Quantitative interference by cysteine and N-acetylcysteine metabolites during the LC-MS/MS bioanalysis of a small molecule.
During LC-MS/MS quantification of a small molecule in human urine samples from a clinical study, an unexpected peak was observed to nearly co-elute with the analyte of interest in many study samples. Improved chromatographic resolution revealed the presence of at least 3 non-analyte peaks, which were identified as cysteine metabolites and N-acetyl (mercapturic acid) derivatives thereof. These metabolites produced artifact responses in the parent compound MRM channel due to decomposition in the ionization source of the mass spectrometer. Quantitative comparison of the analyte concentrations in study samples using the original chromatographic method and the improved chromatographic separation method demonstrated that the original method substantially over-estimated the analyte concentration in many cases. The substitution of electrospray ionization (ESI) for atmospheric pressure chemical ionization (APCI) nearly eliminated the source instability of these metabolites, which would have mitigated their interference in the quantification of the analyte, even without chromatographic separation. These results 1) demonstrate the potential for thiol metabolite interferences during the quantification of small molecules in pharmacokinetic samples, and 2) underscore the need to carefully evaluate LC-MS/MS methods for molecules that can undergo metabolism to thiol adducts to ensure that they are not susceptible to such interferences during quantification.
Barricklow
Jason
J
Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340, USA.
Ryder
Tim F
TF
Furlong
Michael T
MT
D000111:Acetylcysteine / Q000737:chemistry / Q000493:pharmacokinetics / Q000652:urine*; D016477:Artifacts; D001711:Biotransformation; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry / Q000493:pharmacokinetics / Q000652:urine*; D006801:Humans; D015394:Molecular Structure; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D016482:Urinalysis / Q000379:methods*
D003545:Cysteine; D000111:Acetylcysteine