高效液相色谱法测定复方庚酸炔诺酮注射液的有关物质

Determination of Related Substances of Compound Norethisterone Enanthate Injection by HPLC

目的建立复方庚酸炔诺酮注射液有关物质检查的方法。方法采用HPLC梯度洗脱法,Phenomenex Gemini C18柱(4.6mm×250 mm,5μm),流动相A为水,流动相B为乙腈,流速为1.0 mL.min-1,检测波长220 nm,柱温35℃。结果主成分庚酸炔诺酮和戊酸雌二醇与各有关物质分离度良好。已知杂质炔诺酮和雌二醇分别在5.02～45.2和0.505～4.55μg.mL-1内线性关系良好,相关系数分别为1.000 0(n=5)和0.999 2(n=5),平均回收率(n=3)分别为99.4%和96.2%,RSD分别为0.9%和3.3%;庚酸炔诺酮和戊酸雌二醇自身对照溶液分别在2.42～121和0.242～12.1μg.mL-1内线性关系良好,相关系数均为1.000 0(n=6);炔诺酮、雌二醇、庚酸炔诺酮和戊酸雌二醇的检测限分别为7.3、2.0、0.9和1.2 ng。结论建立的HPLC能准确、灵敏、专属地测定复方庚酸炔诺酮注射液中的有关物质,经对不同生产日期的4批产品进行检验,本品有关物质的量与储存时间呈明显的相关性。

OBJECTIVE To establish an HPLC method for the determination of related substances of compound norethisterone enanthate injection. METHODS A Phenomenex Gemini C18 column （4.6 mm × 250 mm,5 μm） was used with water as mobile phase A and acetonitrile as mobile phase B. Gradient elution was used for the determination and the flow rate was 1.0 mL · min-1. The de- tection wavelength was 220 nm. The column temperature was maintained at 35 ℃ o RESULTS Norethisterone enanthate, estradiol valerate and their related substances were separated well. The calibration curves of the known related substances, namely norethisterone and estradiol, were linear within the concentration ranges of 5.02 - 45.2 and 0. 505 - 4.55 μg·mL^-1 with correlation coefficients of 1. 000 0 （n = 5） and 0.9992 （ n = 5）, respectively. The average recoveries of norethisterone and estradiol were 99.4% and 96.2% （n = 3）, with RSDs of 0.9% and 3.3%, respectively. The self-reference standard solution of norethisterone enanthate and estradiol valerate were linear within the concentration ranges of 2.42 - 121 and 0.242 - 12.1 μg·mL^-1 , and the correlation coefficients were both 1. 000 0 （ n = 6）. The LODs of norethisterone, estradiol, norethisterone enanthate, and estradiol valerate were 7.3, 2.0, 0.9 and 1.2 ng, respectively. CONCLUSION The method is accurate, sensitive, and specific. The analsis results of four batches of compound norethisterone enanthate injection showed that there is an evident correlationship between the amount of related substances and storage time of the injection.