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A_Practical_Approach_to_Validation_of_HPLC_Methods_Under_Current_Good_Manufacturing_Practices_0
A_Practical_Approach_to_Validation_of_HPLC_Methods_Under_Current_Good_Manufacturing_Practices_0
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Introduction Regulatory analytical procedures are of two types: com-
Analytical methods validation is an important regulatory pendial and noncompendial. The noncompendial analytical
requirement in pharmaceutical analysis. High-Performance procedures in the USP are those legally recognized as regu-
Liquid Chromatography (HPLC) is commonly used as an latory procedures under section 501(b) of the Federal Food,
analytical technique in developing and validating assay Drug and Cosmetic Act. When using USP analytical meth-
methods for drug products and drug substances. Method val- ods, the guidance recommends that information be provided
idation provides documented evidence, and a high degree of for the following characteristics: specificity of the method,
assurance, that an analytical method employed for a specific stability of the analytical sample solution, and intermediate
test, is suitable for its intended use. Over recent years, regu- precision. Compendial analytical methods may not be stabil-
latory authorities have become increasingly aware of the ne- ity indicating, and this concern must be addressed when de-
cessity of ensuring that the data submitted to them in appli- veloping a drug product specification, because formulation
cations for marketing authorizations have been acquired based interference may not be considered in the monograph
using validated analytical methodology. The International specifications. Additional analytical tests for impurities may
Conference on Harmonization (ICH) has introduced guide- be necessary to support the quality of the drug substance or
1,2
lines for analytical methods validation. The U.S. Food and drug product. Noncompendial analytical methods must be
Drug Administration (FDA) methods validation draft guid- fully validated. The most widely applied validation charac-
3-5
ance document, as well as United States Pharmacopoeia teristics are accuracy, precision (repeatability and intermedi-
(USP) both refer to ICH guidelines.
6
ate precision), specificity, detection limit,
These draft guidances define regulatory quantitation limit, linearity, range, and sta-
and alternative analytical procedures and Figure 1 bility of analytical solutions.
stability-indicating assays. The FDA has
____________________ The parameters that require validation
The chemical structure of
proposed adding section CFR 211.222 on and the approach adopted for each partic-
ethyl 4-hydroxybenzoate.
analytical methods validation to the cur- ular case are dependent on the type and
rent Good Manufacturing Practice applications of the method. Before under-
7
(cGMP) regulations. This would require taking the task of method validation, it is
pharmaceutical manufacturers to estab- necessary that the analytical system itself
lish and document the accuracy, sensitiv- is adequately designed, maintained, cali-
8
ity, specificity, reproducibility, and any brated, and validated. The first step in
other attribute (e.g., system suitability, method validation is to prepare a proto-
stability of solutions) necessary to vali- col, preferably written with the instruc-
date test methods. tions in a clear step-by-step format. This
8
approach has been reported previously. In
this paper, it is intended to review and Figure 2
______________________________________________
demonstrate practical approaches to ana- Graph measured peak area versus ethyl 4-hydroxyben-
lytical method validation in detail with zoate concentration demonstrating linearity.
reference to an HPLC assay of ethyl 4-hy-
droxybenzoate (Figure 1). Ethyl 4-hy-
droxybenzoate alone or in combination
with other esters of p-hydroxybenzoic
acid, or with other antimicrobial agents, is
used as a preservative in pharmaceutical
formulations.
Experimental
✔ Chemicals and reagents.
All chemicals and reagents were of the
highest purity. HPLC-grade acetonitrile was
obtained from Merck (Darmstadt, Ger-
many). Water was purified with a Millipore
Milli-Q system (Watford, UK). Ethyl 4-hy-
droxybenzoate (Batch #1005425) was sup-
plied by Lancaster Synthesis (Morecambe, England). ✔ Preparation of mobile phase.
The mobile phase was prepared by adding 650 ml of
✔ HPLC instrumentation. HPLC-grade acetonitrile in 1000 ml of water (65:35, v/v).
The HPLC system used for the validation studies con- The mobile phase was filtered under a vacuum through 0.45
sisted of a Waters Alliance 2690 Separations Module to a mm nylon filters and degassed before use. Also, the mobile
996 photodiode-array (PDA) detector. The control of the phase continuously was degassed with an on-line degasser.
HPLC system and data collection was by a Compaq com-
puter equipped with Waters® Millennium32 software (ver- ✔ Preparation of standard and sample solutions.
sion 3.20). The second HPLC system used for intermediate Ethyl 4-hydroxybenzoate (100 mg) was weighed accu-
precision studies consisted of Perkin Elmer: model series rately and added to a 100 ml volumetric flask before being
200 UV visible detector, series 200 LC pump, series 200 au- dissolved in acetonitrile. A 2.0 ml aliquot of stock solution
tosampler, and a series 200 peltier LC column oven were was diluted to 100 ml in the mobile phase, yielding a final
used to chromatograph the solutions. The data was acquired concentration of 20 µg/ml. Standard solutions for the eval-
via PE TotalChrom Workstation data acquisition software, uation of ethyl 4-hydroxybenzoate linearity were prepared
(Version 6.2.0) using PE Nelson series 600 LINK interfaces. over a concentration range of 5.0-40 µg/ml, to 25, 50, 75,
Both HPLC systems including software (Food and Drug Ad- 100, 150, and 200% in the mobile phase.
ministration (FDA), 21 Code of Federal Regulations (CFR)
Part 11) were validated prior to use for the test method vali- Results and Discussion
dation.
All chromatographic experiments were performed in the ✔ Validation of the chromatographic method:
isocratic mode. A C18 symmetry analytical column from Linearity and range
Waters (located in Milford, MA, United States) 3.9 x 150 The linearity of the method should be tested in order to
mm, 5 mm particle size was used. The mobile phase con- demonstrate a proportional relationship of response versus
sisted of a mixture of acetonitrile, water solution (65:35, analyte concentration over the working range. The linearity
v/v). The flow rate was set to 1.0 ml/min, and the oven tem- range for evaluation depends on the purpose of the analytical
perature to 25˚C. The injection volume was 20 µl, and the test method. The ICH guidelines specified a minimum of
detection wavelength was set at 254nm. five concentration levels, along with certain minimum spec-
30 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y
Ghulam A. Shabir
Figure 5
________________________________________________________________
HPLC chromatogram of ethyl 4-hydroxybenzoate.
32 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y
Ghulam A. Shabir
Figure 6
________________________________________________________________
HPLC chromatogram for placebo. The analyte peak was eluted at 1.58 minutes.
Figure 8
_____________________________________________________________
Demonstration of the intermediate precision of the HPLC assay for ethyl
4-hydroxybenzoate results in relative percent purity area
Sample S1 S2 S3 S1 S2 S3
(50%) (100%) (150%) (50%) (100%) (150%)
Operator 1, day 1 99.83 99.79 99.76 99.76 99.83 99.83
Operator 1, day 2 99.76 99.74 99.74 99.82 99.80 99.78
Operator 2, day 1 99.71 99.76 99.77 99.75 99.76 99.69
Operator 2, day 2 99.53 99.62 99.57 99.79 99.81 99.82
Mean 99.71 99.73 99.71 99.78 99.80 99.78
(HPLC systems)
Mean (Operators) 99.79 99.79 99.78 99.70 99.74 99.71
RSD (criteria ≤ 2%) S1+S1 S2+S2 S3+S3
HPLC systems 0.05 0.05 0.05
Operators 0.06 0.04 0.05
ation, coefficient of variation, and confidence interval. In this cent purity mean values at 50, 100, and 150% of the test con-
study, precision of the method was evaluated through the re- centration. The RSD values presented in Figure 8 were less
peatability of the method (intra-assay precision) by assaying than 1% for both systems and operators, and illustrated the
ten replicate injections of ethyl 4-hydroxybenzoate at the good precision of the analytical method.
same concentration (20 µg/ml), during the same day, under
the same experimental conditions. The RSD values of the re- Reproducibility
tention time, area, and height of ethyl 4-hydroxybenzoate
peak were found to be < 0.3%, as presented in Figure 7. Reproducibility1 is determined by testing homogeneous
samples in multiple laboratories, often as part of inter-labora-
Intermediate Precision tory crossover studies. An example of reproducibility criteria
for an assay method could be that the assay results obtained
Intermediate precision (inter-day variation) is the results in multiple laboratories will be statistically equivalent, or the
from within lab variations, due to random events, such as dif- mean results will be within 2% of the value obtained by the
ferent days, analysts, equipment, etc. In determining interme- primary testing lab. For an impurity method, results obtained
diate precision, experimental design should be employed, so in multiple laboratories will be statistically equivalent, or the
that the effects (if any) of the individual variables can be mon- mean results will be within 10% (relative) of the value ob-
itored. Precision criteria for an assay method is that the intra- tained by the primary testing lab for impurities. Repro-
assay precision will be ≤ 2%, and for impurity assay, at the ducibility is not normally expected if intermediate precision
limit of quantitation, the instrument precision will be ≤ 5%, is performed.
and the intra-assay precision will be ≤10%. In this study, in-
termediate precision (within-laboratory variation) was Limit of Detection and Quantitation
demonstrated by two operators, using two HPLC systems,
and evaluating the relative percent purity data across the two The Limit of Detection (LOD) and Limit of Quantitation
HPLC systems at three concentration levels (50%, 100%, (LOQ) tests for the procedure are performed on samples
150%) that cover the ethyl 4-hydroxybenzoate assay method containing very low concentrations of analyte. LOD is de-
range (5.0-40 µg/ml). The mean and RSD across the systems fined as the lowest amount of analyte that can be detected
and analysts were calculated from the individual relative per- above baseline noise; typically, three times the noise level.
34 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y
Ghulam A. Shabir
Figure 9
________________________________________________________________
HPLC chromatogram for limit of detection (2 ng/ml)
Figure 10
________________________________________________________________
HPLC chromatogram for limit of quantitation (5 ng/ml).
Figure 11
________________________________________________________________________
Stability of ethyl 4-hydroxybenzoate in solution (n = 6)
Figure 12
_______________________________________________________
Demonstration of the system suitability of the HPLC assay for ethyl
4-hydroxybenzoate
Results
System Suitability Acceptance
Parameter Criteria
HPLC system 1 HPLC system 2
LOQ is defined as the lowest amount of analyte which can mal lighting conditions for 48 hours, and were shown to be
be reproducibly quantitated above the baseline noise, that stable with no significant change in ethyl 4-hydroxybenzoate
gives S/N = 10. concentrations over this period (Figure 11). This is indicated
In this study, LOD for a 20 µl injection of ethyl 4-hy- by <1% changes in area between T = 0 hours and T = 48
droxybenzoate standard (signal to noise = 3) was 2.0 ηg/ml hours. Based on these data that show quantitative recovery
(Figure 9), and the LOQ (signal to noise = 10) was 5 ηg/ml through 48 hours, ethyl 4-hydroxybenzoate solutions can be
(Figure 10) and RSD < 2% (n = 6). assayed within 48 hours of preparation.
36 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y
Ghulam A. Shabir
Conclusion
Article Acronym Listing
It is clear from the various guidelines issued by regula-
tory authorities that analytical methodology should be thor- CFR: Code of Federal Regulations
oughly validated under Current Good Manufacturing Prac- cGMP: current Good Manufacturing Practice
tice (cGMP). HPLC assay of active ingredients in pharma- FDA: Food and Drug Administration
ceutical products, and subsequent method validation, can be HPLC: High Performance Liquid Chromatography
complex and time-consuming. However, a well-defined pro- ICH: International Conference on Harmonization
tocol and documented validation plan simplifies and short- LOD: Limit of Detection
ens the process, while also providing regulatory agencies LOQ: Limit of Quantitation
with evidence that the analytical system and method is suit- PDA: Photodiode Array
able for its intended use. This paper is intended to provide RSD: Relative Standard Deviation
guidance on how to perform method validation for HPLC USP: United States Pharmacopoeia
that generates both useful and meaningful data that meets all UV: Ultra Violet
FDA, USP, and ICH validation requirements for pharmaceu-
tical analysis. ❏
Acknowledgements References
I thank Abbott Laboratories and MediSense for permis- 1. Text on Validation of Analytical Procedures. ICH, Q2A, FDA,
sion to publish this article. I would also thank to Dr Nigel Federal Register, Vol. 60, (March), 1995. p. 11260.
Forrow for his comments on the text. 2. Validation of Analytical Procedures: Methodology. ICH, Q2b.
FDA, Federal Register, Vol. 62, (May), 1997. p. 27463.
3. Analytical Procedures and Methods Validation: Chemistry,
About the Author Manufacturing and Controls Documentation, FDA, Federal
Register (Notices) 65 (169), August, 2000, p. 52776.
4. Validation of Chromatographic Methods, Reviewer Guid-
Ghulam Shabir, BSc (Hons), MSc, FIQA, CIM, is a
ance, Centre for Drug Evaluation and Research, FDA, 1994.
Principal Scientist in Analytical R&D at Abbott Lab-
5. Guideline for Submitting Samples and Analytical Data for
oratories, MediSense Products UK, where he is re-
Methods Validation. FDA, February 1987.
sponsible for analytical methods development and 6. Validation of Compendial Methods. USP 25-NF 20, (1225),
validation and instrument qualification. He has over United States Pharmacopeal Convention, Rockville, MD,
17 years of experience in the pharmaceutical indus- 2002, p. 2256.
tries, and has received the Technical Excellence 7. Current Good Manufacturing Practice for Finished Pharma-
and the Spot Award from Abbott Laboratories. He ceuticals, Proposed Rules, FDA, HHS, 21 CFR Part 211,
has also won the Education Award from the Insti- Federal Register, Vol. 61, 87 (May), 1996. p. 20106.
tute of Manufacturing UK. Mr. Shabir has written for 8. Shabir, G.A., “Validation of HPLC Methods for Pharmaceuti-
over 26 industry publications, and his work has cal Analysis: Understanding the Differences and Similarities
been presented at the world’s premier international between Validation Requirements of the U.S. FDA, USP and
conferences. He is a Fellow of the Institute of Qual- ICH.” Journal of Chromatography A. 987 (2003). pp. 57-66.
ity Assurance, Companion of the Institute of Manu- 9. Chromatography. USP 23 (621), United States Pharmaco-
facturing, and Committee member of the Society of peal Convention, Rockville, MD, 1994, p. 1776.
Chemical Industry Separation Science & Technol-
ogy UK.
Originally published in the May, 2004 issue of the Journal of Validation Technology