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参照美国药典USP37-NF32版,采用柱前衍生化-HPLC法测定庆大霉素C1a(1)对照品及生产阶段的不同样品。在检测经离子交换树脂HZ-732富集后的洗脱液样品B时出现1个新的杂质峰,且检测所得1的浓度明显偏低。经LCMS分析,推测该杂质为邻苯二甲醛(OPA)不足的情况下1与巯基乙酸在碱性条件下的结合产物。经方法优化,通过稀释样品、增加衍生化试剂量及增加衍生化试剂中OPA的量,均可准确检测不同样品中1的浓度和纯度。向衍生化试剂中增加OPA,更适用于1发酵生产过程中不同阶段样品的检测。
Reference to the United States Pharmacopoeia USP37-NF32 version, pre-column derivatization-HPLC method for the determination of gentamicin C1a (1) reference substance and different samples of the production stage. A new impurity peak appeared on the elution sample B after the ion exchange resin HZ-732 was enriched, and the concentration of the test 1 was obviously lower. LCMS analysis suggested that this impurity was a binding product of thioglycolic acid under basic conditions in the case of insufficient phthalaldehyde (OPA). By method optimization, the concentration and purity of 1 in different samples can be accurately detected by diluting the sample, increasing the amount of derivatized reagent, and increasing the amount of OPA in the derivatization reagent. OPA is added to the derivatization reagent, which is more suitable for the detection of samples in different stages in a fermentative production process.