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Bio Analytical Method Development and Validation: A Review

 


Priyanka Patel*, Nilesh Patel, Ashok Patel, Ajay Patel, Amit Vyas 
B.K. Mody government pharmacy college, Rajkot, Gujarat, India

Abstract

Bio analytical methods are used for the quantitative analysis of drugs and their metabolites  in  the  biological  media  like  saliva,  urine,  plasma,  serum.  Development  and validation of Bio analytical method is important to understand the pharmacokinetics of any drug and/or its metabolites. Bio analytical method development consists of three interrelated parts sample  preparation,  chromatographic  separation  and  detection  by  using  proper  analytical method.

Validation of a Bio analytical method is the process by which it is established that the performance characteristics of the method meet the requirements for the intended Bio analytical application. The validation is further divided into 3 segments full validation, partial validation, and  cross  validation.  This  review  describes  mainly the  various  aspects  for development  of Bio analytical method and for the validation of Bio analytical methods.

Keywords: Biological matrix, Method stability, Matrix effect, Extraction.

Introduction

A Bio analytical method is a set of procedures involved in the collection, processing, storage, and analysis of a biological matrix for a chemical compound.  Bio analysis is the method used to determine the concentration of drugs, their metabolites and/or endogenous substances in the biological matrices such as blood, plasma, serum, cerebrospinal fluid, urine, and saliva.   A Properly validated Bio analytical method for quantitative determination of drugs and their metabolites (analytes) plays quite important role in the effective performance of bioequivalence. The basic parameters of validation comprises all criteria determining data quality such as selectivity, sensitivity, calibration model, accuracy, precision, stability, lower limit of quantification (LLOQ), recovery, linearity, limit of detection, reproducibility, and ruggedness 1.

Why Validate Bio analytical Methods?

The reason for validating a Bio analytical procedure is to demonstrate the performance and reliability of a method and hence the confidence that can be placed on the results.Validation involves documenting, through the use of specific laboratory investigations, that the performance characteristics of the method are suitable and reliable for the intended analytical applications 2 

Extraction procedures for drugs and metabolites from biological samples

Extraction of analyte from biological matrix is traditionally carried out by (a) Liquid- Liquid extraction (LLE), (b) Solid-phase extraction (SPE) and (c) Protein precipitation.

Liquid/Liquid Extraction

It is based on the principle of differential solubility and partitioning equilibrium of the analytes

between the aqueous and organic phases. It generally involves the extraction of analyte from one phase into another phase and the distribution of the analyte molecules between two immiscible phases.3 After extraction of analyte the organic layer is separated from aqueous phase and it is evaporated in presence of nitrogen gas so as to get the dry form of sample.

Solid phase extraction (SPE)

Uses a small volume of a chromatographic stationary phase to isolate desired analytes from a sample SPE is a selective method for sample preparation where the analyte is bound onto a solid support, interferences are washed off and the analyte is selectively eluted. different choices of sorbents,  SPE  is  a  very  powerful  technique  (Wells,  2003).  SPE consists of four steps; conditioning, sample loading, washing and elution 4 

Figure 1: Solid phase extraction (SPE) 

Conditioning: The column is activated with an organic solvent that acts as a wetting agent on the packing material and solvates the functional groups of the sorbent. Water or aqueous buffer is added to activate the column for proper adsorption mechanisms.

Sample loading: After adjustment of pH, the sample is loaded on the column by gravity feed, pumping or aspirating by vacuum.

Washing: Interferences from the matrix are removed while retaining the analyte.

Elution: Disruption of analyte-sorbent interaction by appropriate solvent, removing as little of the remaining interferences as possible.

The biological samples are loaded onto SPE cartridges and the biological matrices are then washed out of the cartridges. The retained analytes are often eluted using small aliquots of an organic solvent so that the desired analytes are concentrated enough for analysis without further evaporation and concentration procedures.

Protein Precipitation

Protein precipitation can be applied to extraction of plasma and blood samples. Principle of PP is based on precipitation of the proteins by using various reagents like acid (trichloroacetic acid and perchloric acid), organic solvents (methanol, acetone and Acetonitrile) or by salts (ammonium sulphate). After precipitation the sample is centrifuged, which results in extraction of analyte in the precipitating solvent.5 There are some benefits with the precipitation method as clean-up technique compared to SPE. It is less time consuming, smaller amounts of organic modifier or other solvents are used.

Bio analytical Method Validation:

The guidelines for Bio analytical method validation are published by the United States Food and Drug Administration (USFDA) in May 2001. These guidelines are standard for validation parameters’ evaluation.

The main objective of method validation is to demonstrate the reliability of a particular method developed for the quantitative determination of an analyte in a specific biological matrix.

Full Validation

Full validation is necessary when developing and implementing an analytical method for analysis of a new drug entity, when developing and implementing a Bio analytical method for the first time and when an existing assay method is modified- metabolites are added to an existing assay for quantification of a drug 6.

Partial Validation

Partial validations are modifications of existing validated Bio analytical methods. Partial validation can range from as little as one intra-assay accuracy and precision determination to a nearly full validation. Bio analytical method changes that require partial validation are:

  • Method transfers between laboratories or analysts
  • Modification of analytical methodology (e.g., change in detection systems)
  • Addition of different anticoagulant in harvesting biological f
  • Changes in matrix within the same species (e.g., human plasma to human urine)
  • Alteration of sample processing procedures
  • Changes in species within matrix (e.g., rat plasma to mouse plasma)
  • Variation in relevant concentration range
  • Changes in instruments and/or software platforms
  • Limited sample volume (e.g., pediatric study)

Cross-Validation

In cross-validation two Bio analytical methods for the same drug are compared. Original validated Bio analytical method serves as the reference and the revised Bio analytical method is the comparator. Cross validation is required when two or more Bio analytical methods are used to generate data within the same study. When sample analyses within a single study are conducted at more than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples should be conducted to establish inter-laboratory reliability. Cross-validation should also be considered when data generated using different analytical techniques in different studies 7.

Validation Parameters

The basic parameters for the validation of a chemical assay comprises of all criteria determining data quality such as selectivity, accuracy, precision, recovery, linearity, calibration model, limit of detection (LOD), lower limit of quantification (LLOQ), stability, reproducibility, and ruggedness.  

Selectivity

Selectivity  exercise  is  carried  out  to  assess  the  ability  of  the  Bio analytical  method  to differentiate and quantify the analyte(s) in presence of other components in the sample. For selectivity, analyses of blank samples of appropriate biological matrix (plasma, urine, or other matrix) obtained from at least six sources should be carried  out.  Each  blank  sample  should  be  tested  for  interference  and  selectivity should  be ensured at the lower limit of quantification (LLOQ).

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.

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, intrabatch precision or repeatability, which assesses precision during a single analytical run, and between-run, interbatch precision or repeatability, which measures precision with time and may involve different analysts, equipment, reagents and laboratories.

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 analyte in solvent. 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 unextracted standards that represent 100% recovery 7 

Limit of Detection (LOD)

It is the lowest amount of analyte in a sample that can be detected but not necessarily quantitated under the stated experimental conditions.

Lower Limit of Quantification (LLOQ)

It is the lowest amount of analyte in a sample that can be detected but necessarily quantitated under the stated experimental conditions with acceptable accuracy and precision.

Ruggedness

Ruggedness is a measure for the susceptibility of a method to small changes that might occur during routine analysis due to pH, mobile phase composition, temperature etc.  Ruggedness should be tested if a method is supposed to be transferred to another laboratory. Ruggedness is not mandatory under full validation, but it would be helpful during the method development as problems that may occur during validation are often detected in advance 8 

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. Recovery comparison sample at LQC, MQC and HQC concentration level along with internal standard are prepared and screened 8 

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

Stock solution stability

The stability of the stock solution should be evaluated at room temperature for at least 6 hours.

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 9.

Freeze Thaw Stability

During freeze thaw stability evaluations, the freezing and thawing of stability samples should mimic the intended sample handling conditions to be used during sample analysis. Stability should be assessed for a minimum of three freeze-thaw cycles.

Long-Term Stability

The storage time in a long-term stability evaluation should equal or exceed the time between the date of first sample collection and the date of last sample analysis.

Conclusion

This review describes the various aspects for the Bio analytical method development and its validation. Validation of a Bio analytical method is the process by which it is established that the performance characteristics of the method meet the requirements for the intended Bio analytical application. The development of Bio analytical method and its validation helps to determining the pharmacokinetics of drugs and their metabolites10. 

Acknowledgement

I  express  my  gratitude  and  thanks  to  my  esteemed  honourable  guide  Dr.  Nilesh  K.  Patel, Assistant Professor, BKMGPC for valuable advice. I consider myself extremely fortunate to have had a chance to work under his guidance.

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