Mass spectrometry (MS) analysis of ADMA

Fasting blood samples were collected in ethylenediaminetetraacetic acid (lavender top) tubes and isolated plasma stored at −80°C. Samples were thawed on ice the day of analysis. Plasma ADMA levels were quantified using stable isotope dilution LC/ESI/MS/MS assays using an upgraded ABI 365 triple quadrupole mass spectrometer (Applied Biosystems Inc., Foster City, CA, USA) with Ionics EP 10+ redesigned source (Concord, Ontario, Canada) and electrospray ionization (ESI) needle connected to an Aria LX4 series multiplexed HPLC system with Flux pumps (Cohesive Technologies, Franklin, MA, USA), as previously described¹⁰. MS analyses were performed online using LC/ESI/MS/MS in the positive ion mode with multiple reaction monitoring using unique parent → daughter ion transition and retention time unique for ADMA, and its stable isotope labeled internal standard. Intra-assay and inter-assay coefficients of variance were <10%.