more than
one is present will be denoted as Ωi, Ωii, Ωiii and so on.
An important factor for eye drops is that they should be sterile when dispensed in a
multiple-application container. The preservative should be effective against
accidental introduction of micro-organisms and contaminants. The formulations were
manufactured in a clean environment in a laminar-flow hood using aseptic technique.
62
3.3.1 Materials
The hotplate magnetic stirrer utilized during the manufacturing process was a
Heidolph® MR 3002 (Labotec®, Midrand, SA), the storage chamber (BINDER® KMF
series, BINDER Inc, New York, USA) and the autoclave (Hirayama, New York,
USA), Natural dropper bottle 15 ml (LDPE) polyethylene container with closure
component (plugs with cap).Eeye drop bottles 13 MM CTRL dropper tip 0.031
needle natural (Amcor pharmaceutical packaging, New Jersey, USA), beakers and
graduated glass bottles (SCHOTT® North America, Inc. New York, USA), membrane
filter paper 0.45 µm (Millipore Corporation, Bedford, Massachusetts, USA), alcohol
preparation swabs WEBCOLTM, plastipak syringes (10 and 5 mL), HiCare int. latex
gloves large, avacare hypodermic needles (green) 21 g, alcohol 70% (particle free)
were all supplied by Alpha Pharm. Pty (Ltd), Port Elizabeth, South Africa.
63
Table 5: Formulation summary of active pharmaceutical ingredient and excipients used in the
manufacturing of products I–V
Lot Size: 1L
Material Product Product Product Product Product
I II III IV V
Phenylephrine Hydrochloride 10% w/v 10% w/v 10% w/v 10% w/v 10% w/v
Sodium citrate 0.1% w/v 0.1% w/v - - -
Dihydrate
Sodium - 1% w/v - - -
Metabisulfite
Boric Acid - - - 1.9% w/v -
Benzalkonium 0.1% v/v 0.1% v/v - - -
Chloride
Hydroxypropyl methylcellulose 0.3% w/v 0.3% w/v - - 0.3% w/v
QS to
Methyl hydroxybenzoate - - 0.18% w/v - 0.18% w/v
Propyl hydroxybenzoate - - 0.02% w/v - 0.02% w/v
Sodium carboxy Methylcellulose - - - 0.2 % w/v -
to
Water Purified (RO) 10% v/v 10% v/v 10% v/v 10% v/v 10% v/v
Ethylenediaminetetraacetic - - - 0.1% w/v -
Acid
Glycerol to - - 100% v/v -
1 M Phosphoric acida a a
1 M Sodium Hydroxidei i i i
i and a – For pH adjustment only, ( - ) means it was absent from the formulation
The API and excipients were sterilized as follows: The heat sensitive PE was
steamed in a water bath at 100 °C for 30 minutes. Autoclave is not
recommended as phenylephrine hydrochloride has tendency to decompose
on heating.
All other equipments and excipients were autoclaved as follows: 121 °C for 15
minutes at 1 atm (200 kPa or 15 psi).
After sterilization the bottles containing products were shaken every 15
minutes until cool, to make sure HPMC and SCMC stayed fully mixed and
hydrated.
The manufacturing processes followed the same general method, with slight
variations in the processes based on certain excipeints. The few exceptions are seen
with the preservatives methyl and propyl hydroxybenzoate. Ingredients were added
64
sequentially to the main portion of solution while mixing with the glass rod. The
formulation was mixed until visually homogenous before addition of the next
ingredient. The methyl and propyl hydroxybenzoate were dissolved in ultra-pure
water with the aid of gentle heat and mixing with a glass rod. Addition of the
dissolved preservatives to the bulk solution was done to ensure complete
quantitative transfer of all preservatives to the bulk formulation. The solution was
then transferred to a calibrated plastic bottle, the dropper inserted and the bottle
capped. The stability of the formulations was determined in the primary plastic
packaging. The laboratory scale manufacturing processes for the five products
(products I–V) can be seen in figures 5 to 9.
4) Cooled to room
iii) Disperse and mix with a glass temperature of 25 °C
rod until all dissolve
5) Aseptically and
iv) Autoclave mixture at 121 °C for quantitatively mixed
15 minutes
6) A volume of 15 ml was
aseptically and quantitatively
transferred to the calibrated
plastic eye-drop bottle which
was capped.
65
1) Ultra pure water 10%v/v
4) Cooled to room
iii) Disperse and mix with a glass
temperature of 25 °C
rod until all dissolve
5) Aseptically and
iv) Autoclave mixture at 121 °C for quantitatively mixed
15 minutes
6) A volume of 15 ml was
aseptically and quantitatively
transferred to the calibrated
plastic eye-drop bottle which
was capped
66
1) Ultra pure water 10% v/v
5) Aseptically and
iv) Autoclave mixture at 121 °C for quantitatively mixed
15 minutes
6) A volume of 15 ml was
aseptically and quantitatively
transferred to the calibrated
plastic eye-drop bottle which
was capped.
67
1) Ultra pure water 10% v/v
4) Cooled to room
iii) Disperse and mix with a glass rod temperature of 25 °C
until all dissolve
5) Aseptically and
iv) Autoclave mixture at 121 °C for 15 quantitatively mixed
minutes
6) A volume of 15 ml was
aseptically and quantitatively
transferred to the calibrated
plastic eye-drop bottle which
was capped.
68
1)Ultra pure water 10% v/v
5) Aseptically and
iv) Autoclave mixture at 121 °C for quantitatively mixed
15 minutes
6) A volume of 15 ml was
aseptically and quantitatively
transferred to the calibrated
plastic eye-drop bottle which
was capped.
Testing for stability is crucial in product development. It usually involves at least two
stages: firstly, accelerated tests on a prototype and secondly, storage under
probable conditions of the manufactured product. In SA, the submission of stability
data is compulsory before sale is permitted, result from accelerated stability studies
are usually acceptable for this purpose (Zahn, 2008).
Stability studies on the dosage forms were conducted at specific temperatures and
relative humidities representing storage conditions experienced in the climatic zones
69
of South Africa (climatic zones I and II). The formulations were stored at 40
°C/75%RH, 25 °C/40%RH and 30 °C/65%RH. The five formulations were stored at
all three of the above mentioned conditions and tested in triplicate at 0, 3, and 6
months. The most appropriate and stable formulation was chosen based on the level
of degradation of the phenylephrine hydrochloride, pH change and appearance.
The bulk appearance of the prepared products was visually examined for colour,
homogeneity, and for the appearance of any precipitate. The pH of the solutions
was measured using a pH/mV/ °C meter (744 pH meter, metrohm). Each solution
was stirred with a magnetic stirrer (Hanna instruments supplied by Tecnilab, SA) for
30 seconds before pH measurement.
The unknown peaks have to be less than or equal to 1% while the phenylephrine
hydrochloride left must be between 95%–105% to be accepted as a successful
formulation. The mean peak area was divided by the theoretical amount of drug
found in the formulation multiplied as a percentage to give the final result of
phenylephrine hydrochloride left.
The test challenged the formulation in its final primary container, with a prescribed
inoculum of suitable micro-organisms, storing the inoculated product at a prescribed
temperature, withdrawing the samples from the container at specified intervals of
time and counting the organisms in the samples removed. The two methods used
for determining bacterial numbers were the standard (viable) plate count method and
spectrophotometric (turbidimetric) analysis. The standard plate method reveals
information related to live bacteria while indirectly measuring cell density. The
70
spectrophotometric method is based on turbidity and indirectly measures all bacteria
(cell biomass), dead or alive (Harley and Prescott, 1998).
Materials: Cultures of Escherichia coli (ATCC No. 38218), Candida albicans (ATCC
No. 66027), Pseudomonas aeruginosa (ATCC No. 27853), Staphylococcus aureus
(ATCC No. 43300) were obtained from the Department of Biomedical Technology at
the Nelson Mandela Metropolitan University. The bacteria were chosen because of
their classification as well as pathogenicity. Pipettes, petri plates, curvettes
(Hellma®, precision cells, Lasec, Johannesburg, SA) were obtained from Lasec
(Johannesburg, SA). Bunsen burner, platinum loop wire dispensers (1/200 mL)
(Lasec, Johannesburg, SA), water was produced by Ultra Clear TWF/El-Ion® system
which was pre-treated and made ultrapure (reverse osmosis) (Separations,
Johannesburg, SA). Tryptone soya broth and agar, Sabouraud broth and agar were
supplied by Sigma-Aldrich (Pty) Ltd (Kempton Park, SA). Sodium chloride was
obtained from Merck Laboratory Supplies (Pty) Ltd (Midrand, SA). Hockey stick was
obtained from Lasec (Johannesburg, SA). Tecam® SB-16 Shaking Water bath was
obtained from Spellbound Laboratory solutions (Port Elizabeth, SA). Vacutec
(Johannesburg, SA) supplied the P Spectra colony counter, Shimadzu® UV
spectrophotometer (UV-1800) (Shimadzu, Tokyo, Japan).
Media preparations: All broths and agars poured into 1000 ml Schott bottles with a
screw cap and autoclaved at 121 °C for 15 minutes. Tryptone soya broth was made
by weighing 25 g and dissolving 1000 ml of distilled water. Tryptone soya agar was
made by weighing 40 g which was dissolved in 1000ml of distilled water. Sabouraud
broth was made by weighing 30 g which was dissolved in 1000 ml of distilled water.
Sabouraud agar was made by weighing 45 g which was dissolved in 1000 ml of
distilled water.
All Petri dishes and saline bottles were labelled accordingly. Using aseptic
technique, 0.1 ml was added to a 9.9 ml sterile saline and was shaken vigorously to
71
distribute the bacteria. A volume of 0.1 ml of this was aseptically transferred to a
second 9.9 ml sterile saline, it was then capped and the second dilution was shaken
vigorously. The process was repeated until 10-8 dilution was attained. Volumes of 0.1
ml and 0.01 ml from each dilution of microbes were aseptically transferred to petri
plates. The tryptone soya agar at 48 °C was aseptically poured into a petri dish. The
agar and sample were immediately mixed by gently moving the plate in a figure-eight
motion. The process was repeated for the other plates which were then left to cool;
they were inverted and incubated at 35 °C for 24 hours. The plates were counted
using the P Spectra® colony counter. Plates with more than 250 colonies were not
counted and were labelled too numerous to count (TNTC). Plates with fewer than 25
colonies were designated too few to count (TFTC). The colony forming unit per
millilitre was calculated by dividing the number of colonies by the dilution factor
(Harley and Prescott, 1998).
One empty tube and five tubes containing 3 ml of sterile tryptone soya broth were
placed in a test-tube rack and labelled A–E, with the exception of the empty tube.
The five tubes of broth were used to make five serial dilutions of the culture. With the
aid of a spectrophotometer (Shimadzu® UV spectrophotometer (UV-1800) and
curvettes (Hellma®, precision cells) the different cultures were standardized at a
wavelength of 600 nm; the blank was a sterile broth without the culture. From the
culture broth, different absorbance concentrations of 0.200 to 1.0 were achieved with
sterile saline using the serial dilution method. Each absorbance concentration was
plated and the colony counted in triplicate. A straight line graph was constructed by
plotting the absorbance of the culture broth at 600 nm versus the number of colony
forming units, and a linear regression trendline was fitted to the plot (Microsoft
Excel® 2007, Microsoft Corporation). A linear relationship was found with all the
organisms, as indicated by the linear correlation coefficient (R2), which was greater
than 0.99, even though the y-intercepts were significantly different to zero (Harley
and Prescott, 1998). This method was applied to the different microorganism.
72
3.6.4 Preservative efficacy
The preservative properties of the formulation are adequate if, in the conditions of
the test, there is a significant fall or no increase in the number of micro-organisms in
the inoculated product after the times and at the temperatures prescribed. Negative
controlled samples were prepared by excluding preservatives, and inoculums of 100
µl of 106 CFU/ml were added to both control and products I–V. The inoculated
samples were stored away from light at 25 °C for 6 hours, 24 hours, 7 days, 14 days
and 28 days. Aliquots of 1 ml were removed from each sample at zero, six and 24
hours and after 7, 14 and 28 days intervals. The sampled were then enumerated.
One milliliter aliquots were transferred to a sterile 10 ml nutrient broth and plated in
duplicate on tryptone soya agar (for bacteria) or Sabouraud dextrose agar (for fungi).
Plates were incubated at 35 °C for 48 hours for bacteria and 25 °C for 72 hours for
fungi. Raw data counts were converted to log10 values and the reduction from
inoculum values was calculated for evaluation against compendial requirements
found in Table 6 below.
Table 6: Criteria for tested microorganisms (USP, 2004)
The samples were diluted in a 1:10 ratio at the time of testing, 10 CFU (or 1.0 log
reduction) is the lowest sensitivity allowed by the test.
All measurements were performed with an Anton Paar RheolabQC rheometer with
cylinder measuring CC27 according to ISO 3219. ISO 3219 describes the
dimensions of the cylinder geometry and defines the ratio of measuring cup radius to
measuring bob radius as 1.0847. This guarantees an industrial standard for shearing
73
the sample in the measuring gap, independent of the measuring system size and
manufacturer. The temperature of the measuring system was controlled using a
thermostat (Anton Paar ViscoTherm VT2) directly connected to the Antor Paar
rheometer (Šklubalova & Zatloukal, 2011). For all measurements temperature was
23 °C this was kept constant as determination of the viscosity η and yield point is
very dependent on the temperature.
The testing conditions for the determination of the yield point were as follows:
Descriptive and inferential statistics were used. Standard deviation (+/-) and mean
were calculated from measured replicates using the software package GraphPad
Prism version 6.01 (GraphPad Software Inc., San Diego, USA). Comparisons of
means were achieved using one-way ANOVA. Statistically significant differences
were tested using Student’s t-test with significance in difference defined as p<0.05.
The experimental design was based on a non-matching or pairing of mean, with the
mean of each observation being compared with the mean of every other observation.
The Bonferroni test was chosen as it corrected for multiple comparisons and
indicated confidence intervals and significance.
74
4. RESULTS AND DISCUSSION
4.1 Validation of the stability indicating assay
The method used to analyse the formulations was obtained from the USP. Though it
was a pharmacopoeial method its suitability for use on the present system had to be
validated. The validation process followed ICH guidelines and the results are
indicated below.
4.1.1 Linearity
600000
400000
200000
0
0 0.05 0.1 0.15 0.2
concentration (mg/ml)
Figure 10: Graph showing a mean peak area versus concentration of replicate samples of
phenylephrine hydrochloride standards. Linear regression equation: y = 8541.1x + 438.55, R2 =
0.9999.
75
4.1.2 Accuracy
Accuracy of the method was within acceptable range as indicated in Table 7 below.
The percentage relative standard deviations calculated for the samples at the lower,
middle and upper limits of the concentration range were all below 0.5%. This means
by applying the analytical method of a known purity concentration (linearity) and
comparing the results of accuracy the differences were not greater than 0.5%.
The recovery percentage for the samples was between the limits of 99.00 to
100.10%.
4.1.3 Precision
Precision was within acceptable range as seen below in Table 8. The percentage
relative standard deviations calculated for the samples at the lower, middle and
upper limits of the concentration range were all below 0.5%. The recovery
percentage for the samples was between the limits of 99.00 to 100.00%. The method
being validated showed precision was achieved as the degree of agreement among
individual test results was demonstrated by the relative standard deviation.
76
4.1.4 Limit of detection (LOD) and quantification (LOQ)
The LOD was determined by serial dilutions of the lowest concentration (0.0125
mg/ml) of the attained phenylephrine hydrochloride standard and found to be 12.3
μg/ml. The amount stated is the lowest amount of phenylephrine hydrochloride in a
sample that can be detected. The LOD is the lowest concentration for which the
relative standard deviation of multiple injections is less than 5.0%. The LOQ was
found to be 41 μg/ml. The amount shows the lowest amount of analyte in a sample
that can be determined with acceptable precision and accuracy. By convention, the
LOD value is taken as 0.3 times the LOQ (Armbruster & Pry, 2008) while LOQ = 3.33
LOD (Thomsen et al., 2003). The results of the two did fit this mathematical
assumption. These methods are HPLC specific and are dependent on the type of
HPLC conditions; therefore re-determination in each laboratory is necessary. The
range was 0.0125 to 0.15 mg/ml.
The mean peak area for the 6 replicate injections of phenylephrine hydrochloride had
a percentage relative standard deviation of 0.14 %. The tailing factor was 2.0. The
analytical system thus complied with the requirements specified by the system
suitability as the percentage relative standard deviation of the peak responses due to
phenylephrine hydrochloride for six injections was less than 1.0 %. The column was
thus deemed suitable for analysis. There is a peak tailing as potrayed in the
chromatograms, some reasons could be due to more than one retention mechanism
is present in a separation, and one of those mechanisms is overloaded. At the
surface, the polymer terminates in silanol groups. There are different possible silanol
configurations which are abundant at the surface of the columns as free silanols, but
77
silicon atoms with two hydroxyl groups in a geminal configuration also are present. If
the silanol groups are positioned next to each other, an associated silanol group can
share a proton with an adjacent group. The free silanol groups are more acidic than
the geminal and associated groups, and they interact more strongly with basic
solutes. The result is peak tailing commonly associated with the separation of bases.
To further complicate matters, trace metals (e.g., iron and aluminium) can be present
in the silica matrix. These trace metals can act as ion-exchange sites or, when
adjacent to free silanols, can withdraw electrons, which makes the silanols even
more acidic. To reduce the incidence of tailing or fronting, concentrations should be
reduced proportionally by appropriate dilutions (Dolan, 2003).
78
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 12: HPLC Chromatogram for phenylephrine hydrochloride dissolved in mobile phase with a
retention time of 7.80 minutes.
mAU
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 13: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 1.00000; Single point threshold = 0.999999.
Products I–V without any stress are indicated below in figures 14–23. They show
purity indexes of 1.0 and chromatograms showing no co-elution or impurities. The
average retention time for PE was 7.85 minutes.
mA U
60 280nm,4nm (1.00)
55
50
45
40
35
30
25
20
15
10
-5
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 14: HPLC Chromatogram for product I dissolved in mobile phase with a retention time of 7.87
minutes.
79
mA U
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 15: Peak purity profile calculated using PDA data (from 190–800 nm) for Product I prepared in
mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point
threshold = 0.999054
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 16: HPLC Chromatogram for product II dissolved in mobile phase with a retention time of 7.82
minutes.
mA U
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 17: Peak purity profile calculated using PDA data (from 190–800 nm) for Product II prepared
in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single
point threshold = 0.996482.
80
mA U
280nm,4nm (1.00)
50
45
40
35
30
25
20
15
10
Figure 18: HPLC Chromatogram for product III dissolved in mobile phase with a retention time of
7.83 minutes.
mAU
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 19: Peak purity profile calculated using PDA data (from 190–800 nm) for Product III prepared
in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single
point threshold = 0.998178.
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 20: HPLC Chromatogram for product IV dissolved in mobile phase with a retention time of
7.89 minutes.
81
mAU
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 21: Peak purity profile calculated using PDA data (from 190–800 nm) for Product IV prepared
in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single
point threshold = 0.996947.
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 22: HPLC Chromatogram for product V dissolved in mobile phase with a retention time of
7.84 minutes.
mA U
0.8 Purity Curv e Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 23: Peak purity profile calculated using PDA data (from 190–800 nm) for product V prepared
in mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single
point threshold = 0.999587.
82
Stress under UV light. Products I–V were stressed using UV light, their
chromatograms did not show interference or co-elution with phenylephrine
hydrochloride. Figures 24–35 below indicate that the excipients did not react with the
phenylephrine hydrochloride and no degradants co-eluted with the phenylephrine
hydrochloride. The plastic bottle did not change in colour as the solutions were still
clear and homogenous. The average retention time for the products was 7.85
minutes. Peak purity index was 1.0 for products I, II, IV and V. Product III had a peak
purity index of 0.999999.
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 24: HPLC Chromatogram for phenylephrine hydrochloride stressed under UV light dissolved
in mobile phase with a retention time of 7.81 minutes.
mA U
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 25: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride prepared in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 1.00000; Single point threshold = 0.999999.
83
mA U
60 280nm,4nm (1.00)
55
50
45
40
35
30
25
20
15
10
-5
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 26: HPLC Chromatogram for product I stressed under UV light dissolved in mobile phase with
a retention time of 7.86 minutes.
mAU
Purity Curve Peak
Zero Line
60
0.75
50
40
0.50
30
0.25 20
10
0.00
0
7.75 8.00 8.25 min
Figure 27: Peak purity profile calculated using PDA data (from 190–800 nm) for product I prepared in
mobile phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point
threshold = 0.999054.
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 28: HPLC Chromatogram for product II stressed under UV light dissolved in mobile phase with
a retention time of 7.82 minutes.
84
mAU
Purity Curve Peak
Zero Line 30.0
0.75
25.0
20.0
0.50
15.0
0.25 10.0
5.0
0.00
0.0
7.75 8.00 8.25 min
Figure 29: Peak purity profile calculated using PDA data (from 190–800 nm) for product II in mobile
phase. Peak shown in pink and purity curve in black. Peak purity index = 1.00000; Single point
threshold = 0.999116.
mA U
280nm,4nm (1.00)
50
45
40
35
30
25
20
15
10
Figure 30: HPLC Chromatogram for product III stressed under UV light dissolved in mobile phase
with a retention time of 7.85 minutes.
mA U
Purity Curve Peak
Zero Line 35.0
0.5
30.0
0.4
25.0
0.3 20.0
15.0
0.2
10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 31: Peak purity profile calculated using PDA data (from 190–800 nm) for product III in mobile
phase. Peak shown in pink and purity curve in black. Peak purity index = 0.999999; Single point
threshold = 0.997116.
85
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 32: HPLC Chromatogram for product IV stressed under UV light dissolved in mobile phase
with a retention time of 7.81 minutes.
mAU
Purity Curve Peak
Zero Line 35.0
0.75
30.0
25.0
0.50
20.0
15.0
0.25
10.0
5.0
0.00
0.0
7.75 8.00 8.25 min
Figure 33: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV in mobile
phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point
threshold = 0.997916.
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 34: HPLC chromatogram for product V stressed under UV light dissolved in mobile phase with
a retention time of 7.87 minutes.
86
mAU
0.5 Purity Curve Peak
Zero Line
35.0
0.4
30.0
0.3 25.0
20.0
0.2
15.0
0.1 10.0
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 35: Peak purity profile calculated using PDA data (from 190–800 nm) for product V in mobile
phase. Peak shown in pink and purity curve in black. Peak purity index = 1.000000; Single point
threshold = 0.997475.
No colour change was observed with products I–V and phenylephrine hydrochloride
remained stable under the UV light stress. If it was unstable a colour chang would be
observed from clear to dark brown. The average concentrations of the products
remained at 99.01%. Using the above mentioned statistical analysis (one-way
ANOVA), there were no significant difference in the API concentration among the
products (I–V) manufactured at time zero (T0). They had a P-value of p > 0.9999, this
meant that the products during formulation and testing maintained their API at T0.
Stress with HCl: Products I–V were stressed with HCl, their chromatograms did not
show interference or co-elution with phenylephrine hydrochloride. Figures 36–47
below indicates that the excipients did not react with the phenylephrine hydrochloride
and no degradants co-eluted with the phenylephrine hydrochloride. Discolorations
were observed during the reflux of products I–V with HCl, the colours ranged from
87
very light yellow to yellow. The greatest change was observed in product III, as it
turned bright yellow. These could mean glycerol, HCl and phenylephrine
hydrochloride were undergoing a reaction of oxidation or reduction. The average
retention time for the products was 7.85 minutes. Peak purity index was 1.0 for
products I–V however; the average API concentration had reduced to 79.64 %.
mA U
280nm,4nm ( 1.00)
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 36: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M HCl dissolved in
mobile phase with a retention time of 7.86 minutes.
mA U
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 37: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
1.000000; Single point threshold = 0.999574.
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 38: HPLC chromatogram of Product I stressed with 0.2 M HCl dissolved in mobile phase with
a retention time of 7.82 minutes.
88
mA U
Purity Curve Peak
Zero Line 35.0
0.5
30.0
0.4
25.0
0.3 20.0
15.0
0.2
10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 39: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed
with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
0.999999; Single point threshold = 0.997116.
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 40: HPLC chromatogram for product II stressed with 0.2 M HCl dissolved in mobile phase with
a retention time of 7.83 minutes.
mA U
Purity Curve Peak
Zero Line 30.0
0.75
25.0
0.50 20.0
15.0
0.25 10.0
5.0
0.00
0.0
7.75 8.00 8.25 min
Figure 41: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed
with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
1.000000; Single point threshold = 0.996296.
89
mA U
280nm,4nm ( 1.00)
45
40
35
30
25
20
15
10
Figure 42: HPLC chromatogram for product III stressed with 0.2 M HCl dissolved in mobile phase
with a retention time of 7.91 minutes.
mAU
Purity Curve Peak
0.75 Zero Line
25.0
20.0
0.50
15.0
0.25 10.0
5.0
0.00
0.0
7.75 8.00 8.25 min
Figure 43: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed
with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index
=0.999999; Single point threshold = 0.995179.
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 44: HPLC chromatogram for product IV stressed with 0.2 M HCl dissolved in mobile phase
with a retention time of 7.86 minutes.
90
mA U
Purity Curve Peak
Zero Line 35.0
0.75
30.0
25.0
0.50
20.0
15.0
0.25
10.0
5.0
0.00
0.0
7.75 8.00 8.25 min
Figure 45: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed
with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index
=1.000000; Single point threshold = 0.997097.
Figure 46: HPLC chromatogram of product V stressed with 0.2 M HCl dissolved in mobile phase with
a retention time of 7.80 minutes.
mA U
Purity Curve Peak
Zero Line
60
0.75
50
40
0.50
30
0.25 20
10
0.00
0
7.75 8.00 8.25 min
Figure 47: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed
with 0.2 M HCl in mobile phase. Peak shown in pink and purity curve in black. Peak purity index
=1.000000; Single point threshold = 0.999578.
Stress with NaOH: Products I–V were stressed with NaOH, their chromatograms
did not show interference or co-elution with phenylephrine hydrochloride. Figures
91
48–59 below indicate that the excipients did not react with the phenylephrine
hydrochloride nor did degradation products of products I–V co-elute with the
phenylephrine hydrochloride. Discolorations were observed during the reflux of
products I–V with NaOH, the colours changed from clear to brown. The average
retention time for the products was 7.84 minutes. Due to the caustic nature of NaOH
the products had a reduced average API concentration of 42.36%.
mA U
280nm,4nm (1.00)
40
35
30
25
20
15
10
Figure 48: HPLC chromatogram of phenylephrine hydrochloride stressed with 0.2 M NaOH dissolved
in mobile phase with a retention time of 7.82 minutes
mAU
Purity Curve Peak 30.0
Zero Line
0.6
25.0
0.5
20.0
0.4
0.3 15.0
0.2 10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 49: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride stressed with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in
black. Peak purity index =0.999999; Single point threshold = 0.996847.
92
mA U
280nm,4nm (1.00)
40
35
30
25
20
15
10
Figure 50: HPLC chromatogram of product I stressed with 0.2 M NaOH dissolved in mobile phase
with a retention time of 7.89 minutes.
mAU
Purity Curve Peak 30.0
Zero Line
0.6
25.0
0.5
20.0
0.4
0.3 15.0
0.2 10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 51: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed
with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index
=1.000000; Single point threshold = 0.995815.
mA U
280nm,4nm ( 1.00)
45
40
35
30
25
20
15
10
Figure 52: HPLC chromatogram of product II stressed with 0.2 M NaOH dissolved in mobile phase
with a retention time of 7.9 minutes.
93
mAU
Purity Curve Peak
Zero Line
17.5
0.75
15.0
12.5
0.50
10.0
7.5
0.25
5.0
2.5
0.00
0.0
7.75 8.00 8.25 min
Figure 53: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed
with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
0.999999; Single point threshold = 0.996370.
mA U
37.5 280nm,4nm (1.00)
35.0
32.5
30.0
27.5
25.0
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
Figure 54: HPLC chromatogram for product III stressed with 0.2 M NaOH dissolved in mobile phase
with a retention time of 7.79 minutes.
mAU
Purity Curve Peak
Zero Line
0.75 15.0
12.5
0.50 10.0
7.5
0.25
5.0
2.5
0.00
0.0
7.75 8.00 8.25 min
Figure 55: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed
with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
0.999999; Single point threshold = 0.998771.
94
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 56: HPLC chromatogram of product IV stressed with 0.2 M NaOH dissolved in mobile phase
with a retention time of 7.91 minutes.
mAU
Purity Curve Peak 35.0
0.6 Zero Line
30.0
0.5
25.0
0.4
20.0
0.3
15.0
0.2
10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 57: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed
with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
0.999999; Single point threshold = 0.996930.
mA U
45 280nm,4nm (1.00)
40
35
30
25
20
15
10
Figure 58: HPLC chromatogram for product V stressed with 0.2 M NaOH dissolved in mobile phase
with a retention time of 7.85 minutes.
95
mA U
Purity Curve Peak
0.75 Zero Line
25.0
20.0
0.50
15.0
0.25 10.0
5.0
0.00
0.0
7.75 8.00 8.25 min
Figure 59: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed
with 0.2 M NaOH in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
0.999999; Single point threshold = 0.999587.
Stress with H2O2: Products I–V were stressed with H2O2, their chromatograms did
not show interference or co-elution with phenylephrine hydrochloride. Figures 60–71
below indicates that the excipients did not react with the phenylephrine hydrochloride
or produce degradants found in products I–V which co-eluted with the phenylephrine
hydrochloride. Discolorations were observed during the reflux of products I–V with
H2O2, the colours changed from clear to pale yellow (products I, II, IV and V) and
from clear to lime green (product III). The average retention time for the products
was 7.82 minutes. Due to the oxidizing nature of H2O2 the products had a reduced
average API concentration of 26.43%.
mA U
280nm,4nm (1.00)
55
50
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 60: HPLC chromatogram for phenylephrine hydrochloride stressed with 0.2 M H2O2 dissolved
in mobile phase with a retention time of 7.82 minutes.
96
mAU
Purity Curve Peak
Zero Line
0.75 15.0
12.5
0.50 10.0
7.5
0.25
5.0
2.5
0.00
0.0
7.75 8.00 8.25 min
Figure 61: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride stressed with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black.
Peak purity index = 1.000000; Single point threshold = 0.999987.
mA U
280nm,4nm (1.00)
55
50
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 62: HPLC chromatogram for product I stressed with 0.2 M H2O2 dissolved in mobile phase
with a retention time of 7.81 minutes.
mAU
0.6 Purity Curve Peak 15.0
Zero Line
0.5 12.5
0.4
10.0
0.3
7.5
0.2
5.0
0.1
2.5
0.0
0.0
7.75 8.00 8.25 min
Figure 63: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stressed
with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
1.000000; Single point threshold = 0.999522.
97
mA U
280nm,4nm ( 1.00)
55
50
45
40
35
30
25
20
15
10
Figure 64: HPLC chromatogram for product II stressed with 0.2 M H2O2 dissolved in mobile phase
with a retention time of 7.9 minutes.
mAU
Purity Curve Peak
Zero Line
17.5
0.75
15.0
12.5
0.50
10.0
7.5
0.25
5.0
2.5
0.00
0.0
7.75 8.00 8.25 min
Figure 65: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stressed
with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
0.999999; Single point threshold = 0.999722.
mA U
60 280nm,4nm (1.00)
55
50
45
40
35
30
25
20
15
10
-5
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 66: HPLC chromatogram of product III stressed with 0.2 M H2O2 dissolved in mobile phase
with a retention time 7.94 minutes.
98
mA U
Purity Curve Peak
Zero Line 17.5
0.75 15.0
12.5
0.50 10.0
7.5
0.25
5.0
2.5
0.00
0.0
7.75 8.00 8.25 min
Figure 67: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stressed
with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
0.999999; Single point threshold = 0.999706.
mA U
280nm,4nm (1.00)
55
50
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 68: HPLC chromatogram for product IV with 0.2 M H2O2 dissolved in mobile phase with a
retention time of 7.81 minutes.
mA U
0.7 Purity Curve Peak
Zero Line 12.5
0.6
0.5 10.0
0.4
7.5
0.3
5.0
0.2
0.1 2.5
0.0
0.0
7.75 8.00 8.25 min
Figure 69: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stressed
with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
1.000000; Single point threshold = 0.999896.
99
mA U
50 280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 70: HPLC chromatogram for product V stressed with 0.2 M H2O2 dissolved in mobile phase
with a retention time of 7.88 minutes.
mAU
Purity Curve Peak 35.0
0.6 Zero Line
30.0
0.5
25.0
0.4
20.0
0.3
15.0
0.2
10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 71: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stressed
with 0.2 H2O2 in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
0.999999; Single point threshold = 0.999536.
Stress with heat and humidity: Products I–V were stressed with heat and humidity;
their chromatograms did not show interference or co-elution of impurities with
phenylephrine hydrochloride. Figures 72–98 below indicates that the excipients did
not react with the phenylephrine hydrochloride. In Figure 72, phenylephrine
hydrochloride stressed at 100 °C for 24 hours in a stability chamber showed a
reduction of its API to 95% while showing no physical discolorations. Heat therefore
has an effect in reducing the potency of phenylephrine hydrochloride.
Chromatograms of products I–V stressed at the 65 °C for one month are shown in
figures 74–85 below. The API concentration was reduced and products I and III
100
showed degardants or impurities eluting at 2, 5 and 10 minutes. Tailing was
observed in the all products stressed under this condition, which could mean the API
had reduced however the peak purity profile indicated that it was still pure.
Degradants did not interfere or co-elute with phenylephrine hydrochloride. Changes
in physical appearance were recorded in Table 9 below.
Table 9: Physical appearance of phenylephrine hydrochloride and products I–V before and after
storage conditions 40 °C/75%RH for 1 month
Table 9 above shows changes in appearance at 65 °C for all products. The same did
not occur at the 45 °C/75%RH storage condition where none of the samples
underwent any physical change.
Chromatograms of products I–V stressed at the 40 °C/75%RH for one month are
indicated in figures 86–97 below. The API concentration was reduced, however all
concentrations were above the 95%, except for product V which was at 94.97%.
Products I–V were able to withstand the heat and humidity without showing any
change in physical appearance. Tailing was observed in the all products stressed
under this condition, which could mean the API had slightly reduced however the
peak purity profile indicated that it was still pure. Degradants did not interfere or co-
elute with phenylephrine hydrochloride.
101
mAU
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 72: HPLC chromatogram for phenylephrine hydrochloride stored at 100 °C for 24 hours
dissolved in mobile phase with a retention time of 7.8 minutes.
mA U
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 73: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride stored at 100 °C for 24 hours in mobile phase. Peak shown in pink and purity curve in
black. Peak purity index = 1.000000; Single point threshold = 0.999999.
102
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 74: HPLC chromatogram for phenylephrine hydrochloride stored at 65 °C for 1 month
dissolved in mobile phase with a retention time of 7.83 minutes.
mAU
Purity Curve Peak
Zero Line
0.5 30.0
25.0
0.4
20.0
0.3
15.0
0.2
10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 75: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride stored at 65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in
black. Peak purity index = 1.000000; Single point threshold = 0.999989.
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 76: HPLC chromatogram for product I stored at 65 °C for 1 month dissolved in mobile phase
with a retention time of 7.92 minutes.
103
mAU
Purity Curve Peak
Zero Line 35.0
0.5
30.0
0.4
25.0
0.3 20.0
0.2 15.0
10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 77: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stored at
65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
0.999998; Single point threshold = 0.999956.
mA U
280nm,4nm ( 1.00)
60
55
50
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 78: HPLC chromatogram for product II stored at 65 °C for 1 month dissolved in mobile phase
with a retention time of 7.84 minutes.
mAU
Purity Curve Peak
Zero Line 35.0
0.5
30.0
0.4
25.0
0.3 20.0
0.2 15.0
10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 min
Figure 79: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stored at
65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
1.000000; Single point threshold = 0.999056.
104
mA U
280nm,4nm (1.00)
55
50
45
40
35
30
25
20
15
10
Figure 80: HPLC chromatogram for Product III stored at 65 °C for 1 month dissolved in mobile phase
with a retention time of 7.89 minutes.
mAU
Purity Curve Peak
0.5 Zero Line 35.0
30.0
0.4
25.0
0.3
20.0
0.2 15.0
10.0
0.1
5.0
0.0
0.0
7.75 8.00 min
Figure 81: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stored at
65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
1.000000; Single point threshold = 0.999720.
mA U
280nm,4nm (1.00)
40.0
37.5
35.0
32.5
30.0
27.5
25.0
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
Figure 82: HPLC chromatogram for Product IV stored at 65 °C for 1 month dissolved in mobile phase
with a retention time of 7.87 minutes.
105
mA U
Purity Curve Peak
Zero Line
0.6 25.0
0.5 20.0
0.4
15.0
0.3
10.0
0.2
0.1 5.0
0.0
0.0
7.75 8.00 min
Figure 83: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stored at
65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index
=0.999999; Single point threshold = 0.999803.
mA U
50 280nm,4nm ( 1.00)
45
40
35
30
25
20
15
10
-5
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 84: HPLC chromatogram for Product V stored at 65 °C for 1 month dissolved in mobile phase
with a retention time of 7.90 minutes.
mAU
Purity Curve Peak 70
0.7 Zero Line
60
0.6
50
0.5
0.4 40
0.3 30
0.2 20
0.1
10
0.0
0
7.5 8.0 8.5 min
Figure 85: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stored at
65 °C for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
1.000000; Single point threshold = 0.999752.
106
mA U
280nm,4nm (1.00)
35.0
32.5
30.0
27.5
25.0
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
-2.5
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 86: HPLC chromatogram for phenylephrine hydrochloride stored at 40 °C/75%RH for 1 month
dissolved in mobile phase with a retention time of 7.86 minutes.
mA U
Purity Curve Peak
0.6 Zero Line
20.0
0.5
0.4 15.0
0.3
10.0
0.2
0.1 5.0
0.0
0.0
7.50 7.75 8.00 8.25 min
Figure 87: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine
hydrochloride stored at 40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 1.000000; Single point threshold = 0.999056.
mA U
280nm,4nm (1.00)
35.0
32.5
30.0
27.5
25.0
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
-2.5
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 88: HPLC chromatogram for Product I stored at 40 °C/75%RH for 1 month dissolved in mobile
phase with a retention time of 7.87 minutes.
107
mAU
Purity Curve Peak
Zero Line
0.6
25.0
0.5
20.0
0.4
15.0
0.3
0.2 10.0
0.1 5.0
0.0
0.0
7.50 7.75 8.00 8.25 min
Figure 89: Peak purity profile calculated using PDA data (from 190–800 nm) for product I stored at 40
°C/75% RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 0.999999; Single point threshold = 0.995566.
mA U
280nm,4nm (1.00)
100
90
80
70
60
50
40
30
20
10
Figure 90: HPLC chromatogram for Product II stored at 40 °C/75%RH for 1 month dissolved in
mobile phase with a retention time of 7.84 minutes.
mAU
0.8 Purity Curve Peak
Zero Line
0.7
25.0
0.6
20.0
0.5
0.4 15.0
0.3
10.0
0.2
0.1 5.0
0.0
0.0
7.50 7.75 8.00 8.25 min
Figure 91: Peak purity profile calculated using PDA data (from 190–800 nm) for product II stored at
40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 1.000000; Single point threshold = 0.999579.
108
mA U
50 280nm,4nm (1.00)
45
40
35
30
25
20
15
10
Figure 92: HPLC chromatogram for product III stored at 40 °C/75%RH for 1 month dissolved in
mobile phase with a retention time of 7.81 minutes.
mAU
Purity Curve Peak
Zero Line 25.0
0.75
20.0
0.50
15.0
10.0
0.25
5.0
0.00
0.0
7.50 7.75 8.00 8.25 min
Figure 93: Peak purity profile calculated using PDA data (from 190–800 nm) for product III stored at
40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 1.000000; Single point threshold = 0.994609.
mA U
280nm,4nm (1.00)
50
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 94: HPLC chromatogram for Product IV stored at 40 °C/75%RH for 1 month dissolved in
mobile phase with a retention time of 7.85 minutes.
109
mAU
Purity Curve Peak
Zero Line 25.0
0.75
20.0
0.50
15.0
10.0
0.25
5.0
0.00
0.0
7.75 8.00 8.25 min
Figure 95: Peak purity profile calculated using PDA data (from 190–800 nm) for product IV stored at
40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 1.000000; Single point threshold = 0.994609.
mA U
280nm,4nm (1.00)
60
55
50
45
40
35
30
25
20
15
10
Figure 96: HPLC chromatogram for Product V stored at 40 °C/75%RH for 1 month dissolved in
mobile phase with a retention time of 7.83 minutes.
mAU
Purity Curve Peak
Zero Line
0.75 25.0
20.0
0.50
15.0
0.25 10.0
5.0
0.00
0.0
7.50 7.75 8.00 8.25 min
Figure 97: Peak purity profile calculated using PDA data (from 190–800 nm) for product V stored at
40 °C/75%RH for 1 month in mobile phase. Peak shown in pink and purity curve in black. Peak purity
index = 0.999999; Single point threshold = 0.995332.
110
Peak purities of all samples of stressed and unstressed phenylephrine hydrochloride
and finished product solutions are indicated in Table 10 below. Phenylephrine
hydrochloride alone showed loss to the combination of heat and humidity.
Table 10: Results showing absence of impurity from a series of stressed and unstressed samples of
phenylephrine hydrochloride (API) and products.
Number Sample name Co-eluting Image reference
impurities found
1 Mobile phase only None Figure 11
2 Active: unstressed None Figure 12
3 Product I, unstressed None Figure 14
4 Product II, unstressed None Figure 16
5 Product III, unstressed None Figure 18
6 Product IV, unstressed None Figure 20
7 Product V, unstressed None Figure 22
8 Active, stressed under UV for 17 None Figure 24
hours
9 Product I, stressed under UV for None Figure 26
17 hours
10 Product II, stressed under UV for None Figure 28
17 hours
11 Product III, stressed under UV for None Figure 30
17 hours
12 Product IV, stressed under UV for None Figure 32
17 hours
13 Product V, stressed under UV for None Figure 34
17 hours
14 Active, stressed with HCl None Figure 36
15 Product I, stressed with HCl None Figure 38
16 Product II, stressed with HCl None Figure 40
17 Product III, stressed with HCl None Figure 42
18 Product IV, stressed with HCl None Figure 44
19 Product V, stressed with HCl None Figure 46
20 Active, stressed with NaOH None Figure 48
21 Product I, stressed with NaOH None Figure 50
22 Product II, stressed with NaOH None Figure 52
23 Product III, stressed with NaOH None Figure 54
24 Product IV, stressed with NaOH None Figure 56
25 Product V, stressed with NaOH None Figure 58
26 Active, stressed with H2O2 None Figure 60
27 Product I, stressed with H2O2 None Figure 62
28 Product II, stressed with H2O2 None Figure 64
29 Product III, stressed with H2O2 None Figure 66
30 Product IV, stressed with H2O2 None Figure 68
31 Product V, stressed with H2O2 None Figure 70
32 Active, stressed at 100 °C None Figure 72
33 Active, stressed at 65 °C None Figure 74
34 Product I, stressed at 65 °C None Figure 76
35 Product II, stressed at 65 °C None Figure 78
36 Product III, stressed at 65 °C None Figure 80
37 Product IV, stressed at 65 °C None Figure 82
38 Product V, stressed at 65 °C None Figure 84
39 Active, stressed at 40 °C/75%RH None Figure 86
111
40 Product I, stressed at 40 stressed None Figure 88
at 40 °C/75%RH
41 Product II, stressed at 40 None Figure 90
°C/75%RH
Table 11: Results showing phenylephrine hydrochloride left with samples stressed and unstressed
(API and Products)
112
33 Active, stressed at 65 °C 92.42%
34 Product I, stressed at 65 °C 86.92%
35 Product II, stressed at 65 °C 90.74%
36 Product III, stressed at 65 °C 91.47%
37 Product IV, stressed at 65 °C 80.52%
38 Product V, stressed at 65 °C 88.21%
39 Active, stressed at 40 °C/75%RH 90.20%
40 Product I, stressed at 40 °C/75%RH 96.57%
41 Product II, stressed at 40 °C/75%RH 98.48%
42 Product III, stressed at 40 °C/75%RH 95.99%
43 Product IV, stressed at 40 °C/75%RH 95.37%
44 Product V, stressed at 40 °C/75%RH 94.97%
The HPLC method used for the phenylephrine hydrochloride and finished products
was found to be suitable because the degradants showed no interference. The range
retention time for phenylephrine was 7.78–7.92 minutes. The impurities did not
interfere or co-elute with phenylephrine hydrochloride, as its peak was distinct and
clear.
Three replicates of active and excipient mixtures at a ratio of 1:1 were stored at 40
ºC/75%RH in plastic containers for three months. They were then assayed using the
HPLC method. The percentage of phenylephrine hydrochloride remaining in each
sample was calculated from the linear regression curve, y = 8541.1x + 438.1 taking
the initial quantity of phenylephrine hydrochloride in each container to be 100%. The
potency of samples stored at 40 ºC/75%RH and the unstressed samples were within
95–105%. Figures 98–121 show the chromatograms of phenylephrine hydrochloride
alone and phenylephrine hydrochloride:combined with excipients; there was no
interference observed between the phenylephrine hydrochloride and excipients.
Table 12 below shows that all the samples were within the required potency range of
95–105%.
Table 12: Assay result showing phenylephrine hydrochloride and excipients in a 1:1 ratio after
storage conditions 40 °C/75%RH for 1 month
Phenylephrine hydrochloride and excipients Percentage of phenylephrine
hydrochloride
(API (left) % ± % RSD)
40 °C / 75 % RH
Phenylephrine hydrochloride alone 98.15 ± 0.41
Boric acid 99.48 ± 1.53
Benzalkonium Chloride 98.63 ± 0.25
EDTA 97.73 ± 0.48
113
Glycerol 95.79 ± 0.22
Methyl Paraben 96.33 ± 1.86
Propyl Paraben 96.73 ± 0.36
Sodium metabisulfite 97.58 ± 0.53
Sodium citrate dehydrate 95.10 ± 0.39
Carboxymethylcellulose Sodium 96.24 ± 1.44
Hydroxypropyl methylcellulose 98.59 ± 1.33
Table 13: Physical appearance of active–excipients samples before and after storage conditions 40
°C/75%RH for 4 weeks
Phenylephrine hydrochloride and Initial Physical appearance
excipients physical appearance after 4 weeks of
storage
40 °C/75%RH
Phenylephrine hydrochloride alone Clear solution, no precipitate or No change
residue, no colour change
Benzalkonium chloride and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
Boric acid and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
Sodium carboxy methylcellulose and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
EDTA and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
Glycerol and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
HPMC and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
Methyl paraben and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
Propyl paraben and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
Sodium citrate dihydrate and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
Sodium metabisulfite and Clear solution, no precipitate or No change
phenylephrine hydrochloride residue, no colour change
The physical appearance of the phenylephrine hydrochloride and excipient blends
can be found in Table 13 above. No colour change and precipitate formation were
observed in any of the 1:1 mixtures after four weeks of storage. There were no
reductions or increase in liquid level after storage, meaning that the plastic container
was not permeable to moisture. The data also proved that the active and excipients
did not interfere or interact with each other.
114
mA U
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 98: HPLC chromatogram for phenylephrine hydrochloride alone dissolved in mobile phase
with retention of 7.82 minutes.
mAU
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 99: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine
hydrochloride in mobile phase. Peak shown in pink and purity curve in black. Peak purity index =
1.000000; Single point threshold = 0.999999.
Figure 100: HPLC chromatogram for phenylephrine hydrochloride with sodium citrate dihydrate (1:1)
dissolved in mobile phase with a retention time of 7.92 minutes.
115
mAU
Purity Curve Peak
0.6 Zero Line
200
0.5
0.4 150
0.3
100
0.2
0.1 50
0.0
0
8.0 8.5 min
Figure 101: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride with sodium citrate dihydrate (1:1) in mobile phase. Peak shown in pink and purity
curve in black. Peak purity index = 1.000000; Single point threshold = 0.999896.
mA U
280nm4nm (1.00)
90
80
70
60
50
40
30
20
10
mA U
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 103: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride with carboxymethycellulose sodium (1:1) in mobile phase. Peak shown in pink and
purity curve in black. Peak purity index = 1.000000; Single point threshold = 0.999999.
116
mA U
280nm4nm (1.00)
90
80
70
60
50
40
30
20
10
Figure 104: HPLC chromatogram for phenylephrine hydrochloride with hypromellose (1:1) dissolved
in mobile phase with a retention time of 7.81 minutes.
mA U
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 105: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine
hydrochloride with hypromellose (1:1) in mobile phase. Peak shown in pink and purity curve in black.
Peak purity index = 1.000000; Single point threshold = 0.999999.
mA U
280nm4nm (1.00)
90
80
70
60
50
40
30
20
10
Figure 106: HPLC chromatogram for phenylephrine hydrochloride with glycerol (1:1) dissolved in
mobile phase with a retention time of 7.85 minutes.
117
mA U
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 107: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride with glycerol (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 1.000000; Single point threshold = 0.999989.
mA U
280nm4nm (1.00)
90
80
70
60
50
40
30
20
10
Figure 108: HPLC chromatogram for phenylephrine hydrochloride with benzalkonium chloride (1:1)
dissolved in mobile phase with a retention time of 7.88 minutes.
mA U
0.8 Purity Curve Peak
Zero Line
0.7 60
0.6 50
0.5
40
0.4
30
0.3
0.2 20
0.1 10
0.0
0
7.5 8.0 min
Figure 109: Peak purity profile calculated using PDA date (from 190–800 nm) for phenylephrine
hydrochloride with benzalkonium chloride (1:1) in mobile phase. Peak shown in pink and purity curve
in black. Peak purity index = 0.999999; Single point threshold = 0.999918.
118
Figure 110: HPLC chromatogram for phenylephrine hydrochloride with EDTA (1:1) dissolved in
mobile phase with a retention time of 7.86 minutes.
mA U
Purity Curve Peak
Zero Line 250
0.7
0.6
200
0.5
0.4 150
0.3
100
0.2
0.1 50
0.0
0
8.0 8.5 min
Figure 111: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride with EDTA (1:1) in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 0.999999; Single point threshold = 0.999991.
Figure 112: HPLC chromatogram for phenylephrine hydrochloride with boric acid (1:1) dissolved in
mobile phase with a retention time of 7.92 minutes.
119
mAU
Purity Curve Peak
0.6 Zero Line
20.0
0.5
0.4 15.0
0.3
10.0
0.2
0.1 5.0
0.0
0.0
7.50 7.75 8.00 8.25 min
Figure 113: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride with boric acid (1:1) in mobile phase. Peak shown in pink and purity curve in black.
Peak purity index = 1.000000; Single point threshold = 0.999999.
mA U
280nm,4nm (1.00)
80
70
60
50
40
30
20
10
-10
Figure 114: HPLC chromatogram for phenylephrine hydrochloride with sodium metabisulfite (1:1)
dissolved in mobile phase with a retention time of 7.82 minutes.
mA U
0.5 Purity Curve Peak
Zero Line
175.0
0.4
150.0
0.3 125.0
100.0
0.2
75.0
0.1 50.0
25.0
0.0
0.0
8.0 8.5 min
Figure 115: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride with sodium metabisulfite (1:1) in mobile phase. Peak shown in pink and purity curve in
black. Peak purity index = 1.000000; Single point threshold = 0.999870.
120
Figure 116: HPLC chromatogram for phenylephrine hydrochloride with disodium edetate (1:1)
dissolved in mobile phase with a retention time of 7.82 minutes.
mAU
0.8 Purity Curve Peak
Zero Line 50
0.7
0.6 40
0.5
30
0.4
0.3
20
0.2
0.1 10
0.0
0
8.00 8.25 8.50 8.75 min
Figure 117: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride with disodium edetate (1:1) in mobile phase. Peak shown in pink and purity curve in
black. Peak purity index = 1.000000; Single point threshold = 0.998891.
Figure 118: HPLC chromatogram for phenylephrine hydrochloride with propyl paraben (1:1) dissolved
in mobile phase with a retention time of 7.83 minutes.
121
mA U
Purity Curv e Peak
Zero Line 175.0
0.6
150.0
0.5
125.0
0.4
100.0
0.3
75.0
0.2
50.0
0.1
25.0
0.0
0.0
7.5 8.0 8.5 min
Figure 119: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride with propyl paraben (1:1) in mobile phase. Peak shown in pink and purity curve in
black. Peak purity index = 1.000000; Single point threshold = 0.999948.
Figure 120: HPLC chromatogram for phenylephrine hydrochloride with methyl paraben (1:1)
dissolved in mobile phase with a retention time of 7.82 minutes.
mAU
1.00 Purity Curve Peak 500
Zero Line
0.75 400
300
0.50
200
0.25
100
0.00
0
9.0 9.5 10.0 min
Figure 121: Peak purity profile calculated using PDA data (from 190–800 nm) for phenylephrine
hydrochloride with methyl paraben (1:1) in mobile phase. Peak shown in pink and purity curve in
black. Peak purity index = 1.000000; Single point threshold = 0.999982.
122
4.3 Stability study
The results for storage time zero and one month 40 °C/75%RH (accelerated
condition) were stated in specificity above.
-1
-2
-3
-4
-5
-6
Figure 122: HPLC chromatogram for product I stored at 30 °C/65%RH for 3 months dissolved in
mobile phase with a retention time of 7.87 minutes.
123
mA U
0.65 Purity Curv e Peak 27.5
Zero Line
0.60
25.0
0.55
22.5
0.50
0.45 20.0
0.40 17.5
0.35
15.0
0.30
12.5
0.25
0.20 10.0
0.15 7.5
0.10
5.0
0.05
2.5
0.00
0.0
7.75 8.00 8.25 8.50 min
Figure 123: Peak purity profile calculated using PDA date (from 190–800 nm) for product I stored at
30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 0.999999; Single point threshold = 0.999950.
mA U
280nm,4nm (1.00)
7.5
5.0
2.5
0.0
-2.5
-5.0
Figure 124: HPLC chromatogram for product II stored at 30 °C/65%RH for 3 months dissolved in
mobile phase with a retention time of 7.85 minutes.
mA U
Purity Curv e Peak
0.60 Zero Line
27.5
0.55
25.0
0.50
22.5
0.45
20.0
0.40
0.35 17.5
0.30 15.0
0.25 12.5
0.20 10.0
0.15
7.5
0.10
5.0
0.05
2.5
0.00
0.0
7.75 8.00 8.25 8.50 min
Figure 125: Peak purity profile calculated using PDA date (from 190–800 nm) for product II stored at
30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 1.000000; Single point threshold = 0.996473.
124
mA U
280nm,4nm (1.00)
7.5
5.0
2.5
0.0
-2.5
-5.0
Figure 126: HPLC chromatogram for product III stored at 30 °C/65%RH for 3 months dissolved in
mobile phase with a retention time of 7.87 minutes.
mA U
Purity Curv e Peak
Zero Line 32.5
0.8
30.0
0.7
27.5
25.0
0.6
22.5
0.5
20.0
17.5
0.4
15.0
0.3
12.5
10.0
0.2
7.5
0.1 5.0
2.5
0.0
0.0
7.75 8.00 8.25 8.50 min
Figure 127: Peak purity profile calculated using PDA date (from 190–800 nm) for product III stored at
30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 0.999999; Single point threshold = 0.996955.
mA U
15.0 280nm,4nm (1.00)
12.5
10.0
7.5
5.0
2.5
0.0
-2.5
-5.0
-7.5
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 128: HPLC chromatogram for product IV stored at 30 °C/65% RH for 3 months dissolved in
mobile phase with a retention time of 7.88 minutes.
125
mA U
0.7 Pur ity Curv e Peak
Z er o Line 55.0
50.0
0.6
45.0
0.5
40.0
35.0
0.4
30.0
0.3 25.0
20.0
0.2
15.0
0.1 10.0
5.0
0.0
0.0
7.75 8.00 8.25 8.50 min
Figure 129: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at
30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 0.999998; Single point threshold = 0.998694.
mA U
280nm,4nm (1.00)
7
-1
-2
-3
-4
-5
-6
Figure 130: HPLC chromatogram for product V stored at 30 °C/65%RH for 3 months dissolved in
mobile phase with a retention time of 7.87 minutes.
mA U
0.65 Pur ity Curv e Peak
Z er o Line
0.60 25.0
0.55
22.5
0.50
20.0
0.45
0.40 17.5
0.35 15.0
0.30
12.5
0.25
10.0
0.20
0.15 7.5
0.10 5.0
0.05
2.5
0.00
0.0
7.75 8.00 8.25 8.50 min
Figure 131: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at
30 °C/65%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 0.999998; Single point threshold = 0.994533.
Products I–V stored for 3 months at 25 °C/60%RH: The products showed no co-
elution, no interference, and the API left in products I–V was 82.77%, 96.84%,
126
86.78%, 97.06%, and 70.88% respectively. There was a statistic difference of
p<0.05. The products III and V failed aesthetically as they turned slightly yellow and
brown respectively, with no residue, precipitate and loss of solution. The remaining
products remained clear and aesthetically pleasing. The figures 132–141 below
show the chromatograms of the products.
mAU
17.5 280nm,4nm (1.00)
15.0
12.5
10.0
7.5
5.0
2.5
0.0
-2.5
mA U
Purity Curv e Peak
Zero Line
0.9
45.0
0.8
40.0
0.7
35.0
0.6
30.0
0.5
25.0
0.4
20.0
0.3
15.0
0.2
10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 8.50 min
Figure 133: Peak purity profile calculated using PDA date (from 190–800 nm) for product I stored at
25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 1.000000; Single point threshold = 0.995873.
127
mA U
280nm,4nm (1.00)
100
90
80
70
60
50
40
30
20
10
-10
-20
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 134: HPLC chromatogram for product II stored at 25 °C/60%RH for 3 months dissolved in
mobile phase with a retention time of 7.89 minutes.
mA U
0.45 Purity Curv e Peak
Zero Line 400
0.40
350
0.35
300
0.30
250
0.25
0.20 200
0.15 150
0.10
100
0.05
50
0.00
0
7.50 7.75 8.00 8.25 8.50 min
Figure 135: Peak purity profile calculated using PDA date (from 190–800 nm) for product II stored at
25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 0.999998; Single point threshold = 0.999950.
mA U
15.0 280nm,4nm (1.00)
12.5
10.0
7.5
5.0
2.5
0.0
-2.5
-5.0
-7.5
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 136: HPLC chromatogram for product III stored at 25 °C/60%RH for 3 months dissolved in
mobile phase with a retention time of 7.92 minutes.
128
mA U
Purity Curv e Peak
Zero Line 55.0
0.6
50.0
45.0
0.5
40.0
0.4 35.0
30.0
0.3
25.0
0.2 20.0
15.0
0.1
10.0
5.0
0.0
0.0
7.75 8.00 8.25 8.50 min
Figure 137: Peak purity profile calculated using PDA date (from 190–800 nm) for product III stored at
25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 0.999999; Single point threshold = 0.998742.
mA U
280nm,4nm (1.00)
7.5
5.0
2.5
0.0
-2.5
-5.0
Figure 138: HPLC chromatogram for product IV stored at 25 °C/60%RH for 3 months dissolved in
mobile phase with a retention time of 7.92 minutes.
mA U
0.8 Purity Curv e Peak
Zero Line 32.5
30.0
0.7
27.5
0.6 25.0
22.5
0.5
20.0
0.4 17.5
15.0
0.3
12.5
0.2 10.0
7.5
0.1
5.0
2.5
0.0
0.0
7.75 8.00 8.25 8.50 min
Figure 139: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at
25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 0.999999; Single point threshold = 0.996396.
129
mA U
12.5 280nm,4nm (1.00)
10.0
7.5
5.0
2.5
0.0
-2.5
-5.0
Figure 140: HPLC chromatogram for product V stored at 25 °C/60%RH for 3 months dissolved in
mobile phase with a retention time of 7.84 minutes.
mA U
Purity Curv e Peak
0.60 Zero Line 45.0
0.55
40.0
0.50
0.45 35.0
0.40
30.0
0.35
25.0
0.30
0.25 20.0
0.20
15.0
0.15
0.10 10.0
0.05
5.0
0.00
0.0
7.75 8.00 8.25 8.50 min
Figure 141: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at
25 °C/60%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 1.000000; Single point threshold = 0.998093.
Products I–V stored for 3 months at 40 °C/75%RH: The products showed no co-
elution and no interference. The API left from product I–V were 61.63%, 96.50%,
53.10%, 77.16%, and 54.79% respectively. There was a statistical difference as
p<0.0001. The products I, III and V failed aesthetically as they turned slightly yellow,
dark yellow and brown in colour respectively, with no residue, precipitate and loss of
solution. The remaining products II and IV remained clear and aesthetically pleasing.
The figures 143–152 below show the chromatograms of the products.
130
mA U
280nm,4nm (1.00)
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
Figure 142: HPLC chromatogram for product I stored at 40 °C/75%RH for 3 months dissolved in
mobile phase with a retention time of 7.84 minutes.
mA U
0.65 Purity Curv e Peak
Zero Line
0.60 45.0
0.55
40.0
0.50
35.0
0.45
0.40 30.0
0.35
25.0
0.30
0.25 20.0
0.20
15.0
0.15
0.10 10.0
0.05
5.0
0.00
0.0
7.75 8.00 8.25 8.50 min
Figure 143: Peak purity profile calculated using PDA date (from 190 – 800 nm) for product I stored at
40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 1.000000; Single point threshold = 0.998093.
mA U
280nm,4nm (1.00)
7.5
5.0
2.5
0.0
-2.5
-5.0
Figure 144: HPLC chromatogram for product II stored at 40 °C/75%RH for 3 months dissolved in
mobile phase with a retention time of 7.84 minutes.
131
mA U
Purity Curv e Peak
0.60 Zero Line
27.5
0.55
25.0
0.50
22.5
0.45
20.0
0.40
0.35 17.5
0.30 15.0
0.25 12.5
0.20 10.0
0.15
7.5
0.10
5.0
0.05
2.5
0.00
0.0
7.75 8.00 8.25 8.50 min
Figure 145: Peak purity profile calculated using PDA date (from 190 – 800 nm) for product II stored
at 40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 1.000000; Single point threshold = 0.998093.
mA U
280nm,4nm (1.00)
7.5
5.0
2.5
0.0
-2.5
-5.0
Figure 146: HPLC chromatogram for product III stored at 40 °C/75%RH for 3 months dissolved in
mobile phase with a retention time of 7.84 minutes.
mA U
Purity Curv e Peak 35.0
0.8 Zero Line
0.7 30.0
0.6
25.0
0.5
20.0
0.4
15.0
0.3
0.2 10.0
0.1
5.0
0.0
0.0
7.75 8.00 8.25 8.50 min
Figure 147: Peak purity profile calculated using PDA date (from 190–800 nm) for product III stored at
40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 1.000000; Single point threshold = 0.998093.
132
mA U
280nm,4nm (1.00)
6
-1
-2
-3
-4
-5
-6
Figure 148: HPLC chromatogram for product IV stored at 40 °C/75%RH for 3 months dissolved in
mobile phase with a retention time of 7.84 minutes.
mA U
Purity Curv e Peak
0.9 Zero Line 22.5
0.8 20.0
0.7 17.5
0.6
15.0
0.5
12.5
0.4
10.0
0.3
7.5
0.2
5.0
0.1
2.5
0.0
0.0
7.75 8.00 8.25 8.50 min
Figure 149: Peak purity profile calculated using PDA date (from 190–800 nm) for product IV stored at
40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 1.000000; Single point threshold = 0.998093.
mA U
280nm,4nm (1.00)
6
-1
-2
-3
-4
-5
-6
-7
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 150: HPLC chromatogram for product V stored at 40 °C/75%RH for 3 months dissolved in
mobile phase with a retention time of 7.91 minutes.
133
mA U
Purity Curv e Peak
1.0 Zero Line
22.5
0.9
20.0
0.8
0.7 17.5
0.6 15.0
0.5 12.5
0.4 10.0
0.3
7.5
0.2
5.0
0.1
2.5
0.0
0.0
7.75 8.00 8.25 8.50 min
Figure 151: Peak purity profile calculated using PDA date (from 190–800 nm) for product V stored at
40 °C/75%RH for 3 months in mobile phase. Peak shown in pink and purity curve in black. Peak
purity index = 0.999999; Single point threshold = 0.998093.
Figure 152 below summarises the API remaining in the given storage conditions.
Product II was stable and within the potency range for all storage conditions while
product IV was stable and potent within two storage conditions. The remaining
Products (I, III and V) failed to retain their potency at various conditions. According to
the results obtained, product II and IV were stable and with the most API remaining
in the product. Products II and IV were also the most aesthetically pleasant. This
could be due to the presence of protectors and anti-oxidants such as sodium citrate
dihydrate, EDTA, sodium metabisulfite, boric acid.
120
100 Product V
Product IV
80
product
Product III
60
Product II
40 Product I
20
0
1 2 3
1 - 30 °C / 65 % RH
2 - 40 °C / 75 % RH
3 - 25 °C / 60 % RH
Figure 152: A graph showing standard error and phenylephrine hydrochloride left in product I–V after
12 weeks at 30 °C/65%RH, 40 °C/75%RH, 25 °C/60%RH.
134
Table 14: Results of one-way ANOVA analysis for products I–V stored at 25 °C/60%RH, 30
°C/65%RH and 40 °C/75%RH for 3 months.
Products I II III IV V I II III IV V I II III IV V
°C/%RH 25/ 25/ 25/ 25/ 25/ 30/ 30/ 30/ 30/ 30/ 40/ 40/ 40/ 40/ 40/
60 60 60 60 60 65 65 65 65 65 75 75 75 75 75
I 25/60 **** **** **** **** **** **** **** **** **** **** **** **** **** ****
II 25/60 **** **** ns **** **** ns **** ** **** **** ns **** **** ****
III 25/60 **** **** **** **** **** **** **** **** **** **** **** **** **** ****
IV 25/60 **** ns **** **** **** ns **** * **** **** ns **** **** ****
V 25/60 **** **** **** **** *** **** **** **** **** **** **** **** **** ****
I 30/65 **** **** **** **** *** **** **** **** **** **** **** **** **** ****
II 30/65 **** ns **** **** **** **** **** ** **** **** ns **** **** ****
III 30/65 **** **** **** **** **** **** **** **** **** **** **** **** **** ****
IV 30/65 **** ** **** **** **** **** ** **** **** **** *** **** **** ****
V 30/65 **** **** **** **** **** **** **** **** **** ns **** **** **** ****
I 40/75 **** **** **** **** **** **** **** **** **** ns **** **** **** ****
II 40/75 **** ns **** **** **** **** **** **** *** **** **** **** **** ****
III 40/75 **** **** **** **** **** **** **** **** **** **** **** **** **** **
IV 40/75 **** **** **** **** **** **** **** **** **** **** **** **** **** ****
V 40/75 **** **** **** **** **** **** **** **** **** **** **** **** ** ****
ns - Not Significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001
Above is Table 14 which shows results of statistical comparisons of all the products.
The quantity of phenylephrine hydrochloride in product II did not change significantly
when the remaining amounts of API for the various storage conditions were
compared. The quantity of API present for the other products changed significantly,
this means that product II was stable at all three storage conditions.
Summarised below in tables 15 and 16 are the changes in pH for products I–V.
Table 15 below shows pH change in products I–V stored at 40 °C/75%RH for one
month, Table 16 below shows pH changes in products I–V stored at various storage
conditions after three months of storage. Changes were observed for some products,
as they deviated from the pH of four they were initially formulated at. This could be
due to oxidation or reduction reactions of the API or excipients; it should be noted
that phenylephrine hydrochloride is stable in the pH range of 3.5–8 and none of the
products exceeded this limits even though changes were noted (Giahi et al., 2009).
Over time products I, III and V became more basic, product II and IV were stable and
remained close to their original pH for all storage conditions. According to the pH
results obtained, product II and product IV showed favourable stability.
135
Table 15: Changes in pH of phenylephrine hydrochloride and products I–V before and after storage
conditions 40 °C/75%RH for 1 month.
Table 16: Changes in pH for products I–V before and after varying storage conditions for 3 months
Statistical differences were determined using the one-way ANOVA analysis. All pH
results were significantly different from each other as p<0.0001; similarities were
summarized in Table 17 below.
Table 17: Results of one-way ANOVA analysis for similarities in pH of products II 25 °C/60%RH, 40
°C/75%RH and IV 25 °C/60%RH after 3 months.
136
Table 18: Physical appearance of products I–V before and after varying storage conditions for 3
months.
137
TAU
0
30 10 hpmc product 5 30/65 3 t0 2
Pa CC27-SN25781; d=0 mm
Shear Stress
26 Viscosity
Pa·s
glycerol product 3 30/65 3 t0 2
24
CC27-SN25781; d=0 mm
22 Shear Stress
Viscosity
-1
20 10 hpmc product 1 30/65 3 t0 2
CC27-SN25781; d=0 mm
18
Shear Stress
16 Viscosity
Shear Stress
12
Viscosity
-2
10 10 scmc product 4 30/65 3 t0 1
CC27-SN25781; d=0 mm
8
Shear Stress
Viscosity
6
Prefrin 3 1
4 CC27-SN25781; d=0 mm
Shear Stress
2
Viscosity
-3
0 10
0 100 200 300 400 500 600 1/s 700
.
Shear Rate
Anton Paar GmbH
Figure 153: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition
30 °C/65% RH.
Figures 153–158 show variance in viscosity between the different viscosity modifiers
(glycerol, hydroxy propyl methylcellulose and sodium carboxy methyl cellulose). At
time zero, Prefrin® and product I–V display slightly similar flow. After three months
glycerol had a marked increase in its viscosity, this is seen in the figures 154, 156
and 158 below. Other viscosity modifiers show slight changes.
TAU
0
30 10 glycerol product 330/65 3 1
Pa CC27-SN25781; d=0 mm
Shear Stress
26 Viscosity
Pa·s
hpmc product 2 30/65 3 1
24
CC27-SN25781; d=0 mm
22 Shear Stress
Viscosity
-1
20 10 hpmc product 1 30/65 3 1
CC27-SN25781; d=0 mm
18
Shear Stress
16 Viscosity
Shear Stress
12
Viscosity
-2
10 10 scmc product 4 30/65 3 1
CC27-SN25781; d=0 mm
8
Shear Stress
Viscosity
6
Prefrin 3 1
4 CC27-SN25781; d=0 mm
Shear Stress
2
Viscosity
-3
0 10
0 100 200 300 400 500 600 1/s 700
.
Shear Rate
Anton Paar GmbH
Figure 154: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage
condition 30 °C/65% RH.
138
TAU
0
30 10 hpmc product 1 40/75 3 t0 1
Pa CC27-SN25781; d=0 mm
Shear Stress
26 Viscosity
Pa·s
hpmc product 2 40/75 3 t0 1
24
CC27-SN25781; d=0 mm
22 Shear Stress
Viscosity
-1
20 10 hpmc product 5 40/75 3 t0 1
CC27-SN25781; d=0 mm
18
Shear Stress
16 Viscosity
Shear Stress
12
Viscosity
-2
10 10 scmc product 4 40/75 3 t0 1
CC27-SN25781; d=0 mm
8
Shear Stress
Viscosity
6
Prefrin 3 1
4 CC27-SN25781; d=0 mm
Shear Stress
2
Viscosity
-3
0 10
0 100 200 300 400 500 600 1/s 700
.
Shear Rate
Anton Paar GmbH
Figure 155: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition
40 °C/75% RH.
TAU
0
30 10 scmc product 4 40/75 3 1
Pa CC27-SN25781; d=0 mm
Shear Stress
26 Viscosity
Pa·s
hpmc product 2 40/75 3 1
24
CC27-SN25781; d=0 mm
22 Shear Stress
Viscosity
-1
20 10 hpmc product 1 40/75 3 1
CC27-SN25781; d=0 mm
18
Shear Stress
16 Viscosity
Shear Stress
12
Viscosity
-2
10 10 glycerol product 3 40/75 3 1
CC27-SN25781; d=0 mm
8
Shear Stress
Viscosity
6
Prefrin 3 1
4 CC27-SN25781; d=0 mm
Shear Stress
2
Viscosity
-3
0 10
0 100 200 300 400 500 600 1/s 700
.
Shear Rate
Anton Paar GmbH
Figure 156: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage
condition 40 °C/75% RH.
139
TAU
0
30 10 hpmc product 1 25/60 3 t0 1
Pa CC27-SN25781; d=0 mm
Shear Stress
26 Viscosity
Pa·s
hpmc product 2 25/60 3 t0 1
24
CC27-SN25781; d=0 mm
22 Shear Stress
Viscosity
-1
20 10 hpmc product 5 25/60 3 t0 1
CC27-SN25781; d=0 mm
18
Shear Stress
16 Viscosity
Shear Stress
12
Viscosity
-2
10 10 scmcproduct 4 25/60 3 t0 1
CC27-SN25781; d=0 mm
8
Shear Stress
Viscosity
6
Prefrin 3 1
4 CC27-SN25781; d=0 mm
Shear Stress
2
Viscosity
-3
0 10
0 100 200 300 400 500 600 1/s 700
.
Shear Rate
Anton Paar GmbH
Figure 157: Flow and viscosity curve of Prefrin® and products I–V at time zero for storage condition
25 °C/60%RH.
TAU
0
30 10 glycerol product 3 25/60 3 1
Pa CC27-SN25781; d=0 mm
Shear Stress
26 Viscosity
Pa·s
hpmc product 1 25/60 3 1
24
CC27-SN25781; d=0 mm
22 Shear Stress
Viscosity
-1
20 10 hpmc product 5 25/60 3 1
CC27-SN25781; d=0 mm
18
Shear Stress
16 Viscosity
Shear Stress
12
Viscosity
-2
10 10 hpmc product 2 25/60 3 1
CC27-SN25781; d=0 mm
8
Shear Stress
Viscosity
6
Prefrin 3 1
4 CC27-SN25781; d=0 mm
Shear Stress
2
Viscosity
-3
0 10
0 100 200 300 400 500 600 1/s 700
.
Shear Rate
Anton Paar GmbH
Figure 158: Flow and viscosity curve of Prefrin® and products I–V after 3 months for storage
condition 25 °C/60% RH.
140
Prefrin® was choosen as the product against which others would be measured
because polvinyl alcohol was used as its VMA, while phenylephrine hydrochloride as
its API. This made it a good chioce for comparison as it had a VMA and a similar
API. It proved to be not significantly as different to the rest as seen in figures 154–
159 as p>0.05. Products I–V did not lose water from the containers during the
stability study, thus the change in viscosity cannot be attributed to loss of volume.
1
0.9
0.8
0.7
Viscosity (mPa.s)
0.6
0.5 T1 40/75
0.4 T0 40/75
0.3
0.2
0.1
0
Product I Product II Product III Product IV Product V Prefrin®
Figure 159: Graph showing viscosity of products I–V stored in a stability chamber of 40 °C/75%RH
tested at time zero (T0), three months later (T1) and compared to an original marketed product
Prefrin®.
0.9
0.8
0.7
Viscosity (mPa.s)
0.6
0.5
t1 25/60
0.4
T0 25/60
0.3
0.2
0.1
0
Product I Product II Product III Product IV Product V Prefrin®
Figure 160: Graph showing viscosity of products I–V stored in a stability chamber of 25 °C/60%RH
tested at time zero (T0), 3 months later (T1) and compared to an original marketed product Prefrin®.
141
1
0.9
0.8
0.7
0.6
Viscosity (mPa.s)
0.5 T1 30/65
T0 30/65
0.4
0.3
0.2
0.1
0
Product I Product II Product III Product IV Product V Prefrin®
Figure 161: Graph showing viscosity of products I–V stored in a stability chamber of 30 °C/65%RH
tested at time zero (T0), 3 months later (T1) and compared to an original marketed product Prefrin®.
Using the above graphs in figures 159–161 product II showed stability and good
rheology properties. The higher the value is to 1 mPa.s, the more viscous a product
was.
Statistically no difference was found among most products manufactured and the
marketed product. However, product III had significant differences (p<0.05) in
viscosity at various storage conditions and time frames as seen in Table 19 below.
Product III had a significantly higher viscosity than the other products after the 3
months stability period. The products with “ns” showed that the formulations were the
same in mean flow and yield points.
142
Table 19: Results of one-way ANOVA analysis for viscosity of products I–V stored at 25 °C/60%RH,
30 °C/65%RH and 40 °C/75%RH for 3 months. The values shown indicate differences in p-values and
significance in differences of mean was defined as p < 0.05.
mPa PI PII PIII PI PV PI PII PIII PI V I II III IV V Prefr
.s 25/ 25/ 25/ V 25/ 30/ 30/ 30/ V 30/ 40/ 40/ 40/ 40/ 40/ in®
60 60 60 25/ 60 65 65 65 30/ 65 75 75 75 75 75
60 65
PI ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
25/6
0
PII ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
25/6
0
PIII ns ns ns ns ns ns ns ns ns ns ns ns ns ns **
25/6
0
PIV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
25/6
0
PV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
25/6
0
PI ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
30/6
5
PII ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
30/6
5
PIII ns ns ns ns ** ns ns * ns *** * ns ** * ****
30/6
5
PIV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
30/6
5
PV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
30/6
5
PI ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
40/7
5
PII ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
40/7
5
PIII ns ns ns ns ns ns ns ns ns ns ns ns ns ns **
40/7
5
PIV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
40/7
5
PV ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
40/7
5
Prefr ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
in®
ns - Not Significant, *= p < 0.05, ** = p < 0.01, ***=p < 0.001, **** = p < 0.0001
143
4.5 Effectiveness of the ophthalmic solution preservatives
Ophthalmic drops are sterile formulations which are usually packed in multi-dose
containers. Microbial contamination from multiple uses may lead to product
degradation or result in ocular infection (Sutton et al., 2002). Protection of these
multiple dose products against microbial contamination is usually achieved by
addition of a suitable preservative system. The antimicrobial effectiveness test is
designed to provide a laboratory test that gauges the level of antimicrobial activity by
a pharmaceutical product and to evaluate how well a product withstands microbial
contamination while being used (Nostro et al., 2004 and Sutton et al., 2002).
After 14 days, all the eye-drops had varied log reduction against all the challenging
organisms which were all within acceptable limits. In all cases the number of fungi
after 7, 14 and 28 days were acceptable as they were reduced by 3 and 4 logs which
surpassed the requisite of fungi count.
144
Table 20: Antimicrobial preservative efficacy of the eye-drop products I–V challenged with E. coli,
S.aureus, P. aeruginosa, C.albicans.
Microorganism Eye-drop Sampling time/Log reduction
0 6 hours 24 7 days 14 days 28 days
hour hours
Product I 2.9 x 3 2 - - NR
106
E. coli Product II 2.7 x 3 3 - - NR
106
ATCC 38218
Product III 2.7 x 4 1 - - NR
106
Product IV 2.9 x 2 2 - - NR
106
Product V 2.9 x 4 2 - - NR
106
Product I 1.1 x 3 2 - - NR
106
Product II 1.1 x 3 3 - - NR
S.aureus
106
Product III 2.1 x 3 2 - - NR
ATCC 43300
106
Product IV 2.0 x 2 2 - - NR
106
Product V 1.1 x 4 2 - - NR
106
Eye drops are classified under category 1 in the USP (2007). The criteria for
category one under the USP (2007) goes as thus, “bacteria, not less than 1.0 log
reduction from the initial calculated count at seven days, not less than 3.0 log
145
reduction from the initial count at 14 days, and no increase from 14 days’ count at 28
days”. The fungus that was specified showed no increase from initial calculated
count at 7, 14 and 28 days. No increase was defined as not more than 0.5 log10 unit
higher than the previous value measured. The requirements for antimicrobial
effectiveness of all the products were met.
146
5. CONCLUSION AND RECOMMENDATIONS
The HPLC method used in the present study permitted rapid and precise
determination of phenylephrine hydrochloride and complied with the requirements for
specificity, linearity, accuracy, precision and range. The method was accepted as
valid, and was therefore deemed suitable for quantitative assay of phenylephrine
hydrochloride in the finished products during initial formulation analysis and for the
ensuing stability studies.
147
storage condition whereas product IV was stable and potent in two storage
conditions namely 25 °C/60%RH and 30 °C/65%RH. The remaining Products (I, III,
V) failed to retain their potency at most storage conditions.
Colour and pH changes were observed mostly with no loss or increase of solution.
Observations with regard to no loss of solution meant that the container was suitable
for the formulations and it was impervious to moisture. Further observations were
that products III and V showed degradation of phenylephrine hydrochloride. This was
clearly depicted as phenylephrine hydrochloride within the product formulation of III
and V was brown or reddish in colour. This meant the phenylephrine hydrochloride
had oxidized fully over the period of three months in which the products were
stressed in variable storage conditions. It shows that phenylephrine hydrochloride
formulations which are in solutions require antioxidants or protective agents and
buffering agents to reduce its quick breakdown, product II and IV had both.
The pH of the products varied from 3.5 to 8, all within the stable range of
phenylephrine hydrochloride. From the assay results of HPLC, phenylephrine
hydrochloride loses its potency when reacted with hydrogen peroxide and sodium
hydroxide. As the pH of the products increases there is a decrease in the
concentration of phenylephrine hydrochloride. Product II an IV stayed within the pH
of 3 meaning acidity kept PE from degrading. Any future research could focus on
optimizing buffers and chemicals that protect phenylephrine from degrading.
148
(Parsons, 2006). Generally, increased temperature (autoclaving and storage
conditions) decreases the viscosity of the stated three viscosity modifying agents,
the important function needed is the ability to return back to their original state. With
this being noted, product IV recovered its viscosity during rheological tests of time
zero and three months later while product II also recovered but not completely.
Product I failed to recover at the storage condition 40 °C/75%RH. This could mean
that SCMC and HPMC could have lost some of their viscosity due to cellulose gum
depolymerization. This in turn adds to the reason why it in many cases needs
additives and preservatives. Another fact that was observed from literature written by
Junyan and colleagues (2009) was that EDTA protects cellulose from loss of
viscosity; this could have enabled SCMC and HPMC in achieving their return to initial
viscosity. Product II, III and IV showed best performances with regard to their flow.
According to the results obtained, product II and IV were favourable. Any future
research could focus on optimizing favourable viscosity-modifying range for
hydroxylpropyl methylcellulose and carboxy methylcellulose sodium. The optimal
value for phenylephrine hydrochloride protectors such as sodium citrate dihydrate,
EDTA, sodium metabisulfite, boric acid should be studied.
Another aspect from the objectives was to find whether the products were provided
with preservatives which were effective to inhibit microbial growth. The result showed
a positive answer. All products reduced the microbial load that they were inoculated
with. Also they passed the compendia requisite for category 1 products (eye drops).
Product I, II, III and V were successful in fulfilling the compendial requirement of
preservative efficacy.
Overall, product II was selected as the best among the five products. It would be
beneficial to approach its development on a more intensive and rigorous scale as to
determine if it would be passed as a generic product for the market at large.
Some limitations of the study were due to reduced sample size. It could have been
increased to ensure a representative distribution in differences and possibly
similarities among products. An increase in formulations samples could lead to
149
increased chances of a more favourable and stable solution. This allows for wider
research to find the best fit ingredients to be mixed together to provide the desired
solution. Reliable reports on phenylephrine hydrochloride in solutions would enhance
quicker decisions in stabilization, as those available are patented and require
substantial amount of money to acquire and use. Extra methods could have been
applied in testing the available samples. An example is the full factorial experimental
design, this design makes it possible to examine all possible combinations of
ingredient variant are served with equal probability.
150
REFERENCES
151
Amin, A. Chauhan, S. Dare, M. Bansal, K. A. 2010. Degradation of parabens by
pseudomonas beteli and burkholderia latens. European Journal of Pharmaceutics
and Biopharmaceutics, vol. 75, Issue 2, pp 206–212.
152
Baudouin, C. Labbe, A. Liang, H. Pauly, A. 2010. Preservatives in eye drops: the
good, the bad and the ugly. Progress in retinal and eye research, vol.29, pp 312-334.
Bielory, L. 2000. Allergic and immunologic disorders of the eye Part II: ocular allergy.
Journal of Allergy and Clinical Immunology, vol. 106, Issue 6, pp 1019–1032.
Bielory, L. 2006. Allergic diseases of the eye. Medical Clinics of North America, vol.
90, Issue 1, pp 129–148.
Bielory, L. 2008. Ocular allergy overview. Immunology and Allergy Clinics of North
America, vol. 28, Issue 1, pp 1–23.
153
Brechu, W. F. Maren, T. H. 1993. pH and drug ionization affects ocular pressure
lowering of topical carbonic anhydrase inhibitors. Investigative Ophthalmology Visual
Science, vol. 34, pp 2581–2587.
154
Chien, D. S. Schoenwald, R. D. 1990. Ocular pharmokinetics and
pharmacodynamics of phenylephrine and phenylephrine oxazolidine in rabbit eyes.
Pharmacological Research, vol. 7, no. 5, pp 476–483.
Choi, S. H. Bielory, L. 2008. Late – phase reaction in ocular allergy. Current Opinion
in Allergy and Clinical Immunology, vol. 8, Issue 5, pp 438–444.
155
Das Gupta, V. Parasrampuria, J. 1987. Quantitation of phenylephrine hydrochloride
in pharmaceutical dosage forms. Drug Development and Industrial Pharmacy, vol.
13, Issue 3, pp 473–486.
Del Amo, E. M. Urtti, A. 2008. Current and future ophthalmic drug delivery systems:
a shift to the posterior segment. Drug Discovery Today, vol. 13, no.4, pp 135–143.
Dolan, J. W. 2003. ‘Why Do Peaks Tail?’, LCGC North America, vol. 21, no. 7, pp.
612-616.
156
Eggleston, P. A. Wood, R. A. 1992. Management of allergies to animals. Asthma
Proceedings, vol. 13, Issue 6, pp 289–292.
157
Gad, S. 2008. Pharmaceutical manufacturing handbook: production and processes.
Volume 5 of pharmaceutical development series, volume 1 of pharmaceutical
manufacturing handbook. Illustrated. New York: John Wiley & Sons, pp 726–747.
Ghosh, TK. Jasti, BR. 2005. Theory and practice of contemporary pharmaceutics.
Florida, CRC Press, pp 481–494.
158
Gould, G. W. 2004, ‘Sterilization’, in A. D. Russell, W. B. Hugo, A. P. Fraise, G. A. J.
Ayliffe, P. A. Lambert, J. –Y. Maillard (ed.). Russell, Hugo and Ayliffe’s Principles
and Practice of Disinfection, Preservation and Sterilization, 4th Edition, Wiley-
Blackwell, Oxford, pp 361–383.
Gotzche, P. C. Johansen, H. K. 2008. House dust mite control measures for asthma:
systemic review. Allergy, vol. 63, Issue 6, pp 646–659.
Grosvenor, T. 2007. Primary care optometry. 5th edition. Elsevier Health Sciences,
New York, pp 137–182.
159
Harwood, R. J. 2006. ‘Hypromellose’, in A. H. Kibbe (ed), Handbook of
Pharmaceutical Excipients, 5th Edition, Pharmaceutical Press, Chicago, pp 252–349.
Holladay, J. T. 2006. Quality of vision: essential optics for the cataract and refractive
surgeon. Thorofare, Slack Incorporated, pp 6–14.
160
Huynh-Ba, K. 2009. ‘Stability operating practices’, in K. Huynh-Ba (ed), Handbook for
stability testing in pharmaceutical development: regulations, methodologies and best
practices, Springer, Chicago, pp 303–323.
Jho, C. Carreras, M. 1984. The effect of viscosity on the drop weight technique for
the measurement of dynamic surface tension. Journal of Colloid and Interface
Science, vol. 99, pp 543–548.
161
Johnson, D. Chandrasekhar, S. S. Mautone, A. J. 2008. Intranasal phenylephrine-
surfactant treatment is not beneficial in otitis media with effusion. International
Journal of Pediatric Otorhinolaryngology, vol. 72, Issue 7, pp 1085–1089.
162
Lang, J. C. 1995. Ocular drug delivery conventional ocular formulations. Advanced
Drug Delivery Reviews, vol. 16, no. 1, pp 39–43.
Lee, V. Robinson, J. 1986. Topical ocular drug delivery: Recent developments and
future challenges (Review). Journal of Ocular Pharmacology, vol. 2, pp 67–108.
Leonardi, A. Motterle, L. Bortolotti, M. 2008. Allergy and the eye. Clinical and
Experimental Immunology, vol. 153, pp 17–21.
163
Liaw, J. Robinson, J. R. 1992. The effect of polyethylene glycol molecular weight on
corneal transport and the related influence of penetration enhancers. International
Journal of Pharmaceutical Sciences, vol. 88, pp 125–140.
Liaw, J. Rojanasakul, Y. Robinson, J. R. 1992. The effect of drug charge type and
charge density on corneal transport. International Journal of Pharmaceutical
Sciences, vol. 88, pp 111–124.
164
McDonnell, G. E. 2007. ‘Physical sterilization’, antisepsis, disinfection, and
sterilization: types, action, and resistance, Wiley-Blackwell, Washington D. C, pp
165–188.
McDonald, F. Parkin, J. 1995. The effect of disodium edetate and sodium chloride on
the stability of ethylmercury arising from the decompostion of thimerosal
(thiomersal). Drug Development and Industrial Pharmacy, vol. 21, pp 2403–2410.
165
Muhammad, N. Bodnar, J. A. 1980. Quantitative determination of guaifenesin,
phenylpropanolamine hydrochloride, sodium benzoate and codeine phosphate in
cough syrups by hplc. Journal of Liquid Chromatography, vol. 3, pp 113–122.
166
Palmares, J. Delgado, L. Cidade, M. Quadrado, M. J. Filipe, H. P. 2010. Allergic
conjunctivitis: a national cross – sectional study of clinical characteristics and quality
of life. European Journal of Ophthalmology, vol. 20, Issue 2, pp 257–264.
167
Podder, S. Moy, K. Lee., V. 1992. Improving the safety of topically applied timolol
in the pigmented rabbit through manipulation of formulation composition.
Experimental Eye Research, vol. 54, pp 747–757.
168
Reddy, I. K. Ganesan, M. G. 1996. Ocular therapeutics and drug delivery: An
Overview. In Ocular Therapeutics and Drug Delivery: A Multi-Disciplinary Approach,
ed. I.K. Reddy. Technomic Publishing Co., Inc. Lancaster, Pennsylvania, USA, pp 3–
29.
Rossiter, South African Medicines Formulary. 2010. Rossiter, D. 9th Edition. Health
and Medical Publishing Group. Cape Town.
169
Association, London, pp 89 - 92, 97- 99, 108 -110, 271-273, 343-346, 390-394, 479,
483, 508-512, 521-523, 526-528, 532- 534, 577-578, 596-598, 622-625, 691-693.
170
Scheikh, A. Hurwitz, B. Nurmatov, U. Van Schayck, C. P. 2010. House dust mite
avoidance measures for perennial allergic rhinitis. Cochrane Database of Systamatic
Reviews, vol. 7, pp CD001563.
171
Stahl, J. L. Cook, E. B. Barney, N. P. Graziano, F. M. 2002. Pathophysiology of
ocular allergy: the roles of conjunctival mast cells and epithelial cells. Current Allergy
and Asthma Reports, vol. 2, Issue 4, pp 322–339.
Sutton, S. Dunn, M. Dunn, D. 1998. In-use stability testing: what data are required
and when. The Regulatory Affairs Journal, vol. 8, pp 728–733.
Sweetman, CS (ed.) 2002, Martindale - The Complete Drug Reference, 33rd Edition,
The Pharmaceutical Press, London, pp 1097–1098.
Taylor, P. 2002. ‘Nasal Drug Delivery’, in M.E. Aulton (ed), Pharmaceutics – The
Science of Dosage Form Design, 2nd Edition, Churchill Livingstone, Edinburgh, pp,
489–498.
172
Tkaczyk, C. Okayama, Y. Woolhiser, M. R. Hagaman, D. D. Gilfillan, A. M. Metcalfe,
D. D. 2002. Activation of human mast cells through the high affinity IgG receptor.
Molecular Immunology, vol. 38, Issue (16 – 18), pp 1289–1293.
173
Van Ooteghem, M., 1987. Factors influencing the retention of ophthalmic solutions
on the eye surface. In: Saettone, M., Bucci, M., Speiser, P. (Eds.), Ophthalmic Drug
Delivery. Biopharmaceutical, Technological and Clinical Aspects. Fidia Research
Series, vol. 11. Liviana Press, Padova, pp 7–17.
Veys, J. 2004. Managing the contact lens wearing allergy sufferer. Optician, vol.
5950, Issue 227, pp 22–26.
Wilson, C. G. 2004. Topical Drug Delivery in the Eye, Experimental Eye Research,
vol. 78, pp 737–743.
174
Wong, J. Wiseman, L. Al-Mamoon, S. Cooper, T. Zhang, L. –K. Chan, T. –M. 2006.
Major degradation product identified in several pharmaceutical formulations against
the common cold. Analytical Chemistry, vol. 78, Issue 22, pp 7891–7895.
Zide, B. M. 2006. Surgical Anatomy Around the orbit (The System of Zones),
Lippincott, Williams and Wilkins, Philadelphia, pp 1–128.
175
APPENDIX A
CONCEPT ARTICLE
* E-mail: Mbali.Keele@nmmu.ac.za
Abstract:
Simple, accurate and reproducible high performance liquid chromatography (HPLC)
method was validated for the estimation of phenylephrine hydrochloride in
pharmaceutical eye drop formulations. Phenylephrine hydrochloride (PE) was
assayed using HPLC at concentration range of 0.0125 to 0.15 mg/ml. Linearity, y =
8541.1 x + 438.55 was achieved as the range were directly proportional to the
concentration of phenylephrine hydrochloride within a given range (r2 = 0.9999). The
method was tested and validated for various parameters according to the ICH
(International Conference on Harmonization) guidelines. The detection and
quantification limits were found to be 12.3 and 41 μg/ml respectively. The proposed
method was successfully applied for the determination of phenylephrine
hydrochloride in pharmaceutical eye drop formulations. The results demonstrated
that the procedure is accurate, precise and reproducible, while being simple, cheap
176
and less time consuming, and hence can be suitably applied for the estimation of
phenylephrine hydrochloride in eye drops.
Introduction
The drainage of phenylephrine hydrochloride into the nasal mucosa could result in
systemic absorption of this agent and produce many unwanted systemic side effects
including tachycardia, hypertension, and headache (Bartlett and Jaanus, 2008).
Also, the eye drop solutions were either blinked out or only a small portion of the
drug reached its site of action. The use of viscosity modifying agents is included in
solutions with the aim of obtaining thickening effects. However, these components
may have other effects, whether independently or as a consequence of interactions
with other components, these effects being mostly due to electrostatic, steric,
electrosteric, or depletion mechanisms (Duro et al., 1999).
Various methods have been reported in the literature for the analysis of
phenylephrine hydrochloride including spectrophotometer (Collado et al., 2000; Erk,
2000; Solich et al., 2000; Shama, 2002; Knochen & Giglio 2004), spectrophotometry
with chromogenic reagent (Ahmed and Amin, 2007), fluorometry (Martin et al.,
1993), High-performance liquid chromatography (Marin et al., 2002; Olmo et al.,
2005; Galmier et al., 2000) spectro-fluorimetric and derivative spectrophotometric
methods (Sabry et al., 2000), have also been reported for the determination of
phenylephrine hydrochloride.
The aim of the study was is to use and validate a HPLC analytical method for the
estimation of phenylephrine hydrochloride in pure form and in pharmaceutical eye
177
drop formulations. The method was validated as per ICH (International Conference
on Harmonization) guidelines and MCC requirements.
Materials
Instruments
The HPLC system consisted of a complete FPLC Shimadzu® HPLC system which
has a SPD-M20A Prominence diode array detector, SIL-20A Prominence auto-
sampler, DGU-20A5 Prominence degasser, LC-20AB Prominence liquid
chromatography and CTO-10AS vp Prominence column oven ( Shimadzu, Tokyo,
Japan). Column was a reverse phase Phenomenex® Luna C18 (2) column 250 mm
× 4.60 mm, 5 μm particle size (Separations, Johannesburg, SA).
178
Calibrations
1.1g of octane-1-sulfonic acid sodium salt was dissolved in one litre mixture of
methanol and water (1:1) and the pH was adjusted to 3.0 with pyrophosphoric acid.
The resulting solution was mixed, degassed by ultrasonication (Ultrasonic LC 130,
Labotec, Germany) and vacuum filtered through a 0.45 μm Millipore filter (Millipore
Corporation, Bedford, Massachusetts, USA) prior to use. Dilution solvent was
prepared as a mixture of HPLC grade methanol and water (1:1) and adjusted to a pH
of 3. The stock solution was prepared by accurately weighing 200 mg of
phenylephrine hydrochloride material into a 100 ml volumetric flask, dissolving it and
making up to volume with dilution solvent. Calibration standards containing 0.0125,
0.025, 0.05, 0.075, 0.1 and 0.15 mg / ml were prepared by making appropriate
solvent dilutions of the working stock solution. Each calibration standard was filtered
through a 0.45 μm Millex® syringe driven filter unit prior to injection.
Sample preparation
Eye drop formulations had PE concentration of 100 mg / ml. The eye drop
formulations were filtered and 0.3 ml was dissolved into 10ml of dilution solvent to
get a final concentration of 0.03 mg / ml. The samples were analyzed using the
following analytical method.
Methods
The samples were analyzed using the following analytical method:
Linearity
Linearity of an analytical procedure is its ability, within a given range, to obtain test
results that are directly proportional to the concentration of analyte in the sample
(ICH Harmonized Tripartite Guideline Q2A, 2005). A calibration curve was prepared
and linearity demonstrated over a phenylephrine hydrochloride concentration range.
A stock solution was prepared having a known concentration of 2 mg / ml (defined as
100%) and dilution of the stock solution to final concentrations of 0.0125 to 0.15 mg /
ml were prepared with reverse osmosis ( RO) water and filtered through 23 mm 0.45
179
µm PVDF syringe filters (Millex-HV, Millipore, Billerica, USA). Each of the standards
was assayed in triplicate. The calibration curve was constructed by plotting the peak
areas of phenylephrine hydrochloride versus the respective phenylephrine
hydrochloride concentrations and a linear regression trend line was fitted to the plot
on Microsoft Excel® 2007, Microsoft Corporation.
Specificity
All five products and the phenylephrine hydrochloride were subjected to the following
stress conditions after which they were manipulated and analysed:
180
0.2 M H2O2 for 30 minutes (reflux system);
UV lights (17 hours inside a stability chamber);
100 °C (24 hours inside a stability chamber);
65 °C (1 month inside a stability chamber);
40 °C/75%RH (1 month inside a stability chamber);
Unstressed batch of phenylephrine hydrochloride and Products I–V
Linearity
Linearity indicates that the method a calibration curve was constructed by plotting
the area of the phenylephrine hydrochloride peak versus phenylephrine
hydrochloride concentration. The figure below shows linearity over the concentration
181
range. The linear regression equation for the concentration range of 0.0125 to 0.15
mg/ml was y = 8541.1x + 438.55, with a correlation coefficient, R2, equal to 0.9999.
The requirements for linearity were attained, as the correlation coefficient of the
regression line was greater than 0.999 and the percentage relative standard
deviations for the phenylephrine hydrochloride peak areas of multiple injections were
all less than 1.5 %. The y-intercept was found to be 2 > z > -2. This means that the
results achieved are directly proportional to the concentration of phenylephrine
hydrochloride within a given range.
1400000
1200000
1000000
800000
peak area
600000
400000
200000
0
0 50 100 150 200
concentration (mg/ml)
Figure 1: Graph showing a mean peak area versus concentration of replicate samples of
phenylephrine hydrochloride standards. Linear regression equation: y = 8541.1 x + 438.55, R2 =
0.9999.
182
Accuracy
Accuracy was within acceptable range as noted in Table 1. The percentage relative
standard deviations calculated for the samples at the lower, middle and upper limits
of the concentration range were all below 0.5%. The recovery percentage for the
samples was between the limits of 99.00 to 100.10%. This means by applying the
analytical method of a known purity concentration (linearity) and comparing the
results of a second, well characterised method, the difference should not be greater
than 0.5%.
Precision
Precision was within acceptable range as seen above in Table 2. The percentage
relative standard deviations calculated for the samples at the lower, middle and
upper limits of the concentration range were all below 0.5%. The recovery
percentage for the samples was between the limits of 99.00 to 100.00%. The
precision method had the degree of agreement among individual test results when
the method was applied repeatedly to the three concentrations chosen of
phenylephrine hydrochloride. As above it is expressed in RSD, showing that it can
be reproduced or repeated under normal operating conditions.
The LOD was found to be 12.3 μg/ml. The amount stated is the lowest amount of
phenylephrine hydrochloride in a sample that can be detected. The LOD is the
lowest concentration for which the relative standard deviation of multiple injections is
less than 5.0%. By convention, the LOD value is taken as 0.3 times the LOQ
(Armbruster & Pry, 2008).
183
Limit of quantification (LOQ)
The LOQ was found to be 41 μg/ml. The amount shows the lowest amount of analyte
in a sample that can be determined with acceptable precision and accuracy. LOQ =
3.33 LOD (Thomsen et al., 2003).
Specificity
184
hydrochloride showed loss to the combination of heat and humidity. It was, however,
potent against heat alone. There was decomposition by acid, peroxide and base.
Phenylephrine hydrochloride was stable to UV light.
Conclusion
Reference
Armbruster, D.A. & Pry, T. 2008. Limit of Blank, Limit of Detection and Limit of
Quantitation. Clinical Biochemical Review, vol. 29, Issue 1, pp 49–52.
185
Disperse Systems of Pharmaceutical Interest, Drug Development and Industrial
Pharmacy, vol. 25, no. 7, pp 817-829.
186
Olmo, B. Garcıa, A. Marın, A. Barbas. C. 2005. New approaches with two cyano
columens to the separation of acetaminophen, phenylephrine, chlorpheniramine and
related compounds, Journal of Chromatography. Issue B 817, pp 159–165
187
Table 1: Accuracy data for quantification of phenylephrine hydrochloride
188
Figure 2: Chromatogram of mobile phase alone
mAU
280nm,4nm (1.00)
45
40
35
30
25
20
15
10
189
mA U
60 280nm,4nm (1.00)
55
50
45
40
35
30
25
20
15
10
-5
0.0 2.5 5.0 7.5 10.0 12.5 min
40
35
30
25
20
15
10
190
mAU
280nm,4nm (1.00)
50
45
40
35
30
25
20
15
10
191
APPENDIX B
LIST OF EQUIPMENT
Autoclave Hirayama Manufacturing Corp, Japan
192
APPENDIX C
LIST OF SOLUTIONS
Nutrient agar
Nutrient agar 16 grams
RO water to 1000 ml
1 M sodium hydroxide
Sodium hydroxide 40 grams
193