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Institutional SupervisorInstitutional SupervisorMr. Praveen ChaudharyMr. Praveen ChaudharyDeptt.of Quality Assurance Deptt.of Quality Assurance TechniquesTechniquesSGRRITS, DehradunSGRRITS, Dehradun

Industrial supervisor-Industrial supervisor-Mr. Ajay Kumar SinghMr. Ajay Kumar SinghGroup LeaderGroup LeaderCPP, Ranbaxy Research Lab.CPP, Ranbaxy Research Lab.GurgaonGurgaon

SUBMITTED BY-SUBMITTED BY-Rahul Naudiyal (M. PHARM)Rahul Naudiyal (M. PHARM)Quality Assurance Techniques Quality Assurance Techniques (QAT)(QAT)

Bioanalytical Method Development And Validation

Introduction Objective Method Development Method Validation Validation Parameter Plan of Work Reference

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Bioanalytical Method relates specifically to determine the concentration of drug or its metabolite or both in biological matrix such as plasma, serum, urine , etc

Bioanalytical information used in human clinical pharmacology, bioavailablity (BA) and bioequivalence (BE) studies requiring pharmacokinetic evaluation

Bioanalytical method is also used for non human pharmacology/ toxicology studies (preclinical studies)

LC/MS/MS is a hyphenated technique that combines physical separation power of liquid chromatography with detection power of Mass spectrometry.

LC/MS/MS is a powerful technique used for many application which have very high sensitivity and specificity

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• To develop and validate highly specific, reliable and cost effective LC-MS/MS method for determination of drug in human plasma

• The scope of developing and validating the bioanalytical method is to get a suitable method which is more accurate and precise for the analyte of interest under given set of lab condition by using resources available

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Steps In Method Development

Literature search for drugs.

Identification of analytical techniques and optimization

Reference standard preparation.

Selection of internal standard

Sample pre- treatment (Extraction procedure)

Sample storage

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Literature Search For Drugs

Mode of separation Selection of column Selection of mobile phase Role of pH, Buffer Role of Temperature Role of flow rate

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Liquid-Liquid Extraction Solid phase Extraction Reduced organic solvent consumption Easier collection of the total analyte fraction More efficient separation of interferences Protein precipitation

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Validation

What is Validation and why should it be done ?

Validation is the process of determining the suitability of a given methodology for providing useful analytical data

Validation is required for any new method to ensure that it is capable to give reproducible and reliable results, when used by different operators employing the same equipment in the same or different laboratories

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Definition of Bioanalytical Method Validation

Bioanalytical method validation include all the procedure that demonstrate that a particular method used for quantitative measurement of analyte in given biological matrix are reliable and reproducible for intended use

Types of Method validation

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Validation parametersSpecificity/selectivity

Sensitivity

Accuracy

Precision

Linearity

Recovery

Matrix Effect

Stability

Ability of an analytical method to differentiate and quantify the analyte in the presence of other components in the sample

Selectivity is evaluated by injecting extracted blank plasma and comparing with the response of extracted LLOQ samples processed with internal standard.

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Accuracy

Closeness of determined value to the true value.

The mean value should be within ± 15% of the The mean value should be within ± 15% of the theoretical value, except at LLOQ, where it theoretical value, except at LLOQ, where it should not deviate by more than ± 20%.should not deviate by more than ± 20%.

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Accuracy (%) = 100 x Accuracy (%) = 100 x Found value - Theoretical valueFound value - Theoretical value

Theoretical valueTheoretical value

The closeness of replicate determinations of a sample by an assay.

The acceptance criteria is ≤ 15% CV. At LOQ, 20% deviation is acceptable.

% CV (precision) =100 x Standard deviation/Mean

It Includes

Repeatability

Intermediate Precision

Reproducibility

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

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The effect on an bio-analytical method caused by all other components of the sample except the specific compound to be quantified.

The difference in response between a neat sample solution and the post-extraction spiked sample is called the absolute matrix effect.

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Due to ion suppression/enhancement by the others ions present in the biological matrix which might get ionized during detection and will give false results.

Matrix effect studied by comparing the response of extracted samples spiked before extraction with response of the blank matrix sample to which analyte has been added at the same nominal concentration just before injection

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chemical stability of an analyte in a given matrix under specific conditions for given time intervals.

Analyte change in any respect affect the chromatographic behavior which may complicate the method development the following activities should be considered:•Analyte and IS stock stability in solvent•Short Term Stability in matrix•Bench top stability in matrix•Freeze-thaw stability in matrix•In-injector stability in matrix•Long-term stability in matrix

literature survey Selection of analytical technique. Method Development Method Validation Compilation of data Result & conclusion

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Food and Drug Administration, FDA, Guidance for Industry: Bioanalytical Method Validation, Rockville, MD: US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, 2013.

Peters F.T., Review: Bioanalytical method validation – How, how much and why, Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Saarland.

Pranay W, Bioanalytical Method Development –Determination of Drugs in Biological Fluids 2010.

Skoog DA, West DM, Holler FJ, Crouch SR. Fundamentals of Analytical chemistry. 18 th ed. Thomson Asia pvt Ltd. Chapter 1. The nature of analytical chemistry. 2004; 2-5 & 973-996.

Chatwal R. G., Anand K. S. Instrumental method of chemical analysis, Himalaya Publishing House , Mumbai, 2007, 3rd edition, pp. 2.566 – 2.587, 2.624 – 2.639, 2.272- 2.302.

ICH, Validation of analytical procedure, International conference on Harmonization, IFPMA, Q2B Validation of Analytical Procedures: Methodology

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