321 P a g e International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 3(4): July-August 2014 INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093*** ICV 5.13*** Pharmaceutical Sciences REVIEW ARTICLE!!! BIOANALYTICAL METHOD DEVELOPMENT AND ITS VALIDATION Rahul Naudiyal*, Praveen Kumar, Preeti Kothiyal Shri Guru Ram Rai Institute of Technology & Sciences, Patel Nagar, Dehradun. KEYWORDS: LC-MS/MS bioanalysis, Method development, Method validation. For Correspondence: Rahul Naudiyal* Address: Shri Guru Ram Rai Institute of Technology & Sciences, Patel Nagar, Dehradun. E-mail: naudiyalr@yahoo.com ABSTRACT One of the major challenges facing the pharmaceutical industry today is finding new ways to increase productivity, decrease cost while still ultimately developing new therapies to enhance health. The objective of this paper is to review the sample preparation of drug in biological matrix and to provide practical approaches for determining selectivity, specificity, limit of detection, lower limit of quantitation, linearity, range, accuracy, precision, recovery, stability, ruggedness, and robustness of liquid chromatographic methods to support pharmacokinetic (PK), toxicokinetic, bioavailability, and bioequivalence studies. This review discusses the conceptual aspects of method validation, its management, processes and schemes and mainly deals with its important parameters and their significance. Liquid chromatography-tendam mass spectrometry (LC-MS/MS) is a technique that uses liquid chromatography (or HPLC) with the mass spectrometry. (LC-MS/MS) is commonly used in laboratories for the qualitative and quantitative analysis of drug substances, drug products and biological samples. Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine is reliable and reproducible for the intended use.
322 P a g e International Standard Serial Number (ISSN): 2319-8141 INTRODUCTION: Bioanalytical methods employed for the quantitative determination of drugs and their metabolites in biological matrix (plasma, urine, saliva, serum etc) play a significant role in evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data (Bressolle, 1996). Both HPLC and LCMS-MS can be used for the bioanalysis of drugs in plasma. Each of the instruments has its own merits [4]. Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine, is reliable and reproducible for the intended use (Eric Reid, 1990) (U.S. FDA, Guidance for industry, 2001) [4,5]. These studies generally support regulatory filings [6]. The quality of these studies is directly related to the quality of the underlying bioanalytical data. It is therefore important that guiding principles for the validation of these analytical methods be established and disseminated to the pharmaceutical community [7]. Method development: Analytical method development is the process of creating a procedure to enable a compound of interest to be identified and quantified in a matrix. A compound can often be measured by several methods and the choice of analytical method involves many considerations, such as: chemical properties of the analyte, concentrations levels, sample matrix, cost of the analysis, speed of the analysis, quantitative or qualitative measurement, precision required and necessary equipment. The analytical chain describes the process of method development and includes sampling, sample preparation, separation, detection and evaluation of the results [4]. Sample collection and sample preparation: The biological media that contain the analyte are usually blood, plasma, urine, serum etc. Blood is usually collected from human subjects by vein puncture with a hypodermic syringe up to 5 to 7 ml (depending on the assay sensitivity and the total number of samples taken for a study being performed). The venous blood is withdrawn into tubes with an anticoagulant, e.g. EDTA, heparin etc. Plasma is obtained by centrifugation at 4000 rpm for 15 min. About 30 to 50% of the original volume is collected (Rosing, 2000) [4,8]. The purpose of sample preparation is to clean up the sample before analysis and/or to concentrate the sample. Material in biological samples that can interfere with analysis, the chromatographic column or the detector includes proteins, salts, endogenous macromolecules, small molecules and metabolic byproducts [9]. Steps during method development Compound, its nature, structure and physicochemical properties. Literature survey, various already developed methods and their drawbacks. Selection of analyte concentration level, C max, LOQ, ULOQ, LQC, MQC, HQC. Selection of suitable non reactive and stable Biological matrix.
323 P a g e International Standard Serial Number (ISSN): 2319-8141 Suitable LC-MS/MS system with suitable conditions. Scanning and optimisation. Optimization of chromatographic conditions: mobile phase, column, flow rate, injection volume. Selection of Internal Standard similar to Analyte properties. Optimization of sampling processing techniques. System suitability testing. Validation (accuracy, precision, linearity, selectivity, recovery, reproducibility, stability studies). Documentation. Need of Bioanalytical Method Validation It is essential to used well-characterized and fully validated bioanalytical methods to yield reliable results that can be satisfactorily interpreted. It is recognized that bioanalytical methods and techniques are constantly undergoing changes and improvements; they are at the cutting edge of the technology. It is also important to emphasize that each bioanalytical technique has its own characteristics, which will vary from analyte to analyte, specific validation criteria may need to be developed for each analyte [26]. Moreover, the appropriateness of the technique may also be influenced by the ultimate objective of the study. When sample analysis for a given study is conducted at more than one site, it is necessary to validate the bioanalytical method(s) at each site and provide appropriate validation information for different sites to establish inter-laboratory reliability [3, 27]. Method validation: Method validation is a process used to verify/confirm that an analytic method developed is suitable for its intended purpose, that it provides reliable and valid data for a specific analyte. Typical parameters to validate are; include selectivity, accuracy, precision, linearity and range, limit of detection, limit of quantification, recovery, robustness and stability. General recommendation for analytical method validation, i.e. for pharmaceutical methods, can be found in The US Food and Drug Administration (FDA) guideline [4,5]. Selectivity/Specificity: The terms selectivity and specificity generally refers to a method that produces a response for a single analyte only, while the term selective refers to a method that provides responses for a number of chemical entities that may or may not be distinguished from each other. Since there are very few methods that respond to only one analyte, the term selectivity is
324 P a g e International Standard Serial Number (ISSN): 2319-8141 usually more appropriate. Selectivity studies should also assess interferences that may be caused by the matrix, e.g., urine, blood, soil, water or food. Optimized sample preparation can eliminate most of the matrix components. The absence of matrix interferences for a quantitative method should be demonstrated by the analysis of at least five independent sources of control matrix [10]. System Suitability: System suitability is routinely assessed before an analytical run. Data generated from system suitability checks should be maintained in a specific file on-site and should be available for inspection. System suitability samples should be different from the study samples, standards, and QCs to be analyzed in the run. Therefore, study samples, standards, or QCs should not be used as their own system suitability samples within the analytical run [5]. Accuracy: The accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. Accuracy is determined by replicate analysis of samples containing known amounts of the analyte. Accuracy should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The mean value should be within 15% of the actual value except at LLOQ, where it should not deviate by more than 20%. The deviation of the mean from the true value serves as the measure of accuracy [4,5]. Precision: The precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix. Precision should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV. Precision is further subdivided into within-run, intra-batch precision or repeatability, which assesses precision during a single analytical run, and between-run, inter batch precision or repeatability, which measures precision with time, and may involve different analysts, equipment, reagents, and laboratories [4,5,10]. Linearity: The ability of the bioanalytical procedure to obtain test results that are directly proportional to the concentration of analyte in the sample within the range of the standard curve [3,11]. The concentration range of the calibration curve should at least span those concentrations expected to be measured in the study samples. If the total range cannot be described by a single calibration curve, two calibration ranges can be validated. It should be kept in mind that the accuracy and precision of the method will be negatively affected at the extremes of the range by extensively expanding the range beyond necessity. Correlation coefficients were most widely used to test
325 P a g e International Standard Serial Number (ISSN): 2319-8141 linearity. The deviation should not exceed more than 20% from the nominal concentration of the LLOQ and not more than 15% from the other standards in the curve [4]. Carry-Over test: During validation carry-over should be assessed by injecting blank samples after a high concentration sample or calibration standard at the upper limit of quantification. Carry-over blank following the high concentration should not be greater than 20% of the lower limit of quantification and 5% for the internal standard. This test is performed to check either the concentration of one sample injection is showing its effect on next sample concentration or not. If it appears that carry-over is unavoidable, study samples should not be randomised [13]. Limit of detection: The limit of detection (LOD) is a characteristic for the limit test only. It is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated under the stated experimental conditions. The detection is usually expressed as the concentration of the analyte in the sample, for example, percentage, parts per million (ppm), or parts per billion (ppb) [4,10] Limit of quantification Lower limit of quantification: LLOQ is the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. Determining LLOQ on the basis of precision and accuracy is probably the most practical approach and defines the LLOQ as the lowest conc. of the sample that can still be quantified with acceptable precision and accuracy. LLOQ based on signal and noise ratio (s/n) can only be applied only when there is baseline noise, for example to chromatographic methods. Upper limit of quantification: ULOQ is the maximum analyte conc. of a sample that can be quantified, with acceptable precision and accuracy. The ULOQ is identical with the conc. of the highest calibration standards. Recovery: The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard. Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Recovery of the analyte need not be 100%, but the extent of recovery of an analyte and of the internal standard should be consistent, precise, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high) with un-extracted standards that represent 100% recovery [4]. Absolute Recovery= Response of analyte spiked into matrix (processed) Response of analyte spiked into matrix (unprocessed) x 100
326 P a g e International Standard Serial Number (ISSN): 2319-8141 Matrix effect: Matrix effect is investigated to ensure that selectivity and precision are not compromised within the matrix screened. Three blank samples from each of at least six batches of matrix under screening are extracted. For matrix effect LQC (lower quality control), MQC (middle quality control) and HQC (higher quality control) spiking dilutions and internal standard dilution are spiked in the above extracted blank samples [4,5]. Matrix factor: It is the quantitative estimation of matrix effect to calcute the intensity of the error produced. Robustness: According to ICH guidelines, The robustness of an analytical procedure is the measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Robustness can be described as the ability to reproduce the (analytical) method in different laboratories or under different circumstances without the occurrence of unexpected differences in the obtained result(s), and a robustness test as an experimental set-up to evaluate the robustness of a method. Stability: The stability of the analyte under various conditions should also be studied during method validation. The conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. The following stability conditions are required by FDA and are advisable to investigate; [5] Stock solution stability: The stability of the stock solution should be evaluated at room temperature for at least 6 hours [5]. Short-term temperature stability: The stability of the analyte in biological matrix at ambient temperature should be evaluated. Three aliquots of low and high concentration should be kept for at least 24 hours and then analysed [5]. Long-term temperature stability: The stability of the analyte in the matrix should exceed the time period from sample collection until the last day of analysis [5]. Freeze and thaw stability: The stability of the analyte should be determined, after three freeze and thaw cycles. Three aliquots of low and high concentration should be frozen for 24 hours and then thawed at ambient temperature [5]. Ruggedness: This includes different analysts, laboratories, columns, instruments, sources of reagents, chemicals, solvents. Ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test condition. The ruggedness of the method was studied by changing the experimental condition such as, [12]. a. Changing to another column of similar type b. Different operation in the same laboratory
327 P a g e International Standard Serial Number (ISSN): 2319-8141 Incurred sample reanalysis: ISR is conducted by repeating the analysis of a subset of subject samples from a given study in separate runs on different days to critically support the precision and accuracy measurements established with spiked QCs; the original and repeat analysis is conducted using the same bioanalytical method procedures. ISR samples should be compared to freshly prepared calibrators. ISR is expected for all in vivo human BE studies and all pivotal PK or pharmacodynamic (PD) studies [5]. Concomitant Validation: This process include the measurement of analyte response along with some common medications (eg; acetaminophen, diclofenac sodium, etc) to check wether the method is slective and accurate or not in multiple dose condition. For the conduction of Bioequivalence of drug a protocol is made by the PK scientist and clinical department according to the regulatory.the parameter on which the protocol is made are: Reference and Test drug name and dose. Dosage form Duration of action Time interval Study design. Sequence of Reference and Test dosing. Ambulatory visit. Method development & Method Validation strategy Pharmacokinectic parameter determination. Statistical analysis Application of method development and validation: After the protocol is finalized by the authority the clinical study is conducted in clinics and in the the laboratory the method is developed and validated for the conduction of bioanalysis of drug. After the method is finalized the sample is collected from the clinics and come to the laboratory under specified condition mentioned in protocol. After that the sample is analysed on the developed method and the analysis report is send to the Pharmacokinetic department where the pharmacokinetic parameter is calculated by Winolin, version-7 software. The parameters which are determined are: C max t max AUC 0-t AUC 0- AUC extrapolated K el
328 P a g e International Standard Serial Number (ISSN): 2319-8141 After the Pharmacokinetic parameter analysis the analytical record is send to the statistical department where the transform the numerical value into logarithm form and by applying ANOVA and SAS software they calculate the confidence interval and proof the bioequivalence and bioavailability of drug. Finally all the reports are sent to DRA department for filling of the bioequivalence of the drug and getting the license for marketing of that drug. Conclusion: This review summarizes the method development and validation parameters that are required according; to the requirements of ICH and US FDA. The method validation process and; the minimum requirements to be included in a regulatory method are also discussed. The concepts and relatively new technology covered in this review article can be used to enhance LC-MS/MS bioanalytical method development. It provides information for the bioavailability, bioequivalence and therapeutic drug monitoring studies. An overview of phase appropriate method validation are presented to stimulate ideas and the; thought process to follow when such situations are encountered. An attempt has been made to understand and explain the bioanalytical method development and validation from basic point of view. References: 1. Bressolle F, Bromet P, Audran M. Validation of liquid chromatography and gas chromatographic methods application to pharmacokinetics. Journal of Chromatography B. 686, 1996, 3-10. 2. Shah V.P., The History of Bioanalytical Method Validation and regulation: Evolution of a Guidence Document on Bioanalytical Methods Validation, The AAPS Journal 9(1), 2007, 43-47. 3. Tiwari G, Tiwari R, Bioanalytical Method Validation: A Updated Review, Pharmaceutical Methods, 1(1) 2010, 25-38. 4. Murugan. S., Pravallika. N., Sirisha. P., A review on bioanalytical method development and validation by using LC-MS/MS, Journal of Chemical and Phamaceutical Sciences, Department of Pharmaceutical Analysis, Andhra Pradesh, India., Issue: 1, Vol: 6, March 2013. 5. U.S Department of Health and Human Services, Food and Drug Administration, Guidance for Industry, Bioanalytical Method Validation, May 2001. 6. Ludwig. H., Validation of Analytical Methods. Agilent Technology, 1-65. March 2010. 7. Lalit. V. Sonawane, Bhagwat. N. Poul, Sharad. V. Usnale, Bioanalytical Method Validation and Its Pharmaceutical Application- A Review, Pharmaceutica Analytica Acta, Issue: 3, Vol: 5, 2014.
329 P a g e International Standard Serial Number (ISSN): 2319-8141 8. Rosing H, Man WY, Doyle E, Bult A, Beijnen J H., Bioanalytical liquid chromatographic method validation- A review of current practices and procedures, Journal of Liquid Chromatography and Related Technology, Vol: 23, 2000, 329-354. 9. Wells DA. High throughput bioanalytical sample preparation : methods and automation strategies, Progress in pharmaceutical and biomedical analysis, Amsterdam, London, Elsevier science B, 2003, 610. 10. Sharma. G., Jain. A, Bioanalytical Technologies: A Review To Method Validation, International Journal of Pharmaceutical Research and Development, Vol: 3, May 2011. 11. Singh UK, Pandey S, Pandey P, Keshri PK, et al. (2008), Bioanalytical Method Development and Validation, Express Pharma. 12. Sekar. V., Jayaseelan. S, Subash. N., Bioanalytical Method Development and Validation of Letrozole by Rp-HPLC method, International Journal of Pharmaceutical Response and Development, June 2009. 13. Committee for Medicinal Products for Human Use, Guideline on bioanalytical method validation, European Medicines Agency, July 2011. 14. Guidelines for bioavailability and bioequivalence studies,by central drugs standard control organization,directorate general of health services,ministry of health and family welfare,govt. of India,New Delhi 2005. 15. Guidance for Industry Bioavailability and BioequivalenceStudies for Orally Administered Drug,U.S. Department of Health and Human ServicesFood and Drug Administration Center for Drug Evaluation and Research (CDER)March 2003 BP. 16. SADC Guideline for bioavailability and bioequivalence studies 2007. 17. Chinese Regulatory Guideline for bioavailability and bioequivalence studies 2009. 18. Vinod P. Shah, Kamal K. Midha, Analytical methods validation: Bioavailability, bioequivalence, and pharmacokinetic studies, Journal of Pharmaceutical Sciences, Issue-3, page no.309-312, March 1992. 19. Mie-lang Chen,Bioavailability and Bioequivalence: An FDA Regulatory Overview, Pharmaceutical Research, Vol.18, Issue-12, December 2001. 20. D.M.Brahmandkar, Jaiswal S.B, Bio-pharmaceutics and Pharmacokinetics, edtion-5, VallabhPrakashan, Pg no.315-389. 21. Leon Shargel, Applied Bio-pharmaceutics and Pharmacokinetics, edition-5, McGraw Hill Professional, chapter-15,year-2005.
330 P a g e International Standard Serial Number (ISSN): 2319-8141 22. Putheti RR, Okigbo RN, Patil SC, Advanapu MS, Leburu R, Method Development and Validations: Characterization of Critical Elements in the Development of Pharmaceuticals, International Journal of Health Research, 1(1), june 2008, pg: 11-20. 23. Wells DA. High throughput bioanalytical sample preparation : methods and automation strategies, Progress in pharmaceutical and biomedical analysis, Amsterdam, London, Elsevier, Oct 2003, pg: 610. 24. Silvia I, Laurian V, Daniela LM (2008) Bioanalytical method validation. Revista Romana de Medicina de Laborator 10: 13-21. 25. Tur F1, Tur E, Lentheric I, Mendoza P, Encabo M, et al. (2013) Validation of an LC-MS bioanalytical method for quantification of phytate levels in rat, dog and human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 928: 146-154. 26. Yadav AK, Singh SK, Yashwant, Verma S (2012) Bioanalytical Method Validation How, How Much and Why: A Reaseach Perspective. International Journal of Natural Product Science 1: 123. 27. www.cpharmaguide.com.