C-13(4)-secobarbital as the internal standard for the quantitative determination of secobarbital - A critical evaluation

In this study, C-13(4)-secobarbital was used as an exemplar compound to ill ustrate the mechanism based on which the effectiveness of a proposed intern al standard (IS) could be evaluated. A deuterated analog. H-2(5)-secobarbit al, 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 designate d for the analyte (secobarbital) by the proposed IS, and similarly, the con tribution of the intensity of an ion designated for the IS dy the analyte-a phenomenon termed "cross-contribution"-were evaluated based on a "direct m easurement" procedure in which equimolar amounts of the analyte and the IS were used to generate intensity darn. These intensity data were then used a s the basis for the calculation of "cross-contribution" (in percentages) of ions designated for the analyte and the IS. Cross-contribution data were c ompared with the linearity data resulting from two series of standards cont aining 25 to 9600 ng/mL secobarbital using two sets of quantitation ion pai rs-m/z 196/200 and 195/199 with C-13(4)-secobarbital as the IS and m/z 196/ 201 and 195/200 with H-2(5)-secobarbital as the IS. C-13(4)-secobarbital wa s found to be much less problematic and thus can serve as a very effective IS. Cross-contribution data alone cannot fully explain the observed differe nces resulting from the use of these two ISs; further systematic study is n eeded to provide better understanding of the underlying interference mechan ism.