目的: 建立一种超临界流体色谱方法同时分离测定骨化三醇软胶囊中骨化三醇及抗氧剂叔丁羟甲苯(BHT)和丁基羟基苯甲醚(BHA)。方法: 采用ACQUITY UPC2 Torus DIOL(100 mm×3.0 mm,1.7 μm)色谱柱,以超临界二氧化碳-甲醇为流动相,进行梯度洗脱,流速为1.0 mL·min-1,背压为13.8 MPa,柱温为40 ℃,进样量为2 μL,在检测波长265 nm处同时检测骨化三醇及BHT、BHA。结果: 抗氧剂BHT、BHA和主成分骨化三醇在5 min内分离完全,辅料中油酯不影响分离测定。BHT、BHA和骨化三醇质量浓度分别在17.0~85.1、17.1~85.3和0.34~1.71 μg·mL-1范围内与峰面积呈良好线性关系,相关系数分别为0.999 9、0.999 7和0.999 6 (n=5);三者的定量限分别为3.5、2.5和0.2 ng (S/N为10);低、中、高3个浓度水平的平均回收率(n=9)分别为101.5%、101.8%和98.3%,RSD分别为1.8%、2.1%和1.2%。3批样品,平均每粒含骨化三醇0.266、0.264和0.272 μg,含BHT 15.6、15.6和15.8 μg,含BHA 17.0、16.9和17.0 μg。结论: 该方法绿色环保,专属性强,准确性高,分析速度快,可有效用于脂溶性软胶囊中主成分和抗氧剂的同时质量控制。
Objective: To develop a supercritical fluid chromatography method for the simultaneous determination of active pharmaceutical ingredient (API)-calcitriol, as well as two antioxidants-butylated hydroxy toluene (BHT) and butylated hydroxyanisole (BHA), in calcitriol soft capsules. Methods: Calcitriol, BHT and BHA were separated and determined on an ACQUITY UPC2 Torus DIOL column (100 mm×3.0 mm,1.7 μm) maintained at 40 ℃ with mobile phase containing a mixture of CO2 and methanol at 1.0 mL·min-1 in gradient mode. The detection wavelength was set at 265 nm, the back pressure was set at 13.8 MPa and injection volume was 2 μL. Results: BHT, BHA and calcitriol were successfully separated within 5 min with satisfied resolutions. Good linear relationships were established by plotting the peak area versus concentration in the range of 17.0-85.1 μg·mL-1 for BHT, 17.1-85.3 μg·mL-1 for BHA and 0.34-1.71 μg·mL-1 for calcitriol, respectively (r=0.999 9, 0.999 7 and 0.999 6, respectively, n=5). The quantitation limits (LOQs) were 3.5 ng for BHT, 2.5 ng for BHA and 0.2 ng for calcitriol (S/N=10), respectively. The average recoveries (n=9) of BHT, BHA and calcitriol were 101.5%, 101.8% and 98.3% with RSDs of 1.8%, 2.1% and 1.2%, respectively. The average contents were 0.266, 0.264 and 0.272 μg per capsule for calcitriol,15.6, 15.6 and 15.8 μg per capsule for BHT, and 17.0, 16.9 and 17.0 μg per capsule for BHA in three batches of calcitriol soft capsules. Conclusion: The proposed environmentally friendly method demonstrates high efficiency, accuracy, repeatability, and stability. It can be employed for the quality control and stability research of calcitriol, BHT and BHA in calcitriol soft capsules, which might provide a reference for the quality control for other lipophilic drugs.
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