Selecting an appropriate isotopic internal standard for gas chromatography/mass spectrometry analysis of drugs of abuse pentobarbital example

Internal standards are commonly used for the quantitative determination of drags of abuse and their metabolites (drug/metabolite) in biological fluids and tissues by the selective ion monitoring (SIM) gas chromatography/mass spectrometry (GC/MS) procedure. Analogs of drugs/metabolites that are labeled with three or more deuterium atoms (isotopic analog) at appropriate positions are considered to be the most effective internal standards for these applications. Before a specific deuterated analog can be adopted as an internal standard in a GC/MS assay, the mass spectrum of the compound or its derivative must be evaluated along with the corresponding spectrum from the parent drug/metabolite. There should be an adequate number of sufficiently high-mass ions (typically three for the drug/metabolite and two for the isotopic analog) that can be attributed to each analyte, and these ions should be sufficiently free of interference from the other analyte of the pair (cross-contribution). Interferences may be caused by the presence of an isotopic impurity in the deuterated analog (extrinsic factor) or may be due to the ion fragmentation characteristics of the compound (intrinsic factor). The extrinsic factor may be corrected by the manufacturer with different synthetic methods and purification procedures, while the intrinsic factor may be partially or wholly corrected through the use of different chemical derivatives (sample preparation stage) or different ionization (GC/MS assay stage) procedures. In this study, pentobarbital/d₅-pentobarbital is used as the exemplar analyte/deuterated analog pair to illustrate the ion selection and evaluation procedures. Full-scan mass spectra were employed for preliminary ion selection. SIM data were then used to calculate the extent, if any, of cross-contributions. SIM ion chromatograms obtained under a lower GC oven temperature were used to differentiate sources (ion fragmentation mechanism versus isotopic impurity) of cross-contributions.