INTRODUCTION:
The LC/MS data may be used to provide information about the molecular weight, structure, identity and quantity of particular sample components. The study determine of the selectivity, specificity, LOD , LOQ, linearity, range, accuracy, precision, recovery, stability, ruggedness, and robustness of liquid chromatographic studies1,2.
Drug Discovery & Development
The goal of the drug discovery stage is to generate a novel lead candidate with suitable pharmaceutical properties (i.e., efficacy, bioavailability, toxicity) for preclinical evaluation.
Potential lead compounds contained in natural product sources or from the extensive database of a synthetic amalgam library are screened for activity. Lead compounds identify field from screening efforts are optimized in closed collaboration with exploratory metabolism programs and drug safety evaluations. In 1997, it was estimated that the synthesis and screening of ca. 100,000 compounds is typically required for the discovery a single quality lead compound.
The process of identifying a lead compound can take up to 2-4 years. Optimization of the resulting lead may take an additional 1-2 years. The drug discovery stage involve three primary analysis activities: target identification; lead identification; and lead optimization 3-9.
LC/MS applications and advances for discovery chemistry accessing compound identity and purity
The medicinal chemist’s objective is to develop drug-like molecules with high affinity/activity to test in the clinic. This is met by building a broad understanding of the structure–activity and structure–property relationships (SAR, SPR) through an iterative process of molecular design, synthesis and hypothesis testing (activity or molecular properties). The goal of the analytical scientist is to implement LC/MS systems that yield maximum capability for laboratories challenged by throughput requirements. LC/MS open access service (analysis carried out by chemists with minimum training on sample submission) has become the standard technique to monitor the progress of synthetic reactions in real time and/or verify the identity and purity of compounds from structure–activity relationship studies. Maximizing purification through put purification of compounds typically takes a medicinal chemist from 25 to 50% of their laboratory time.
Many pharmaceutical companies have developed purification laboratories to build a technology platform that integrates processes, technologies, methodologies and data management. Centralized purification laboratories with experimental knowledge in analytical/medicinal chemistry and a high level of automation are becoming a powerful line of attack to sustain small-scale and large-scale achiral/chiral purification at different levels of throughput. The leading technology involved in this scenario is LC with UV and/or MS-guided collection, which can deliver thousands of purified samples/week
Drug Discovery Environment10
In order to be useful, information from pharmaceutical profiling must match the needs and preferences of discovery scientists. Drug discovery research is an increasingly complicated and challenging field. Tried and true strategies are always the primary focus and new strategies must prove themselves in order to be accepted. Four elements of discovery research that are critical to consider in developing a pharmaceutical profiling program are:
Primary focus on high affinity ligands. The need for information to guide decisions. The need for speed. Diversity and numbers. The use of high-performance liquid chromatography joined with mass spectrometry (HPLC–MS) or tandem mass spectrometry (HPLC–MS–MS) has proven to be the analytical technique of choice for most assays used in various stages of new drug discovery . New drug discovery can be defined as the process whereby compound libraries are screened, then hits are selected and modified to become leads that are optimized until a compound emerges that can be developed into a drug candidate. HPLC–MS and HPLC–MS–MS are used for the analysis of newly synthesized compounds that become part of a compound library. These assays check that the correct compound has been synthesized and that the purity is sufficient to be used in the library. In a second stage, various physical and chemical properties (e.g. physiological solubility, permeability and chemical stability) of these new chemical entities (NCEs) are assessed and HPLC–MS is often used for these assays. Furthermore, there are also a series of drug metabolism and pharmacokinetics (DMPK) tests that are performed as part of new drug discovery; these tests measure the absorption, distribution, metabolism and excretion (ADME) properties of the NCE, as well as the pharmacokinetic (PK) parameters of the molecule. Most of these assays rely on HPLC–MS or HPLC–MS–MS for the measurement step. This review will provide an overview of the various ways in which LC–MS (which will be used as a term that includes both HPLC–MS and HPLC–MS–MS) can be used in the new drug discovery process. The review will also provide an introduction into the various types of mass spectrometers that can be selected for the multiple tasks that can be performed using LC–MS as the analytical.
Fig 2: The elements of an LC–MS system. (a) Auto sampler (loads the samples onto the HPLC); (b) HPLC; (c) ionization source (interface for LC to MS); (d) Mass spectrometer (various types, see Figure 3).
Principles of LC–MS (11-13)
As shown in Figure2, the elements of an LC–MS system include the auto sampler, the HPLC system, the ionization source (which interfaces the LC to the MS) and the mass spectrometer. Ideally, these elements are all under the control of a single computer system. HPLC is a common technique, so it will not be described here. It should be noted that to interface HPLC with MS, there are some restrictions on the flow rate and mobile phases that can be used. Typical reversed phase HPLC systems connected to MS would use some combination of water and either methanol or acetonitrile as the mobile phase. There are limitations on the mobile phase modifiers; for example, in most cases the modifiers have to be volatile. Mobile phase modifiers are chemicals added to the mobile phase that are used primarily to improve the chromatography of the analytes of interest. Typical mobile phase modifiers would include ammonium acetate, acetic acid and formic acid. There are multiple articles that focus on the HPLC parameters that are important in LC–MS assays. There are various types of ionization sources that can be used as the interface between the HPLC eluant and the mass spectrometer. The two most general sources are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI); both of these source types are now standard equipment on mass spectrometers that are used for LC–MS applications.
Fig 3 Types of mass spectrometers that can be used in LC–MS systems
INTRODUCTION TO BIOANALYTICAL METHOD DEVELOPMENT
Quantitative determination of drugs and their metabolites in biological fluids is crucial between drug discovery and development. LC-MS is the preferred methodology for that purpose.
The LC-MS method development is carried out by the following steps:
1. Considering the physicochemical properties of the analyte, such as chemical structure, functional group (s), molecular weight, purity, solubility, stability
2. Determination of the solubility in the required solutions
3. MS or MS/MS scanning and optimization, e.g. Electron Spray Ionization (ESI) or Atmospheric Pressure Chemical Ionization (APCI)
4. Development and optimization of the LC method
a. Selection of the best chromatography method (RP- (reversed phase) or NP- (normal phase) chromatography), selecting of a suitable column, temperature.
b. Mobile phase selection and optimization (choosing best buffers, pH, flow rate)
c. Selection of the best internal standard, which possesses similar ionization response and similar chromatographic retention time as the requested substance
5. Development of sample preparation method - Selection of extraction method and Optimization: The most critical step in the development of LC-MS methods is the sample Preparation to obtain homogenous solutions suitable for injection onto column, as well as Low ion suppression for reliable MS detection. The approaches of sample preparation in bio analytical processes are: Solid-phase extraction (SPE), liquid-liquid extraction (LLE), Protein precipitation techniques (PPT), filtration etc.
Optimization of LC-MS method
During the optimization stage, the initial sets of conditions that have evolved from the first stages of development are improved or maximized in terms of resolution and peak shape, plate counts asymmetry, capacity factor, elution time, detection limits, limit of quantification and overall ability to quantify the specific analyte of interest. This type of method development is time consuming, labour and instrument intensive and costly when several different LC-MS/MS methods for various types of analytes requirement to be development. A concept of simultaneous developmental 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 evaluation 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.
The various parameters to be optimized during method development are:
I) Selection of Mode of Separation
Modes of acquiring and visualizing LC/MS data: 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. A single mass scan can take anywhere from 10 most 1 s depending on the type of scan. Umpteen scans are acquired during an LC/MS analysis.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 point against time.
The most general modes of acquiring LC/MS data are:
(1) Full scanning acquisition resulting in the typical total ion current plot (TIC)
(2) Selected Ion Monitoring (SIM)
(3) Selected Reaction Monitoring (SRM) or multiple reactions monitoring (MRM).
The nature of the analyte is the primary factor in the selection of the mode of separation. For the separation of polar or moderate polar compounds, the most preferred mode is reverse phase. In reverse phase mode, the mobile phase is comparison more polar than the stationary phase. The capability to rapidly switch between ionization modes is expected to increase system productivity and flexibility. Detection was performing on electrospray ionization (ESI), triple quadrupole mass spectrometer equipped with an ESI interface operate in positive and negative ionization mode. The selective reaction monitoring mode (SRM/MRM) is used to provide MS/MS detection. It generally improves detestability and reduces ion suppression effect. Both acetonitrile and methanol was test as organic mobile phases for the LC separation. In order to obtain sufficient retention for acidic drugs and reproducible retention times the use of a buffer in the eluent or acidification of the mobile phase was recommended, although it caused the reduction of the signal intensities due to suppressing effects in the MS interface.LC separation of blood–lipid regulators and _blockers in extract obtained from environmental waters has been carried out mainly using C18 column and the mobile phase consisting of water and methanol or acetonitrile as organic mobile phase at different pH. used a mixture of acetonitrile and methanol as organic mobile phase to lead to short retention times and better resolution of the analytes. The use of ammonium acetate as additive in the mobile phase is general in the reported methods to improved ESI performance in the NI mode. Acids (e.g. acetic acid, formic acid), TrBA and methyl ammonium acetate have been also used to improve the sensitivity of MS detection. The selection of ion source is based on the chemical properties of the compound. If the drug is acid, then negative mode is selected. If the drug is a basic compound, then positive ion mode is selected. Best suited is negative ion mode due to acidic nature of the compound.
II) Selection of column
The heart of the chromatographic system is the column. The high capability into separation, of column material (micro-particles, 5-10μm size) packed in such a way that highest number of theoretical plates is possible. Silica (SiO2, H2O) is the most widely used compound for the manufacture of packing materials. It consists of a network of siloxane linkages (Si-O-Si) in an erect three dimensional structure containing inter connecting pores. Thus a wide range of trade products is available with surface areas ranging from 100to 800 m2/g. and particle sizes from 3 to 50μm. The sialon groups on the surface of silica give it a polar character, which is exploited in adsorption chromatography using non-polar organic eluents. The attachment of hydrocarbon chains to silica produces a non-polar surface suitable for reversed phase chromatography where mixtures of water and organic solvents are used as eluents. The most popular material is octadecyl-silica (ODS-Silica), which contains C18, chains, but materials with C4 and C8 chains are also available.
In LC, generally two types of columns are used, normal phase columns and reversed phase columns. Using normal phase chromatography, particularly of nonpolar and moderate polar drugs can make excellent separation. Reversed phase chromatography is carry out using a polar mobile phase such as methanol, Acetonitrile, water, buffers etc., over a nonpolar stationary phase. Ranges of stationary phases (C18, C8, -NH2, -CN, -phenyl etc.) are accessible and very selective separations can be achieved.
III) Selection of mobile phase
In LC, selectivity is generally altered by changing mobile phase composition or stationary phase functionality. The most successfully and conveniently option in the fast, particularly for analysis of ion compounds, has been to optimization the mobile phase composition by changing solvent type and strength, buffer type and concentration, and pH.
Solvents
The most common solvents used in LC/ESI-MS are water, Isopropanol, Methanol, Acetonitrile admixtures of the above. The solvent composition (aqueous-to-organic ratio) is particularly important in ionization process. The effectiveness of the spraying process depends on and surface tension of the liquid being vaporized. If more aqueous solvent issued, it is difficult to produce stable spray because conductivity and/or the surface area are too high, vaporization of droplet formed by the action of high voltage and nebulizing gas is also difficult.
Buffers and Additives
Volatile buffers are required for faster evaporation.
1. To act as a buffer for the chromatographic process in the traditional way, i.e. control and maintain the pH of the mobile phase in order to keep the ionization state of analyte constant.
2. To adjust the pH of the carrier solvent (mobile phase) in such a way as to present the anal ytes to the MS already in ionic form. The most compatible buffer mobile phase are ammonium format, ammonium acetate, and ammonium hydroxide and format at concentrations of 10 to 20 mM in ESI and up to 50 mM in APCI, Ammonium adducts can be frequently seen in positive ion mode and format or acetate ions adduct in negative ion mode. Basic compound will usually show enhanced signal by low pH of mobile phase in LC/MS/MS. Other additives occasionally used include Trifluoroacetic acid (TFA), Triethylamine (TEA) Formic acid and Diethyl amine (DEA) but these need to be used at low concentrations (< 0.1% v/v) since they may cause ionization suppression. Non-volatile buffers such as Phosphates, citrates, borate buffers and ion pairing agents, inorganic acids are generally avoided (may block the orifice and get deposited in ion source, analysers and detectors) in LC-MS method development.
Instrumentation
Mass Spectrometer contains the following components two type: -
Ø Ion source/Ionization sources
Ø Mass analysers
Ion source / Ionization source
The sample is introduced to the ionisation source either by manual direct infusion (DI) or via A variety of ionization techniques are used for mass spectrometry. Most ionization techniques excite the neutral analyte molecule which then excretes an electron to form a radical cation. Other ionization techniques implicate ion molecule reactions that produce adduct ions. The most important thought is physical state of the analyte and the ionization energy. The instruments atmospheric pressure ionization (API) technique are used to solvent elimination and ionization steps are combining in the source and take place at atmospheric pressure. When electron impact ionization (EI) is the choice, the solvent elimination and ionization steps are different. The interface is a particle beam type, which different the sample from the solvent, and allows the introduction of the sample in the form of dry particles into the high lacuna region. Electron impact is of interest for molecules which do not ionize with API technique, or when an electron aftermath spectrum is mandatory, since it gives spectra information of independent on a sample introduction technique (GC or LC, or direct introduction) and instrument provide.
Electron ionization (EI) and chemical ionization (CI) are only due to for gas phase ionization. Fast atom bombardment (FAB), secondary ion mass spectrometry, electro spray (ESI), and matrix aid laser desorption (MALDI) are used to ionize condensed phase samples. The ionization energy is important because it controls the amount of fragmentation observed in the mass spectrum. Although this fragmentation pattern of complicates the mass spectrum, it provides structural information for the identification of unknown compounds. Some ionization techniques are very soft and only producer molecular ions; other techniques are very energetic and cause ions to undergo extensive fragmentation. Although this fragmentation complicated the mass spectrum, it provides structural information for the identification of unknown compounds.
Application
API-electrospray (API-ES) is useful in analyzing samples that become multiply charged such as proteins, peptides, and oligonucleotides, as well as in analyzing samples that are individually charged, such as benzodiazepines and sulphated conjugates. API-ES can be used to measure molecular weights of most polymers, peptides, proteins, and oligonucleotides up to150, 000 Daltons fast and with high mass accuracy. In biopharmaceutical applications, chemists use API-ES to speed protein characterization, to accurately identify and characterize post-translational modifications, and to quick confirm the molecular weight of synthetic peptides.
Process
API-ES is a process of ionization followed by evaporation. It occurs in three basic
Nebulization
The HPLC effluent is pump through a nebulising needle which is at ground potential. The jet goes through a semi-cylindrical electrode which is at a high potential. The potential difference between the needle and the electrode produces strong electrical fields. This field charges the surface of the liquid and forms a spray of charged droplets. There is a concentration flow of gas which assists in the nebulisation process.
Desolation
The charged droplets are attracted toward the capillary sampling leak. There is a Counter flow of heated nitrogen drying gas which shrinks the droplets and carries away the uncharged material.
Ionization
As the droplets shrink, they approach a point where the electrostatic (columbic) forces exceed the cohesive forces. This process continues until the analytes ions are ultimately desorbed into the gas phase. These gas-phase ions pass through the capillary sampling orifice into the low-pressure region of the ion source and the mass analyser.
Figure: 4 API-electrospray ionization
Atmospheric Pressure Chemical Ionization
Application
Atmospheric pressure chemical ionization (APCI) is an ionization technique that is related
To a wide range of polar and nonpolar analytes that have moderate molecular weights.
Process
APCI, a process of evaporation followed by ionization, is complementary to API-ES. Nebulisation and Desolation. APCI nebulisation is similar to that in API-ES. However, APCI nebulisation occurs in a hot (Typically 250°C–400°C) vaporizer chamber. The heated rapidly evaporates the spray droplets, resulting in gas-phase HPLC solvent and analyte molecules.
Ionization
The gas-phase solvent molecules are ionized by the ejected from a corona need. In APCI there is charge transference from the ionized solvent reagent ions to the analyte molecules in a way that is similar to chemical ionization in GC/MS. These analyte ions then are transported through the ion optics to the filter and detector.
Fig 5: Atmospheric pressure chemical ionisation
B) MASS ANALYSERS
The m/z analyzer (mass analyzer) is used to separate the ions according to their m/z ratio based on their characteristic behaviour in electric and/or magnetic fields. With the advent of ionization sources that can vaporize and ionize bio molecules, it has become necessary to improve mass analyzer performance with respect to speed, accuracy, and resolution. In the most common terms, a mass analyzer measures gas phase ions with respect to their m/z, where the charge is produced by the addition or loss of a proton(s), cation(s), anion(s) or electron(s). The addition of charge allows the molecule to be affected by electric field thus allowing its mass measurements. This is important facet to recall about mass analyzers that they measure the m/z ratio, not the mass itself. The performance of a mass analyzers can be typical be defined by the following characteristics such as accuracy, resolution, mass range, tandem analysis capabilities, and scan speed.
Table no: -2
|
Sr. No |
Types of Analysers |
Basis of Separation |
|
1 |
Electric sector |
Kinetic energy |
|
2 |
Magnetic sector |
Momentum |
|
3 |
Quadrople/Ion trap |
m/z |
|
4 |
Time of flight |
Flight time |
|
5 |
FT-ion cyclotron resonance |
m/z (resonance activities) |
Quadrople/ Ion trap
The Quadrupole ion storage trap mass spectrometer (QUISTOR) is a recently developed mass analyzer with some particular capabilities which are very sensitive, relatively inexpensive, and scan speed enough for GC/MS experiments. As the name indicates, quadrupole mass analyzers consist of four parallel rods just as indicated in a quadrupole mass analyzer, direct current (DC) and alternate current (AC) voltages are applied to the rods in such a way that two opposite rods have the same voltage, while the perpendicular ones have a voltage with opposite sign (+ and –, respectively). To be able to interact with this vibrating electromagnetic field in between the rods, the ions should enter the quadrupolar field with low velocity (e.g., with a few eV kinetic energy). Accordingly, no high voltage (HV) is necessary to accelerate the ions before the mass analysis. This is especially useful when ESI or APCI is used as there is relatively high pressure in the source region (which may result in HV discharge). Two opposite rods have positive potential excluding for a short period of time when the negative RF voltage exceeds the positive DC voltage (Fig. 6). Because of this small period of negative potential, only the lighter positively charged ions will be defocused by these rods. This also means that these rods will focus only relatively large positively charged ions so that they can be considered as high mass filters. With similar consideration, the other two rods will defocus the positively charged ions most of the time and will focus only the lighter (positively charged) ions during a small period while the voltage is positive on these rods (Fig. 6). Thus, the other pair of rods works as low-mass filter. By applying the AC and DC voltages to all of the rods a band filter is created, i.e., a filter that permitted an ion with a given m/z to move through the rods and, subsequently, to reach the detector. (Comment that in this case the ions are ejected axially from the quadrupolar field.) By changing the absolute values of the AC and DC voltages, but keeping their ratio constant, the mass spectrum can be acquired. Comment that in the so-called “RF only” operation, no DC voltage is applied to the rods. In this case, all ions with higher m/zthan the short masscult-off will pass the quadrupole analyzer. This RF-only operation is fully often used to focus an ion beam consisting of ions with different m/z ratios.
Fig. 6: - Quadrupole ionization
SAMPLE PREPARATION METHODS
Sample preparation is very important step for analysis of drugs and metabolites in bio analytical study. The “unwanted substances” can interfere with small concentrations of the drug during analysis. The main objective of sample preparation is to eliminate all possible undesirable substances without significant loss of analyte of interest. Currently several techniques are available for extraction of analyte from the biological matrices like; solid phase extraction (SPE), liquid-liquid extraction (LLE) and protein precipitation (PP). One strategy for high-through put into bioanalytical analysis is to use well-established instrumentation; rigorous, standardized techniques; and automation, wherever possible, to replace manual tasks. Automation results in greater performance constancy over time and in more reliable methods transfer from site to site. Automated 96-well plate technology is well established and received and has been shown to effectively replace manual tasks.
Protein Precipitation (PP)
Protein precipitation is often used as the initial sample preparation scheme in the analysis of a Drug substance since it does not require any method development. It can be applied to extraction of plasma and blood samples. Principle of PP is based on precipitation (denaturation) of the proteins by using various reagents like acid (trichloroacetic acid and perchloric acid), organic solvent precipitants lowers the dielectric constant of the plasma protein solution and facilitate the precipitation (methanol, acetone and acetonitrile) or by salts (ammonium sulphate, ammonium chloride). The binding of positively charged metal ions reduces protein solubility and facilitate precipitation (zinc sulphate). After denaturation the sample is centrifuged, which results in extraction of analyte in the precipitating solvent. Methanol is commonly preferred solvent amongst the organic solvent as it can produce clear supernatant which is appropriate for direct injection into LC-MS/MS. Salts are other alternative to acid and organic solvent used to be precipitation. This technique is called as salt-induced precipitation. As the salt concentration of a solution is increased, proteins aggregate and precipitate from the solution It is simple, universal, inexpensive popular procedure. This method can be applied for extraction of both hydrophobic and hydrophilic substances. Matrix components are not efficiently removed and may be present in supernatant which may reduce the efficiency of ionisation process, loss in response and this phenomenon is referred as ionization suppression. Protein precipitation may clog the column.
Liquid-Liquid Extraction (LLE)
Liquid-Liquid Extraction, also called solvent extraction, is a technique used to separate analytes from interferences in the sample matrix by partitioning the analytes between two immiscible liquids or phases (aqueous and organic). Extraction of analyte occurs from aqueous phase into organic phase (when analytes are unionized and solubility in that organic solvent). Analytes get extracted into the organic phase (organic solvents eg., hexane, diethyl ether, methyl tert-butyl ether MBTH, ethyl acetate) are easily recovered by evaporation of solvent in presence of nitrogen gas so as to get the dry form of sample and reconstituted (mobile phase) prior to chromatographic analysis, while analytes extracted into the aqueous phase can often be injected directly onto a reverse-phase HPLC column. Now-a-days traditional LLE has been replaced with advanced and improved techniques like liquid phase micro extraction (LPME), single drop-liquid phase micro extraction (DLPME) and supported membrane extraction (SME). Clean extracts can be obtained. Technique is simple, rapid and has relatively less cost per sample. pH control of the sample necessary for extraction. During evaporation, since the temperature is increased. Method cannot be used for thermo labile substances.
Solid Phase Extraction (SPE)
The principle of SPE is based on the partitioning of the analytes between two phases. The analyte of interest must have higher affinity to solid phase than the matrix components. Now- days, SPE employs a small plastic disposable column or cartridge (syringe-barrel format with 0.1 to 0.5 g of sorbent). Sorbent is commonly a reversed phase material (e.g., C18-silica) pack is held in syringe barrel by two fritted disks in the cartridge made up of polypropylene, PTFE and fibre glass based is for SPE disks. Silica-base with chemically bonded functional groups or highly cross-linked polymers such as styrene divinylbenzene and polymethacrylate are generally used materials for preparation of SPE cartridges. These cartridges can be utilized only once so as to avoid any interference and mostly to eliminate carry-over effects. Different types of SPE sorbents used are non-polar, polar and ion exchange sorbents. For reverse- phase SPE, the stationary phase is usually silica bonded with alkyl and/aryl functional groups. C18 and C8 are the most common sorbents used. The packing materials required for SPE and liquid chromatography are comparable; only particle size is more in SPE as compared to that of liquid chromatography. SPE technique is modified by solid phase micro extraction. SPE method development involves generally four main steps includes conditioning the packing, sample application (loading), washing the packing (removal of interferences), recovery of analyte. No need of phase separation (as in LLE), total analyte fraction is easily collected, more recovery of analyte. Low concentration of drug can be detected. Effective in selective removal of interferences, different types of adsorbents can be used, extending the analytical column life, reduced system maintenance, minimizing ion suppression. Extraction is difficult for high-density materials, extraction processes a number of steps are to be carried out making it a time consuming process, variability of SPE Cartridges, irreversible adsorption of some analytes on cartridges.
Method validation(16-28)
Owing to the importance of method validation in the whole field of analytical chemistry, a number of guidance documents on this subject have been issued by various international organizations and conferences. All the documents are important and potentially helpful for any method validation. Besides these official guidance documents, a number of review articles have been published on the topic of analytical method validation. All of these papers are interestedness and further some for any methods validation, while only part of them specifically addresses analysis of drugs, poisons, and/or their metabolites in body fluids or tissues. This includes the excellent review on validation of bio analytical chromatographic methods which includes detailed information discussions of theoretical and practical aspects The other two deal with the implications of bio analytical method validation in clinical and forensic toxicology and with theoretical and practical aspects in method validation using LC–MS (LC/MS). The latter also describes a proposed experimental design for validation experiments, as well as the statistical procedures for calculating validation parameters.
Validation parameters
Analytical methods in clinical and forensic toxicology may be used for screening and identification of drugs, poisons and/or their metabolites in biological fluids or tissues, for their quantification in these matrices, or for both. For quantitative bioanalytical procedures, there is a common agreement, that at least the following validation parameters should be evaluated: selectivity, calibration model (linearity), stability, accuracy (bias), precision (repeatability, intermediate precision) and the lower limit of quantification (LLOQ). Additional parameters which may be relevant include limit of detection (LOD), recovery, reproducibility, and ruggedness (robustness). For qualitative procedures, a general validation guideline is currently not available, but there seems to be agreement that at least selectivity and the LOD should be evaluated and that is additional parameters like precision, recovery and ruggedness (robustness) might also be important. For methods using LC–MS, experiments for assessment of possible matrix impact (ME), i.e. ion suppression or ion enhancement, should always be part of the validation process, particularly if they employ electro spray ionisation (ESI). The guidelines for bio analytical method validation are published by the United States Food and Drug Administration (USFDA) in May 2001. These guidelines are standard for validation parameters’ evaluation and requirement. Bioanalytical method validation is the approaches employed to indicate that the analytical method used to assess an analyte in biological matrix is reliable and also reproducible. There are three types of method validations, including “Full Validation, Partial Validation, and Cross Validation”. This is different types of bioanalytical method validations are defined and characterized as follows: Full Validation, Partial Validation, Cross Validation
Full Validation should be performed to support pharmacokinetic, bioavailability, bioequivalence and drug interaction studies in a new drug application (NDA) or an abbreviated new drug application (ANDA). Full validation is necessary when developing and implementing a bioanalytical method for the first time and mandatory for any new drug entity. Partial validations are usually modifications of validated bioanalytical methods which do not essentially require complete revalidations. In partial validation either one intra-assay of precision and accuracy is carried out or “approximately” full validation is done. Partial validation can also be carried out when there is alteration in species within matrix (e.g. rat plasma to mouse plasma), changes in matrix within a species (e.g., human plasma to human urine), change in analytical methodology (e.g., change in detection systems), change in sample processing procedure(s), change in anticoagulant in harvesting biological fluid. Cross validation is comparison of two bioanalytical methods. Cross validations are essential. when two or more bio analytical methods are applied to generate information within same study. The evaluations should be done by considering an innovative validated bioanalytical method as the reference and the repeated bioanalytical method as the comparator and vice versa. Cross validation with spiked matrix and subject samples should be carrie out at each site or laboratory to establish the inter-laboratory reliability when sample analyses within a single study conducted more than one site laboratory. This should be taken into consideration when data is generated by using various analytical techniques (e.g. LC-MS-MS vs ELISA) in different studies are included in a regulatory submission.
Selectivity
In the Conference Report II, selectivity was defined as ‘‘the ability of the bioanalytical method to measure unequivocally and to differentiate the analyte(s) in the presence of components, which may be expected to be present. Typically, these might include metabolites, impurities, degradant, matrix components. Selectivity is an important component of method validation. “The method must be able to quantify the analyte in the presence of endogenous compounds, degradation products, other medicine likely to be present in study samples, and metabolites of the medicine(s) under study there are several items to be considered when evaluating the selectivity of the assay. Evaluation of a minimum of 6 different lots or origin of matrix must be performed as matrix blanks (containing no analyte or IS). Additional lots beyond the six required lots should bead when needed in order to test each of the expected selectivity scenarios. For nonclinical studies with large animals, six individual plenties are recommended, while studies with small animals (i.e., rat or mouse) may use pooled lots. For clinical assay, lots from individual donors are suggested. Choice of the matrix lots should be based on the expected constitution of study samples. Forexample, if specific limitation is placed on the study samples, then the choice of selectivity lots should reflect this limitation. Some analytes generate dissimilar results in separate genetic populations, different genders, or different age groups. For clinical studies in which there are fasted and fed component, the choice of selectivity lots during validation should include a choice of lots from fasted subjects and fed subjects in order to assess the potential effect of fed/fasted state on the matrix. In addition, haemolytic and lipemic plasma should be evaluated to determine the impact on quantitation. For evaluation of haemolytic and lipemic lots, it is recommending that QC samples be prepared at high and low levels of haemolysis/lipemia and prepared per the analytical method. Acceptance criteria for these samples should imitate that of the assay (relative error ±15%, relative standard deviation ≤15%).
Calibration of standard curve (Linearity and range)
The choice of a suitable calibration model is necessary for reliable quantification. Therefore, the relationship between the concentration of analyte in the sample and the corresponding to response (in bioanalytical methods mostly the area ratio of analyte versus IS) must be investigate. The linearity of the method was determined by using standard plots associated with 8-point standard curve including LLOQ and ULOQ. Concentration of calibration curve standards was calculated against the calibration curve and the linearity of the method was evaluated by protect the acceptance of precision and accuracy of calibration curve standards. Two consecutive calibration curve standards should not be beyond the acceptance criteria. The lower limit of quantification (LLOQ) was the lowest concentration at which the precision expressed by relative standard deviations (RSD, CV %) is better than 20% and the accuracy (bias) expressed by relative difference of the measured and true value was also lower than 20%.
Accuracy (bias) and precision
The purpose of validation is to ensure that the methods developed are sufficiently accurate and precise to quantify the actual concentrations of analyte which will be present in the study samples. The accuracy of an analytical method recounts the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. The QC samples are prepared by spiking analyte into matrix at Low (three times the LLOQ), Middle (around 50%of the logarithmic curve range) and High (at about 75% of the upper limit of quantitation (ULOQ)) concentrations. Dilution QC samples are prepared at 5 to 100 times ULOQ for assessment of ability of the method to accurately quantitative study samples which are initially above the limit of quantitation (ALQ) between sample analysis (this is also known as dilution integrity). QC samples are also prepared at the LLOQ to validate the sensitivity of the assay. Samples with concentrations below the LLOQ are below the limit of quantitation (BLQ). “The QC samples are analyzed against the calibration curve, and the obtained concentrations are contrast with the nominal value”. An accuracy value of not more than 100±15% should be achieve for Low, Middle, and High QC samples, and not more than 100±20% for QC samples at the LLOQ with at least 50% at each level gathering acceptance.
Precision
The precision of an analytical method recounts the closeness of individual measure of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix. Precision should be measure using a minimum of five determinations per concentrations. A minimum of three concentrations in the range of Expected concentrations are recommended. Precision is the degree of reproducibility and is usually reported in terms of %CV or %RSD over the range of quantification for a single experiment. A value of not more than 15% for Low, Middle, and High QC samples and not more than 20% for the QC samples at the LLOQ is acceptable. Accuracy and precision should both be contrast within independent runs (i.e., intra-run), and between at least two different runs (i.e., inter-run). It is highly recommended that at least one of the runs accommodate the same number of samples as will be expected in the longest expect sample run. Tracking the IS response difference is also critical to consistent assay performance. There is an assumption that the IS will correct for variability and recompense for differences between different matrix sources. The minimum IS answer and the maximum IS answer should be monitoring between a run sequence.
‘‘Repeatability conveys the precision under the same operating conditions over a small interval of time. Repeatability is also term intra-assay precision’’. Within-run or within day precision are also frequently used to describe repeatability.
‘‘Intermediate precision convey within-laboratories variations: different days, different analysts, different equipment, etc.’’. In a strict sense intermediate precision is the total precision under varied conditions, whereas so-called interassay, between-run or between-day precision only measure the precision components caused by the respective factors (see below). However, the latter terms not clearly defined and obviously frequently used interchangeably with each other and also with the term intermediate precision.
‘‘Reproducibility convey the precision between laboratories (collaborative studies, usually applied to standardization of methodology)’’. Reproducibility only has to be studied, if a method is supposed to be use in different laboratories. Unfortunately, some authors also use the term reproducibility for within laboratory studies at the level of intermediate precision, which should be avoided to prevent confusion.
Lower limit of quantification (LLOQ)
The LLOQ is defined as the lowest concentration of a sample that can still be quantified with acceptable precision and accuracy (bias). In the Conference Reports, the acceptance criteria for these two parameters at LLOQ are 20% R.S.D. for precision and _20% for bias. In the second and also very common approach, the LLOQ is estimated based on the signal-to-noise ratio (S/N). S/N can be defined as the height of the analyte peak (signal) and the amplitude between the highest and lowest point of the baseline (noise) in a certain area around the analyte peak. For LLOQ, S/N is usually requiringbeing equal to or greater than 10. A third approach to estimate the LLOQ is the concentration that corresponds to a response that is k-times greater than the estimated S.D. of blank samples, where a k-factor of 10 is usually applied. The concentration is obtained by dividing this response by the slope of the calibration curve
Upper limit of quantification (LLOQ)
The ULOQ is the maximum analyte concentrations of a sample that can be qantified with acceptable precision and accuracy (bias). In general, the ULOQ is identcal with the concentration of the highest calibration standard.
Limit of detection (LOD)
Quantification below LLOQ is by definition not acceptable. Therefore, below this value a method can only produce semi quantitative or qualitative data. However, particularly in analytical toxicology, it can be very important to know the LOD of the method, which can be defined as the lowest concentration of analyte in a sample which can be detected but not necessarily quantified as an exact value or as the lowest concentration of an analyte in a sample, that the bioanalytical procedure can reliably differentiate from background noise. The approaches most often applied for estimation of the LOD are basically the same as those described for LLOQ with the exception of the approach using precision and accuracy data, which cannot be used here for obvious reasons. In contrast to the LLOQ determination, for LOD a S/N or k-factor equal to or greater than three is usually chosen. At this point it must be noted that all these approaches only evaluate the pure response of the analytes. In toxicology, however, unambiguous identification of an analyte in a sample requires more complex acceptance criteria to be fulfilled. Such criteria have recently been reviewed by River. Especially in forensic toxicology and doping control, it would certainly be more appropriate to define the LOD as the lowest concentration of analyte in a sample, for which specific identification criteria can still be fulfilled.
Recovery
As already mentioned above, recovery is not among the validation parameters regarded as essential for method validation. Most authors agree, that the value for recovery is not important, as long as the data for LLOQ, (LOD), precision and accuracy (bias) are acceptable. It can be calculated as the percentage of the analyte response after sample workup collate to that of a solution containing the analyte at a concentration corresponding to 100% recovery. Therefore, whole recoveries can usually not be determined if the sample workup includes a derivatization step, as the derivatives are often not available as reference substances. Nevertheless, some guidance documents request the determination of the recovery at high and low concentrations or even specify that the recovery should be greater than 50%. In LC–MS(–MS) analysis, a different experimental design must be used to determine recovery, because part of the change of the response in prepared samples in comparison to respective standard solutions might be attributable to ME. In the validation of LC–MS (–MS), it is therefore more appropriate to perform the recovery experiments together with ion suppression/ enhancement experiments.
Ruggedness
One precision and accuracy batch will be processed and analysed with different analyst and Another precision and accuracy batch will be processed and analysed using different column or with different sets of reagents. It should meet the acceptance criteria of precision and accuracy batch.
Stability29
Evaluation of stability should be carried out to assure that each step taken during sample preparation of sample analysis, as well as the storage conditions used do not affect the concentration of the analyte. The stability of the samples should be assessed under conditions that are as close as possible to those under which the samples are actually stored or analyzed. Careful consideration should be given to the solvent or matrix type, container materials, and storage condition used in the stability-determination process.
a. Freeze and Thaw Stability: During freeze carry out of thaw stability evaluations, the freezing and thaw of stability samples should mimic the intended sample handling condition to be used during the sample analysis. Stability can be assesses for a minimum of three freeze-thaw cycles.
b. Bench-Top Stability: Bench top stability experiments should be designed and conducted to cover the laboratory handling conditions that are expected for study samples.
c. Long-Term Stability: The storage time in a long-term stability evaluation should equal or excitedly the time between the date of first sample collection and the date of last sample analysis.
d. Stock Solution Stability: It should be stability of stock solutions of drug and internal standard should be evaluated. When the stock solution exists in a different between state (solution vs. solid) or in a different buffer composition (generally the case for macromolecules) from the certified reference standard, the stability data on this stock solution should be generally to justify the duration of stock solution storage stability.
e. Processed Sample Stability: The stability of processed samples, including the reticently time in the auto sampler, should be determined.
Applications of LC-MS
1. Molecular Pharmacognosy 30: LCMS determines the contents and categories of different groups of cultured plant cells and select the pair of groups with the biggest different content of ingredient for the study ingredient difference phenotypic cloning.
2. Characterization and Identification of Compounds
Carotenoids31: Because carotenoids are not thermally stable, separation of mixtures and removals of impurities is usually carry out by reversed phase HPLC (particularly HPLC) instead of gas chromatography The mini samples of carotenoids which were isolated from biological matrices such as human serum or tissue stop structural analysis by Nuclear Magnetic Resonance. Hence, only the most sensitive analytical methods are adequate such as Liquid Chromatography / Mass Spectrometry and HPLC with photodiode-array UV / visible absorbance detection. At the minimum level, carotenoid identification may be confirmed by combining data such as HPLC retention times, photodiode-array absorbance spectroscopy, mass spectrometry and tandem mass spectrometry. Up to date, five LC/MS techniques have been used for carotenoid analysis including moving belt, particle beam, continuous flow fast atom bombardment, electrospray and Atmospheric Pressure Chemical Ionization (APCI). Among these LC/MS interfaces, electrospray and APCI are probably the easiest to used and are rapidly becoming the most widely available. These techniques supply comparable sensitivity (at the low p mol level) and produce enormous molecular ions.
1. Proteomics (32, 33)
Liquid Chromatography / Mass Spectrometry (LC/MS) has become a powerful technology in proteomics studies in drug discovery which includes target protein characterization and the discovery of biomarkers.
A. Glycopeptides Characterization
MS-based glyco-proteomic studies are used to characterize the glycopeptides under examination. This involves pinpointing the glycosylation site, the type of glycan involved and the peptide backbone core. In present, with MS-based strategies, tandem MS fragmentation and data analysis problems provide efficient characterization of intact glycopeptides and then analysis of the peptides is done via Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS
b. Peptide Mapping
In earlier days’ protein drugs were made from proteins refined from living organisms. However, they are recently produced using recombinant technology. Insulin, interferon, and erythropoietin are some of the protein drugs made by recombination which are available in the market.
Confirmation of the expression of recombinant proteins is important from the quality control viewpoint. Some of the methods applied for this include analysis of amino acid sequence by peptide sequencer and other simpler methods such as peptide mapping by HPLC or mass mapping by MALDI-TOF MS. For example, Protein analysis and peptide mass mapping of a model sample of horse heart myoglobin is done by LC/MS using a quadrupole mass spectrometer.
3. Automated Immunoassay in Therapeutic Drug Monitoring
Therapeutic drug monitoring (TDM) of certain drugs with a narrow therapeutic index helps in improving patient outcome. The need for accurate, precise, and standardized measurement of drugs poses a major challenge for clinical laboratories and the diagnostics industry. Different techniques had developed in the past to meet these requirements. Nowadays liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based methods and immunoassays seem to be the most widespread approaches in clinical laboratories. Mass spectrometry–based assays can be analytically sensitive, specific and capable of measuring several compounds in a single process. This is a cost-effective approach in monitoring patients receiving multidrug therapy (e.g., antibacterial therapy for Tuberculosis patients). The selectivity supply by successive mass filtrations is an added advantage of tandem mass spectrometry over immunoassays, as is shown for immunosuppressant drugs.
4. Two Dimensional (2-D) Hyphenated Technology (34,35)
The use of LCMS has become a powerful two dimensional (2D) hyphenated technology for the use in a wide variety of analytical and bio analytical techniques for the analysis of proteins, amino acids, nucleic acids, amino acids, carbohydrates, lipids, peptides, etc and/or in the main classification in the field of genomics, lipid omics, metabolomics, proteomics, etc. LCMS was preferred originally and it can be intensified by the need of more powerfully analytical and bio analytical techniques that can absolutely distinguish the target analytes with high complexity mixtures in a sensitive and particular way. The combination of this hybrid class of HPLC and MS to perform both routine qualitative discovery and quantitative directed analysis of complex mixtures is conceivably one of the most significant combinations in developments and separations, where mass spectrometry plays a major role in the field of science by detecting various analytical & bio analytical techniques in the past decade. It gives an increased level of robustness and accuracy out of their LC systems and improved detection abilities when coupled with a MS system.
CONCLUSION:
In this LC-MS technique it provides the analysis of the various chemical constituents of new chemical moieties, impurities analysis in the various formulations. In this technique it gives the results for the elucidation of the molecules. This review provides the detailed information about the LC-MS technique.
REFERENCES:
1. Saibaba SV, Kumar MS, Pandiyan PS. Mini Review on LC/MS Techniques.
2. Verma PK, Kamboj VK, Ranjan S. Spectrophotometric estimation of olmesartan medoxomil in tablet dosage form with stability studies. Int J ChemTech Res. 2010;2:1129-34.
3. Swamy G, Agasimundin YS. Synthesis and antimicrobial activity of some novel chalcones containing 3-hydroxy benzofuran. Acta Pharmaceutica Sciencia. 2008;50(3).
4. Sarvani V, Elisha Raju P, Sreekanth Nama, Leela Madhuri Pola, Chandu Babu. Rao.Role of LC-MS in drug discovery process. International Journal of Pharmacy & Therapeutics on 2013 March 4,148-153
5. Espada A, Molina-Martin M, Dage J, Kuo MS. Application of LC/MS and related techniques to high-throughput drug discovery. Drug Discovery Today. 2008 May 1;13(9-10):417-23.
6. Molina‐Martin M, Marin A, Rivera‐Sagredo A, Espada A. Liquid chromatography‐mass spectrometry and related techniques for purity assessment in early drug discovery. Journal of Separation Science. 2005 Sep;28(14):1742-50.
7. Kingston J, O’connor DE, Sparey T, Thomas S. 6 Hyphenated techniques in drug discovery: purity assessment, purification, quantitative analysis and metabolite identification. Sample Preparation for Hyphenated Analytical Techniques. 2004 Sep 27:114.
8. Schaffrath M, von Roedern E, Hamley P, Stilz HU. High-throughput purification of single compounds and libraries. Journal of Combinatorial Chemistry. 2005 Jul 11;7(4):546-53.
9. Kerns EH. High throughput physicochemical profiling for drug discovery. Journal of Pharmaceutical Sciences. 2001 Nov; 90(11): 1838-58.
10. Korfmacher WA. Foundation review: principles and applications of LC-MS in new drug discovery. Drug Discovery today. 2005 Oct 15;10(20):1357-67.
11. Tiller PR, Romanyshyn LA, Neue UD. Fast LC/MS in the analysis of small molecules. Analytical and Bioanalytical Chemistry. 2003 Nov 1;377(5):788-802.
12. Fang AS, Miao X, Tidswell PW, Towle MH, Goetzinger WK, Kyranos JN. Mass spectrometry analysis of new chemical entities for Pharmaceutical Discovery. Mass Spectrometry Reviews. 2008 Jan;27(1):20-34.
13. MKM MR, Agilandeswari D, Madhu BH, Math MM, Sojwanya PJ. Aphrodisiac activity on ethanolic extract Nymphaea stellate leaves in male rats. Contemporary Investment Observe Pharmacology. 2012; 1: 24-30.
14. Snyder LR, Kirkland JJ, Glajch JL. Practical HPLC method development. John Wiley & Sons; 2012 Dec 3.
15. Michely JA, Meyer MR, Maurer HH. Power of Orbitrap‐based LC‐high resolution‐MS/MS for comprehensive drug testing in urine with or without conjugate cleavage or using dried urine spots after on‐spot cleavage in comparison to established LC–MSn or GC–MS procedures. Drug Testing and Analysis. 2018 Jan; 10(1):158-63.
16. Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A, Layloft T, Viswanathan CT, Cook CE, McDowall RD, Pitman KA. Conference report: analytical methods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies. Pharm Res. 1992;9(4):588-92.
17. Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ, McKay G, Miller KJ, Patnaik RN, Powell ML, Tonelli A. Bioanalytical method validation—a revisit with a decade of progress. Pharmaceutical Research. 2000 Dec 1;17(12):1551-7.
18. F.T. Peters, M. Hartung, M. Herbold, G. Schmitt, T. Daldrup, F. Musshoff, Anlage zu den Richtlinien der GTFCh zur Qualita¨ tssicherung bei forensisch- Untersuchungen, Anhang C: Anforderungen an die Durchfu ¨hrung von Analysen, 1.Validierung, Toxichem. Krimtech. 71 (2004) 146–154. http://www.gtfch.org/tk/tk71_3/Peters1.pdf.
19. Hartmann C, Smeyers-Verbeke J, Massart DL, McDowall RD. Validation of bioanalytical chromatographic methods. Journal of Pharmaceutical and Biomedical Analysis. 1998 Jun 1;17(2):193-218.
20. Peters FT, Maurer HH. Bioanalytical method validation and its implications for forensic and clinical toxicology—a review. In Validation in Chemical Measurement 2002 (pp. 1-9). Springer, Berlin, Heidelberg.
21. Maurer HH. Current role of liquid chromatography–mass spectrometry in clinical and forensic toxicology. Analytical and Bioanalytical Chemistry. 2007 Aug 1;388(7):1315-25.
22. Guideline IH. Validation of analytical procedures: text and methodology Q2 (R1). In International conference on harmonization, Geneva, Switzerland 2005 Nov 10 (pp. 11-12).
23. Zhou S, Song Q, Tang Y, Naidong W. Critical review of development, validation, and transfer for high throughput bioanalytical LC-MS/MS methods. Current Pharmaceutical Analysis. 2005 Jan 1;1(1):3-14.
24. Petrović M, Hernando MD, Díaz-Cruz MS, Barceló D. Liquid chromatography–tandem mass spectrometry for the analysis of pharmaceutical residues in environmental samples: a review. Journal of Chromatography A. 2005 Mar 4;1067(1-2):1-4.
25. Jemal M, Xia YQ. LC-MS development strategies for quantitative bioanalysis. Current Drug Metabolism. 2006 Jul 1;7(5):491-502.
26. PG S, Tripathi AS, Dewani AP, Bakal RL, Mohale DS, Chandewar AV. Liquid Chromatography in conjunction with Mass Spectrometry (LC-MS).
27. Xu RN, Fan L, Rieser MJ, El-Shourbagy TA. Recent advances in high-throughput quantitative bioanalysis by LC–MS/MS. Journal of Pharmaceutical and Biomedical Analysis. 2007 Jun 28;44(2):342-55.
28. Kumar PR, Dinesh SR, Rini R. LCMS—A review and a recent update. J Pharm Pharm Sci. 2016 Mar 1;5:377-91.
29. Gupta VK, Nalwad DN, Pere KL, Gite A, Shinde P, Kendre A. Bioanalytical method Development and Validation by LC-MS/MS: A Review.
30. He CM, Cheng ZH, Chen DF. Qualitative and quantitative analysis of flavonoids in Sophora tonkinensis by LC/MS and HPLC. Chinese Journal of Natural Medicines. 2013 Nov;11(6):690-8.
31. Wu SW, Pu TH, Viner R, Khoo KH. Novel LC-MS2 product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides. Analytical Chemistry. 2014 May 13;86(11):5478-86.
32. Lauber MA, Koza SM, Fountain KJ. Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C18 and TFA-Free Mobile Phases. Waters Application Note 720004571en. 2013.
33. Barua AB, Furr HC, Olson JA, van Breemen RB. Vitamin A and carotenoids. Chromatographic Science Series. 2000 Apr 18;84:1-74.
34. Y. B. Zambare, S. S. Chitlange, R. P. Bhole. Design and Screening of PPAR-γ agonist based Isatin derivatives and its remarkable activity as Anti-cancer and Anti-diabetic. Research J. Pharm. and Tech. 2019; 12(4):2017-2026.
35. Wu AH, French D. Implementation of liquid chromatography/mass spectrometry into the clinical laboratory. Clinica Chimica Acta. 2013 May 1;420:4-10.
Received on 19.06.2019 Modified on 28.08.2019
Accepted on 20.09.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(1): 505-516.
DOI: 10.5958/0974-360X.2020.00097.9