This tutorial applies to the quantitation of small molecules using mass spectrometry in pharmacokinetic analysis, also known as PK/MS. In addition these very same principles can be applied to the quantitation of peptides and proteins in biological matrices.
Traditionally before the advent of modern-day mass spectrometry quantitation was accomplished using HPLC and UV detection. HPLC PK analysis relied on; retention time, peak area and UV spectral character. Unfortunately the HPLC assay suffered from lack of sensitivity and specificity. We have seen examples where a molecule was extensively metabolized and yet the retention time and UV spectral character remained the same as the parent compound. This lack of specificity will from time to time mislead the investigator. MS characterization is now a vital new tool in pharmacokinetic analysis.
The accepted way of performing mass spec quantitation is by using a mass spectrometer capable of MS/MS fragmentation. MS/MS used in conjunction with quantitation is commonly accomplished with a triple quadrupole or ion trap mass spectrometer. The reason MS/MS is required is because many compounds have the same intact mass. While many researchers use the first dimension of MS to perform quantitation, that technique again suffers from lack of specificity especially in very complex matrices like blood. The second dimension of MS fragmentation in the majority of cases provides a unique fragment. The combination of the specific parent mass and the unique fragment ion is used to selectively monitor for the compound to be quantified. Below we will discuss the method for acquiring and visualizing LC/MS data
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