APPLICATION OF LC-MS

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About Authors:
Jatin  Patel1*, Prof. Rajesh Kumar Dholpuria2, Dhiren Shah1
2(Professor, Head of Department of pharmacognosy),
1Seth G.L. Bihani S.D. College of Technical Education,
Institute of Pharmaceutical Sciences and Drug Research,
Sri Ganganagar, Rajasthan, INDIA
*Patelj313@yahoo.com

ABSTRACT:
Liquid chromatography is a fundamental separation technique in the life sciences and related fields of chemistry. Unlike gas chromatography, which is unsuitable for nonvolatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins. Traditional detectors for liquid chromatography include refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-Vis) detectors. Some of these generate two- dimensional data; that is, data representing signal strength as a function of time. Others, including fluorescence and diode- array UV-Vis detectors, generate three-dimensional data. Three-dimensional data include not only signal strength but spectral data for each point in time. Mass spectrometers also generate three- dimensional data. In addition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample. Mass spectral data add specificity that increases confidence in the results of both qualitative and quantitative analyses. For most compounds, a mass spectrometer is more sensitive and far more specific than all other LC detectors. It can analyze compounds that lack a suitable chromophore. It can also identify components in unresolved chromatographic peaks, reducing the need for perfect chromatography. Mass spectral data complements data from other LC detectors. While two compounds may have similar UV spectra or similar mass spectra, it is uncommon for them to have both. The two orthogonal sets of data can be used to confidently identify, confirm, and quantify compounds.

Reference Id: PHARMATUTOR-ART-1502

INTRODUCTION:
The separation power of modern chromatography combined with the selectivity and very low detection limits of modern mass spectrometers enables us to analyze very complex samples with a high degree of confidence.

The development of combinations of liquid chromatography with mass spectroemtry in an on line fashion has come to a stage where such combinations become part of routine procedures for the analysis of a broad  variety of samples. Since several technically very different interfaces have been devised in recent years, the major types arebriefly discussed, even though it becomes more and more clear that only a few will survive.

The most outstanding device appears to be the atmospheric pressure sources with electrospray and chemical ionization, since it covers many areas of chemical analysis in terms of polarity, lability and molecular weights.[Michael Linscheid, Westmorel, D.G,]

Why Liquid Chromatography/ Mass Spectrometryfi
Liquid chromatography is a fundamental separation technique in the life sciences and related fields of chemistry. Unlike gas chromatography, which is unsuitable for nonvolatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins.

Traditional detectors for liquid chromatography include refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-Vis) detectors. Some of these generate two- dimensional data; that is, data representing signal strength as a function of time. Others, including fluorescence and diode- array UV-Vis detectors, generate three-dimensional data. Three-dimensional data include not only signal strength but spectral data for each point in time.

Mass spectrometers also generate three- dimensional data. In addition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample. Mass spectral data add specificity that increases confidence in the results of both qualitative and quantitative analyses.

For most compounds, a mass spectrometer is more sensitive and far more specific than all other LC detectors. It can analyze compounds that lack a suitable chromophore. It can also identify components in unresolved chromatographic peaks, reducing the need for perfect chromatography. Mass spectral data complements data from other LC detectors. While two compounds may have similar UV spectra or similar mass spectra, it is uncommon for them to have both. The two orthogonal sets of data can be used to confidently identify, confirm, and quantify compounds.

Some mass spectrometers have the ability to perform multiple steps of mass spectrometry on a single sample. They can generate a mass spectrum, select a specific ion from that spectrum, fragment the ion, and generate another mass spectrum; repeating the entire cycle many times. Such mass spectrometers can literally deconstruct a complex molecule piece by piece until its structure is determined. [agilent.com]

Who Uses MSfi
Researchers and practitioners from various disciplines and sub disciplines within chemistry, biochemistry, and physics regularly depend upon MS analysis. Pharmaceutical industry workers involved in drug discovery and development rely on the specificity, dynamic range, and sensitivity of MS to differentiate closely related metabolites in a complex matrix and, thus, identify and quantify metabolites. Particularly in drug discovery, where compound identification and purity from synthesis and early pharmacokinetics are determined, MS has proved indispensable.

Biochemists expand the use of MS to protein, peptide, and oligonucleotide analysis. Using mass spectrometers, they monitor enzyme reactions, confirm amino acid sequences, and identify large proteins from databases that include samples derived from proteolytic fragments. They also monitor protein folding, carried out by means of hydrogen–deuterium exchange studies, and important protein–ligand complex formation under physiological conditions.

Clinical chemists, too, are adopting MS, replacing the less-certain results of immunoassays for drug testing and neonatal screening. So are food safety and environmental researchers. They and their allied industrial counterparts have turned to MS for some of the same reasons: PAH and PCB analysis, water quality studies, and to measure pesticide residues in foods. Determining oil composition, a complex and costly prospect, fueled the development of some of the earliest mass spectrometers and continues to drive significant advances in the technology.

Interface between LC/MS:
The combination of high-performance liquid chromatography and mass spectrometry (LC/MS) has had a significant impact on drug development over the past decade. Continual improve- ments in LC/MS interface technologies combined with powerful features for structure analysis, qualitative and quantitative, have resulted in a widened scope of application. These improvements coincided with breakthroughs in combinatorial chemistry, molecular biology, and an overall industry trend of accelerated development.

The interfacing of a liquid chromatograph to a mass spectrometer proved much more difficult than interfacing a gas chromatograph since each mol of solvent introduce into the instrument produce 22.4 l of solvent vapour, even at atmospheric pressure. The techenique has made huge advance in the last 10 years and there are many types of interface available , the most successful of which are the electrospray and atmospheric pressure ionization. [Watson, D.G]

The Interface MS detectors manipulate and detect ions in the gaseous phase; so for the MS to be useful as an HPLC detector, the mobile phase must be evaporated and sample ions must be generated. This is the function of the MS inter- face. The mobile phase must be converted from liquid to gas, an expansion in volume of  1000-fold; at the same time, the pressure must be reduced from atmos- pheric pressure (760 torr) to 10-5 to 10-6torr in the 10–20 cm fiow path of the inter- face. Pressure is reduced by pumping most of the vaporized sample and mobile phase to waste (no concentration takes place); only a tiny fraction of the sample is drawn into the MS itself.

The two most popular interfaces are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

The ESI interface (Fig. 1) adds a charge to analytes in the mobile phase by placing a potential (e.g., 3–5 kV) on the stainless-steel nebulizer-spray-tip (“capillary” in Fig. 1). Mobile phase is sprayed into the heated interface, where solvent evaporates, leaving ions in the gaseous state. ESI is the most commonly used interface for bioanalytical applications because it is a “softer” ionization technique and it is less likely to cause undesirable analyte degradation.

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