Journal of Pharmaceutical Analysis

2022, 12(5): ii-ii. doi: 10.1016/S2095-1779(22)00086-7

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Multimodal integrated strategy for the discovery and identification of quality markers in traditional Chinese medicine

Xiaoyan Lu, Yanyan Jin, Yuzhen Wang, Yunlong Chen, Xiaohui Fan

2022, 12(5): 701-710. doi: 10.1016/j.jpha.2022.05.001

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With the modernization and internationalization of traditional Chinese medicine (TCM), the requirement for quality control has increased. The quality marker (Q-marker) is an important standard in this field and has been implemented with remarkable success in recent years. However, the establishment of Q-markers remains fragmented and the process lacks systematicity, resulting in inconsistent quality control and insufficient correlation with clinical efficacy and safety of TCM. This review introduces four multimodal integrated approaches that contribute to the discovery of more comprehensive and accurate Q-markers, thus aiding in the establishment of new quality control patterns based on the characteristics and principles of TCM. These include the whole-process quality control strategy, chemical-activity-based screening method, efficacy, safety, and consistent combination strategy, and TCM theory-guided approach. Furthermore, methodologies and representative examples of these strategies are described, and important future directions and questions in this field are also proposed.

Potential therapeutic effects and applications of Eucommiae Folium in secondary hypertension

Mengyuan Li, Yanchao Zheng, Sha Deng, Tian Yu, Yucong Ma, Jiaming Ge, Jiarong Li, Xiankuan Li, Lin Ma

2022, 12(5): 711-718. doi: 10.1016/j.jpha.2021.10.004

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Eucommiae Folium (EF), a traditional Chinese medicine, has been used to treat secondary hypertension, including renal hypertension and salt-sensitive hypertension, as well as hypertension caused by thoracic aortic endothelial dysfunction, a high-fat diet, and oxidized low-density lipoprotein. The antihypertensive components of EF are divided into four categories: flavonoids, iridoids, lignans, and phenylpropanoids, such as chlorogenic acid, geniposide acid and pinoresinol diglucoside. EF regulates the occurrence and development of hypertension by regulating biological processes, such as inhibiting inflammation, regulating the nitric oxide synthase pathway, reducing oxidative stress levels, regulating endothelial vasoactive factors, and lowering blood pressure. However, its molecular antihypertensive mechanisms are still unclear and require further investigation. In this review, by consulting the relevant literature on the antihypertensive effects of EF and using network pharmacology, we summarized the active ingredients and pharmacological mechanisms of EF in the treatment of hypertension to clarify how EF is associated with secondary hypertension, the related components, and underlying mechanisms. The results of the network pharmacology analysis indicated that EF treats hypertension through a multi-component, multi-target and multi-pathway mechanism. In particular, we discussed the role of EF targets in the treatment of hypertension, including epithelial sodium channel, heat shock protein70, rho-associated protein kinase 1, catalase, and superoxide dismutase. The relevant signal transduction pathways, the ras homolog family member A (RhoA)/Rho-associated protein kinase (ROCK) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/eNOS/NO/Ca²⁺ pathways, are also discussed.

Visualizing the spatial distribution and alteration of metabolites in continuously cropped Salvia miltiorrhiza Bge using MALDI-MSI

Chenglong Sun, Li Cui, Bingqian Zhou, Xiao Wang, Lanping Guo, Wei Liu

2022, 12(5): 719-724. doi: 10.1016/j.jpha.2021.09.011

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Salvia miltiorrhiza Bge (SMB) has long been used in traditional Chinese medicine to treat cardiovascular and cerebrovascular diseases. Growing clinical usage has led to a huge demand for artificial planting of SMB. Thus, continuous cropping of SMB is an important challenge that needs to be addressed. Continuous cropping can alter the metabolic profile of plants, resulting in poor growth and low yield. In this study, we tried to image the spatial location and variation of endogenous metabolites in continuously cropped SMB using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Spatially resolved expressions of tanshinones, salvianolic acids, polyamines, phenolic acids, amino acids, and oligosaccharides in normal and continuously cropped SMB roots were compared. The expressions of dihydrotanshinone I, tanshinone II A, dehydromiltirone, miltirone, dehydrotanshinone IIA, spermine, salvianolic acid B/E, tetrasaccharide, and pentasaccharide in continuously cropped SMB roots were much lower than those in normal roots. There was little difference in the expressions of caffeic acid and salvianolic acid A in normal and continuously cropped SMB roots. Ferulic acid was more widely distributed in xylem of normal SMB but strongly expressed in xylem, phloem, and cambium of continuously cropped SMB. The spatially resolved metabolite information enhances our understanding of the metabolic signature of continuously cropped SMB and also provides insights into the metabolic effects of continuous cropping in other plants.

Screening of immune cell activators from Astragali Radix using a comprehensive two-dimensional NK-92MI cell membrane chromatography/C₁₈ column/time-of-flight mass spectrometry system

Xinyi Chai, Yanqiu Gu, Lei Lv, Chun Chen, Fei Feng, Yan Cao, Yue Liu, Zhenyu Zhu, Zhanying Hong, Yifeng Chai, Xiaofei Chen

2022, 12(5): 725-732. doi: 10.1016/j.jpha.2022.05.006

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Astragali Radix (AR) is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer (NK) cells. However, owing to the complexity of its composition, the specific active ingredients in AR that act on NK cells are not clear yet. Cell membrane chromatography (CMC) is mainly used to screen the active ingredients in a complex system of herbal medicines. In this study, a new comprehensive two-dimensional (2D) NK-92MI CMC/C₁₈ column/time-of-flight mass spectrometry (TOFMS) system was established to screen for potential NK cell activators. To obtain a higher column efficiency, 3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column. In total, nine components in AR were screened from this system, which could be washed out from the NK-92MI/CMC column after 10 min, and they showed good affinity for NK-92MI/CMC column. Two representative active compounds of AR, isoastragaloside I and astragaloside IV, promoted the killing effect of NK cells on K562 cells in a dose-dependent manner. It can thus suggest that isoastragaloside I and astragaloside IV are the main immunomodulatory components of AR. This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.

Evaluation of the chemical profile from four germplasms sources of Pruni Semen using UHPLC-LTQ-Orbitrap-MS and multivariate analyses

Zihan Zhao, Yue Liu, Yushi Zhang, Zeyu Geng, Rina Su, Lipeng Zhou, Chao Han, Zhanjun Wang, Shuangcheng Ma, Weidong Li

2022, 12(5): 733-742. doi: 10.1016/j.jpha.2022.06.007

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Pruni Semen, the seed of several unique Prunus plants, is a traditional purgative herbal material. To determine the authentic sources of Pruni Semen, 46 samples from four species were collected and analyzed. Ten compounds including multiflorin A (Mul A), a notable purative compound, were isolated and identified by chemical separation and nuclear magnetic resonance spectroscopy. Seventy-six communal components were identified by ultra-high performance liquid chromatography with linear ion trap-quadrupole Orbitrap mass spectrometry, and acetyl flavonoid glycosides were recognized as characteristic constituents. The flavonoids were distributed in the seed coat and cyanogenic glycosides in the kernel. Based on this, methods for identifying Pruni Semen from different sources were established using chemical fingerprinting, quantitative analysis of the eight principal compounds, hierarchical cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis. The results showed that the samples were divided into two categories: one is the small seeds from Prunus humilis (Ph) and Prunus japonica (Pj), and the other is the big seeds from Prunus pedunculata (Pp) and Prunus triloba (Pt). The average content of Mul A was 3.02, 6.93, 0.40, and 0.29 mg/g, while the average content of amygdalin was 18.5, 17.7, 31.5, and 30.9 mg/g in Ph, Pj, Pp, and Pt, respectively. All the above information suggests that small seeds might be superior sources of Pruni Semen. This is the first comprehensive report on the identification of chemical components in Pruni Semen from different species.

Caenorhabditis elegans deep lipidome profiling by using integrative mass spectrometry acquisitions reveals significantly altered lipid networks

Nguyen Hoang Anh, Young Cheol Yoon, Young Jin Min, Nguyen Phuoc Long, Cheol Woon Jung, Sun Jo Kim, Suk Won Kim, Eun Goo Lee, Daijie Wang, Xiao Wang, Sung Won Kwon

2022, 12(5): 743-754. doi: 10.1016/j.jpha.2022.06.006

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Lipidomics coverage improvement is essential for functional lipid and pathway construction. A powerful approach to discovering organism lipidome is to combine various data acquisitions, such as full scan mass spectrometry (full MS), data-dependent acquisition (DDA), and data-independent acquisition (DIA). Caenorhabditis elegans (C. elegans) is a useful model for discovering toxic-induced metabolism, high-throughput drug screening, and a variety of human disease pathways. To determine the lipidome of C. elegans and investigate lipid disruption from the molecular level to the system biology level, we used integrative data acquisition. The methyl-tert-butyl ether method was used to extract L4 stage C. elegans after exposure to triclosan (TCS), perfluorooctanoic acid, and nanopolystyrene (nPS). Full MS, DDA, and DIA integrations were performed to comprehensively profile the C. elegans lipidome by Q-Exactive Plus MS. All annotated lipids were then analyzed using lipid ontology and pathway analysis. We annotated up to 940 lipids from 20 lipid classes involved in various functions and pathways. The biological investigations revealed that when C. elegans were exposed to nPS, lipid droplets were disrupted, whereas plasma membrane-functionalized lipids were likely to be changed in the TCS treatment group. The nPS treatment caused a significant disruption in lipid storage. Triacylglycerol, glycerophospholipid, and ether class lipids were those primarily hindered by toxicants. Finally, toxicant exposure frequently involved numerous lipid-related pathways, including the phosphoinositide 3-kinase/protein kinase B pathway. In conclusion, an integrative data acquisition strategy was used to characterize the C. elegans lipidome, providing valuable biological insights into hypothesis generation and validation.

The dynamic metabolic profile of Qi-Yu-San-Long decoction in rat urine using UPLC-QTOF-MS^E coupled with a post-targeted screening strategy

Ting Zheng, Yue Zhao, Ruijuan Li, Mengwen Huang, An Zhou, Zegeng Li, Huan Wu

2022, 12(5): 755-765. doi: 10.1016/j.jpha.2022.05.005

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Qi-Yu-San-Long decoction (QYSLD) is a traditional Chinese medicine that has been clinically used in the treatment of non-small-cell lung cancer (NSCLC) for more than 20 years. However, to date, metabolic-related studies on QYSLD have not been performed. In this study, a post-targeted screening strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight full information tandem mass spectrometry (UPLC-QTOF-MS^E) was developed to identify QYSLD-related xenobiotics in rat urine. The chemical compound database of QYSLD constituents was established from previous research, and metabolites related to these compounds were predicted in combination with their possible metabolic pathways. The metabolites were identified by extracted ion chromatograms using predicted m/z values as well as retention time, excimer ions, and fragmentation behavior. Overall, 85 QYSLD-related xenobiotics (20 prototype compounds and 65 metabolites) were characterized from rat urine. The main metabolic reactions and elimination features of QYSLD included oxidation, reduction, decarboxylation, hydrolysis, demethylation, glucuronidation, sulfation, methylation, deglycosylation, acetylation, and associated combination reactions. Of the identified molecules, 14 prototype compounds and 58 metabolites were slowly eliminated, thus accumulating in vivo over an extended period, while five prototypes and two metabolites were present in vivo for a short duration. Furthermore, one prototype and five metabolites underwent the process of “appearing-disappearing-reappearing” in vivo. Overall, the metabolic profile and characteristics of QYSLD in rat urine were determined, which is useful in elucidating the active components of the decoction in vivo, thus providing the basis for studying its mechanism of action.

Evaluation of the metabolism of PEP06, an endostatin-RGDRGD 30-amino-acid polypeptide and a promising novel drug for targeting tumor cells

Liyun Niu, Huiyu Zhou, Yueru Lian, Ya Gao, Yulu Liu, Ruolan Gu, Zhuona Wu, Xiaoxia Zhu, Hui Gan, Zhiyun Meng, Guifang Dou

2022, 12(5): 766-773. doi: 10.1016/j.jpha.2022.03.002

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PEP06 is a novel endostatin-Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) 30-amino-acid polypeptide featuring a terminally fused RGDRGD hexapeptide at the N terminus. The active endostatin fragment of PEP06 directly targets tumor cells and exerts an antitumoral effect. However, little is known about the kinetics and degradation products of PEP06 in vitro or in vivo. In this study, we investigated the in vitro metabolic stability of PEP06 after it was incubated with living cells obtained from animals of different species; we further identified the degradation characteristics of its cleavage products. PEP06 underwent rapid enzymatic degradation in multiple types of living cells, and the liver, kidney, and blood play important roles in the metabolism and clearance of the peptides resulting from the molecular degradation of PEP06. We identified metabolites of PEP06 using full-scan mass spectrometry (MS) and tandem MS (MS²), wherein 43 metabolites were characterized and identified as the degradation metabolites from the parent peptide, formed by successive losses of amino acids. The metabolites were C and N terminal truncated products of PEP06. The structures of 11 metabolites (M6, M7, M16, M17, M21, M25, M33, M34, M39, M40, and M42) were further confirmed by comparing the retention times of similar full MS spectrum and MS² spectrum information with reference standards for the synthesized metabolites. We have demonstrated the metabolic stability of PEP06 in vitro and identified a series of potentially bioactive downstream metabolites of PEP06, which can support further drug research.

Effects of polyol excipient stability during storage and use on the quality of biopharmaceutical formulations

Min-Fei Sun, Jia-Ning Liao, Zhen-Yi Jing, Han Gao, Bin-Bin Shen, You-Fu Xu, Wei-Jie Fang

2022, 12(5): 774-782. doi: 10.1016/j.jpha.2022.03.003

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Biopharmaceuticals are formulated using a variety of excipients to maintain their storage stability. However, some excipients are prone to degradation during repeated use and/or improper storage, and the impurities generated by their degradation are easily overlooked by end users and are usually not strictly monitored, affecting the stability of biopharmaceuticals. In this study, we evaluated the degradation profile of polyol excipient glycerol during repeated use and improper storage and identified an unprecedented cyclic ketal impurity using gas chromatography with mass spectrometry (GC-MS). The other polyol excipient, mannitol, was much more stable than glycerol. The effects of degraded glycerol and mannitol on the stability of the model biopharmaceutical pentapeptide, thymopentin, were also evaluated. The thymopentin content was only 66.4% in the thymopentin formulations with degraded glycerol, compared to 95.8% in other formulations after the stress test. Most glycerol impurities (i.e., aldehydes and ketones) reacted with thymopentin, affecting the stability of thymopentin formulations. In conclusion, this work suggests that more attention should be paid to the quality changes of excipients during repeated use and storage. Additional testing of excipient stability under real or accelerated conditions by manufacturers would help avoid unexpected and painful results.

Rapid fabrication of zwitterionic sulfobetaine vinylimidazole-based monoliths via photoinitiated copolymerization for hydrophilic interaction chromatography

Qiqin Wang, Lingjue Sun, Huihui Wu, Ning Deng, Xianglong Zhao, Jingwei Zhou, Tingting Zhang, Hai Han, Zhengjin Jiang

2022, 12(5): 783-790. doi: 10.1016/j.jpha.2022.05.008

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Zwitterionic sulfobetaine-based monolithic stationary phases have attracted increasing attention for their use in hydrophilic interaction chromatography. In this study, a novel hydrophilic polymeric monolith was fabricated through photo-initiated copolymerization of 3-(3-vinyl-1-imidazolio)-1-propanesulfonate (SBVI) with pentaerythritol triacrylate using methanol and tetrahydrofuran as the porogenic system. Notably, the duration for the preparation of this novel monolith was as little as 5 min, which was significantly shorter than that required for previously reported sulfobetaine-based monoliths prepared via conventional thermally initiated copolymerization. Moreover, these monoliths showed good morphology, permeability, porosity (62.4%), mechanical strength (over 15 MPa), column efficiency (51,230 plates/m), and reproducibility (relative standard deviations for all analytes were lower than 4.6%). Mechanistic studies indicated that strong hydrophilic and negative electrostatic interactions might be responsible for the retention of polar analytes on the zwitterionic SBVI-based monolith. In particular, the resulting monolith exhibited good anti-protein adhesion ability and low nonspecific protein adsorption. These excellent features seem to favor its application in bioanalysis. Therefore, the novel zwitterionic sulfobetaine-based monolith was successfully employed for the highly selective separation of small bioactive compounds and the efficient enrichment of N-glycopeptides from complex samples. In this study, we prepared a novel zwitterionic sulfobetaine-based monolith with good performance and developed a simpler and faster method for preparation of zwitterionic monoliths.

Effective extraction of fluoroquinolones from water using facile modified plant fibers

Nan Zhang, Yan Gao, Kangjia Sheng, Wanghui Jing, Xianliang Xu, Tao Bao, Sicen Wang

2022, 12(5): 791-800. doi: 10.1016/j.jpha.2022.06.004

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In this study, ecofriendly and economic carboxy-terminated plant fibers (PFs) were used as adsorbents for the effective in-syringe solid phase extraction (IS-SPE) of fluoroquinolone (FQ) residues from water. Based on the thermal esterification and etherification reaction of cellulose hydroxy with citric acid (CA) and sodium chloroacetate in aqueous solutions, carboxy groups grafted onto cotton, cattail, and corncob fibers were fabricated. Compared with carboxy-terminated corncob and cotton, CA-modified cattail with more carboxy groups showed excellent adsorption capacity for FQs. The modified cattail fibers were reproducible and reusable with relative standard deviations of 3.2%–4.2% within 10 cycles of adsorption-desorption. A good extraction efficiency of 71.3%–80.9% was achieved after optimizing the extraction condition. Based on carboxylated cattail, IS-SPE coupled with ultra-performance liquid chromatography with a photodiode array detector was conducted to analyze FQs in environmental water samples. High sensitivity with limit of detections of 0.08–0.25 μg/L and good accuracy with recoveries of 83.8%–111.7% were obtained. Overall, the simple and environment-friendly modified waste PFs have potential applications in the effective extraction and detection of FQs in natural waters.

Fluorescent intracellular imaging of reactive oxygen species and pH levels moderated by a hydrogenase mimic in living cells

Xin-Yuan Hu, Jia-Jing Li, Zi-Wei Yang, Jun Zhang, Huai-Song Wang

2022, 12(5): 801-807. doi: 10.1016/j.jpha.2022.05.007

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The catalytic generation of H₂ in living cells provides a method for antioxidant therapy. In this study, an [FeFe]-hydrogenase mimic [Ru + Fe₂S₂@F127(80)] was synthesized by self-assembling polymeric pluronic F-127, catalytic [Fe₂S₂] sites, and photosensitizer Ru(bpy)₃²⁺. Under blue light irradiation, hydrated protons were photochemically reduced to H₂, which increased the local pH in living cells (HeLa cells). The generated H₂ was subsequently used as an antioxidant to decrease reactive oxygen species (ROS) levels in living cells (HEK 293T, HepG2, MCF-7, and HeLa cells). Our findings revealed that the proliferation of HEK 293T cells increased by a factor of about six times, relative to that of other cells (HepG2, MCF-7, and HeLa cells). Intracellular ROS and pH levels were then monitored using fluorescent cell imaging. Our study showed that cell imaging can be used to evaluate the ability of Ru + Fe₂S₂@F127 to eliminate oxidative stress and prevent ROS-related diseases.

Sensitive detection of microRNAs using polyadenine-mediated fluorescent spherical nucleic acids and a microfluidic electrokinetic signal amplification chip

Jun Xu, Qing Tang, Runhui Zhang, Haoyi Chen, Bee Luan Khoo, Xinguo Zhang, Yue Chen, Hong Yan, Jincheng Li, Huaze Shao, Lihong Liu

2022, 12(5): 808-813. doi: 10.1016/j.jpha.2022.05.009

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The identification of tumor-related microRNAs (miRNAs) exhibits excellent promise for the early diagnosis of cancer and other bioanalytical applications. Therefore, we developed a sensitive and efficient biosensor using polyadenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) for miRNA analysis based on strand displacement reactions on gold nanoparticle (AuNP) surfaces and electrokinetic signal amplification (ESA) on a microfluidic chip. In this FSNA, polyA-DNA biosensor was anchored on AuNP surfaces via intrinsic affinity between adenine and Au. The upright conformational polyA-DNA recognition block hybridized with 6-carboxyfluorescein-labeled reporter-DNA, resulting in fluorescence quenching of FSNA probes induced by AuNP-based resonance energy transfer. Reporter DNA was replaced in the presence of target miRNA, leading to the recovery of reporter-DNA fluorescence. Subsequently, reporter-DNAs were accumulated and detected in the front of with Nafion membrane in the microchannel by ESA. Our method showed high selectivity and sensitivity with a limit of detection of 1.3 pM. This method could also be used to detect miRNA-21 in human serum and urine samples, with recoveries of 104.0%–113.3% and 104.9%–108.0%, respectively. Furthermore, we constructed a chip with three parallel channels for the simultaneous detection of multiple tumor-related miRNAs (miRNA-21, miRNA-141, and miRNA-375), which increased the detection efficiency. Our universal method can be applied to other DNA/RNA analyses by altering recognition sequences.

2022 Vol. 12, No. 5