Capillary electrophoresis with dual diode array detection and tandem mass spectrometry to access cardiovascular biomarkers candidates in human urine: Trimethylamine-N-Oxide and l-carnitine.

A capillary electrophoresis with diode array and tandem mass spectrometry detection (CE-UV-MS/MS) method has been developed for the targeted assessment of cardiovascular biomarkers candidates, trimethylamine-N-Oxide (TMAO) and l-carnitine, and creatinine in human urine samples. The dual detection was applied due to the high concentration of creatinine (monitored by UV detection at 200 nm) in relation to TMAO and l-carnitine (quantified by selected reaction monitoring (SRM) mass spectrometry), in human urine. All instrumental parameters, sheath liquid (SHL) and background electrolyte (BGE) compositions were optimized with a pool of urine provided by adult healthy volunteers and evaluated by signal-to-noise ratio (SNR) and peak shape of TMAO. The compositions for the optimized BGE was formic acid at concentration of 0.10 mol L-1, and for SHL was 70:30 MeOH:H2O containing 0.05% (v/v) formic acid, delivered at a flow rate of 5 μL min-1. Limits of detection for TMAO, l-carnitine and creatinine were 0.76, 0.54 and 303 μmol L-1, respectively. Limits of quantification were 2.5, 1.8 and 1000 μmol L-1, respectively. Linearity was evaluated by ANOVA and presented R2 from 0.993 to 0.997. Precision and accuracy were evaluated at three concentration levels. Coefficients of variation (CV) from 1 to 21% were obtained for the intra-day precision evaluation and from 2 to 16% for the inter-day precision evaluation. The recovery ranged from 75 to 116%. Quantitation of TMAO and l-carnitine in infarcted patients urine in comparison to healthy individuals indicated a 2.2 fold increase of TMAO and a 7.0 fold increase of l-carnitine. These results showed the potential applicability of the proposed method for the evaluation of TMAO and l-carnitine in urine within a panel of candidate metabolites in targeted metabolomics studies of cardiovascular diseases among other conditions.