酸性ホスファターゼによるリボフラビンへの変換に基づく前処理法を利用したリン酸リボフラビンナトリウムのHPLC定量

概要

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A simple and rapid method for high-performance liquid chromatographic determination of riboflavin sodium phosphate (FMN) based on its conversion to riboflavin (FR) with acid phosphatase was developed. A mixture for the conversion of FMN to FR contained, in a final volume of 4.0 ml, 1.0 ml of 0.8 M sodium acetate buffer (pH 4.8), 20.0 units of acid phosphatase (Type I : from wheat germ, Sigma Chemical Co.) and less than 0.5 mg of FMN. After incubation for 30 min at 37℃ in an amber test tube with a stopper, the reaction mixture was filtered through a 0.45 μm membrane filter. The filtrate was injected to reversed-phase high performance liquid chromatography equipped with an octadecylsilyl column (L-column ODS, 4.6 mm i.d.×250 mm) and detected for absorbance at 270 nm. FR in the acidic filtrate was stable for at least 24 h. The recoveries of FMN in drugs and soft drinks by this method were 97.7-100.8% with the coefficients of variation of 0.4-2.8%. Impurity FR was detected in all samples under the same conditions without enzymatic hydrolysis. This method could be applied to the separative determination of FR and FMN.
公益社団法人日本薬学会の論文
1995-10-31