Each year so many drugs loss their patent protection and opens the door for the generic alternatives. In this way Bioavailability and Bioequivalence studies becomes most important.
Bioavailability is defined as “The rate and extent to which the active moiety is absorbed from a drug product and becomes available at the site of action.” Bioavailability can be generally documented by a systematic exposure profile obtained by measuring drug and/metabolite concentration in the systemic circulation over a particular time period.
Scope of Bioavailability studies:
Development of new formulations of the already existing drugs.
Determination of effect of excipients, patient related factors and possible drug
To ensure the of quality of a drug product during the early stages of marketing in order to determine the influence of manufacturing factors, storage and stability factors on drug absorption.
The systemic exposure profile of drug or metabolite obtained by measuring concentration in the systemic circulation over a particular time period during clinical trials in the early stages of drug development can serve as a benchmark for subsequent bioequivalence studies.
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Bioequivalence is a relative term which shows the absence of a significant difference in the rate and extent to which the active ingredient becomes available at the site of drug action when two or more identical dosage forms administered at the same molar dose under similar conditions. Bioequivalence mainly focus release of drug substances from its dosage forms and subsequent absorption into the systemic circulation i.e. test dose plasma concentration-time will be identical with reference dose plasma concentration-time without showing any significant statistical differences ,then test dosage form will consider as therapeutically equivalent to the reference dosage form .
Scope of Bioequivalence studies:
To establish relativity between different formulations used during the development of a new product.
The therapeutic equivalence of a generic product and the reference product can be demonstrated.
Development of a modified release form of a product which has already approved as an immediate release formulation.
Development of alternative salt form for pharmaceutically equivalent drugs.
Bioequivalence studies are designed to establish equivalence between the test and reference products. If test and reference products are found to be bioequivalent,by this one can expect that the test product will also be therapeutically effective.
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.
Bioanalytical method validation parameters:
Validation documentation is done, by using specific laboratory investigations ,which ensures that the performance characteristics of the method is suitable for the intended analytical use. The analytical method is applicable only when the validation parameters are in acceptable range.
Types of validation:
A. Full Validation
Developing and implementing a bioanalytical method for the first time.
Full validation is required for a new drug entity.
If metabolites are added to an existing assay for quantification full validation is required.
B. Partial Validation:
These are modifications of already validated bioanalytical methods validation. Typical bioanalytical method changes includes:
Transfers of bioanalytical method between laboratories/analysts
Change in the process of analytical methodology
Change in anticoagulant in the collected biological fluid
Alteration in matrix within species (e.g., human plasma to human urine)
Alteration in sample processing, extraction procedures
Change in species within matrix (e.g., rat plasma to mouse plasma)
Any change in linearity range
Changes in instruments or software version
When two or more bioanalytical methods are used to generate data within the same study cross-validation is required. When data generated using different analytical techniques (e.g., LC-MS-MS vs.ELISA) in different studies are included in a regulatory submission cross validation should be considered.
Mass spectrometer is an instrument used to separate the ions in gas phase according to mass/charge ratio,when the analytical compound is bombarded with an electron beam which produce fragments of the original compound
Method development consists of the following steps:
Tuning of Analyte/metabolites/ISTD
Optimisation of Mass parameters
Optimisation of chromatographic conditions
Optimisation of extraction procedure
Tuning of Analyte/Metabolites:
To ensure the mass spectrum for a particular Analyte/Metabolite tuning is performed .To achieve the maximum mass spectral sensitivity tuning is an essential process which involve optimising voltages(capillary ,cone ,extractor and RF lens voltages),currents ,ion source and flow parameters. Perflourotributylamine (PFTBA) is used as a standard to tune the mass spectrometer.
Tuning of mass spectrometer provides the information regarding:
Ion sources parameters (no of ions produced and no ions directed towards the mass filter).
Mass filter parameters (sensitivity, mass resolution, peak widths and mass assignments).
Detector parameters(detector sensitivity ,magnitude of the signal)
Optimisation of mass parameters:
Optimisation of mass parameters involves setting of proper mass range, proper threshold, and verification of system performance and maintaining of system performance. In order to get the proper mass spectral resolution threshold setting is done which involves adjusting the capillary voltage, cone voltage, extractor voltage and RF lens voltage. Verification of system performance ensures system sensitivity, chromatographic performance and back ground signal. Maintenance of system performance ensures temperature in MS unit continuous carrier-gas flow into the MS and maintenance of vacuum.
Optimisation of chromatographic conditions:
Selection of column
Selection mobile phase
Optimisation of mobile phase composition
Column oven temperature
Auto sample temperature
Optimisation of extraction procedure (sample preparation):
Objective of sample preparation is removal of interfering compounds, Extraction of sample in to a suitable solvent and pre -concentration of the sample. Sample preparation improves specificity, reproducibility, accuracy, precision, recovery and stability of the sample and instrument life during sample analysis.
Sample preparation method:
Protein precipitation method
Liquid -liquid Extraction method
Solid-Phase Extraction procedure method
Hybrid Extraction method
ï¶ Protein precipitation: Involves denaturation of proteins by using water-miscible organic solvents (methanol, ACN, ethanol……), acid. Denaturation of proteins involves changing the pH of the sample, addition of organic solvent and increase the salt concentration of sample).
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Procedure: I part of the sample was diluted with a two-three parts of precipitating agent, then samples were vertexed and fallowed by addition of extraction solvent. Then the samples were centrifuged at high rpm then collection of supernatant liquid and the sample were directly analysed. If required, concentrated samples the supernatant sample were evaporated to dryness and reconstituted before analysis.
Advantages: Simple, inexpensive, universal method for sample extraction procedure.
Disadvantages: matrix components are not separated efficiently and it will decrease the efficiency of Ionization process, analytical column and instrument life, and effects the sample recovery, accuracy, linearity and specificity.
ï¶Liquid-liquid extraction: It involves by partitioning of sample (matix) between two immiscible solvents (i.e., organic and aqueous phase). Liquid-liquid extraction mainly base on the solubility and partition coefficient of the sample between the two phases.
Procedure: Addition of mobile phase, organic solvent to the biological matrix and vertexed fallowed by addition of extraction solvent. The samples were centrifuged at specified rpm, then collection of supernatant liquid. And the samples were directly analysed. If required, concentrated samples the supernatant samples were evaporated to dryness and reconstituted before analysis. Commonly used extraction solvents are (tBMA, n-hexane, dichloro methane …..).It is inexpensive method compare with SPE, it can efficiently extracts the samples and decreases the analytical problems during analysis.
ï¶Solid-phase extraction: It involves that adsorption of the targeted analyt on the solid phase support. By using suitable organic solvents (methanol, ACN, t-BMA……) the targeted analyte can be collected.
Basic steps involved the solid phase extraction:
1. Conditioning: The SPE cartridges to be conditioned with diluents organic solvents (methanol, ACN ……)
Avoid excessive drying of stationary phase bed
To activate the sites of stationary phase and removes dust, moisture from the stationary phase.
2. Sample-pre treatment: It involves addition of recommended amount of mobile phase, ISTD and organic solvent to the sample, vortex and addition of suitable buffering agent to the sample.
3. Sample application: From the top the SPE cartridges at a slow rate the sample to be applied, the vacuum pump place a role and collect the matrix from the cartridges. The targeted analyte bounded to the Stationary phase.
4. Washing/rinsing of stationary bed: This is mainly for the removal of interferences and matrix components from the cartridges by using solvents (water, buffers, and very dilute organic solvents).
5. Drying: It can be done by applying vacuum for recommended time(2-3min).it is mainly for the removal of excess washing solvents, avoid air bubble formation which leads to blockage of cartridges.
6. Elution: By passing elution solvents (methanol, ACN, t-BMA, dichloromethane……) from the cartridges the sample. Here organic solvent place a role to weaken the bonds between the Analyte to the sorbent. In each step applying recommended vacuum place a key role during extraction.
ï³ Buffering agent: Buffering agent selection mainly based on pKa of drug. If the pH of the buffer 1.5 units above its pKa value, the analyte will ionized and selects aqueous phase, only less polar interferences are goes to organic solvents. If the pH drugs below its pKa, the analyte wii unionized and extracted in to the organic phase by leaving most polar interferences in the aqueous phase.
ï³Mobile phase buffering agent: For sample analysis buffer pH should be selected as Â±2 of its pKa value. Some times higher buffer concentration may affect the instrument parts. Mostly used Buffers are ammonium formate, ammonium acetate….. some volatile buffers…..etc. they maintain the sample pH and also increases the extraction