测定外周血核黄素及其衍生物含量的HPLC方法研究

DETERMINATION OF RIBOFLAVIN,FMN,AND FAD IN HUMAN PLASMA AND ERYTHROCYTES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

目的：建立测定外周血核黄素及其衍生物FMN、FAD含量的高效液相色谱方法。方法：血浆和红细胞样品用乙腈、三氯甲烷处理，离心后取上清液进样分析。采用Waters Atlantis^TM C18色谱柱（150mm×4．6mmi．d．，5μm）分离，以甲醇-5mmol／L乙酸铵（体积比为35：65）为流动相，流速1．0ml／min，荧光检测器检测（激发波长450nm，发射波长520nm）。结果：本法测定核黄素、FMN、FAD的线性范围均为1-400nmol／L，最低检测限依次分别为0．2、0．25、0．25nmol／L（S／N=3）；日内和日间测定的峰面积的相对标准偏差（RSD）核黄素分别为1．3％，2．2％，FMN分别为2．9％，3．7％，FAD分别为2．0％，3．6％。三者在血浆样品和红细胞样品中的加标回收率均达到要求。结论：本法灵敏、快速、准确，可用于测定血浆和红细胞中核黄素、FMN、FAD含量。【营养学报，2006，28（1）：79-82】

Objective： To develop a high performance liquid chromatographic （HPLC） method for determination of riboflavin, FMN, and FAD in human plasma and erythrocytes. Method： Waters 600 model HPLC pump and an AtlantisTM C18 column （150 mm×4.6 mm i.d. 5 μm） were used. The mobile phase consisted of 35% methanol and 65% 5 mmol/L ammonium acetate solution. The flow rate was 1.0ml/min. The spectro-photofluorimeter was set at wavelength of 450 nm for excitation and 520 nm for emission. Plasma and erythrocyte hemolyzed samples were treated with acetonitrile and chloroform, and the supernatant was analyzed. Results： A good linear correlation existed between riboflavin, FMN and FAD concentration （from 1 to 400 nmol/L） and fluorescence intensity. The detection limits were 2.0 nmol/L, 2.5nmol/L and 2.5nmol/L at a signal to noise ratio of 3, respectively. The intra-assay and inter-assay relative standard deviations were in the range of 1.3% to 3.7%. The recoveries for riboflavin, FMN and FAD in both plasma and erythrocytes were satisfactory. Conclusion. This method for determination of riboflavin, FMN, and FAD in human plasma and erythrocytes is sensitive, rapid and accurate. [ACTA NUTRIMENTA SINICA, 2006, 28（1）： 79-82]