Profiling of new modified nucleosides from urine plays an important role in exploring biomarkers for cancer. However, limitations from the nature of the compound, bio-sample, instrumentation, and analytical method pose great challenges to achieving a comprehensive analysis of urinary nucleosides. Herein, a method of BH+/MH+-matching (BH+, protonated nucleobase ion; MH+, protonated precursor ion) was developed to discover novel modified nucleosides from human urine samples based on solid phase extraction targeted toward specific modified nucleosides combined with ultra-performance liquid chromatography coupled with dual-mass spectrometry platforms. Firstly, nucleosides containing 2,3-diol structure on ribose were effectively enriched by PBA (Phenylboronic Acid) cartridges. Secondly, a novel method, "BH+/MH+-matching" was established to achieve rapid screening of modified nucleosides. Based on the in-source fragmentation pattern of nucleosides, a series of putative modified nucleosides were rationally designed and characterized by matching the daughter ion BH+ and its parent ion MH+ in UPLC-MSE spectra. Finally, as a complement to UPLC Q-TOF/MS, UPLC Q-Trap/MS was employed to validate the structure of putative compounds by MRM-IDA-EPI mode. Using the strategy, 12 new cis-diol-containing nucleoside analogs were successfully characterized, which were formed by modified base (m1A, m6A, m2,2,7G, ac4C) and modified ribose containing C5'-O-formylation or C5'-O-methylation. Taken together, the results demonstrated our strategy could efficiently support the rapid discovery of cis-diol-containing nucleosides with modifications on either ribose or base moiety (or both), which exhibited a promising perspective in the future application of biochemical analysis and clinical diagnosis.
从尿液中分析新的修饰核苷在探索癌症生物标志物方面起着重要作用。然而，由于化合物的性质、生物样品、仪器和分析方法的限制，对实现尿核苷的全面分析提出了巨大的挑战。本文提出了一种质子化核碱基离子BH+/MH+匹配方法;采用超高效液相色谱-双质谱联用技术，建立了以特定修饰核苷为目标的固相萃取技术——质子化前体离子MH+ (proton - protonated precursor ion)，从人尿样品中发现新型修饰核苷。首先，在核糖上含有2,3-二醇结构的核苷被PBA (Phenylboronic Acid)试剂有效富集。其次，建立了“BH+/MH+-匹配”的新方法，实现了修饰核苷的快速筛选。基于核苷的源内破碎模式，合理设计了一系列可能的修饰核苷，并在UPLC-MSE光谱中匹配子离子BH+和母离子MH+进行了表征。最后，作为UPLC Q-TOF/MS的补充，利用UPLC Q-Trap/MS通过MRM-IDA-EPI模式对推测化合物的结构进行验证。利用该策略，通过修饰碱基(m1A, m6A, m2,2,7G, ac4C)和含有C5'- o -甲酰化或C5'- o -甲基化的修饰核糖，成功表征了12个新的含顺式二醇核苷类似物。综上所述，结果表明我们的策略可以有效地支持快速发现含有顺式二醇的核苷，这些核苷在核糖或碱基部分(或两者)上进行修饰，这在未来的生化分析和临床诊断中具有广阔的应用前景。
{"title":"BH+/MH+-Matching Method for Discovery of Cis-Diol-Containing Modified Nucleosides in Urine by Ribose-Targeted Solid Phase Extraction Followed by Dual-Mass Spectrometry Platform Identification","authors":"Zhiwei Lu, Xinyue Liu, Xiaoyan Gao","doi":"10.2139/ssrn.3929854","DOIUrl":"https://doi.org/10.2139/ssrn.3929854","url":null,"abstract":"Profiling of new modified nucleosides from urine plays an important role in exploring biomarkers for cancer. However, limitations from the nature of the compound, bio-sample, instrumentation, and analytical method pose great challenges to achieving a comprehensive analysis of urinary nucleosides. Herein, a method of BH+/MH+-matching (BH+, protonated nucleobase ion; MH+, protonated precursor ion) was developed to discover novel modified nucleosides from human urine samples based on solid phase extraction targeted toward specific modified nucleosides combined with ultra-performance liquid chromatography coupled with dual-mass spectrometry platforms. Firstly, nucleosides containing 2,3-diol structure on ribose were effectively enriched by PBA (Phenylboronic Acid) cartridges. Secondly, a novel method, \"BH+/MH+-matching\" was established to achieve rapid screening of modified nucleosides. Based on the in-source fragmentation pattern of nucleosides, a series of putative modified nucleosides were rationally designed and characterized by matching the daughter ion BH+ and its parent ion MH+ in UPLC-MSE spectra. Finally, as a complement to UPLC Q-TOF/MS, UPLC Q-Trap/MS was employed to validate the structure of putative compounds by MRM-IDA-EPI mode. Using the strategy, 12 new cis-diol-containing nucleoside analogs were successfully characterized, which were formed by modified base (m1A, m6A, m2,2,7G, ac4C) and modified ribose containing C5'-O-formylation or C5'-O-methylation. Taken together, the results demonstrated our strategy could efficiently support the rapid discovery of cis-diol-containing nucleosides with modifications on either ribose or base moiety (or both), which exhibited a promising perspective in the future application of biochemical analysis and clinical diagnosis.","PeriodicalId":19880,"journal":{"name":"PharmSciRN EM Feeds","volume":"57 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79234947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Marcela Hollá, D. Šatínský, F. Švec, H. Sklenářová
Natural sweeteners are in high demand as a part of a healthy lifestyle. Among them, sweeteners with decreased caloric value and suitability for diabetes patients are most requested. Extension in their consumption extends the need for their quality control. A fast gradient UHPLC coupled with charged aerosol detection enabling quantitation of stevioside, rebaudioside A-D, and steviolbioside in commercial sweeteners and Stevia rebaudiana plant extracts has been developed. The method was developed to achieve high efficiency, simplicity, versatility, and low solvent consumption. All steviol glycosides were baseline-separated in less than 4 min with a total run time of 7 min. Buffer-free eluents were used in the separations and only 2.45 mL solvent were needed per analysis. The Luna Omega Polar column featuring polar modification of the C18 stationary phase was employed with mobile phases composed of water and acetonitrile for the excellent separation of polar steviol glycosides. The flow rate of the mobile phase 0.35 mL/min, column temperature 50 °C and injection volume 2 µL were used. Critical pair of glycosides, stevioside and rebaudioside A, were baseline separated with a resolution of 2.41. The universal charged aerosol detector allowed quantitation of steviol glycosides with a limit of detection and quantitation 0.15 and 0.5 µg/mL, respectively. Method intra-day precision was less than 2% (RSD), and the recovery was 89.6-105.0% and 93.8-111.4% for plant material and sweetener tablets, respectively. The quantity of steviol glycosides in three out of four commercial sweeteners was 3.0-12.3% higher than declared. The content was about 12.4% less than declared in one sample. But the difference from the labeled content corresponded to trueness and precision of the developed method together with variability of sweeteners production. The most abundant glycoside detected in sweeteners was stevioside followed by rebaudioside A. A leaf-to-stem ratio describing the dominant accumulation of steviol glycosides in leaves affected the differences in the amount of steviol glycosides among plant samples.
{"title":"UHPLC Coupled with Charged Aerosol Detector for Rapid Separation of Steviol Glycosides in Commercial Sweeteners and Extract of Stevia Rebaudiana","authors":"Marcela Hollá, D. Šatínský, F. Švec, H. Sklenářová","doi":"10.2139/ssrn.3875187","DOIUrl":"https://doi.org/10.2139/ssrn.3875187","url":null,"abstract":"Natural sweeteners are in high demand as a part of a healthy lifestyle. Among them, sweeteners with decreased caloric value and suitability for diabetes patients are most requested. Extension in their consumption extends the need for their quality control. A fast gradient UHPLC coupled with charged aerosol detection enabling quantitation of stevioside, rebaudioside A-D, and steviolbioside in commercial sweeteners and Stevia rebaudiana plant extracts has been developed. The method was developed to achieve high efficiency, simplicity, versatility, and low solvent consumption. All steviol glycosides were baseline-separated in less than 4 min with a total run time of 7 min. Buffer-free eluents were used in the separations and only 2.45 mL solvent were needed per analysis. The Luna Omega Polar column featuring polar modification of the C18 stationary phase was employed with mobile phases composed of water and acetonitrile for the excellent separation of polar steviol glycosides. The flow rate of the mobile phase 0.35 mL/min, column temperature 50 °C and injection volume 2 µL were used. Critical pair of glycosides, stevioside and rebaudioside A, were baseline separated with a resolution of 2.41. The universal charged aerosol detector allowed quantitation of steviol glycosides with a limit of detection and quantitation 0.15 and 0.5 µg/mL, respectively. Method intra-day precision was less than 2% (RSD), and the recovery was 89.6-105.0% and 93.8-111.4% for plant material and sweetener tablets, respectively. The quantity of steviol glycosides in three out of four commercial sweeteners was 3.0-12.3% higher than declared. The content was about 12.4% less than declared in one sample. But the difference from the labeled content corresponded to trueness and precision of the developed method together with variability of sweeteners production. The most abundant glycoside detected in sweeteners was stevioside followed by rebaudioside A. A leaf-to-stem ratio describing the dominant accumulation of steviol glycosides in leaves affected the differences in the amount of steviol glycosides among plant samples.","PeriodicalId":19880,"journal":{"name":"PharmSciRN EM Feeds","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76612557","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In this study, a supercritical fluid chromatography (SFC) method based on ion pair reagents was used to separate polar alkaloids. The chromatographic parameters containing stationary phases, additive types, additive concentrations, back pressures, temperatures and flow rates, were optimized. Baseline separation of the compounds were completed in 20 min on an Agilent Pursuit 5 PFP column (4.6 × 150 mm) by using carbon dioxide as mobile phases and 7.5 mM sodium 1-pentanesulfonate as additive with a gradient elution at 140 bar, 60 °C, and a flow rate of 1.5 mL/min. The retention rate and resolution of the analytes are satisfactory. The limits of detection were 27.04−298.03 ng/mL, and the limits of quantification were 90.15−993.42 ng/mL. The recoveries of low and high concentrations were 77.46%−111.86% and 83.84%−111.00%, respectively. This ion pair additive greatly improved the separation efficiency of polar compounds. Consequently, this SFC method was successfully applied to the separation of alkaloids from rhizoma corydalis.
{"title":"Ion Pair-Based Mobile Phase Additives to Improve the Separation of Polar Compounds in Supercritical Fluid Chromatography","authors":"Zicheng Yue, Jun Cao","doi":"10.2139/ssrn.3888168","DOIUrl":"https://doi.org/10.2139/ssrn.3888168","url":null,"abstract":"In this study, a supercritical fluid chromatography (SFC) method based on ion pair reagents was used to separate polar alkaloids. The chromatographic parameters containing stationary phases, additive types, additive concentrations, back pressures, temperatures and flow rates, were optimized. Baseline separation of the compounds were completed in 20 min on an Agilent Pursuit 5 PFP column (4.6 × 150 mm) by using carbon dioxide as mobile phases and 7.5 mM sodium 1-pentanesulfonate as additive with a gradient elution at 140 bar, 60 °C, and a flow rate of 1.5 mL/min. The retention rate and resolution of the analytes are satisfactory. The limits of detection were 27.04−298.03 ng/mL, and the limits of quantification were 90.15−993.42 ng/mL. The recoveries of low and high concentrations were 77.46%−111.86% and 83.84%−111.00%, respectively. This ion pair additive greatly improved the separation efficiency of polar compounds. Consequently, this SFC method was successfully applied to the separation of alkaloids from rhizoma corydalis.","PeriodicalId":19880,"journal":{"name":"PharmSciRN EM Feeds","volume":"88 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78618733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Asari Radix et Rhizoma (AR) is a well-known Chinese herbal medicine which has been used to treat cold headache, toothache, runny nose, rhinorrhea, rheumatism and cough in China. However, the existing Pharmacopoeia standard of AR is still unable to comprehensively evaluating the overall efficacy and safety of AR, whose traditional HPLC detection method has great limitations for the detection of trace aristolochic acids. In order to improving the quality control method of AR, a rapid, simple, and reliable chromatographic analytic method based on ultra performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QQQ-MS) was established for the analysis of 8 target compounds, including 6 aristolochic acids derivates aristolochic acid (AA)-I, AA-II, AA-III, AA-IVa, AA-VIIa, aristololactam I (AL-I), and 2 lignans asarinin and sesamin. Chromatographic separation has been achieved on an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, id 1.8 μm) with a mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B) with gradient elution manner. Positive electrospray ionization mode with multiple reaction monitoring (MRM) was applied for the detection of the eight analytes. Method validation results indicated that UPLC-QQQ-MS was precise, accurate and sensitive for quantitative determination of the eight analytes. Finally, the method was successfully used to the analysis of each component in 15 batches of AR herb samples. In addition to existing marker ingredients AA-I and asarinin, considerable amounts of AA-IVa, AA-VIIa, AL-I and sesamin were also found in each AR sample. The result suggesting that AA-IVa, AA-VIIa, AL-I should be limited and sesamin should be added as a quality marker for the quality control of AR.
细辛是一种著名的中草药，在中国被用来治疗感冒、头痛、牙痛、流鼻涕、鼻漏、风湿病和咳嗽。然而，现有的AR药典标准仍无法全面评价AR的整体功效和安全性，其传统的HPLC检测方法对痕量马兜铃酸的检测存在很大的局限性。为了改进AR的质量控制方法，建立了一种基于超高效液相色谱-三重四极杆串联质谱(UPLC-QQQ-MS)的快速、简便、可靠的色谱分析方法，对马兜铃酸衍生物马兜铃酸(AA)-I、AA- ii、AA- iii、AA- iva、AA- viia、马兜铃内酯I (AL-I)、木脂素asarinin和sesamin等8个目标化合物进行分析。采用ACQUITY UPLC BEH C18色谱柱(50 mm × 2.1 mm, id 1.8 μm)，流动相为含有0.1%甲酸(a)和乙腈(B)的水，梯度洗脱。采用多反应监测的正电喷雾电离模式(MRM)对8种分析物进行检测。方法验证结果表明，UPLC-QQQ-MS方法精密度高、准确度高、灵敏度高。最后，该方法成功应用于15批AR药材样品中各成分的分析。在每个AR样品中，除了现有的标记成分AA-I和细辛素外，还发现了相当数量的AA-IVa、AA-VIIa、AL-I和芝麻素。结果提示，应限制AA-IVa、AA-VIIa、AL-I的含量，并添加芝麻素作为质量标记物对AR进行质量控制。
{"title":"Rapid and Simultaneously Determination of Six Aristolochic Acids and Two Lignans in Asari Radix Et Rhizoma by Ultra Performance Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry","authors":"Hanze Liu, Huida Guan, Xue Cheng, Changhong Wang","doi":"10.2139/ssrn.3874499","DOIUrl":"https://doi.org/10.2139/ssrn.3874499","url":null,"abstract":"Asari Radix et Rhizoma (AR) is a well-known Chinese herbal medicine which has been used to treat cold headache, toothache, runny nose, rhinorrhea, rheumatism and cough in China. However, the existing Pharmacopoeia standard of AR is still unable to comprehensively evaluating the overall efficacy and safety of AR, whose traditional HPLC detection method has great limitations for the detection of trace aristolochic acids. In order to improving the quality control method of AR, a rapid, simple, and reliable chromatographic analytic method based on ultra performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QQQ-MS) was established for the analysis of 8 target compounds, including 6 aristolochic acids derivates aristolochic acid (AA)-I, AA-II, AA-III, AA-IVa, AA-VIIa, aristololactam I (AL-I), and 2 lignans asarinin and sesamin. Chromatographic separation has been achieved on an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, id 1.8 μm) with a mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B) with gradient elution manner. Positive electrospray ionization mode with multiple reaction monitoring (MRM) was applied for the detection of the eight analytes. Method validation results indicated that UPLC-QQQ-MS was precise, accurate and sensitive for quantitative determination of the eight analytes. Finally, the method was successfully used to the analysis of each component in 15 batches of AR herb samples. In addition to existing marker ingredients AA-I and asarinin, considerable amounts of AA-IVa, AA-VIIa, AL-I and sesamin were also found in each AR sample. The result suggesting that AA-IVa, AA-VIIa, AL-I should be limited and sesamin should be added as a quality marker for the quality control of AR.","PeriodicalId":19880,"journal":{"name":"PharmSciRN EM Feeds","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75264990","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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