Nierenberg D W, Lester D C
J Chromatogr. 1985 Dec 13;345(2):275-84. doi: 10.1016/0378-4347(85)80165-1.
A method of sample clarification and high-performance liquid chromatography specifically developed to permit precise and rapid determination of vitamin A (retinol) and vitamin E (alpha-tocopherol) in serum and plasma is reported. Serum proteins were denatured by the addition of acetonitrile containing alpha-tocopherol acetate, the internal standard; the vitamins were subsequently extracted into an organic matrix consisting of ethyl acetate-butanol (1:1); no solvent evaporation step was required. The three vitamins of interest were eluted from a reversed-phase C18 column with an isocratic mobile phase methanol-water (95:5); detection was accomplished by measuring ultraviolet absorption at 280 nm. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from spiked aqueous samples averaged 100.0, 100.0 and 98.8%, respectively. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from plasma and serum relative to water were 102.6, 96.9 and 96.5%, respectively. Retinol and alpha-tocopherol were stable in the extraction matrix for up to 3.5 h, and were stable in heparinized plasma stored at room temperature for two days. Oxalate, citrate and EDTA caused significant losses of retinol and alpha-tocopherol, while vitamin levels in serum and heparinized plasma were similar. Limits of detection for retinol and alpha-tocopherol were 60 ng/ml and 0.9 micrograms/ml, respectively. Each run required 12 min. Same-day coefficients of variation were 3.5 and 3.6% for retinol and alpha-tocopherol, respectively (n = 11). Between-day coefficients of variation for retinol and alpha-tocopherol were 4.8 and 5.5%, respectively (n = 5). This method permits simple, rapid, sensitive, selective and precise determination of retinol and alpha-tocopherol using 0.5 ml of serum or heparinized plasma.
报道了一种专门开发的样品澄清和高效液相色谱法，用于精确快速地测定血清和血浆中的维生素A（视黄醇）和维生素E（α-生育酚）。通过加入含有内标α-生育酚乙酸酯的乙腈使血清蛋白变性；随后将维生素萃取到由乙酸乙酯-丁醇（1:1）组成的有机基质中；无需溶剂蒸发步骤。用甲醇-水（95:5）等度流动相从反相C18柱上洗脱三种目标维生素；通过测量280nm处的紫外吸收进行检测。加标水样中视黄醇、α-生育酚和α-生育酚乙酸酯的回收率分别平均为100.0%、100.0%和98.8%。相对于水，血浆和血清中视黄醇、α-生育酚和α-生育酚乙酸酯的回收率分别为102.6%、96.9%和96.5%。视黄醇和α-生育酚在萃取基质中长达3.5小时稳定，在室温下储存两天的肝素化血浆中也稳定。草酸盐、柠檬酸盐和乙二胺四乙酸会导致视黄醇和α-生育酚显著损失，而血清和肝素化血浆中的维生素水平相似。视黄醇和α-生育酚的检测限分别为60ng/ml和0.9μg/ml。每次运行需要12分钟。视黄醇和α-生育酚当日变异系数分别为3.5%和3.6%（n = 11）。视黄醇和α-生育酚日间变异系数分别为4.8%和5.5%（n = 5）。该方法使用仅0.5ml血清或肝素化血浆即可简单、快速、灵敏、选择性且精确地测定视黄醇和α-生育酚。
