Bioanalysis Zone

Learn with Lin about LC–MS bioanalysis: part II – get to know the system


In this Learn with Lin article, Zone Leader Lin Wang (Covance, NJ, USA) explores the different components of a LC–MS and explains how they work.

Lin's WangLin Wang received her Bachelor of Science degree in Chemistry from Xiamen University in China and Master of Science in Chemical Engineering from Rutgers University in New Jersey, USA. She is also enrolled in the Business and Science Program at Rutgers University to develop a comprehensive understanding of the interface between science and business and look at the revolution of analytical industry from different perspectives.

She started her career with Envigo (NJ, USA) in the department of Formulation and Inhalation Analysis and is currently the Research Associate II at Covance. She has been working in the CRO industry since 2015 and focusing on method development and validation on both small and large molecules.

Liquid chromatography–mass spectrometry (LC–MS) is a technique that combines the separation capability of high-performance liquid chromatography with the detection advantages of mass spectrometry. It is preferred for bioanalysis by virtue of its great selectivity, sensitivity and linear range for detection of analytes of interest in complex matrices. During the 5 years in dose formulation analysis, the instrument which I use almost every day is HPLC-UV. Compared with a MS detector, there are not a lot of adjustments to be made for a UV detector, other than setting the right wavelength. However, when it comes to the mass spectrometer, there is a lot more to look at. For method development, each additional parameter will add a variable that may have a significant impact on the final outcome. As the saying goes, ‘you can’t make bricks without straw’. So, in this piece, we’ll look at the components of a LC–MS and get to know how they work.

For a typical LC–MS system, the complete entity can be divided into four basic components (shown in Figure 1): liquid chromatograph, ion source, mass analyzer and detector.

  • Liquid chromatograph (comprised of pump, autosampler and degasser) will carry the work for compound separation.
  • Ion source serves as a device to create charged species in the gas phase for the analyte of interest for subsequent mass analysis.
  • Mass analyzer filters out the analyte ions and fragments of analyte ions by their mass-to-charge (m/z) ratio.
  • Detector will convert the quantity of ions into signal output to a computer.
Figure 1: overview of an LC/MS/MS System
Fig 1
Ion source[1, 2]

The ion source is crucial as it is considered the ‘bridge’ to directly couple LC and MS. What comes out of an LC is a flow of liquid, whereas the mass spectrometer requires gas phase conditions and necessitates high vacuum in the analyzer region beyond the interface. Patrick[3] had a very good metaphor for LC–MS, as ‘a difficult courtship’ between a fish and a bird. However, the invention of atmospheric pressure ionization (API) techniques successfully solved this incompatibility. Without that, we would not be able to witness the continuation of this ‘love story’. The most common API techniques are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

Read ‘Learn with Lin: part 1’ here
Electrospray ionization (ESI)

ESI is a technique used to produce gaseous ions by applying high voltage to an ionic liquid eluent, in ionspray with the aid of nebulization to enhance aerosol formation. The nebulization is pivotal to cope with regular HPLC flow rates. It is considered as ‘soft ionization’ as it generates little fragmentation, which benefits the selection of precursor ions and maximizing sensitivity. This advantage is particularly significant when analyzing large molecules, because it overcomes the propensity of these molecules to fragment during ionization[4].

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