Measurement of S-nitrosoalbumin by gas chromatography mass spectrometry - II. Quantitative determination of S-nitrosoalbumin in human plasma using S-[N-15]nitrosoalbumin as internal standard

A gas chromatographic-mass spectrometric method for the quantitative determ ination of S-nitrosoalbumin (SNALB) in human plasma is described. The metho d is based on selective extraction of SNALB and its N-15-labeled SNALB anal og (S(15)NALB) used as internal standard on HiTrapBlue Sepharose affinity c olumns, Hg2+-catalysed conversion of the S-nitroso groups to nitrite and [N -15]nitrite, respectively, followed by their derivatization to the pentaflu orobenzyl derivatives and quantification by CC-MS. Mean recovery of SNALB a nd S15NALB from plasma was 45%. Mean precision and accuracy within the rang e 0-10 mu M was 95% and 99%, respectively. The limit of quantitation was de termined as 100 nM at a precision of 93.8% and an accuracy of 94.8%. Consid erable improvement of method sensitivity is possible by eliminating nitrite present in the elution buffer. The limit of detection was 0.2 nM correspon ding to 67 amol of S(15)NALB. In 0.4-ml aliquots of plasma samples from hea lthy humans, endogenous SNALB was determined at concentrations of 181 +/- 1 50 nM (mean +/- SD, n=23). External addition of SNALB to these plasma sampl es at 2 mu M and 5 mu M serving as quality control samples resulted in quan titative recovery of SNALB. Our results show that SNALB occurs in human pla sma at concentrations at least one-order of magnitude smaller than those re ported in the literature from measurements using chemiluminescence. (C) 199 9 Elsevier Science BN. All rights reserved.