Liquid chromatography-mass spectrometry (LC-MS) is a routinely employed tool for quantifying analytes in bioanalytical experiments. With the advent of the electrospray ionization technique, multiplex assay services are increasingly employing LC-MS assays due to their robust and easy interface. With robust LC-MS method development utilizing stable isotopes and tandem mass spectrometry, LC-MS testing provides sensitive and accurate analysis of a broad spectrum of biomolecules. However, achieving sensitivity and specificity will require LC-MS method validation and optimization efforts.
The feature of rapid scanning speeds offers the benefit of multiplexing several compounds in a single assay run. With the availability of more reliable and affordable instruments, LC-MS labs will be expanding their applications in several domains of clinical biochemistry to compete with other techniques, such as conventional liquid chromatography and ligand-binding assays. However, a robust LC-MS method development and validation will remain crucial for developing reliable, accurate, reproducible results. Let us understand the analytical precision of LC-MS sample testing.
LC-MS assays for bioanalytical studies
LC-MS assay is an ideal technique to identify, quantify, and analyze mixture components and to determine their purity and chemical composition. With high sensitivity, LC-MS assays can perform reproducible and precise quantitative assessments. This technique is ideal for ionizable analytes such as proteins or food contaminants.
The liquid chromatography component connects with the mass spectrometer through an interface that allows the separated components from the chromatography column into the ion source of mass spectrometers. This interface is crucial for LC-MS testing as liquid chromatography requires high pressure, while mass spectrometers operate at high vacuum.
The HPLC unit has a mobile and stationary phase. Both these phases can be modified to achieve the desired assay properties. Usually, modifications in the mobile phase are done for the sample of interest, while the stationary phase is modified according to the mobile phase. The degree of compound retention and separation in the chromatography column is based on the compound’s relationship with the stationary and mobile phases.
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Moreover, based on the properties of the mobile and stationary phases, the LC component can be divided into normal phase and reverse phase chromatography. A nonpolar mobile phase and polar stationary phase combine to form normal phase chromatography, while a polar mobile phase and a nonpolar stationary phase combine to form reverse phase chromatography.
Once the liquid chromatography component separates the compound, the individual components are then identified and determined using mass spectrometry. The MS unit develops a unique mass spectrum for each compound. These compounds are ionized and fragmented using chemical or electron ionization, and a mass analyzer then accelerates them through an ion trap or a quadrupole system to identify the generated ions based on their mass-to-charge ratio.
Liquid chromatography units can also be combined with other detector systems, for example, HPLC-DAD systems that use a diode ion detector. The choice of the detection unit often depends on the experimental goal and the analyte of interest. Generally, DAD detectors work best while quantifying known components, whereas MS units are ideal for unknown analytes.
In Conclusion
LC-MS assays offer precise and accurate estimations of analytes in complex study samples.
