Liquid Chromatography-Mass Spectrometry LC-MS: Fundamentals and Applications

An LC-MS method employs physical separation of analytes followed by detection based on their mass-to-charge ratio. LC-MS analysis is sensitive, selective, and accurate and can detect analytes at the microgram and nanogram levels. The LC-MS has a broad range of applications including, environmental testing, pesticides, and food analysis, drug development, and medical device testing.

LC-MS method is fundamentally an HPLC assay with mass spectrometry detection units. LC-MS is HPLC with one mass spectrometry unit, whereas LC-MS/MS analysis uses two mass spectrometry detector units. The LC component separates the compounds while the MS unit quantifies them based on their mass-to-charge ratio. However, similar to HPLC method validation, LC-MS/MS and LC-MS method validation are paramount for robust assay results. Let us learn more about the principles and applications of the LC-MS method.

LC-MS fundamentals

As the name suggests, liquid chromatography separates target compounds in a sample solution. The sample solution is injected into the mobile phase. Depending on the experimental requirement, sample volume varies from 0.1 µL to 100 µL. The instrument continuously pumps the mobile phase onto a silica particle-coated stationary phase column. When the mobile phase-sample solution mixture reaches the stationary phase, the components will react differentially, depending on their chemical and physical properties.

Some analytes may interact strongly with the stationary phase. The analyte with the least interaction will emerge first from the stationary phase. As the mixture continues to flow through the column, the remaining analytes will sequentially elute based on the strength of their interaction. The time spent in the stationary phase is called the retention time.

The eluted mobile phase is then transferred to the MS detector unit. However, the eluent is not directly allowed into the MS unit. MS operates under a vacuum, and therefore, the eluent has to pass through an interface before entering MS units. The interface primarily removes the liquid mobile phase before transferring it to the MS detection unit. The interface nebulizes the liquid into a fine spray, ionizes it, and then allows it to enter the MS unit. Also, The MS unit exposes the ions to electric or magnetic fields for detecting the analytes.

This exposure alters ion movements and segregates them according to their masses. The detector then amplifies the ion current and quantifies the segregated ions. Finally, the mass-to-charge data is converted into a graphical format for visualizing results. Such accurate mass-to-charge data has made LC-MS the primary bioanalytical tool in several industrial applications. Let us now dive deep into the diverse applications of the LC-MS method.

Applications of LC-MS analysis

Being highly sensitive and accurate, the LC-MS method has an extensive range of bioanalytical method and analytical applications. Some examples are outlined below:

Analysis of both large and small protein molecules in complex biological matrices

Determining and quantifying genotoxic impurities in active drug compounds

Detection of twelve model compounds representing specific doping agents

Quantifying drug metabolite in sample matrices

Determining adulterants or contaminants in dietary supplements and food materials

Determining alkylphenol ethoxylates in tannery sediments

Analysis of river and swimming pool samples

Quantifying proteome

Quantifying nucleotides

Rapid detection of Sars-CoV-2

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