Kohei Ohnuma, Misato Hirano-Kodaira, Michiko Bannai, Yoshibumi Shimizu, Masayuki Yamada, Kenji Kinoshita, Gary Ngai-Wa Leung and Hideaki Ishii
{"title":"利用优化的固相萃取和液相色谱-高场非对称离子迁移谱-质谱法筛查马尿中兴奋剂的广谱多肽方法。","authors":"Kohei Ohnuma, Misato Hirano-Kodaira, Michiko Bannai, Yoshibumi Shimizu, Masayuki Yamada, Kenji Kinoshita, Gary Ngai-Wa Leung and Hideaki Ishii","doi":"10.1039/D4AY01477D","DOIUrl":null,"url":null,"abstract":"<p >The abuse of prohibited peptide-based drugs with a broad spectrum of chemical characteristics poses a significant concern for the horseracing industry. Recently, there has been a notable increase in positive cases of small-peptide drugs reported in equine and canine sports. In addition to small peptides, large peptides (over 2 kDa) with structural diversity have also entered the market in increasing numbers as drugs for humans and livestock. However, the simultaneous analysis of both small- and large-peptide-based drugs is still challenging. In this study, a screening method was developed to cover 74 analytes, including peptides, their catabolites, and/or their mimetics, with molecular weights ranging from 0.3 kDa to greater than 5 kDa. The simultaneous extraction of both small and large peptides was achieved using a weak cation-exchange solid-phase extraction cartridge with a mixture of different pore sizes (suitable for large peptides), followed by analysis using liquid chromatography high-field asymmetric ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS). For method validation, the limits of detection (LoDs), reproducibility, recovery, matrix effect, selectivity, and carryover were evaluated. Remarkably, the LoDs of ∼80% of the analytes were less than or equal to 50 pg ml<small><sup>−1</sup></small>, with the lowest LoD (1 pg ml<small><sup>−1</sup></small>) being observed for selected peptides in horse urine. These results indicate a substantial advancement in achieving comprehensive coverage for both small and large peptides with high sensitivity for the purpose of doping control in horseracing and equestrian sports.</p>","PeriodicalId":64,"journal":{"name":"Analytical Methods","volume":" 47","pages":" 8250-8267"},"PeriodicalIF":2.7000,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A broad-spectrum peptide screening method using an optimized solid-phase extraction and liquid chromatography-high-field asymmetric ion mobility spectrometry-mass spectrometry for doping control in equine urine†\",\"authors\":\"Kohei Ohnuma, Misato Hirano-Kodaira, Michiko Bannai, Yoshibumi Shimizu, Masayuki Yamada, Kenji Kinoshita, Gary Ngai-Wa Leung and Hideaki Ishii\",\"doi\":\"10.1039/D4AY01477D\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >The abuse of prohibited peptide-based drugs with a broad spectrum of chemical characteristics poses a significant concern for the horseracing industry. Recently, there has been a notable increase in positive cases of small-peptide drugs reported in equine and canine sports. In addition to small peptides, large peptides (over 2 kDa) with structural diversity have also entered the market in increasing numbers as drugs for humans and livestock. However, the simultaneous analysis of both small- and large-peptide-based drugs is still challenging. In this study, a screening method was developed to cover 74 analytes, including peptides, their catabolites, and/or their mimetics, with molecular weights ranging from 0.3 kDa to greater than 5 kDa. The simultaneous extraction of both small and large peptides was achieved using a weak cation-exchange solid-phase extraction cartridge with a mixture of different pore sizes (suitable for large peptides), followed by analysis using liquid chromatography high-field asymmetric ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS). For method validation, the limits of detection (LoDs), reproducibility, recovery, matrix effect, selectivity, and carryover were evaluated. Remarkably, the LoDs of ∼80% of the analytes were less than or equal to 50 pg ml<small><sup>−1</sup></small>, with the lowest LoD (1 pg ml<small><sup>−1</sup></small>) being observed for selected peptides in horse urine. These results indicate a substantial advancement in achieving comprehensive coverage for both small and large peptides with high sensitivity for the purpose of doping control in horseracing and equestrian sports.</p>\",\"PeriodicalId\":64,\"journal\":{\"name\":\"Analytical Methods\",\"volume\":\" 47\",\"pages\":\" 8250-8267\"},\"PeriodicalIF\":2.7000,\"publicationDate\":\"2024-10-30\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical Methods\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://pubs.rsc.org/en/content/articlelanding/2024/ay/d4ay01477d\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Methods","FirstCategoryId":"92","ListUrlMain":"https://pubs.rsc.org/en/content/articlelanding/2024/ay/d4ay01477d","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
A broad-spectrum peptide screening method using an optimized solid-phase extraction and liquid chromatography-high-field asymmetric ion mobility spectrometry-mass spectrometry for doping control in equine urine†
The abuse of prohibited peptide-based drugs with a broad spectrum of chemical characteristics poses a significant concern for the horseracing industry. Recently, there has been a notable increase in positive cases of small-peptide drugs reported in equine and canine sports. In addition to small peptides, large peptides (over 2 kDa) with structural diversity have also entered the market in increasing numbers as drugs for humans and livestock. However, the simultaneous analysis of both small- and large-peptide-based drugs is still challenging. In this study, a screening method was developed to cover 74 analytes, including peptides, their catabolites, and/or their mimetics, with molecular weights ranging from 0.3 kDa to greater than 5 kDa. The simultaneous extraction of both small and large peptides was achieved using a weak cation-exchange solid-phase extraction cartridge with a mixture of different pore sizes (suitable for large peptides), followed by analysis using liquid chromatography high-field asymmetric ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS). For method validation, the limits of detection (LoDs), reproducibility, recovery, matrix effect, selectivity, and carryover were evaluated. Remarkably, the LoDs of ∼80% of the analytes were less than or equal to 50 pg ml−1, with the lowest LoD (1 pg ml−1) being observed for selected peptides in horse urine. These results indicate a substantial advancement in achieving comprehensive coverage for both small and large peptides with high sensitivity for the purpose of doping control in horseracing and equestrian sports.