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 (13)C, (15)N, and (18)O are expected to behave more closely to their corresponding unlabeled analytes, compared with that of the more classically used (2)H-labeled ISs. This study has investigated the behavior of amphetamine, (2)H3-, (2)H5, (2)H6-, (2)H8-, (2)H11-, and (13)C6-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 (2)H-labeled amphetamines increased with the number of (2)H-substitutes. For chromatographic studies we also included seven additional (13)C6-amphetamines and their analytes. All the (13)C6-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 (2)H-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 (13)C-labeled ISs are superior to (2)H-labeled ISs, for analytical purposes.