9-芴甲氧羰酰氯柱前衍生化RP-HPLC法测定蒜氨酸含量及有关物质

目的: 采用9-芴甲氧羰酰氯(FMOC)为柱前衍生化试剂,建立了FMOC柱前衍生化反相高效液相色谱法用于测定蒜氨酸及其有关物质的含量。 方法: 采用Kromasil C₁₈柱(250 mm×4.6 mm,5 μm),流动相为乙腈-0.1 mol·L^-1的醋酸铵缓冲溶液(冰醋酸调pH为4.2),线性梯度洗脱,流速1.0 mL·min^-1,紫外波长265 nm检测,并采用LC-MS/MS法鉴定主要有关物质。 结果: 蒜氨酸及其有关物质衍生物色谱分离度良好,保留适宜;蒜氨酸浓度在1.0~200 μg·mL^-1范围内,线性关系良好(r=0.9995),检测限0.1 μg·mL^-1,进样精密度良好(RSD=0.28%,n=6);根据LC-MS/MS初步推断了蒜氨酸中杂质的结构。 结论: 方法专属性高、准确度和耐用性好,适用于蒜氨酸的含量测定及有关物质检测。

Objective: To establish a reversed-phase high performance liquid chromatographic method with 9-fluorenylmethyl chloroformate pre-column derivatization for the determination of alliin and its related substances. Methods: The analysis was carried out on a Kromasil C₁₈ column (250 mm×4.6 mm, 5 μm) by using acetonitrile-0.1 mol·L^-1 ammonium acetate (adjust to pH 4.2 with glacial acetic acid) as the mobile phase with linear gradient elution mode at a flow rate of 1.0 mL·min^-1, and detection at 265 nm. LC-MS/MS method was employed for the identification of the major related substances. Results: Good retention and separations were achieved for derivatives of alliin and its related substances. The linear calibration was found for alliin within the range of 1.0-200 μg·mL^-1(r=0.9995).The limit of detection was 0.1 μg·mL^-1. The precision was good with RSD of 0.28%(n=6). The structures of major impurities in alliin were identified by LC-MS/MS. Conclusion : The established method is specific and accurate with very good ruggedness and suitable for the determination of alliin and its related substances.