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

Oct 11, 2017Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

Fast urine test using a special column and liquid chromatography to measure trimethylamine N-oxide and trimethylamine at the same time

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Abstract

A new assay can simultaneously measure trimethylamine N-oxide (TMAO) and trimethylamine (TMA) in urine with a limit of quantitation of 1.0μg/mL for TMAO and 0.025μg/mL for TMA.

The method uses a simple dilute and shoot approach with a 100-fold dilution and a small sample volume of 10μL.
Analyses are completed in 4.0 minutes using LC-MS/MS detection without the need for sample cleanup or derivatization.
The assay demonstrated wide linearity for TMAO (1.0-400μg/mL) and TMA (0.025-10μg/mL).
No interference from 41 major urinary components or related drugs was observed during quantitation.
A significant correlation was found between the new spot urine sampling method and traditional 24-hour urine collection (p<0.0001).
The reliability of the assay was confirmed through reference intervals established in a cohort of 118 healthy individuals.

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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.

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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

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