Biomedical Chromatography

Tao, Yi; Yang, Ying; Zhu, Fei; Wu, Mei; Kong, Xiangjun; Wang, Ping

doi: 10.1002/bmc.5706pmid: 37491783

Anoectochilus roxburghii (Wall.) Lindl. (AR) has been traditionally used to treat inflammatory diseases, but the specific mechanism underlying its hepatoprotective effect remains unclear. Here, serum metabolomics and network pharmacology were employed to investigate the hepatoprotective mechanism of AR. Thirty male Sprague–Dawley rats were divided into six groups: normal, model, positive, high‐dose AR, middle‐dose AR, and low‐dose AR. The positive group received therapeutic doses of silibinin, whereas the AR‐treated groups received different doses of AR extract once daily. After 10 days of intragastric administration, the rats were intraperitoneally injected with a 50% CCl4 olive oil solution (2 mL/kg) to induce liver injury. Serum and liver samples were obtained, and GC–MS was utilized to monitor changes in serum metabolome. The levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and hydrooxproline in serum significantly increased in the model group. On the contrary, AR‐treated group showed a significant decrease in the levels of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and hydrooxproline. Histopathological observation also revealed that the extent of liver injury was alleviated in the AR‐treated group. Fifty differential metabolites were identified, suggesting that AR may prevent liver damage by modulating carbohydrate and amino acid metabolism.

Tiris, Gizem; Genc, Asena Ayse; Oven, Elifnaz; Erk, Nevin

doi: 10.1002/bmc.5712pmid: 37641431

Two spectrophotometric techniques and a novel HPLC method were consecutively applied for the simultaneous quantification of the active ingredients of emtricitabine (EMC), tenofovir (TNF), and bictegravir (BIC). The first spectrophotometric method is the dual amplitude difference method coupled with the ratio difference method. TNF was determined using the dual amplitude difference method, while BIC and EMC were determined using the ratio difference method. The second spectrophotometric method was the constant multiplication with absorbance extraction method, and was applied for the determination of active substances used in the treatment of human immunodeficiency virus (HIV) infection. BIC was determined by the constant multiplication method, whereas EMC and TNF were determined using the absorbance extraction method. For the HPLC method, the XBridge C18 column was used. The solvent system comprised acetonitrile:phosphate buffer (pH 6.8; 30:70 v/v). All active ingredients were detected at 260.0 nm, and the flow rate was 0.5 mL/min. The experiment was completed within 5.5 min. The experiments carried out enabled the simultaneous analysis of the three active substances and they were economical, fast, environmentally friendly, and simple. The methods have been successfully applied to prepare mixtures and tablets without matrix interference. The methods were evaluated in terms of green chemistry. The methods have been validated according to International Council for Harmonisation (ICH) guidelines.

Cardoso, Mariana Silva; Rocha, Andrea Rebouças; Souza‐Júnior, José Antônio; Menezes‐Filho, José Antonio

doi: 10.1002/bmc.5699pmid: 37427763

Homovanillic acid (HVA) and 5‐hydroxyindoleacetic acid (5‐HIAA) are the urinary metabolites of dopamine (DA) and serotonin (5‐HA), respectively. We aimed to develop an extraction method for the determination of HVA and 5‐HIAA, using strong anionic exchange cartridges combined with HPLC with electrochemical detection, and apply it to measure the levels of HVA and 5‐HIAA in children living near a ferro‐manganese alloy plant in Simões Filho, Brazil. The validated method showed good selectivity, sensitivity, precision, and accuracy. The limits of detection (LOD) were 4 and 8 μmol/L for 5‐HIAA and HVA, respectively, in urine. Recoveries ranged from 85.8 to 94%. The coefficients of determination (R2) of the calibration curves were greater than 0.99. Spot urine samples of 30 exposed children and 20 nonexposed ones were processed accordingly. The metabolite levels in exposed and reference children were within the physiological ranges. The medians (range) for 5‐HIAA and HVA of the exposed ones were 36.4 μmol/L (18.4–58.0) and 32.9 μmol/L (<LOD—91.9), respectively. There was no significant difference between the values presented by children in the reference group: 25.7 μmol/L (19.9–81.4) and 35.2 μmol/L (<LOD—67.6) for 5‐HIAA and HVA, respectively. These results suggest that the quantification of the urinary metabolites possibly does not reflect the interference of manganese in the metabolism of DA and 5‐HA in the central nervous system.

Shi, Qingxin; Lin, Yuqi; Huang, Lu; Jin, Shuna; Huang, Rongzeng; Zhang, Lijun; Song, Chengwu; Xu, Lei; Zhang, Shiying

doi: 10.1002/bmc.5707pmid: 37496197

Hyperlipidemia is a chronic metabolic disorder characterized by alterations in lipid metabolism as well as other pathways. Laportea bulbifera, an indigenous medicinal plant of Chinese herbal medicine, exhibits therapeutic effects on hyperlipidemia, but the mechanisms remain unclear. This study investigated the potential mechanisms underlying the anti‐hyperlipidemic effects of L. bulbifera using an integrated strategy based on metabolomics and network pharmacology methods that were established to investigate the potential mechanism of anti‐hyperlipidemia effect of L. bulbifera. First, the therapeutic effects of L. bulbifera on body weight reduction and biochemical indices were assessed. Next, 18 significant metabolites distinguishing the control and model groups were identified based on serum metabolomics and multivariate analyses. Then, a compound–target network was constructed by linking L. bulbifera and hyperlipidemia using network pharmacology. Three metabolic pathways involved in treating hyperlipidemia were identified. Finally, five crucial targets were selected by constructing a bionetwork starting from the compounds and ending in the metabolites. This study established an integrated strategy based on metabolomics coupled with network pharmacology and revealed the mechanism underlying the protective effects of L. bulbifera against hyperlipidemia for the first time.

Jan, Ishrat; Dar, Alamgir A.; Mukhtar, Malik; Shah, Mehraj D.; Wani, Ashraf A.; Dar, Shahnawaz R.; Dar, Irshad H.; Sofi, Javid A.

doi: 10.1002/bmc.5705pmid: 37525473

The present study on “acephate persistence on green pea” was conducted in SKUAST‐Kashmir. The study aimed to determine the persistence, dissipation kinetics and waiting period of acephate on green pea. Acephate was sprayed at 75% soluble powder (SP) at 560 g a.i.ha−1 at the fruiting stage followed by another application at a 10 day interval. A rapid and accurate method (quick, easy, cheap, effective, rugged and safe, QuEChERS) was used for extraction and the residue was determined by gas chromatography–electron capture detection on a CPSIL‐8CB capillary column (0.25um film thickness, 0.25 mm i.d, 30 m length). At the fortification levels of 0.05, 0.1 and 0.5 mg kg−1, the percentage recovery of acephate on green pea was found in the range of 71–107%. The initial deposit of green pea was estimated to be 0.37 mg kg−1. At the indicated dose, the residue of acephate on green pea dissipated below the limit of quantification of 0.05 mg kg−1 after 10 days. Acephate degradation was quick in green pea, with a half‐life of 4.07 days. For safe eating of green peas, a 10 day waiting period is recommended. The gas chromatography–electron capture detection technique was validated by following the SANTE standards.

Tang, Yang; Van Parys, Michael; Walker, Abigail; Drelick, Alexandra; Liang, Xiaorong; Dean, Brian; Chen, Liuxi

doi: 10.1002/bmc.5713pmid: 37544926

In pharmacokinetic studies for respiratory diseases, urea is a commonly used dilution marker for volume normalization of various biological matrices, owing to the fact that urea diffuses freely throughout the body and is minimally affected by disease states. In this study, we developed a convenient liquid chromatography–tandem mass spectrometry (LC–MS/MS) surrogate matrix assay for accurate urea quantitation in plasma, serum and epithelial lining fluid. Different mass spectrometer platforms and ionization modes were compared in parallel. The LC method and mass spectrometer parameters were comprehensively optimized to reduce interferences, to smooth the baseline and to maximize the signal‐to‐noise ratio. Saline was selected as the surrogate matrix, and its suitability was confirmed by good parallelism and accurate quality control sample measurements. Reliable and robust assay performance was demonstrated by precision and accuracy, dilution integrity, sensitivity, recovery and stability, all of which met bioanalysis requirements to support clinical studies. The assay performance was also verified and better understood by comparing it with a colorimetric assay and to a surrogate analyte assay. The newly developed surrogate matrix assay has the potential to be further expanded for urea quantitation in numerous physiological matrices.

Wang, Lili; Li, Zhengyan; Lu, Tulin; Su, Lianlin; Mao, Chunqin; Zhang, Yiting; Zhang, Xinrui; Jiang, Xiaofeng; Xie, Hui; Yu, Xiaoling

doi: 10.1002/bmc.5709pmid: 37533317

Choulingdan mixture (CLDM) is an empirical clinical prescription for the adjuvant treatment of acute lung injury (ALI). CLDM has been used for almost 30 years in the clinic. However, its mechanism for improving ALI still needs to be investigated. In this study, high‐performance liquid chromatography–quadrupole/time‐of‐flight mass spectrometry (HPLC–Q‐TOF–MS/MS) was applied to characterize the overall chemical composition of CLDM. A total of 93 ingredients were characterized, including 25 flavonoids, 20 organic acids, 11 saponins, nine terpenoids, seven tannins and 21 other compounds. Then network pharmacology was applied to predict the potential bioactive components, target genes and signaling pathways of CLDM in improving ALI. Additionally, molecular docking was performed to demonstrate the interaction between the active ingredients and the disease targets. Finally, animal experiments further confirmed that CLDM significantly inhibits pulmonary inflammation, pulmonary edema and oxidative stress in lipopolysaccharide‐induced ALI mice by inhibiting the PI3K–AKT signaling pathway. This study enhanced the amount and accuracy of compounds of CLDM and provided new insights into CLDM preventing and treating ALI.

Tang, Xiao; Zhang, Yu; Zhang, Yue Yi; Zhang, Zunjian; Tian, Yuan

doi: 10.1002/bmc.5714pmid: 37574765

Clopidogrel (CLP) and simvastatin (SV) are commonly used in combination therapies as anti‐cardiovascular drugs. However, the effect of coadministration on the absorption and metabolism of the two drugs in vivo is not clear. This study developed and validated an LC‐MS/MS method for the simultaneous determination of CLP, clopidogrel carboxylic acid (CLPCA), 2‐oxo‐clopidogrel (2‐O‐CLP), SV, and simvastatin hydroxy acid (SVA) in beagle plasma. Chromatographic separation was achieved on an InfinityLab Poroshell 120 SB‐C8 column (2.1 × 100 mm, 2.7 μm) using methanol and 0.1% formic acid in water as the mobile phase at a flow rate of 0.3 mL/min in gradient mode. The lower limits of quantification are 0.1, 0.8, 0.05, 0.05, and 0.05 ng/mL for CLP, CLPCA, 2‐O‐CLP, SV, and SVA, respectively. The selectivity, linearity, accuracy, precision, extraction recovery, matrix effect, and stability were validated within acceptable criteria. This method was successfully applied to the pharmacokinetic drug interaction study between CLP and SV, and the results revealed that combined administration affected the metabolic rate of CLP, SV, and their metabolites. This study is the first to detect CLP, CLPCA, 2‐O‐CLP, SV, and SVA simultaneously.

Fan, Zhuoyu; Guan, Jiao; Tang, Xinmiao; Ge, Shengyu; Shen, Guanghai; Li, Lele; Zhu, Heyun; Feng, Bo

doi: 10.1002/bmc.5715pmid: 37607558

Huangqi Guizhi Wuwu decoction (HGWD) is an effective traditional Chinese medicine prescription, which is used for treating blood arthralgia in the clinic. However, its material basis has not been studied yet. Herein, a new and highly sensitive ultra‐high‐performance liquid chromatography–quadrupole‐time of flight–MS (UHPLC–Q‐TOF–MS) technique is proposed and used for the high‐resolution and accurate identification of the material basis of HGWD. Seventy‐eight compounds have been identified in HGWD. The advantages of information‐dependent acquisition (IDA), sequential window acquisition of all theoretical fragment‐ion spectra (SWATH), and MSALL in the quantitative and qualitative analyses of compounds were compared. For the identification of compounds, the best mode with the highest accuracy is the IDA. For the quantification of compounds, MSALL shows the best repeatability and linearity. This research provides a theoretical basis for the study of quality control of traditional Chinese medicine preparations.

Liu, Meiqi; Liu, Guoqiang; Ma, Zicheng; Wen, Jin Li; Liu, Yi; Sun, Lili; Ren, Xiaoliang

doi: 10.1002/bmc.5701pmid: 37406673

Physalis Calyx seu Fructus (PCF) is a herb widely used in China for its function of clearing heat and detoxifying, benefitting the pharynx and reducing phlegm, both in health care and in tea drinking. However, the quality of its fruit and calyx is uneven and the storage period is short. Therefore, it is crucial to develop other parts of PCF with longer storage periods and obvious medicinal effects. Firstly, high‐performance liquid chromatography was used to develop the fingerprint of different parts of PCF, and various chemometric analyses were conducted to screen out chemical markers. The calyxes of PCF were found to cluster together, distinct from the fruits, roots, stems and leaves. The active components of PCF were concentrated in the persistent calyxes, and flavonoids were mainly found in the persistent calyxes and leaves. Secondly, the extraction of persistent calyxes showed the strongest scavenging ability of DPPH and ABTS. Finally, the important chemical markers were verified by network pharmacological analysis and molecular docking. It provides a reference for the clinical application of PCF, and the obtained chemical markers offer a scientific basis for quality evaluation.