目的: 建立一种氨基固相萃取-HPLC法测定艾地骨化醇软胶囊中3个同分异构体的有关物质。方法: 采用氨基固相萃取法,经乙酸乙酯-正己烷洗脱,无水乙醇提取,提取液经减压蒸干后用乙腈复溶,经ODS色谱柱(250 mm×4.6 mm,5 μm)分析,以水-乙腈为流动相,梯度洗脱,流速为1.0 mL·min-1,进样量为50 μL,采用紫外检测器,在检测波长265 nm处检测有关物质。结果: 固相萃取前处理可有效去除空白油干扰,艾地骨化醇与各杂质均可基线分离;在低温4 ℃条件下考察溶液稳定性,24 h时供试品溶液中各杂质面积百分比为98.6%~102.3%;杂质对照品溶液中,24 h时Tachy杂质、前体、艾地骨化醇和Trans杂质的面积百分比分别为101.2%、96.9%、98.5%和96.2%;艾地骨化醇、trans杂质、前体和tachy杂质的定量限分别为0.107、0.102、0.128、0.063 μg·mL-1,检测限分别为0.054、0.051、0.064、0.031 μg·mL-1;四者质量浓度分别在0.1~6.3 μg·mL-1、0.1~1.2 μg·mL-1、0.1~5.6 μg·mL-1和0.1~1.1 μg·mL-1范围内与峰面积呈良好线性关系,相关系数(n=5)分别为0.999 9、1.000、1.000和0.999 4;tachy杂质、前体和trans杂质回收率(n=6)分别为106.6%、88.6%和102.8%,RSD(n=6)分别为6.7%、3.1%和2.1%。3批样品含前体5.0%、5.0%和5.1%,杂质tachy、杂质Trans和未知单杂均未检出。结论: 该方法操作简单,经济实用,安全性高,专属性强,重复性好,准确度高,为艾地骨化醇软胶囊及类似油性基质的药物质量研究提供可靠依据。
Objective: To establish an amino solid-phase extraction-HPLC method for the determination of three related substances in eldecalcitol soft capsules. Methods: The eldecalcitol soft capsules were extracted by an amino solid-phase extraction method, followed by eluting with ethyl acetate: n-hexane and anhydrous ethanol. The residue was then dissolved with acetonitrile and water after vacuum evaporation. An ODS column (250 mm×4.6 mm, 5 μm) was used. Water-acetonitrile with gradient elution was employed. The flow rate was 1.0 mL·min-1, the injection volume was 50 μL, and the detection wavelength was set at 265 nm. Results: Interference from blank excipients was eliminated with pretreatment. Baseline separation and good selectivity of eldecalcitol and other impurities was achieved. The stability of the test and reference solution was investigated at 4 ℃. The area change percentage of the impurities in the test solution were 98.6%-102.3% at 24 h. The area change percentage of the impurities in the reference solution were 101.2%, 96.9%, 98.5% and 96.2% at 24 h, respectively. The quantitation limits of eldecalcitol, trans impurity, pre-eldecalcitol and tachy impurity were 0.107 μg·mL-1, 0.102 μg·mL-1, 0.128 μg·mL-1 and 0.063 μg·mL-1, respectively. The detection limits were 0.054 μg·mL-1, 0.051 μg·mL-1, 0.064 μg·mL-1, 0.031 μg·mL-1, respectively. The linear ranges of 4 components were 0.1-6.3 μg·mL-, 0.1-1.2 μg·mL-, 0.1-5.6 μg·mL-and 0.1-1.1 μg·mL-1, respectively. The correlation coefficients(n=5) were 0.999 9, 1.000, 1.000 and 0.999 4, respectively. The average recoveries (n=6)of tachy impurity, pre-eldecalcitol and trans impurity were 106.6%, 88.6% and 102.8% with RSDs of 6.7%, 3.1% and 2.1%, respectively. In the three batches of samples, no impurities were detected except pre-eldecalcitol. The contents of pre-eldecalcitol were 5.0%, 5.0% and 5.1%, respectively. Conclusion: The method is simple to operate, economical and practical with high safety, strong specificity, good repeatability and high accuracy. The method provides a reliable basis for the study of drug quality such as eldecalcitol soft capsules and drugs with similar oily substrate.
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