INTRODUCTION:

Modern physical methods of analysis are so sensitive that they provide accurate and detailed information even from small samples. These are mainly applied and generally flexible to automation¹.

For these reasons, they are now used in product development, manufacturing and formulation control, as storage stability control, and in monitoring drug and drug use. It is an analytical technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications that has very high sensitivity and selectivity. It is commonly used in pharmacokinetic studies of pharmaceutical products and is the most used technique in the field of bioassay².

LC-MS also plays a role in pharmacognosy, especially in the field of molecular pharmacognosy when it comes to differences between ingredients in aspects of phenotypic cloning. The most important factor to consider is how to make the most active ingredient difference in plant cells between the plant test group and the controlled ones³. In LC-MS-based proteomics, complex mixtures of proteins are first subjected to enzymatic cleavage, then the resulting peptide products are analyzed using a mass spectrometer; this is in contrast to "top-down" proteomics, which deals with intact proteins and is limited to single protein mixtures⁴. A standard bottom-up experiment involves the following key steps:

· Extraction of proteins from a sample.

· Fractionation to remove contaminants and proteins that are not of interest, in particular high maintenance proteins. Which are usually not indicative of the disease under study.

· Digestion of proteins into peptides⁵.

· Post-digestion separations to obtain a more homogeneous mixture of peptides.

· Analysis by MS.

The two fundamental challenges in analyzing MS-based proteomics data are, therefore, identifying the proteins present in a sample and quantifying the abundance levels of those proteins. There are a large number of processing tasks associated with each of these challenges. The first step in protein identification is the identification of the constituent peptides⁶. This is done by comparing the observed characteristics with entries in a database of theoretical or previously identified peptides. In tandem mass spectrometry (indicated by MS / MS), a parent ion is selected that possibly matches a peptide in MS1 for further fragmentation in MS2. The resulting fragmentation spectra are compared with the fragmentation spectra in a database, using software such as SEQUEST, Mascot Tandem Alternatively, high resolution MS tools can be used to obtain highly accurate mass measurements and these can be compared with mass measurements in a previously identified peptide database with high reliability via MS/MS using the same software tools as above⁷. In both cases, a statistical evaluation of the level of confidence in the identification of the peptides is desirable. Protein identification can be accomplished by accumulating levels of confidence in identification at the peptide level at the protein level, a process that is associated with a number of problems and complexities. The goal of the identification process is generally to identify as many proteins as possible by checking the number of false identifications to a tolerable level⁸. There is a wide variety of options for the exact identification method used, including:

a. Choosing a statistic to assess the similarity between an observed spectral pattern and a database entry⁹.

b. The choice of how to model the null distribution of the similarity metric. There are two other methods of protein identification: de novo and de novo hybrids and database comparison. The purpose of this article is to provide an accessible overview of LC-MS-based proteomics. Our model for this article was a 2002 biometrics article with a similar focus on the configuration of the DNA microarray. We hope that this, like the 2002 paper on DNA microarrays, will serve as an entry point for more statisticians to join the exciting research that is underway in the field of LC-MS-based proteomics¹⁰.

Basic Principle of LC-MS:

Liquid Chromatography:

High Performance Liquid Chromatography Current liquid chromatography generally uses very small particles packed and operating at relatively high pressure and is called high performance liquid chromatography (HPLC); Modern LC-MS methods use HPLC instrumentation essentially exclusively for the sample¹¹. The basic principle of HPLC is adsorption. In HPLC, the sample is forced by a liquid at high pressure (the mobile phase) through a column which is filled with a stationary phase generally composed of irregularly shaped or spherical particles chosen or derivatized to obtain particular types of separations. HPLC methods have historically been divided into two different subclasses based on stationary phases and the corresponding required polarity of the mobile phase¹².

Flow Splitting:

Flow is often split at a -10: 1 ratio when using standard diameter (4.6mm) columns. It is useful to use other techniques together, such as MS and UV detection. However, the sensitivity of the spectrophotometric detectors will decrease if the flux division is towards UV rays. Mass spectrometry will also show improved sensitivity at flow rates of 200 μL/min or less¹³.

Mass Spectrometry:

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used to determine the masses of particles, to determine the elemental composition of a sample or molecule, and to clarify the chemical structures of molecules, such as peptides and other chemical compounds¹⁴. MS works by ionizing chemical compounds to generate charged molecules or molecule fragments and by measuring their charge-to-mass ratios. In a typical MS procedure, a sample is loaded onto the MS instrument and vaporized. The components of the sample are ionized by one of several methods (for example, by impacting them with an electron beam), resulting in the formation of charged particles (ions). Ions are separated based on their mass/charge ratio in an electromagnetic field analyzer. Ions are detected, usually by a quantitative method. The ion signal is processed in mass spectra¹⁵.

Mass Analyzer:

There are many different mass analyzers that can be used in LC/MS. Some of them are Simple Quadrupole, Triple Quadrupole, Ion Trap, Time of Flight (TOF) and Quadrupole Time of Flight (QTOF)¹⁶.

Interface:

The interface between a continuous flow liquid phase technique and a gas phase technique performed under vacuum has been difficult for a long time. The advent of electrospray ionization has changed this. The interface is usually an electrospray ion source or a variant such as a nano-spray source; however, the atmospheric pressure chemical ionization interface is also used. Various deposition and drying techniques have also been used, such as the use of mobile belts; however, the most common is the offline MALDI repository. A new approach still in development called the Direct-EI LC-MS interface that couples a nano HPLC system with a mass spectrometer equipped with electronic ionization¹⁷.

Figure 1: Schematic Representation of Basic Principle and Instrumentation Involved in Liquid Chromatography-Mass Spectrometry (LC-MS)

Combination of HPLC and MS:

HPLC not only separates things, but also provides little additional information on what a chemical might look like. In fact, in HPLC it is difficult to be sure of the purity of a particular peak and whether it contains only one chemical. Adding a mass spectrometry to this will tell you the masses of all the chemicals present in the peak, which can be used to identify them, and an excellent method for verifying purity¹⁸. Even a simple mass specification can be used as a specific mass detector, specific to the chemical under study. More sophisticated mass detectors, such as triple quadrupole and ion trap instruments, can also be used to perform more detailed structure-dependent analysis of what is eluting from the HPLC system¹⁹.

Advantages of LC-MS:

There are several advantages of LCMS over other chromatographic methods of which a few are as follows;

· Selectivity: Coeluted peaks can be isolated by mass selectivity and are not limited by chromatographic resolution²⁰.

· Assignment of peaks: a molecular fingerprint is generated for the compound under study, ensuring the correct assignment of peaks in the presence of complex matrices.

· Molecular weight information: confirmation and identification of known and unknown compounds.

· Structural information: Controlled fragmentation allows for the structural elucidation of a chemical.

· Rapid Method Development: Provides easy identification of eluted analytes without retention time validation.

· Sample matrix adaptability: reduces sample preparation time and thus saves time.

· Quantification: Quantitative and qualitative data can be easily obtained with limited optimization of the tool²¹.

Various LC-MS Applications:

1. Molecular Pharmacognosy:

LCMS determines the contents and categories of different groups of cultured plant cells and selects the pair of groups with the highest content of different ingredients for phenotypic cloning of the difference of study ingredients²².

2. Characterization and Identification of Compounds Carotenoids:

Since carotenoids are not thermally stable, separation of mixtures and removal of impurities is generally done by reverse-phase HPLC (especially HPLC) rather than gas chromatography. Structural analysis using Nuclear Magnetic Resonance. Therefore, only the most sensitive analytical methods, such as liquid chromatography/mass spectrometry and HPLC with photodiode matrix UV-Visible absorbance detection are suitable²³. At the lowest level, the identification of carotenoids can be confirmed by combining data such as HPLC retention times, photodiode array absorbance spectroscopy, mass spectrometry, and tandem mass spectrometry.

Proteomics:

Liquid chromatography/mass spectrometry (LC/MS) has become a powerful technology in proteomics studies in drug discovery, including characterization of target proteins and biomarker discovery²⁴.

a) Characterization of Glycopeptides:

MS-based glycoproteomic studies are used to characterize the glycopeptides examined. This involves identifying the glycosylation site, the type of glycan involved and the core of the peptide structure. Today, with MS-based strategies, tandem MS fragmentation and data analysis issues provide efficient characterization of intact glycopeptides and then peptide analysis is performed by liquid chromatography-tandem mass spectrometry (LC -MS/MS)²⁵.

b) Peptide Mapping:

In the early days, protein drugs were made from refined proteins of living organisms. However, they have recently been produced using recombinant technology. Insulin, interferon and erythropoietin are some of the recombinantly produced protein drugs available on the market. For example, protein analysis and peptide mass mapping of a horse heart myoglobin model sample is performed by LC/MS using a quadrupole mass spectrometer²⁶.

c) Degradation Products:

LCMS was used to separate, identify and characterize the degradation products under certain conditions of hydrolytic, oxidative, photolytic and thermal stress. A full path of massive drug fragmentation was first established with the help of LC-MS/MS studies. Stressed samples were subjected to LC-MS studies. It is performed to swap mass studies to get the precise mass, fragment pattern and number of unstable hydrogens. The results of the Member States helped to assign temporary structures to degradation products²⁷. Few examples are the identification and characterization of the breakdown products of Irbesartan, the breakdown products prompted by Prulifloxacin²⁸.

3. Quantitative and Qualitative Analysis:

Quantitative Bioanalysis of Various Biological Samples:

The LC-MS/MS methodology includes sample preparation, component separation, and MS/MS detection and applications in various areas, such as biogenic amine quantification, immunosuppressant pharmacokinetics, and doping control²⁹. Advancement that includes automation in LC-MS/MS instruments along with parallel sample processing, column switching and the use of more efficient supports for SPE, driving the trend towards fewer sample cleanup times and total execution times (high performance methodology) in the current area of quantitative bioanalysis³⁰.

Qualitative and Quantitative Analysis of Complex Lipid Mixtures:

It is an LC-MS based methodology for the study of lipid mixtures where it has been described and its application to the analysis of lipids associated with human lipoproteins is demonstrated. After an optional initial fractionation on Silica 60, normal phase HPLC-MS on a YMC PVA-Sil column is first used for class separation, followed by reverse-phase LC-MS or LC-tandem mass spectrometry using one column. Capillary Atlantis dC18 and/or MS nano-spray, to fully characterize the individual lipids³¹.

Phytoconstituents/Plant Metabolomics:

LC-MS provides a tool to differentiate this immense plant biodiversity by the ability of this technique to analyze a wide range of metabolites, including secondary metabolites (eg, alkaloids, glycosides, phenylpropanoids, flavonoids, isoprenes, glucosinolates, terpenes), benzoids) and highly polar and/or higher molecular weight molecules (oligosaccharides and lipids). LC-MS is one of the leading undirected analytical techniques for determining global metabolic profiles, aiding in the identification and relative quantification of all peaks in the chromatogram as ions that are initially defined by retention time and molecular mass³².

4. Automated Immunoassay in the Monitoring of Therapeutic Drugs:

Therapeutic drug monitoring (MDT) of some drugs with a narrow therapeutic index helps improve patient outcome. The need for accurate, precise and standardized drug measurement poses a major challenge for clinical laboratories and the diagnostic industry. In the past, various techniques have been developed to meet these requirements³³. At present, methods and immunoassays based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) appear to be the most popular approaches in clinical laboratories³⁴.

5. Two-dimensional (2-D) Hyphenated Technology:

The use of LCMS has become a powerful two-dimensional (2D) scripted technology for use in a wide variety of analytical and bioanalytical techniques for analyzing proteins, amino acids, nucleic acids, amino acids, carbohydrates, lipids, peptides, etc³⁵. and/or in the main classifications in the field of genomics, lipidomics, metabolomics, proteomics, etc. LCMS was originally preferred and can be intensified by the need for more powerful analytical and bioanalytical techniques that can accurately distinguish target analytes with highly complex mixtures in a sensitive and distinctive way³⁶.

6. Clinical Chemistry and Toxicology:

For some analytes in clinical chemistry and toxicology, liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS) offers eloquent advantages over traditional immunoassays. Analytes analyzed include estradiol, testosterone, thyroid hormones, immunosuppressants, vitamin D, steroids for newborn screening programs, and clinical and forensic toxicology. Although immunoassays are commonly used in clinical laboratories, analytical sensitivity and specificity are lower for many of the analytes tested in routine clinical laboratories. Additionally, LC-MS/MS can be multiplexed for high throughput testing and multi-analyte detection. The application of LC-MS/MS in clinical chemistry and toxicology studies will improve and the benefits will be well known³⁷.

Future Prospects For LC-MS:

Metabolomics:

At present, mass spectrometry (MS) -based metabolomics has been widely used to gain new insights into biomarkers, drug and plant discoveries, nutritional research, food control, and microbial biochemistry in humans and plants. The next 5-10 years will inevitably see increased inter-laboratory cooperation to collect as much data as possible on LC-MS-based metabolites. In-house MS/MS libraries are likely to be more readily available to interested collaborators with similar model and instrumentation samples, increasing the knowledge base of all participating laboratories. Integration of NMR into LC-MS-based metabolic and metabolic profile studies is likely to increase, either by offline analysis of collected LC fractions or by hybrid LCNMR-MS instrumentation. There are two important issues that stand out, which could be better addressed through coordinated and unified actions in the future³⁸.

Only 5-10% of known metabolites were reported in compound-focused databases. A significant increase in MS, MS / MS and MSn spectra from authentic chemical standards should be addressed through an international initiative bringing togetherorganic chemistry and metabolomics groups, and is likely to involve both academia and commercial companies³⁹.

Despite the indisputable presence of unknown (i.e. not previously discovered) natural metabolites from undirected metabolomic studies, it is ambiguous whether this phenomenon is distorted due to errors in elucidation of adducts/fragments and chemical/background noise. This problem can be partially relegated to a new frontier of metabolomic databases characterized by well constructed mass spectra containing all species of adducts and fragments for reference substances. Furthermore, saving full scan (MS1) spectral data of authentic chemical standards showing divergences in adduct formation would allow the development of estimated methods to address the illustration of mzRT characteristics in LC/MS-based undirected metabolomic studies⁴⁰.

Proteomics:

The spectacular development of LC-MS peptide instrumentation over the past decade has nearly left protein sample preparation, including extraction and digestion, as the major sticking point in proteomics workflows in the overall performance of proteomics experiments. Cleaning the samples for non-protein contaminants greatly affects the speed of protein identification.

Pharmacovigilance:

Pharmacovigilance (PV or PhV), known as drug safety, it is one of the pharmacological sciences that is related to the collection, detection, evaluation, monitoring and even prevention of negative side effects with pharmaceuticals. Detection and follow-up can be performed using an LC-MS based disease modification technique that provides detailed profiles⁴¹.

Organic/Inorganic Hybrid Nanoflowering:

The LCMS analytical method can be used for the detection of general nanoflowering. It helps in the development of drug delivery systems, biosensors, biocatalysts and biology-related devices are expected to take multiple directions. New synthesis principles, new types of hybrid nanoflowers and detailed mechanisms are expected to emerge. The application of nanoflowers in biocatalysis and enzyme mimetics, tissue engineering and the design of high sensitivity biosensitivity kits, as well as industrial biological devices with advanced functions, variable and controllable synthesis, biocompatibility and modifications of hybrid nanoflowers. Facilities and properties should receive increasing attention⁴².

1. Basic Biological Principles Underlying Proteomics:

Proteins are the main structural and functional units of any cell. Proteins are made up of amino acids arranged in a linear sequence, which is then folded to form a functional protein. The amino acid sequence in proteins is encoded by genes stored in a DNA molecule.

2. Experimental Procedure:

An LC-MS-based proteomics experiment requires several sample preparation steps, including cell lysis to separate cells, protein separation to distribute protein collection into more homogeneous groups, and protein digestion to break down intact proteins into more manageable peptide components. Once completed, the peptides are further separated, then ionized and fed into the mass spectrometer.

2.1 Sample Preparation:

Whole cell proteome analysis generally involves harvesting intact cells, washing and adding a lysate buffer, which contains a combination of chemicals that destroy the cell membrane and protease inhibitors that prevent protein degradation. Cells are homogenized and incubated with buffer, after which centrifugation is used to separate cell debris and membrane from supernatant or cell lysate. The cell lysis step is not necessary when analyzing body fluids such as blood serum. The blood samples are centrifuged, after which the red blood cells are pelleted in the lower part of the tube and the plasma is collected in the upper part. Fibrinogen and other clotting factors are removed to obtain serum. High-abundance proteins are also removed, as they are not normally involved in the disease⁴³.

2.2 Mass Spectrometry:

A mass spectrometer measures the mass-to-charge ratio (m/z) of ionized molecules. There has been a huge improvement in MS technology in recent years and there are around 20 different mass spectrometers commercially available for proteomics. All mass spectrometers are designed to perform the various functions of ionization and mass analysis. The key components of a mass spectrometer are the ion source, the mass analyzer and the ion detector. The ion source is responsible for assigning charge to each peptide. Mass analyzers take many different forms, but ultimately they measure the mass/charge (m/z) ratio of each ion. The detector captures the ions and measures the intensity of each ion species. In terms of the mass spectrum, the mass analyzer is responsible for the m/z information on the x axis and the detector is responsible for the maximum intensity information on the y axis⁴⁴.

3. Data Acquisition:

In LC-MS, each sample can lead to thousands of scans, each containing a mass spectrum. The mass spectrum for a single MS scan can be summarized by plotting the m / z values against the peak intensities. Buried in these data are signals specific to individual peptides. As a first step towards the identification and quantification of these peptides, the characteristics must be identified in the data and, for example, distinguished from the background noise. The first step in this is detecting the EM spikes. Many approaches to peak detection have been proposed, as this is a longstanding problem in the field of signal processing⁴⁵.

4. Identification of Proteins

In bottom-up proteomics, protein identification is usually achieved by first comparing the observed MS characteristics with a database of previously identified or predicted characteristics (e.g., from MS/MS or based on a previous analysis of a well characterized sample, Figure 5). The most widely used approach is MS in tandem with database search, where peptide fragmentation models are compared with theoretical models in a database using software such as Sequest, Tandem and Mascot. With high resolution LC-MS instruments, identifications can only be made on the basis of mass and elution time or in combination with MS / MS fragmentation standards⁴⁵.

5. Quantification of Proteins:

Quantitative proteomics is concerned with quantifying and comparing the abundance of proteins under different conditions. There are two main approaches: stable isotope labeling and no labeling. In all cases, as in the identification scenario, there is the challenge of passing information at the peptide level down to the protein level.

Proteomics Technologies:

Two-dimensional Polyacrylamide Gel Electrophoresis:

For three decades, 2-D PAGE has proven to be a reliable and efficient method of separating proteins based on mass and charge. It is possible to achieve the separation of several thousand different proteins on one gel. The high resolution 2-D PAGE can resolve up to 10,000 protein stains per gel. Dyes like Coomassie Blue, Silver, SYPRO Ruby, and Deep Purple can be used to visualize proteins. Proteins are separated by charge (isoelectric point) in the first dimension and by mass in the second dimension. In isoelectric focusing, proteins migrate in a pH gradient towards pH where they have no net charge. The most common proteins are separated by the isoelectric point in the horizontal direction and by size in the vertical direction⁴⁶.

Liquid Chromatography:

LC is a method of separating compounds within a sample so that they can be identified and quantified. There are growing applications in proteomics research due to its ability to analyze large and fragile biomolecules. With advances in ionization methods and instrumentation, LC combined with MS has become a powerful technology for the characterization and identification of peptides and proteins in a complex mixture. The bottom-up LC-MS approach to proteomics generally involves protease digestion followed by LC-MS or LC-MS/MS peptide mass fingerprinting to derive the sequence of the individual peptides. Another significant improvement is the development of multidimensional protein identification technology, which separates peptides in 2-D LC. The separation can be connected directly to the ion source of a mass spectrometer^3,4.

Labeling of Isotope-Encoded Affinity Tags:

Quantitative proteomics has recently emerged as a complementary technology to mRNA profiling with the ability to fully characterize the structural and functional aspects of protein species. Due to the variability of ionization for different peptides, the best internal standard for peptide quantification is the same stable isotope-labeled peptide. There are several protocols for the use of stable isotopes in proteomics, where the ICAT labeling technique has received the greatest attention. Relative quantification is very accurate because it is based on stable isotope dilution techniques⁵.

Mass Spectrometry:

In order to exploit the profiles of proteins expressed in different physiological and pathophysiological conditions, MS protein analysis is generally used as a powerful platform in proteomics. The technology is applied for mass determination and can be adapted for protein identification¹.

Electrospray Ionization:

This technology involves the production of gaseous ions by applying a potential to a flowing liquid, which results in the formation of a spray of small droplets with solvent-containing analyte. The solvent is removed from the drop by heat or another form of energy, such as collision with a gas, and multiply charged ions are formed.

Matrix-Assisted Laser Desorption/Ionization:

MALDI ionization involves a suspended or dissolved protein in a crystalline structure (the matrix) of small organic molecules that absorb UV rays. The crystal absorbs energy at the same wavelength as the laser that is used to ionize the protein⁴⁷.

Surface Enhances Laser Desorption Ionisation:

SELDI is designed to perform mass spectrometric analyzes of protein mixtures retained on chromatographic chip surfaces. Differentially expressed proteins can be determined from these protein profiles by comparing the intensity of the peak.

Quadrupole-Orbital Linear Iontrap Mass Spectrometry:

Linear Ion Trap Quadrupole (LTQ): Orbitrap MS couples a LIT mass spectrometer to an Orbitrap mass analyzer via a radiofrequency capture quadrupole with a curved axis. The latter injects pulsed ion beams into a rapidly changing electric field in the Orbitrap where they are trapped at high kinetic energies around an internal electrode⁴⁸.

Protein Identification:

MS can also be used to identify proteins. Proteolytic digestion of gel-separated proteins into peptides and peptide mass analysis provide a fingerprint of the peptide mass, which can be searched based on theoretical sequence fingerprints in databases⁴⁹.

Bioinformatics:

Bioinformatics is an integration of mathematical, statistical and computational methods for analyzing biological, biochemical and biophysical data. Sophisticated bioinformatics tools are being developed to handle the large amount of data resulting from genomics and proteomics studies⁵⁰.

CONCLUSION:

As we know that liquid chromatography mass spectrometry is an effective technique used in the analysis of pharmaceuticals. The continuous demand of LC-MS in analytical chemistry has been increased day by day. To understand the process involves in liquid chromatography mass spectrometry we designed a precise review on the major key aspects of LC-MS. The study includes the basic principle, applications, advantages, future aspects of LC-MS. In which we must say that the use of this technique were effective in analytical chemistry. Also, the main important highlight of our study is proteomics based LC-MS, which is used in the characterization, and quantification of compounds in pharmaceutical industries. So, the study concluded that liquid chromatography mass spectroscopy is a wonderful technique used in the analytical chemistry, and proteomics experimentation.

ACKNOWLEDGEMENT:

The author wishes to acknowledge Laureate Institute of Pharmacy, Jawalamukhi, Himachal Pradesh (176031) for providing their support, and other required facilities in the preparation of this review article.

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Received on 08.02.2021 Modified on 01.04.2021

Asian J. Pharm. Res. 2021; 11(3):194-201.

DOI: 10.52711/2231-5691.2021.00035