Understanding LC/MS/MS

Mass spectrometry is a detection method that relies on the mass-to-charge ratio of a compound. Following the chromatographic separation, molecules are ionized using an energy source. These ionized molecules then pass through a set of magnets, which further separates molecules based on their mass-to-charge ratio. Detection is performed based on charged particles striking a detector and creating a current. The detector only detects charges and cannot differentiate molecules based on mass. The magnetic separation provides the selectivity of the method. Thus, two molecules that are ionized at the same time, but have different masses can be detected independently using MS detection. Similarly, two molecules that are ionized at the same time and have the same masses, but have different charges, can be detected independently using MS detection.

Further specificity can be obtained when multiple ionization events occur. The second ionization process fragments the molecules into pieces that each have their own mass-to-charge ratio. These pieces are called daughter ions and can be detected. Each parent molecule fragments in a predictable fashion. Thus, even if two parent molecules have the same mass-to-charge ratio, if they break into different daughter ions, these can be differentiated. The multiple ionization event method is called tandem mass spectrometry (or MS/MS). The MS/MS technology is only used when a single ionization is not adequate to achieve the desired sensitivity.

The unique specificity of mass spectrometry detection permits more flexibility with the liquid chromatographic separations. Because molecules can be uniquely identified by MS, even if they exit chromatography together, the liquid chromatography becomes less important for LC/MS/MS than it is for HPLC/UV. Often, crude chromatographic techniques are adequate for LC/MS/MS, where complete separation is required for HPLC/UV. The results are that run times can be much shorter with LC/MS/MS, and precision and accuracy are increased. It really is the best of both worlds!

The primary challenges in MS detection are controlling the ionization process. Problems with incomplete ionization and interference can lead to poor precision and accuracy with the MS detector. These problems can be resolved with a variety of different ionization techniques, solvents, and even chromatographic separations. Additional techniques include time of flight MS, ion capture MS, and others. These special methods use different MS detector setups to improve the precision, accuracy, and sensitivity of MS detection.

It is unlikely that you will be an expert in mass spectrometry after reading this post. However, you should be able to understand the fundamental tenets of the technology and how it is used in bioanalysis.

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