The need for adequate chromatographic separation in the quantitative determination of drugs in biological samples by high performance liquid chromatography with tandem mass spectrometry

This work describes real examples of classes of drugs (drug candidates) whose quantitative determination in biological matrices by high performance liquid chromatography with electrospray tandem mass spectrometry (LC/MS/MS) may be hampered by interferences from drug-related biotransformation products (or prodrugs), unless the chromatographic conditions used achieve the separation of the drug from drug-related compounds. The classes of drugs investigated include: (a) a lactone drug, with the open-ring acid as the potential biotransformation product; (b) a phenolic drug and its prodrug; (c) an E-isomer methyloxime drug, with its Z-isomer as the potential biotransformation product; (d) a carboxylic acid drug, with its acylglucuronide as the potential biotransformation product; and (e) a thiol drug, with its disulfide as the potential biotransformation product. For each pair of a drug and the associated compound (except for the isomeric pair), the full-scan electrospray single mass spectrum of the associated compound gave an insource collisionally induced dissociation (CID) generated fragment ion identical to the parent ion of the drug. Thus, the associated compound, if present in the biological sample, would interfere with the quantitative determination of the drug by LC/MS/MS using the selected reaction monitoring (SRM) transition of the parent ion of the drug, unless the LC conditions utilized achieve clearcut separation of the drug from the associated compound. Thus, LC/MS/MS bioanalytical methods with very short retention times, where there are minimal chromatographic separations, should be used only for biological samples which are known to not contain analyte-related compounds that can undergo the SRM transition used for the drug determination.