Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) enables the accurate and precise quantification of various analytes at very low concentrations, but it has room for improvement in analysis throughput. Multiplexing of samples in the same injection could be a promising procedure for enhancing the analysis throughput. This could be achieved by derivatization of the multiple samples with multiple isotopologous reagents. In this study, a sample-multiplexed LC/ESI-MS/MS assay using the 1-[(4-dimethylaminophenyl)carbonyl]piperazine (DAPPZ) isotopologues (2 H0-, 2 H3-and 2 H6-forms) was developed and validated for the simultaneous determination of primary bile acids in three different plasma samples in a single run. The developed method had satisfactory intra-and inter-assay precisions (≤ 2.3% and ≤ 4.2%, respectively) and accuracy (99.0-100.3%), and could reduce the total LC/ESI-MS/MS run time by more than 60% for 42 samples compared to the conventional method. Thus, the derivatization with the DAPPZ isotopologues worked well for enhancing the throughput of the LC/ESI-MS/MS assay of the bile acids.

Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has the great ability to accurately and precisely quantify various biomolecules, but there is a concern about its analysis time, especially during the analysis of a high number of samples. Sample-multiplexing in the same injection is a promising strategy for reducing the total analysis time. This strategy can be accomplished by derivatization of multiple samples with multiple isotopologous reagents. In this study, a sample-triplex LC/ESI-MS/MS assay was developed for quantifying the urinary hexanoylglycine (HG), a diagnostic marker of medium-chain acyl-coenzyme A dehydrogenase deficiency, in three different samples within a single run. For this purpose, the 1-[(4-diethylaminophenyl)carbonyl]piperazine (DEAPPZ) isotopologues ( 2 H 0 -, 2 H 3 -and 2 H 6 -forms) were synthesized. When compared to the non-derivatization method, which analyzed one sample in each run, the analysis time after the sample pretreatment was reduced to 55% (390 min → 210 min) for 30 samples in the sample-triplex method, which also had an acceptable precision (intra-and inter-assay precisions; ≤ 5.9% and ≤ 9.1%, respectively) and accuracy (91.7-97.1%). Thus, the sample-triplex strategy using the DEAPPZ isotopologues could successfully reduce the analysis time in the urinary HG quantification.