Anusha Kodidasu, , , Koushik Acharyya*, , and , Vasanthakumar Ganga Ramu*,
{"title":"利用高分辨率质谱法对多肽药物中溶出杂质的绝对定量:胰高血糖素——药物开发中的一个案例研究。","authors":"Anusha Kodidasu, , , Koushik Acharyya*, , and , Vasanthakumar Ganga Ramu*, ","doi":"10.1021/jasms.5c00105","DOIUrl":null,"url":null,"abstract":"<p >Synthetic peptide-based drugs provide customized therapeutic solutions, but developing a peptide medicine presents various challenges, especially in terms of impurity management. This holds true when traditional techniques like RP-HPLC fail to separate low-abundance coeluting impurities. In this regard, liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) has proven to be effective for identifying and characterizing peptide impurities, although its application for accurate quantitation is still limited. This study developed and validated two quantitation strategies using UPLC-HRMS, employing full scan and pseudo-MRM (<i>p</i>-MRM) modes, to identify and accurately quantify two structurally similar coeluting peptide impurities (des-Gly<sup>4</sup> glucagon and des-Thr<sup>5</sup> glucagon) observed in synthetic glucagon. The methods exhibited high specificity and demonstrated good linearity (<i>R</i><sup>2</sup> > 0.99) across a concentration range of 0.25–25 μg/mL. The limits of detection (LODs) for des-Gly<sup>4</sup>-glucagon and des-Thr<sup>5</sup>-glucagon were determined to be as low as 0.01% and 0.02%, with limits of quantitation (LOQs) at 0.02% and 0.04%, respectively. Precision (RSD%) was recorded at less than 10%, and recovery ranged from 100% to 120%. A comparative analysis of the results indicated that both quantifitation methods performed similarly in terms of accuracy, precision, and recovery, producing comparable impurity estimates in three glucagon API samples. However, <i>p</i>-MRM showed slightly better linearity and sensitivity compared to the full scan EIC-based quantitation method.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2072–2078"},"PeriodicalIF":2.7000,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Absolute Quantitation of Coeluting Impurities in Peptide Drugs Using High Resolution Mass Spectrometry: Glucagon a Case Study in Pharmaceutical Development\",\"authors\":\"Anusha Kodidasu, , , Koushik Acharyya*, , and , Vasanthakumar Ganga Ramu*, \",\"doi\":\"10.1021/jasms.5c00105\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Synthetic peptide-based drugs provide customized therapeutic solutions, but developing a peptide medicine presents various challenges, especially in terms of impurity management. This holds true when traditional techniques like RP-HPLC fail to separate low-abundance coeluting impurities. In this regard, liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) has proven to be effective for identifying and characterizing peptide impurities, although its application for accurate quantitation is still limited. This study developed and validated two quantitation strategies using UPLC-HRMS, employing full scan and pseudo-MRM (<i>p</i>-MRM) modes, to identify and accurately quantify two structurally similar coeluting peptide impurities (des-Gly<sup>4</sup> glucagon and des-Thr<sup>5</sup> glucagon) observed in synthetic glucagon. The methods exhibited high specificity and demonstrated good linearity (<i>R</i><sup>2</sup> > 0.99) across a concentration range of 0.25–25 μg/mL. The limits of detection (LODs) for des-Gly<sup>4</sup>-glucagon and des-Thr<sup>5</sup>-glucagon were determined to be as low as 0.01% and 0.02%, with limits of quantitation (LOQs) at 0.02% and 0.04%, respectively. Precision (RSD%) was recorded at less than 10%, and recovery ranged from 100% to 120%. A comparative analysis of the results indicated that both quantifitation methods performed similarly in terms of accuracy, precision, and recovery, producing comparable impurity estimates in three glucagon API samples. However, <i>p</i>-MRM showed slightly better linearity and sensitivity compared to the full scan EIC-based quantitation method.</p>\",\"PeriodicalId\":672,\"journal\":{\"name\":\"Journal of the American Society for Mass Spectrometry\",\"volume\":\"36 10\",\"pages\":\"2072–2078\"},\"PeriodicalIF\":2.7000,\"publicationDate\":\"2025-08-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of the American Society for Mass Spectrometry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://pubs.acs.org/doi/10.1021/jasms.5c00105\",\"RegionNum\":2,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of the American Society for Mass Spectrometry","FirstCategoryId":"92","ListUrlMain":"https://pubs.acs.org/doi/10.1021/jasms.5c00105","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Absolute Quantitation of Coeluting Impurities in Peptide Drugs Using High Resolution Mass Spectrometry: Glucagon a Case Study in Pharmaceutical Development
Synthetic peptide-based drugs provide customized therapeutic solutions, but developing a peptide medicine presents various challenges, especially in terms of impurity management. This holds true when traditional techniques like RP-HPLC fail to separate low-abundance coeluting impurities. In this regard, liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) has proven to be effective for identifying and characterizing peptide impurities, although its application for accurate quantitation is still limited. This study developed and validated two quantitation strategies using UPLC-HRMS, employing full scan and pseudo-MRM (p-MRM) modes, to identify and accurately quantify two structurally similar coeluting peptide impurities (des-Gly4 glucagon and des-Thr5 glucagon) observed in synthetic glucagon. The methods exhibited high specificity and demonstrated good linearity (R2 > 0.99) across a concentration range of 0.25–25 μg/mL. The limits of detection (LODs) for des-Gly4-glucagon and des-Thr5-glucagon were determined to be as low as 0.01% and 0.02%, with limits of quantitation (LOQs) at 0.02% and 0.04%, respectively. Precision (RSD%) was recorded at less than 10%, and recovery ranged from 100% to 120%. A comparative analysis of the results indicated that both quantifitation methods performed similarly in terms of accuracy, precision, and recovery, producing comparable impurity estimates in three glucagon API samples. However, p-MRM showed slightly better linearity and sensitivity compared to the full scan EIC-based quantitation method.
期刊介绍:
The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role.
Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives