With the development of an electrospray ionization source, LC-MS has become a routine analysis in the drug development process. The LC-MS method has numerous applications. Besides, the use of LC-MS/MS analysis and stable isotopes has further accelerated the development of accurate and sensitive LC-MS testing. LC-MS bioanalysis comprises faster-scanning speed facilitating multiplexing of biological samples. Today LC-MS assays are competing with other techniques, such as immunoassay, due to the development of more reliable and affordable instruments.
In MS systems, the analyte molecules are converted to an ionized state, and the generated ions and fragment ions are analyzed based on their mass-to-charge ratio. Scientists have several different types of instruments available at their disposal. These instruments can have different technologies for ionization and ion analysis. However, some configurations are better than others. Hence, the current article focuses on the five basic principles of LC-MS systems that every researcher should know.
Flow rate
Generally, the standard flow rates are up to 1 ml/min. However, lower flow rates can improve sensitivity. Columns with a diameter of 1.0 to 0.2 mm are well suited for direct coupling. Switching to columns with these reduced diameters is easily manageable. Besides, a drop in diameter size also decreases the amount of mobile phase required for analysis.
Direct injection method
Often with LC-MS/MS analysis, researchers can directly inject the samples and measure the analytes. Direct injection into the ion sources usually requires multiple reaction monitoring coupled with internal standards. Sometimes, with study samples such as urine, one may dilute and inject the sample into the ion sources. This technique is also called “dilute and shoot.” However, the dilute and shoot method may not be feasible for more complex study samples such as plasma.
Ion suppression
Biological samples are inherently complex. Thus, multiple sample components in the ion sources may reduce the MS signal. This phenomenon, called ion suppression, results due to the competition of several sample components in the ionization process. Researchers often compare the peak area responses for calibrators, spiked samples and spiked sample extracts, and pure standards to evaluate the effects of ion suppression. Besides, stable isotope-labeled internal standards can help minimize ion suppression.
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Mobile phase
Typical LC solvents are compatible with electrospray ionization sources. However, a solvent suitable for LC separation may not be applicable for LC-MS-based analysis. For example, the solvent in LC analysis may be UV readable at 210 nm but may contain compounds affecting LC-MS measurements. Hence, researchers must avoid buffers with inorganic ions. These ions cause considerable ion suppression and may contaminate the ion source. Substituting with buffers compatible with MS systems can help alleviate these challenges.
Through-put and resolution
Conventional LC-UV setup generally requires baseline separation of peaks. However, with LC-MS testing, peaks with independent MS signals will not require complete chromatographic separation. Hence, LC-MS analysis facilitates using lower resolution and shorter columns that reduces assay run times. Another approach employs shorter columns with smaller particle sizes and high pressure to speed up the analytical process. Shorter particle sizes can generate better signal: noise ratios than larger particle-size columns.
