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Chinese Journal of Chromatography
2022, Vol. 40, No. 7
Online: 08 July 2022

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Annual Reviews

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Annual review of capillary electrophoresis technology in 2021 MA Yao, HU Yangyang, ZHENG Liting, CHEN Li, ZHAO Xinying, QU Feng 2022, 40 (7):  591-599.  DOI: 10.3724/SP.J.1123.2022.03040 Abstract ( 280 )   HTML ( 467 )   PDF (881KB) ( 143 )   This paper provides an annual review of capillary electrophoresis (CE) technology in 2021. A total of 291 research papers related to CE technology published in 2021 were retrieved from the ISI Web of Science using the keywords, “capillary electrophoresis-mass spectrometry” “capillary isoelectric focusing” “micellar electrokinetic chromatography”, or “capillary electrophoresis” (not “capillary electrochromatography” “microchip” and “capillary monolithic column”). In addition, nine research papers related to CE technology in Chinese journals were reviewed: Chinese Journal of Chromatography and Chinese Journal of Analytical Chemistry. This review focused on seven papers published in Coordination Chemistry Reviews, Angewandte Chemie-International Edition, Nature Protocols, TrAC-Trends in Analytical Chemistry, and Signal Transduction and Targeted Therapy with impact factors (IFs) greater than 10.0, as well as 42 papers reported in Analytical Chemistry, Analytica Chimica Acta, Talanta, and Food Chemistry with IFs between 5.0 and 10.0. This review also provides a comprehensive overview of representative CE works in Journal of Chromatography A and Electrophoresis with IFs<5.0, as well as important Chinese journals, Chinese Journal of Chromatography and Chinese Journal of Analytical Chemistry. According to the IF, this paper introduces the representative work of CE-related papers to allow readers to quickly understand the important research progress of CE technology in the past year.

Special Column on the National Symposium on Chromatography of China

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Application progress of covalent organic framework materials in extraction of toxic and harmful substances ZHANG Wenmin, LIU Guancheng, MA Wende, FANG Min, ZHANG Lan 2022, 40 (7):  600-609.  DOI: 10.3724/SP.J.1123.2021.12004 Abstract ( 249 )   HTML ( 366 )   PDF (902KB) ( 250 )   Toxic and hazardous substances constitute a category of compounds that are potentially hazardous to humans, other organisms, and the environment. These substances include pesticides (benzoylureas, pyrethroids, neonicotinoids), persistent organic pollutants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls, perfluorinated compounds), plasticizers (phthalate esters, phenolic endocrine disruptors), medicines (sulfonamides, non-steroid anti-inflammatory drugs, tetracyclines, fluoroquinone antibiotics), heterocyclic aromatic amines, algal toxins, and radioactive substances. Discharge of these toxic and harmful substances, as well as their possible persistence and bioaccumulation, pose a major risk to human health, often to the extent of being life-threatening. Therefore, it is important to analyze and detect toxic and hazardous substances in the environment, drinking water, food, and daily commodities. Sample pretreatment is an imperative step in most of the currently used analytical methods, especially in the analysis of trace toxic and harmful substances in complex samples. An efficient and fast sample pretreatment technology not only helps improve the sensitivity, selectivity, reproducibility, and accuracy of analytical methods, but also avoids contamination of the analytical instruments and even damages the performance and working life of instruments. Sample pretreatment techniques widely used in the extraction of toxic and hazardous substances include solid-phase extraction (SPE), solid-phase microextraction (SPME), and dispersed solid-phase extraction (DSPE). The adsorbent material plays a key role in these pretreatment techniques, thereby determining their selectivity and efficiency. In recent years, covalent organic frameworks (COFs) have attracted increasing attention in sample pretreatment. COFs represent an exciting new class of porous crystalline materials constructed via the strong covalent bonding of organic building units through a reversible condensation reaction. COFs present four advantages: (1) precise control over structure type and pore size by consideration of the target molecular structure based on the connectivity and shape of the building units; (2) post-synthetic modification for chemical optimization of the pore interior toward optimized interaction with the target; (3) straightforward scalable synthesis; (4) feasible formation of composites with magnetic nanoparticles, carbon nanotubes, graphene, silica, etc., which is beneficial to enhance the performance of COFs and meet the requirement of diverse pretreatment technologies. Because of the well-defined crystalline porous structures and tailored functionalities, COFs have excellent potential for use in target extraction. However, some issues need to be addressed for the application of COFs in the extraction of toxic and hazardous substances. (1) For the sample matrix, most of the reported COFs are highly hydrophobic, which limits their dispersibility in water-based samples, leading to poor extraction performance. COFs with good dispersibility in water-based samples are urgently required. (2) Besides, COFs rely on hydrophobic interaction, size repulsion, π-π stacking, and Van der Waals forces to extract target substances, but they are not effective for some polar targets. Thus, it is necessary to develop COFs with high affinity for polar toxic and hazardous substances. (3) Methods for the synthesis of COFs have evolved from solvothermal methods to room-temperature methods, mechanical grinding, microwave-assisted synthesis, ion thermal methods, etc. Most of the existing methods are time-consuming, laborious, and environmentally unfriendly. The starting materials are too expensive to prepare COFs in large quantities. More effort is required to improve the synthesis efficiency and overcome the obstacles in the application of COFs for extraction. This article summarizes and reviews the research progress in COFs toward the extraction of toxic and hazardous substances in recent years. Finally, the application prospects of COFs in this field are summarized, which serves as a reference for further research into pretreatment technologies based on COFs.

Communications

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In-site electrophoretic elution of excessive fluorescein isothiocyanate from fluorescent particles in gel for image analysis CHEN Guohong, GUO Zehua, CAO Yiren, FAN Liuyin, LIU Weiwen, MA Yixin, CAO Chengxi, ZHANG Qiang 2022, 40 (7):  610-615.  DOI: 10.3724/SP.J.1123.2022.04023 Abstract ( 110 )   HTML ( 351 )   PDF (2813KB) ( 89 )   Supporting Information The sensitivity, accuracy, and efficiency of fluorescent particle detection can be improved by purifying the fluorescent-dye-labeled particles. In this study, an in-site model of electrophoretic elution (EE) was developed for the facile and efficient removal of unconjugated fluorescent dyes after labeling reactions, thereby facilitating the sensitive fluorescent imaging of proteins captured by microbeads. First, bovine serum albumin (BSA) and magnetic beads (MBs) were chosen as the model protein and particles, respectively, and an MBs-BSA complex was synthesized by mixing the beads with the BSA solution. Second, excessive fluorescein isothiocyanate (FITC) was added to the EP tube with MBs-BSA suspension for the fluorescent labeling of BSA, and a labeled compound was obtained after 8-h incubation in the dark at 4 ℃. The unpurified MBs-BSAFITC was obtained by removing the supernatant, leaving 5 μL of the residual solution in the EP tube. The obtained MBs-BSAFITC solution was added to a 50-μL phosphate buffer solution (PBST, containing 0.01% Triton X-100, pH 7.4). Third, gel suspension was prepared by mixing the MBs-BSAFITC solution with the low-gelling-temperature agarose gel (10 g/L) and filled into an electrophoresis channel. To demonstrate the high efficiency of the in-site model of EE for removing excessive FITC, a 10-mm hydrogel segment was prepared using MBs-BSAFITC sandwiched between two blank hydrogels and filled into a 50-mm-long electrophoresis tube (outer diameter: 5 mm; inner diameter: 3 mm) for the EE. Subsequently, the filled channel was set in an electrophoresis device to construct the in-site EE model. The particle size of the MBs was larger than the pore size of the gel, and the fluorescent beads were physically immobilized in the gel while the excessive FITC was removed from the channel by electrophoresis. Before an EE run, the original fluorescence image of the target gel was captured using a CCD camera. After the 30-min EE (50 V, 6 mA, pH 7.4 PBS), the fluorescence image was also recorded by the CCD camera. The fluorescent images were converted to a grayscale intensity map. To simplify the calculation, a simple fluorescent image analysis method was developed. The side view of the grayscale intensity map is a two-dimensional plot of peaks. Each peak indicates a fluorescent spot at a given position along the length of the channel when the distribution density of the particles is low, and the peak value is the grayscale intensity of the fluorescent spot. The statistical peak numbers and values can be used to approximate fluorescent spots on the image. After image processing and calculations, 27.8% of the average grayscale intensity of the fluorescent spot was retained, comparing the average gray value of the bright spot before and after EE, and 97.6% of excessive FITC in the channel was cleared, obtained by calculating the decreased background fluorescence grayscale intensity after EE. The particle-to-background signal ratio (P/B ratio, PBr) increased from 1.08 to 12.2 after EE with an exposure time of 1.35 s. In addition, different exposure times were explored during the fluorescence detection. Increasing the exposure time from 1.35 to 2.35 s enhanced PBr from 12.2 to 15.5, which could effectively increase the signal-to-noise ratio. An appropriate increase in exposure time also allowed the detection of many weak fluorescent particles that were previously undetectable, indicating increased sensitivity of the fluorescence detection. The EE model has the following advantages: (i) increase in specificity by eluting FITC absorbed to the surface of beads; (ii) high efficiency in the removal of free FITC with more than 97% clearance; (iii) rapid decrease in noise in the mass hydrogel (within 30 min). This method can be used in beads/spots-based immunoassay in gel, immuno-electrophoresis, and fluorescent staining of protein/nucleic acid bands in gel electrophoresis.

Articles

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Nonspecific adsorption evaluation and general minimization strategy in peptide analysis based on ultra-performance liquid chromatography-mass spectrometry ZHANG Ying, YANG Jing, MA Yuexin, CAO Ling, HUANG Qing 2022, 40 (7):  616-624.  DOI: 10.3724/SP.J.1123.2021.12012 Abstract ( 375 )   HTML ( 49 )   PDF (2258KB) ( 138 )   Proteomics technology is being increasingly used in the development of novel therapeutic peptides and protein drugs, and also in the intensive search for clinical biomacromolecule diagnostic biomarkers. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) is a rapid method to analyze peptides and proteins in low abundance. However, the nonspecific adsorption properties of peptides may lead to the loss or interference of the analytes throughout the analytical process. This unique nonspecific adsorption property is the main reason for the false negative and false positive results obtained through quantification, as well as for the poor precision, accuracy, linear range, and sensitivity, all of which impose significant challenges in the development of analytical methods. Accordingly, a general strategy was established to evaluate and reduce the negative impact of the nonspecific adsorption of peptides on UPLC-MS analysis. In this study, bovine serum albumin (BSA) was used as a model protein to explore the correlation between the physicochemical properties of 50 peptides obtained by the enzymatic digestion of BSA, as well as the degree of nonspecific adsorption. First, these peptides were classified into four categories according to their response and the degree of adsorption in the pretreatment containers and LC system. Next, the factors influencing the adsorption of 12 Class Ⅱ peptides, which were highly responsive and susceptible to adsorption, were systematically studied in terms of several aspects, including: (1) time-dependent adsorption on centrifuge tubes of three kinds (Protein-LoBind polypropylene tube and two types of polypropylene tubes); (2) time-dependent adsorption on sample vials of three kinds (Protein-LoBind polypropylene vial, polypropylene vial, and glass vial); (3) carryovers on chromatographic columns with six different stationary phases (Polar C18, Cortecs C18+, PFP, BEH C18, CSH C18, and BEH C8); (4) carryovers at different chromatographic gradients (2%B-30%B, 2%B-40%B, 2%B-50%B, and 2%B-60%B within 3 min), flow rates (0.2, 0.3, and 0.4 mL/min), and column temperatures (30, 40, 50, and 60 ℃); and (5) carryovers using different washing needle solutions (0.2% formic acid in 10% acetonitrile and 0.2% formic acid in 90% acetonitrile). The results showed that parameters such as the HPLC index and amino acid length of peptides were significantly correlated with their degree of adsorption (p<0.05), However, the above parameters can only explain the adsorption degree of 30% of the peptides. The use of the modified polypropylene material resulted in higher recovery (recovery rate>80% within 24 h) of the peptide solution during storage or pretreatment. During protein/peptide pretreatment and storage, good overall recoveries (recovery rate>80% within 24 h) were obtained using centrifuge tubes and sample vials made of the modified polypropylene material. Analysis and optimization of the LC conditions revealed that the use of the C8 chromatographic column, a high flow rate (0.4 mL/min), slow gradient (2%B-50%B within 3 min), and strong needle solution (0.2% formic acid in 90% acetonitrile) could minimize the carryover. However, the effect of the column temperature on the carryover varied considerably from peptide to peptide, and hence, requires further analysis for specific peptides. The combined optimization of the above experimental conditions resulted in minimal (approximately 1/150) or no adsorption of the Class Ⅱ peptides that were susceptible to adsorption in the analytical process. In this study, a workflow was designed to standardize the procedures for evaluating and reducing peptide adsorption. Detailed data were collected to elucidate the key risk factors and corresponding general mechanism of nonspecific adsorption throughout the analysis. Thus, this study serves as a reference for the development of analytical methods for peptides and proteins with different physicochemical properties. In future work, the risk factors should be assessed during the development and validation of protein-based macromolecular analysis methods. In conclusion, it is important to implement adequate and appropriate measures to ensure risk elimination or minimization.

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Determination of five nitroimidazoles and six benzodiazepines in aquatic products using ultra-high performance liquid chromatography-tandem mass spectrometry coupled with dispersive solid-phase extraction YANG Xiao, WAN Yiwen, HUANG Huawei, SUO Wenwen, XIAO Wei, LI Xiaoling 2022, 40 (7):  625-633.  DOI: 10.3724/SP.J.1123.2022.01005 Abstract ( 248 )   HTML ( 75 )   PDF (1179KB) ( 135 )   Nitroimidazoles (NMZs) are a crucial group of antibacterial compounds from a historical perspective. In the past, they were used for treating and preventing parasitic infections in fish. Benzodiazepines (BZDs) are second-generation sedative-hypnotics. Some fish farmers or vendors use them illegally to keep aquatic products fresh during the transportation of aquatic animals. Aquatic products are one of the most common food sources of protein and can be contaminated by NMZs and BZDs, which could impact humans through the food chain. Until recently, there was limited information on the simultaneous determination of NMZs and BZDs. Thus, it is critical to accurately quantify NMZs and BZDs for risk assessment and risk monitoring of food safety. For the simultaneous determination of five nitroimidazoles and six benzodiazepines in aquatic products, a new approach based on the dispersive solid-phase extraction (dSPE) coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed. First, the samples were extracted with acetonitrile containing 1% (v/v) ammonium hydroxide, and the extracts were purified using dSPE with C18 and primary secondary amine sorbents. Second, the extracts were collected and dried at 45 ℃ under nitrogen flow. Finally, the extracts were redissolved in 1 mL methanol-water (1∶9, v/v) mixture, filtered through a nylon-66 microfiltration membrane, and analyzed using UHPLC-MS/MS. The separation of compounds was conducted on a Kinetex F5 column (100 mm×3.0 mm, 2.6 μm) using gradient elution with 1% (v/v) formic acid aqueous solution and methanol as the mobile phase. The analytes were detected using MS/MS with positive electrospray ionization (ESI+) source under the multiple reaction monitoring modes. The matrix-matched external standard approach was used for quantitative analysis. The compounds of five nitroimidazoles and six benzodiazepines could be examined within 8.5 min. Under the optimal conditions, the standard curves were linear in the range of 0.5-20 μg/L, with the correlation coefficients exceeding 0.995. The limits of detection and limits of quantification were 0.2-0.5 μg/kg and 0.5-1.0 μg/kg, respectively. The average recoveries at three spiked levels in blank samples (grass carp, large yellow croaker, and prawn) ranged from 73.2% to 110.6%, with relative standard deviations of less than 15%. The developed approach is simple, sensitive, fast, and inexpensive. It can be used for determining five nitroimidazoles and six benzodiazepines in aquatic products.

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Chiral separation of new chiral insecticide pyraquinil isomers and establishment of analytical methods in vegetables CHEN Yan, HUANG Congling, JIANG Xunyuan, CHEN Zhiting, WANG Gang, WAN Kai, TANG Xuemei 2022, 40 (7):  634-643.  DOI: 10.3724/SP.J.1123.2022.01011 Abstract ( 256 )   HTML ( 48 )   PDF (1776KB) ( 77 )   Pyraquinil (Pyr), with a new skeleton of pyrazolo[1,5-a]quinazoline fused heterocycle, is a new chiral insecticide independently developed by South China Agricultural University in 2017. In previous studies, we found that pyraquinil can effectively control the lepidopteran pest population on cruciferous crops. Remarkably, the insecticidal activity of pyraquinil was 64-fold better than that of fipronil against the fipronil-resistant Plutella xylostella field population. Pesticides with new active mechanisms should be developed in the future to cope with the development of resistance to Plutella xylostella. Therefore, pyraquinil with new active sites has the potential to be the main rotation variety for the control of Plutella xylostella. Thus, pyraquinil has a broad prospect for application in the future. However, a chiral separation and analysis method for pyraquinil and oxidation products using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has not been established yet. Based on the physical and chemical properties of the new chiral pesticide pyraquinil, we screened chiral chromatographic columns and optimized the mobile phase ratio in this study. The separation and analysis methods for pyraquinil were developed based on the QuEChERS (quick, easy, cheap, effective, rugged, safe) system. Simultaneously, we also established oxidation metabolites. These methods were used for the simultaneous determination of the chiral isomers of pyraquinil and oxidation products in pakchoi (Brassica rapa ssp. chinensis L.) and water spinach (Ipomoea aquatica Forsk) via HPLC-MS/MS. The Chiral INC column (250 mm×4.6 mm, 5 μm) was used for separation. Ammonium formate aqueous solution (2 mmol/L) and acetonitrile were used as the mobile phases. The column temperature was 28 ℃. The injection volume was 1 μL, and the flow rate was 0.5 mL/min. MS analysis was performed using an electrospray ionization source in the negative and multiple reaction monitoring modes. We found that under the optimized conditions, the resolution of the four isomers of pyraquinil were 1.63, 2.83, and 1.74, respectively, and the resolution of the isomers of the pyraquinil oxidation product was 5.82, which achieved baseline separation. Then, the absolute configuration and peak order of pyraquinil and oxidation product isomers were determined by derivatization. The order of the peaks was RS-Pyr, SS-Pyr, RR-Pyr, SR-Pyr, S-Pyr+O, and R-Pyr+O. The purification conditions of sample pretreatment were optimized; 1 g (0.835 g MgSO4+0.150 g PSA+0.015 g GCB) was determined to be the optimal purification agent; and the average recoveries ranged from 80% to 110%. The chiral isomers of pyraquinil and oxidation products showed good linearity in the concentration range of 1.25 to 1250 μg/L and 2.5 to 2500 μg/L respectively. The square of the regression coefficient of the linear equation (R2) was greater than 0.99. The matrix effects of the pyraquinil and oxidation product isomers in pakchoi ranged from 6.1 to 30.6. In the water spinach, the matrix effect of the pyraquinil and oxidation product isomers were in the range of 0.7-26.8. The average recoveries of pyraquinil isomer at three spiked levels of 0.25, 5, 100 μg/kg in samples (pakchoi and water spinach) ranged from 90.2% to 110.6%. The oxidation product isomer average recoveries in samples (pakchoi and water spinach) spiked with 0.5, 10, 200 μg/kg ranged from 72.6% to 100.1%. Further, the relative standard deviations (RSDs) were 0.5%-9.4%. In water spinach, the intra-day and inter-day repeatability RSDs ranged from 0.5% to 8.7% and 1.0%to 8.6%, respectively. In pakchoi, the intra-day and inter-day repeatability RSDs ranged from 0.6% to 9.4% and 1.0% to 7.6%, respectively. These results indicate that the proposed method has satisfactory sensitivity, accuracy, and precision. This study can provide analytic technology for a novel chiral pesticide for environmental behavior studies, quality control, and pharmacodynamics evaluation, as well as significant technical support for the development and application of new pesticides.

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Determination of polychlorinated naphthalenes in ambient air by isotope dilution gas chromatography-triple quadrupole mass spectrometry LIU Hongyuan, JIN Jing, GUO Cuicui, CHEN Jiping, HU Chun 2022, 40 (7):  644-652.  DOI: 10.3724/SP.J.1123.2021.12006 Abstract ( 214 )   HTML ( 52 )   PDF (1194KB) ( 136 )   Polychlorinated naphthalenes (PCNs) have a structure similar to that of polychlorinated biphenyls (PCBs) and represent a new type of persistent organic pollutants (POPs) that are widely present in the environment and biological communities. PCNs can migrate and transform via different environmental media, which severely affects the health of humans and organisms. Researchers have devoted considerable focus on ambient air pollution. Although the current ambient air quality has not yet limited the concentration of PCNs, the Stockholm Convention has required parties to prohibit and eliminate their production and use. As one of the contracting parties, China is obligated to improve its environmental monitoring. In other words, the development of a method for monitoring PCNs in ambient air is important for understanding ambient air quality and safeguarding human health. PCNs are generally present at trace levels (pg/m3) in ambient air. To achieve accurate quantification of PCNs, high demands are raised on the methods for extraction, purification, and instrumental analysis, which can directly affect the efficiency, accuracy, and sensitivity of a method. Considering the trace-level presence of PCNs in ambient air and the high efficiency and accuracy of the analytical method, accelerated solvent extraction (ASE), combined with column chromatography using a multilayer silica gel column and a neutral alumina column, was established for the extraction and purification of PCNs in ambient air. The important parameters involved in the aforementioned steps, such as the type of extraction and volume of elution solvent, were optimized. The results indicated that dichloromethane-hexane (1∶1, v/v) was the best extraction solvent for the recovery of PCNs. Hexane and dichloromethane-hexane (5∶95, v/v) were used as the elution solvents for the multi-silica gel column and neutral alumina column, respectively. Isotope dilution gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) was used to quantify the target compounds. Gas chromatographic parameters, such as temperature program conditions and inlet temperature, were also optimized. The oven temperature program was as follows: 80 ℃ for 1 min, 80 ℃ to 160 ℃ at 15 ℃/min, 160 ℃ to 265 ℃ at 3 ℃/min, and 265 ℃ to 280 ℃ at 5 ℃/min, followed by holding the temperature at 280 ℃ for 10 min. The inlet temperature was set at 260 ℃. The optimal characteristics of ion pair, collision energy, and ion source temperature were determined by optimizing the key mass spectrometry parameters. The developed instrumental method, combined with suitable sample preparation techniques, was used to determine the concentrations of PCNs in ambient air samples. Quality control (QC) and quality assurance (QA) were performed by adding isotope internal standards before sampling, extraction, and injection analysis to monitor the entire analysis process. The relative standard deviations (RSDs) of the relative response factors (RRFs) for trichloronaphthalene to octachloronaphthalene were less than 16% in the concentration range of 2-100 ng/mL. The method detection limits (MDLs) for PCN homologues were in the range of 1-3 pg/m3(calculated using a sample volume of 288 m3). The precision and accuracy of this method for determining PCNs in ambient air samples were evaluated using a spiked matrix. The average spiked recoveries of trichloronaphthalene to octachloronaphthalene were 89.0%-119.4%, 98.6%-122.5% and 93.7%-124.5% at low, medium, and high spiked concentrations (20, 50, and 90 ng/mL), respectively. The RSDs of the assay results were 1.9%-7.0%, 1.6%-6.6%, and 1.0%-4.8%, respectively. During the entire analysis process, the average recoveries of the sampling and extracted internal standards were 136.2%-146.0% and 42.4%-78.1%, respectively, and the corresponding RSDs were 5.6%-7.5% and 2.7%-17.5%. Thus, this method meets the requirements of trace analysis and exhibits good parallelism, high sensitivity, high accuracy, and good precision, and it is suitable for the accurate quantitative determination of trichloronaphthalene to octachloronaphthalene in ambient air.

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Rapid analysis of serum components and metabolites of Sanzi San by high performance liguid chromatography-quadrupole/ electrostatic field orbitrap high resolution mass spectrometry ZHANG Huiwen, XIA Huimin, LIU Hong, LIU Yanyan, JIU Xin, ZHANG Minhui, HE Chunlong, WANG Huanyun 2022, 40 (7):  653-660.  DOI: 10.3724/SP.J.1123.2021.09022 Abstract ( 143 )   HTML ( 43 )   PDF (912KB) ( 61 )   Sanzi San, a Mongolian medicine, comprises three herbs: Terminalia chebula, Melia toosendan, and Gardenia jasminoides. Clinically, Sanzi San is administered orally and distributed via blood to the action site, which implies that the absorption, distribution, metabolism, and excretion (ADME) are closely related to the pharmacological action and curative effect. Therefore, possible explanations for the material basis of Sanzi San were explored in this study preliminarily. A strategy based on serum pharmacochemistry was first applied to explore the absorbed bioactive components and metabolites of Sanzi San. Wistar rats were randomly divided into normal and dosing groups, which were provided with the Sanzi San’s water extract for three days. Then, the rat’s blood samples were obtained from their abdomiral aorta using a sterile blood collection tube after administering the medicine. The blood samples were then centrifuged at 3500 r/min for 10 min to obtain the serum samples. A practical method based on high performance liquid chromatography coupled with quadrupole and electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q/Orbitrap HRMS) was developed to screen and analyze numerous bioactive components and metabolites adsorbed in the serum of the dosing rats after oral administration of the Sanzi San’s water extract. Chromatographic separation was achieved on a SHIMADZU GIST C18 chromatographic column (150 mm×4.6 mm, 5 μm). The temperature of the column was maintained at 30 ℃. The flow rate was 0.5 mL/min, and the injection volume was 10 μL. The mobile phase comprised an aqueous solution of 0.1% formic acid and methanol under gradient elution. A heated electrospray ion (HESI) source was used with positive and negative ion scanning modes. To rapidly screen out and identify the absorbed bioactive components and metabolites of Sanzi San in the rat serum samples, a simple three-step approach was developed. First, the known components in Sanzi San were listed systematically by exploring various databases, such as the Web of Science, PubMed, and Chinese National Knowledge Infrastructure. In addition, relevant information on drug biotransformation and the characteristic fragmentation patterns of parent compounds were summarized. Second, the absorbed components and metabolites were ascertained using the Xcalibur 3.0 software. Based on the information related to the parent compound’s structure, the software could be used to identify the unique peaks by comparing the chromatograms of the normal and dosing samples. Consequently, the total ion chromatograms of serum samples were established. Finally, the Compound Discover 3.0 software was used to predict the metabolic pathways and fragmentation of the absorbed compounds. Using this approach, 55 compounds were characterized, including 41 prototype components and 14 metabolites. The main prototype components in the serum sample were tannins, iridoids, and phenolic acids. The details of these compounds have been summarized and presented. Regarding the absorbed bioactive components and metabolites in the serum samples of rats administered with Sanzi San, phase Ⅰ and phase Ⅱ biochemical reactions were involved in the biotransformation pathways. The phase Ⅰ reaction modified the components and created sites for the phase Ⅱ reaction, involving reduction and hydrolysis. The phase Ⅱ reaction coupled groups to existing conjugation sites, including glucuronide to glucuronic acid, sulfate, and methyl. MS/MS spectra indicated that methylation, demethylation, and dehydroxylation are the metabolic pathways of procyanidins. Additionally, glucuronidation, deglucosidation, hydration, and demethylation are the metabolic pathways of iridoids in Sanzi San. This study comprehensively analyzed the components of the Sanzi San’s water extract absorbed in the rat’s serum. Our results revealed information regarding the pharmacodynamic substances and the major pathways involved in the ADME of Sanzi San. Further, potential medicinal ingredients for the pharmacological effects and clinical use of Sanzi San were explored at the serum pharmacochemistry level.

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Determination of dazomet and its metabolite methyl isothiocyanate residues in plant-derived foods by gas chromatography-triple quadrupole mass spectrometry RONG Jiefeng, XU Meizhu, ZHANG Zhiyong, ZOU Qiang, XU Dunming, ZHONG Jianhai, ZHANG Songyan, LE Youdong, SHE Ziwen 2022, 40 (7):  661-668.  DOI: 10.3724/SP.J.1123.2021.12021 Abstract ( 167 )   HTML ( 67 )   PDF (1247KB) ( 99 )   Dazomet is a kind of crystal solid that is stable at room temperature and acts as a fumigant. It is commonly used to control soil fungi, as an insecticide, and in sterilization and weeding. It can effectively kill root-knot nematodes, soil pests, weeds, and many soil-borne disease-causing organisms, to provide clean and healthy soil. Dazomet slowly decomposes and releases methyl isothiocyanate, methylamine, carbon disulfide, and hydrogen sulfide in acidic soil, and diffuses upward through the spaces in the soil to kill contact organisms. When agricultural crops are planted in soil treated with cotton wool, the residues in the grown crop can cause harm to human body when consumed. To ensure the quality and safety of food crops, it is important to develop a detection method for dazomet and its metabolites in plant-derived foods. Hence, in this study, a rapid and simultaneous determination method was developed for dazomet and its metabolite methyl isothiocyanate residues in plant-derived foods by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). The sample pretreatment and chromatographic conditions were optimized in the experiment. Subsequently, dazomet and its metabolite methyl isothiocyanate residues in vegetables, fruits, grains, nuts, tea, and spices were extracted with ethyl acetate, and purified using graphitized carbon, a primary-secondary amine, stearyl-bonded silica gel, and anhydrous magnesium sulfate as dispersive solid-phase extraction sorbents. After centrifugation and filtration, the target compounds were analyzed in the multiple reaction monitoring (MRM) mode by GC-MS/MS, and quantified by matrix matching external standard method. The matrix effects of the samples were also evaluated. The matrix effect was found to be in the range of 2.5% to 13.6% for methyl isothiocyanate in 16 matrices. As this matrix effect was weak, there was no need for compensatory measures. In contrast, the matrix effect of dazomet in 16 matrices was in the range of 240.3% to 331.2%. This matrix effect was strong and required compensation. Finally, a matrix matching calibration method was used to compensate the matrix effects. The relative matrix effects of other tested substrates were analyzed using lettuce as the representative substrate; it was found that all showed weak matrix effects. Therefore, the use of lettuce as a representative matrix to prepare a matrix standard curve can effectively correct the matrix effects of dazomet and methyl isothiocyanate in other substrates. Under the optimal conditions, the calibration curves were linear in the range of 0.005-1 mg/L with correlation coefficients higher than 0.99. Recovery tests were conducted by adding mixed standards to blank samples at four levels. The recoveries were in the range of 74.2%-117.2% with relative standard deviations (RSDs, n=6) of 2.8%-9.0%. The limits of quantification (LOQs) of dazomet and methyl isothiocyanate were 0.01 mg/kg. The accuracy and precision of this method met the requirements of pesticide residue determination. The established method was used to detect dazomet and its metabolite methyl isothiocyanate residues in six samples of Chinese cabbage, Chinese chives, cowpea, lettuce, eggplant, ginger, celery, potato, orange, kiwifruit, tomato, chili, rice, tea, almond, and Cuminum cyminum L. in the laboratory, and nothing was detected. The method is simple, rapid, and sensitive; overcomes the shortcomings of existing methods that require two pretreatment steps and two sets of equipment; and meets the requirements for the detection of dazomet and its metabolite methyl isothiocyanate residues in plant-derived foods.

Technical Notes

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Simultaneous determination of nine estrogens in bullfrogs using filtered solid phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry QIU Qiaoli, CHEN Xiaohong, PAN Shengdong, JIN Micong 2022, 40 (7):  669-676.  DOI: 10.3724/SP.J.1123.2022.01017 Abstract ( 203 )   HTML ( 82 )   PDF (2053KB) ( 107 )   Due to the harmful effects of estrogens and their prevalence in animal foods, accurate analysis of estrogen levels in animal foods is imperative in order to effectively assess food safety risks and ensure consumer safety. Therefore, a rapid and accurate method based on PRiME HLB solid phase extraction (SPE) cartridge purification and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to determine nine estrogen residues in bullfrogs. The nine estrogens included estriol (E3), 17β-estradiol (β-E), 17α-estradiol (α-E), 17α-ethinylestradiol (EE2), estrone (EI), diethylstilbestrol (DES), dienestrol (DE), hexestrol (HEX), and dienestrol diacetate (DD). This study optimized the mobile phase system, extraction solvent, and SPE cartridges. Because estrogens present weak alkalinity, adding a small amount of alkaline substance to the mobile phase benefits estrogen ionization into the ionic state, eliminates the peak trailing phenomenon, and enhances the signal response of estrogens to improve sensitivity. Estrogens have one or more hydroxyl groups in their chemical structures. According to the principle of similar solubility, polar solvents are chosen as extraction solvents. Based on the complex matrix composition of meat samples, SPE is required for purification to reduce matrix effects. The liquid chromatographic conditions were optimized, and the 0.5 mmol/L ammonium fluoride aqueous solution-acetonitrile system as mobile phases showed better sensitivity than the ammonium acetate aqueous solution-acetonitrile system and the ammonia-acetonitrile system for the nine estrogens. When acetonitrile was used as the extraction solvent, the extraction rates of all nine estrogens exceeded those of methanol and ethyl acetate and increased by 15%-40%. By focusing on the matrix purification effect of four different SPE cartridges, the results showed that the matrix purification ability of the PRiME HLB cartridge outperformed that of the HLB, C18, and Silica SPE cartridges. After purification by the PRiME HLB cartridge, the recoveries of all compounds were in the range of 70%-125%, and the DD recovery was increased from 47% to 74%, whereas the HEX recovery was reduced from 180% to 123%. Therefore, the PRiME HLB SPE cartridge was selected as the cleanup material for this experiment. Finally, the sample was extracted using acetonitrile, purified by PRiME HLB SPE cartridge, and separated on a Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) with a mobile phase of 0.5 mmol/L ammonium fluoride aqueous acetonitrile solution at a flow rate of 0.3 mL/min. The detection was conducted in positive and negative ion switching mode (ESI+/ESI-) and multiple reaction monitoring (MRM) scanning, and it was quantified using a matrix-matched external standard method. Under the optimal experimental conditions, the linear ranges were 0.5-100.0 μg/L for E3, β-E, α-E, EI, DE, HEX, and DD, and 1.0-100.0 μg/L for EE2 and DES. The nine estrogens showed good linearity in all linear ranges, with correlation coefficients of 0.9953-0.9994. The limits of detection were 0.17-0.33 μg/kg, and the limits of quantification were 0.5-1.0 μg/kg. The recoveries of the nine estrogens spiked at the three spiked levels of low (2.0 μg/kg), medium (10.0 μg/kg), and high (80.0 μg/kg) were 107.4%-125.3%, 67.0%-123.3%, and 65.1%-128.2%, respectively. The relative standard deviations were 1.9%-17.6%. The method established in this study was applied to detect nine estrogen residues in 50 commercially available bullfrog samples, and the results showed that HEX, EI, and DES were detected in few samples. The method is simple, rapid, sensitive, and reproducible, and can be used for the simultaneous, rapid and accurate determination of large quantities of samples.

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Detection of drugs in urine by ambient direct ionization mass spectrometry XIONG Shiling, HONG Huanhuan, WEN Luhong, HU Shundi, CHEN Anqi, XIONG Wei, CHEN La 2022, 40 (7):  677-683.  DOI: 10.3724/SP.J.1123.2022.01013 Abstract ( 204 )   HTML ( 58 )   PDF (970KB) ( 81 )   According to the Report of Drug Situation in China (2020), the growth rate of the number of drug abusers in China has decreased, but the number of drug abusers is still large. An efficient screening method is necessary for controlling drug abuse. As an important type of biological sample, urine is widely used for the rapid screening of drug addicts. However, because of the complex composition, low content, and strong interference from the body’s metabolism, the detection of drugs in urine remains a challenge. Traditional rapid screening techniques such as immunocolloidal gold analysis have a high false positive rate and insufficient quantitative capability. In addition, laboratory mass spectrometry methods require complicated time-consuming sample pre-processing and strict environmental conditions, and hence, are unsuitable for on-site rapid analysis. In recent years, various direct ionization mass spectrometry techniques such as direct analysis in real time (DART), desorption electrospray ionization (DESI), and dielectric barrier discharge ionization (DBDI) have advanced rapidly. These techniques have been applied to public safety, food safety, environmental detection, etc. In contrast to traditional ionization mass spectrometry methods, these direct ionization techniques allow for the in situ analysis of samples with simple or no pretreatment; moreover, they have the advantages of high analytical efficiency and sensitivity. In particular, pulsed electrospray ionization has the characteristics of less sample demand, compact, lightweight equipment, and no carrier gas. This paper presents a rapid method based on pulsed electrospray ionization mass spectrometry for the detection of urine samples. A rapid detection platform comprising a probe electrospray ionization source, a portable linear ion trap mass spectrometer (MS), and their coupling interface is adopted. The probe electrospray ion source includes a conducting metal wire, plastic handle, and silica glass capillary, whose tip has an inner diameter of 50 μm. The guide rail at the coupling interface is used to align the probe with the sample inlet of the portable mass spectrometer and maintain a distance of 10 mm between the probe tip and the sample inlet of the MS. The spray voltage of the probe electrospray ion source and the temperature of the MS inlet capillary are optimized at 1.8 kV and 205 ℃, respectively. In addition, rapid and efficient pretreatment techniques for urine samples have been developed. Buffer salts used for pH regulation and liquid-liquid extraction based on ethyl acetate were adopted for the pretreatment process. The linearity of the detection ability and the linear ranges of various drug-spiked solutions were also investigated. The results showed that the correlation coefficients for the quantitative detection of methamphetamine, ketamine, methylenedioxymethamphetamine (MDMA), and cocaine were greater than 0.99 at concentrations ranging from 1 to 100 ng/mL. Moreover, the limits of detection (LODs) for the five conventional drug-spiked urine were 0.5-30 ng/mL. The spiked recoveries ranged from 56.1% to 103.7%, with relative standard deviations (RSDs) of 9.0%-27.8%, implying that the combination of the instruments and the pretreatment method can lead to good accuracy. To validate the performance of the rapid detection method, 40 positive and 110 negative urine samples were tested and analyzed. The overall accuracy was over 99%, and the five conventional drugs in urine samples could be detected within 20 s. The research findings of this work could promote the development of rapid detection technology, accelerate the popularization and application of ambient direct ionization mass spectrometry, and improve the services of on-site law enforcement.