硫酸卡那霉素及注射液有关物质高温型及低温型蒸发光检测器方法建立及比对

目的：建立硫酸卡那霉素及注射液有关物质高温型及低温型蒸发光检测器方法并进行结果比对。方法：高温型蒸发光检测器方法为采用Waters XSelect^® HSS T3（5 μm，4.6 mm×250 mm）色谱柱，以0.2 mol·L^-1三氟醋酸溶液为流动相，流速0.3 mL·min^-1，柱温30℃；蒸发光散射检测器漂移管温度110℃，载气流量2.5 L·min^-1。低温型蒸发光检测器方法为采用Agilent ZORBAX SB-C₁₈（5 μm，4.6 mm×250 mm）色谱柱，以0.2 mol·L^-1三氟醋酸溶液-甲醇（98∶2）为流动相，流速0.3 mL·min^-1，柱温30℃；蒸发光散射检测器漂移管温度65℃，喷雾器比率40%，压力0.207 MPa。结果：采用高温型蒸发光检测器，线性范围为0.003~0.12 mg·mL^-1（r=0.999 6），检测下限和定量下限分别为0.031 μg和0.062 μg；采用低温型蒸发光检测器，线性范围为0.003~0.12 mg·mL^-1（r=0.999 1），检测下限和定量下限分别为0.022 μg和0.051 μg。2种方法的专属性，流动相比例和色谱柱的耐用性均符合要求。4批硫酸卡那霉素和6批注射液的检测结果比对表明卡那霉素B的检测结果差异不大，其他单个杂质和杂质总量低温型检测器检测结果均高于高温型检测器。结论：本文建立的2种有关物质检测方法均适用于硫酸卡霉素及其注射液的有关物质检查，低温型检测器的最大杂质为相对保留时间约为0.85的未知杂质，高温型检测器的最大杂质也为主峰前杂质，但与其他杂质峰的未达到基线分离。对杂质的分离能力，低温型检测器更有优势。同时，低温型检测器杂质检出量和检出个数均优于高温型检测器，2种方法的建立为进一步制定合理限度进行有关物质控制以及杂质解析奠定了研究基础。

Objective: To establish and compare two HPLC-ELSD[high temperature (high TEMP)/low temperature] (low TEMP)methods for related substance analysis in kanamycin sulfate and its injection. Methods: For the HPLC-ELSD (high TEMP) method, a Waters XSelect^® HSS T3 (5 μm, 4.6 mm×250 mm) was used as chromatographic column at the column temperature of 30, 0.2 mol·L^-1 trifluoroacetic acid solution was used as the mobile phase at a flow rate of 0.3 mL·min^-1;the temperature of ELSD drift tube was set at 110℃, and the carrier gas flow was 2.5 L·min^-1. For the HPLC-ELSD (low TEMP) method, an Agilent ZORBAX SB-C₁₈ (5 μm, 4.6 mm×250 mm) was used as chromatographic column at the column temperature of 30, a mixture of 0.2 mol·L^-1 trifluoroacetic acid solution-methyl alcohol (98:2) was used as the mobile phase at a flow rate of 0.3 mL·min^-1;the temperature of ELSD drift tube was set at 65℃, the spray ratio was 40%, and the pressure was 0.207 MPa. Results: For the HPLC-ELSD (high TEMP) method, it reflected a good linearity in the range of 0.003-0.12 mg·mL^-1 (r=0.999 6), and the LOD and LOQ were 0.031μg and 0.062 μg, respectively. For the HPLC-ELSD (low TEMP) method, it reflected a good linearity in the range of 0.003-0.12 mg·mL^-1 (r=0.999 1), and the LOD and LOQ were 0.022 μg and 0.051 μg, respectively. The specificity, mobile phase ratio, and chromatographic column durability of the two methods all met the requirements. The detection results of four batches of kanamycin sulfate and six batches of kanamycin sulfate injection showed that the difference between detection results of kanamycin B was small. However, for other single impurities and the impurity amount, the detection results of HPLC-ELSD (low TEMP) method were all higher than those of HPLC-ELSD (high TEMP) method. Conclusion: The two methods established in this paper are both appropriate for the detection of related substance in kanamycin sulfate and its injection. The impurity separation effect of HPLC-ELSD (low TEMP) method is better than that of HPLC-ELSD (high TEMP) method. The biggest impurity of HPLC-ELSD (low TEMP) is the unknown impurity with a relative retention time of 0.85. The biggest impurity of HPLC-ELSD (high TEMP) is also the impurity before the main peak, which doesn't reach the baseline separation with peaks of other impurities. The quantity and number of impurities detected by HPLC-ELSD (low TEMP) method are superior to or more than those of impurities detected by HPLC-ELSD (high TEMP) method. This paper will lay a foundation for further establishment of a reasonable limit for impurity control and analysis.