D9 tmao

Fasting blood samples were drawn within 24 hours of patients' hospital admission. Plasma samples were separated at clinical laboratories of each participating hospital and immediately frozen at -80 °C. Choline pathway nutrients and metabolites were quantified on an Ultimate 3000 ultra-high performance liquid chromatograph coupled with Q Exactive quadrupole-Orbitrap high-resolution mass spectrometer system (Thermo Scientific) using d9-choline, d11-betaine, and d9-TMAO as internal standards (Cambridge Isotope Laboratories, Inc, Andover). The metabolites were chromatographically resolved on an Acquity HILIC amide column (Waters, Co; 1.7 μm, 2.1×100 mm) after 2 μL aliquots of metabolites extract injected, and 10% water in acetonitrile as weak eluent and 50% acetonitrile in water as strong eluent which were both added ammonium formate and formic acid as buffer salt to improve separation. The chromatographic gradient ramped from 100% weak elution to 30% in 9 minutes; meanwhile, the flow rate was set at 0.3 mL/min. After separation, the metabolites were ionized by a heated electrospray ionization source and then detected by parallel reaction monitoring mode. The main parameters were optimized as follows: positive

Plasma levels of l-carnitine and trimethylamine N-oxide (TMAO) in mice were analyzed using liquid chromatography-mass spectrometry (LC-MS) as previously described by Koeth et al., 2013 [8] 8. Koeth, R.A. . In brief, 50 μL of the mouse plasma samples were pipetted into a 2 mL centrifuge tube, where 50 μL of HPLC grade acetonitrile and 100 μL of internal standard solution were then added. All samples were targeted to contain 1 μg/mL synthetic d9 -l-carnitine hydrochloride (Sigma Aldrich) and 0.1 μg/mL d9-TMAO (Cambridge Isotope Laboratories, Tewksbury, MA). The mixture was vortexed for 2 min, followed by centrifugation. The upper supernatant was taken for HPLC-MS/MS analysis, using an Agilent 1200 HPLC and 6410 triple quadrupole MS system. The elution was carried out on a Phenomenex Synergi 4 μm Polar-RP column (150 × 4.6 mm, 4 μm, part No. 00F-4336-E0). All analyses was performed at a column temperature of 30 ± 1 °C with a mobile phase of 10 mM ammonium formate and methanol. Detection and quantification of l-carnitine and TMAO levels (ppm) was achieved by ESI-MS/MS operating in the positive ion mode and the developed method was validated as per the requirements for the ICH guidelines.