Biomedical Chromatography

Zhang, Ye; Lu, Jingkun; Ma, Yuheng; Sun, Lijun; Wang, Suwei; Yue, Xin; Yu, Jiuwang; Xue, Peifeng

doi: 10.1002/bmc.5475pmid: 35947036

This study established the fingerprint of Syringa pinnatifolia Hemsl. (SP), analyzed the SP ingredients absorbed into the rats blood, and evaluated its anti‐myocardial ischemic effect to provide a scientific basis for the follow‐up development and research of SP and lay a foundation for its clinical application using ultra‐performance liquid chromatography‐Q Exactive‐mass spectrometry and GC–MS. Myocardial infarction was induced in rat by ligating the left anterior descending branch of the rat coronary artery, and SP alcohol extract was administered to evaluate its anti‐myocardial ischemic effect. We analyzed the SP ingredients absorbed into the rats blood, screened the active compounds, established a database of SP anti‐myocardial ischemic targets, and explored the possible mechanism of SP in treating myocardial infarction using bioinformatics. The rats were examined using echocardiography, serum biomarkers were determined, and pathological changes were observed by histopathological examination. TUNEL staining was performed to detect the apoptotic level of cells, and Western blot and quantitative real‐time polymerase chain reaction were performed to detect the expression levels of Bcl‐2, Bax, and Caspase‐3 in heart tissues. In the fingerprint of SP, 24 common peaks were established, and the similarity evaluation results of 10 batches of SP were all >0.9. Ultra‐performance liquid chromatography‐Q Exactive‐mass spectrometry and GC–MS detected 17 active ingredients in the drug‐containing serum, including terpenoids, flavonoids, phenols, phenylpropanoids, and phenolic acids, the most abundant of which was resveratrol. Enrichment analysis of SP targets against myocardial ischemia revealed that key candidate targets of SP were significantly enriched in multiple pathways associated with apoptosis. Resveratrol was administered to the successfully modeled rats, and the results showed that the resveratrol group significantly decreased left ventricular end‐diastolic diameter and left ventricular end‐systolic diameter and significantly increased ejection fraction and fractional shortening in all groups compared with the model group. Resveratrol significantly decreased the levels of creatine kinase isoenzyme and lactate dehydrogenase in serum compared to the model group (P < 0.001). Hematoxylin–eosin staining of rat myocardial tissue showed that all lesions were reduced under microscopic observation in the resveratrol group compared with the model group. Real‐time polymerase chain reaction and Western blot results showed that the resveratrol group downregulated the expression of the proapoptotic factor Bax, upregulated the expression of the antiapoptotic factor Bcl‐2, and decreased the expression of Caspase‐3. The established fingerprints are accurate, reliable, and reproducible and can be used as an effective method for quality control of the herbs. The anti‐myocardial ischemia effect of SP is that resveratrol improves cardiac function and inhibits cardiomyocyte apoptosis to protect cardiomyocytes. The present study provides ample evidence for the clinical use of SP, suggesting that this drug has great potential in the treatment of ischemic heart disease.

Wu, Jiaofeng; Duan, Rong; Deng, Haoran; Li, Lele; Zhao, Yunli; Yu, Zhiguo

doi: 10.1002/bmc.5453pmid: 35853731

Aconiti Radix [Chuanwu (CW)] is widely used to treat chronic and intractable diseases due to its remarkable curative effect. CW has been combined with honey for thousands of years to reduce toxicity and enhance efficacy. This study first determined the compatibility mechanism of CW with honey using a comparative pharmacokinetic concept. We developed and validated a simple, sensitive, specific, and accurate UHPLC–MS/MS method to simultaneously determine five Aconitum alkaloids in rat plasma after the oral administration of CW decoction and CW–honey concentrated solution. Pharmacokinetic parameters were significantly different between the two groups (P < 0.01 and P < 0.05). Compared with the CW group, Cmax and AUC0 → t decreased in the CW–honey group for three diester‐diterpenoid alkaloids (hypaconitine, mesaconitine, and aconitine); Tmax and T1/2 were prolonged. However, Cmax and AUC0 → t increased in the CW–honey group for two monoester‐diterpenoid alkaloids (benzoylaconine and benzoylmesaconine); Tmax was shortened, and T1/2 was prolonged. These findings suggest that honey affected the pharmacokinetic behaviors of five Aconitum alkaloids. We speculate that the detoxification and synergism of honey might result from reducing the toxicity of diester‐diterpenoid alkaloids and promoting the biological activity of monoester‐diterpenoid alkaloids in vivo. This study provides a theoretical basis for the clinical use of CW combined with honey.

Yu, Xinwei; Wu, Yuchen; Wang, Yuzhen; Cheng, Yu; Xiang, Zheng

doi: 10.1002/bmc.5473pmid: 35916265

Soyasaponin Bb is one of the bioactive oleanolic acid–type triterpenoid saponins mainly isolated from soybean. It possesses significant antithrombosis, hypolipidemic, anticancer, and antioxidant activities. However, the metabolic profiles of soyasaponin Bb are still unknown. The present study investigated the metabolites of soyasaponin Bb in plasma, bile, urine, and feces samples after intragastric administration using ultra‐performance liquid chromatography coupled with quadrupole‐time‐of‐flight‐mass spectrometry, and its possible metabolic pathways were subsequently proposed. Using the metabolite profiling strategy, 11 metabolites were first identified from urine, plasma, bile, and feces of rats after intragastric administration of soyasaponin Bb. Hydroxylation and hydrolysis were the major metabolic pathways of soyasaponin Bb in rat. The results expand our knowledge of the metabolism of soyasaponin Bb, which could provide valuable information for better comprehension of future pharmacological research.

Zheng, Dayong; Li, Xiao; Chu, Yang; Li, Yazhuo; Li, Dekun; Ju, Aichun; Wu, Yi; Xie, Yuesheng; Li, Wei

doi: 10.1002/bmc.5463pmid: 35895507

Salvianolic acids for injection (SAI) is developed from traditional Chinese medicine and approved for the treatment of cardiovascular and cerebrovascular diseases. Clopidogrel is an inhibitor of platelet aggregation, which is often prescribed for patients in combination with SAI. This present study aimed to assess the effects of SAI on the pharmacogenomics, pharmacokinetics, and pharmacodynamics of clopidogrel, thereby ensuring the safety and efficacy of coadministration. In vitro cytochrome P450 isoenzyme assays were performed in human liver microsomes using LC–MS/MS method to assess the metabolites of CYPs substrates. The effects of SAI on the pharmacokinetic and pharmacodynamic behaviors of clopidogrel were investigated in rats. The main pharmacokinetic parameters were analyzed using LC–MS/MS. Prothrombin time, activated partial thromboplastin time, bleeding time, and inhibition of platelet aggregation were measured to evaluate the effects of pharmacodynamics. Our study revealed that the clinical dose of SAI has no significant inhibitory effect on clopidogrel‐related liver microsome metabolic CYP450 isoenzymes. Moreover, SAI did not affect the pharmacokinetics of clopidogrel when rats were administered both single and multiple doses. In pharmacodynamic study, SAI has no effect on platelet aggregation rate, prothrombin time, and activated partial thromboplastin time of clopidogrel but could significantly prevent the risk of bleeding caused by clopidogrel.

Long, Jia‐Yi; Hu, Ya‐Hui; Xia, Ying; Du, Fei‐Fei; Dai, Hao‐Ran; Tian, Man; Xu, Jing; Cheng, Rui; Ding, Xuan‐Sheng; Guo, Hong‐Li; Chen, Feng

doi: 10.1002/bmc.5462pmid:

Meng, Xianshuang; Bai, Hua; Ma, Qiang; Zhang, Peng; Ma, Hong; Deng, Yulin

doi: 10.1002/bmc.5464pmid: 35899750

Neuroinflammatory injury is one of the typical brain injuries after the body is exposed to radiation. It is mainly characterized by the release of inflammatory factors by activated microglia and peripherally invading lymphocytes. To provide early warning for nerve injury and early diagnosis of neurodegenerative diseases, it is of great significance to explore the biomarker candidates of neuroinflammatory injury. This study focused on the screening of small molecular biomarker candidates in peripheral blood from rats with neuroinflammatory injury induced by whole‐brain irradiation. The rats were exposed to 0, 10, 10 × 3, and 30 Gy of cobalt‐60 γ‐rays. Serum was collected on the 30th day after exposure and analyzed using reversed‐phase liquid chromatography and hydrophilic interaction liquid chromatography coupled with high‐resolution mass spectrometry based on untargeted metabolomics. Biomarker candidates were investigated by comparing the 0‐Gy group and three irradiation groups using univariate statistical analysis, principal component analysis, and orthogonal partial least squares discriminant analysis. Eleven biomarker candidates were putatively identified, and four major altered metabolic pathways were found. The screened small molecular biomarker candidates could be used as a useful supplement to traditional biomacromolecule markers and may be valuable for radiation protection, target therapy of inflammatory injury, and discovery of new target drugs for the prevention and cure of related neurodegenerative diseases.

Qi, Bing; Bao, Yongrui; Wang, Shuai; Li, Tianjiao; Meng, Xiansheng

doi: 10.1002/bmc.5460pmid: 35903874

Qizhiweitong is a famous traditional Chinese prescription medicine. It has been used to treat various stomach disorders, such as functional dyspepsia, chronic gastritis and intestinal stress syndrome, for a long time and gives favorable therapeutic effects in clinical settings. However, its chemical composition and possible bioactive components are not completely known. In the present study, we used ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (UPLC–QTOF–MS) and qualitatively analyzed the chemical composition of Qizhiweitong tablet extract and the absorbed prototype constituents along with corresponding metabolites in rat plasma following oral administration of Qizhiweitong tablet on the basis of our self‐developed component database that was established accurately and rapidly. We detected a total of 119 compounds and 61 xenobiotics in the Qizhiweitong tablet, which included 32 prototypes and 28 metabolites. The results of the present study laid a solid foundation for quality marker screening and an integrative pharmacology‐based study on the Qizhiweitong tablet.

AL‐Hashimi, Nabil N.; Al‐Degs, Yahya S.; Jaafreh, Sawsan; Al‐Khatib, Hatim S.; El‐Sheikh, Amjad H.; Abdelghani, Jafar I.; Jaber, Mai R.

doi: 10.1002/bmc.5476pmid: 35918842

A sensitive and simple sample pretreatment method based on a two‐phase solvent bar microextraction (SBME) technique coupled with HPLC–diode array detector (DAD) was developed for simultaneous extraction and determination of trace amounts of furosemide and carbamazepine in human urine and plasma samples. The significance of operational factors on carbamazepine and furosemide extraction efficiency % (EE%) was screened using full factorial design (FFD) while central composite design (CCD) was used to model the entire process. A quadratic model was found convenient to correlate the extraction EE% of selected drugs with dominant experimental factors. A Pareto chart was also used to examine the importance of factors on drugs’ EE%. The analytical performance of the method in urine and plasma samples demonstrated good linearity (R2 ˃ 0.992) with detection limits ranging from 4.2 to 10.9 μg L−1, and extraction recovery over 89.45% for both drugs in urine and plasma samples. A comparison against published methods was also performed and the results revealed that the developed method exhibits a confident sensitivity, feasible operation, and simple analysis for both drugs. Finally, the practicability of the validated SBME–HPLC–DAD method was demonstrated by successfully applying it to the analysis of furosemide and carbamazepine in real patient urine samples.

Kiesel, Brian F.; Parise, Robert A.; Krishnamurthy, Anuradha; Gore, Steven; Beumer, Jan H.

doi: 10.1002/bmc.5455pmid: 35876841

Ataxia‐telangiectasia‐mutated and Rad3‐related (ATR) is master regulator of the DNA‐damage response that, through multiple mechanisms, can promote cancer cell survival in response to replication stress from sources, including chemotherapy and radiation. Elimusertib (BAY‐1895344) is an orally available small‐molecule ATR inhibitor currently in preclinical and clinical development for cancer treatment. To support these studies and define elimusertib pharmacokinetics, we developed a HPLC–MS method for its quantitation. A 50‐μL volume of plasma was subjected to acetonitrile protein precipitation and then chromatographic separation using a Phenomenex Polar‐RP column (2 × 50 mm, 4 μm) and a gradient mobile phase consisting of 0.1% formic acid in acetonitrile and water during a 7‐min run time. Mass spectrometric detection was achieved using a SCIEX 4000 triple‐stage mass spectrometer with electrospray positive‐mode ionization. With a stable isotopic internal standard, the assay was linear from 30 to 5000 ng/mL and proved to be both accurate (93.5–108.2%) and precise (<6.3% coefficient of variation) fulfilling criteria from the Food and Drug Administration guidance on bioanalytical method validation. This LC–MS/MS assay will support several ongoing clinical studies by defining elimusertib pharmacokinetics.

Zhou, Zehua; Liu, Wangzhenzu; Li, Xue; Li, Chan; An, Rui; Liang, Kun; Wang, Xinhong

doi: 10.1002/bmc.5458pmid: 35883246

Chronic gastritis (CG) has become a major threat to human health. Banxia Xiexin Decoction (BXXXD) has been used clinically to treat gastritis by acting on the spleen and stomach for thousands of years. Baicalin, wogonoside, liquiritin and liquiritigenin are the main bioactive flavonoids of BXXXD. A rapid, sensitive and selective HPLC–triple quadrupole (TQ)–MS/MS method was developed to simultaneously quantify the four flavonoids in rat plasma in this study. With salidroside as internal standard (IS), plasma samples were extracted and separated on a Welch HPLC XB‐C18 column (2.1 × 50 mm, 1.8 μm) using gradient elution. The optimized gradient of the mobile phase consisted of water (containing 0.1% formic acid) (A) and methanol (B) was used. Detection was implemented in multiple reaction monitoring mode with an electrospray negative ionization source. The comparative pharmacokinetics of four analytes in normal and CG rats after oral administration of BXXXD or its different compatibilities were first investigated. The results indicated that the pharmacokinetic behaviors of analytes were obviously changed in CG rats. From the comparison between the whole prescription group and the compatibility groups, it was found that the pharmacokinetic behavior of analytes also changed to some extent. The pharmacokinetic alterations of analytes might be due to the pathological conditions of CG.