Tmao d9

In this study, serum TMAO was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample was prepared by adding 10 uL of TMAO d9 (Toronto Research Chemicals Inc., Toronto, Canada) to 100 uL serum; 300 uL of acetonitrile precipitated the protein, vortexed for 1 min, centrifuged at 1,000 rpm, 4°C for 5 min; and 200 uL of the remaining supernatant was injected into a Waters Atlantis HILIC Silica column for analysis.

Sigma Aldrich (St Quentin Fallavier, France) provided indole-3-acetic acid, indoxyl sulfate, hippuric acid, kynurenic acid, TMAO, phenylalanine, kynurenine, indole-3-acetic acid-d5, tryptophan, tyrosine, sodium hydrogen phosphate, formic acid, and dimethylsulfoxide (DMSO). Phenylacetyl-L-glutamine, Phenylacetyl-L-glutamine-d5, PCS, kynurenine-d4, p-cresyl-sulfate-d7, p-cresyl glucuronide, hippuric acid-d5, p-cresyl glucuronide-d7, TMAO-d9, tyrosine-d4, tryptophan-d5, phenylalanine-d5, kynurenic acid-d5, and 3-carboxy-4-methyl-5-propyl-2-furan were provided by Toronto Research Chemicals (North York, Toronto, ON, Canada). VWR Chemicals (Radnor, PA, USA) supplied the sodium dihydrogen phosphate and sodium chloride. Fisher Scientific (Illkirch, France) provided the LC-MS-grade methanol, water, and acetonitrile.

Reference standards, i.e., ADMA, SDMA, and TMAO, as well as internal standards ADMA-d6 and TMAO-d9 were purchased from Toronto Research Chemicals (Toronto, Canada).
Uremic toxins were determined with validated high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) using multiple-reaction-monitoring (MRM) mode on Agilent 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA) coupled to QTRAP 4000 (AB Sciex, Framingham, MA, USA). MRM transitions, declustering potential (DP), and collision energy (CE) were: ADMA, m/z 203 > 46 (DP = 61V, CE = 41V); SDMA, m/z 203 > 172 (DP = 61V, CE = 19V); ADMA-d6, m/z 209 > 77 (DP = 66V, CE = 45V); TMAO, m/z 76 > 42 (DP = 66V, CE = 53V); and TMAO-d9, m/z 85 > 46 (DP = 61V, CE = 59V), respectively. Chromatographic separation was achieved using a SeQuant^® ZIC^®-HILIC (50 × 2.1 mm, 5 μm, Merck (Darmstadt, Germany)) column. The column was maintained at 25 °C at a flow rate of 0.5 mL min⁻¹. The mobile phases consisted of 20 mM ammonium acetate as eluent A and acetonitrile with 0.2% formic acid as eluent B. The gradient (%B) was as follows: 0 min, 95%; 1 min, 95%; 7 min, 50%; and 8 min, 50%. The injection volume was 5 μL. Urine samples (0.1 mL) prior to injection with LC were mixed with internal standards (0.1 mL, 6 μg/mL) and acetonitrile (0.6 mL), vortexed at high speed (3 min), and centrifuged (5 min at 10,000 g).