Biomedical Chromatography

Fan, Yanna; Zhang, Mengwei; Ma, Jiamin; Zhang, Yannan; Yang, Jianjun

doi: 10.1002/bmc.5562pmid: 36480472

Metabolic disorders accompany nonalcoholic fatty liver disease (NAFLD), associated with prediabetes. Lycium barbarum polysaccharides (LBP) seem to be a potential prebiotic, and aerobic exercise has shown protective effects on NAFLD with prediabetes. However, their combined effects on NAFLD and prediabetes remain unclear. This study investigated the effects of LBP and aerobic exercise alone, and their combined effects on the metabolomics of serum, and explored the potential mechanisms utilizing a high‐fat diet‐induced rat model of NAFLD and prediabetes. It provided the metabolic basis for the pathogenesis and early diagnosis of prediabetes complicated with NAFLD. Untargeted metabolomics profiling was performed using ultra‐high‐performance liquid chromatography coupled with quadrupole Orbitrap mass spectrometry to analyze the changes in overall metabolites in each group of samples. An orthogonal partial least squares‐discriminant analysis model with variable importance on projection >1 and p < 0.05 were used as the screening criteria to screen the significant differential metabolites and analyze the expression changes and functional pathways. Different intervention treatments showed clear discrimination by univariate and multivariate analyses. The model group had a high relative level of expression of lipids. Comparison between the two groups showed steroids with high expression after LBP and aerobic exercise treatment separately and alkaloids and fatty acyls with high expression after aerobic exercise and the combination intervention, respectively. Comparison of the five groups showed some of the metabolites to be differently expressed after the intervention improved lipid and fatty acid metabolism. The three types of intervention had sound effects on the changes in liver index for the diseases studied. Furthermore, the combination treatment may be a better choice for disease prevention and treatment than a single treatment. Our analysis of metabolomics confirmed that the different treatments had significant regulatory effects on the metabolic pathways. Our findings strongly support the possibility that aerobic exercise combined with LBP can be regarded as a potential therapeutic method for NAFLD in prediabetics.

Bandaru, Lova Gani Raju; Konduru, Naresh; Kowtharapu, Leela Prasad; Regulagadda, Suryanarayana; Kanuparthy, Phani Raja; Gundla, Rambabu

doi: 10.1002/bmc.5576pmid: 36573285

A related‐substances method was developed for the anticancer drug formulation apalutamide 60 mg tablets and validated using a liquid chromatography gradient elution method. All of the impurities and degradants were separated using the Luna Omega 5 μm Polar C18, (250 × 4.6) mm HPLC column with a 1.0 ml min−1 flow rate. The detection was done at 225 nm by injecting the 10 μl of injection volume, controlling the sample temperature at 10°C and maintaining the column compartment temperature at 30°C. The total run time was 85 min. A 0.01 m disodium phosphate dihydrate pH 4.20 ± 0.05 buffer mixed with acetonitrile in the ratio of 73:27 (v/v) was used as mobile phase A. Mobile phase B consisted of water and acetonitrile in the ratio 30:70 (v/v). The proposed method was validated as per the current regulatory guidelines. The method precisions (RSD) at 100% specification level were 1.41, 1.74, 1.84, and 1.66% for the four impurities. The accuracy results were obtained between 96.0 and 106.3% for the limit of quantitation to the 150% level. The standard and sample solutions stability were established for 44 h at 10°C. The correlation coefficient (r) value was >0.999 for all four impurities, indicating good linearity between the concentration and peak response: 0.9999, 0.9999, 0.9999 and 1.0000. These results show the method’s linearity. The three filter compatibility was proved and it was concluded that 0.45 μm Nylon, PTFE and PVDF filters are suitable. The robustness of the method was established by varying the conditions. The method specificity was proved and the forced degradation data reveal the method’s stability‐indicating nature.

Zhou, Yan; Shan, Hu; Lü, Haitao

doi: 10.1002/bmc.5579pmid: 36602095

An efficient method was established by high‐speed countercurrent chromatography (HSCCC) for the separation and purification of three flavonoids from Oroxylum indicum. Optimized by single‐factor and orthogonal experiments, the optimal extraction conditions were an extraction temperature of 50°C, a solid‐to‐liquid ratio of 1:50 (g/ml), an ethanol concentration of 75% and an extraction time of 45 min. Using a two‐phase solvent system composed of chloroform–methanol–water (6:10:5, v/v/v), the preparative separation was successfully performed by HSCCC in head‐to‐tail elution mode. Totals of 12.63 mg of oroxin A at a purity of 97.61% with 96.46% recovery, 10.96 mg oroxin B at a purity of 98.32% with 98.81% recovery, and 9.34 mg baicalein at a purity of 98.64% with 97.87% recovery were obtained in one‐step separation from 200 mg crude extract. Their chemical structures were confirmed by melting points, HPLC, UV, FTIR, MS, 1H and 13C NMR data. Furthermore, they were efficient scavengers of 1,1‐diphenyl‐2‐picrylhydrazyl and hydroxyl free radicals in a concentration‐dependent manner.

Hu, Aizhen; Liu, Qingwang; Ouyang, Jing

doi: 10.1002/bmc.5573pmid: 36529812

Moscatilin, a bibenzyl derivative from the stem of Dendrobium loddigesii, has been shown to have anticancer activity. The aim of this study was to identify and characterize the possible in vitro metabolites of moscatilin generated from hepatocytes. The metabolites generated in the hepatocytes of mouse, rat, dog, monkey and human were identified and characterized employing ultra‐high‐performance liquid chromatography coupled with quadrupole Orbitrap tandem mass spectrometry (LC–Orbitrap–MS/MS) based on diagnostic fragment ions and accurate mass measurements. A total of 18 metabolites were identified, among which seven were phase I and 11 were phase II metabolites. The plausible structures of the metabolites and the probable biotransformation pathways were proposed based on the diagnostic fragment ions, chemical formula and mass fragmentation pattern, as well as the accurate masses. The majority of phase I metabolites were generated by demethylation and hydroxylation, while phase II metabolites were mainly generated by glucuronidation, glutathione conjugation and sulfation. Our study first expounded the metabolites of moscatilin in mouse, rat, dog, monkey and human hepatocytes and provided a foundation for a further pharmacokinetic and toxicity study. More importantly, LC–Orbitrap–MS/MS combined with diagnostic fragment ions and accurate mass measurements has been proved to be an effective method for the rapid identification of bibenzyl derivatives and their metabolites.

Dudwal, Ramgopal; Jakhar, Bhanwar Lal; Pathan, Abdul Rashid Khan; Jan, Ishrat; Kakralya, Bajrang Lal; Dhaka, Sis Ram; Kataria, Alka; Yadav, Amit Kumar; Choudhary, Sandeep Kumar

doi: 10.1002/bmc.5577pmid: 36573415

doi: 10.1002/bmc.5399pmid: N/A

Yang, Xiao‐Yun; Zhu, Yi‐Wen; Fan, Li; Yi, Shan‐Yong

doi: 10.1002/bmc.5592pmid: 36733235

Xiao’er Qingre Zhike Oral Solution (XQZS) is a commonly used TCM formula to treat cough in children in China. Its complicated composition renders its chemical analysis and mechanism elucidation difficult. To evaluate the bioactive components and mechanism of XQZS against cough, we used a combination strategy of chemical analysis and network pharmacology. A UHPLC/Q‐Orbitrap–MS method was established for the identification and qualitative analysis of components of XQZS, and a total of 33 components were unambiguously identified. Aiming at identifying the components, network pharmacology revealed 107 potential targets related to cough. Using protein–protein interactions analysis, nine core targets were selected. Several cough‐related pathways were enriched using the Kyoto Encyclopedia of Genes and Genomes, including neuroactive ligand–receptor interaction, serotonergic synapse and dopaminergic synapse. The herb–compound–target–pathway network indicated that PTGS2 (COX‐2) was the core target of XQZS against cough. To demonstrate the inhibition effects of the major components against the key target, a COX‐2 inhibitor screening assay was used. Compounds P2, P4, P23 and P49 exhibited promising inhibition effects on COX‐2 at 20 μm, with inhibitory rates of 55.80–69.87%. In conclusion, this study demonstrates that XQZS may alleviate cough via the inhibition of PTGS2 (COX‐2) and the regulation of the serotonergic synapse pathway. The chemical analysis and network pharmacology integrated evaluation provided an efficient strategy for discovering the key pharmacological mechanism of XQZS.

Zakkula, Ashok; Tripathy, Harsha K.; Bestha, Rama Murthi; Vinod, A. B.; Kiran, Vinay; Dittakavi, Sreekanth; Mullangi, Ramesh

doi: 10.1002/bmc.5587pmid: 36680551

Paxalisib is a pan‐PI3K and mTOR inhibitor, currently entering into Phase II clinical trials as a potential drug to treat glioblastoma patients. We report the development and validation of a high‐performance liquid chromatography (HPLC) method for the quantitation of paxalisib in mouse plasma as per the US Food and Drug Administration regulatory guidelines. From the mouse plasma, paxalisib and the internal standard (IS; filgotinib) were extracted using ethyl acetate as an extraction solvent. The chromatographic separation of paxalisib and the IS was accomplished on a Symmetry C18 (250 × 4.6 mm, 5.0 μm) column maintained at 40°C using 10 mm ammonium formate and acetonitrile in gradient conditions at a 0.8 ml/min flow‐rate. The injection volume was 20 μl. The elution was monitored using a photo‐diode array detector set at λmax 280 nm. Paxalisib and the IS eluted at 6.5 and 5.9 min, respectively with a total run time of 10 min. The calibration curve was linear over the range of 111–4,989 ng/ml. Inter‐ and intraday precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated and the results met the acceptance criteria. The validated HPLC method was extended to assess the pharmacokinetic parameters of paxalisib in mice.

Lv, Pan; Zhu, Fei; Lv, Meiyan; Bao, Jiaqi; Lv, Linying; Xu, Xiangwei; Tao, Yi

doi: 10.1002/bmc.5578pmid: 36601730

In this study, we used a serum metabolomics methodology based on GC coupled with MS (GC–MS) to investigate the liver‐protective effects of raw and stir‐fried semen of Hovenia dulcis in rats models of carbon tetrachloride–induced liver injury. Multivariate statistical analysis, such as principal component analysis and orthogonal partial least squares discriminant analysis, were performed to examine changes in the metabolic state of rats with carbon tetrachloride–induced liver injury, as well as the recovery pattern of rats pretreated with the raw and stir‐fried semen of H. dulcis. Liver tissues were subjected to histopathological examination. A total of 47 biomarkers were predicted to contribute to the dynamic pathological processes in the liver injury, such as phenylalanine, glutamic acid, glycine, arachidonic acid and linoleic acid. Further analysis revealed that pathways associated with phenylalanine, tyrosine and tryptophan biosynthesis, and linoleic acid metabolism were altered in the injured liver, and that pretreatment with raw and stir‐fried semen of H. dulcis abolished the changes in the aforementioned metabolic pathways.

Liu, Chenchen; Chen, Huiling; Zhang, Yida; Li, Meng; Jiang, Qiyao; Wang, Zhendong; Yu, Liangwen; Wang, Qi; Pan, Huafeng; Zhuo, Yue

doi: 10.1002/bmc.5589pmid: 36689998

Li‐Zhong‐Xiao‐Pi granules (LZXP) are effective for treating gastric precancerous lesions (GPL) in traditional Chinese medicine. However, the active compounds of LZXP and their potential therapeutic mechanism in GPL remained unclarified. The purpose of this study is to investigate the chemical composition and potential targets of LZXP. Based on the accurate masses, ion fragments, and literature data, a total of 128 compounds were identified in the LZXP sample using ultra‐performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry (UPLC–Q‐TOF–MS) in both positive and negative ion modes, and 28 of these compounds were exactly determined by comparison with authentic reference standards. Meanwhile, 11 typical components were quantified via UPLC during a 24 min period. The linearity, accuracy, stability and recovery of the method were all proven. Through the network pharmacological analysis, six chemicals (quercetin, 4′‐hydroxywogonin, sinensetin, 5, 7, 8, 3′, 4′‐pentamethoxyflavanone, 8‐gingerdione and quercetin) were identified as the active ingredients, and five LZXP targets (AKT1, CYP1B1, PTGS2, MMP9 and EGFR) were found to be the crucial molecules in the treatment of GPL. This study provides a systematic and applicable method for the rapid screening and identification of the chemical constituents from LZXP, and an effective understanding the mechanism of LZXP in the treatment of GPL.