Biomedical Chromatography

Tantawy, Mahmoud A.; Weshahy, Soheir A.; Wadie, Mina; Rezk, Mamdouh R.

doi: 10.1002/bmc.4850pmid: 32302430

A promising combination of tamsulosin HCl and tadalafil has recently been introduced for treating two prevalent and associated urological disorders: benign prostate hyperplasia and erectile dysfunction. Novel HPTLC methods were designed and validated for assaying the cited drugs in their challenging combined formulation. Separation was achieved using HPTLC silica gel 60 F254 plates as a stationary phase with a densitometric measurement at 280 nm. The proposed methods with two different chromatographic systems were successfully applied: a conventional mixture (method I) of ethyl acetate–toluene–methanol–ammonia (5:3:2:0.5, by volume) and a greener one (method II) with ethyl acetate–ethanol–ammonia (8:2:0.1, by volume). The two methods were evaluated through a comparative study in terms of selectivity, tailing factor, developing time and concentration ranges. The greenness profile for each method was then appraised with several green guides, namely GlaxoSmithKline solvent sustainability guide, Environmental, Health and Safety (EHS) tool, National Environmental Method Index (NEMI) and Eco‐scale. Moreover, method specificity and peak homogeneity were evaluated by peak purity assessment using the winCATS® software spectral correlation tool. The methods have potential for being simple, fast, economic and selective, and the greener one could be a good option for sustainable analysis of the drugs.

Ji, Yubin; Luo, Hongjun; Li, Hui; Lin, Zhexuan; Luo, Wenhong

doi: 10.1002/bmc.4845pmid: 32267542

An ultra‐high performance liquid chromatography tandem mass spectrometry method was developed for determination of homocysteine (HCY) in human plasma. The HCY was derivatized with 2‐chloro‐1‐methylquinolinium tetrafluoroborate and isolated using solid‐phase extraction. Derivatization, isolation and detection procedures were optimized. Satisfactory linearity was obtained with determination coefficients (r2) >0.999. The intra‐ and inter‐day precisions were in the interval of 1.2–5.1% and accuracy was within ±7%. Mean recoveries were close to 100%. The limit of detection and the limit of quantification were 0.46 and 1.38 μmol/L, respectively. The method was then applied to investigate the relationship between plasma HCY and whole blood 5‐methyltetrahydrofolate levels in healthy volunteers. The results revealed that the plasma level of HCY was significantly negatively correlated to whole blood 5‐methyltetrahydrofolate in volunteers.

Chen, Jing; Guan, Zhenhua; Dong, Na; Li, Xueliang

doi: 10.1002/bmc.4863pmid: 32329073

Ziritaxestat is a first‐in‐class autotoxin inhibitor. The purpose of this study was to develop a liquid chromatography/electrospray ionization tandem mass spectrometric (LC–MS/MS) method for the determination of ziritaxestat in rat plasma. The plasma sample was deproteinated using acetonitrile and then separated on an Acquity BEH C18 column with water containing 0.1% formic acid and acetonitrile as mobile phase, which was delivered at 0.4 ml/min. Ziritaxestat and the internal standard (crizotinib) were quantitatively monitored with precursor‐to‐product transitions of m/z 589.3 > 262.2 and m/z 450.1 > 260.2, respectively. The total running time was 2.5 min. The method showed excellent linearity over the concentration range 0.5–2000 ng/ml, with correlation coefficient >0.9987. The extraction recovery was >82.09% and the matrix effect was not significant. Inter‐ and intra‐day precisions (RSD) were <11.20% and accuracies were in the range of −8.50–7.45%. Ziritaxestat was demonstrated to be stable in rat plasma under the tested conditions. The validated LC–MS/MS method was successfully applied to study the pharmacokinetic profiles of ziritaxestat in rat plasma after intravenous and oral administration. Pharmacokinetic results demonstrated that ziritaxestat displayed a short half‐life (~3 h) and low bioavailability (20.52%).

Zhao, Ling‐Kun; Zhao, Yun‐Bo; Yu, Peng‐Cheng; Zhang, Peng‐Xia

doi: 10.1002/bmc.4847pmid: 32285481

Ultra‐performance liquid chromatography/mass spectrometry‐based metabolomics can been used for discovery of metabolite biomarkers to explore the metabolic pathway of diseases. Identification of metabolic pathways is key to understanding the pathogenesis and mechanism of disease. Myocardial dysfunction induced by sepsis (SMD) is a severe complication of septic shock and represents major causes of death in intensive care units; however its pathological mechanism is still not clear. In this study, ultrahigh‐pressure liquid chromatography with mass spectrometry‐based metabolomics with chemometrics anaylsis and multivariate pattern recognition analysis were used to detect urinary metabolic profile changes in a lipopolysaccharide‐induced SMD mouse model. Multivariate statistical analysis including principal component analysis and orthogonapartial least squares discriminant analysis for the discrimination of SMD was conducted to identify potential biomarkers. A total of 19 differential metabolites were discovered by high‐resolution mass spectrometry‐based urinary metabolomics strategy. The altered biochemical pathways based on these metabolites showed that tyrosine metabolism, phenylalanine metabolism, ubiquinone biosynthesis and vitamin B6 metabolism were closely connected to the pathological processes of SMD. Consequently, integrated chemometric analyses of these metabolic pathways are necessary to extract information for the discovery of novel insights into the pathogenesis of disease.

Bala, Veenu; Chhonker, Yashpal S.; Sleightholm, Richard L.; Crawford, Ayrianne J.; Hollingsworth, Michael A.; Murry, Daryl J.

doi: 10.1002/bmc.4859pmid: 32307720

A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (MS/MS) method was developed and validated for the quantitation of the novel CDK5 inhibitor ‘20–223' in mouse plasma. Separation of analytes was achieved by a reverse‐phase ACE Excel C18 column (1.7 μm, 100 × 2.1 mm) with gradient elution using 0.1% formic acid (FA) in methanol and 0.1% FA as the mobile phase. Analytes were monitored by MS/MS with an electrospray ionization source in the positive multiple reaction monitoring mode. The MS/MS response was linear over the concentration range 0.2–500 ng/mL for 20–223. The within‐ and between‐batch precision were within the acceptable limits as per Food and Drug Administration guidelines. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. Compound 20–223 was highly bound to mouse plasma proteins (>98% bound). Utilizing mouse S9 fractions, in vitro intrinsic clearance (CLint) was 24.68 ± 0.99 μL/min/mg protein. A total of 12 phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. The validate method required a low sample volume, was linear from 0.2 to 500 ng/mL, and had acceptable accuracy and precision.

Tiwari, Shristy S.; Mukesh, Sumit; Sangamwar, Abhay T.; Talluri, M.V.N. Kumar

doi: 10.1002/bmc.4860pmid: 32311767

Cetilistat (CET) is a pancreatic lipase inhibitor approved for management of obesity after the serious adverse effects exhibited by its analogue orlistat. Exhaustive literature review reveals lack of comprehensive reports on its biotransformation. With a view to study the same, the present study reports the identification and characterization of metabolites of CET in rats using UPLC–MS/MS. As the small intestine is the site of action for CET, it is important that the role of microbial flora in the metabolism of CET be explored. To achieve this, the metabolic profile of CET was compared between normal and pseudo‐germ‐free rats. The study involved the administration of a drug suspension to male Sprague–Dawley pseudo‐germ‐free and normal untreated rats followed by collection of urine, feces, and blood at specific intervals. Sample preparation was performed using liquid–liquid extraction and concentration of samples followed by analysis using LC–MS/MS. Finally, an in silico study was performed on the drug and metabolites to predict their toxicological properties using ADMET PredictorTM software. Four metabolites of CET were observed in in vivo matrices. As expected, significant changes were observed both qualitatively and quantitatively, implying that formation of metabolites was both CYP enzymes and gut microflora mediated.

Wu, Chao; Liu, Hanze; Rong, Xiaojuan; Liu, Jiahao; Ding, Wenzheng; Cheng, Xuemei; Xing, Jianguo; Wang, Changhong

doi: 10.1002/bmc.4865pmid: 32330321

The aerial parts of Dracocephalum moldavica L. are extensively used in traditional ethnic medicines in China as a remedy for cardiovascular and cerebrovascular damage. However, the chemical composition and the accumulation of main secondary metabolites of D. moldavica in different natural environments remain unclear. This study aimed to conduct a qualitative and quantitative analysis of the main secondary metabolites to explore the quality variation of D. moldavica in markets. The evaluation of space–time accumulation of main secondary metabolites in D. moldavica was carried out during different growth periods and in different geographical locations. A total of 35 ingredients were detected and 24 identified, including 21 flavonoids, two phenolic acids and one coumarin by UPLC–QTOF–MS method. Furthermore, a simple and convenient HPLC method was successfully developed for the simultaneous determination of lutelin‐7‐O‐glucuronide and tilianin and rosmarinic acid in D. moldavica. The results of space–time accumulation analysis showed the distinct variation of secondary metabolites of D. moldavica with the growth period and geographical location. Finally, the current study provided a meaningful and useful approach for comprehensively evaluating the quality of D. moldavica.

Shigeta, Kensuke; Kikuchi, Masafumi; Tanaka, Masaki; Takasaki, Shinya; Oishi, Hisashi; Sado, Tetsu; Matsuda, Yasushi; Noda, Masafumi; Okada, Yoshinori; Mano, Nariyasu; Yamaguchi, Hiroaki

doi: 10.1002/bmc.4853pmid:

Guo, Nan; Zhang, Aiying; Zhuang, Hui; Zhang, Changzhen

doi: 10.1002/bmc.4862pmid: 32307722

A simple and sensitive ultra‐high‐performance liquid chromatography tandem mass spectrometric method was developed and validated for the determination of foretinib in rat plasma. The analyte and internal standard were extracted from the bio‐samples with acetonitrile and then separated on an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm) using 0.1% formic acid aqueous and acetonitrile as mobile phase, at a flow rate of 0.4 ml/min. The mass detection was performed in positive selected reaction monitoring mode with precursor‐to‐product transitions at m/z 317.1 > 128.1 for foretinib and m/z 502.2 > 323.1 for internal standard. The assay was demonstrated to be linear in the concentration range of 0.5–1000 ng/ml, with correlation coefficient >0.999. The mean extraction recovery of foretinib from rat plasma was within the range of 84.55–88.09%, while the matrix effect was in the range of 88.56–99.21%. The intra‐ and inter‐day precisions were <12.95% and the accuracy ranged from −7.55 to 8.57%. Foretinib was stable in rat plasma under the tested storage conditions. The validated assay was successfully applied to the pharmacokinetic study of foretinib in the rats. The results revealed that foretinib showed moderate elimination half‐life, low clearance and dose‐independent pharmacokinetic profiles inrats.

Wang, Jing; Zhang, Yong; Zhou, Meiqi; Zheng, Meizhu; Cui, Jing; Liu, Zhiqiang; Liu, Chunming; Liu, Shu

doi: 10.1002/bmc.4852pmid: 32302005

Superoxide anion radical scavenger and xanthine oxidase inhibitor play an important role in the treatment of several relevant human diseases. In the present study, ultrafiltration liquid chromatography–mass spectrometry coupled to microplate reader was applied to screen and identify superoxide anion radical scavengers and xanthine oxidase inhibitors from total flavonoids of Ginkgo biloba leaves. As a result, four compounds (quercetin, apigenin, kaempferol and isorhamnetin) were screened as xanthine oxidase inhibitors by ultrafiltration LC–MS, and the 50% scavenging concentration values of the screened flavonoids were lower than those for allopurinol. Lineweaver–Burk plot results indicated that kaempferol was a competitive xanthine oxidase inhibitor; the other flavonoids were all anticompetitive inhibitors. Four flavonoids—rutin, quercetin, kaempferol and isorhamnetin—were screened as superoxide anion radical scavengers by LC–MS. The results demonstrate that the method for screening and evaluation of superoxide anion radical scavenger and xanthine oxidase inhibitor from a complex mixture system is feasible and efficient.