6460 series triple quadrupole lc ms

Plasma aliquots were stored at −40 °C prior to analysis. The plasma TMAO concentrations were quantified using stable isotope dilution liquid chromatography tandem mass spectrometry (6460 Series Triple Quadrupole LC/MS; Agilent) as previously described^{52 (link)}.
The molecular weight of TMAO is 75.22 g/mol. The extracted plasma aliquots were spiked with internal standards comprised of d9-TMAO in methanol. The samples were mechanically vortexed for 1 minute and centrifuged at 15,000 × g for 25 minutes at 4 °C. The analytes were separated on a phenomenex Luna Silica column (4.6 mm × 250 mm, 5 μm particle size) at room temperature. The mobile phase consisted of 80% solvent A (0.1% formic acid in water) and 20% solvent B (methanol) at a flow rate of 0.5 mL/min. The MS/MS analyses for TMAO and d9-TMAO were conducted in the positive multiple reaction monitoring mass spectrometry mode at m/z 76 → 58 and m/z 85 → 66, respectively. To calculate the TMAO concentration, various concentrations of a TMAO standard were added to control plasma to prepare calibration curves. The data were collected and analysed using the chemistry station provided by Agilent Technology.

Overnight fasting (≥8 h) venous blood samples were collected using vacuum tubes containing EDTA. The levels of TMAO and L-carnitine were determined using stable isotope dilution liquid chromatography–tandem mass spectrometry (6460 Series Triple Quadrupole LC/MS; Agilent, Santa Clara, CA, USA). Serum levels of fasting lipids including alanine aminotransferase (ALT), albumin (ALB), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), uric acid (UA), and creatinine obtained at baseline were measured using automatic clinical analyzers (Beckman Coulter) at the Shenzhen Tailored Medical Laboratory.
A standard questionnaire was used to collect information on the socioeconomic status, lifestyle behaviors, and medical history of participants and their family members. Body measurements were conducted for all participants by trained medical staff.

The collected blood samples were immediately processed and stored at −80°C until analysis. Plasma TMAO levels were quantified by stable isotope dilution liquid chromatography tandem mass spectrometry (6460 Series Triple Quadrupole LC/MS; Agilent) using a d9-(trimethyl)-labeled internal standard, as previously described (23 (link)).
In total, 50 μL of plasma was added to a 1.5 mL Eppendorf tube and mixed with 200 μL of 10 μmol/L internal standard composed of d9-TMAO in methanol. The samples were vortexed for 1 min, and then the supernatant was aliquoted following centrifugation at 15,000 g and 4°C for 25 min. Two microliters of supernatant was injected directly into a silica column (4.69 × 250 mm, 5 μm Luna silica, catalog no. 00G-4274-E0; Phenomenex) at a flow rate of 0.5 mL/min⁻¹ with 80% solvent A (0.1% formic acid in water) and 20% solvent B (methanol). TMAO and d9-TMAO were monitored in the positive multiple reaction monitoring mass spectrometry mode using characteristic precursor–production transitions including m/z 76/58 and m/z 85/66, respectively. To calculate TMAO concentrations, serial concentrations of TMAO standard were added to control plasma to prepare a calibration curve.