Measurement of Plasma Metabolites

Plasma TMAO, choline, betaine, carnitine, and butyrobetaine were measured at the Cleveland Clinic using stored samples from baseline (1989-1990 or 1992-1993) and follow-up examination (1996-1997). Measurements were performed with stable-isotope dilution liquid chromatography with tandem mass spectrometry using high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry (4000 QTRAP LC-MS/MS System; SCIEX) and corresponding internal standard of d9(trimethyl)TMAO mass spectrometry (LCMS-8050 Triple Quadrupole Liquid Chromatograph Mass Spectrometer; Shimadzu Corporation).^{23 (link)} Concentrations of TMAO and d9(trimethyl)TMAO were monitored using electrospray ionization in positive ion mode with multiple reaction monitoring of parent ion transition m/z 76→58 and characteristic daughter ion transition m/z 85→66. Laboratory coefficients of variation varied by batch throughout the study period and by metabolite; the maximum coefficients of variation observed were 5.8% for TMAO, 4.1% for choline, 5.7% for carnitine, 7.0% for betaine, and 7.9% for butyrobetaine concentrations.