Biomedical Chromatography

Zhao, Xihong; Lv, Chunjing; Chen, Hong; Qin, Feng; Lu, Xiumei

doi: 10.1002/bmc.5780pmid: 38071752

A rapid and sensitive ultra‐high performance liquid chromatography–tandem mass spectrometry method was developed to determine flurbiprofen in rat plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used in negative ion mode. Acetonitrile precipitation was selected to prepare samples. Flurbiprofen and internal standard flurbiprofen‐d5 were analyzed on an Acquity UPLC BEH C18 column with the mobile phase consisting of acetonitrile and water, and a gradient procedure was used for separation. The retention time of flurbiprofen was 0.67 min, and the whole running time was only 1.2 min. The detection was performed on a triple quadrupole tandem mass spectrometer using multiple reaction monitoring mode via an ESI source with optimized mass spectrometry parameters. The calibration curve was linear in the range of 25.0–1.00 × 104 ng/mL (r ≥ 0.99). The within‐run and between‐run relative standard deviations were not more than 13.9%. The within‐run and between‐run relative errors were from −9.0% to 3.4%. There was no significant matrix effect, and recovery was high. This method was fully validated, including whole blood stability in rat plasma, and successfully applied to the pharmacokinetic study in which 100% incurred sample reanalysis met the criteria.

Zhang, Tao; Xie, Yating; Li, Tao; Deng, Yaling; Wan, Quan; Bai, Tingting; Zhang, Qing; Cai, Zhongxi; Chen, Mingxia; Zhang, Jinlian

doi: 10.1002/bmc.5768pmid: 38087457

Polygoni Multiflori Radix (PMR) is a medicinal herb commonly used in China and Eastern Asia. Recently, the discovery of hepatotoxicity in PMR has received considerable attention from scientists. Processing is a traditional Chinese medicine technique used for the effective reduction of toxicity. One uncommon technique is the braising method—also known as ‘Wen‐Fa’ in Chinese—which is used to prepare tonics or poisonous medications. Braised PMR (BPMR)—also known as ‘Wen‐He‐Shou‐Wu’—is one of the processed products of the braising method. However, the non‐volatile components of BPMR have not been identified and examined in detail, and therefore, the hepatotoxic advantage of BPMR remains unknown. In this study, we compared the microscopic characteristics of different samples in powder form using scanning electron microscopy (SEM), investigated the non‐volatile components, assessed the effects of different processed PMR products on the liver, and compared the differences between BPMR and PMR Praeparata recorded in the Chinese Pharmacopoeia (2020 edition). We found that the hepatotoxicity of BPMR was dramatically decreased, which may be related to an increase in polysaccharide content and a decrease in toxic substances. The present study provides an important foundation for future investigations of the processing mechanisms of BPMR.

Davis, Michael T.; Anders, Nicole M.; Colevas, A. Dimitrios; Messick, Troy E.; Rudek, Michelle A.

doi: 10.1002/bmc.5775pmid: 37942577

EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV‐associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK‐2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV‐associated carcinomas. A sensitive and reliable method was developed to quantify VK‐2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK‐2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK‐2019, two purported metabolites, and the internal standard, VK‐2019–d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK‐2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5–500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK‐2019 was stable in plasma at −70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK‐2019 in a patient who received a single and multiple oral daily doses of 120 mg.

Xiao, Tingyu; Lin, Huaqing; Lao, Jiekeng; Hu, Xin; Chen, Yucheng; Lei, Yicong; Xu, Mingzhi

doi: 10.1002/bmc.5790pmid: 38158853

In the production of doxofylline, the common occurrence of toxic p‐toluene sulfonate generation prompted the development and validation of a method using HPLC with ultraviolet detection (HPLC‐UV). This method is designed for detecting four potential genotoxic impurities (PGIs) present in both doxofylline drug substance and tablets, with a focus on the UV‐absorbing group p‐toluene sulfonate. The four impurities were methyl 4‐methylbenzenesulfonate (PGI‐1), ethyl 4‐methylbenzenesulfonate (PGI‐2), 2‐hydroxyethyl 4‐methylbenzenesulfonate (PGI‐3), and 2‐(4‐methylphenyl)sulfonyloxyethyl 4‐methylbenzenesulfonate (PGI‐4). In this method, chromatographic separation was achieved using a Waters Symmetry C18 column (250 mm × 4.6 mm, 5 μm). The mobile phases consisted of 20% acetonitrile as mobile phase A and pure acetonitrile as mobile phase B, operating in gradient elution mode at a flow rate of 1.0 mL/min. According to the guidelines of the International Conference on Harmonization, it was determined that this method could quantify four PGIs at 0.0225 μg/mL in samples containing 60 mg/mL. The validated approach demonstrated excellent linearity (R2 > 0.999) across the concentration range of 30%–200% (relative to 0.075 μg/mL doxofylline) for the four PGIs. The accuracy of this method for the four PGIs ranged from 94.8% to 100.4%. The reverse‐phase‐HPLC‐UV analytical method developed in this study is characterized by its speed and precision, making it suitable for the sensitive analysis of benzene sulfonate PGIs in doxofylline drug substances and tablets.

Girgin, Gozde; Sanajou, Sonia; Meric‐Deliveli, Sinem; Baydar, Terken

doi: 10.1002/bmc.5791pmid: 38031497

Colostrum, the first breast fluid produced by mammals after giving birth, is followed by breast milk, which serves as the sole source of nutrients for breastfed newborns and infants. Tryptophan, an essential amino acid, plays a crucial role in the development and maturation of the central nervous system in infants. Tryptophan is primarily degraded through the kynurenine pathway. Owing to its sensitivity to dietary intake, immune‐mediated tryptophan degradation is assessed by the kynurenine‐to‐tryptophan ratio, with a focus on one of the rate‐limiting enzymes in the pathway. This study involved the validation of the simultaneous determination of tryptophan and kynurenine using HPLC. The validated method was then used to detect levels of tryptophan and kynurenine, as well as to calculate the kynurenine‐to‐tryptophan ratio in colostrum samples. Simultaneously, these results were compared with colostrum neopterin levels measured using commercial enzyme‐linked immunosorbent assay kits. The mean levels for tryptophan, kynurenine, and neopterin were 17.3 ± 62.4 μM, 0.45 ± 0.03 μM, and 28.9 ± 2.6 nM, respectively. This study is among the few that have evaluated these parameters in colostrum samples. Neopterin levels secreted by the mammary gland were found not to be correlated with tryptophan degradation, a process influenced by the mother’s nutritional status.

Wang, Jing; Ouyang, Bingchen; Cao, Rui; Xu, Yu

doi: 10.1002/bmc.5784pmid: 38009806

Yinchenhao decoction (YCHD), a famous traditional Chinese medicine formula, has been applied for relieving jaundice in China for more than 1800 years. However, the material basis for YCHD is still unclear, and the chemical composition and metabolism characteristic in vivo are undefined, making the potential effective constituents and mechanism of action unclear. Herein, an ultrahigh‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (UHPLC–QTOF–MS)‐based strategy was applied for the chemical profiling of YCHD, as well as their in vivo prototypes and global metabolites that defined the metabolome. Our results showed that a total of 139 chemicals were identified in YCHD, including 28 organic acids, 12 monoterpenoids, five diterpenes, three triterpenoids, 17 iridoids, 23 anthraquinones, 26 flavonoids, four coumarins and 21 other types. Moreover, 58 prototypes and 175 metabolites were found in rat biological samples after oral administration of YCHD; those distributed in plasma, liver, intestine and feces were suggested to be potentially effective substances. Oxidation, hydrogenation, decarboxylation and conjugations with methyl, sulfate and glucuronate were considered as the predominant metabolic pathways in vivo. In conclusion, this is a systemic study of chemical constituents and in vivo metabolome profiles of YCHD, contributing to the material basis understanding and further mechanism research.

Chatki, Pankaj Kisan; Mahajan, Ujwala N.

doi: 10.1002/bmc.5792pmid: 38017613

The objective of this study was to quantitatively determine Bruton's tyrosine kinase inhibitor ibrutinib in its capsule dosage form and assess the homogeneity of the dosage form using green chromatography. The chromatographic method using gradient elution mode was optimized and validated in accordance with the International Council for Harmonization guidelines. The analysis was conducted on a Zodiac C18 column (75 × 4.6 mm and 3.5 μm) using a mobile phase consisting of pH 5.5 potassium phosphate buffer (mobile phase A) and 90% ethanol in milli‐Q water (mobile phase B), with a flow rate set at 0.6 mL/min. Based on the validation data, the accuracy results fell within the range of 99.1%–100.6%. The relative standard deviation (% RSD) from precision for both the assays and the uniformity of dosage by content uniformity were determined to be 0.82 and 1.16, respectively. The correlation coefficient obtained from the linearity experiment was 0.999, indicating a strong linear relationship. The greenness of the developed method was assessed using various tools, including the National Environmental Methods Index (NEMI) pictogram, Modified NEMI, Analytical Eco‐score calculation, Green Analytical Procedure Index (GAPI) pictogram, Analytical GREEnness (AGREE), and AGREE preparation (AGREEprep). The obtained greenness profile suggests that the optimized LC method is an excellent greener method, supported by the analytical eco‐score of 86.

Zamira, Djumayeva; Khaydarov, Khislat; Zafar, Muhammad; Ramadan, Mohamed Fawzy; Ahmad, Mushtaq; Aziza, Nozimova; Ochilov, Ulugbek; Zebiniso, Umurzakova; Farzona, Davronkulova

doi: 10.1002/bmc.5774pmid: 37972935

Considering the limited data available on tree species in Uzbekistan, this research aimed to provide new insights. We gathered plant samples from different locations within Samarkand city and thoughtfully selected 15 tree species that represent the country’s flora. Using scanning electron microscopy, we conducted comprehensive analyses of pollen morphology, revealing a diverse range of variations in the shapes, dimensions, and surface characteristics displayed by pollen grains. Distinct ornamentations such as micro‐echinate, reticulate, rugulate, gemmate–verrucate, and verrucate–scabrate patterns facilitated the differentiation of species. These scanning electron microscopy findings enhance our comprehension of tree species diversity, adaptation, and ecological roles. In addition, leaf extracts were analyzed using HPLC and GC–MS, revealing a plethora of bioactive compounds, including catechins, chlorogenic acid, vanillic acid, and others. Furthermore, GC–MS analysis revealed the presence of seven key compounds, including 1‐hexadecyne, 2‐chloroethanol, 1,6‐heptadiene, 2‐methyl‐, 5‐bromoadamantan‐2‐one, ethyl 3‐(3‐pyridyl) propenoate, bis (2‐ethylhexyl) phthalate, and quercetin. This study demonstrates the effectiveness of this method in assessing the quality of leaf extracts from tree species by examining both microscopic characteristics and chemical composition. This multifaceted approach has deepened our understanding of the characteristics and chemical compositions of these trees, thus contributing to a more profound appreciation of their ecological significance and potential applications.

Zhang, Mengyu; Jin, Ying; Li, Wanling; He, Chaoqun; Di, Xiangjie; Duan, Yifei; Chen, Lei; Wang, Zhenlei

doi: 10.1002/bmc.5777pmid: 37990827

Although levetiracetam (LEV) has favorable linear pharmacokinetic properties, therapeutic drug monitoring (TDM) is necessary for pregnant women with epilepsy. This study aims to build a simple, reliable, and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for determining LEV concentrations in plasma and saliva samples, to support the routine TDM of LEV in Chinese pregnant women with epilepsy. The stable isotope‐labeled LEV‐d6 was used as the internal standard. The extracted samples were analyzed using a UPLC–MS/MS system with positive electrospray ionization. Mobile phase A was water containing 5 mM ammonium acetate and 0.1% formic acid, and phase B was 1:1 methanol–acetonitrile with 0.1% formic acid. The method was validated and utilized to determine LEV concentrations in non‐pregnant and pregnant patients with epilepsy. The developed method was validated in both plasma and saliva samples over a concentration range of 0.1–50 μg/mL. The intra‐ and inter‐batch accuracy for LEV ranged from −7.0% to 2.9%, with precisions between 2.7% and 9.3%. In pregnant patients, the mean dose‐standardized LEV trough plasma concentrations were significantly lower than those in non‐pregnant patients (4.73 ± 2.99 vs. 7.74 ± 3.59 ng/mL per mg/day; P < 0.0001). It is recommended that the TDM of LEV should be routinely performed during the different stages of pregnancy.

doi: 10.1002/bmc.5670pmid: N/A