Journal of Pharmaceutical Analysis

2022, 12(1): ii-ii. doi: 10.1016/S2095-1779(22)00007-7

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Screening strategies for quorum sensing inhibitors in combating bacterial infections

Lan Lu, Mingxing Li, Guojuan Yi, Li Liao, Qiang Cheng, Jie Zhu, Bin Zhang, Yingying Wang, Yong Chen, Ming Zeng

2022, 12(1): 1-14. doi: 10.1016/j.jpha.2021.03.009

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Interference with quorum sensing (QS) represents an antivirulence strategy with a significant promise for the treatment of bacterial infections and a new approach to restoring antibiotic tolerance. Over the past two decades, a novel series of studies have reported that quorum quenching approaches and the discovery of quorum sensing inhibitors (QSIs) have a strong impact on the discovery of anti-infective drugs against various types of bacteria. The discovery of QSI was demonstrated to be an appropriate strategy to expand the anti-infective therapeutic approaches to complement classical antibiotics and antimicrobial agents. For the discovery of QSIs, diverse approaches exist and develop in-step with the scale of screening as well as specific QS systems. This review highlights the latest findings in strategies and methodologies for QSI screening, involving activity-based screening with bioassays, chemical methods to seek bacterial QS pathways for QSI discovery, virtual screening for QSI screening, and other potential tools for interpreting QS signaling, which are innovative routes for future efforts to discover additional QSIs to combat bacterial infections.

Capillary electrophoresis methods for impurity profiling of drugs: A review of the past decade

Mansi Shah, Nrupesh Patel, Nagja Tripathi, Vivek K. Vyas

2022, 12(1): 15-28. doi: 10.1016/j.jpha.2021.06.009

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Capillary electrophoresis (CE) is widely used for the impurity profiling of drugs that contain stereochemical centers in their structures, analysis of biomolecules, and characterization of biopharmaceuticals. Currently, CE is the method of choice for the analysis of foodstuffs and the determination of adulterants. This article discusses the general theory and instrumentation of CE as well as the classification of various CE techniques. It also presents an overview of research on the applications of different CE techniques in the impurity profiling of drugs in the past decade. The review briefly presents a comparison between CE and liquid chromatography methods and highlights the strengths of CE using drug compounds as examples. This review will help scientists, fellow researchers, and students to understand the applications of CE techniques in the impurity profiling of drugs.

Antiviral, immunomodulatory, and anticoagulant effects of quercetin and its derivatives: Potential role in prevention and management of COVID-19

Souparnika H. Manjunath, Rajesh K. Thimmulappa

2022, 12(1): 29-34. doi: 10.1016/j.jpha.2021.09.009

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The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has caused a devastating health crisis worldwide. In this review, we have discussed that prophylactic phytochemical quercetin supplementation in the form of foods or nutraceuticals may help manage the COVID-19 pandemic. The following evidence supports our argument. First, nuclear factor erythroid-derived 2-like 2 (NRF2) agonists abrogate replication of SARS-CoV-2 in lung cells, and quercetin is a potent NRF2 agonist. Second, quercetin exerts antiviral activity against several zoonotic coronaviruses, including SARS-CoV-2, mainly by inhibiting the entry of virions into host cells. Third, inflammatory pathways activated by nuclear factor kappa B, inflammasome, and interleukin-6 signals elicit cytokine release syndrome that promotes acute respiratory distress syndrome in patients with COVID-19, and quercetin inhibits these pro-inflammatory signals. Fourth, patients with COVID-19 develop thrombosis, and quercetin mitigates coagulation abnormalities by inhibiting plasma protein disulfide isomerase. This review provides a strong rationale for testing quercetin for the management of COVID-19.

Anti-inflammatory and analgesic properties of Moroccan medicinal plants: Phytochemistry, in vitro and in vivo investigations, mechanism insights, clinical evidences and perspectives

Abdelhakim Bouyahya, Fatima-Ezzahrae Guaouguaou, Nasreddine El Omari, Naoual El Menyiy, Abdelaali Balahbib, Mohamed El-Shazly, Youssef Bakri

2022, 12(1): 35-57. doi: 10.1016/j.jpha.2021.07.004

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Moroccan medicinal plants exhibit several pharmacological properties such as antimicrobial, anticancer, antidiabetic, analgesic, and anti-inflammatory effects, which are related to the presence of numerous bioactive compounds, including phenolic acids, flavonoids, and terpenoids. In the present review, we systematically evaluate previously published reports on the anti-inflammatory and analgesic effects of Moroccan medicinal plants. The in vitro investigations revealed that Moroccan medicinal plants inhibit several enzymes related to inflammatory processes, whereas in vivo studies noted significant anti-inflammatory and analgesic effects as demonstrated using different experimental models. Various bioactive compounds exhibiting in vitro and in vivo anti-inflammatory and analgesic effects, with diverse mechanisms of action, have been identified. Some plants and their bioactive compounds reveal specific secondary metabolites that possess important anti-inflammatory effects in clinical investigations. Our review proposes the potential applications of Moroccan medicinal plants as sources of anti-inflammatory and analgesic agents.

Assessment of the binding interactions of SARS-CoV-2 spike glycoprotein variants

Deepa Raghu, Pamela Hamill, Arpitha Banaji, Amy McLaren, Yu-Ting Hsu

2022, 12(1): 58-64. doi: 10.1016/j.jpha.2021.09.006

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Severe acute respiratory syndrome-associated coronavirus 2 is a major global health issue and is driving the need for new therapeutics. The surface spike protein, which plays a central role in virus infection, is currently the target for vaccines and neutralizing treatments. The emergence of novel variants with multiple mutations in the spike protein may reduce the effectiveness of neutralizing antibodies by altering the binding activity of the protein with angiotensin-converting enzyme 2 (ACE2). To understand the impact of spike protein mutations on the binding interactions required for virus infection and the effectiveness of neutralizing monoclonal antibody (mAb) therapies, the binding activities of the original spike protein receptor binding domain (RBD) sequence and the reported spike protein variants were investigated using surface plasmon resonance. In addition, the interactions of the ACE2 receptor, an anti-spike mAb (mAb1), a neutralizing mAb (mAb2), the original spike RBD sequence, and mutants D614G, N501Y, N439K, Y453F, and E484K were assessed. Compared to the original RBD, the Y453F and N501Y mutants displayed a significant increase in ACE2 binding affinity, whereas D614G had a substantial reduction in binding affinity. All mAb-RBD mutant proteins displayed a reduction in binding affinities relative to the original RBD, except for the E484K-mAb1 interaction. The potential neutralizing capability of mAb1 and mAb2 was investigated. Accordingly, mAb1 failed to inhibit the ACE2-RBD interaction while mAb2 inhibited the ACE2-RBD interactions for all RBD mutants, except mutant E484K, which only displayed partial blocking.

Identifying the molecular basis of Jinhong tablets against chronic superficial gastritis via chemical profile identification and symptom-guided network pharmacology analysis

Danfeng Shi, Lingxian Liu, Haibo Li, Dabo Pan, Xiaojun Yao, Wei Xiao, Xinsheng Yao, Yang Yu

2022, 12(1): 65-76. doi: 10.1016/j.jpha.2021.01.005

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Chronic superficial gastritis (CSG) is a common disease of the digestive system that possesses a serious pathogenesis. Jinhong tablet (JHT), a traditional Chinese medicine (TCM) prescription, exerts therapeutic effects against CSG. However, the molecular basis of its therapeutic effect has not been clarified. Herein, we employed ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based chemical profile identification to determine the chemical components in JHT. Further, we applied network pharmacology to illustrate its molecular mechanisms. A total of 96 chemical constituents were identified in JHT, 31 of which were confirmed using reference standards. Based on the bioinformatics analysis using the symptom-guided pharmacological networks of “chi,” “blood,” “pain,” and “inflammation,” and target screening through the interaction probabilities between compounds and targets, matrix metalloproteinase 2 (MMP2), dopamine d2 receptor (DRD2), and Aldo-keto reductase family 1 member B1 (AKR1B1) were identified as key targets in the therapeutic effect exhibited by JHT against CSG. Moreover, according to the inhibitory activities presented in the literature and binding mode analysis, the structural types of alkaloids, flavonoids, organic acids, including chlorogenic acid ( 10 ), caffeic acid ( 13 ), (−)-corydalmine ( 33 ), (−)-isocorypalmine ( 36 ), isochlorogenic acid C ( 38 ), isochlorogenic acid A ( 41 ), quercetin-3-O-α-l-rhamnoside ( 42 ), isochlorogenic acid B ( 47 ), quercetin ( 63 ), and kaempferol ( 70 ) tended to show remarkable activities against CSG. Owing to the above findings, we systematically identified the chemical components of JHT and revealed its molecular mechanisms based on the symptoms associated with CSG.

MTBSTFA derivatization-LC-MS/MS approach for the quantitative analysis of endogenous nucleotides in human colorectal carcinoma cells

Huixia Zhang, Yan Li, Zheng Li, Christopher Wai-Kei Lam, Peng Zhu, Caiyun Wang, Hua Zhou, Wei Zhang

2022, 12(1): 77-86. doi: 10.1016/j.jpha.2021.01.001

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Endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs) are important metabolites related to the pathogenesis of many diseases. In light of their physiological and pathological significances, a novel and sensitive pre-column derivatization method with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was developed to determine RNs and dRNs in human cells using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). A one-step extraction of cells with 85% methanol followed by a simple derivatization reaction within 5 min at room temperature contributed to shortened analysis time. The derivatives of 22 nucleoside mono-, di- and triphosphates were retained on the typical C₁₈ column and eluted by ammonium acetate and acetonitrile in 9 min. Under these optimal conditions, good linearity was achieved in the tested calibration ranges. The lower limit of quantitation (LLOQ) was determined to be 0.1–0.4  μM for the tested RNs and 0.001–0.1  μM for dRNs. In addition, the precision (CV) was <15% and the RSD of stability was lower than 10.4%. Furthermore, this method was applied to quantify the endogenous nucleotides in human colorectal carcinoma cell lines HCT 116 exposed to 10-hydroxycamptothecin. In conclusion, our method has proven to be simple, rapid, sensitive, and reliable. It may be used for specific expanded studies on intracellular pharmacology in vitro.

Long-chain alkanol–alkyl carboxylic acid-based low-viscosity hydrophobic deep eutectic solvents for one-pot extraction of anthraquinones from Rhei Radix et Rhizoma

Anqi Huang, Wenwen Deng, Xiao Li, Qutong Zheng, Xuanxuan Wang, Yuxiu Xiao

2022, 12(1): 87-95. doi: 10.1016/j.jpha.2021.03.002

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Natural long-chain alkanol and alkyl carboxylic acid were used to prepare novel hydrophobic deep eutectic solvents (HDESs). These HDESs are liquid at room temperature and have low viscosity (<12.26 mPa‧s), low polarity (lower than that of methanol, ChCl-based deep eutectic solvents and other reported HDESs), and low density (<0.928 g/mL). A simple one-pot method based on a novel HDES–water two-phase extraction system was constructed for the extraction of weak-polarity bioactive components, anthraquinones, from Rhei Radix et Rhizoma. This HDES-based new extraction method does not consume hazardous organic solvents and can obtain a total anthraquinone yield of 21.52 mg/g, which is close to that obtained by the Chinese pharmacopoeia method (21.22 mg/g) and considerably higher than those by other reported HDESs-based extraction methods (14.20–20.09 mg/g, p < 0.01). The high extraction yield can be mainly attributed to the severe destruction of the RRR cell walls by the extraction system and the excellent dissolving ability of novel HDESs for anthraquinones.

Carbon dots derived from Poria cocos polysaccharide as an effective “on-off” fluorescence sensor for chromium (VI) detection

Qianqian Huang, Qianqian Bao, Chengyuan Wu, Mengru Hu, Yunna Chen, Lei Wang, Weidong Chen

2022, 12(1): 104-112. doi: 10.1016/j.jpha.2021.04.004

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Chromium is a harmful contaminant showing mutagenicity and carcinogenicity. Therefore, detection of chromium requires the development of low-cost and high-sensitivity sensors. Herein, blue-fluorescent carbon quantum dots were synthesized by one-step hydrothermal method from alkali-soluble Poria cocos polysaccharide, which is green source, cheap and easy to obtain, and has no pharmacological activity due to low water solubility. These carbon quantum dots exhibit good fluorescence stability, water solubility, anti-interference and low cytotoxicity, and can be specifically combined with the detection of Cr(VI) to form a non-fluorescent complex that causes fluorescence quenching, so they can be used as a label-free nanosensor. High-sensitivity detection of Cr(VI) was achieved through internal filtering and static quenching effects. The fluorescence quenching degree of carbon dots fluorescent probe showed a good linear relationship with Cr(VI) concentration in the range of 1–100 μM. The linear equation was F₀/F = 0.9942 + 0.01472 [Cr(VI)] (R² = 0.9922), and the detection limit can be as low as 0.25 μM (S/N = 3), which has been successfully applied to Cr(VI) detection in actual water samples herein.

Reversible regulation of enzyme-like activity of molybdenum disulfide quantum dots for colorimetric pharmaceutical analysis

Juan Tan, Shiyue Wu, Qingqing Cai, Yi Wang, Pu Zhang

2022, 12(1): 113-121. doi: 10.1016/j.jpha.2021.03.010

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Regulating the catalytic activity of nanozymes is significant for their applications in various fields. Here, we demonstrate a new strategy to achieve reversible regulation of the nanozyme's activity for sensing purpose. This strategy involves the use of zero-dimensional MoS₂ quantum dots (MQDs) as the building blocks of nanozymes which display very weak peroxidase (POD)-like activity. Interestingly, such POD-like activity of the MQDs largely enhances in the presence of Fe³⁺ while diminishes with the addition of captopril thereafter. Further investigations identify the mechanism of Fe³⁺-mediated aggregation-induced enhancement of the POD-like activity and the inhibitory effect of captopril on the enhancement, which is highly dependent on their concentrations. Based on this finding, a colorimetric method for the detection of captopril is developed. This sensing approach exhibits the merits of simplicity, rapidness, reliability, and low cost, which has been successfully applied in quality control of captopril in pharmaceutical products. Moreover, the present sensing platform allows smartphone read-out, which has promising applications in point-of-care testing devices for clinical diagnosis and drug analysis.

Regulation of drug release performance using mixed doxorubicin-doxorubicin dimer nanoparticles as a pH-triggered drug self-delivery system

Jiagen Li, Xinming Li, Pengwei Xie, Peng Liu

2022, 12(1): 122-128. doi: 10.1016/j.jpha.2021.03.001

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A mixed drug self-delivery system (DSDS) with high drug content (>50%) was developed to regulate pH-triggered drug release, based on two doxorubicin (DOX)-DOX dimmers: D-DOX_ADH and D-DOX_car conjugated with acid-labile dynamic covalent bonds (hydrazone and carbamate, respectively) and stabilized with PEGylated D-DOX_ADH (D-DOX_ADH-PEG). Owing to the different stability of the dynamic covalent bonds in the two dimers and the noncovalent interaction between them, pH-triggered drug release could be easily regulated by adjusting the feeding ratios of the two DOX-DOX dimers in the mixed DSDS. Similar in vitro cellular toxicity was achieved with the mixed DSDS nanoparticles prepared with different feeding ratios. The mixed DSDS nanoparticles had a similar DOX content and diameter but different drug releasing rates. The MTT assays revealed that a high anti-tumor efficacy could be achieved with the slow-release mixed DSDS nanoparticles.

An integrated strategy for comprehensive characterization of metabolites and metabolic profiles of bufadienolides from Venenum Bufonis in rats

Wen-Long Wei, Hao-Jv Li, Wen-Zhi Yang, Hua Qu, Zhen-Wei Li, Chang-Liang Yao, Jin-Jun Hou, Wan-Ying Wu, De-An Guo

2022, 12(1): 136-144. doi: 10.1016/j.jpha.2021.02.003

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Comprehensive characterization of metabolites and metabolic profiles in plasma has considerable significance in determining the efficacy and safety of traditional Chinese medicine (TCM) in vivo. However, this process is usually hindered by the insufficient characteristic fragments of metabolites, ubiquitous matrix interference, and complicated screening and identification procedures for metabolites. In this study, an effective strategy was established to systematically characterize the metabolites, deduce the metabolic pathways, and describe the metabolic profiles of bufadienolides isolated from Venenum Bufonis in vivo. The strategy was divided into five steps. First, the blank and test plasma samples were injected into an ultra-high performance liquid chromatography/linear trap quadrupole-orbitrap-mass spectrometry (MS) system in the full scan mode continuously five times to screen for valid matrix compounds and metabolites. Second, an extension-mass defect filter model was established to obtain the targeted precursor ions of the list of bufadienolide metabolites, which reduced approximately 39% of the interfering ions. Third, an acquisition model was developed and used to trigger more tandem MS (MS/MS) fragments of precursor ions based on the targeted ion list. The acquisition mode enhanced the acquisition capability by approximately four times than that of the regular data-dependent acquisition mode. Fourth, the acquired data were imported into Compound Discoverer software for identification of metabolites with metabolic network prediction. The main in vivo metabolic pathways of bufadienolides were elucidated. A total of 147 metabolites were characterized, and the main biotransformation reactions of bufadienolides were hydroxylation, dihydroxylation, and isomerization. Finally, the main prototype bufadienolides in plasma at different time points were determined using LC-MS/MS, and the metabolic profiles were clearly identified. This strategy could be widely used to elucidate the metabolic profiles of TCM preparations or Chinese patent medicines in vivo and provide critical data for rational drug use.

Pharmacokinetics, distribution, and excretion of sodium oligomannate, a recently approved anti-Alzheimer's disease drug in China

Jiaojiao Lu, Qiongqun Pan, Jieqiang Zhou, Yan Weng, Kaili Chen, Lv Shi, Guanxiu Zhu, Chunlin Chen, Liang Li, Meiyu Geng, Zhenqing Zhang

2022, 12(1): 145-155. doi: 10.1016/j.jpha.2021.06.001

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The National Medical Products Administration has authorized sodium oligomannate for treating mild-to-moderate Alzheimer's disease. In this study, an LC-MS/MS method was developed and validated to quantitate sodium oligomannate in different biomatrices. The plasma pharmacokinetics, tissue distribution, and excretion of sodium oligomannate in Sprague-Dawley rats and beagle dogs were systematically investigated. Despite its complicated structural composition, the absorption, distribution, metabolism, and excretion profiles of the oligosaccharides in sodium oligomannate of different sizes and terminal derivatives were indiscriminate. Sodium oligomannate mainly crossed the gastrointestinal epithelium through paracellular transport following oral administration, with very low oral bioavailability in rats (0.6%–1.6%) and dogs (4.5%–9.3%). Absorbed sodium oligomannate mainly resided in circulating body fluids in free form with minimal distribution into erythrocytes and major tissues. Sodium oligomannate could penetrate the blood-cerebrospinal fluid (CSF) barrier of rats, showing a constant area under the concentration-time curve ratio (CSF/plasma) of approximately 5%. The cumulative urinary excretion of sodium oligomannate was commensurate with its oral bioavailability, supporting that excretion was predominantly renal, whereas no obvious biliary secretion was observed following a single oral dose to bile duct-cannulated rats. Moreover, only 33.7% (male) and 26.3% (female) of the oral dose were recovered in the rat excreta within 96 h following a single oral administration, suggesting that the intestinal flora may have ingested a portion of unabsorbed sodium oligomannate as a nutrient.

Biological analysis of an innovative biodegradable antibiotic eluting bioactive glass/gypsum composite bone cement for treating experimental chronic MRSA osteomyelitis

Surajit Mistry, Subhasish Burman, Subhasis Roy, Nilendu Jyoti Maitra, Rajiv Roy, Abhijit Chanda

2022, 12(1): 164-177. doi: 10.1016/j.jpha.2021.02.005

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A multi-barrier antibiotics loaded biodegradable composite bone cement for resolving chronic osteomyelitis has been studied to understand the physico-mechanical properties, drug loading/eluting efficiency, and different merits and demerits prior to clinical application. After successful induction of bone infection in 28 rabbits using methicillin-resistant Staphylococcus aureus (MRSA) strains, calcium sulfate/bioactive glass based composite cement was implanted in 12 defects to assess its performance over parenteral therapy with microscopic and radiological examination for 90 days. The composite cement revealed acceptable physico-mechanical properties and controlled drug elution kinetics. Furthermore, the antibiotics concentrations in bone up to 42 days were sufficient to kill MRSA without eliciting adverse drug reactions. The striking feature of platelets aggregation by composite cement could assist bone healing. The controlled degradation with simultaneous entrapment of composite cement within the osteoid tissues and complete repair of infected cortical defects (holes) in rabbit tibia at 6 weeks indicated the excellent anti-infective and osteoconductive properties of composite cement. Thus, the animal study demonstrated the superiority of composite over injectable antibiotic therapy based on infection resolution and bone regeneration. We thereby conclude that the composite cement can be effectively applied in the treatment of resistant cases of chronic osteomyelitis.

Erythrocyte sphingolipid species as biomarkers of Alzheimer's disease

Jericha Mill, Vihar Patel, Ozioma Okonkwo, Lingjun Li, Thomas Raife

2022, 12(1): 178-185. doi: 10.1016/j.jpha.2021.07.005

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Diagnosing Alzheimer's disease (AD) in the early stage is challenging. Informative biomarkers can be of great value for population-based screening. Metabolomics studies have been used to find potential biomarkers, but commonly used tissue sources can be difficult to obtain. The objective of this study was to determine the potential utility of erythrocyte metabolite profiles in screening for AD. Unlike some commonly-used sources such as cerebrospinal fluid and brain tissue, erythrocytes are plentiful and easily accessed. Moreover, erythrocytes are metabolically active, a feature that distinguishes this sample source from other bodily fluids like plasma and urine. In this preliminary pilot study, the erythrocyte metabolomes of 10 histopathologically confirmed AD patients and 10 patients without AD (control (CTRL)) were compared. Whole blood was collected post-mortem and erythrocytes were analyzed using ultra-performance liquid chromatography tandem mass spectrometry. Over 750 metabolites were identified in AD and CTRL erythrocytes. Seven were increased in AD while 24 were decreased (P<0.05). The majority of the metabolites increased in AD were associated with amino acid metabolism and all of the decreased metabolites were associated with lipid metabolism. Prominent among the potential biomarkers were 10 sphingolipid or sphingolipid-related species that were consistently decreased in AD patients. Sphingolipids have been previously implicated in AD and other neurological conditions. Furthermore, previous studies have shown that erythrocyte sphingolipid concentrations vary widely in normal, healthy adults. Together, these observations suggest that certain erythrocyte lipid phenotypes could be markers of risk for development of AD.

Fabrication of edge-curled petals-like covalent organic frameworks and their properties for extracting indole alkaloids from complex biological samples

Fanrong Sun, Ligai Bai, Mingxue Li, Changqing Yu, Haiyan Liu, Xiaoqiang Qiao, Hongyuan Yan

2022, 12(1): 96-103. doi: 10.1016/j.jpha.2020.12.006

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In this study, a functionalized covalent-organic framework (COF) was first synthesized using porphyrin as the fabrication unit and showed an edge-curled, petal-like and well-ordered structure. The synthesized COF was then introduced to prepare porous organic polymer monolithic materials (POPMs). Two composite POPM/COF monolithic materials with rod shapes, referred to as sorbent A and sorbent B, were prepared in stainless steel tubes using different monomers. Sorbents A and B exhibited relatively uniform porous structures and enhanced specific surface areas of 153.14 m²/g and 80.01 m²/g, respectively. The prepared composite monoliths were used as in-tube solid-phase extraction (SPE) sorbents combined with HPLC for the on-line extraction and quantitative analytical systems. Indole alkaloids (from Catharanthus roseus G. Don and Uncaria rhynchophylla (Miq.) Miq. Ex Havil.) contained in mouse plasma were extracted and quantitatively analyzed using the online system. The two composite multifunctional monoliths showed excellent clean-up ability for complex biological matrices, as well as superior selectivity for target indole alkaloids. Method validation showed that the RSD values of the repeatability (n=6) were ≤ 3.46%, and the accuracy expressed by the spiked recoveries was in the ranges of 99.38%–100.91% and 96.39%–103.50% for vinca alkaloids and Uncaria alkaloids, respectively. Furthermore, sorbents A and B exhibited strong reusability, with RSD values ≤ 5.32%, which were based on the peak area of the corresponding alkaloids with more than 100 injections. These results indicate that the composite POPM/COF rod-shaped monoliths are promising media as SPE sorbents for extracting trace compounds in complex biological samples.

Deciphering bioactive compounds of complex natural products by tandem mass spectral molecular networking combined with an aggregation-induced emission based probe

Zhenzhong Yang, Jun Li, Xuechun Chen, Xiaoping Zhao, Yi Wang

2022, 12(1): 129-135. doi: 10.1016/j.jpha.2020.11.007

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Natural products are great treasure troves for the discovery of bioactive components. Current bioassay guided fractionation for identification of bioactive components is time- and workload-consuming. In this study, we proposed a robust and convenient strategy for deciphering the bioactive profile of natural products by mass spectral molecular networking combined with rapid bioassay. As a proof-of-concept, the strategy was applied to identify angiotensin converting enzyme (ACE) inhibitors of Fangjihuangqi decoction (FJHQD), a traditional medicine clinically used for the treatment of heart failure. The chemical profile of FJHQD was comprehensively revealed with the assistance of tandem mass spectral molecular networking, and a total of 165 compounds were identified. With characterized constituents, potential clinical applications of FJHQD were predicted by Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine, and a range of cardiovascular related diseases were significantly enriched. ACE inhibitory activities of FJHQD and its constituents were then investigated with an aggregation-induced emission based fluorescent probe. FJHQD exhibited excellent ACE inhibitory effects, and a bioactive molecular network was established to elucidate the ACE inhibitory profile of constituents in FJHQD. This bioactive molecular network provided a panoramic view of FJHQD's ACE inhibitory activities, which demonstrated that flavones from Astragali Radix and Glycyrrhizae Radix et Rhizoma, saponins from Astragali Radix, and sesquiterpenoids from Atractylodis Macrocephalae Rhizoma were principal components responsible for this effect of FJHQD. Among them, four novel ACE inhibitors were the first to be reported. Our study indicated that the proposed strategy offers a useful approach to uncover the bioactive profile of traditional medicines and provides a pragmatic workflow for exploring bioactive components.

Global characterization of modifications to the charge isomers of IgG antibody

Xinling Cui, Wei Mi, Zhishang Hu, Xiaoyu Li, Bo Meng, Xinyuan Zhao, Xiaohong Qian, Tao Zhu, Wantao Ying

2022, 12(1): 156-163. doi: 10.1016/j.jpha.2020.11.006

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Posttranslational modifications of antibody products affect their stability, charge distribution, and drug activity and are thus a critical quality attribute. The comprehensive mapping of antibody modifications and different charge isomers (CIs) is of utmost importance, but is challenging. We intended to quantitatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity. The CIs of antibodies were fractionated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection, followed by stepwise structural characterization at three levels. First, the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach; this showed differences in glycoforms and deamidation status. Second, at the peptide level, common modifications of oxidation, deamidation, and glycosylation were identified. Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs. In total, 10 N-glycoforms were detected by peptide mapping. Finally, an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides. Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms. The results revealed that sialic acid modification is a critical factor accounting for charge heterogeneity, which is otherwise missed in peptide mapping and intact molecular weight analyses. This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.

Time-course monitoring of in vitro biotransformation reaction via solid-phase microextraction-ambient mass spectrometry approaches

Karol Jaroch, Janusz Pawliszyn

2022, 12(1): 186-191. doi: 10.1016/j.jpha.2021.08.001

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The solid-phase microextraction technique quantifies analytes without considerably affecting the sample composition. Herein, a proof-of-concept study was conducted to demonstrate the use of coated probe electrospray ionization (coated-PESI) and coated blade spray (CBS) as ambient mass spectrometry approaches for monitoring drug biotransformation. The ability of these methods was investigated for monitoring the dephosphorylation of a prodrug, combretastatin A4 phosphate (CA4P), into its active form, combretastatin A4 (CA4), in a cell culture medium supplemented with fetal bovine serum. The CBS spot analysis was modified to achieve the same extraction efficiency as protein precipitation and obtained results in 7 min. Because coated-PESI performs extraction without consuming any samples, it is the preferred technique in the case of a limited sample volume. Although coated-PESI only extracts small quantities of analytes, it uses the desorption solvent volume of 5–10 pL, resulting in high sensitivity, thus allowing the detection of compounds after only 1 min of extraction. The biotransformation of CA4P into CA4 via phosphatases occurs within the simple matrix, and the proposed sample preparation techniques are suitable for monitoring the biotransformation.

2022 Vol. 12, No. 1