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目的:对维U颠茄铝胶囊中维生素U含量测定的方法进行改进,根据维生素U的特殊结构,建立柱前衍生化HPLC检测法测定其含量。方法:以二硝基氟苯(FDNB)为柱前衍生剂,在碳酸钠-碳酸氢钠缓冲液(pH 10.0)介质中进行衍生化反应;以HPLC法测定含量,采用Agilent TC-C18(250 mm×4.6 mm,5μm)色谱柱,以0.01 mol·L~(-1)乙酸钠溶液(用乙酸调pH至6.5)-乙腈(70∶30)为流动相,流速1.0m L·min~(-1),检测波长360 nm。结果:2种结构维生素U(氯甲基蛋氨酸和碘甲基蛋氨酸)的衍生产物色谱分离效果均良好,氯甲基蛋氨酸和碘甲基蛋氨酸质量浓度分别在为20.12~201.20和29.23~292.31μg·m L~(-1)范围内线性关系良好,氯甲基蛋氨酸和碘甲基蛋氨酸的加样回收率分别为97.33%~98.74%和97.71%~99.24%,RSD(n=6)分别为1.95%~2.52%和1.85%~2.78%;以本文的柱前衍生化HPLC法测得的不同厂家的3批样品中维生素U含量分别为99.06%、99.73%、100.52%,传统滴定法测得的含量分别为101.07%、102.26%、99.79%,两法结果基本一致。结论:方法学验证结果表明,本文建立的方法可以用于测定维U颠茄铝胶囊制剂中维生素U的含量。
OBJECTIVE: To improve the method for the determination of vitamin U in Victoria U belladonna capsule, according to the special structure of vitamin U, a pre-column derivatization HPLC assay was established to determine its content. Methods: The derivatization reaction was carried out in the medium of sodium carbonate-sodium bicarbonate buffer (pH 10.0) with dinitrofluorobenzene (FDNB) as the pre-column derivatization reagent. The content of the product was determined by HPLC with Agilent TC-C18 mm × 4.6 mm, 5 μm) with a mobile phase of 0.01 mol·L -1 sodium acetate (adjusted to pH 6.5 with acetic acid) -acetonitrile (70:30) at a flow rate of 1.0 mL · min -1 -1), detection wavelength of 360 nm. Results: The chromatographic separation results of the two derivatives of vitamin U (chloromethyl methionine and iodomethyl methionine) were good. The concentrations of chloromethyl methionine and iodomethyl methionine were in the range of 20.12 ~ 201.20 and 29.23 ~ 292.31μg · The linear recoveries of chloromethyl methionine and iodomethyl methionine were 97.33% ~ 98.74% and 97.71% ~ 99.24%, respectively, and RSD (n = 6) were 1.95 % ~ 2.52% and 1.85% ~ 2.78%. The vitamin U contents in three batches of samples from different manufacturers measured by this method were 99.06%, 99.73% and 100.52%, respectively. Content were 101.07%, 102.26%, 99.79%, the results of the two methods are basically the same. Conclusion: The results of methodological verification show that the method established in this paper can be used to determine the content of vitamin U in the preparation of U-belladonna aluminum capsule.