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

• Several new 13C labeled internal standards (ISs) used for the first time as ISs.
• 13C labeled ISs better suited than 2H labeled ISs in RP LC–MS/MS analysis.
• 13C labeled ISs behave as their analytes in chromatographic separations.
• ISs with few 2H are better suited than those with many 2H substitutes.
• No differences between extraction recovery of 13C- and 2H ISs were observed.

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