Pub Date : 2024-10-03DOI: 10.1016/j.jpba.2024.116501
Evan M. Hetrick, Brian W. Pack, Chad N. Wolfe, Meng Zhao
The concept of mass balance is discussed as it pertains to the pharmaceutical development of therapeutic peptides. Case studies are presented demonstrating how to perform a mass balance assessment on solid drug substance and solution drug product, and the role of mass balance in the context of the overall product control strategy is discussed. Utilizing mass balance as a specification test where the result is calculated from other critical quality attribute tests, each with their own specification, offers little value as a formalized quality acceptance criterion and may create more deviations, non-value added investigations, and potential batch failures. While useful in characterizing the performance of analytical methods and as part of a rigorous understanding of the manufacturing process and control strategy development, mass balance should not be required as a specification control and should instead be demonstrated during method development and through well-designed forced degradation experiments. Analytical method variability is discussed in relation to the analytical target profile, and the overall impact of sources of variability on the mass balance calculation is described in support of this position.
{"title":"Mass balance analysis for therapeutic peptides: Case studies, applications, and perspectives","authors":"Evan M. Hetrick, Brian W. Pack, Chad N. Wolfe, Meng Zhao","doi":"10.1016/j.jpba.2024.116501","DOIUrl":"10.1016/j.jpba.2024.116501","url":null,"abstract":"<div><div>The concept of mass balance is discussed as it pertains to the pharmaceutical development of therapeutic peptides. Case studies are presented demonstrating how to perform a mass balance assessment on solid drug substance and solution drug product, and the role of mass balance in the context of the overall product control strategy is discussed. Utilizing mass balance as a specification test where the result is calculated from other critical quality attribute tests, each with their own specification, offers little value as a formalized quality acceptance criterion and may create more deviations, non-value added investigations, and potential batch failures. While useful in characterizing the performance of analytical methods and as part of a rigorous understanding of the manufacturing process and control strategy development, mass balance should not be required as a specification control and should instead be demonstrated during method development and through well-designed forced degradation experiments. Analytical method variability is discussed in relation to the analytical target profile, and the overall impact of sources of variability on the mass balance calculation is described in support of this position.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116501"},"PeriodicalIF":3.1,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502442","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-03DOI: 10.1016/j.jpba.2024.116503
Gengpeng Xiao , Lu Yuan , Dandan Liao , Yousheng Huang , Xiang Luo , Jianglian Huo
Based on precolumn derivatization, an analytical method has been developed for the determination of six high boiling point polyhydric alcohols (HBPAs, b.p. > 300 ℃) in cosmetics and toothpaste, including erythritol, xylitol, Pro-Xylane-S, inositol, mannitol, and sorbitol. The water dispersion and oil in water samples were extracted by distilled water. The water in oil sample was firstly pre-dispersed with acetone, and then extracted by distilled water. The extract was concentrated to dry under nitrogen, and derivatized with acetic anhydride under the dispersion and catalysis of anhydrous pyridine. The derivatives were detected by gas chromatography-tandem mass spectrometry in the selected reaction monitoring mode, and quantified using arabinitol as internal standard. The experimental conditions such as the selection of columns, extraction procedures, and derivative conditions were optimized. This method was properly validated under the optimized conditions, and obtained excellent analytical features. Specifically, the correlation coefficients in the range of 0.02 ∼ 0.5 mg/L all exceed 0.992. The method limits of detection and quantification were 0.25 and 0.8 mg/kg, respectively. The average recoveries in toothpaste, cosmetics with oil in water and water in oil were 81.8 ∼ 107.1 %, with the relative standard deviation were 3.1 ∼ 7.2 %. The established method was successfully applied to commercial samples of different matrices, showing the advantages of simplicity, sensitivity, and good reproducibility, and can be used for the determination of HBPAs in cosmetics and toothpaste. The proposed methodology solves the problem that there is no detection method for HBPAs in cosmetics.
{"title":"Implementation of gas chromatography tandem mass spectrometry for the analysis of six high boiling point polyhydric alcohols in cosmetics and toothpaste based on precolumn derivatization","authors":"Gengpeng Xiao , Lu Yuan , Dandan Liao , Yousheng Huang , Xiang Luo , Jianglian Huo","doi":"10.1016/j.jpba.2024.116503","DOIUrl":"10.1016/j.jpba.2024.116503","url":null,"abstract":"<div><div>Based on precolumn derivatization, an analytical method has been developed for the determination of six high boiling point polyhydric alcohols (HBPAs, b.p. > 300 ℃) in cosmetics and toothpaste, including erythritol, xylitol, Pro-Xylane-S, inositol, mannitol, and sorbitol. The water dispersion and oil in water samples were extracted by distilled water. The water in oil sample was firstly pre-dispersed with acetone, and then extracted by distilled water. The extract was concentrated to dry under nitrogen, and derivatized with acetic anhydride under the dispersion and catalysis of anhydrous pyridine. The derivatives were detected by gas chromatography-tandem mass spectrometry in the selected reaction monitoring mode, and quantified using arabinitol as internal standard. The experimental conditions such as the selection of columns, extraction procedures, and derivative conditions were optimized. This method was properly validated under the optimized conditions, and obtained excellent analytical features. Specifically, the correlation coefficients in the range of 0.02 ∼ 0.5 mg/L all exceed 0.992. The method limits of detection and quantification were 0.25 and 0.8 mg/kg, respectively. The average recoveries in toothpaste, cosmetics with oil in water and water in oil were 81.8 ∼ 107.1 %, with the relative standard deviation were 3.1 ∼ 7.2 %. The established method was successfully applied to commercial samples of different matrices, showing the advantages of simplicity, sensitivity, and good reproducibility, and can be used for the determination of HBPAs in cosmetics and toothpaste. The proposed methodology solves the problem that there is no detection method for HBPAs in cosmetics.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116503"},"PeriodicalIF":3.1,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391317","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-03DOI: 10.1016/j.jpba.2024.116500
Kitiara Griffin , Lindsee Miller , Yijia Yang , Elizabeth Sharp , Lane Young , Liza Garcia , John Griswold , Dimitri Pappas
Sepsis is a life-threatening immune response to infection in the body, eventually resulting in fatal organ failure. Current methods utilize blood cultures and quick-Sequential-Organ-Failure-Assessment (qSOFA), but there is a need for more accurate and time-sensitive diagnostic methods to improve survival rates. We present a 3D-printed microfluidic chip that bioconjugates antibodies CD69, CD64, and CD25 to channel surfaces to capture sepsis cells in blood samples and validate it with clinical samples (n = 125 septic, n = 10 healthy). Other variables were taken such as healthy volunteer blood samples and patient demographics to validate and confirm our device’s diagnostic ability. Statistical differences were found between healthy volunteer and sepsis patient antigen cell counts (CD69 p-value < 0.001, CD64 p-value < 0.004, CD25 p-value < 0.0009), and were confirmed using principal component analysis. Demographics such as length of stay, age, culture results, and need for surgery also factored into sepsis detection on a smaller scale than the antigen cell counts. The receiver operating characteristic (ROC) analysis showed an area under the curve (AUC) of 0.989, 0.988, and 0.992 for CD69, CD64, and CD25, respectively, and a combined biomarker panel of 0.997. Overall, the device performed within a shorter time frame of 4 h compared to standard blood culture tests and was validated for use in detecting sepsis in patients.
{"title":"Affinity-based 3D-printed microfluidic chip for clinical sepsis detection with CD69, CD64, and CD25","authors":"Kitiara Griffin , Lindsee Miller , Yijia Yang , Elizabeth Sharp , Lane Young , Liza Garcia , John Griswold , Dimitri Pappas","doi":"10.1016/j.jpba.2024.116500","DOIUrl":"10.1016/j.jpba.2024.116500","url":null,"abstract":"<div><div>Sepsis is a life-threatening immune response to infection in the body, eventually resulting in fatal organ failure. Current methods utilize blood cultures and quick-Sequential-Organ-Failure-Assessment (qSOFA), but there is a need for more accurate and time-sensitive diagnostic methods to improve survival rates. We present a 3D-printed microfluidic chip that bioconjugates antibodies CD69, CD64, and CD25 to channel surfaces to capture sepsis cells in blood samples and validate it with clinical samples (n = 125 septic, n = 10 healthy). Other variables were taken such as healthy volunteer blood samples and patient demographics to validate and confirm our device’s diagnostic ability. Statistical differences were found between healthy volunteer and sepsis patient antigen cell counts (CD69 p-value < 0.001, CD64 p-value < 0.004, CD25 p-value < 0.0009), and were confirmed using principal component analysis. Demographics such as length of stay, age, culture results, and need for surgery also factored into sepsis detection on a smaller scale than the antigen cell counts. The receiver operating characteristic (ROC) analysis showed an area under the curve (AUC) of 0.989, 0.988, and 0.992 for CD69, CD64, and CD25, respectively, and a combined biomarker panel of 0.997. Overall, the device performed within a shorter time frame of 4 h compared to standard blood culture tests and was validated for use in detecting sepsis in patients.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116500"},"PeriodicalIF":3.1,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-02DOI: 10.1016/j.jpba.2024.116498
Anja Gummesson, Fred Zack, Andreas Buettner
Lethal intoxications can only very rarely be recognized during an external examination of corpses, as poisoning does not leave any characteristic findings on the deceased. The present study is a retrospective review on 2,4-dinitrophenol (2,4-DNP) intoxications in human subjects from the beginning of the 20th century until today, as well as a case report on a fatal intoxication of a 50-year old obese man in Rostock (Germany) and an introduction for toxicological analysis in post-mortem specimens of the substance ingested in these rare cases.
Via selective literature search, the information on occurrence and localization of abnormal pathomorphological external and/or internal findings in cases of 2,4-DNP ingestion/ intoxication was gathered. By 2021, a total of 13 case reports with information on morphological findings due to 2,4-DNP ingestion/intoxication were found. The external findings were dominated by yellowing of the skin, followed by exanthemas/rashes and yellowing of the sclera. The internal findings included yellowing of the internal organs, yellow color of the stomach contents, yellowing of the mucous membranes and an intense yellow color of the urine. Yellowish discoloration of the skin, sclera, mucous membranes, internal organs, sweat and/or an intensive yellow discoloration of the urine are not observed in every 2,4-DNP intoxication. However, when they do occur, they are a characteristic indication of 2,4-DNP ingestion and, if localized to the skin, indicate prolonged consumption. A fatal case from Rostock in 2016 due to prolonged intake of 2,4-DNP for weight loss is exemplified. A simple, fast and cost-effective workup combined with HPLC-DAD for post-mortem toxicology ultimately delivers reliable analysis results.
{"title":"2,4-dinitrophenol intoxication and its morphological findings as an indication of substance intake","authors":"Anja Gummesson, Fred Zack, Andreas Buettner","doi":"10.1016/j.jpba.2024.116498","DOIUrl":"10.1016/j.jpba.2024.116498","url":null,"abstract":"<div><div>Lethal intoxications can only very rarely be recognized during an external examination of corpses, as poisoning does not leave any characteristic findings on the deceased. The present study is a retrospective review on 2,4-dinitrophenol (2,4-DNP) intoxications in human subjects from the beginning of the 20th century until today, as well as a case report on a fatal intoxication of a 50-year old obese man in Rostock (Germany) and an introduction for toxicological analysis in post-mortem specimens of the substance ingested in these rare cases.</div><div>Via selective literature search, the information on occurrence and localization of abnormal pathomorphological external and/or internal findings in cases of 2,4-DNP ingestion/ intoxication was gathered. By 2021, a total of 13 case reports with information on morphological findings due to 2,4-DNP ingestion/intoxication were found. The external findings were dominated by yellowing of the skin, followed by exanthemas/rashes and yellowing of the sclera. The internal findings included yellowing of the internal organs, yellow color of the stomach contents, yellowing of the mucous membranes and an intense yellow color of the urine. Yellowish discoloration of the skin, sclera, mucous membranes, internal organs, sweat and/or an intensive yellow discoloration of the urine are not observed in every 2,4-DNP intoxication. However, when they do occur, they are a characteristic indication of 2,4-DNP ingestion and, if localized to the skin, indicate prolonged consumption. A fatal case from Rostock in 2016 due to prolonged intake of 2,4-DNP for weight loss is exemplified. A simple, fast and cost-effective workup combined with HPLC-DAD for post-mortem toxicology ultimately delivers reliable analysis results.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116498"},"PeriodicalIF":3.1,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.jpba.2024.116492
Yujiao Hua , Juan Lv , Yan Zhang , Yongjuan Ding , Jinghua Chen
Oxapliplatin-induced peripheral neuropathy (OIPN) is a significant adverse effect encountered in patients with colorectal cancer undergoing oxaliplatin therapy. However, the pathogenesis of OIPN remains unclear. This study aimed to identify potential diagnostic biomarkers for OIPN and discover the metabolic pathways associated with the disease. Serum samples were collected from 218 subjects, including patients with OIPN and control (CONT). The metabolite profiles were analyzed using nontargeted liquid chromatography-mass spectrometry (LC-MS) serum metabolomics method. Subsequently, differentially altered metabolites were identified and evaluated through multivariate statistical analyses. In this study, patients with OIPN and CONT were distinguished by ten significant metabolites. The levels of racemethionine, O-acetylcarnitine, stearolic acid, aminoadipic acid, iminoarginine, galactaric acid, and all-trans-retinoic acid were increased, whereas the levels of 3-methyl-L-tyrosine, 5-aminopentanoic acid, and erythritol compared were found to be diminished in patients with OIPN when compared to the CONT. Through receiver operating characteristic (ROC) curve analysis, racemethionine, stearolic acid, 5-aminopentanoic acid, erythritol, aminoadipic acid, and all-trans-retinoic acid were pinpointed as promising biomarkers for OIPN. Significantly altered pathways included amino acids (arginine biosynthesis, beta-alanine metabolism, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, lysine degradation, and phenylalanine, tyrosine and tryptophan biosynthesis), lipid (linoleic acid metabolism and the biosynthesis of unsaturated fatty acids), and energy metabolism. This study, by identifying serum biomarkers and dissecting metabolic pathways, offers a groundbreaking perspective on the susceptibility mechanisms underlying OIPN. It stands as an invaluable resource for the adjunctive diagnosis of OIPN, with the potential to diminish the incidence of adverse reactions and to enhance the objectivity and reliability of clinical diagnoses of OIPN.
{"title":"LC-MS-based serum metabolomics analysis and potential biomarkers for oxaliplatin induced neurotoxicity in colorectal cancer","authors":"Yujiao Hua , Juan Lv , Yan Zhang , Yongjuan Ding , Jinghua Chen","doi":"10.1016/j.jpba.2024.116492","DOIUrl":"10.1016/j.jpba.2024.116492","url":null,"abstract":"<div><div>Oxapliplatin-induced peripheral neuropathy (OIPN) is a significant adverse effect encountered in patients with colorectal cancer undergoing oxaliplatin therapy. However, the pathogenesis of OIPN remains unclear. This study aimed to identify potential diagnostic biomarkers for OIPN and discover the metabolic pathways associated with the disease. Serum samples were collected from 218 subjects, including patients with OIPN and control (CONT). The metabolite profiles were analyzed using nontargeted liquid chromatography-mass spectrometry (LC-MS) serum metabolomics method. Subsequently, differentially altered metabolites were identified and evaluated through multivariate statistical analyses. In this study, patients with OIPN and CONT were distinguished by ten significant metabolites. The levels of racemethionine, O-acetylcarnitine, stearolic acid, aminoadipic acid, iminoarginine, galactaric acid, and all-trans-retinoic acid were increased, whereas the levels of 3-methyl-L-tyrosine, 5-aminopentanoic acid, and erythritol compared were found to be diminished in patients with OIPN when compared to the CONT. Through receiver operating characteristic (ROC) curve analysis, racemethionine, stearolic acid, 5-aminopentanoic acid, erythritol, aminoadipic acid, and all-trans-retinoic acid were pinpointed as promising biomarkers for OIPN. Significantly altered pathways included amino acids (arginine biosynthesis, beta-alanine metabolism, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, lysine degradation, and phenylalanine, tyrosine and tryptophan biosynthesis), lipid (linoleic acid metabolism and the biosynthesis of unsaturated fatty acids), and energy metabolism. This study, by identifying serum biomarkers and dissecting metabolic pathways, offers a groundbreaking perspective on the susceptibility mechanisms underlying OIPN. It stands as an invaluable resource for the adjunctive diagnosis of OIPN, with the potential to diminish the incidence of adverse reactions and to enhance the objectivity and reliability of clinical diagnoses of OIPN.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116492"},"PeriodicalIF":3.1,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142375552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.jpba.2024.116495
Jasmin Schairer , Jennifer Römer , Christian Neusüß
The analysis of product-related substances and impurities is a critical step in the biopharmaceutical quality control of multiattribute monoclonal antibodies (mAbs), as posttranslational modifications or other variants can influence the product's biological activity. Many approaches are available for variant analysis; however, they are either variant-specific, mAb-specific, time-consuming, or require expensive equipment. Here, we present a generic capillary electrophoretic method based on a neutral-coated capillary which was coupled to mass spectrometry (MS) via the nanoCEasy interface for mAb variant analysis at the subunit level (enzymatically digested and reduced mAb). The method enabled the separation of several (i) size variants (e.g. glycosylation variants) and (ii) charge variants (e.g. c-terminal lysin clipping) as well as (iii) multiple other proteoforms (e.g. additional glycation) and (iv) incompletely reduced subunits. Separated variants were confirmed by MS/MS fragmentation even for small mass deviations like deamidation or open disulfide bridges. The system, initially developed for one mAb, was tested with nine other IgG1s to show the general applicability of the system. The presented multiattribute method enables fast and detailed characterization of mAb variants with little sample preparation and relatively simple separation equipment enabling the separation of a large set of mAb variants.
产品相关物质和杂质分析是多属性单克隆抗体（mAbs）生物制药质量控制的关键步骤，因为翻译后修饰或其他变体会影响产品的生物活性。目前有许多方法可用于变异体分析，但这些方法要么针对特定变异体，要么针对特定 mAb，要么耗时长，要么需要昂贵的设备。在此，我们介绍一种基于中性涂层毛细管的通用毛细管电泳方法，该方法通过 nanoCEasy 接口与质谱（MS）联用，用于亚单位水平的 mAb 变异分析（经酶消化和还原的 mAb）。该方法可分离多种(i)尺寸变体（如糖基化变体）和(ii)电荷变体（如 c 端赖氨酸剪切）以及(iii)多种其他蛋白形式（如额外糖化）和(iv)未完全还原的亚单位。即使是质量偏差较小的变体，如脱氨或开放的二硫桥，也能通过 MS/MS 片段分析得到确认。该系统最初是为一种 mAb 开发的，后来用其他九种 IgG1 进行了测试，以显示该系统的普遍适用性。所介绍的多属性方法只需很少的样品制备和相对简单的分离设备，就能快速、详细地鉴定 mAb 变体，并分离出大量 mAb 变体。
{"title":"CE-MS and CE-MS/MS for the multiattribute analysis of monoclonal antibody variants at the subunit level","authors":"Jasmin Schairer , Jennifer Römer , Christian Neusüß","doi":"10.1016/j.jpba.2024.116495","DOIUrl":"10.1016/j.jpba.2024.116495","url":null,"abstract":"<div><div>The analysis of product-related substances and impurities is a critical step in the biopharmaceutical quality control of multiattribute monoclonal antibodies (mAbs), as posttranslational modifications or other variants can influence the product's biological activity. Many approaches are available for variant analysis; however, they are either variant-specific, mAb-specific, time-consuming, or require expensive equipment. Here, we present a generic capillary electrophoretic method based on a neutral-coated capillary which was coupled to mass spectrometry (MS) via the nanoCEasy interface for mAb variant analysis at the subunit level (enzymatically digested and reduced mAb). The method enabled the separation of several (i) size variants (e.g. glycosylation variants) and (ii) charge variants (e.g. c-terminal lysin clipping) as well as (iii) multiple other proteoforms (e.g. additional glycation) and (iv) incompletely reduced subunits. Separated variants were confirmed by MS/MS fragmentation even for small mass deviations like deamidation or open disulfide bridges. The system, initially developed for one mAb, was tested with nine other IgG1s to show the general applicability of the system. The presented multiattribute method enables fast and detailed characterization of mAb variants with little sample preparation and relatively simple separation equipment enabling the separation of a large set of mAb variants.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116495"},"PeriodicalIF":3.1,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142377988","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-01DOI: 10.1016/j.jpba.2024.116494
Ji Yeon Jang, Chulmin Moon, Kyuran Kim, Chi Soo Park, Leeseul Jang, Chang Myeong Jeong, Han Seul Lee, Haeun Byeon, Ha Hyung Kim
MIN6, a mouse pancreatic beta cell line, is used in diabetes research, and the cellular N-glycoproteins in membrane are important in regulating the metabolism of insulin secretion. However, the identities of N-glycans in MIN6 cells are yet to be fully elucidated. In this study, the structures of N-glycans were analyzed using liquid chromatography-electrospray ionization-higher energy collisional dissociation-tandem mass spectrometry. The abundances (%) of each N-glycan relative to the total N-glycans (100 %) were also obtained. Fifty N-glycans (with relative abundance of each > 0.5 %) were obtained, revealing 22 bisecting N-acetylglucosamine (GlcNAc; associated with cell adhesion and growth; sum of relative abundance of each: 27.1 %), 21 core-fucosylated (associated with glucose sensing and insulin secretion regulation; 28.3 %), and 16 sialylated (N-acetylneuraminic acid; related to the expression of glucose transporters and diabetes;15.5 %) N-glycans. Membranes contained higher bisecting GlcNAc and core-fucosylation, similar sialylation, but less high-mannosylation than the lysate (the cellular contents). Notably, all bisecting GlcNAc N-glycans were categorized into structures with (16.6 %) or without (10.5 %) core-fucosylation and with (6.9 %) or without (20.2 %) sialylation. The bisecting GlcNAc structures were not found in human islets; moreover, sialylation levels were 6.9 times higher than for human islets. These structural characteristics of N-glycans affect their cell adhesion and distribution through homologous interactions between beta cells, leading to increased insulin secretion efficiency. This study is the first to identify the structures and quantities of 50 N-glycans in MIN6 cell membranes that may play an important role in regulating the functions of pancreatic beta cells.
{"title":"Structural and quantitative characterization of membrane N-glycans from MIN6 mouse pancreatic beta cells using liquid chromatography-quadrupole-Orbitrap tandem mass spectrometry","authors":"Ji Yeon Jang, Chulmin Moon, Kyuran Kim, Chi Soo Park, Leeseul Jang, Chang Myeong Jeong, Han Seul Lee, Haeun Byeon, Ha Hyung Kim","doi":"10.1016/j.jpba.2024.116494","DOIUrl":"10.1016/j.jpba.2024.116494","url":null,"abstract":"<div><div>MIN6, a mouse pancreatic beta cell line, is used in diabetes research, and the cellular <em>N</em>-glycoproteins in membrane are important in regulating the metabolism of insulin secretion. However, the identities of <em>N</em>-glycans in MIN6 cells are yet to be fully elucidated. In this study, the structures of <em>N</em>-glycans were analyzed using liquid chromatography-electrospray ionization-higher energy collisional dissociation-tandem mass spectrometry. The abundances (%) of each <em>N</em>-glycan relative to the total <em>N</em>-glycans (100 %) were also obtained. Fifty <em>N</em>-glycans (with relative abundance of each > 0.5 %) were obtained, revealing 22 bisecting <em>N</em>-acetylglucosamine (GlcNAc; associated with cell adhesion and growth; sum of relative abundance of each: 27.1 %), 21 core-fucosylated (associated with glucose sensing and insulin secretion regulation; 28.3 %), and 16 sialylated (<em>N</em>-acetylneuraminic acid; related to the expression of glucose transporters and diabetes;15.5 %) <em>N</em>-glycans. Membranes contained higher bisecting GlcNAc and core-fucosylation, similar sialylation, but less high-mannosylation than the lysate (the cellular contents). Notably, all bisecting GlcNAc <em>N</em>-glycans were categorized into structures with (16.6 %) or without (10.5 %) core-fucosylation and with (6.9 %) or without (20.2 %) sialylation. The bisecting GlcNAc structures were not found in human islets; moreover, sialylation levels were 6.9 times higher than for human islets. These structural characteristics of <em>N</em>-glycans affect their cell adhesion and distribution through homologous interactions between beta cells, leading to increased insulin secretion efficiency. This study is the first to identify the structures and quantities of 50 <em>N</em>-glycans in MIN6 cell membranes that may play an important role in regulating the functions of pancreatic beta cells.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116494"},"PeriodicalIF":3.1,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142381135","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-30DOI: 10.1016/j.jpba.2024.116489
Tingting Liu , Xiaomin Chen , Guanxuanzi Zhang , Jing Zhao , Qian Lu , Fang Wang , Hongxia Li , Bing Liu , Ping Zhu
Significant pharmacokinetic variation occurs in critically ill patients, leading to underexposure to antibiotics and poor prognosis. In this study, we developed a simple, sensitive, and fast liquid chromatography tandem mass spectrometry (LCMS/MS) platform for the simultaneous quantification of 8 antibacterial and 2 antifungal drugs, which is optimally suited for clinically efficient, real-time therapeutic drug monitoring (TDM). Multiple reaction monitoring (MRM) mass spectrometry was used in this method, and samples were prepared via protein precipitation with methanol. Chromatographic separation was accomplished on a BGIU Column-U02 (2.1ｘ50 mm, 3 µm), with six stable isotopes and one analog as an internal standard. The overall turnaround time of the assay was 5 minutes. All the drugs tested (piperacillin, cefoperazone, meropenem, levofloxacin, moxifloxacin, daptomycin, linezolid, vancomycin, fluconazole and voriconazole) were linear in the test concentration range (r ≥ 0.9900), the accuracy was 95 %-111 %, the precision variation coefficient was greater than or equal to 10 %, the lower limit of quantitation was 0.31–7.51 mg/L, and the coefficient of variation of the matrix factor was less than 10 %. The recovery rates ranged from 85 % to 115 %, and the antibiotics were stable at 4°C and −20°C for 6 days, with an offset of greater than or equal to 15 %. This method was successfully applied to routine TDM in 252 elderly critically ill patients.
{"title":"An LCMS/MS method for the simultaneous determination of ten antimicrobials and its application in critically ill patients","authors":"Tingting Liu , Xiaomin Chen , Guanxuanzi Zhang , Jing Zhao , Qian Lu , Fang Wang , Hongxia Li , Bing Liu , Ping Zhu","doi":"10.1016/j.jpba.2024.116489","DOIUrl":"10.1016/j.jpba.2024.116489","url":null,"abstract":"<div><div>Significant pharmacokinetic variation occurs in critically ill patients, leading to underexposure to antibiotics and poor prognosis. In this study, we developed a simple, sensitive, and fast liquid chromatography tandem mass spectrometry (LC<img>MS/MS) platform for the simultaneous quantification of 8 antibacterial and 2 antifungal drugs, which is optimally suited for clinically efficient, real-time therapeutic drug monitoring (TDM). Multiple reaction monitoring (MRM) mass spectrometry was used in this method, and samples were prepared via protein precipitation with methanol. Chromatographic separation was accomplished on a BGIU Column-U02 (2.1ｘ50 mm, 3 µm), with six stable isotopes and one analog as an internal standard. The overall turnaround time of the assay was 5 minutes. All the drugs tested (piperacillin, cefoperazone, meropenem, levofloxacin, moxifloxacin, daptomycin, linezolid, vancomycin, fluconazole and voriconazole) were linear in the test concentration range (r ≥ 0.9900), the accuracy was 95 %-111 %, the precision variation coefficient was greater than or equal to 10 %, the lower limit of quantitation was 0.31–7.51 mg/L, and the coefficient of variation of the matrix factor was less than 10 %. The recovery rates ranged from 85 % to 115 %, and the antibiotics were stable at 4°C and −20°C for 6 days, with an offset of greater than or equal to 15 %. This method was successfully applied to routine TDM in 252 elderly critically ill patients.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116489"},"PeriodicalIF":3.1,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-30DOI: 10.1016/j.jpba.2024.116493
Joanna E. Frackowiak , Paweł Kubica , Michał Kosno , Agnieszka Potęga , Katarzyna Owczarek-Grzymkowska , Julia Borzyszkowska-Bukowska , Tomasz Laskowski , Ewa Paluszkiewicz , Zofia Mazerska
Unsymmetrical bisacridines (UAs) represent a novel class of anticancer agents. Their high cytotoxicity towards multiple human cancer cell lines and inhibition of human tumor xenograft growth in nude mice signal their potential for cancer treatment. Therefore, the mechanism of their strong biological activity is broadly investigated. Here, we explore the efflux and metabolism of UAs, as both strongly contribute to the development of drug resistance in cancer cells. We tested two highly cytotoxic UAs, C-2028 and C-2045, as well as their glucuronic acid and glutathione conjugates in human cancer cell lines (HepG2 and LS174T). As a point of reference for cell-based systems, we examined the rate of UA metabolic conversion in cell-free systems. A multiple reaction monitoring (MRM)-mass spectrometry (MS) method was developed in the present study for analysis of UAs and their metabolic conversion in complex biological matrices. Individual analytes were identified by several features: their retention time, mass-to-charge ratio and unique fragmentation pattern. The rate of UA uptake and metabolic transformation was monitored for 24 h in cell extracts and cell culture medium. Both UAs were rapidly internalized by cells. However, C-2028 was gradually accumulated, while C-2045 was eventually released from cells during treatment. UAs demonstrated limited metabolic conversion in cells. The glucuronic acid conjugate was excreted, whereas the glutathione conjugate was deposited in cancer cells. Our results obtained from cell-free and cell-based systems, using a uniform MRM-MS method, will provide valuable insight into the mechanism of UA biological activity in diverse biological models.
不对称双吖啶（UAs）是一类新型抗癌剂。它们对多种人类癌细胞株具有很高的细胞毒性，并能抑制人类肿瘤异种移植在裸鼠体内的生长，这表明它们具有治疗癌症的潜力。因此，人们对其强大生物活性的机制进行了广泛的研究。在此，我们探讨了 UAs 的外流和代谢问题，因为这两个问题在很大程度上导致了癌细胞耐药性的产生。我们在人类癌细胞系（HepG2 和 LS174T）中测试了两种高细胞毒性 UAs（C-2028 和 C-2045）及其葡萄糖醛酸和谷胱甘肽共轭物。作为基于细胞的系统的参考点，我们研究了无细胞系统中 UA 的代谢转化率。本研究开发了一种多反应监测（MRM）-质谱（MS）方法，用于分析复杂生物基质中的UA及其代谢转化。通过保留时间、质量电荷比和独特的碎片模式等几个特征来识别单个分析物。在细胞提取物和细胞培养基中对 UA 的吸收率和代谢转化率进行了 24 小时的监测。两种 UA 都能迅速被细胞内化。然而，C-2028 会逐渐积累，而 C-2045 最终会在处理过程中从细胞中释放出来。UAs 在细胞中的代谢转化有限。葡萄糖醛酸共轭物被排出体外，而谷胱甘肽共轭物则沉积在癌细胞中。我们采用统一的 MRM-MS 方法从无细胞和基于细胞的系统中获得的结果将为我们深入了解 UA 在不同生物模型中的生物活性机制提供宝贵的信息。
{"title":"Distinct cellular uptake patterns of two anticancer unsymmetrical bisacridines and their metabolic transformation in tumor cells","authors":"Joanna E. Frackowiak , Paweł Kubica , Michał Kosno , Agnieszka Potęga , Katarzyna Owczarek-Grzymkowska , Julia Borzyszkowska-Bukowska , Tomasz Laskowski , Ewa Paluszkiewicz , Zofia Mazerska","doi":"10.1016/j.jpba.2024.116493","DOIUrl":"10.1016/j.jpba.2024.116493","url":null,"abstract":"<div><div>Unsymmetrical bisacridines (UAs) represent a novel class of anticancer agents. Their high cytotoxicity towards multiple human cancer cell lines and inhibition of human tumor xenograft growth in nude mice signal their potential for cancer treatment. Therefore, the mechanism of their strong biological activity is broadly investigated. Here, we explore the efflux and metabolism of UAs, as both strongly contribute to the development of drug resistance in cancer cells. We tested two highly cytotoxic UAs, C-2028 and C-2045, as well as their glucuronic acid and glutathione conjugates in human cancer cell lines (HepG2 and LS174T). As a point of reference for cell-based systems, we examined the rate of UA metabolic conversion in cell-free systems. A multiple reaction monitoring (MRM)-mass spectrometry (MS) method was developed in the present study for analysis of UAs and their metabolic conversion in complex biological matrices. Individual analytes were identified by several features: their retention time, mass-to-charge ratio and unique fragmentation pattern. The rate of UA uptake and metabolic transformation was monitored for 24 h in cell extracts and cell culture medium. Both UAs were rapidly internalized by cells. However, C-2028 was gradually accumulated, while C-2045 was eventually released from cells during treatment. UAs demonstrated limited metabolic conversion in cells. The glucuronic acid conjugate was excreted, whereas the glutathione conjugate was deposited in cancer cells. Our results obtained from cell-free and cell-based systems, using a uniform MRM-MS method, will provide valuable insight into the mechanism of UA biological activity in diverse biological models.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116493"},"PeriodicalIF":3.1,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142377989","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Since the safety and efficacy of therapeutic products are strongly related to their stability and purity, impurities including the unavoidable degradation products may affect the pharmacological effect. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q3A requires the identification of process impurities and as well as degradation products in any drug substance to assess the inherent stability of the drug. The present work involves an ICH-guided degradation study for the Brinzolamide (BRZ), a topical ophthalmic drug which is generally used to lower the intraocular pressure (IOP) during glaucoma. Under oxidative stress at room temperature for 20 h, four degradation products (namely BRZ-Pk1, BRZ-PK2, BRZ-Pk3, and BRZ-Pk4) are isolated using advanced chromatographic techniques. Upon confirming the masses of the compounds using High-resolution mass spectrometry (HRMS), functional groups are identified with the help of Fourier-transform infrared spectroscopy (FT-IR). Extensive 1-dimensional (1D) and 2-dimensional (2D) Nuclear Magnetic Resonance spectroscopic (NMR) experiments especially 1D nOe, 1H-13C-HSQC and 1H-13C-HMBC unequivocally confirm the structures. Among the four compounds analyzed, three (BRZ-Pk1, BRZ-Pk2, and BRZ-Pk4) are novel, while BRZ-Pk3 was previously reported solely with mass spectrometric data. Nitrogen-based 2D NMR experiments are crucial for determining the oxidation state of hydroxylamine and oxime products within the molecules, and 1D nOe measurements help confirming E/Z isomerism (geometrical isomerism) for BRZ-Pk2 and BRZ-Pk4. All the proposed structures are justified with appropriate analytical data. The proposed mechanisms are expected to help in identifying the possible degradation pathways for similar pharmaceutical candidates.
{"title":"Oxidative stress-induced degradation of Brinzolamide: Isolation and in-depth characterization of unique hydroxylamine and oxime degradation products","authors":"Mahesh Ranga , Arun Kumar Modini , Raju Doddipalla , Muralidharan Kaliyaperumal , Anandarup Goswami","doi":"10.1016/j.jpba.2024.116491","DOIUrl":"10.1016/j.jpba.2024.116491","url":null,"abstract":"<div><div>Since the safety and efficacy of therapeutic products are strongly related to their stability and purity, impurities including the unavoidable degradation products may affect the pharmacological effect. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q3A requires the identification of process impurities and as well as degradation products in any drug substance to assess the inherent stability of the drug. The present work involves an ICH-guided degradation study for the Brinzolamide (BRZ), a topical ophthalmic drug which is generally used to lower the intraocular pressure (IOP) during glaucoma. Under oxidative stress at room temperature for 20 h, four degradation products (namely BRZ-Pk1, BRZ-PK2, BRZ-Pk3, and BRZ-Pk4) are isolated using advanced chromatographic techniques. Upon confirming the masses of the compounds using High-resolution mass spectrometry (HRMS), functional groups are identified with the help of Fourier-transform infrared spectroscopy (FT-IR). Extensive 1-dimensional (1D) and 2-dimensional (2D) Nuclear Magnetic Resonance spectroscopic (NMR) experiments especially 1D nOe, <sup>1</sup>H-<sup>13</sup>C-HSQC and <sup>1</sup>H-<sup>13</sup>C-HMBC unequivocally confirm the structures. Among the four compounds analyzed, three (BRZ-Pk1, BRZ-Pk2, and BRZ-Pk4) are novel, while BRZ-Pk3 was previously reported solely with mass spectrometric data. Nitrogen-based 2D NMR experiments are crucial for determining the oxidation state of hydroxylamine and oxime products within the molecules, and 1D nOe measurements help confirming E/Z isomerism (geometrical isomerism) for BRZ-Pk2 and BRZ-Pk4. All the proposed structures are justified with appropriate analytical data. The proposed mechanisms are expected to help in identifying the possible degradation pathways for similar pharmaceutical candidates.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"252 ","pages":"Article 116491"},"PeriodicalIF":3.1,"publicationDate":"2024-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142423324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号