A simple dilute and shoot approach incorporated with pentafluorophenyl (PFP) column based LC-MS/MS assay for the simultaneous determination of trimethylamine N-oxide and trimethylamine in spot urine samples with high throughput.

Determination of trimethylamine N-oxide (TMAO) and trimethylamine (TMA) in biological and environmental samples has drawn great attention recently due to their increasing association with human health and disease. It remains a challenge to simultaneously quantify TMAO and TMA in a simple, fast and cost-effective manner due to pre-analytical and analytical constraints. For the first time, we describe a dilute and shoot approach combined with LC-MS/MS detection for the simultaneous measurement of the analytes in spot urine samples with high throughput. Compared to the existing methods, the merits of the proposed assay include the use of a simple dilute and shoot approach (100-fold), small sample volume (10μL), short LC run on a PFP column (4.0min) and multi-analyte MS detection without sample cleanup, derivatization, evaporation and a HILIC column. Dilution, LC and MS parameters were optimized in detail. Method validation yielded a wide linearity for TMAO (1.0-400μg/mL) and TMA (0.025-10μg/mL) with a respective limit of quantitation of 1.0 and 0.025μg/mL. The quantitation was not affected by 41 major urinary components, structurally-related drugs and metabolites. The intra- and inter-day assay precisions were ≤3.6% and recoveries were 93.3%-103.3% for spiked quality control samples. The clinical utility of the alternative spot urine sampling approach compared to conventional 24h urine collection was supported by a significant correlation between the two sampling strategies (n=20, p<0.0001, r=0.757-0.862; slope=0.687-1.170) and no statistical difference in day-to-day biological variability (n=20). The applicability and reliability of the assay was verified by the assessment of reference intervals in a cohort of 118 healthy people. The proposed assay would be beneficial for the rapid and accurate determination of the increasingly important TMAO and TMA demanded in clinical, environmental, pharmaceutical and nutritional fields.