Casoli P, Vérine H
INSERM, Marseille, France.
Biomed Chromatogr. 1990 Sep;4(5):209-13. doi: 10.1002/bmc.1130040508.
A convenient high performance liquid chromatographic method for the determination of methylxanthines in biological samples is described. Separation was achieved by reversed phase chromatography using a mobile phase consisting of tetrahydrofuran + methanol + 0.01M potassium dihydrogen phosphate, pH 3.5 (1:20:79, v/v/v), on a 7 microns C18 column and a C18 Lichrosorb precolumn at a flow rate of 0.8 mL/min. Levels varying from 0.25-16 mg/L could be detected by UV at 280 nm. In this range, standard curves were established for 4 methylxanthines: theobromine, paraxanthine, theophylline and caffeine in 4 media: mobile phase, serum, gastric and pancreatic juices, and were found to be linear (r greater than or equal to 0.9975). Overall characteristics of the method were determined as: percent recovery (89.54%), accuracy (greater than or equal to 99.4%) and reproducibility (greater than or equal to 95%). Retention times ranged from 4.21 +/- 0.01 (1-methyluric acid) to 10.8 +/- 0.03 min (caffeine). Animal experiments (5 and 10 mg/kg boluses) were used to determine caffeine half life in dog's blood (310 +/- 46 and 453 +/- 59 min, respectively) and its secretion into pentagastrin stimulated gastric juice (mean concentrations 2.51 and 6.04 mg/L; mean outputs 351 and 1206 micrograms/2.25 h; both statistically different at p less than 0.001 level).
本文描述了一种用于测定生物样品中甲基黄嘌呤的简便高效液相色谱法。采用反相色谱法进行分离，流动相为四氢呋喃+甲醇+0.01M磷酸二氢钾，pH 3.5（1:20:79，v/v/v），使用7微米C18柱和C18 Lichrosorb预柱，流速为0.8 mL/min。在280 nm处通过紫外检测可检测到浓度范围为0.25 - 16 mg/L的甲基黄嘌呤。在此范围内，针对4种甲基黄嘌呤：可可碱、副黄嘌呤、茶碱和咖啡因，在4种介质：流动相、血清、胃液和胰液中建立了标准曲线，发现呈线性（r大于或等于0.9975）。该方法的总体特性确定为：回收率（89.54%）、准确度（大于或等于99.4%）和重现性（大于或等于95%）。保留时间范围从4.21±0.01（1 - 甲基尿酸）到10.8±0.03分钟（咖啡因）。动物实验（5和10 mg/kg推注剂量）用于测定狗血液中咖啡因的半衰期（分别为310±46和453±59分钟）及其分泌到五肽胃泌素刺激的胃液中的情况（平均浓度2.51和6.04 mg/L；平均输出量351和1206微克/2.25小时；在p小于0.001水平时两者均有统计学差异）。
