Varian 1200 ms

Analysis of ADMA and L-arginine in plasma was performed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a validated and published method [11 (link)]. Briefly, 25 μL of plasma was diluted with stable isotope-labeled internal standards. Proteins were precipitated with methanol; the guanidine compounds were converted to their butyl esters and analyzed by LC–MS/MS (Varian 1200 MS, Agilent Technologies, Santa Clara, CA, USA). Quantification was performed by calculation of peak area ratios and calibration with known concentrations of analytes in dialyzed EDTA plasma. The analytical range of the method was validated from 0.05 to 4 μmol/L for ADMA and SDMA, respectively, and from 0.5 to 250 μmol/L for L-arginine, and mean coefficients of variation were ≤ 5% for all analytes. All other laboratory values were measured using routine clinical laboratory methods.

At baseline, non-fasting blood and CSF samples were drawn from arterial catheters (N = 51) and external ventricular drains (when available, N = 38), respectively. For ethical reasons, no patient received any interventional measures for study-related sample collection. Samples were centrifuged immediately and stored at −20 °C until analysis. ADMA, SDMA, and L-arginine were determined by liquid chromatography-tandem mass spectrometry (LC–MS/MS) using a previously validated method [40 (link)]. In brief, 25 μL of plasma or CSF was diluted with stable isotope-labeled internal standards. Proteins were precipitated with methanol; the guanidine compounds were converted to their butyl esters and analyzed by LC–MS/MS (Varian 1200 MS, Agilent Technologies, Santa Clara, CA, USA). Quantification was performed by calculation of peak area ratios and calibration with known concentrations of analytes in dialyzed EDTA plasma. The analytical range of the method was validated from 0.05 to 4 μmol/L for ADMA and SDMA, respectively, and from 0.5 to 250 μmol/L for L-arginine, and mean coefficients of variation were ≤5% for all analytes. All other laboratory values were measured using routine clinical laboratory methods.

Plasma L-homoarginine was measured from ethylenediaminetetraacetic acid (EDTA) plasma aliquots stored at −80 °C using electrospray ionization-liquid chromatography-mass spectrometry with a high-throughput mass spectrometric assay. Briefly summarized, by adding 100 µL of internal standard (2.5 µmol/L [¹³C₆]-homoarginine) disbanded in methanol to 25 µL of EDTA plasma, proteins were precipitated. The aliquots were centrifuged, vaporized, and afterwards translated to their butyl ester derivatives applying butanolic 1N hydrochloric acid (HCl). After repeated centrifugation, the eluates were dried and again dissolved in 100 µL of methanol:water (25:75) containing 0.1% ammonium format. The plates were positioned in a CTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland)) and 20 µL samples were injected. Further testing was performed with the mass spectrometer system (Varian 1200 MS, Agilent Technologies, Santa Clara, CA, USA). Lower threshold of quantification for homoarginine was set to be 0.01 µmol/L. Intra- and interassay coefficients of variation were ≤7.5%. Creatinine was determined by routine laboratory method.