5.

DRUG PROFILE

DMM

Structure :

Chemical name :

Description :
Solubility :

Molecular formula :
Molecular mass :
pKa :
BCS Classification :
Melting point :
Solubility :
Pregnancy Category :

Elimination Half life :

M.O.A:

Adverse reaction

Dose :

Pharmacokinetics

Pharmacodynamics

1 C.L.Baid Metha College of Pharmacy

6. MATERIALS AND

INSTRUMENTS Instruments used:

 System : HPLC(Shimadzu)

 Pump : I80 ( LC – 10 AT Vp series)

 Detector : UV/Visible E2469

 Column : Intertsil ODS-3 C18 (300mm x 3.9 mm, 5 µ

i.d.)

 Semi-Micro balance : LC/GC

 Analytical balance : Sartorious

 Hot air oven : Technico

 Mechanical shaker : Remi RS-24 Plus

 Sonicator : PCI Analytics

Reagents and Chemicals

 Acetonitrile : HPLC grade

 Water : HPLCgrade

 Hydrogen peroxide : AR

 Sodium hydroxide : AR grade

 Hydrochloric acid : EMPARTA

Reference Standards

1. DMM: : Purity 99.25%

2. DTT : Purity 99.48%

3. Propyl paraben : Purity 100%

Brand Used : Megace

Label claim :
Preparation of buffer :

Preparation of mobile phase :

Standard Preparation:

DMM (10µg/ml) & DTT (5 µg/ml)

50 mg DMM ------->. 100 ml -------> 2ml -------> 100ml

50 mg DTT -------->. 100 ml -------> 1ml -------> 100ml

Sample Preparation: label claim of DMM 1mg and DTT 0.5 mg

1g sample made up to 100 ml sonicated for 15 min and then volume make up with
mobile phase -----> The final concentration of DMM(10 µg/ml) & DTT (5 µg/ml)

METHOD DEVELOPMENT AND OPTIMIZATION OF

CHROMATOGRAPHIC CONDITIONS

SELECTION OF CHROMATOGRAPHIC CONDITION

Proper selection of the method depends upon the nature of the sample (ionic /
ionisable / neutral molecule), its molecular weight and solubility. The drugs selected
in the present study are polar in nature and hence reversed phase or ion-pair or ion
exchange chromatography method may be used. The reversed phase HPLC was
selected for the separation because of its simplicity and suitability.
SELECTION OF WAVELENGTH ( max)

10 mg of DMM and DTT was dissolved in mobile phase. The solution was
scanned from 200-400 nm the spectrum was obtained. The overlay spectrum was
used for selection of wavelength for DMM and DTT. The isobestic point was taken
as detection wavelength. In the entire UV visible region both the drugs were
strongly absorbed at 254 nm.So this wavelength was selected for further studies.

7.1. METHOD DEVELOPMENT TRIALS

Trial –1, 2,3,4,5,6.

6.4 Results Optimized Condition

Chromatographic conditions
 System : HPLC(Shimadzu)

 Column : Intertsil ODS-3 C18 (300mm x 3.9 mm, 5 µ i.d.)

 Flow rate : 1.2 ml/ min

 UV Detection : 254 nm
 Injection Volume : 20 µl
 Run Time : 15 min

 Mobile phase :
 PH :
 Diluents : Mobile phase
VALIDATION

Method validation can be defined as per ICH

Establishing documented evidence, which provides a high degree of

assurance that a specific activity will consistently produce a desired result or product
meeting its predetermined specifications and quality characteristics.

The developed method was validated according to ICH guidelines.

• System suitability

• Specificity

• Linearity

• Range

• Accuracy

• Precision

• LOD, LOQ

• Robustness and ruggedness.

SYSTEM SUITABILITY

System suitability was performed by five replicate injection of standard solution

with the concentration of 10µg/mL of DMM and 5µg/mL of DTT was injected. The
parameters like retention time, theoretical plate and peak area were shown in the
table

Figure 1. System suitability chromatogram of DMM and DTT

Injection DMM DTT

ID USP Plate RT(min) Area USP Plate RT(min) Area

Count Count

1 30563 2.61 2322845 45714 9.64 973418

2 30128 2.65 2303613 45072 9.62 961548

3 31265 2.63 2329012 45639 9.63 972640

4 30987 2.60 2312915 44986 9.62 968365

5 30302 2.66 2298810 45428 9.67 980139

Average 2.63 2313439 9.64 971222

SD 0.03 12667.12 0.02 6587.38

%RSD 0.97 0.55 0.22 0.71

SPECIFICITY

Specificity is the ability to check unequivocally the analyte in the presence of

components which may expect to be present. Typically these might include
impurities, degradant and matrix. There was no interference from excipient and
other component with the drug peak. So, the developed method has been found
to be specific.

Figure 2. chromatogram of Blank

Figure 3. chromatogram of Placebo

 Figure 4. chromatogram of Std

Quantitative estimation

S.no Sample Retention Peak area Amount present

name time

DMM DTT DMM DTT DMM DTT

1 Standard 2.61 9.64 2313439 971222 101.21% 100.58%

(1.01 mg) (0.50 mg)
2 Sample 1 2.65 9.65 2342581 992643 100.73 % 100.18%
(1.01mg) (0.50 mg)
3 Sample 2 2.63 9.63 2331537 988672 100.97% 100.38%
(1.01 mg) (0.50 mg)

ACCEPTANCE CRITERIA:
Assay value should be in the range of 90% to 110%
Result
Test result is showing that the test method is precise. The percentage assay of
DMM and DTT found to be 100.97% and 100.38%.Results are within the limits
Accuracy

The closeness of agreement between the true values which is accepted either
conventional new value or an accepted reference value and the value found.
Based on the comparison to reference standard method.
Procedure:

The accuracy was calculated the present method to the analysis of tablet and
standard at low, medium, and high concentration level. The accuracy was estimate
from three replicate injection and calculated as the µg/mL drug recovered from the
drug matrix.

Acceptance criteria:

The percentage Recovery for each level should be between 98.00 to 102.00%

Figure 5. chromatogram of Low level

 Figure 6. chromatogram of Middle level

Figure 7. chromatogram of High level

Accuracy

Accuracy table of DMM

% Amount Spiked Amount % Mean %

Level (mg) recovered (mg) Recovery Recovery

50 % 1 1.0145 100.72 100.35 %

1 1.0207 101.34

1 0.9971 99.00

100% 2 2.0315 100.85 100.36%

2 2.0088 99.72
 2 2.0245 100.50

150% 3 3.0301 100.28 100.07%

3 3.0156 99.80

3 3.0255 100.13

Accuracy table of DTT

% Amount Spiked Amount recovered % Mean %

Level (µg/ml) (µg/ml) Recovery Recovery

50 % 0.5 0.5024 100.48 100.56%

0.5 0.5070 101.40

0.5 0.4990 99.80

100% 1.0 1.0041 100.41 100.45%

1.0 1.0164 101.64

1.0 0.9931 99.31

150% 1.5 1.5133 100.89 100.51%

1.5 1.5233 101.55

1.5 1.4862 99.08

PRECISION
Precision of an analytical method is the degree of agreement among individual test
results when the procedure is applied repeatedly to multiple sampling of a
homogenous sample. Precision of analytical method is usually expressed as the
standard deviation and relative standard deviation.

Determination:
The precision of the analytical method was determined by assaying sufficient
number of samples and relative standard deviation was calculated.
The precision of the instrument was determined by assaying the samples
consecutively, number of time and relative standard deviation was calculated.
Inject 20μl of the blank solution and the standard solution of for five times and
calculate the %RSD for the area of six replicate injections.

Acceptance Criteria:
The % RSD for the area of six injections results should not be more than 2%.

DMM

S.NO Parameter Mean peak area %RSD Mean RT %RSD

1 System precision 2373733 0.380 2.61 0.69

2 Method precision 2380506 0.728 2.63 0.71

3 Intermediate precision 2318839 0.469 2.64 0.26

DTT

S.NO Parameter Mean peak area %RSD Mean RT %RSD

1 System precision 970997 0.230 9.64 0.58

2 Method precision 978478 1.431 9.61 0.64

3 Intermediate precision 968137 0.812 9.65 0.41

7.2.6 LINEARITY AND RANGE

LINEARITY: Linearity is the ability of the method to obtain test results that are
directly
Proportional to the analyte concentration within a given range.

RANGE: Range of analytical procedure is the interval between the upper and lower
concentration of analyte in the sample (including concentrations) for which it has
been
Demonstrated that the analytical procedure has a suitable level of precision,
accuracy, and linearity.
Preparation of Standard Solution:
The linearity of the method was performed by preparing the concentration range of
40-120µg/mL and 20-60 µg/mL for DMM and DTT, from standard stock solution.
Calibration curves were constructed by plotting concentration versus area of DMM
and DTT.
DMM

DTT
 Linearity Results:

PARAMETER DMM DTT

Concentration 40-120 20-60

Correlation 0.999 0.999

Slope 29061 805.4

Intercept 2855 107

No. of data points 5 5

ROBUSTNESS

The robustness of a method was analysed by changing experimental,

chromatographic condition. Altering in flow rate (1.2±1 mL/min), changes in
column oven temperature (40±5 °C), Changes mobile phase buffer pH (3.5±0.2),
changes in mobile phase composition and changes in wavelength allowable limits
from actual chromatographic condition. It was noted that there is no recognizable
change in mean RT and RSD and parameters fall within the limit of ≤ 2. The
theoretical plate, tailing factor, resolution is found to be good of DMM and DTT.
This method is robust with variability condition. The analytical condition result is
shown in table

DMM

Proposed Mean %RSD Mean %RSD

peak
variations RT
area

Normal 2373733 0.380 2.61 0.69

Variation in flow rate 1.1 ml 2350211 0.776 2.64 0.42

±0.1(optimized
1.2ml/min) 1.3 ml 2371026 0.534 2.65 0.71

Variation in 252 2375364 0.550 2.63 0.63

wavelength 256 2369712 0.670 2.61 0.57
±2(optimized 254)

DTT

Proposed Mean %RSD Mean %RSD

peak
variations RT
area

For Optimized Condition 970997 0.230 9.64 0.66

Variation in flow rate 1.1 980956 1.102 9.65 0.64

±0.1(optimized 1.2ml/min) ml

1.3 984194 0.789 9.64 0.38

ml

Variation in wavelength 252 976556 0.814 9.64 0.41

±2(optimized 254)

256 974081 0.774 9.63 0.53

SOLUTION STABILITY

Evaluate the stability of analytical solution by injecting the standard

and sample solution at 48 hours. The results are summarized in the table (13:14) for
standard and sample solutions.

Table No. 13 Solution stability for standard

Time in hours % Assay % Difference

Initial 100.2 -

48 Hours 100.0 0.2

Table No. 13 Solution stabiity for sample

Time in hours % of Assay % Diff for % of Assay % Diff for

For 1mg sample For 0.5mg sample

[DMM] [DTT]

Initial 99.5 - 100.2 -

48 Hours 99.2 0.3 99.8 0.4

Acceptance Criteria

The % difference for % assay of standard and sample preparation between initial
and different time intervals should be within ±2.0

SUMMARY

An innovative RP-HPLC method was developed for the simultaneous estimation of

DMM and DTT in semisolid dosage forms and validated according to the ICH
guidelines.

OPTIMIZED CHROMATOGRAPHIC CONDITION

 System : HPLC(Shimadzu)

 Column : Intertsil ODS-3 C18 (300mm x 3.9 mm, 5 µ i.d.)

 Flow rate : 1.2 ml/ min

 UV Detection : 254 nm
 Injection Volume : 20 µl
 Run Time : 15 min

 Mobile phase :
 pH :
 Diluents : Mobile phase

QUANTITATIVE ESTIMATION

SAMPLE AMOUNT PRESENT (%) ACCEPTANCE LIMIT

DMM 100.97% 90%-110%

DTT 100.38%

VALIDATION REPORTS OF OPTIMIZED METHOD

S.NO PARAMETERS ACCEPTANCE RESULTS OBTAINED

CRITERIA

1 System Theoretical plate NLT DMM - 30649

suitability 2000
DTT - 45368

2 specificity No interference was No interference was

observed between the observed between the
placebo and blank placebo and blank with the
principal peak

3 Accuracy 90-110 % DMM - 100.26%

DTT - 100.51%

4 Precision % RSD NMT 2 % %RSD NMT 2 %

5 Linearity Correlation co efficient DMM - 0.999

NLT 0.996 DTT - 0.999

6 Solution % RSD NMT 2 % Stable for 48 hours
stability

7 Assay 90-110 % DMM - 100.97%

DTT - 100.38%

8 Robustness %RSD NMT 2 % %RSD NMT 2 %