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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KromasilC18柱(250mm×4.6mm,5μm),流动相为乙腈-0.1mol·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1.0mL.min-1,紫外波长265nm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1.0～200μg·mL-1范围内,线性关系良好(r=0.9995),检测限0.1μg·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 an FMOC pre-column derivatization reversed-phase high performance liquid chromatography (RP-HPLC) for the determination of alliin and its related substances using 9-fluorenylmethoxycarbonyl chloride (FMOC) as pre-column derivatization reagent. Methods: Kromasil C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase consisted of acetonitrile-0.1 mol·L-1 ammonium acetate buffer (adjusted to pH 4.2 with glacial acetic acid) by linear gradient elution at a flow rate of 1.0 mL · min- 1, UV wavelength 265nm detection, and the use of LC-MS / MS identified major related substances. Results: The chromatographic resolution of alliin and its related derivatives was good and the retention was suitable. The linearity was good (r = 0.9995), the detection limit was 0.1μg · mL-1 for alliin in the range of 1.0 ~ 200μg · mL- 1, the precision of injection was good (RSD = 0.28%, n = 6); The structure of impurities in alliin was preliminarily inferred by LC-MS / MS. Conclusion: The method has high specificity, good accuracy and durability, and is suitable for the determination of alliin and related substances.