Measurement of S-nitrosoalbumin by gas chromatography-mass spectrometry. II. Quantitative determination of S-nitrosoalbumin in human plasma using S-[15N]nitrosoalbumin as internal standard.
A gas chromatographic-mass spectrometric method for the quantitative determination of S-nitrosoalbumin (SNALB) in human plasma is described. The method is based on selective extraction of SNALB and its 15N-labeled SNALB analog (S15NALB) used as internal standard on HiTrapBlue Sepharose affinity columns, Hg2+ -catalysed conversion of the S-nitroso groups to nitrite and [15N]nitrite, respectively, followed by their derivatization to the pentafluorobenzyl derivatives and quantification by GC-MS. Mean recovery of SNALB and S15NALB from plasma was 45%. Mean precision and accuracy within the range 0-10 microM was 95% and 99%, respectively. The limit of quantitation was determined as 100 nM at a precision of 93.8% and an accuracy of 94.8%. Considerable improvement of method sensitivity is possible by eliminating nitrite present in the elution buffer. The limit of detection was 0.2 nM corresponding to 67 amol of S15NALB. In 0.4-ml aliquots of plasma samples from healthy humans, endogenous SNALB was determined at concentrations of 181+/-150 nM (mean +/- SD, n = 23). External addition of SNALB to these plasma samples at 2 microM and 5 microM serving as quality control samples resulted in quantitative recovery of SNALB. Our results show that SNALB occurs in human plasma at concentrations at least one-order of magnitude smaller than those reported in the literature from measurements using chemiluminescence.
Tsikas
D
D
Institute of Clinical Pharmacology, Hannover Medical School, Germany. tsikas.dimitros@mh-hannover.de
Sandmann
J
J
Gutzki
F M
FM
Stichtenoth
D O
DO
Frölich
J C
JC
D002138:Calibration; D002846:Chromatography, Affinity; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D009587:Nitrogen Isotopes; D009603:Nitroso Compounds; D010949:Plasma; D010974:Platelet Aggregation; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis*
D009587:Nitrogen Isotopes; D009603:Nitroso Compounds; C091665:S-nitrosoalbumin; D012710:Serum Albumin, Bovine