Varian l1200 ms ms

After thawing of frozen PRP and PPP samples, the level of S1P has been measured by liquid chromatography-tandem mass spectrometry as previously described [87 (link)]. In brief, 20 μL of PRP and PPP respectively were incubated with 20 μL of the internal standard (1 μM [16,17,18-²H₇]-S1P (S1P-d₇, Avanti Polar Lipids, Alabaster, AL, USA). Subsequently, proteins were precipitated with 350 μL of acetonitrile/water, 80/20, vol/vol. After centrifugation at 10,000 g for 15 min the extracts were subjected to reverse-phase chromatography on a Zorbax SB-C8 column (2.1 × 50 mm; Agilent Technologies, Santa Clara, CA, USA) at a flow rate of 0.35 mL/min. S1P was eluted with a binary gradient for 6 min (methanol/acetonitrile/0.1% formic acid, 2.5/2.5/95, vol/vol/vol to methanol/acetonitrile/0.1% formic acid, 30/30/40, vol/vol/vol) and measured by tandem mass spectrometry (Varian L1200 MS/MS, Agilent Technologies, Waldbronn, Germany) in the multiple reaction mode, monitoring the [M+H]⁺ S1P parent ion (m/z = 380) fragmentation to the daughter ion m/z = 264. The internal standard S1P-d₇ with the m/z 387 to 271 transition was used to correct for variations in sample preparation and instrument response. Calibration curves (four levels of S1P: 0; 0.1; 0.3; 1; 3 μmol/L) were generated to calculate absolute S1P concentrations in PRP and PPP samples.

S1P was measured in serum of 141 AF patients whereas 21 had FU samples and data accessible. S1P was determined by LC-MS/MS as previously described [24 (link)]. In brief, 20 µL of serum sample were incubated with 20 µL of internal standard 1 µM [16,17,18-²H₇]-S1P (S1P-d₇, Avanti Polar Lipids, Alabaster, AL, USA) and subsequently proteins were precipitated by addition of 350 µL of acetonitrile/water, 80/20 (vol/vol). The supernatant was subjected to reverse-phase chromatography on a Zorbax SB-C8 (2.1 × 50 mm; Agilent Technologies, Santa Clara, CA, USA). S1P was eluted with 0.35 ml/min as a binary gradient for 6 min (methanol/acetonitrile/0.1% formic acid: 2.5/2.5/95–30/30/40, vol/vol/vol) and quantified by tandem mass spectrometry (Varian L1200 MS/MS, Agilent Technologies, Waldbronn, Germany).