evaluation of an analytical method

INTERNATIONAL JOURNAL OF PHARMACEUTICAL CHEMISTRY RESEARCH

Volume 1 Issue 1 2012 www.earthjournals.org

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Review Article

EVALUATION OF AN ANALYTICAL METHOD Shah, K.*1, Kumar, S.2, Upmanyu, N.3 and Mishra, P.1

1Institute of Pharmaceutical Research, GLA University, Mathura, UP, India, 281406 2Department of Pharmacy, Barkatullah University, Bhopal M.P. India

3RKDF College of Pharmacy, Bhopal M.P. India

Email: [email protected]

Abstract:

Pharmaceutical analysis plays an important role right from testing of raw materials, in-

process quality checks to the analysis of finished products. Pharmaceutical analysis is

considered to determine identity, strength, quality and purity of drug samples. Pharmacists

have made important contributions in the field of medicinal chemistry, both in discovering or

isolating new therapeutic agents and in developing methods for standardizing and controlling

medicinal. In small laboratories the responsibility for performing analysis may be delegated

entirely to pharmacist staff members. But whether or not pharmacists may have occasion to

conduct analysis, they at least should understand the basic principles involved in the

standardization and control of the medicinal agents dispensed . The paper is focusing on the

parameters required to validate an analytical method.

Keywords : analytical chemistry, validation, limit of detection, limit of quantification,

robustness and ruggedness.

INTRODUCTION:

Analytical chemistry is as old as chemistry itself. In many respect analytical chemistry acts as a foundation for other branches of chemistry. In fact, the science of chemistry came into being as a result of human inquisitiveness to understand the nature of an extraordinary variety of matter that surrounds him. In analytical chemistry it is

of prime importance to gain information about the qualitative and quantitative compositions of substances and chemical species, that is, to find out what a substance is composed of and exactly how much. Analytical chemistry may be defined as the science and art of determining the composition of materials



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in terms of elements or compounds contained.[1]The resolution of a chemical compound into its proximate or ultimate parts. The determination of its elements or of the foreign substances it may contain. Thus reads a dictionary definition.[2] In general terms, then pharmaceutical analysis comprises those procedure necessary to determine the identity, strength, quality and purity of drugs.[3] Analytical Chemistry can also be said that it deals with the methods for determining the chemical composition of samples of matter. A qualitative method yields information about the atomic or molecular species or the functional groups that exist in the sample; a quantitative method in contrast, provides numerical information as to the relative amount of or more of these compounds [4-6]. Method validation can be defined as (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.

Method validation is an integral part of the method development; it is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, quality, purity, and potency of the drug substances and drug products. Simply, method validation is the process of proving that an analytical method is acceptable for its intended purpose.

OBJECTIVE OF VALIDATION [7] The primary objective of validation is to form a basis for written procedure for production and process control which are designed to assure that the drug products have the identity, strength, quality and purity they purport or are represented to

possess quality, safety and efficacy must be designed to build into the product. Each step of the manufacturing process must be controlled to maximize the probability that the finished products meet all quality and design specification.

Benefits of Validation

a) Produces quality products b) Helps in process improvement

technology transfer, related product validation, failure investigation, and increased employee awareness.

c) Cost reduction by increasing efficacy, few reject, longer equipment life, production of cost effective products

d) Helps in optimization of process or method.

e) Regulatory affairs-produces approved products and increased ability to export.

The US Food and Drug Administration (FDA) [8-9] and US Pharmacopoeia (USP) [10] both refer to ICH guidelines. Validation as Defined by Different Agencies[11] USFDA: According to this Validation is the process of establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. WHO: Defines Validation as an action of providing any procedure, process, equipment, material, activity or system actually leads to the expected results.

EUROPEANCOMMITTEE:

Defines Validation as an action of providing in accordance with the principles of GMP that any procedure.



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Process, material, activity or system actually leads to expected results. This process consists of establishment of the performance characteristics and the limitations of the method. Method Validation is required when A new method is been developed. Revision established method. When established methods are used in different laboratories and different analysts. Comparison of methods. When quality control indicates method changes. Method Validation, however, is generally a one-time process performed after the method has been developed to demonstrate that the method is scientifically sound and that it serves the intended analytical purpose [12-16]. Performance characteristics examined when carryout method validation are: Accuracy / Precision. Repeatability / Reproducibility. Linearity / Range. Limit of detection (LOD)/ Limit of quantification (LOQ). Selectivity / Specificity. Robustness / Ruggedness. Accuracy It relates to the closeness of test results to true value i.e. measure of exactness of analytical method. Determination of accuracy The accuracy may be determined by application of the analytical method to an analyte of known purity (example: reference standard) and also by comparing the results of the method with those obtained using an alternate procedure that has been already validated. Accuracy is calculated as the percentage of recovery by the assay of the known added amount of the analyte in the sample or the difference between the mean and

accepted true value together with confidence intervals. The ICH guidelines recommended to take minimum of 3 concentration levels covering the specified range and 3 replicates of each concentration are analyzed (totally 3* 3=9 determinations.) It is expressed as percent recovery by the assay of known/added amount of analyte in the linearity range. Accuracy can also be determined by comparing the results with those obtained using an alternative method which has already been validated. Precision It expresses as degree of agreement among individual test results when method/procedure is applied to homogenous sample usually expressed as SD/RSD. It is measure of degree of repeatability or reproducibility under normal conditions. Determination of precision The procedure is applied repeatedly to separate identical samples drawn from the homogeneous batch of material and measured by the scatter of individual results from the mean and expressed as the standard deviation or as the coefficient of variation (relative standard deviation). Precision may be the measure of either the degree of reproducibility or of repeatability of the analytical method under normal operating conditions. According to ICH guidelines repeatability should be assessed using minimum of 9 determinations covering the specified range for the procedure. Precision should be measured for repeatability (intra-assay precision), intermediate precision and reproducibility.



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Linearity and Range Linearity Linearity is the ability of the method to obtained test results that are directly proportional to the analyte concentration within a given 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. Measurement A range of standards should be prepared containing at least 5 different concentrations of analyte which are approximately evenly spaced and span 50-150% of the label claim. At least 6 replicates per concentration to be studied. Plot a graph of concentration (on x - axis) Vs mean response (on Y -axis). Calculate the regression equation, Y- intercept and correlation coefficient. Plot another graph of concentration (on X-axis) Vs response ratio (replicate response divided by concentration, on Y-axis). The range of the method is validated by verifying that the analytical method provides acceptable precision, accuracy and linearity when applied to samples containing analyte at the extreme of the range as well as within the range. Specificity ICH document divides specificity into two categories. a) Identification tests b) Assay / impurity tests ICH defines specificity of an assay is its ability to measure accurately and specifically the analyte of interest in the presence of other components that might be expected to present in the sample matrix.

It is the degree of interference from excipients, impurities or degradation products ensuring that a peak response is due to a single component only i.e., no co-elutions. Determination of Specificity When chromatographic procedures are used representative chromatograms should be presented to demonstrate the degree of selectivity and peak should be appropriately labeled. Peak purity test (e.g. using diode assay or mass spectrometry) may be useful to show that the analyte chromatographic peak is not attributable to more than one component. Selectivity It is a procedure to detect qualitatively the analyte in the presence of components that may be expected to be present in the sample matrix or the ability of a separative method to resolve different compounds. It is the measure of the relative method location of two peaks. Determination of Selectivity It is determined by comparing the test results obtained on the analyte with and without addition of potentially interfering material. When such components are either unidentified or unavailable a measure of selectivity can be obtained by determining the recovery of a standard addition of pure analyte to a material containing a constant level of the other components.

Ability of the method to measure accurately and specifically the analyte of interest in presence of matrix and other components likely to be present in the sample matrix as impurities, degradation products and other related substances. For this, one may compare the test results of analysis of sample containing other ingredients, impurities, degradation



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product, related substances, placebo ingredients with those obtained from analysis of sample without these, i.e., the method must allow distinct analytical measurement of analyte of interest and exclusion of all other relevant interferences. If the impurities/degradation products or potential contaminants are not available, one can apply a proposed method to the strained and stressed (heat, light, humidity) samples, degree of agreement among results will explain specificity of the method. Limit of Detection (LOD) and Limit of Quantitation (LOQ) Lowest concentration of the analyte in the sample that the method can detect but not necessarily quantify under the stated experimental conditions simply indicates that the sample is below as above certain level. The LOD will not only depend on the procedure of analysis but also on type of instrument.

Measurement is based on

1. Signal to noise ratio

2. Visual evaluation (relevant chromatogram acceptable)

3. The standard deviation of the response and the slope.

LOD =

S3.3

= Standard deviation of the response.

S = Slope of the calibration curve of the analyte

Limit of Quantification The LOQ is the lowest concentration of analyte in a sample which can quantitative determined that may be measured with an acceptable level of accuracy and precision

under the stated operational conditions of the method. LOQ can vary with the type of method employed and the nature of the sample. Based on the standard deviation of the response and the quantitation limit QL may expressed as

S10LOQ =

= Standard deviation of the response.

S = Slope of the calibration curve of the analyte

Measurement For instrumental and non instrumental methods the quantitation limit is generally determined by the analysis of the samples with known concentration of the analyte and by establishing the minimum level at which the analyte can be determined with acceptable accuracy and precision. In case of instrumental methods that exhibit back ground noise the ICH document describes to compare measured signals from samples with known concentration of analyte with those of blank samples. A typically acceptable signal to noise ratio is 10:1 Ruggedness Degree of reproducibility of test results obtained by analyzing the same sample under variety of normal test conditions such as different.

Analysts Instruments Days Reagents Columns and TLC plates

i.e. lack of influence of environmental variables on the method. Comparison of reproducibility of test results to the



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precision of assay is the direct measure of ruggedness of the method. Robustness It is the measure of the capacity of the analytical method to remain unaffected by small but deliberate variations in procedure to provide an indication about variability of the method during normal laboratory conditions. Simplicity Simplicity refers to when the analysis can be carried out in a minimum number of step and need only easily available reagents and equipments. As with the increase in number of steps, there are more chances of personal errors creeping in. Cost of Analysis Cost of analysis is based on the simplicity of method, number of steps involved, time required, cost of reagent, requirement of instrument and the skilled and experienced analyst etc. These are the some of the factors that contributes to the cost of analysis. Validation of the proposed analytical method and its acceptability depends on the above factors. REFERENCES

1. Jeffery GH, Bassett J, Mendham J, Denney RC, In : Vogels Text book of Quantitative Chemical Analysis, 5th edn., Longman Group UK Ltd., England; 1989. P. 3.

2. Sharma BK, In : Instrumental Methods of Chemical Analysis, 20th edn., Goel Publishing House, Meerut; 2001. P. 3.

3. Beckett AH and Stenlake JB, In : Practical Pharmaceutical Chemistry, 3rd edn., Vol. II, CBS Publishers and Distributors, New Delhi ; 1986. P. 131.

4. United State Pharmacopoeia, National Formulary, US Pharmacopial conention Inc, MD 2006. p. 2109,107.

5. British Pharmacopoeia, Volume I, (2005). p. 106-107.

6. Harsono T, Yuwono M, Indrayanto G, Journal of AOAC International. Vol. 88 (4), 2005, 1093-1098.

7. FDA, "Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls Documentation; Availability," Federal Register (Notices) 65(169), 5277652777 (2000).

8. www.fda.gov/cder/guidance/cmc3.pdf

9. USP 25NF 20, Validation of Compendial Methods Section (1225) (United States Pharmacopeal Convention, Rockville, Maryland, USA, 2002) p 2256.

10. Skoog DA, Holler FJ and Crouch, SR, Pharmaceutical Analysis, 5th edition, Brooks/cole ; 2007. 32-35

11. Shabir A, Journal of Chromatography A. Vol. 987 (1-2), 2003, 57-66.

12. Sethi PD, In : Quantitative Analysis of Drugs in Pharmaceutical Formulations, 3rd edn., CBS Publishers and Distributors, New Delhi, 1997.xi and 53-57.

13. ICH Harmonised Tripartite Guidelines (Nov 2005) Validation of Analytical Procedures: Text and Methodology Q2 (R1).

14. Willard HH, Merritt LL and Dean, JA, Settle, FA. In : Instrumental



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Methods of Analysis, 4th edn., Affiliated East West Press Pvt. Ltd., New Delhi; 1986. P. 30-32.

15. Crosby NT, Merritt LL, Dean JA, Hardcastle WA, Holcombe DG, Treble RD, Prichard FE, Quality in Analytical Chemistry Laboratory. John Wiley and Sons; 1995. P. 67-77.

16. Christian, G.D., Analytical Chemistry, 6th edn., John Wiley and Sons; 2004. p. 126-132.

evaluation of an analytical method

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