目的: 建立高效液相色谱-电喷雾检测器法测定熊去氧胆酸中的10个有关物质。方法: 采用SHISEIDO Capcell PAK C18 MGII(150 mm×4.6 mm,5 μm)色谱柱,以0.1%甲酸-甲醇-乙腈(30∶45∶25)为流动相A,乙腈为流动相B,梯度洗脱,流速1.0 mL·min-1,柱温40 ℃,检测器温度35 ℃,采集频率5 Hz,过滤常数3.6 s。采用主成分自身对照法计算已知杂质和其它杂质的含量,并对建立的方法进行方法学验证。结果: 熊去氧胆酸与各杂质分离度良好;熊去氧胆酸、杂质A、杂质B、杂质C、杂质E的质量浓度均在1.0~100.0 μg·mL-1范围内与峰面积呈良好的线性关系,定量限均为1.0 μg·mL-1,检测限均为0.5 μg·mL-1,杂质的平均回收率在99.3%~100.1%,RSD(n=9)不高于2.0%;供试品溶液在10 ℃条件下放置24 h内稳定;微调液相色谱参数后,对有关物质的检测结果无影响。3批样品有关物质结果显示,杂质A的含量均小于1.0%,其他单一杂质的含量均小于0.1%,杂质总量均小于1.5%。结论: 本文所建立的HPLC-CAD法简便、灵敏、准确,可用于熊去氧胆酸的有关物质检测。
Objective: To establish an high performance liquid chromatography-charged aerosol detector method for the determination of the 10 related substances in ursodeoxycholic acid. Methods: A SHISEIDO Capcell PAK C18MGII (150 mm×4.6 mm,5 μm) column was adopted. The mobile phase A was composed of 0.1%formic acid-methanol-acetonitrile (30∶45∶25), and the mobile phase B was acetonitrile. The gradient elution was performed. The flow rate was 1.0 mL·min-1, the column temperature was 40 ℃, the detector temperature was 35 ℃, the sampling frequency was 5 Hz, and the filtration constant was 3.6 s. The contents of known impurities and other impurities were calculated by principal component self-comparison method, and the established method was validated. Results: Ursodeoxycholic acid was well separated from impurities. The mass concentrations of ursodeoxycholic acid, impurity A, impurity B, impurity C and impurity E all showed good linear relationship within the range of 1.0-100.0 μg·mL-1, the lower limit of quantification was 1.0 μg·mL-1, the lower limit of detection was 0.5 μg·mL-1, and the impurity recovery rates were between 99.3% and 100.1%, and the RSDs (n=9) of each recovery were less than 2.0%. The test solution was stable within 24 h after being placed at 10 ℃. Fine tuning of liquid chromatographic parameters had no effect on the detection results of related substances. The results of three batches of samples showed that the contents of impurity A were less than 1.0%, the contents of other single impurities were less than 0.1%, and the total amount of impurities were less than 1.5%. Conclusion: The established method is simple, sensitive and accurate, which can be used to determinate the related substances of ursodeoxycholic acid.
[1] 尤梅桂.熊去氧胆酸的研究概况[J]. 药学研究, 2021, 40(3):199
YOU MG. Overview of research on ursodeoxycholic acid[J]. J Pharm Res, 2021, 40(3):199
[2] LI H, GUAN Y, HAN C, et al. The pathogenesis, models and therapeutic advances of primary biliary cholangitis[J]. Biomed Pharmacother, 2021,140:111754
[3] 帅俊芳,韩子岩.对熊去氧胆酸应答不佳或不耐受的原发性胆汁性胆管炎患者应用奥贝胆酸和贝特类药物治疗的研究进展[J]. 临床肝胆病杂志, 2022, 38(4):913
SHUAI JF,HAN ZY.Research advances in obeticholic acid and fibrates in treatment of primary biliary cholangitis with poor response or intolerance to ursodeoxycholicacid[J]. J Clin Hepatol, 2022, 38(4):913
[4] 林锦燕,缪晓峰.治疗非酒精性脂肪性肝病应用熊去氧胆酸的临床效果分析[J]. 中外医疗, 2022, 14:103
LIN JY, MIAO XF. Analysis of clinical effect of ursodeoxycholic acid in the treatment of non-alcoholic fatty liver disease[J]. China Foreign Med Treat, 2022, 14:103
[5] ZENG L, LI M, CEN Y, et al. Effects of S-adenosyl-L-methionine combined with ursodesoxycholic acid on serum endotoxin, MMP-9 and IL-18 in neonates with Cholestasis[J]. J Coll Physicians Surg Pak, 2021, 31(12):1445
[6] 兰静,何秉燕.熊去氧胆酸对极低出生体重儿胃肠外营养相关性胆汁淤积的疗效[J]. 武汉大学学报(医学版), 2022, 43(3):491
LAN J, HE BY. Effect of ursodeoxycholic acid on parenteral nutrition associated cholestasis in very low birth weight infants[J]. Med J Wuhan Univ, 2022, 43(3):491
[7] 中华人民共和国药典2020年版.二部[S]. 2020: 1788
ChP 2020. Vol Ⅱ [S]. 2020: 1788
[8] USP-NF2022[S]. 2022:ISSUE1
[9] JP 18[S]. ⅩⅧ:1891
[10] BP 2022. Vol Ⅱ[S]. 1261
[11] EP 10.8. Vol Ⅲ[S]. 4152
[12] 丁劲松,王安娜,黄亮,等.人体熊去氧胆酸代谢及其生物等效性研究的技术挑战[J]. 药学学报, 2020, 55(9):2070
DING JS, WANG AN, HUANG L, et al. Metabolism of ursodeoxycholic acid in human raises challenges to its bioequivalence studies[J]. Acta Pharm Sin, 2020, 55(9):2070
[13] EL-KAFRAWYDS, BELALTS, MAHROUSMS, et al. Validated spectrophotometric and RP-HPLC-dad methods for the determination of ursodeoxycholic acid based on derivatization with 2-nitrophenylhydrazine[J]. J AOAC Int, 2017, 100(3):677
[14] 黄诺哲,李智颖.HPLC 法测定熊去氧胆酸片有关物质的含量[J]. 中国药品标准,2017, 18(5):388
HUANG NZ, LI ZY. Determination of related substances in ursodeoxycholic acid tablets by HPLC[J]. Drug Stand China, 2017, 18(5):388
[15] 谭菊英,颜其双,王娟,等. HPLC-CAD法测定人工牛黄及其制剂小儿氨酚黄那敏颗粒中胆酸和猪去氧胆酸的含量[J]. 中国药师, 2019, 22(12):2321
TAN JY, YAN QS, WANG J, et al. Determination of cholalic acid and hyodeoxycholic acid in bovis calculus artifactus and its preparation pendiatricparacetamol,artificial cow-bezoar and chlorphenamine maleate granules by HPLC-CAD[J]. China Pharm, 2019, 22(12):2321
[16] 王恩,兰公剑,万国盛,等. HPLC-CAD法测定6-乙基鹅去氧胆酸起始物料中有关物质[J]. 山东化工,2021, 50(19):142
WANG E, LAN GJ, WAN GS, et al. Determination of related substances in the startiing material of 6-ethylchenodeoxycholic acid by HPLC-CAD[J]. Shandong Chem Ind, 2021, 50(19):142
[17] 石岩,魏峰,林瑞超,等.熊胆粉中主要胆汁酸类成分的测定及分析[J]. 中国药学杂志,2016, 51(22):1958
SHI Y, WEI F, LIN RC, et al. Quantification of the major bile acids in bear bile powder by HPLC-ELSD[J]. Chin Pharm J, 2016, 51(22):1958
[18] 刘菁,吴燕.新型通用型检测器-电喷雾检测器在药物分析种的应用进展[J]. 天津药学,2022, 34(3):61
LIU J,WU Y. The application progress of a new common detector-charged aerosol detector in pharmaceutical analysis[J]. Tianjin Pharm, 2022, 34(3):61
[19] 南易, 郑伟, 马凤霞, 等.HPLC-CAD同时测定知母中芒果苷和知母皂苷BⅡ的含量[J]. 药物分析杂志,2021,41(1):111
NAN Y,ZHENG W,MA FX, et al. Simultaneous determination of mangiferin and timosaponin BⅡ of Anemarrhenae Rhizoma by HPLC-CAD[J]. Chin J Pharm Anal, 2021, 41(1):111
