13C4-secobarbital as the internal standard for the quantitative determination of secobarbital--a critical evaluation

In this study, 13C4-secobarbital was used as an exemplar compound to illustrate the mechanism based on which the effectiveness of a proposed internal standard (IS) could be evaluated. A deuterated analog, 2H5-secobarbital, was also studied in parallel for comparison purposes. Well-established solid-phase extraction and methylation procedures were used prior to the GC/MS measurement step. The contribution of the intensity of an ion designated for the analyte (secobarbital) by the proposed IS, and similarly, the contribution of the intensity of an ion designated for the IS by the analyte--a phenomenon termed "cross-contribution"-were evaluated based on a "direct measurement" procedure in which equimolar amounts of the analyte and the IS were used to generate intensity data. These intensity data were then used as the basis for the calculation of "cross-contribution" (in percentages) of ions designated for the analyte and the IS. Cross-contribution data were compared with the linearity data resulting from two series of standards containing 25 to 9600 ng/mL secobarbital using two sets of quantitation ion pairs--m/z 196/200 and 195/199 with 13C4-secobarbital as the IS and m/z 196/201 and 195/200 with 2HS-secobarbital as the IS. 13C4-secobarbital was found to be much less problematic and thus can serve as a very effective IS. Cross-contribution data alone cannot fully explain the observed differences resulting from the use of these two ISs; further systematic study is needed to provide better understanding of the underlying interference mechanism.