Evaluation of 13C- and 2H-labeled internal standards for the determination of amphetamines in biological samples, by reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry

Stable isotope-labeled internal standards (SIL-ISs) are often used when applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze for legal and illegal drugs. ISs labeled with C-13, N-15, and O-18 are expected to behave more closely to their corresponding unlabeled analytes, compared with that of the more classically used H-2-labeled ISs. This study has investigated the behavior of amphetamine, H-2(3)-, H-2(5,) H-2(6)-, H-2(8)-, H-2(11)- and C-13(6)-labeled amphetamine, during sample preparation by liquid-liquid extraction and LC-MS/MS analyses. None or only minor differences in liquid-liquid extraction recoveries of amphetamine and the SIL-ISs were observed. The chromatographic resolution between amphetamine and the H-2-labeled amphetamines increased with the number of H-2-substitutes. For chromatographic studies we also included seven additional C-13(6)-amphetamines and their analytes. All the C-13(6)-labeled ISs were co-eluting with their analytes, both when a basic and when an acidic mobile phase were used. MS/MS analyses of amphetamine and its SIL-ISs showed that the ISs with the highest number of H-2-substitutes required more energy for fragmentation in the collision cell compared with that of the ISs with a lower number. The findings, in this study, support those of previous studies, showing that C-13-labeled ISs are superior to H-2-labeled ISs, for analytical purposes. (C) 2014 Elsevier B.V. All rights reserved.