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Bioanalytical method employed for the quantitative determination of drug and their metabolites in biological matrix plasma, urine, saliva, sputum etc. play significant role in evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data. Chromatographic methods are commonly used in regulatory laboratories for the qualitative and quantitative analysis of drug substances, drug products, raw materials and biological samples throughout all phases of drug development in research and quality and control. Bioanalytical method validation is carried out to ensure that method is accurate, precise, specific, reproducible and rugged over the specified range in which analyte will be analyzed. This dissertation deals with the studies carried out on the development of bioanalytical method used for the estimation of antihyperglycemic agent Metformin, in human plasma using LC-MS/MS and its validation. Before discussing the experimental results, a brief introduction to bio pharmaceutical analysis, analysis of drug in biological media, preliminary treatment of biological samples, estimation procedures for drugs and metabolites from biological samples by LC-MS/MS, is presented.
1.1 Biopharmaceutical analysis
Bioavailability is rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action.Â For drug products that are not intended to be absorbed into the bloodstream, bioavailability may be assessed by measurements intended to reflect the rate and extent to which the active ingredient or active moiety becomes available at the site of action. Â
Bioequivalence is the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administered at the same molar dose under similar conditions in an appropriately designed study.
1.1.3 Need for biopharmaceutical analysis1:
Methods of measuring drugs in biological media are becoming increasingly important and the following studies are highly dependent on biopharmaceutical analytical methodology.
Bioavailability & Bioequivalence studies
New drug development
Research in basic biomedical and pharmaceutical sciences
Therapeutic drug monitoring.
1.1.4 Analysis of drug in various biological media2.
The most common samples obtained for biopharmaceutical analysis are blood and urine. Faeces are also utilized, especially if the drug or metabolite is poorly absorbed. The choice of sampling media is determined largely by the nature of the drug study. Separation or isolation of drugs and metabolites from biological samples is performed in order to partially purify a sample. In this manner, an analyst can obtain the selectivity and sensitivity needed to detect a particular compound and can do so with minimum interference from components of the more complex biological matrix. The number of steps in separation procedure should be kept to a minimum to prevent loss of drug or metabolites. Sometimes the separation steps are preceded by a sample pretreatment.
184.108.40.206 Choice of body fluid for assay:
Concentration of drug is important element in determining individual or population pharmacokinetics; it is measured mainly in plasma and urine. In few cases, measurement of drug concentration in the other body fluids may be warranted. Choice of the sampling is determine largely by the nature of the drug under study.
i) Drug concentration in blood, plasma or serum:
Measurement of drug concentration (level) in blood, plasma or serum is the most direct approach to assessing the pharmacokinetics of the drug in the body. Assuming that drug in the plasma is in dynamic equilibrium with tissue changes in the drug concentration in plasma will reflect changes in tissue drug concentration.
ii) Drug concentration in Tissue:
Tissue biopsies are occasionally done for diagnostic purpose, such as identification of malignancy. Usually only small sample of tissue is removed, making estimation of drug concentration difficult. Drug concentration in tissue biopsies may not reflect drug concentration in the other tissue or the drug concentration in all parts other than from which the biopsy material has been removed. The measurement of drug concentration in tissue biopsy material may be used to ascertain if the selected drug reached the target tissue and reached the proper concentration within the tissue.
iii) Drug concentration in Urine:
Measurement of drug concentration in urine is an indirect method to ascertain the bioavailability of drug. The rate and extent of drug excreted in the urine reflects the rate and extent of systemic drug absorption.
iv) Drug concentration in Faeces:
Measurement of drug concentration in feces may reflect that the drug has not been absorbed after oral dose or may reflect that drug has been expelled by biliary secretion after systemic absorption. Fecal drug level estimation is often done in mass balance studies.
v) Drug concentration in Saliva:
Saliva is a component of oral fluid. Oral fluid is composed of many components and concentration of drug typically parallel to those found in blood. Drugs that are highly protein bound in blood will have a lower concentration in oral fluid. The use of salivary drug concentration as therapeutic indicator should be used with the caution and preferably as a secondary indicator.
220.127.116.11 Significance of Measurement of drug concentration in plasma:
The intensity of the pharmacological or toxic effect of drug is often related to the concentration of drug at receptor site. As most of the tissue cells are richly perfused with tissue fluids or plasma, measuring the plasma drug level is a responsive method of monitoring the course of therapy. Monitoring of plasma drug concentration allows for the adjustment of the dosage of drug in order to individualize and optimize therapeutic regimens. In case of alteration in physiological functions due to disease, monitoring plasma drug concentration may guide to progress of the disease state and enable the investigator to modify the drug dosage accordingly. Mathematical analysis of plasma level verses time curve permits estimation of half-lives, absorption and excretion rates, extent of absorption (AUC) and other constants that are useful in describing fate of given drug in humans. Comparative bioavailability studies permits judgments as to the bioequivalence of drugs.
1.2 Primary treatment of biological sample 3- 6 :
Detection of drug or its metabolite in biological media is usually complicated by the matrix effects. Because of this, various types of cleanup procedures involving solvent extraction and chromatography are employed to effectively separate the drug component from endogenous biological material. Primary treatment of biological samples is an essential part of various analytical techniques, intended to provide a reproducible and homogeneous sample.
Sample preparation prior to chromatographic separation has the major objectives :
The dissolution of analyte in suitable solvent.
Removal of interfering compound as far as possible.
For primary treatment of biological samples various methods used are :
Liquid-liquid extraction (LLE)
Solid phase extraction (SPE)
1.2.1 Protein precipitation :
Biological sample such as plasma contains significant amount of protein which can bind a drug. The drug may have to be freed from protein before further manipulation. Protein precipitation is important because the presence of lipids, salts and other endogenous materials can cause rapid deterioration of HPLC columns and interfere with the assay. In protein precipitation acid or water miscible organic solvent are used to remove protein by denaturation and precipitation.
Generally used solvents for protein precipitation are methanol, Acetonitrile, trichloro acetic acid, perchloro acetic acid, ammonium sulphate and Tungstic acid.
The acids which are in their cationic form at low pH, form insoluble salts with acids. Organic solvents methanol, acetone, acetonitrile, ethanol have relatively low efficiency in removing plasma protein but are widely used in bioanalysis because of their compatibility with HPLC mobile phase. These organic solvents which lower the solubility of proteins and precipitate them from solution have an effectiveness inversely related to their polarity.
Methanol and acetonitrile are preferred because, they provide
Prevent drug entrapment.
Produce a flocculent precipitate.
The analyst can remove a drug or metabolite from larger concentration.
With denaturation of proteins, denaturation of analyte may also take place.
This method cannot be used if the analyte is a protein.
In this technique, the elevation in the pH of the sample may create problem.
1.2.2 Liquid-liquid extraction (LLE):
It is based on the principles of differential solubility and partitioning of analyte molecules between aqueous (the original sample) and the organic phases. LLE initially involves pH adjustment of the sample with an appropriate buffer. This pH adjustment is intended to neutralize the molecule, making it more amenable to extraction. The next step is the addition of an immiscible organic extraction solvent, followed by the agitation (vortexing) to facilitate portioning of analyte molecules between phases. The phases are separated and the aqueous component is subjected to re-extraction and then discarded. The organic phases is evaporated to dryness and resultant residue is dissolved in suitable solvent and used for analysis
The analyst can extract a drug or metabolite from larger sample with low concentration of analyte.
The technique is simple, rapid and has relatively small cost factor per sample.
The extracted material can be redissolved in small volume (e.g.100 to 500Âµl).
It is possible to extract more than one sample concurrently.
Accuracy is less as compared to other method.
Formation of emulsion may occur.
Sometime pH control of the sample necessary for extraction.
During evaporation temperature is increased, so the method cannot be used for thermolabile substances.
1.2.3 Solid phase extraction (SPE):
In solid phase extraction the analyte is retained on solid phase sorbent while rest of the sample passes through it which is followed by elution of analyte with appropriate solvent.
A typical SPE sorbent consist of 40-60Âµm silica particles to which has been bonded a hydrocarbon phase. This bonding is achieved by reaction of chlorosilane with the hydroxyl group of silica gel to form silicon-oxygen-silicon link.
Solid phase extraction is particularly suitable for polar compounds that would otherwise tend to remain in the aqueous phase. The method is also useful for amphoteric compounds that cannot be extracted easily from water.
Biological samples can be prepared for cleanup by passing the sample through the resin bed where drug components are adsorbed and finally eluted with an appropriate solvent.
Low concentration of drug can be detected.
Effective in selective removal of interferences.
A number of samples can be extracted simultaneously and eluted selectively.
Different types of adsorbents can be used.
Extending the analytical column life, reduced system maintenance, minimizing ion suppression.
Internal standard is required.
Extraction is difficult for high-density materials.
As in extraction processes, a number of steps are to be carried out making it a time consuming process.
Biological sample even after primary treatment are partially purified and are still a complex mixture of various ingredients and the concentration of analyte of interest in such mixture is very low ranging from ng/ml to Âµg/ml. Hence for analysis of biological samples, sophisticated techniques such as UV, HPLC, MS, GPC etc. are employed singly or in combination.
Hyphenating chromatographic technique such as GC/HPLC with UV, NMR, MS are preferred techniques of bioanalytical chemists.
1.3 Chromatographic method :
The presence of metabolites or more than one drug in a biological sample usually demands a more sophisticated separation for their measurement especially, when two or more drugs have similar physical and chemical nature. Chromatography is a separation technique that is based on differing affinities of a mixture of solutes between two phases. The result is a physical separation of the mixture into its various components. The affinities or interactions can be classified in terms of a solute adhering to the surface of a polar solid (adsorption), a solute dissolving in a liquid (partition) and a solute passing through or impeded by a porous substance based on its molecular size (exclusion).
This technique has been employed suitably modified and have been employed in pharmaceutical analysis.
These techniques are :
In all these techniques, the separation is ensured using a stationary phase and mobile phase. The elution of analyte is achieved by passing the mobile phase. The speed and efficiency of such techniques is limited and flow rate of mobile phase determines the speed.
Increasing the flow rate of mobile phase results in faster separation. Reduction in particle size, chemical composition of stationary phase brings in efficiency in separation. These have been combined in HPLC advantageously.
1.3.1 High Performance Liquid Chromatography 7, 8 :
The heart of a HPLC system is the column. The column contains a stationary phase. The mobile phase is pumped through the column by a pump. The mixture to be separated is injected into the flowing mobile phase by an injector. When the mobile phase passes through the column that contains the stationary phase, the molecules that adsorbs most to the stationary phase migrates slowest through the column. The mobile phase on passing through the column enters the detector where different molecules are detected as they pass through it. Signals from the detector are recorded as a chromatogram.
Figure 1.1: Schematic diagram of HPLC
a) Stationary phase (Adsorbents):
HPLC separations are based on the surface interactions and depend on the types of the adsorption sites. Modern HPLC adsorbents are the small rigid porous particles with high surface area. Important features of adsorbent parameters are:
Particle size: 3 to 10 Î¼m
Particle size distribution: as narrow as possible, usually within 10% of the mean
Pore size: 70 to 300 Å
Surface area: 50 to 250 m2/g
Bonding phase density (number of adsorption sites per surface unit): 1 to 5 per 1 nm2
The last parameter in the list represents an adsorbent surface chemistry. Depending on the type of the ligand attached to the surface, the adsorbent could be normal phase (-OH,-NH2), or reversed-phase (C8, C18, Phenyl), and even anion (NH4 +), or cation (-COO-) exchangers.
b) Mobile phase (eluents):
In HPLC type and composition of the mobile phase (eluent) is one of the variables influencing the separation. Despite of the large variety of solvents used in HPLC, there are several common properties like purity, detector compatibility, solubility of the sample, low viscosity, Chemical inertness, reasonable price which are required.
Each mode of HPLC has its own requirements. For normal phase mode solvents are mainly nonpolar, for reversed-phase eluents are usually a mixture of water with some polar organic solvent such as acetonitrile. Size-exclusion HPLC have special requirements, SEC eluents has to dissolve polymers, but the most important is that SEC eluent has to suppress all possible interactions of the sample molecule with the surface of the packing material.
Usual detection systems for HPLC are :
1.4 Mass Spectrometer 9, 10 :
Mass spectrometer is an analytical instrument used for measuring the molecular mass of a sample. The technique is based on generation of positive charged ion by extraction of an electron and measuring mass to charge ratio using various analytical/detection system. The response is then recorded as relative abundance of base peak.
Mass spectrometer are divided into following main parts
a) Sample inlet system b) Ionizer c) Mass analyzer
d) Detector e) Recorder f) Vacuum system
1.4.1 Sample inlet system 11 :
There are mainly two types of sample inlet system. The sample introduced as neutral species through a controlled vacuum leak followed by ionization in vacuum chamber. Create the ion at atmospheric pressure and then introduced the ion in to the mass spectrometer through a controlled vacuum leak with aid of electrostatic field, this process is called API (atmospheric pressure ionization) it provide best way when a dynamic coupling of liquid chromatography done.
Figure 1.2: Sample inlet system
1.4.2 Ionization source 12, 13 :
Ionization proceeds by two fundamental processes:
Loss/gain of electron
Loss/gain of charged particle
An odd electron ion is generated by the loss/gain of an electron. In vacuum generating method ionic species of identical nominal molecular weight differ only by the mass of an electron to the neutral species from which it was generated.
An even electron is produced by gain or loss of even electron specie from a molecule.
The following are a few of the important ionization techniques are :
Atmospheric Pressure Ionization (API)
Electron Ionization (EI)
Matrix-Assisted Laser Desorption Ionization (MALDI)
Fast Atom Bombardment (FAB)
Field Desorption/Field Ionization (FD/FI)
Types of API source :
Atmospheric Pressure Electro Spray Ionization (ESI)
Atmospheric Pressure Chemical Ionization (APCI)
18.104.22.168 Atmospheric Pressure Electro Spray Ionization (ESI) :
It is an atmospheric ionization technique in which ions are generated in the solution phase by evaporation of solvent and followed by ionization in gaseous phase.
Figure 1.3: Electrospray Ionization
An appropriate solvent from LC (liquid chromatography) system is passed through a metal capillary to which a static DC voltage is applied to create ionization of effluents. When the solvent evaporates the charge density increases creating columbic repulsion and subsequent dissociation of droplet. Further evaporation of droplet creates an environment in which charge transfer takes place from the solvent to the analyte.
Typically a voltage of 2.5 to 5 kV is applied to generate an even electron ion in gas phase. This method is commonly used for high molecular weight compounds. Presently most of the designs rely on coaxial gas flow (nitrogen) which improves desolvation. These sources with coaxial gas flow are called nebulization assisted electro spray ionization. Low ionization is observed in this technique due to solvent clustering and analyte adduct formation, so this is most applicable for LC-MS/MS system.
Droplet size reduction in ESI :
Droplet size reduction occurs by the continual repetition of two processes:
Desolvation (evaporation of neutral solvent and volatile buffers)
Droplet fission caused by electric repulsion between like charges.
Figure 1.4: Droplet size reduction & fission in ESI
22.214.171.124 Atmospheric Pressure chemical Ionization (APCI) :
It is basically a soft ionization technique in which the ionization occurs not in vacuum but at atmospheric pressure. It is gas phase ionization process whereby gas molecules are isolated from the carrier solvent before ionization.
Figure 1.5: APCI Interface
Generally less polar compounds are ionized by this method. Typically the mobile phase containing eluting analyte is heated to relatively high temperature (above 4000c), sprayed with high flow rates of nitrogen and the entire aerosol cloud is subjected to a corona discharge that creates ions.
1.4.3 Mass analyzer 14, 15 :
The main function of the mass analyzer is to separate, or resolve the ions formed in the
ionization source of the mass spectrometer according to mass-to-charge (m/z) ratios. There are a number of analyzers currently available, the better known of which include quadrupoles, time-of-flight (TOF) analyzers, magnetic sectors, and both Fourier transform and quadrupole ion traps. But quadrupole analyzers are commonly used in case of LC-MS/MS.
126.96.36.199 Quadrupole mass analyzer :
It consists of two pair of electrically connected rods/electrodes on to which a concurrent radio frequency (RF) and direct current (DC) voltage is applied. The rod consists of Molybdenum or Gold with a diameter of 1cm & 30cm in length. Opposite Quadrupole rods pairs are connected electrically such that they carry identical RF/DC ratio. For a given RF/DC voltage ratio the Quadrupole analyzer filter only the ion within a selected mass/charge ratio which have a stable trajectory as they pass from one end to other.
Figure 1.6: Quadrupole mass analyzer
If RF/DC is held constant only a narrow m/z range will transmit the Quadrupole. In this configuration Quadrupole mass analyzer acts as a mass filter or Quadrupole filter.
Ionization polarity :
In API method either positive or negative charge ion can be generated. The type of ions generated is dependent on the gain or loss of electron during ionization. A positive DC voltage on the capillary will generate positive ion from the basic compound such as Amine. A negative DC voltage on the capillary will generate negative ion from the acidic compound such as Carboxylic acid.
188.8.131.52 Tandem mass spectrometry :
The basic modes of data acquisition for tandem mass spectrometer experiments are as follows.
Figure 1.7: MS/MS Analysis
Tandem mass spectrometry is also called as mass spectrometry- mass spectrometry because the instrument contains two mass analyzers. A collision cell is placed between the two analyzers. The tandem arrangement allows the m/z relating to the analyte to be selected at first Quadrupole and the m/z characteristics of daughter ions to be selected at the second Quadrupole.
This arrangement provides good selectivity and sensitivity. The first Quadrupole is used to select the parent ion and fragmentation occurs in the collision cell. The fragmented ion enters to second Quadrupole, which selects the ions of the specific reaction product only. In this process the internal energy of analyte is increased which induces the fragmentation. Collision with neutral gas molecules is called collision induced dissociation (CID).
Q0 Q1 Q2 Q3
RF only Scanning RF/DC RF only collision cell Scanning RF/DC
Figure 1.8: Triple Quadrupole mass analyzer
Collision Induced Dissociation (CID) :
CID is a process in which first collision when ion translation energy is converted into internal energy to obtain an ion in excited state and a secondary slow unimolecular decomposition which yield various ion products through a number of competitive reactions. As a result there is increase in internal energy which leads to the fragmentation of ions. This fragmentation is induced by high pressure.
In principle, two mass analyzers are required; one is for selecting the precursor ion from the ions generated in the ion source and other for analyzing the product ions after the collision. This approach is called tandem mass spectrometry.
1.4.4 Detector16 :
The detector either measures the ion current directly or with possible single amplification or using ion conversion followed by amplification. The detectors are
Multichannel plate detector
These are the modern type of detectors mostly used in MS system. It utilizes the dynode to convert ions signal into secondary particle before amplification. Signal response is a function of ions mass and kinetic energy.
i. Photomultiplier :
It provides signal detection through conversion and subsequent amplification. The conversion dynodes of a multiplier detector generate electron that impinge on a phosphorescent screen, which subsequently generate photon that are detected and amplified. The mass spectrometer that utilizes the photomultiplier must be isolated from ambient light because it is sensitive to light background.
Advantage: Lifetime of photomultiplier is relatively long about 10 year as photomultiplier is encased in glass and is not susceptible to the environmental corrosion.
1.4.5 Recorder :
Computers are an integral part of modern mass spectrometers. The molecules which are fragmented into different ions, leads to a discrete spectral peak. For structural determination, the heights and mass-to-charge ratios of each peak must be determined, stored, and ultimately displayed in computer system.
1.4.6 Vacuum system :
All mass spectrometer need a vacuum to allow ions to reach the detector without colliding with other gaseous molecules or atoms. If such collisions occur, the instruments suffer from reduced resolution and sensitivity. Higher pressure may also cause high voltage discharge which can damage the instrument, its electronics and/or the computer system. In general, an efficient vacuum system is crucial for MS.
Mass spectrometer and MS/MS are being used for determination of molecular weight, isotopes study, structural elucidation etc. Though highly sophisticated, MS/MS technique needs the sample to be as pure as possible. Hence samples from biological origin need an efficient separation technique for removal of interfering substances. Hyphenating LC/MS ensures combined effect of excellent separation technique of LC with high selectivity and sensitivity of MS. Hence LC/MS and LC-MS/MS methods are choice of bioanalytical chemists' world over.
1.5 Liquid Chromatography with Mass Spectrometer 17 :
The main purpose of interface between LC and MS is to evaporate the mobile phase and transfer the analyte from the higher pressure or atmospheric pressure at which chromatographic separation is achieved to the negative pressure required for the mass analysis. Mass spectrometer is used as a detector system while liquid chromatography ensures effective separation.
Figure 1.9: Instrumentation of LC-MS/MS
1.5.1 Modes of LC/MS/MS monitoring 17 :
Typically the mass spectrometer is set to scan a specific mass range. This mass scan can be wide as in the full scan analysis or can be very narrow as in selected ion monitoring. Many scans are acquired during a single LC/MS analysis.
First analyzer allows the transmission of all simple ions, whilst the second analyzer is set to monitor specific fragment ions, which are generated by bombardment of the sample ions with the collision gas in the collision cell. This type of experiment is particularly useful for monitoring groups of compounds contained within a mixture which fragment to produce common fragment ions e.g. glycosylated peptides in a tryptic digest mixture.
LC/MS data is represented by adding up the ion current in the individual mass scans and plotting that total ion current as an intensity plot against time. The most common modes of acquiring LC/MS data are:
Precursor or Parent ion scanning mode.
Product or Daughter ion scanning mode.
Constant neutral ion scanning mode.
Selected / Multiple Reaction Monitoring mode. (SRM) or (MRM)
184.108.40.206 Precursor or Parent ion scanning mode :
In precursor or parent ion scanning, total ion current (TIC) of parent ion having high intensity is measured. As a molecule elutes from the HPLC column the relative intensity goes up and a peak appears in the total ion current plot as the points (TIC of scan) are plotted against time.
Compounds of every mass are plotted in the TIC plot above. Finding the compound of interest can be difficult since many compounds have the same mass. The intact mass of a compound is not a unique identifier. Using the data set above a specific mass can be selectively plotted; however the sensitivity will be less than what is observed in the SRM experiment described below,
Ion accumulation Parent ion
Figure 1.10: Precursor or Parent ion scanning
220.127.116.11 Product or Daughter ion scanning mode :
In product or daughter ion scanning, the mass spectrometer is set to scan over a very small mass range, typically one mass unit. The first analyzer is used to select user specified sample ions arising from a particular component; usually (i.e. (M+H) + OR (M-H) - ions. These chosen ions pass into the collision cell and are bombarded by the gas molecules which cause fragment ions and these fragment ions are analyzed by the second analyzer.
Fragmentation Linear ion trap
Figure 1.11: Product or Daughter ion scanning mode
The product or daughter ion plot is a plot of the ion current resulting from very small mass range. The reason is that the peaks seen in the product or daughter ion scanning may be very minor components in the plot. The product ion scanning is more sensitive than the Parent ion scanning because the mass spectrometer can dwell for a longer time over a smaller mass range and highly selective as a particular daughter ion is produced only by particular compound and not by other compounds of same mass.
18.104.22.168 Constant neutral ion scanning mode :
This involves both analyzers scanning, and collecting data, across the whole m/z range, but the two are off-set so that the second analyzer allows only those ions which differ by a certain number of mass units (equivalent to a neutral fragment) from the ions transmitted through the first analyzer.
22.214.171.124 Selected or Multiple reaction monitoring mode (SRM) or (MRM) :
Selected reaction monitoring or multiple reaction monitoring is the method used by the majority of scientists performing mass spectrometric quantitation. SRM delivers a unique fragment ion that can be monitored and quantified in the midst of a very complicated matrix. SRM plots are very simple, usually containing only a single response. This characteristic makes the SRM plot ideal for sensitive and specific quantification. The MRM experiment is accomplished by specifying the parent mass of the compound for MS/MS fragmentation and then specially monitoring for a single fragment ion.
Ion accumulation Daughter ion
Parent ion Fragmentation Linear ion trap
Figure 1.12: Selected or multiple reaction monitoring
1.6 Bioanalytical method development and validation 18, 19 :
Bioanalytical chemistry involves the qualitative and quantitative analysis of drug substances in biological fluids (mainly plasma, serum and urine) or tissue. It plays a significant role in the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data. The main analytical phases that comprise bioanalytical services are method development, method validation and sample analysis (method application).
A bioanalytical method is a set of all the procedures involved in the collection, processing, storing, and analysis of a biological matrix for an analyte (Shah et al., 1992). Analytical methods employed for quantitative determination of drugs and their metabolites in biological fluids are the key determinants in generating reproducible and reliable data that in turn are used in the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetics (Shah et al., 2000).
Method development is a trial and error procedure. It involves evaluation and optimization of the various stages of sample preparation, chromatographic conditions, detection and quantification. An extensive literature survey is carried out on the analyte. Literature survey aids in selecting the techniques of sampling, primary treatment, separation and quantification.
1.6.1 Method development :
An analytical method development involves following stages :
Study of properties of compound of interest
Selection of analytical instrument
Selection and optimization of instrumental parameters like chromatographic condition in case of LC, ionization technique in MS etc.
In case of development of bioanalytical method using LC-MS/MS, the following stages are of prime importance.
1.6.2 LC-MS/MS method development :
Various stages involved in method development are :
Stock solution preparation and tuning of analyte
Selection and optimization of Chromatographic conditions
Selection and optimization of Extraction method
Selection and tuning of Internal standard
a) Literature survey :
Literature survey is carried out to gain knowledge and information about the sample and analyte which helps in choice of instrumental conditions, pretreatment, analytical method, specifying the methods to be employed alongwith parameter such as mobile phase, temperature, flow rate, column, internal standard etc.
b) Stock solution preparation and Tuning of analyte :
Prepare the stock solution of analyte and test solution of suitable concentration using appropriate solvent and diluent. The diluted solutions are used to set tuning process.
A syringe infusion pump ensures continuous infusion which is necessary for tuning.
Syringe infusion pumps provides :
Accurate, low flow rate.
Allows continuous sample introduction for tuning and optimization.
Figure 1.13: Continuous infusion of target compound by syringe infusion pump
Select an ion source (ESI or APCI) and ion mode (positive or negative) based on chemical properties of the compound.
Infuse suitable stock dilution in Parent ion Scanning Mode. Select the m/z of the parent ion based on the molecular weight of the compound. (If molecular weight of the compound is 200 then m/z of the parent ion in positive mode is 201 and 199 in negative mode). Then for fragmentation of parent ion allow the parent ion in Product Ion Mode and check for m/z of various daughter ions obtained. Select the prominent and suitable daughter ions by altering the various tuning parameter as indicated below,
` Compound dependent parameters :
Declustering Potential (DP)
Focusing Potential (FP)
Entrance Potential (EP)
Collision Energy (CE)
Cell entrance potential(CEP)
Cell Exit Potential (CXP)
Source dependent parameters :
Ion spray voltage
Then using solution of suitable concentration, MS is obtained with m/z of selected parent and daughter ions in multiple reactions monitoring (MRM) mode. Then MRM conditions are optimized by altering above parameters using solution of various concentrations for quantification.
c) Selection and optimization of Chromatographic conditions :
Choice of columns depends on the properties and chemical nature of the analyte such as,
Column of different length ranging from 50mm to 250mm with internal diameter ranging from 3.5 to 4.6 are used. The column best suited for the analyte is selected based on retention time, peak shape and response.
Optimize the mobile phase composition by altering the following :
Solvent, Solvent proportion
Ionic samples (acidic or basic) can be separated only, if they are present in undissociated form. Dissociation of ionic samples is suppressed by selection of proper pH.
Linearity of the response is check by analysing a set of serially diluted calibration curve samples at 6-8 points. The linearity is accepted only if co-efficient of regression is ï‚³ 0.9800.
d) Selection and optimization of Extraction method :
Process blank matrix samples along with spiked middle point of calibration curve range by following extraction techniques :
Analyze the processed samples using optimized chromatographic conditions and compare the sample processing techniques for the interference at analyte retention time, peak shape of spiked sample and recovery of spiked sample. If the chromatography and/or response are poor re-optimize the chromatographic conditions and processing method. Choose a column and sample processing techniques which gives best possible retention time, peak shape, response, maximum recovery and no significant interference.
Process and inject blank matrix sample to check for any late eluting interference. Optimize the run time to avoid presence of late eluting interference in consecutive injections. Check the linearity of spiked sample using selected chromatographic conditions and processing method. Calibration curve should be linear for a required regression coefficient (r) ï‚³ 0.9800.
e) Selection and tuning of internal standard :
Select the I.S. based on following criteria
Detectable under chromatographic conditions and getting extracted in extraction procedure of the main compound.
No significant interference at the retention time of internal standard in the processed blank matrix sample (blank sample processed by selected extraction procedure).
Check the linearity of spiked sample using selected internal standard. Select the Quality Control (QC) concentration and spiked six sets of QC sample of each concentration in blank matrix.
Performance of selected method is checked by running three or more precision & accuracy batches and evaluating the results for meeting acceptance criteria.
Finally selected method is validated to see whether it does what it was intended to do. Then the validated method is applied for quantitation of drug.
1.6.3 Bio-analytical method validation 18 :
Method validation can be defined (as per ICH) "Establishing documented evidences, which provides a high degree of assurance that a specific method or activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics".
Method validation is an integral part of method development; it is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, quality, purity and potency of the drug substances and drug products. Simply method validation is the process of proving that an analytical method is acceptable for its intended purpose.
Selective and sensitive analytical methods for the quantitative evaluation of drug and their metabolites (analytes) are critical for the successful conduct of preclinical, biopharmaceutical and pharmacological studies.
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 and urine is reliable and reproducible for the intended use.
The process by which a specific bioanalytical method is developed, validated, and used in routine sample analysis can be divided into :
1) Reference standard preparation,
2) Bioanalytical method development and establishment of assay procedure,
3) Application of validated bioanalytical method to routine drug analysis and
acceptance criteria for the analytical run and/or batch.
126.96.36.199 Types of method validation 19 :
a) Full Validations :
Full validation is important when developing and implementing a bioanalytical method for the first time and is also important for a new drug entity.
A Full validation of the revised assay is important if metabolites are added to an existing assay for quantification.
b) Partial Validations :
Partial validations are modifications of already validated bioanalytical methods. Partial validation can range from as little as one intra-assay accuracy and precision determination to a nearly full validation.
Typical bioanalytical method changes that fall into this category include, but are not limited to :
Bioanalytical method transfers between laboratories or analysts
Change in analytical methodology (e.g., change in detection systems)
Change in anticoagulant in harvesting biological fluid
Change in matrix within species (e.g., human plasma to human urine)
Change in sample processing procedures
Change in species within matrix (e.g., rat plasma to mouse plasma)
Change in relevant concentration range
Limited sample volume (e.g., pediatric study)
Selectivity demonstration of an analyte in the presence of concomitant medications or of specific metabolites
c) Cross validation :
Cross-validation is a comparison of validation parameters when two or more bioanalytical methods are used to generate data within the same study or across different studies. An example of cross validation would be a situation where an original validated bioanalytical method serves as the reference and the revised bioanalytical method is the comparator.
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 at each site or laboratory to establish inter laboratory reliability. Cross-validation should also be considered when data generated using different analytical techniques (e.g., LC-MS-MS vs.ELISA4) in different studies are included in a regulatory submission. The bioanalytical method for human BA, BE, PK, and drug interaction studies must meet the criteria in 21 CFR 320.29.
The analytical laboratory should have a written set of standard operating procedures (SOPs) to ensure a complete system of quality control and assurance. The SOPs should cover all aspects of analysis from the time the sample is collected and reaches the laboratory until the results of the analysis are reported. The SOPs also should include record keeping, security and chain of sample custody, sample preparation, and analytical tools such as methods, reagents, equipment, instrumentation, and procedures for quality control and verification of results.
Each method developed is validated for the following fundamental parameters:
Accuracy & Precision
Intra-day or within batch
Inter-day or between batch
Standard Stock Solution Stability
Bench Top Stability in Human plasma
Short term Stability at -20Â°C
Long-term stability at -20Â°C
Selectivity is the ability of an analytical method to differentiate and quantify the target analyte in the presence of other components in the sample. It is also defined as the lack of significant interfering peaks at the retention time of analyte and internal standard.
Selectivity is checked by injecting extracted blank biological matrix (plasma, serum, urine, etc.) and comparing any interference at the retention time of analyte peak by proposed extraction procedure and chromatographic conditions. Blank matrix lots are compared with LLOQ (Lower limit of quantification) samples processed with internal standard.
A method is said to be sensitive if small changes in concentration cause large changes in the response function. Sensitivity of an analytical method is determined from the slope of calibration curve. The sensitivity required for a specific response depends on the concentration to be measured in the biological specimens generated in the specific study.
Linearity (Calibration curve) :
Calibration (standard) curve is the relationship between instrument response and known concentrations of the analyte. A calibration curve should be generated for each analyte in the sample and also for analytical tools such as methods, reagents, equipments, instrumentation and procedures for quality control and verification of results.
A calibration curve should be prepared in the same biological matrix as the samples in the intended study by spiking the matrix with known concentrations of the analyte. The number of standards used in constructing a calibration curve will be a function of the anticipated range of analytical values and the nature of the analyte/response relationship. A calibration curve should consist of a blank sample (matrix sample processed without internal standard), a zero sample (matrix sample processed with internal standard), and six to eight non-zero samples covering the expected range, including LLOQ.
The obtained values of slope "m" and intercept "c" are used in the linear regression equation:
y = mx + c
To calculate the concentration of the quality controls (x) by interpolating the peak area ratios (y), from the corresponding standard curve.
As an appropriate weighing model, the standard curves were calculated with 1/x2 weighing factor.
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 should be measured using a minimum of five determinations per concentration.
(%) Nominal =
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.
(%) CV =
The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added and extracted from the biological matrix, compared to 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 unextracted standards that represent 100% recovery. It may be desirable to intentionally sacrifice high recovery in order to achieve better selectivity with some sample extraction procedure. Solvents such as ethyl acetate normally give rise to high recovery of analyte; however these solvents simultaneously extract many interfering compounds.
% Recovery =
Mean Response of Extracted Standards
Mean Response of Aqueous Standards
Matrix effect is studied by comparing the response of extracted samples spiked before extraction with the response of the extracted blank matrix to which analyte has been added at the same nominal concentration just before injection. Matrix effect is evaluated for six lots of plasma which were processed and then spiked post extraction in duplicate as per extraction procedure at LQC, MQC, and HQC range along with internal standard.
Drug stability in a biological fluid is a function of the storage conditions, the chemical properties of the drug, the matrix, and the container system. Stability of analyte in biological samples is sometimes critical due to degradation of analyte in storage period. Therefore it is important to verify that there is no sample degradation between the time of collection of the sample and their analysis that would compromise the result of the study. Stability evaluation is done to show that the concentration of analyte at the time of analysis corresponds to the concentration of the analyte at the time of sampling.
1.7 Specific Recommendations for Bioanalytical Method Validation as per US-FDA guidelines9 :
Table 1.1: Specific Recommendations for Bioanalytical Method Validation as per US-FDA guidelines
Bioanalytical Method Validation
Matrix-based standard curve(Calibration curve)
-Should consist of a minimum of six standard points, excluding blanks, using single or replicate samples.
-Out of eight-point calibration curve a minimum of 6 standards should meet the acceptance criteria.
LLOQ (Lower limit of quantification)
-Should serve as the lowest concentration on the standard curve.
Accuracy (% Nominal)
-Should be determined using a minimum of five determinations per concentration level (excluding blank samples).
-The mean value should be within 15% of the theoretical value, except at LLOQ, where it should not deviate by more than 20%.
-Should be determined using a minimum of five determinations per concentration level (excluding blank samples).
- The precision around the mean value should not exceed
15%, except for LLOQ, it should not exceed 20%.
-Stability of the analyte in biological matrix at intended storage temperatures should be established.
1) Freeze-thaw cycles
-A minimum of three cycles at two concentrations in triplicate should be studied.
2) Stability of the analyte in matrix at ambient temperature
-Should be evaluated over a time period equal to the typical sample preparation, sample handling, and analytical run times.
-Should be evaluated to determine if an analytical run could be reanalyzed in the case of instrument failure.
-Should be established using a minimum of six independent sources of the same matrix. For hyphenated mass spectrometry-based methods, however, testing six independent matrices for interference may not be important.
In LC-MS and LC-MS/MS based procedure
-Matrix effects should be investigated to ensure that precision, selectivity, and sensitivity will not be compromised.