An LC/ESI-MS/MS Method for Quantification of Hexanoylglycine in Three Different Urine Samples Within a Single Run Using Triplex 1-[(4- Diethylaminophenyl)carbonyl]piperazine Isotopologues

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.