Objective: To establish an HPLC gradient elution method for the determination of the related substances of alfacalcidol,and to identify the key related substance D. Methods: The HPLC method was carried out using a Waters XBridge C18（250 mm×4.6 mm,5 μm）;the mobile phase A was acetonitrile-water-ammonia （80:20:0.1）,and the mobile phase B was acetonitrile;the flow rate was 1.0 mL·min-1;the column temperature was 30 ℃ and the detection wavelength was 265 nm．The column and detection wavelength of HPLC-MS/MS method were the same as those of HPLC method;the mobile phase was acetonitrile-water-ammonia（80:20:0.1）; the flow rate was 2.6 mL·min-1;the column temperature was 30 ℃．The HPLC-MS/MS system was equipped with an APCI source in positive ionization mode;the spray capillary voltage was 6.0 kV;capillary temperature was at 275 ℃ ;capillary voltage was 35 V;Tube lens was 125 V;atomizer temperature was at 450 ℃. Results: Alfacalcidol and its related substances were separated completely under the above chromatographic conditions． The standard curves of alfacalcidol and its related substances were linear within the range 0.1-2.0 μg·mL-1,with the correlation of 0.999．LOQs of related substances were all below 5 ng and LODs were below 1.5 ng．Impurity D was identified as（1α,3β,5Z,7E）-9,10-secocholesta-5,7,10（19）- triene-1,3-diol by means of HPLC-MS/MS analysis. Conclusion: The established method is specific,accurate and better than the existing standard method．Impurity D is identified and set into the quality standard．It is a supplement to the present standards.
ZHANG Yuan-yuan, ZHANG Wen-xuan, WU Song, ZHANG Jie, FENG Jing, WANG Yue, YANG Qing-yun
. Determination of related substances in alfacalcidol by HPLC*[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(1)
: 160
-168
.
DOI: 10.16155/j.0254-1793.2021.01.20
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