1. INTRODUCTION
Each and every 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 (amount) of adsorption of unchanged drug from its dosage form" Bioavailability can be generally documented by a systematic exposure profile obtained by measuring drug/metabolite concentration in the systemic circulation over a particular time period.
Scope of Bioavailability studies [1-2]
Development of new formulations of the already existing drugs.
Determination of effect of excipients, patient related factors and possible interaction on drug absorption.
To ensure the 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 can obtain by measuring concentration in the systemic circulation over a particular time period. The systemic exposure profile of drug during the clinical trials in the early stages of drug development can serve as a benchmark for subsequent bioequivalence studies.
Bioequivalence is a relative term which denotes that the drug substance in two or more drug products of identical dosage forms reaches the systemic circulation at the same relative rate and to the same relative extent. Bioequivalence are the comparative studies and mainly focus on the 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
Several types of studies can be performed to determine the Bioequivalence as follows:
Pharmacokinetic studies
Pharmacodynamic studies
Clinical studies
In vitro studies
A Bioequivalence study ensures the 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 therapeutically effective.
Bio analytical method validation gives the information regarding all the validation parameters that specifies the method is suitable for the Quantitation of analytes/metabolite in the biological matrix. Bioanalytical method validation parameters:
Accuracy
Precision
Selectivity
Sensitivity
Reproducibility
Stability
Documentation of validation is done, by using specific laboratory investigations, which ensures that the characteristics of the method are suitable for the intended analytical use. The analytical method is applicable only when the validation parameters within the acceptable range.
1.1 INSTRUMENTATION [3-8]
Combined liquid chromatography/Mass spectrometry:
LC/MS is a highly hyphenated technique, having combination power of HPLC with detection power of mass spectrometer and provides qualitative and quantitative information of a particular compound. Ms provides a finger print mass spectrum, which contain the information regarding molecular weight and is structure specific fragmentation ion. Interfering of HPLC with MS thus mutually from the high resolution separation capability of HPLC and highly sensitive and structure specific detection capability of MS.
Mass spectrometer is an analytical instrument capable of generating ions from neutral organic molecules, and separating these ions according to m/z values, and detecting the resulting mass separated ions to produce a "mass spectrum". The mass spectrum gives the information by concerning the molecular structure of organic, inorganic compounds.
Mass spectrometer consists:
Sample Inlet unit
Ionisation chamber
Mass analyser(ion separator)
Detector and readout system
Vacuum system
In the mass spectrometer mass analyser is the heart of the instrument, it separates the gas phase ions in the presences electric/magnetic fields or both. These ions will produce the signal and it get amplified in the detector finally gives the mass spectrum. Organic mass spectrometers must have sample inlet systems (e.g., HPLC), processes for generating ions (i.e., "ion sources"), mass analyzers (e.g., quadrupole mass filters, magnetic sectors, quadrupole ion traps, time-of-flight (TOF) analyzers, etc.), and ion detectors (e.g., electron multipliers, photomultipliers, etc.).
Table: 1.1A Ionisation process
Ionization processes
Electron impact ionization
EI
Chemical ionization
CI
Fast atom bombardment
FAB
Plasma desorption
PD
Laser desorption
LD
Field ionization
FI
Field desorption
FD
Secondary ion
SIMS
Thermospray
TSP
Electrospray
ESI
Atmospheric pressure chemical ionization
APCI
Atmospheric pressure photo ionization
APPI
Table: 1.1B Types of Mass analysers
Mass analyzers
Magnetic sector/electric sector
BE (or EB)
Quadrupole mass filter (hexapole/octapole)
Q
Triple quadrupole
QQQ
Time-of-flight
TOF
Quadrupole ion trap (cubic or linear)
Trap
Hybrid quadrupole/time-of-flight
QTOF
Hybrid time-of-flight
TOF/TOF
Hybrid quadrupole/ion trap
Q-trap
MS/MS:
Tandem mass spectrometry (MS/MS) means two or more mass analysers either of different type (TOF/Q) or same type (Q/Q). First MS is used to isolate an ion and a second stage is then used to ensure the relationship of first MS ion with others from which it generated. The two stages of mass spectrometry provide the desired analytical information. It improves the selectivity and sensitivity of the Quantitative method.
Different types of MS-MS experiments.
The product-ion scan
The precursor-ion scan
The constant-neutral-loss scan
Selected decomposition monitoring
Modes of acquiring LC-MS/MS data:
Total ion current plot(TIC)
Selected Ion Monitoring(SIM)
Selected Reaction Monitoring(SRM)
Multiple Reactions Monitoring(MRM)
Total ion current plot (TIC):
The MS- total ion current plot is just like HPLC UV trace but mass spectrometer has an advantage of detecting many UV- transparent components. It is the plot of total ion current in each MS vs Intensity point. When a small molecule elutes from the column then the peaks appear in the plot of total ion current vs time. The main disadvantage of TIC is difficult because many compounds posses the same mass. It is not a unique identifier when compared to SIM experiment.
Selected Ion Monitoring (SIM):
SIM can scan very small mass range. To get more specific SIM assay mass range should be very narrow.SIM plot are very widely used for scanning serum (or) plasma samples. The reason behind more sensitive of SIM than full TIC plot is that it can dwell for a longer time over narrow mass range.
Selected Reaction Monitoring (SRM):
SRM is set to scan the fragment ion, typically one fragment ion. Quantitation of that particular fragment ion can be done plots obtained fragment ion. The obtained plot is very simple and continues a single peak. SRM shows more selectivity and Specific Quantification.
Multiple Reactions Monitoring (MRM): In this scan type Q1 is the 1st Quad which allows the parent ion filtration, Q2 is the collision cell, in this parent ion get fragmented and focus the fragment ions to the Q3.
Figure: 1.1C Multiple reactions monitoring
Electrospray Interface
Electro spray is one type of ionisation process in which the solution phase ions are turned to gas phase ions and these ions get separated in the mass analyzer based on their m/z values and reaches the detector of mass spectrometer.
Steps involved in the Electron spray ionization as below,
Production of charged droplets.
Droplet size reduction and Fission.
Gas phase ion formation
Liquid which the analyte(s) have been dissolved is passed through a capillary, at which atmospheric pressure maintained at high voltage. Highly charged droplets are formed by liquid stream breaks up. These are desolvated and pass through the atmospheric-pressure region of the source towards a counter electrode. Nitrogen gas plays a key role for the Desolvation of ions passed into the spraying region.
Analyte ions are obtained from these droplets pass through two differentially pumped regions into the source of the mass spectrometer. A schematic of an electro spray system is shown in Figure I.
Figure: 1.1D Schematic of an electro spray LC-MS interface
From solution ionization takes place directly, thermally labile molecules may be ionized without degradation. The ions produced by Electrospray are multiply charged majorly. This is great significance as the mass spectrometer measures the m/z (mass-to-charge) ratio of an ion and the 'mass' range of an instrument may therefore be effectively extended by a factor equivalent to the number of charges residing on the analyte molecule.
Figure: 1.1D Electrospray ionization probe mounted on source enclosure
Figure: 1.1 E Electrospray ionization probe
The Triple Quadrupole
This is the most widely used in MS-MS instrument. It consists three sets of quadrupole rods in series. The first set of rods (Q1) for parent ion selection. The second set of rods is not used for any mass separation and used as a collision cell (Q2), in this collision cell the fragmentation of parent ion in the will takes place. And these fragment ions focusing into the third set of quadrupole rods (Q3). Both sets of rods may be controlled to allow the transmission of ions of a single m/z ratio or a range of m/z values to give the desired analytical information.
Figure: 1.1D Triple quadrupole
Q2 acts a collision cell where parent ion fragmentation will occurs. It does not the filter ions. It accepts all ions sent to it by Q1 and passes all ions formed by collision to Q3.
ZSPRAY interface: ZSPRAY is the patented ionization source in the waters micro mass spectrometer. In this at atmospheric pressure ions were generate and accelerate towards the two orthogonal orifice in order to get in to the vacuum system of the mass spectrometer, so it named as ZSPRAY. Applications of ZSPRAY interface: It provides the more information regarding CID (collision induced deviations) which produces fragmented ions. And it increases the sensitivity and contains a vacuum isolation valve for easy removal of sample cone to clean without venting the instrument. And it contain cone shied which protects the sample cone from the contamination.
Figure1.1E: ZSPRAY interface
1.2 METHOD DEVELOPMENT [12-18]
Method development consists of the following steps:
Literature review
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 can performed .To achieve the maximum mass spectral sensitivity tuning is an essential process, which involves optimising the 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.
Tuning parameters:
Table1.2A: Tuning parameters
Source parameters
Analyzer parameters
Capillary (kV)
LM Resolution 1
Cone (V)
HM Resolution 1
Extractor (V)
Ion Energy 1
RF Lens (V)
Entrance
Source Temp (0 C)
Collision
Desolvation Temp (0 C)
Exit
Cone Flow (L/h)
LM Resolution 2
Desolvation Flow (L/h)
HM Resolution 2
Collision gas Pressure
Ion Energy 2
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 the 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, etc.) and acids. Denaturation of proteins involves by changing the pH of the sample, addition of organic solvent and increase the salt concentration of sample.
Procedure: I part of the sample can be 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 fallows the 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 cannot be separated efficiently and it will decrease the efficiency of Ionization process, analytical column and instrument life, and affects the sample recovery, accuracy, linearity and specificity.
ï¶Liquid-liquid extraction: It involves by partitioning of sample (matrix) between two immiscible solvents (i.e., organic and aqueous phase).Liquid-liquid extraction mainly based on the solubility (partition coefficient) of the sample between the two phases.
Procedure: Mobile phase and organic solvent were added to the biological matrix and vertexed fallowed by addition of extraction solvent. The samples were centrifuged at specified rpm, the supernatant liquid was collected. The extracted samples were directly analysed. If required concentrated samples, the supernatant samples were evaporated to dryness and reconstituted before analysis. Commonly used extraction solvents are (t-BMA, 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 the adsorption of the targeted analyte on the solid phase support. By using suitable organic solvents (methanol, ACN, t-BMA, etc.) the targeted analyte can be collected.
Basic steps involved the solid phase extraction:
1. Conditioning: The SPE cartridges to be conditioned by using dilute organic solvents (methanol, ACN, etc.).
The main purpose of conditioning is as fallows
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 the addition of recommended amount of mobile phase, ISTD and organic solvent to the sample 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 will bounded to the Stationary phase itself.
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: Can be performed by passing elution solvents (methanol, ACN, t-BMA, dichloromethane, etc.) 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.
Extraction solvents:
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 will unionized and extracted in to the organic phase by leaving most polar interferences in the aqueous phase. So the buffering agent mainly used to maintain the pH of the Analyte.
ï³ Mobile phase buffering agent:
For sample analysis buffer pH should be selected as ±2 of its pKa value. Some times higher buffer concentration may affect the instrument parts. Mostly used Buffers are ammonium formate, ammonium acetate, etc. They will maintain the sample pH and also increases the extraction efficiency, sensitivity, linearity of the sample during analysis.
1.3 BIOANALYTICAL METHOD VALIDATION
Method validation can be defined as according to ICHQ.2B guidelines "Establishing documented evidence, which provides 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 can be performed after the method development and it gives documented information regarding the linearity, accuracy, specificity and stability parameters of the analyte. And it also demonstrates the specific method is applicable for the Quantitation of analyte in the biological matrices which are reproducible for intended and long term use.
Validation parameters have been proved by considering the sample preparation, sample extraction procedure, chromatographic parameters. If the method proving all the validation parameters are within the acceptance range according to guide lines (US FDA,ANVISA,ICHQ.2B) ,then it is a validated method to demonstrate the Analyte concentrations in the sample.
Method Validation parameters:
System suitability:
The main purpose to perform the system suitability is to ensure that all analysing parameters of the method (reagents, samples, columns, instruments, glass ware, etc.) are suitable for the intended method. This experiment was performed by using injecting 6 subsequent injections of Aq MQC from a single vial.
Auto sampler carryover:
This parameter mainly used to ensure the carry over effect of initial injection to the subsequent injections during analysis. And can be performed by injecting the samples in the order of RS, AQ ULOQ, RS and AQ LLOQ of unextrated samples against STD BLK, AQ ULOQ, STD BLK and AQ LLOQ of extracted samples.
Linearity:
Linearity demonstrates the relationship between the experimental response values against analytical response values. Linearity graph can be plotted by using calibration curve standards. The number of standards used for constructing the calibration curve gives the information regarding the linearity range. The calibration consists of standard zero, eight or ten none zero standards. The calibration cure can plot by spiking the matrix with known concentration of the analyte. The calibration curve concentration range based on the expected concentration range of the particular study. The concentration of unknown sample can be identified by insert them in the calibration range.
The calibration curve of Analyte, Metabolites and ISTD was plotted by peak area ratio (Drug (r) Metabolites/ISTD) on Y-axis Vs the nominal concentrations on X-axis (first-order y = ax + b, where a=slope, b=intercept, x=concentration and y=peak area ratio of Analyte/ISTD).
LLOQ (Lower limit of Quantification): It is the lowest standard on the calibration curve.
ULOQ (Upper limit of Quantification): It is the higher standard on the calibration curve.
Precision, Accuracy:
Precision is defined as the "Degree of the reproducibility or repeatability while doing the (experiment) measurements or calculations shows the similar the results.
Precision method is represented by % CV (coefficient of variation.
%CV= (SD/Mean) Ã-100
SD=Standard deviation
Accuracy is defined as the "degree of closeness of the experimental value to the true value.
% Accuracy = Obtained concentration of QC/Nominal concentration Ã- 100
% Mean Accuracy = mean of obtained Con.for QC/Nominal concentration Ã- 100
Both Accuracy and precision determined by two ways:
Within batch precision/ Accuracy
Between batch precision/ Accuracy
Acceptance criteria:
The % Accuracy for (STD2-STD10) should be within 85.00-115.00 %, for LLOQ should be within 80.00-120.00 %. ULOQ, LLOQ should pass and 75 % of CC standards (STD2-STD9) should meet the acceptance criteria. Response of interfering peaks in STD Blk at the retention time of ISTD should be ï‚£ 5.00 % of LLOQ.
For precision at least 67 % (16 out of 24) of total QC samples and 50 % (3 out of 6) at each level should pass .And the % CV ≤ 15% & for LLQC it should be ≤20%.
%Accuracy =obtained concentration of QC / Nominal concentration of QC Ã-100
%Mean Accuracy = mean of obtained concentration of QC / Nominal concentration
Of QC Ã-100
Selectivity:
This parameter can be determined by performing specificity, matrix effect experiment.
Specificity:
It ensures that the intended method can able to differentiate the targeted analyte in the presences of other interfering substances.
Matrix effect:
It involves determination of any direct or indirect interference may alter the analytical response of the analyte. It can be performed by screening the different plasma lots. Reinjection Reproducibility:
This experiment can be carried out by injecting all ready passed P&A batch (1 set of CC and 6 sets of QC's) to identify again the P&A batches are giving same analytical results are not.
Effect of potential interfering drugs:
This experiment can preformed to ensure, any OTC drugs (e.g.: paracetamol, Ibuprofen, caffeine, diphinhydramine, diclofenacsodium and chlorfinaramine maleate.) may alter the Analytical response of the targeted Analyte.
Recovery:
This experiment can be preformed to ensure the extraction efficiency of the Analytical process and to know the recovery of the sample, by comparing the extracted sample area ratio against unextrated sample area ratio and reported as % recovery.
% recovery= Extracted analyte or ISTD peak ratio / unextrated analyte or ISTD peak area ratioÃ-100
Analyte peak area ratio= Area of analyte/Area of ISTD
ISTD peak area ratio=Area of ISTD/Area of Analyte
Ruggedness:
It defined as the degree of reproducibility of the method under a variety of normal method conditions.
Different Analyst
Different column
Different equipment
Stability:
Stability experiment procedure ensures the stability of analyte during sample collection, sample extraction; sample storage (i.e., Bench top, Freeze thaw, auto sampler, dry extract stability and solution stability).It can be performed by analysis of the Stability samples against comparative (freshly prepared) samples.
Stability parameters:
Analyte stability
Stability in solutions
Short term stability
Long term stability
Matrix stability
Bench top stability
Freeze-thaw stability
Auto sampler stability
Wet extract stability
Stability of analyte in blood
Long term stock stability
