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Article(id=1210516641985729462, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1210516638089212895, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2022-0408, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1649520000000, receivedDateStr=2022-04-10, revisedDate=1658160000000, revisedDateStr=2022-07-19, acceptedDate=null, acceptedDateStr=null, onlineDate=1766539257761, onlineDateStr=2025-12-24, pubDate=1662912000000, pubDateStr=2022-09-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1766539257761, onlineIssueDateStr=2025-12-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1766539257761, creator=13701087609, updateTime=1766539257761, updator=13701087609, issue=Issue{id=1210516638089212895, tenantId=1146029695717560320, journalId=1189982191388893191, year='2022', volume='57', issue='9', pageStart='1', pageEnd='2888', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1766539256832, creator=13701087609, updateTime=1766539546411, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1210517852726096743, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1210516638089212895, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1210517852726096744, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1210516638089212895, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2805, endPage=2810, ext={EN=ArticleExt(id=1210516643894136849, articleId=1210516641985729462, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=HPLC assay of epinephrine sulfonate in epinephrine injection, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

An HPLC method was established for the assay of epinephrine sulfonate (impurity F) in epinephrine injection. The determination was performed on an AQUASIL C18 (100 mm × 4.6 mm, 3 μm) column with a gradient elution system, and the mobile phase was consisted of monopotassium phosphate solution (mobile phase A) and acetonitrile (mobile phase B). The injection volume was 40 μL. The detection wavelength was at 210 nm and the column temperature was 25 ℃. The total analytical time was 40 min. The results showed that the standard cure of epinephrine sulfonate (impurity F) between peak area and concentration exhibited good linear relationship within the ranges of 0.520-12.480 μg·mL-1 and the R2 = 0.999 8. The average recovery rate was 103.04% and the RSD was 2.00%. The limit concentration of detection was 0.104 μg·mL-1 and the limit concentration of quantitation was 0.520 μg·mL-1. The method could be applied to the determination of epinephrine sulfonate in epinephrine injection with high accuracy and precision, as well as good sensitivity. It could also enhance the quality standards of epinephrine-related products.

, correspAuthors=Wen-sheng ZHENG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2022 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Chu-chu ZHOU, Peng DU, Ya MENG, Zhi-hui LI, Li ZHANG, Xiu-ping GUO, Yu-jia ZHANG, Qiu-shi HAO, Wen-sheng ZHENG), CN=ArticleExt(id=1210516644766552146, articleId=1210516641985729462, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=肾上腺素注射液中肾上腺素磺化物的HPLC检测方法研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

本研究建立高效液相色谱法, 用于肾上腺素注射液中肾上腺素磺化物(杂质F) 的测定。采用AQUASIL C18色谱柱(100 mm × 4.6 mm, 3 μm), 流动相A为磷酸二氢钾溶液, 流动相B为乙腈, 梯度洗脱, 进样量40 μL, 检测波长为210 nm, 柱温25 ℃, 分析时间40 min。结果表明, 肾上腺素磺化物(杂质F) 在0.520~12.480 μg·mL-1质量浓度内与峰面积呈良好的线性关系, R2 = 0.999 8; 平均回收率为103.04%, RSD为2.00%; 肾上腺素磺化物的最小检测限为0.104 μg·mL-1, 定量限为0.520 μg·mL-1。该检测方法准确度和精密度高, 灵敏度好, 可用于肾上腺素注射液中肾上腺素磺化物的分析测定, 提高肾上腺素相关产品的质量标准。

, correspAuthors=郑稳生, authorNote=null, correspAuthorsNote=
*郑稳生, E-mail:
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Acta Pharm Sin (药学学报), 2022, 57: 467-473., articleTitle=Determination of six effective ingredients in Imperatae Rhizoma concentrated granules by ultra-performance liquid chromatography using two internal references, refAbstract=null), Reference(id=1210516657185887155, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210516641985729462, doi=null, pmid=null, pmcid=null, year=2019, volume=54, issue=null, pageStart=1661, pageEnd=1666, url=null, language=null, rfNumber=[26], rfOrder=25, authorNames=null, journalName=Acta Pharm Sin (药学学报), refType=null, unstructuredReference=Zhang Y, Xu HR, Li JS, et al. Simultaneous quantitative analyses of eight components in Artemisia capillaris Thunb standard decoction based on a quantitative method of multi-components with a single-marker[J]. 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A: H<sub>2</sub>O; B: Blank excipient; C: Epinephrine sulfonate reference (impurity F) solution; D: Epinephrine reference solution; E: Mixed solution; F: Mixed solution. 1, 2: Gradient peak; 3: The peak of blank excipient; 4: The peak of epinephrine sulfonate; 5: The peak of epinephrine , figureFileSmall=9YFMPshORbbCSAD6T5/xFA==, figureFileBig=wINtIDUs6NRui+2mngop9A==, tableContent=null), ArticleFig(id=1210516652576346721, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210516641985729462, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Time/minMobile phase A (%, v/v)Mobile phase B (%, v/v)Flow rate /mL·min-1
0-59820.8-1
5-3098-552-451
30-3555-9845-21
35-409820.8
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The gradient elution system of mobile phase A and B

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Time/minMobile phase A (%, v/v)Mobile phase B (%, v/v)Flow rate /mL·min-1
0-59820.8-1
5-3098-552-451
30-3555-9845-21
35-409820.8
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PreparationMass/g
Sodium chloride3.264
Sodium metabisulfite0.184
Citric acid, anhydrous0.852
Sodium citrate0.164
H2O400
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The preparation of blank excipient

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PreparationMass/g
Sodium chloride3.264
Sodium metabisulfite0.184
Citric acid, anhydrous0.852
Sodium citrate0.164
H2O400
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Batch123456
Concentration/μg·mL-10.5201.0403.1205.20010.40012.480
Peak area83 749164 808480 916845 9491 709 2672 041 598
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Linearity and range test results of epinephrine sulfonate (impurity F)

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Batch123456
Concentration/μg·mL-10.5201.0403.1205.20010.40012.480
Peak area83 749164 808480 916845 9491 709 2672 041 598
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BatchSpiked/μg·mL-1Found/μg·mL-1Recovery rate/%
Quantification limit 10.520.55105.77
Quantification limit 20.520.53101.92
Quantification limit 30.520.56107.69
Low concentration 18.328.55102.76
Low concentration 28.328.41101.08
Low concentration 38.328.34100.24
Medium concentration 110.4010.61102.02
Medium concentration 210.4010.58101.73
Medium concentration 310.4010.81103.94
High concentration 111.9612.25102.42
High concentration 211.9612.41103.76
High concentration 311.9612.33103.09
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The recovery rate of epinephrine sulfonate (impurity F)

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BatchSpiked/μg·mL-1Found/μg·mL-1Recovery rate/%
Quantification limit 10.520.55105.77
Quantification limit 20.520.53101.92
Quantification limit 30.520.56107.69
Low concentration 18.328.55102.76
Low concentration 28.328.41101.08
Low concentration 38.328.34100.24
Medium concentration 110.4010.61102.02
Medium concentration 210.4010.58101.73
Medium concentration 310.4010.81103.94
High concentration 111.9612.25102.42
High concentration 211.9612.41103.76
High concentration 311.9612.33103.09
), ArticleFig(id=1210516653536842401, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210516641985729462, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
BatchConcentration/μg·mL-1
20210724-11.17
20210724-21.00
20210728-10.76
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The assay of epinephrine sulfonate (impurity F) in epinephrine injection with low concentration

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BatchConcentration/μg·mL-1
20210724-11.17
20210724-21.00
20210728-10.76
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肾上腺素注射液中肾上腺素磺化物的HPLC检测方法研究
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周楚楚 1 , 杜鹏 1 , 孟雅 1 , 李志辉 2 , 张礼 3 , 郭修平 1 , 张宇佳 1 , 郝秋实 4 , 郑稳生 1, *
药学学报 | 研究论文 2022,57(9): 2805-2810
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药学学报 | 研究论文 2022, 57(9): 2805-2810
肾上腺素注射液中肾上腺素磺化物的HPLC检测方法研究
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周楚楚1, 杜鹏1, 孟雅1, 李志辉2, 张礼3, 郭修平1, 张宇佳1, 郝秋实4, 郑稳生1, *
作者信息
  • 1.北京协和医学院、中国医学科学院药物研究所, 药物传输技术及新型制剂北京市重点实验室, 北京 100050
  • 2.广西医科大学, 广西 南宁 530021
  • 3.安庆医药高等专科学校, 安徽 安庆 246052
  • 4.河北金牛原大药业科技有限公司, 河北 定州 073000

通讯作者:

*郑稳生, E-mail:
HPLC assay of epinephrine sulfonate in epinephrine injection
Chu-chu ZHOU1, Peng DU1, Ya MENG1, Zhi-hui LI2, Li ZHANG3, Xiu-ping GUO1, Yu-jia ZHANG1, Qiu-shi HAO4, Wen-sheng ZHENG1, *
Affiliations
  • 1. Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing City Key laboratory of Drug Delivery Technology and Novel Formulation, Beijing 100050, China
  • 2. Guangxi Medical University, Nanning 530021, China
  • 3. Anqing Medical College, Anqing 246052, China
  • 4. Hebei Jinniuyuanda Pharmaceutical Technology Co., Ltd., Dingzhou 073000, China
出版时间: 2022-09-12 doi: 10.16438/j.0513-4870.2022-0408
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摘要
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本研究建立高效液相色谱法, 用于肾上腺素注射液中肾上腺素磺化物(杂质F) 的测定。采用AQUASIL C18色谱柱(100 mm × 4.6 mm, 3 μm), 流动相A为磷酸二氢钾溶液, 流动相B为乙腈, 梯度洗脱, 进样量40 μL, 检测波长为210 nm, 柱温25 ℃, 分析时间40 min。结果表明, 肾上腺素磺化物(杂质F) 在0.520~12.480 μg·mL-1质量浓度内与峰面积呈良好的线性关系, R2 = 0.999 8; 平均回收率为103.04%, RSD为2.00%; 肾上腺素磺化物的最小检测限为0.104 μg·mL-1, 定量限为0.520 μg·mL-1。该检测方法准确度和精密度高, 灵敏度好, 可用于肾上腺素注射液中肾上腺素磺化物的分析测定, 提高肾上腺素相关产品的质量标准。

高效液相色谱法  /  肾上腺素注射液  /  肾上腺素磺化物  /  含量测定  /  质量标准

An HPLC method was established for the assay of epinephrine sulfonate (impurity F) in epinephrine injection. The determination was performed on an AQUASIL C18 (100 mm × 4.6 mm, 3 μm) column with a gradient elution system, and the mobile phase was consisted of monopotassium phosphate solution (mobile phase A) and acetonitrile (mobile phase B). The injection volume was 40 μL. The detection wavelength was at 210 nm and the column temperature was 25 ℃. The total analytical time was 40 min. The results showed that the standard cure of epinephrine sulfonate (impurity F) between peak area and concentration exhibited good linear relationship within the ranges of 0.520-12.480 μg·mL-1 and the R2 = 0.999 8. The average recovery rate was 103.04% and the RSD was 2.00%. The limit concentration of detection was 0.104 μg·mL-1 and the limit concentration of quantitation was 0.520 μg·mL-1. The method could be applied to the determination of epinephrine sulfonate in epinephrine injection with high accuracy and precision, as well as good sensitivity. It could also enhance the quality standards of epinephrine-related products.

HPLC  /  epinephrine injection  /  epinephrine sulfonate  /  assay  /  quality standards
周楚楚, 杜鹏, 孟雅, 李志辉, 张礼, 郭修平, 张宇佳, 郝秋实, 郑稳生. 肾上腺素注射液中肾上腺素磺化物的HPLC检测方法研究. 药学学报, 2022 , 57 (9) : 2805 -2810 . DOI: 10.16438/j.0513-4870.2022-0408
Chu-chu ZHOU, Peng DU, Ya MENG, Zhi-hui LI, Li ZHANG, Xiu-ping GUO, Yu-jia ZHANG, Qiu-shi HAO, Wen-sheng ZHENG. HPLC assay of epinephrine sulfonate in epinephrine injection[J]. Acta Pharmaceutica Sinica, 2022 , 57 (9) : 2805 -2810 . DOI: 10.16438/j.0513-4870.2022-0408
正文
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肾上腺素是一种肾上腺素类受体激动剂, 可以直接作用于肾上腺素能α、β受体[1]。临床上常作为急救用药物, 用于过敏性休克或心脏骤停的抢救, 也可用于荨麻疹等过敏性疾病的治疗[2, 3]。
肾上腺素属于儿茶酚胺类化合物, 在空气中易发生氧化反应生成肾上腺素红[4-6], 所以在肾上腺素注射液制备过程中, 常加入辅料焦亚硫酸钠作为抗氧剂[7, 8]。但是研究表明, 肾上腺素会和焦亚硫酸钠在水中解离生成的亚硫酸根离子反应, 生成无生理活性的肾上腺素磺化物(杂质F)[9, 10], 进而影响药效(图 1), 所以控制肾上腺素注射液中肾上腺素磺化物的含量在产品的质量控制及临床使用中具有很重要的意义。
《中华人民共和国药典》2020年版[11]、《欧洲药典》 (EP10.3)[12]、《美国药典》 (USP43-NF38)[13]及《日本药局方》 (JP17)[14]中均收录了肾上腺素及肾上腺素注射液(含盐酸肾上腺素注射液)。但是《中华人民共和国药典》2020年版、《美国药典》 (USP43-NF38) 及《日本药局方》 (JP17) 中均没有对肾上腺素磺化物(杂质F) 的含量进行控制; 《欧洲药典》 (EP10.3) 中虽然控制了肾上腺素磺化物(杂质F) 的含量, 但标准方法灵敏度有限, 肾上腺素磺化物和辅料之间的分离度达不到要求, 其有关物质的检查方法不适用于本实验室开发的低浓度肾上腺素注射液。目前只有极少的报道研究了肾上腺素注射液中肾上腺素磺化物的含量测定[15-19], 宋冬梅等[20-22]采用HPLC-QTOF MS法可以有效地检出肾上腺素磺化物, 但是该检测方法成本较高且前处理较为复杂, 所以亟需开发出一种准确度高且简单易行的检测方法, 用于肾上腺素注射液的质量评价。
本文根据药品杂质分析指导原则和ICH相关规定[23, 24], 参考《欧洲药典》 (EP10.3) 中肾上腺素有关物质的检查方法并对该方法进行了进一步的改进和优化, 建立了适用于本实验室开发的低浓度肾上腺素注射液中肾上腺素磺化物含量的测定方法。方法学验证结果表明, 该方法操作方便、准确度和精密度高、灵敏度好, 可以用于本实验室自制的低浓度肾上腺素注射液上市后的质量控制和评价。
材料与方法
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实验仪器  Waters 2695高效液相色谱仪(美国Waters公司); Agilent 1100高效液相色谱仪(美国Agilent公司); 电子天平[型号: ME104, 梅特勒-托利多仪器(上海) 有限公司]; 纯水机(上海和泰仪器有限公司); pH计(型号: PHS-3SW, 上海般特仪器制造有限公司)。
对照品与试剂  肾上腺素对照品(批号: 100154-201605, 中国食品药品检定研究院, 纯度: 99.7%); 氯化钠(批号: 20191105, 中盐云虹湖北制药股份有限公司); 焦亚硫酸钠(批号: 20210201, 成都华邑药用辅料制造有限公司); 无水枸橼酸(批号: 21022402, 湖南新绿方药业有限公司); 枸橼酸钠二水合物(批号: 20200501, 湖南华日制药有限公司); 磷酸二氢钾(批号: C2013061, Aladdin); 辛烷磺酸钠(批号: 2054572, Fisher); 磷酸(批号: 20170506, 北京化工厂); 盐酸(批号: 200310011A, 南京化学试剂股份有限公司); 乙腈(批号: 201304R, Fisher); 肾上腺素磺化物(杂质F) 对照品(批号: C4X-184931-2007, CATO, 纯度: 100.0%); 低浓度肾上腺素注射液(批号: 20210724-1、20210724-2、20210728-1, 自制); 纯水(批号: 20210726等, 杭州娃哈哈集团有限公司)。
色谱条件  参考《欧洲药典》 (EP10.3) 中肾上腺素有关物质的检查方法并对该方法进行了进一步的改进和优化, 建立了适用于本实验室开发的低浓度肾上腺素注射液中肾上腺素磺化物含量的测定方法: 色谱柱为AQUASIL C18柱(100 mm × 4.6 mm, 3 μm); 流动相A为磷酸二氢钾溶液(取磷酸二氢钾5 g, 辛烷磺酸钠2.6 g, 加水溶解并稀释至2 L, 然后用磷酸调节pH值为2.8), 流动相B为乙腈; 进样量为40 μL; 检测波长为210 nm; 柱温为25 ℃; 分析时间40 min, 按照表 1进行梯度洗脱。
肾上腺素对照品溶液的制备  取肾上腺素对照品10 mg, 精密称定, 置于100 mL量瓶中, 向其中加入0.1 mol·L-1盐酸溶液5 mL使溶解, 用流动相A稀释至刻度, 摇匀, 即得106 μg·mL-1肾上腺素储备液; 再取1 mL肾上腺素储备液于10 mL量瓶中, 加流动相A定容至刻度, 即得10.6 μg·mL-1肾上腺素对照品溶液。
肾上腺素磺化物(杂质F) 对照品溶液  取10 mg肾上腺素磺化物(杂质F) 对照品, 精密称定, 置于100 mL量瓶中, 加入0.1 mol·L-1盐酸溶液5 mL使溶解, 用流动相A稀释至刻度, 摇匀, 即得104 μg·mL-1肾上腺素磺化物(杂质F) 储备液; 取104 μg·mL-1肾上腺素磺化物(杂质F) 储备液1 mL于10 mL量瓶中, 加流动相A定容至刻度, 即得10.4 μg·mL-1的肾上腺素磺化物(杂质F) 对照品溶液。
空白辅料溶液的制备  按照表 2称取相应处方量的辅料于广口瓶中, 加入400 mL的注射用水, 100 r·min-1磁力搅拌, 同时立即伴随在液面下充氮, 控制溶解氧在0.8~1.0 ppm内, 观察辅料完全溶解即得空白辅料溶液。
混合溶液的制备  精密量取肾上腺素储备液、肾上腺素磺化物(杂质F) 储备液及空白辅料溶液各1 mL于10 mL量瓶中, 向其中加入流动相A定容至刻度, 摇匀, 即得混合溶液。
专属性  分别取空白辅料溶液、肾上腺素磺化物(杂质F) 对照品溶液、肾上腺素对照品溶液及混合溶液, 进样考察, 记录色谱图, 分析空白辅料及肾上腺素是否会干扰肾上腺素磺化物(杂质F) 的含量测定[25]。
系统适用性  取肾上腺素磺化物(杂质F) 对照品溶液, 重复进样5次, 记录色谱图, 并计算5次进样结果中肾上腺素磺化物(杂质F) 峰面积的RSD值。同时取混合溶液进样, 肾上腺素磺化物(杂质F) 峰与辅料峰之间的分离度应不小于1.5。
线性关系与范围  分别取肾上腺素磺化物(杂质F) 储备液1、3、5、10和12 mL于100 mL量瓶中, 向其中加流动相A定容至刻度, 即得质量浓度分别为1.040、3.120、5.200、10.400和12.480 μg·mL-1的肾上腺素磺化物(杂质F) 溶液; 再分别取1.040 μg·mL-1肾上腺素磺化物(杂质F) 溶液5 mL于10 mL量瓶中, 定容至刻度, 即得0.520 μg·mL-1肾上腺素磺化物(杂质F) 溶液; 取上述浓度的肾上腺素溶液分别进样测定, 考察峰面积与肾上腺素磺化物(杂质F) 溶液浓度之间的关系[26]。
定量限及检测限  分别取肾上腺素磺化物(杂质F) 对照品溶液适量, 稀释至不同的浓度并进样考察, 记录信噪比 > 3时对应的浓度即为检测限; 信噪比 > 10时对应的浓度即为定量限[27]。
以空白辅料溶液为溶剂进行检测限和定量限的确证实验: 精密量取适量肾上腺素磺化物(杂质F) 对照品溶液于量瓶中, 向其中加入空白辅料溶液定容至刻度, 得到浓度分别与检测限与定量限相当的肾上腺素磺化物(杂质F) 溶液, 其中与定量限相当的样品平行配制3份, 进样分析, 记录信噪比。
重复性  取20210724-1批肾上腺素注射液样品, 制备6份供试品溶液, 分别进样分析, 记录肾上腺素磺化物(杂质F) 面积, 按照外标法计算含量, 并计算RSD值。
中间精密度  由本实验室的另一名分析人员取同一批次(20210724-1批) 样品, 按照同法配制6份供试品溶液, 用另一台高效液相色谱仪进行检测分析, 记录肾上腺素磺化物(杂质F) 面积, 按照外标法计算含量, 并结合重复性实验的结果, 计算RSD值。
稳定性  取供试品溶液, 分别于0、3、6、12和24 h进样, 记录不同时间点肾上腺素磺化物(杂质F) 峰面积, 并计算RSD值。
回收率  精密量取适量不同浓度的肾上腺素磺化物(杂质F) 溶液于量瓶中, 加入空白辅料溶液稀释并定容至刻度, 制成相当于肾上腺素磺化物(杂质F) 限度(10 μg·mL-1) 的80%、100%、115%及定量限浓度的回收率测定溶液, 每个浓度的样品平行制备三份, 分别进样考察, 每份样品中肾上腺素磺化物(杂质F) 的含量使用外标法计算得到, 得到的数值与实际加入量的比值即为每份样品的回收率。
方法耐用性  分别调整高效液相色谱方法中流动相pH (±0.2)、流速(±0.1 mL·min-1) 及使用不同批次的色谱柱, 取肾上腺素磺化物(杂质F) 对照品溶液进样考察, 考察该方法的耐用性[28]。
样品测定  取3批本实验室自制的低浓度肾上腺素注射液样品, 分别进样考察, 记录色谱图, 同时计算每批样品中肾上腺素磺化物(杂质F) 的含量。
结果
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1 专属性考察
空白辅料溶液、肾上腺素磺化物(杂质F) 对照品溶液、肾上腺素对照品溶液及混合溶液的进样结果见图 2, 结果表明, 空白辅料不干扰肾上腺素磺化物(杂质F) 的测定, 且肾上腺素磺化物(杂质F) 与相邻峰之间的分离度也符合要求, 说明本实验室建立的高效液相色谱法的专属性良好。
2 系统适用性实验
根据《中华人民共和国药典》2020年版“0512高效液相色谱法通则”和“9101分析方法验证指导原则”要求[11], 由于肾上腺素注射液中待测定成分肾上腺素磺化物(杂质F) 的限度为10 μg·mL-1 (即10 μg·g-1), 故本实验中将系统适用性实验中连续进样5次峰面积测量值的RSD放宽至6%。计算5次进样肾上腺素磺化物(杂质F) 的峰面积的RSD为0.86%, 符合要求; 混合溶液进样后色谱图见图 2E和2F, 肾上腺素磺化物(杂质F) 与辅料峰之间的分离度为2.7, 符合要求, 说明该方法的系统适用性较好。
3 线性关系与范围
肾上腺素磺化物(杂质F) 对照品溶液进样浓度与峰面积的关系如表 3所示, 以肾上腺素磺化物(杂质F) 对照品溶液浓度为横坐标, 峰面积为纵坐标, 绘制标准曲线, 实验结果表明肾上腺素磺化物(杂质F) 在0.520~12.480 μg·mL-1内, 线性关系良好, 标准曲线方程为A = 164 722C – 11 667, R2 = 0.999 8。
4 检测限与定量限
在空白溶剂中, 肾上腺素磺化物(杂质F) 浓度为0.104 μg·mL-1时, 进样考察, 信噪比为3.1, 故肾上腺素磺化物(杂质F) 的检测限为0.104 μg·mL-1; 肾上腺素磺化物(杂质F) 浓度为0.520 μg·mL-1时, 进样考察, 信噪比为10.3, 故肾上腺素磺化物(杂质F) 的定量限为0.520 μg·mL-1。
以空白辅料溶液作为溶剂, 进行该方法检测限和定量限的确证实验: 肾上腺素磺化物(杂质F) 浓度为0.104 μg·mL-1时, 进样考察, 信噪比为3.3, 确证以上检测限; 肾上腺素磺化物(杂质F) 浓度为0.520 μg·mL-1时(平行配制3份), 进样考察, 信噪比均值为10.4, 信噪比RSD为0.55%; 回收率分别为105.77%、101.92%和107.69%, 回收率均值为105.13%, 回收率RSD为2.79%, 确证以上定量限。
5 重复性
6份供试品溶液的测量结果表明, 供试品溶液中肾上腺素磺化物(杂质F) 的平均含量为1.18 μg·mL-1, RSD计算结果为4.06%, 根据《中华人民共和国药典》2020年版“通则9101分析方法验证指导原则”要求[11], 本供试品中杂质F精密度要求RSD应小于6%。本方法精密度满足要求, 说明所建立的用于肾上腺素磺化物含量测定的高效液相色谱方法重复性良好。
6 中间精密度
合并统计重复性实验与中间精密度实验结果(共12组), 供试品溶液中肾上腺素磺化物(杂质F) 的平均含量为1.17 μg·mL-1, 综合RSD为4.00%, 说明所建立的用于肾上腺素磺化物含量测定的高效液相色谱方法中间精密度良好。
7 稳定性
供试品溶液在室温放置不同时间后分别进样, 肾上腺素磺化物(杂质F) 峰面积的RSD为1.58%, 说明供试品溶液在室温条件下24 h内稳定。
8 回收率
计算每份样品的回收率结果如表 4所示, 平均回收率为103.04%, RSD为2.00%。
9 方法耐用性
结果表明, 当色谱条件发生微小变动时, 对肾上腺素磺化物(杂质F) 比例的测定结果没有显著性影响, 表明本实验室建立的肾上腺素磺化物(杂质F) 测定方法耐用性良好。
10 样品测定
分别取3批自制肾上腺素注射液进样测定, 计算肾上腺素磺化物(杂质F) 含量, 结果如表 5所示。
讨论
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本实验室首先参考2020版《中华人民共和国药典》二部中肾上腺素有关物质的检测方法并进行优化, 优化内容包括调整流动相A/B比例、流动相的pH值及流动相中离子对的用量。实验结果表明, 肾上腺素磺化物(杂质F) 与辅料峰的分离度偏低, 不符合要求, 说明该方法不适用于本实验室自制的低浓度肾上腺素注射液。继续考察能否通过优化《欧洲药典》 (10.3) 中肾上腺素有关物质检测方法准确测定肾上腺素注射液中肾上腺素磺化物(杂质F) 的含量。实验结果表明, 适当优化色谱条件中流动相的比例, 提高流动相A的比例为98%, 可以延长肾上腺素磺化物(杂质F) 保留时间, 且该条件下杂质F和辅料峰的分离度为2.7, 肾上腺素、肾上腺素酮(杂质C) 及肾上腺素红均出锋。分别配制浓度约为10 µg·mL-1的肾上腺素对照品溶液和肾上腺素磺化物(杂质F) 对照品溶液、空白辅料溶液和自制肾上腺素注射液, 于200~400 nm波长内进行紫外扫描。结果显示对照品溶液和自制肾上腺素注射液均在210 ± 2 nm处有较强的紫外吸收, 且在该波长条件下溶剂、辅料均无影响, 所以选用210 nm作为检测波长, 该波长与《欧洲药典》 (10.3) 中肾上腺素原料药有关物质检查项波长一致。
实验结果证明, 该方法准确度和精密度高、灵敏度好, 可以用于肾上腺素注射液中肾上腺素磺化物(杂质F) 的测定。参比制剂的统计回顾报告表明, 参比制剂在长期条件下放置12个月后, 肾上腺素磺化物(杂质F) 的比例为3.8%~4.3%。由于参比制剂的有效期不少于24个月, 暂定本产品中肾上腺素磺化物(杂质F) 的限度为10.0%。本实验室开发的有关物质检查方法检测限为0.520 μg·mL-1, 可以实现肾上腺素磺化物的准确定量。
进一步研究表明, 该方法可以同时检出肾上腺素红和肾上腺素酮(杂质C), 有望用于肾上腺素注射液中肾上腺素红和肾上腺素酮(杂质C) 的分析测定, 对于肾上腺素注射液的质量控制具有重要的意义, 为儿茶酚胺类药物及其代谢产物的检测提供了新的研究思路。
作者贡献: 周楚楚进行了本文的撰写、实验设计和数据处理; 杜鹏、孟雅主要进行了技术指导和实验记录的整理; 李志辉、张礼和郭修平完成了所有的实验操作; 张宇佳、郝秋实和郑稳生在选题、实验思路和文章修改方面进行了专业指导。
利益冲突: 本文无任何利益冲突。
基金
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  • 中国医学科学院医学与健康科技创新工程基金资助(2021-I2M-1-028)
  • 河北省科技厅《河北金牛原大药业科技有限公司儿童异烟肼糖浆新药研发创新团队》资助项目(205A7606D)
文献
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2022年第57卷第9期
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doi: 10.16438/j.0513-4870.2022-0408
  • 接收时间：2022-04-10
  • 首发时间：2025-12-24
  • 出版时间：2022-09-12
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  • 收稿日期：2022-04-10
  • 修回日期：2022-07-19
基金
中国医学科学院医学与健康科技创新工程基金资助(2021-I2M-1-028)
河北省科技厅《河北金牛原大药业科技有限公司儿童异烟肼糖浆新药研发创新团队》资助项目(205A7606D)
作者信息
    1.北京协和医学院、中国医学科学院药物研究所, 药物传输技术及新型制剂北京市重点实验室, 北京 100050
    2.广西医科大学, 广西 南宁 530021
    3.安庆医药高等专科学校, 安徽 安庆 246052
    4.河北金牛原大药业科技有限公司, 河北 定州 073000

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2种不同金属材料的力学参数
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species		 占总种数比例
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total species (%)		 属
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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