What is the Working Principle of LC-MS Mass Spectrometry?
What is LC-MS?
Liquid chromatography-mass spectrometry (LC-MS) is an analytical technique that integrates the technology of physical separation of components in a mixture of compounds (i.e., liquid chromatography, LC or high-performance liquid chromatography, HPLC) with mass analysis technology (i.e., mass spectrometry). Integrating the two techniques synergistically enhances the individual techniques’ capacity. Hence, LC-MS mass spectrometry has high sensitivity. It allows the separation of compounds and selective identification of the components, thus eliminating the need for complete chromatographic separation.
The mass spectrometry assay has diverse applications, including omics-based approaches, food processing, pharmaceutical analysis, cell proliferation assay, forensic examination, environmental monitoring, agrochemicals, cosmetics, and other biotech applications.
Principle of LC-MS Mass Spectrometry
The liquid chromatography (LC) system separates a mixture of compounds based on the difference in the affinity, i.e., retention of the individual components of the mixture on the stationary phase upon elution with the mobile phase. In the reverse phase LC, the hydrophobic compounds get retained in the column for longer durations.
Once the components get isolated, the analytes enter the ion source section of the mass spectrometer, where they are ionized. Electrospray ionization (ESI) is a common method for generating analyte ions. Other means of ionizing analytes include atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). Upon entering the mass spectrometer through the inlet capillary, electrodes direct the ions away from the source towards a mass filter (quadrupole mass analyzer). The quadrupole consists of four cylindrical rods to which a direct current and radio frequency is applied. This exposes the ions to a combination of electric and magnetic fields. The specific combination of the voltage and radio frequency allows ions with a particular mass-to-charge (m/z) ratio to travel toward the detector at any time. Thus, the detector detects ionized analytes based on their m/z ratio and amplifies the signal. This significantly reduces noise and enhances sensitivity.
Upon LC-MS bioanalysis, a mass spectrum is generated, where the signal intensity (abundance of the ions) is represented as a function of the m/z ratio. The peaks in the mass spectrum correspond to the ions arriving at the detector. The mass spectrum of a sample can determine the concentration of analytes and the purity of a sample and obtain an insight into the molecular structure of the analyte.
Although a wide variety of detectors, such as UV-visible, fluorescence, refractive index, and evaporative light scattering are available, mass spectrometry is frequently used since it helps to analyze compounds that may be optically inactive due to the absence of chromophores. Mass spectrometry assay offers unique, invaluable, and orthogonal information.
LC-MS bioanalysis involves LC-MS method development for quantitative analysis of drugs and their metabolites or other compounds of interest. Thereafter, the method developed is validated for good laboratory practice. It helps to ensure that the mass spectrometry assay developed is optimized to produce accurate and reliable results for the intended application. In addition to pharmaceutical applications, LC-MS is used extensively in proteomics, metabolomics, biomarker identification, and pharmacokinetics.
LC-MS/MS Analysis
Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) is an advanced technique based on the principles of LC-MS. In contrast to LC-MS, which integrates an HPLC with a mass spectrometry detector, LC-MS/MS couples an HPLC to two mass spectrometry detectors. In the LC-MS/MS technique, the ion produced in the first mass spectrometer gets further fragmented through collision, dissociation, or other methods. A second mass spectrometry unit detects these fragmented ions.
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LC-MS/MS is a highly sensitive and selective technique capable of quantifying clinically relevant compounds effectively. Furthermore, due to the possibility of using internal standards, LC-MS/MS displays an extensive linear dynamic range, high accuracy, and enhanced precision, along with the capacity to quantify multiple analytes simultaneously.
LC-MS/MS finds extensive bioanalytical applications; hence, method development and LC-MS/MS method validation are routine tasks in bioanalytical laboratories. LC-MS/MS is used to assess the pharmacokinetics of small molecules due to its high sensitivity and specificity. In peptide mapping, LC-MS/MS helps to verify the amino acid sequence of a protein using top-down and bottom-up approaches. LC-MS/MS analysis helps to identify glycosylation or other post-translational modifications. In terms of quality control, this technique helps to identify the chemical structure of a compound and assess its purity. It is also used in extractable and leachable studies to determine the substances that can leach into the product from the packaging material, thus causing adverse effects.
Conclusion
Mass spectrometry (both LC-MS and LC-MS/MS) is an advanced technique with high sensitivity, selectivity, precision, high throughput, and depth of coverage. The high sensitivity allows the method to quantify analytes at very low concentrations. Despite these advantages, mass spectrometry is not without limitations. This technique is not well-suited for identifying hydrocarbons that produce similar ions. Furthermore, it is challenging to separate geometrical and optical isomers using this technique. However, one can easily overcome these limitations by integrating gas chromatography with mass spectrometry.
