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Chinese Journal of Chromatography
2021, Vol. 39, No. 3
Online: 08 March 2021

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Synthesis of zwitterionic dual-functional metal-organic framework nanocomposite with ultra-hydrophilicity for selective enrichment of glycopeptides LI Dapeng, XIE Guangshan, XIE Peisi, ZHU Lin, CAI Zongwei 2021, 39 (3):  205-210.  DOI: 10.3724/SP.J.1123.2020.11006 Abstract ( 261 )   HTML ( 75 )   PDF (1310KB) ( 152 )   Protein glycosylation is a ubiquitous and important biological process involved in various molecular functions and biological pathways. It also yields important biomarkers for clinical diagnoses. However, glycopeptide analysis is challenging due to low abundance, low ionization efficiency, and glycan heterogeneity. In the present study, a method based on hydrophilic interaction liquid chromatography (HILIC) was developed for the selective enrichment of glycopeptides using a novel metal-organic framework (MOF) nanocomposite (AuGC/ZIF-8). Dual functionalization with glutathione and cysteine has resulted in an ultra-hydrophilic MOF, with synergistic effects and lower steric hindrance, providing more affinity sites for the glycopeptide enrichment. Horseradish peroxidase (HRP) was used as a model glycoprotein, and AuGC/ZIF-8 was used to enrich glycopeptides prior to analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). AuGC/ZIF-8 displayed outstanding performance at enriching HRP glycopeptides, with high enrichment capacity (250 μg/mg), high selectivity in mixtures containing bovine serum albumin (BSA) (HRP-BSA (1∶200, mass ratio)), and high sensitivity at very low content (0.3 ng/μL). Thus this MOF holds promise for in-depth, comprehensive glycoproteomic and related analysis.

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Research progress and application of retention time prediction method based on deep learning DU Zhuokun, SHAO Wei, QIN Weijie 2021, 39 (3):  211-218.  DOI: 10.3724/SP.J.1123.2020.08015 Abstract ( 308 )   HTML ( 54 )   PDF (910KB) ( 145 )   In “shotgun” proteomics strategy, the proteome is explained by analyzing tryptic digested peptides using liquid chromatography-mass spectrometry. In this strategy, the retention time of peptides in liquid chromatography separation can be predicted based on the peptide sequence. This is a useful feature for peptide identification. Therefore, the prediction of the retention time has attracted much research attention. Traditional methods calculate the physical and chemical properties of the peptides based on their amino acid sequence to obtain the retention time under certain chromatography conditions; however, these methods cannot be directly adopted for other chromatography conditions, nor can they be used across laboratories or instrument platforms. To solve this problem, in recent years, deep learning was introduced to proteomics research for retention time prediction. Deep learning is an advanced machine-learning method that has extraordinary capability to learn complex relationships from large-scale data. By stacking multiple hidden neural networks, deep learning can ingest raw data without manually designed features. Transfer learning is an important method in deep learning. It improves the learning process a new task through the transfer of knowledge from an already-learned related task. Transfer learning allows models trained using large datasets to be utilized across conditions by fine-tuning on smaller datasets, instead of retraining the whole model. Many retention time prediction methods have been developed. In the process of training the model, the sequences of peptides are encoded to represent peptide information. Deep learning considers the relationship between the characteristics of the peptides and their corresponding retention times without the need for manual input of the physical and chemical properties of the peptides. Compared with traditional methods, deep learning methods have higher accuracy and can be easily used under different chromatography conditions by transfer learning. If there are not enough datasets to train a new model, a trained model from other datasets can be used as a replacement after calibration with small datasets obtained from these chromatography conditions. While the retention times of modified peptides can also be predicted, the predictions are inadequate for complex modifications such as glycosylation, and this is one of the main problems to be solved. The predicted retention times were used to control the quality of peptide identification. With high accuracy, the predicted retention times can be considered as actual retention times. Therefore, the difference between predicted and observed retention times can serve as an effective and unbiased quantitative metric for evaluating the quality of peptide-spectrum matches (PSMs) reported using different peptide identification methods. Combined with fragment ion intensity prediction, retention time prediction is used to generate spectral libraries for data-independent acquisition (DIA)-based mass spectrometry analysis. Generally, DIA methods identify peptides using specific spectrum libraries obtained from data-dependent acquisition (DDA) experiments. As a result, only peptides detected in the DDA experiments can be present in the libraries and detected in DIA. Furthermore, it takes a lot of time and effort to build libraries from DDA experiments, and typically, they cannot be adopted across different laboratories or instrument platforms. In contrast, the pseudo spectral libraries generated by retention times and fragment ion intensity prediction can overcome these shortcomings. The pseudo spectral libraries generate theoretical spectra of all possible peptides without the need for DDA experiments. This paper reviews the research progress of deep learning methods in the prediction of retention time and in related applications in order to provide references for retention time prediction and protein identification. At the same time, the development direction and application trend of retention time prediction methods based on deep learning are discussed.

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Research progress of microextraction by packed sorbent and its application in microvolume sample extraction WEI Jianan, QIN Molin, YANG Junchao, YANG Liu 2021, 39 (3):  219-228.  DOI: 10.3724/SP.J.1123.2020.04024 Abstract ( 229 )   HTML ( 27 )   PDF (2814KB) ( 159 )   Microextraction is a rapidly developing sample preparation technology in the field of analytical chemistry, which is seeing widespread application. Accurate sample preparation can not only save time but also improve the efficiency of analysis, determination, and data quality. At present, sample pretreatment methods must be rapid, allow for miniaturization, automation, and convenient online connection with analytical instruments. To meet the requirements of green analytical methods and improve the extraction efficiency, microextraction techniques have been introduced as suitable replacements to conventional sample preparation and extraction methods. Microextraction using a packed sorbent (MEPS) is a new type of sample preparation technology. The MEPS equipment was prepared using microsyringe with a volume of 50-500 μL, including MEPS syringes and MEPS adsorption beds (barrel insert and needle, BIN), which is essentially similar to a miniaturized solid phase extraction device. The BIN contains the adsorbent and is built into the syringe needle. A typical MEPS extraction procedure involves repeatedly pumping the sample solution in two directions (up and down) through the adsorbent multiple times in the MEPS syringe. The specific operation course of MEPS includes conditioning, loading, washing, elution, and introduction into the analysis instrument. The conditioning process is adopted to infiltrate the dry sorbent and remove bubbles between the filler particles. The adsorption process is accomplished by pulling the liquid plunger of the syringe so that the sample flows through the adsorbent in both directions multiple times. The washing process involves rinsing the sorbent to remove unwanted components after the analyte is retained. The elution process involves the use of an eluent to ensure that the sample flows through the adsorbent in both directions multiple times, so that elution can be realized by the pumping-pushing action. The target analyte is eluted with the eluent, which can be directly used for chromatographic analysis. However, when processing complex biological matrix samples by MEPS, pretreatment steps such as dilution of the sample and removal of proteins are commonly required. At present, the operation modes of the MEPS equipment are classified into three types: manual, semi-automated, and fully automated. This increase in the degree of automation is highly conducive to processing extremely low or extremely high sample volumes. Critical factors affecting the MEPS performance have been investigated in this study. The conditions for MEPS optimization are the operating process parameters, including sample flow rate, sample volume, number of sample extraction cycles, type and volume of the adsorbent, and elution solvents. It is also necessary to consider the effect of the sample matrix on the performance of MEPS. The MEPS sorbent should be cleaned by a solvent to eliminate carryover and reuse. The sorbent is a core aspect of MEPS. Several types of commercial and non-commercial sorbents have been used in MEPS. Commercial sorbents include silica-based sorbents such as unmodified silica (SIL), C2, C8, and C18. Unmodified silicon-based silica is a normal phase adsorption material, which is highly polar and can be used to retain polar analytes. C18, C8, and C2 materials are suitable for reversed-phase adsorption, while SCX, SAX, APS, and M1 (C8+SCX) adsorbents are suitable for the mixed-mode and ion-exchange modes. Noncommercial sorbents include molecularly imprinted materials, restricted-access molecularly imprinted materials, graphitized carbon, conductive polymer materials, modified silicon materials, and covalent-organic framework materials. The performance of MEPS has recently been illustrated by online with LC-MS and GC-MS assays for the analysis of biological matrices, environmental samples, and food samples. Pretreatment in MEPS protocols includes dilution, protein precipitation, and centrifugation in biological fluid matrices. Because of the small sample size, fast operation, etc., MEPS is expected to be more widely used in the analysis of bio-matrix samples. MEPS devices could also play an important role in field pretreatment and analysis.

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Fabrication of nanomaterials incorporated polymeric monoliths and application in sample pretreatment LI Ziling, LI Na, ZHAO Tengwen, ZHANG Ziyang, WANG Manman 2021, 39 (3):  229-240.  DOI: 10.3724/SP.J.1123.2020.05030 Abstract ( 157 )   HTML ( 30 )   PDF (2370KB) ( 118 )   Polymeric monolithic columns are fabricated by in situ polymerization of the corresponding monomer, crosslinkers, porogenic solvents and radical initiators within a mold. Compared with the conventional packed solid phase extraction adsorbents, polymeric monolithic columns with a continuous porous structure process distinctive advantages of rapid mass transfer and excellent permeability, which facilitates the extraction of trace amounts of the target from the matrix even at high flow velocities. Besides, these materials can be easily fabricated in situ within various cartridges, avoiding a further packing step associated with packed particulate adsorbents. Additionally, the abundant monomer availability, flexible porous structure, and wide applicable pH range make monoliths versatile for use in separation science. Thus, polymeric monolithic columns have been increasingly applied as efficient and promising extraction media for sample pretreatment food, pharmaceutical, biological and environmental analyses. However, these materials usually have the difficulty in morphology control and their interconnected porous micro-globular structure, which may result in low porosity, limited specific surface area and poor efficiency. In addition, polymeric monoliths suffer from the swelling in organic solvents, thus decreasing the service life and precision while increasing the cost consumption. Recently, the development of nanomaterial-incorporated polymeric monoliths with an improved ordered structure, enhanced adsorption efficiency and outstanding selectivity has attracted considerable attention. Nanoparticles are considered as particulates within the size range of 1-100 nm in at least one dimension, which endows them with unique optical, electrical and magnetic properties. These materials have a large surface area, excellent thermal and chemical stabilities, remarkable versatility, as well as a wide variety of active functional groups on their surface. With the aim of exploiting these advantages, researchers have shown great interest in applying nanomaterial-incorporated polymeric monoliths to separation science. Accordingly, significant progress has been achieved in this field. Nanomaterials can be entrapped via the direct synthesis of a polymerization solution that contains well dispersed nanomaterials in porogens. In addition, nanoparticles can be incorporated into the monolithic matrix by copolymerization and post-polymerization modification via specific interactions. Therefore, nanomaterial-incorporated polymeric monoliths combined the different shapes, chemical properties, and physical properties of the polymers with those of the nanoparticles. The presence of nanoparticles can improve the structural rigidity as well as the thermal and chemical stabilities of monolithic adsorbents. Besides, nanoparticles are capable of increasing the specific surface area and providing multiple active sites, which leads to enhanced extraction performance and selectivity of polymeric monolithic materials. In recent years, diverse types of nanomaterials, such as carbonaceous nanoparticles, metallic materials and metal oxides, metal-organic frameworks, covalent organic frameworks and inorganic nanoparticles have been extensively explored as hybrid adsorbents in the modes of solid phase extraction, solid phase microextraction, stir bar sorption extraction and on-line solid phase extraction. This review specifically summarizes the fabrication methods for nanomaterial incorporated polymeric monoliths and their application to the field of sample pretreatment. The existing challenges and future possible perspectives in the field are also discussed.

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Research progress in application of immobilized ionic liquid materials to separation by solid-phase extraction WANG Yicong, LIU Leilei 2021, 39 (3):  241-259.  DOI: 10.3724/SP.J.1123.2020.08002 Abstract ( 180 )   HTML ( 28 )   PDF (5305KB) ( 216 )   Ionic liquids are low-temperature molten salts with almost no vapor pressure, and they are composed of organic cations and inorganic anions. Ionic liquids are characterized by the properties of good chemical stability, high solubility, designable structure, high conductivity and so on. The physicochemical properties of an ionic liquid depend on the nature and size of the cation and anion, which confer unique characteristics; hence, these reagents are also termed “designed extractants.” As a new class of green solvents, ionic liquids are potential replacements to traditional volatile organic solvents used for extraction; for this reason, ionic liquids have attracted the attention of scientists. Research on the methods of preparation and applications of ionic liquids is being diversified, and they are extensively used in catalytic chemistry, photoelectron chemistry, materials chemistry, analytical chemistry, etc. By functional guiding design, the structures of ionic liquids, especially the imidazole ring cations, can be easily grafted with active groups such as hydroxyl, amino, carboxyl, and cyano groups, so that interactions between the ionic liquids and target molecules can be promoted via the formation of π-π bonds, hydrogen bonds, ionic bonds, and van der Waals forces. In addition, ionic liquids can be readily immobilized on solid carriers by physical or chemical means in order to obtain a new solid material with ionic liquids embedded internally or decorated on the surface. Furthermore, ionic liquids could be converted into ionic liquid-immobilized composite materials by impregnation, grafting, etc. The resulting composites not only suffer minimal loss of ionic liquids but also retain the typical characteristics of the ionic liquids and solid materials, thus showing improved mass transfer performance and better adsorption performance. Immobilized materials are characterized by high enrichment efficiency, high adsorption capacity, good stability, and strong extraction selectivity, as well as the presence of numerous recognition sites and high utilization rate of ionic liquids. In recent years, they have been widely used as solid-phase extraction adsorption materials for the separation of small organic molecules. This review introduces common immobilization methods and the characteristics of ionic liquid-immobilized materials, as well as their application in solid-phase extraction. In this paper, methods for the immobilization of ionic liquids with solid carriers such as silica gel, molecular sieves, molecularly imprinted polymers, graphene oxide, and magnetic nanomaterials are summarized, and the application of ionic liquid-immobilized materials in solid-phase extraction is reviewed. The target substances include alkaloids, flavonoids, polyphenols, and other natural active components as well as common drug molecules, organic pesticides, and other organic small molecular compounds. The properties, applications, and separation mechanisms of ionic liquids immobilized with various carriers are systematically introduced. Literature survey shows that the distribution of the binding active sites of ionic liquid-immobilized materials to the target molecules is more uniform, which increases the adsorption capacity of the materials. The adsorption efficiency of ionic liquid-immobilized materials is related to the type of ionic liquid, amount of adsorption material, concentration of the sample solution, adsorption temperature, solution pH, flow rate of the eluent, and type and amount of the eluting solvent. The existing disadvantages of ionic liquids, such as simple structures, insufficient basic theoretical research, and unsatisfactory extraction degree in complex matrixes would also be discussed. The corresponding solutions would be presented with the aim of providing guidance for the application of ionic liquid-immobilized materials in the separation and analysis of targets in complex matrices, thus paving the way for a new direction in the field of extraction and separation.

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Highly toxic type Ⅱ ribosome-inactivating proteins ricin and abrin and their detection methods: a review LIANG Longhui, XIA Junmei, LIU Changcai, LIU Shilei 2021, 39 (3):  260-270.  DOI: 10.3724/SP.J.1123.2020.10001 Abstract ( 204 )   HTML ( 21 )   PDF (1674KB) ( 83 )   Type Ⅱ ribosome-inactivating proteins (RIPs) are an important class of protein toxins that consist of A and B chains linked by an interchain disulfide bond. The B-chain with lectin-like activity is responsible for binding to the galactose-containing receptors on eukaryotic cell surfaces, which is essential for A-chain internalization by endocytosis. The A-chain has N-glycosidase activity that irreversibly depurinates a specific adenine from 28S ribosomal RNA (28S rRNA) and terminates protein synthesis. The synergistic effect of the A-B chain inactivates the ribosome, inhibits protein synthesis, and exhibits high cytotoxicity. Ricin and abrin that are expressed by the plants Ricinus communis and Abrus precatorius, respectively, are typical type Ⅱ RIPs. The toxicity of ricin and abrin are 385 times and 2885 times, respectively, more that of the nerve agent VX. Owing to their ease of preparation, wide availability, and potential use as a bioterrorism agent, type Ⅱ RIPs have garnered increasing attention in recent years. Ricin is listed as a prohibited substance under schedule 1A of the Chemical Weapons Convention (CWC). The occurrence of ricin-related bioterrorism incidents in recent years has promoted the development of accurate, sensitive, and rapid detection and identification technology for type Ⅱ RIPs. Significant progress has been made in the study of toxicity mechanisms and detection methods of type Ⅱ RIPs, which primarily involve qualitative and quantitative analysis methods including immunological assays, mass spectrometry analysis methods, and toxin activity detection methods based on depurination and cytotoxicity. Immunoassays generally involve the specific recognition of antigens and antibodies, which is based on oligonucleotide molecular recognition elements called aptamers. These methods are fast and highly sensitive, but for highly homologous proteins in complex samples, they provide false positive results. With the rapid development of biological mass spectrometry detection technology, techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are widely used in the identification of proteins. These methods not only provide accurate information on molecular weight and structure of proteins, but also demonstrate accurate quantification. Enzyme digestion combined with mass spectrometry is the predominantly used detection method. Accurate identification of protein toxins can be achieved by fingerprint analysis of enzymatically digested peptides. For analysis of protein toxins in complex samples, abundant peptide markers are obtained using a multi-enzyme digestion strategy. Targeted mass spectrometry analysis of peptide markers is used to obtain accurate qualitative and quantitative information, which effectively improves the accuracy and sensitivity of the identification of type Ⅱ RIP toxins. Although immunoassay and mass spectrometry detection methods can provide accurate identification of type Ⅱ RIPs, they cannot determine whether the toxins will retain potency. The widely used detection methods for activity analysis of type Ⅱ RIPs include depurination assay based on N-glycosidase activity and cytotoxicity assay. Both the methods provide simple, rapid, and sensitive analysis of type Ⅱ RIP toxicity, and complement other detection methods. Owing to the importance of type Ⅱ RIP toxins, the Organization for the Prohibition of Chemical Weapons (OPCW) has proposed clear technical requirements for the identification and analysis of relevant samples. We herein reviewed the structural characteristics, mechanism of action, and the development and application of type Ⅱ RIP detection methods; nearly 70 studies on type Ⅱ RIP toxins and their detection methods have been cited. In addition to the technical requirements of OPCW for the unambiguous identification of biotoxins, the trend of future development of type Ⅱ RIP-based detection technology has been explored.

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Research progress on chiral separation of amphetamines, ketamine, cathinones TANG Wenchuan, CHANG Jing, WANG Yuanfeng, WANG Aihua, WANG Ruihua 2021, 39 (3):  271-280.  DOI: 10.3724/SP.J.1123.2020.05020 Abstract ( 272 )   HTML ( 37 )   PDF (1016KB) ( 140 )   Enantiomers are ubiquitous in nature, and they are especially important in the field of pharmaceutical chemistry. Although the enantiomers of chiral drugs have identical chemical structures, they differ notably in their pharmacological, toxicological, pharmacokinetic, metabolic, and other biological activities. The same is true for amphetamines, ketamine, and cathinones, as the chiral separation of these three drugs is representative of drugs. Gas chromatography (GC), high performance liquid chromatography (HPLC), and capillary electrophoresis (CE) are widely used for the chiral separation of these three kinds of drugs. There are some similarities among the three methods for the chiral separation of amphetamines, ketamine, and cathinones: n-trifluoroacetyl-L-prolinyl chloride and (+)R-α-methoxy-α-trifluoromethylphenylacetic acid are the two typical chiral derivatization reagents used in GC. In HPLC, three kinds of chiral stationary phases are used: proteins, polysaccharides, and macrocyclic antibiotics. Cyclodextrin and its derivatives are most commonly used in CE. However, these three methods have inherent shortcomings. In the case of GC, impurities produced during chiral derivatization may interfere with the analysis, and high reaction temperatures affect the efficiency of chiral separation. HPLC has limited application scope and is expensive. In CE, there has no established process to determine the appropriate chiral selector. In recent years, research into application of the chiral separation of the above-mentioned three kinds of drugs has its own characteristics in forensic toxicology. The chiral separation of amphetamine drugs is mostly used to infer the prototype and synthesis route of drugs on the market. The chiral separation of ketamine involves a variety of biological samples. For cathinones, chiral separation methods emphasize their wide applicability. In this review, 66 reports published in professional local and overseas magazines during the past decade are collated. The characteristics of the enantiomers of amphetamines, ketamine, and cathinones as well as the mechanism of chiral recognition are briefly introduced. The commonness of the research and the application of chiral separation in forensic toxicology are reviewed. This paper proposes that the chiral separation of drugs can be further investigated from the following three aspects: 1) the use of computer technology to establish a molecular model for exploring the mechanism of chiral recognition; 2) developing new technologies for chiral separation and carrying out commercial research on the supercritical fluid method; 3) applying chiral separation to judicial practice, pharmaceutical research and development, and other practical fields.

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Liquid chromatography-mass spectrometry-based metabolomics study of the efficacy of Chinese medicine asthma-relieving decoction on respiratory syncytial virus infection OUYANG Yang, CHI Lei, XU Chao, ZHAO Xinjie, CUI Zhenze 2021, 39 (3):  281-290.  DOI: 10.3724/SP.J.1123.2020.06013 Abstract ( 195 )   HTML ( 29 )   PDF (3492KB) ( 288 )   Respiratory syncytial virus (RSV) can cause lower respiratory tract infections, such as bronchiolitis in infants. In China, traditional asthma-relieving medicine has numerous clinical applications in the treatment of RSV infections. However, due to the complexity of the traditional Chinese medicine system, its therapeutic mechanism and main pharmacological components remain unclear. Metabolomics can be used to analyze the efficacy of traditional Chinese medicine to provide modern scientific evidence for such treatments. In this study, an animal model experiment was performed with seven groups of three-week-old rats. The model group and five intervention groups were inoculated nasally with RSV for three consecutive days, and the normal group was treated with the same amount of saline for three consecutive days under the same conditions. In parallel, the five intervention groups were treated separately with the following via intragastric administration for seven consecutive days: asthma-relieving traditional Chinese medicine decoction, its three constituent agents (ascending (xuan) therapy, descending (jiang) therapy, pyretic clearing (qing) therapy), and ribavirin. Both normal group and RSV model group were administered with normal saline via intragastric administration as controls for seven consecutive days. The fundus plasma of rats in each group was collected on day 0, day 3, and day 7. Liquid chromatography-mass spectrometry-based untargeted metabolomics analysis was performed to investigate the changes in the metabolome after RSV infection, the effects of the asthma-relieving decoction on the regulation of metabolites related to RSV infection, and the primary source of efficacy. The detected metabolite ions were corrected using internal standards. Multivariate analysis of ions with an RSD value of less than 30% in quality control (QC) samples was used to construct principal component analysis models to monitor the overall metabolic changes of each group. The results showed that, during RSV infection and treatment, the asthma-relieving decoction and the positive control ribavirin had similar effects on the overall metabolic regulation of RSV-infected rats. Among the three asthma-relieving decoction constituent agents, the ascending (xuan) therapy agents which was composed of ephedra and ginkgo had a closer metabolic regulation effect with asthma-relieving decoction, and might be the main source of pharmacological efficacy. Based on the retention time, m/z value and tandem mass spectra in the database established by our laboratory, a total of 150 metabolites were identified. Paired t-tests were performed using data of the identified metabolites before and after RSV infection in each group, and it was found that 83 metabolite levels significantly changed after RSV infection, indicating that RSV infection could lead to disorders of multiple metabolic pathways in rats. The altered pathways included aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, primary bile acid biosynthesis, phenylalanine metabolism and sphingomyelin metabolism. On the third day, the asthma-relieving decoction had regulatory effects on several metabolites such as bile acids, amino acids, organic acids, lipids, etc. Among the three asthma-relieving decoction constituent agents, the ascending (xuan) therapy agents had more similar effects on the regulation of metabolites with the asthma-relieving decoction. On the other hand, the descending (jiang) therapy agents and pyretic clearing (qing) therapy agents down-regulated the abnormal increase in acylcarnitine caused by the RSV infection. Additionally, both asthma-relieving decoction and its constituent agents could maintain the stability of the immune system and metabolism of the intestinal flora in rats. This study used metabolomics to evaluate the efficacy of an asthma-relieving decoction and demonstrate the metabolites and the corresponding changes after asthma-relieving decoction-based treatment. It provides theoretical support for research on the therapeutic mechanism and active ingredients of asthma-relieving decoction.

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Analysis of differences between unifloral honeys from different botanical origins based on non-targeted metabolomics by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry SHEN Shi, YANG Yi, WANG Jingbo, CHEN Xi, LIU Tingting, ZHUO Qin 2021, 39 (3):  291-300.  DOI: 10.3724/SP.J.1123.2020.06029 Abstract ( 202 )   HTML ( 32 )   PDF (2006KB) ( 163 )   Different nectar plants contain various secondary metabolites. Herein, the differences in the contents of endogenous metabolites in honeys from eight botanical origins (i. e., acacia, jujube, vitex, linden, buckwheat, manuka, wolfberry, and motherwort honeys) were investigated by a non-targeted metabolomics-based method. This method involved solid-phase extraction pretreatment and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MSE). An oasis HLB cartridge was used for the removal of many saccharides. Chromatographic experiments were performed on an HSS T3 column (100 mm×2.1 mm, 1.8 μm) using a mobile phase that consisted of 0.1% (v/v) formic acid in acetonitrile and water. Mass spectrometry was conducted in the positive and negative modes by electrospray ionization (ESI). Metabolic information about the honeys from different botanical origins was acquired using a multivariate statistical analysis model. Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were conducted for pattern recognition and difference analysis. PCA was performed for 10557 and 2706 data variables in the positive and negative ion modes, respectively. The distribution of honeys from different botanical origins was investigated in 88 honey samples. The three principal components exhibited 48.05% and 57.88% of the total variance in positive and negative ion modes, respectively. The samples studied were divided into six different groups on the basis of their botanical origins and metabolic compounds: linden, vitex, buckwheat, manuka, jujube, and acacia honeys. A permutation test (n=200) was conducted to verify the fit of the model. The differential metabolites were screened on the basis of variable importance in project (VIP; >1), analysis of variance (ANOVA; p<0.05), and maximum fold change (>1.5) by using the PLS-DA model. The compounds were identified based on the data retrieved from the Chemspider and HMDB databases according to the quality information of precursor ions and fragment ions. Thirty-two differential metabolites were screened and primarily identified according to the characteristic fragmentation rules of specific structure types and data retrieval, including 18 flavonoids, 7 phenolic acids, 6 phenyl and terpenoid glycosides, and 1 steroid. Various flavonoids in buckwheat and manuka honeys, such as quercetin, sakuranetin, 7-hydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one, 5,7-dihydroxy-2-(3-methoxyphenyl)-4H-chromen-4-one, luteolin-7-methyl ether, and pollenitin, were found. In buckwheat honey, the contents of 3-methoxy-2-(4-methylbenzoyl)-4H-chromen-4-one, 2-hydroxy-3,4-diphenylpentanedioic acid, 3'-methoxydihydroformononetin, phenylpyruvic acid, 2-O-p-coumaroyltartronic acid, 2-(3-hydroxy-4,5-dimethoxyphenyl)-4H-chromen-4-one, 7-hydroxy-6-methoxy-3-(4-methoxyphenyl)-4H-chromen-4-one, 4-[(2E)-3-(4-hydroxyphenyl)prop-2-en-1-yl]-3-methoxyphenol, and 7-hydroxy-5-methoxyflavan were the highest; these compounds are the characteristic metabolites of buckwheat honey. In addition, manuka honey possessed the highest contents of gnaphaliin and galangin 3-methyl ether. Moreover, linden honey contained the characteristic phenyl glycosides of (S)-multifidol 2-[apiosyl-(1➝6)-glucoside], 2-phenylethyl-β-D-glucopyranoside, benzyl O-[arabinofuranosyl-(1➝6)-glucoside], crosatoside B, and terpenoid glycosides of isopentyl gentiobioside and 6-O-oleuropeoylsucrose. Vitex honey was found to be rich in quinic acid derivatives such as caffeoyl-3-O-feruloyl-quinic acid/1-feruloyl-5-caffeoylquinic acid, 3-O-caffeoyl-4-O-methyl-quinic acid/3-feruloylquinic acid, and 3-O-caffeoyl-1-O-methyl-quinic acid, in addition to the flavonoids of vitexin, namely, 6″-(3-hydroxy-3-methylglutarate) and apigenin-7-[galactosyl-(1➝4)-mannoside]. Moreover, ponasteroside A was a characteristic marker of jujube honey, and the contents of 6-C-fucosylluteolin and kaempferol 3-(2″-rhamnosylrutinoside) were the highest in acacia honey. In conclusion, the method based on non-targeted metabolomics involving UPLC-Q-TOF-MSE for different unifloral honeys was found to be fast, effective, specific, and accurate. The differences in metabolite contents and the characteristic compounds in various unifloral honeys were preliminarily illustrated. This study provides an effective analytical strategy for honey traceability and quality analysis of unifloral honey.

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Rapid screening and confirmation of 415 pesticide residues in red cabbages by liquid chromatography-quadrupole-time of flight-mass spectrometry XIE Yujie, CHEN Hui, GE Lijuan, HUO Siyu, FAN Chunlin, Lü Meiling 2021, 39 (3):  301-315.  DOI: 10.3724/SP.J.1123.2020.05006 Abstract ( 192 )   HTML ( 42 )   PDF (1327KB) ( 164 )   Supporting Information An analytical method for the simultaneous rapid screening and accurate confirmation of 415 pesticide residues in red cabbages was established using liquid chromatography-quadrupole-time of flight-mass spectrometry (LC-QTOF/MS) with single acquisition. In the established method, the pesticides in red cabbage were extracted using acetonitrile-acetic acid (99∶1, v/v) and salted-out using anhydrous magnesium sulfate and sodium chloride. The resultant solution was then cleaned-up by automatic solid phase extraction using a Carbon/NH2 cartridge. The SPE cartridge was activated with 4 mL acetonitrile-toluene (3∶1, v/v) and the effluents were discarded. The resultant solution was transferred to the Carbon/NH2 cartridge, using 3×2 mL acetonitrile-toluene (3∶1, v/v) to wash the test sample concentrate bottle, and waited until the surface of the test sample concentrate liquid reached the top layer of anhydrous Na2SO4 before transferring the washing liquid to the cartridge. A 30-mL reservoir was attached to the upper part of the SPE cartridge and 25 mL acetonitrile-toluene (3∶1, v/v) was used to wash the SPE cartridge again. The eluent was evaporated in the glass tube in a water bath at 37 ℃ and shaking speed 150 r/min to reduce the volume to 0.5 mL. Nitrogen was used to dry the concentrates, and the residues were dissolved in 1.0 mL acetonitrile-water (3∶2, v/v), homogenized by ultrasonication, and passed through 0.22-μm filtering membrane before determination. The dissolved sample solution was loaded onto a ZORBAX SB-C18 column (100 mm×2.1 mm, 3.5 μm) and separated under gradient elution using 0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium acetate and acetonitrile as the binary mobile phase. The eluent from the column was further detected by QTOF/MS under electrospray positive ionization in the MS/MS scanning mode. A matrix-matched external calibration method was used for quantitation. By optimizing the different parameters under Auto MS/MS and All Ions MS/MS acquisition modes, the optimal conditions for All Ions MS/MS under each acquisition mode were obtained, which were then compared for selection of a better mode. The results demonstrated that the developed method can be used to accurately screen and quantify all 415 pesticides in red cabbage. The linear regression correlation coefficients (r2) for the 415 pesticides were all greater than 0.990 in the corresponding linear concentration range. In addition, the screening detection limits (SDL) of 411 pesticides were no more than 5 μg/kg, and the limits of quantification (LOQs) of 413 pesticides were no more than 10 μg/kg. At the spiked levels of LOQ, two-fold LOQ, and 10-fold LOQ, the recoveries were in the ranges of 65.7%-118.4%, 72.0%-118.8% and 70.2%-111.2%, with relative standard deviations (RSDs) in the ranges of 0.9%-19.7%, 0.2%-19.9% and 0.6%-19.9%, respectively. The method was applied to detect pesticide residues in the red cabbage samples provided by the 2019 European proficiency test project for unknown pesticide screening (EUPT-SM-11) and accurate quantitation (EUPT-FV-21). For EUPT-SM-11, all the spiked and incurred pesticides in red cabbage were qualified accurately, without false positives or false negatives. This is completely consistent with the final results published by the EU official. For EUPT-FV-21, there were 19 non-volatile pesticides that can be detected by LC-MS, which were then accurately quantitated with the corresponding pesticide standard. The results demonstrate that the proposed method is accurate and reliable. It is also rapid and time-saving, and can be used for high-throughput screening and quantitative determination of pesticide residues in cabbage. It can also be extended to other fruits and vegetable matrices.

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Determination of three diphenyl ether herbicides in rice by magnetic solid phase extraction using Fe3O4@MOF-808 coupled with high performance liquid chromatography YAN Meng, JIA Yeqing, QI Peiru, WANG Yahui, JI Qianqian, WANG Manman, WANG Qian, HAO Yulan 2021, 39 (3):  316-323.  DOI: 10.3724/SP.J.1123.2020.06007 Abstract ( 242 )   HTML ( 28 )   PDF (4043KB) ( 167 )   The complex matrix of rice samples and the small amount of the target analytes in the sample necessitate an effective pretreatment process to enrich the target analytes and minimize matrix interference before instrumental analysis. Magnetic solid phase extraction (MSPE) is a dispersive solid phase extraction technique which allows for the rapid separation of sorbents from the sample solution under an external magnetic field. Compared with other traditional solid phase extraction methods, MSPE has the advantages of convenient operation, minimal interference and absence of column pressure. In this work, a metal organic framework composite (Fe3O4@MOF-808) was synthesized by a facile solvothermal method for using as an effective adsorbent to concentrate nitrofen (NIT), oxyfluorfen (OXY) and bifenox (BIF) in rice samples. Based on the pretreatment, a method was developed by coupling with high performance liquid chromatography-ultraviolet detection (HPLC-UV). The prepared material was characterized by Fourier-transform infrared spectroscopy, X-ray diffractometry, scanning electron microscopy and vibrating sample magnetometry measurements for determining its functional groups, morphology and magnetic strength. The results showed that MOF-808 has a regular octahedral morphology and well-dispersed, and the particle size of the material ranged from 400 to 500 nm with a smooth surface. The spherical Fe3O4 particles were uniformly attached to the surface of the octahedral MOF-808 crystals. The maximum saturation magnetization of this composite was 40.35 emu/g which is lower than the saturation magnetization Fe3O4 (78.26 emu/g) but still sufficient for the requirements of MSPE. The prepared Fe3O4@MOF-808 was used in the MSPE of three diphenyl ether herbicides (Des) in rice. As is well known, the key factors influencing MSPE are the adsorption and elution processes. In order to establish the optimal extraction conditions, the adsorption parameters (adsorbent amount, extraction time, elution solvent and elution volume) were investigated in detail. A 15 mL mixed standard solution was used in the experiment, and the concentrations of the three Des were 65 ng/mL. All the experiments were performed in parallel three times. The effects of the dosages of Fe3O4@MOF-808 (10, 15, 20, 25 and 30 mg), adsorption time (2, 4, 6, 8 and 10 min), elution solvents (acetone, acetonitrile and methanol) and elution volume (0.5 mL, 0.5 mL×2, 0.5 mL×3, 0.5 mL×4) were investigated. The Des could be adsorbed completely by using 25 mg of Fe3O4@MOF-808 for no more than 6 min. Elution was performed with 0.5 mL×2 of methanol. Various parameters such as limits of detection (LODs), limits of quantification (LOQs), accuracy and precision of the method were evaluated. The method showed good linearity in the range of 2-300 μg/L (r > 0.998). The LODs and LOQs were 0.6, 0.6, 0.4 μg/kg and 2.0, 2.0,1.5 μg/kg for NIT, OXY, BIF respectively. At spiked levels of 5, 10 and 20 μg/kg, the recoveries ranged from 87.3% to 96.7% with relative standard deviations (RSDs) less than 10.8%. The enrichment factors (EFs) of the method for the three Des were between 25 and 29. The method was applied to the pretreatment of the three Des in real samples, and none of the Des could be detected at any of the samples. This method had a lower LOD than that of the national standard method, but its LOD and recovery were similar to those of other reference methods. In summary, the developed method has the advantages of operational simplicity, rapidity and accuracy, and it is suitable for the analysis of herbicide residues in rice samples.

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Simultaneous determination of 18 chlorinated hydrocarbon organic solvents in cosmetics by gas chromatography-mass spectrometry TANG Juan, FEI Xiaoqing, ZHOU Jia, QIAN Kai, DONG Shaowei, CAO Lihua, DING Youchao 2021, 39 (3):  324-330.  DOI: 10.3724/SP.J.1123.2020.05010 Abstract ( 207 )   HTML ( 30 )   PDF (1148KB) ( 155 )   Organic solvents can be used to dissolve and disperse flavors, bactericides, preservatives, surfactants, oils, and coloring agents during the production of cosmetics. However, harmful chlorinated hydrocarbon organic solvents are found in cosmetics such as manicure products, anti-acne products, and perfumes. Long-term contact with such cosmetics will have an adverse effect on the consumers’ health. Past research has focused on very few chlorinated hydrocarbon organic solvents in cosmetics. Most organic solvents with low boiling points are typically determined by headspace-gas chromatography-mass spectrometry. In this study, a high-boiling-point solvent was used as the injection solvent, and the solvent delay time was cancelled. The compounds that could only peak during the solvent delay time were effectively separated. A method coupling sample pretreatment with gas chromatography-mass spectrometry (GC-MS) was developed for the simultaneous determination of 18 chlorinated hydrocarbon organic solvents in cosmetics: vinylidene chloride, dichloromethane, trans-1,2-dichloroethylene, 1,1-dichloroethane, cis-1,2-dichloroethylene, chloroform, 1,1,1-trichloroethane, carbon tetrachloride, 1,2-dichloroethane, trichloroethylene, 1,1,2-trichloroethane, tetrachloroethylene, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, 1,2,3-trichloropropane, pentachloroethane, hexachloroethane, and hexachloro-1,3-butadiene. These 18 solvents have a wide range of polarities and a notable difference in volatilities, in addition to many isomers and structural analogs, which renders their separation difficult. Therefore, the separation effect of three kinds of GC columns with different polarities was compared. n-Tetradecane, an injection solvent with good solubility, was selected as the extraction solvent. An organic solvent with high polarity has low extraction rate because of its weak polarity. Adding sodium chloride solution to the sample to induce the “salting out” effect could change the partition coefficient of the components, thereby improving the extraction rate. Therefore, the concentration of the sodium chloride solution added to the sample was optimized. In this work, liquid-liquid extraction was the main extraction process, so the effects of different shaking times, temperatures, and frequencies on the extraction rate were discussed. The optimized results are as follows: at normal temperature, the sample dispersed or dissolved in saturated sodium chloride solution was extracted by n-tetradecane at an oscillating speed of 100 r/min for 20 min. Separation was performed on an Agilent J&W DB-624 column (30 m×0.25 mm×1.4 μm) by GC-MS with an electrospray ionization (EI) source in the selected ion monitoring (SIM) mode. The external standard method was used for quantitative determination. The 18 compounds could be analyzed within 19 min. The linear equations, linear correlation coefficients, and linear ranges were obtained by analyzing a series of mixed standard working solutions. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) of the 18 components were determined. The negative lipstick (solid) and mouthwash (liquid) samples were used as the spiked sample matrix at three levels, and the recoveries and precisions were calculated. The calibration curves showed good linearities for the 18 chlorinated hydrocarbon organic solvents in range of 0.2-100 mg/L, with correlation coefficients (R2) not less than 0.9992. The LODs and LOQs were in the range of 0.033-0.049 mg/L and 0.10-0.15 mg/L, respectively. The average recoveries of the 18 chlorinated hydrocarbon organic solvents in lipstick (solid) and mouthwash (liquid) were 92.4%-103.1% and 93.3%-102.4% respectively; the corresponding relative standard deviations (RSDs) were 3.1%-5.3% and 2.8%-5.4% (n=6). This method was used to determine 115 different types of cosmetics, and tetrachloroethylene was detected in three nail polishes. With its advantages of high sensitivity, good precision, and accuracy, the developed method is suitable for the quantitative analysis of the aforesaid 18 compounds in all kinds of cosmetics. The study findings would serve as a reference for the quality and safety monitoring of cosmetics.

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Ensemble hologram quantitative structure activity relationship model of the chromatographic retention index of aldehydes and ketones LEI Bin, ZANG Yunlei, XUE Zhiwei, GE Yiqing, LI Wei, ZHAI Qian, JIAO Long 2021, 39 (3):  331-337.  DOI: 10.3724/SP.J.1123.2020.06011 Abstract ( 112 )   HTML ( 13 )   PDF (903KB) ( 85 )   Chromatographic retention index (RI) is an important parameter for describing the retention behavior of substances in chromatographic analysis. Experimentally determining the RI values of different aldehyde and ketone compounds in all kinds of polar stationary phases is expensive and time consuming. Quantitative structure activity relationship (QSAR) is an important chemometric technique that has been widely used to correlate the properties of chemicals to their molecular structures. Irrespective of whether the properties of a molecule have been experimentally determined, they can be calculated using QSAR models. It is therefore necessary and advisable to establish the QSAR model for predicting the RI value of aldehydes and ketones. Hologram QSAR (HQSAR) is a highly efficient QSAR approach that can easily generate QSAR models with good statistics and high prediction accuracy. A specific fragment of fingerprint, known as a molecular hologram, is proposed in the HQSAR approach and used as a structural descriptor to build the proposed QSAR model. In general, individual HQSAR models are built in QSAR researches. However, individual QSAR models are usually affected by underfitting and overfitting. The ensemble modeling method, which integrate several individual models through certain consensus strategies, can overcome the shortcomings of individual models. It is worth studying whether ensemble modeling can improve the prediction ability of the HQSAR method in order to build more accurate and reliable QSAR models. Therefore, this study investigates the QSAR model for chromatographic RI of aldehydes and ketones using ensemble modeling and the HQSAR method. Two individual HQSAR models comprising 34 compounds in two stationary phases, DB-210 and HP-Innowax, were established. The prediction ability of the two established models was assessed by external test set validation and leave-one-out cross validation (LOO-CV). The investigated 34 compounds were randomly assigned into two groups. Group Ⅰ comprised 26 compounds, and Group Ⅱ comprised 8 compounds. In the validation of the external test set, Group Ⅰ was employed to manually optimize the two fragment parameters (fragment distinction (FD) and fragment size (FS)) and build the HQSAR models. Group Ⅱ was used as the test set to assess the predictive performance of the developed models. For the DB-210 stationary phase, the optimal individual HQSAR model was obtained while setting the FD and FS to “donor/acceptor atoms (DA)” and 1-9, respectively. For the HP-Innowax stationary phase, the optimal individual HQSAR model was obtained by setting the FD and FS to “DA” and 4-7 respectively. The squared correlation coefficient of cross validation ( q_{\mathrm{cv}}^2 ), concordance correlation coefficient (CCC), squared correlation coefficient of external validation ( q_{\mathrm{ext}}^2 ), predictive squared correlation coefficient ( Q_{\mathrm{F}2}^2 and Q_{\mathrm{F}3}^2 ) of the two models for predicting the RI value were 0.935 and 0.909, 0.953 and 0.960, 0.925 and 0.927, 0.922 and 0.918, and 0.931 and 0.927, respectively. The results of the two validations show that there is a quantitative relationship between the molecular structure of these compounds and the RI value, and the HQSAR model is capable of modeling this relationship. Second, the ensemble HQSAR models were established using the four individual HQSAR models with the highest accuracy as the sub-models through arithmetic averaging. The ensemble HQSAR models were validated by external test set validation and LOO-CV. The q_{\mathrm{cv}}^2 , CCC, q_{\mathrm{ext}}^2 , Q_{\mathrm{F}2}^2 , and Q_{\mathrm{F}3}^2 for predicting the RI values of the DB-210 and HP-Innowax stationary phases were 0.927 and 0.919, 0.956 and 0.979, 0.929 and 0.963, 0.927 and 0.958, and 0.935 and 0.963, respectively. Compared to the individual HQSAR models, the established ensemble HQSAR models show better robustness and accuracy, thus establishing that ensemble modeling is an effective approach. The combination of HQSAR and the ensemble modeling method is a practicable and promising method for studying and predicting the RI values of aldehydes and ketones.

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Determination of amanita peptide toxins in human urine by TurboFlow online clean-up-liquid chromatography-tandem mass spectrometry FANG Li, QIU Fengmei, YU Xinwei 2021, 39 (3):  338-345.  DOI: 10.3724/SP.J.1123.2020.06005 Abstract ( 163 )   HTML ( 20 )   PDF (1365KB) ( 89 )   Amanita peptide toxins are cyclic polypeptide mushroom toxins that can cause acute liver damage. The fatality rate associated with these toxins is very high. Monitoring the concentration of amanita peptide toxins in human urine can provide valuable information for early clinical diagnosis and treatment. Therefore, a TurboFlow online clean-up-liquid chromatography-triple quadrupole mass spectrometry (TF-LC-MS/MS) method was established for the simultaneous quantitative determination of five amanita peptide toxins (α-amanitin, β-amanitin, γ-amanitin, phallacidin, and phalloidin) in human urine. After pre-treatment with high-speed centrifugation, urine samples were analyzed using TF-LC-MS/MS. The main factors influencing purification efficiency, including the TF column, loading solution, eluting solution, transfer flow, and transfer time, were optimized in this study. Under the optimized experimental conditions, the analytes were purified using a TurboFlowTM Cyclone column (50 mm×0.5 mm) and separated on a Hypersil GOLD C18 column (100 mm×2.1 mm) using the mobile phases of methanol and 4 mmol/L aqueous ammonium acetate solution with gradient elution. The analytes were detected in selected reaction monitoring (SRM) mode via positive electrospray ionization. Matrix-matched external standard calibration was used for quantitation. The linear range of the method ranged from 1.0 μg/L to 50.0 μg/L for all five amanita peptide toxins, with correlation coefficients (r2) higher than 0.997. The limits of detection were 0.15-0.3 μg/L and the limits of quantification (LOQs) were 0.5-1.0 μg/L for the five amanita peptide toxins in urine. The intra-day and inter-day recoveries of amanita peptide toxins were 87.0%-108.6% and 86.8%-112.7%, respectively, at the spiked levels of 2.0, 5.0, and 10.0 μg/L. The intra-day and inter-day relative standard deviations (RSDs) were less than 14.5%. The method is accurate, rapid, sensitive, easy to operate, and can satisfy the requirements of public health emergency testing or clinical poisoning testing.