Rippin J J
Department of Immunology, Charing Cross and Westminster Medical School, London, UK.
Clin Chem. 1992 Sep;38(9):1722-4.
Fully oxidized D-neopterin in serum can be measured by HPLC. Serum samples were preincubated with ferric nitrate/EDTA solution to remove any dihydroneopterin, which is unstable and may give spuriously high results because of its conversion to D-neopterin. Pterins were extracted onto solid-phase propylbenzenesulfonic acid minicolumns and eluted with a 1:5 (by vol) mixture of ammonia solution (308 g/L) in acetonitrile. Extracts were evaporated and then reconstituted in mobile phase (50 mL of methanol per liter of 50 mmol/L phosphate buffer, pH 6.2) before injection. Separation was performed with a 25-cm ODS2 column (particle size, 5 microns) at 32 degrees C with fluorescence detection (lambda ex 360 nm, lambda em 440 nm). The between-batch CV was 7.1% and 5.6% for neopterin concentrations of 21.7 and 67.3 nmol/L, respectively. The limit of detection was 0.75 nmol/L, and the mean recovery of the extraction procedure was 90% for neopterin and internal standard. Correlation with a radioimmunoassay (x) gave y = 0.99x + 0.64 (r = 0.970, Sy/x = 2.75). The method allows daily analysis of serum D-neopterin in small batches and is currently used to monitor patients undergoing bone-marrow transplant.
血清中完全氧化的D-新蝶呤可用高效液相色谱法测定。血清样本与硝酸铁/乙二胺四乙酸溶液预孵育以去除任何二氢新蝶呤，二氢新蝶呤不稳定，因其转化为D-新蝶呤可能导致结果假性升高。蝶呤用固相丙基苯磺酸微型柱萃取，并用氨溶液（308 g/L）与乙腈按1:5（体积比）的混合物洗脱。提取物蒸发后，在进样前用流动相（每升50 mmol/L磷酸盐缓冲液，pH 6.2中含50 mL甲醇）复溶。使用25 cm的ODS2柱（粒径5微米）在32℃进行分离，采用荧光检测（激发波长360 nm，发射波长440 nm）。新蝶呤浓度为21.7和67.3 nmol/L时，批间变异系数分别为7.1%和5.6%。检测限为0.75 nmol/L，新蝶呤和内标的萃取回收率平均为90%。与放射免疫分析法（x）的相关性为y = 0.99x + 0.64（r = 0.970，Sy/x = 2.75）。该方法可每日对血清D-新蝶呤进行小批量分析，目前用于监测接受骨髓移植的患者。
