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About Authors:
Satya Lakshmi.B*, P.Raju vel, Dr. P. Venkateswara Rao, Sindhu.G, Nikil Kumar.K
A.M Reddy Memorial College of Pharmacy,
Narasaraopet, AN University, Guntur.

The reliability of quantitative assays in determination of drugs in biological fluids using High-performance liquid chromatography with Tandem Mass spectrometric determination (LC-MS/MS) detection methods and the integrity of resulting Pharmacokinetic data may not be absolute, in contrary to common perceptions and possible conjectures. The results may be adversely affected by lack of specificity and selectivity due to ion suppression caused by the sample matrix and interferences from metabolites. The advancements in the past few years and new technologies introduced can be used in enhancing LC-MS/MS Bio-analytical method development by reducing matrix effects. This Article reviews Automated Sample preparation and various extraction techniques like liquid-liquid extraction, Solid phase extraction and protein precipitation which plays an important role in sample preparation and detection by LC-MS/MS. Potential drawbacks during method development and validation are pointed out.

Reference Id: PHARMATUTOR-ART-1975

Method Development, Validation, Transfer is to understand Pharmacokinetics of drug and / or its metabolites in biological matrices. Bioanalytical methods employed for the quantitative determination of drugs and their metabolites in biological matrix (plasma, urine, saliva, serum etc) play a significant role in evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data. The rapid growth in the use of LC-MS/MS in recent years due to its advantages of high Sensitivity, Extreme Selectivity and increased rate of analysis.The other advantages of LCMS/MS include low detection limits, the ability to generate structural information, the requirement of minimal sample treatment and the possibility to cover a wide range of analytes differing in their polarities. 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. The principle of MS is the production of ions from analyzed compounds that are separated or filtered on the basis of their mass-to charge ratio(m/z). Most of applications for quantitative bioanalysis use tandem mass spectrometers (MS/MS) that employs two mass analyzers – one for the precursor ion in the first quadrupole and the other for the product ion in the third quadrupole after the collision -activated dissociation of the precursor ion in a collision cell. The effective interface connection between LC  (operated under atmospheric pressure) and MS (operated under a high- vaccum environment) have made LC congenial with MS. Electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI), collectively called atmospheric pressure ionization (API), have matured into reliable interface necessary for quantitative LC-MS/MS bioanalysis. More recently, atmospheric pressure photo-ionization (APPI) also became an interesting alternative ionization source for quantitative LC-MS/MS. Successful use of LC-MS/MS requires understanding the mechanism of various sample extraction processes and the underlying principles of both chromatography and MS.

This article reviews Method development, various extraction techniques of sample preparations, Validation and Method transfer of robust LC-MS/MS with emphasis on important factors impacting the incurred sample analysis. We focused on the mature and established technologies used in most quantitative bioanalytical laboratories. The fundamental parameters for this validation include selectivity, Accuracy, Precision, Linearity and Range, Limit of detection, Limit of quantification, Recovery, Robustness and Stability.

Analytical method development is the process of creating a procedure to enable a compound of interest to be identified and quantified in a matrix. A compound can often be measured by several methods and the choice of analytical method involves many considerations, such as: chemical properties of the analyte, concentrations levels, sample matrix, cost of the analysis, speed of the analysis, quantitative or qualitative measurement, precision required and necessary equipment. The analytical chain describes the process of method development and includes sampling, sample preparation, separation, detection and evaluation of the results.

Traditional sequential method development requires sequential optimization of mass   spectrometric and chromatographic conditions, sample extraction, recovery of the analyte, and lack of interference and matrix effects for each method. This type of method development is timeconsuming, labor and instrument intensive and costly when several different LC-MS/MS methods for various types of analytes need to be developed. This approach has been identified as the major bottleneck for meeting the everincreasing needs for LC-MS/MS methods. In a Reearch paper ,”A concept of simultaneous development of multiple bioanalytical LCMS/MS methods” was presented. Optimal conditions of mass spectrometry, chromatography, and extraction were screened and developed for six structurally different analytes. Experimental designs for simultaneously determining and evaluating recovery, matrix effects, and chromatographic interference were proposed. In another presentation, processes were optimized so that a robust LCMS/ MS method was developed in a single working day .Two scientists work   simultaneously on the same project in a coordinated way: one focusing on sample preparation and other focusing on instrumentation. Important method parameters such as matrix suppression and recovery were investigated. Janiszewski et al. also proposed highthroughput method development approaches by simultaneous testing multiple SPE chemistries using a custom multiple sorbent 96-well plate with optimized extraction conditions for up to five analytes are determined in a single experiment.


The biological media that contain the analyte are usually blood, plasma, urine, serum etc. Blood is usually collected from human subjects by vein puncture with a hypodermic syringe up to 5 to 7 ml (depending on the assay sensitivity and the total number of samples taken for a study being performed). The venous blood is withdrawn into tubes with an anticoagulant, e.g. EDTA, heparin etc. Plasma is obtained by centrifugation at 4000 rpm for 15 min. About 30 to 50% of the original volume is collected Sadagopan et al. systematically investigated the feasibility of using EDTAanticoagulant in plasma to improve the throughput of LCMS/MS assays.

The purpose of sample preparation is to clean up the sample before analysis and/or to concentrate the sample. Material in biological samples that can interfere with analysis, the chromatographic column or the detector includes proteins, salts, endogenous macromolecules, small molecules and metabolic byproducts. A goal with the sample preparation is also to exchange the analyte from the biological matrix into a solvent suitable for injection into the chromatographic system. General procedures for sample preparation like liquid/liquid extraction, solid-phase extraction (SPE) and protein precipitation.

Liquid-Liquid extraction: It is based on the principles of differential solubility and partitioning equilibrium of analyte molecules between aqueous (the original sample) and the organic phases. Liquid-liquid extraction generally involves the extraction of a substance from one liquid phase to another liquid phase. Now-a-days traditional LLE has been replaced with advanced and improved techniques like liquid phase microextraction (LPME), single drop-liquid phase micro extraction (DLPME) and supported membrane extraction (SME).

Solid-phase extraction: 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. Due to many different choices of sorbents, SPE is a very powerful technique. SPE consists of four steps; conditioning, sample loading, washing and elution.

Column Solvation: 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.

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

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

Fig 1: General Solid Phase Extraction Procedure.



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