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Chinese Journal of Chromatography
2021, Vol. 39, No. 4
Online: 08 April 2021

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Preparation of covalent organic framework based on room temperature solution-suspension approach and its application to solid-phase microextraction of pyrethroids in tea YU Qidong, ZHANG Lan, ZHANG Wenmin, YANG Jiangfan 2021, 39 (4):  349-356.  DOI: 10.3724/SP.J.1123.2020.12012 Abstract ( 169 )   HTML ( 213 )   PDF (5365KB) ( 147 )   Pyrethroids (PYs) have been widely used to control pests and prevent diseases in tea gardens. However, with the increasingly stringent pesticide testing standards in the import and export trade of tea, there is an urgent need for methods to detect trace amounts of PYs in tea. In this study, a covalent organic framework (COF) material TpBD with excellent thermal/chemical stability, high porosity, and a large specific surface area was prepared by a room-temperature solution-suspension approach (SSA). TpBD-coated solid phase microextraction (SPME) fibers were fabricated by coating the material on etched stainless-steel fibers by a simple physical coating method. The fibers were used in combination with gas chromatography-tandem mass spectrometry (GC-MS/MS) to establish a highly sensitive method for the detection of PYs. The enrichment factors of this method for cyfluthrin, cypermethrin, flucythrinate, fenvalerate, and deltamethrin were 702-2687. The method showed low LODs (0.1-0.5 ng/L), wide linear ranges (0.2-800 ng/L), good linearities (correlation coefficients (R)≥0.9991) and acceptable repeatabilities (RSD≤11.0%, n=3). Green tea and oolong tea samples were analyzed using the developed method, and trace levels of the five PYs were successfully detected. The recoveries of the spiked PYs in the real green tea and oolong tea samples were in the range of 80.2%-109.5%. Experimental results showed that the established analytical method is suitable for the determination of PY pesticides in tea. Furthermore, the TpBD material was successfully prepared by the SSA method, demonstrating that the method has good universality and excellent potential for the simple synthesis of other COF materials.

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Recent advances in microchip liquid chromatography WEN Hanrong, ZHU Jue, ZHANG Bo 2021, 39 (4):  357-367.  DOI: 10.3724/SP.J.1123.2020.07031 Abstract ( 263 )   HTML ( 52 )   PDF (1485KB) ( 269 )   Miniaturization is an important trend in modern analytical instrument development, including miniaturized gas chromatography and liquid chromatography, as well as micro bore columns and capillary-to-microfluidics-based platforms. Apart from the miniaturization of the separation column, which is the core part of a chromatographic system, other parts of the system, including the sampler, pumping system, gradient generation, and detection systems, have been miniaturized. Miniaturized liquid chromatography significantly reduces solvent and sample consumption while providing comparable or even better separation efficiency. When liquid chromatography is coupled with mass spectroscopy, a low flow rate can increase the ionization efficiency, leading to enhanced sensitivity of the mass spectrometer. In contrast, normal-scale liquid chromatography suffers from its relatively high volumetric flow rate, which challenges the scanning frequency of the mass spectrometer. On the other hand because of the small sample size, other detection strategies such as spectrometric methods cannot provide sufficient sensitivity and limits of detection. In this sense, mass spectrometry has become the detection method of choice for micro-scale liquid-phase chromatography. Miniaturized liquid chromatography can diminish sample dilution efficiently when extremely small amounts of samples are used. The main driving force for this miniaturization trend, especially in liquid-phase separations, is the desperate need for microscale analyses of biological and clinical samples, given these samples are precious and the sample size is usually very small. At present, microscale liquid-phase chromatography is the only method of choice for such small, precious, and highly informative samples. The miniaturization of liquid chromatography systems, especially chromatographic columns, would be advantageous to the modularization and integration of liquid chromatography instrumental systems. Chip liquid chromatography is an integration of chromatography columns, liquid control systems, and detection methods on a single microfluidic chip. Chip liquid chromatography is an excellent format for the miniaturization of liquid chromatography systems, and it has already attracted significant attention from academia and industry. However, this attempt is challenging, and great effort is required on fundamental techniques, such as the substrate material of the microfluidic chip, structure of the micro-chromatography column, fluid control method, and detection methods, in order to make the chips suitable for liquid chromatography. Currently, the major problem in chip liquid chromatography is that the properties of the chip substrate materials cannot meet the requirements for further miniaturization and integration of chip liquid chromatography. The strength of the existing chip substrate materials is generally below 60 MPa, and the material properties limit further advances in the miniaturization and integration of chromatographic chips. Therefore, new chip substrate materials and the standard of chip channel design such as channel size and channel structure should be the key for further development of chip liquid chromatography. Mainstream instrumentation companies as well as new start-up innovation companies are now undertaking efforts toward the development of microchip liquid chromatographic products. Agilent, the first instrumentation company that introduced commercial microchip liquid chromatographic columns to the market, has led this field. Apart from microchip-based columns, Agilent introduced trap columns for different kinds of biological molecules as well as gradient generation systems for microchip-based liquid phase chromatography. Recently, another start-up company introduced microchip columns based on the in situ microfabrication of the column bed rather than packing the column with a particulate material. Such developments in microfabrication may further propel the advancement of micro-scale liquid-phase chromatography to an unprecedented level, which is beyond the conventional components and materials employed in normal-scale liquid chromatography. This review introduces the recent research progress in microchip liquid chromatography technologies, and briefly discusses the current state of commercialization of microchips for liquid chromatography by major instrumentation companies.

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Research progress in the application of magnetic solid phase extraction based on carbon based magnetic materials in food analysis LIAO Yingmin, HUANG Xiaojia, WANG Zhuozhuo, GAN Rui 2021, 39 (4):  368-375.  DOI: 10.3724/SP.J.1123.2020.05038 Abstract ( 194 )   HTML ( 43 )   PDF (1177KB) ( 133 )   Trace toxic substances in food pose a serious threat to human health, and need to be detected and analyzed to ensure food safety. However, there are many kinds of toxic substances in food, with small amounts and complex matrices, making it necessary to select an appropriate sample pretreatment technology for extraction and purification. There are some disadvantages to sample pretreatment methods such as solid phase extraction and liquid-liquid extraction, in terms of poor selectivity, significant influence of matrix interference, large sample requirement, long extraction time, use of a large amount of harmful organic solvents, and cumbersome and time-consuming operation. Magnetic solid phase extraction (MSPE) combines the advantages of magnetic separation and traditional SPE technology, avoids time-consuming column loading, and can extract the target analyte efficiently. Because of its advantages, in that it has simple operation, is time-saving and fast, requires no centrifugal filtration, and is environmentally friendly, it is considered an efficient sample pretreatment technology and applied in food analysis. The adsorption capacity and selectivity of the magnetic adsorbent used in MSPE are the key factors affecting the extraction efficiency and selectivity of MSPE, and play a key role in the accuracy of the established method. Carbon-based magnetic materials are a type of new functional magnetic materials prepared by the co-precipitation of carbon-based materials (carbon nanotubes, graphene, metal-organic framework-derived carbon, or activated carbon) and magnetic materials. In order to endow carbon-based magnetic materials with the advantages of both, carbon materials and magnetic materials, while also reflecting the advantages of high specific surface area, good stability, low cost, environmental friendliness, excellent physical and chemical properties, high porosity, and high adsorption capacity, proper functional modification is needed. Carbon-based magnetic materials modified by functionalization can efficiently enrich organic and inorganic analytes with different properties, and have seen significant progress in environmental analysis, biological detection, pollution control, and other fields. In recent years, MSPE technology based on carbon-based magnetic materials has been gradually applied in food analysis and pretreatment, but its use is still in infancy and holds immense application potential. Reference to more than 50 papers published in SCI and Chinese core journals over the past four years reveals that carbon-based materials include carbon nanotubes modified by functional groups, reagents, or materials; graphene, graphene oxide, and reduced graphene oxide; carbon derived from a gold organic framework; activated carbon biochar; and nanodiamond. The harmful substances in food samples include esters, mycotoxins, polycyclic aromatic hydrocarbons, antibiotics, alkaloids, phenols, vitamins, and antibiotics. Based on the classification of carbon-based materials, this review reveals that carbon-based magnetic materials have good preconcentration ability for harmful substances in food samples. MSPE can be combined with GC-MS, liquid chromatography-high resolution mass spectrometry (LC-HRMS), ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS), ultra high performance liquid chromatography-Q-Exactive high resolution mass spectrometry (UHPLC-Q-Exactive HRMS), high performance liquid chromatography-diode array detection (HPLC-DAD), gas chromatography micro-electron capture detection (GC-μECD), high performance liquid chromatography fluorescence with post-column photochemical derivatization (HPLC-PCD-FLD), and HPLC-UV to analyze food samples. These combined technologies have high accuracy and recovery. However, the synthesis methods of carbon-based magnetic materials such as carbon nanotubes and graphene, incur high energy consumption and high cost, and involve complex processes, which limit their application. Therefore, a carbon-based magnetic adsorbent with low cost, high selectivity, and high extraction efficiency was developed by further exploring functional modification with biochar as a carbon base. This is a very promising direction to develop MSPE technology utilizing biochar-based magnetic materials for food sample pretreatment. This review provides a theoretical basis and technical support for the wide application of carbon-based magnetic materials in MSPE technology for food analysis.

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Recent advances in stable isotope ratio analysis of common explosives HU Can, MEI Hongcheng, GUO Hongling, SUN Zhenwen, LIU Zhanfang, ZHU Jun 2021, 39 (4):  376-383.  DOI: 10.3724/SP.J.1123.2020.09015 Abstract ( 153 )   HTML ( 35 )   PDF (1190KB) ( 82 )   The ratio of stable isotopes of the elements in explosives differs depending on the raw materials obtained from different geographical sources or the production processes adopted. Hence, this ratio can be used as an important index for the comparison and trace of explosives. Isotope ratio mass spectrometry (IRMS), a high-precision method for the analysis of stable isotope ratios, has evolved into a mature tool in this regard. In combination with elemental analysis, gas chromatography, liquid chromatography, etc., IRMS is widely used in food safety, environmental protection, forensic science, and other fields. IRMS also plays an important role in the comparison and trace of explosives. Since its application to distinguish trinitrotoluene (TNT) produced in different countries in 1975, IRMS has been successfully used in the analysis of various explosives. However, there is no systematic summary on the research progress on the stable isotope ratio analysis of common explosives. This paper provides a brief description of the related principle, instrumental composition, and characteristics of stable isotope ratio analysis. Methods for the stable isotope ratio analysis of common explosives such as ammonium nitrate, black powder, TNT, pentaerythritol tetranitrate (PETN), and cyclotrimethylene trinitroamine (RDX) are reviewed. The bulk stable isotopic ratio analysis method was used in most of the studies to determine the total isotope ratio of the sample. A compound-specific isotope analysis method was also employed to determine the isotope ratio of organic explosives in a complex matrix. The reported stable isotope ratios of explosives such as ammonium nitrate, black powder, and TNT produced in different countries are summarized. The discrimination ability of the stable isotope ratio for explosives is discussed. Based on the stable isotope ratio, explosives from different sources can be distinguished effectively. By combining the results of elemental analysis with the ICP-MS results, the discrimination efficiency of different samples could be further improved. The influence of relevant factors on the isotope ratio during the production and storage of explosives are collated. There is a strong correlation between the stable isotope ratios of explosives and raw materials. The stable isotope ratios of TNT, PETN, and other explosives are related to that of nitric acid used in the production. The stable isotope ratios of nitrogen and oxygen in the explosive are relatively stable and almost unchanged within one year of production. The complexity of the environmental matrix at the explosion site and the low concentration of explosive residues make the stable isotope analysis of explosive residues challenging. However, the changes in the stable isotope ratio before and after the explosion are discussed. Since there is no information on the application of stable isotope analysis to the traceability of explosives, the paper mentions that the standardized explosive sample pretreatment, stable isotope analysis method, collection and analysis of large amounts of explosive samples, and explosive stable isotope database are the basis of explosive traceability. This paper also outlines the existing challenges in the analysis of the stable isotope ratios of explosives, including the small number of explosive samples, lack of a stable isotope explosive analysis database, and difficulty in the stable isotope analysis of explosive residues. Possible solutions to these problems are proposed, followed by suggestions for the future development of the stable isotope ratio analysis of common explosives. The suggestions include establishing an effective extraction and enrichment method for explosive residues, combining IRMS with GC or LC for analyzing explosives, establishing a comprehensive process for the analysis of the stable isotope ratios of inorganic and organic explosives, and comparison and analysis of the stable isotope analysis data using statistical methods.

Articles

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Preparation of luminescent silica nanoparticles with immobilized metal ion affinity for labeling phosphorylated proteins in Western Blot MAO Yuxiao, ZHENG Mengmeng, LIU Guizhen, AN Baoli, KANG Jingwu 2021, 39 (4):  384-390.  DOI: 10.3724/SP.J.1123.2020.05024 Abstract ( 107 )   HTML ( 28 )   PDF (5138KB) ( 66 )   Protein phosphorylation is an important type of post-translational protein modification. In Western Blot experiment, the assay of phosphoproteins need special phospho antibodies, which are expensive, difficult to preserve, poorly reproducible. To this end, the immobilized metal ion affinity luminescent silica nanoparticles for instead of phospho antibodies were prepared. A layer of polymer was created on the surface of the silica nanoparticles via co-polymerization to protect the nanoparticles and to functionalize them with the immobilized metal ion affinity property to specifically label the phosphorylated proteins in Western Blot assays. The affinity luminescent silica nanoparticles were prepared with the following procedure. First, the sol-gel precursor fluorescein isothiocyanate-3-aminopropyltriethoxysilane (FITC-APTES) with the fluorescent moiety was prepared by modifying APTES with FITC. The luminescent silica nanoparticles (FITC@SiO2) were synthesized using the Stöber synthesis method in a reversed microemulsion. Briefly, 123.2 mL of cyclohexane, 25.6 mL of n-hexanol, and 5.44 mL of deionized water were ultrasonically mixed, and then 28.3 g of Triton X-100 were added and the mixture was magnetically stirred for 15 min to form a clear and transparent microemulsion system. Within 10 min, 0.8 mL of FITC-APTES precursor, 1.6 mL of tetraethoxysilane (TEOS), and 0.96 mL of concentrated ammonia (25%-27%, mass fraction) were added to the microemulsion, and the mixture was stirred at 24 ℃ for 24 h. After the reaction, the microemulsion system was destroyed by adding 200 mL of ethanol. The resulting FITC@SiO2 luminescent silica nanoparticles were centrifuged, and washed three times with ethanol. After dryness, the FITC@SiO2 nanoparticles were modified with methacryloxy-propyltrimethoxysilane (MPS) to introduce the double bonds for further modification. The functional monomer nitrilotriacetic acid (NTA) and glycidyl methacrylate (GMA) were copolymerized on the surface of the nanoparticles to convert FITC@SiO2-MPS to FITC@SiO2-MPS-GMA-NTA. The polymer coating of the silica nanoparticles was not only able to protect the silica from hydrolysis, but also to introduce the functional groups of nitrilotriacetic acid, which can chelate with metal ions. Elemental analysis demonstrated that the NTA groups had been bonded to the surface of the nanoparticles via copolymerization. The polymerization did not affect the morphology and fluorescence properties of the nanoparticles. The FITC@SiO2-MPS-GMA-NTA nanoparticles were activated with three different metal ions Zr4+, Fe3+, and Ti4+, for the enrichment of phosphorylated peptides derived form α-casein tryptic digestion. HPLC-MS analysis indicated that the FITC@SiO2-MPS-GMA-NTA-Ti 4+ nanoparticles are the best for the enrichment of phosphorylated peptides. The FITC@SiO2-MPS-GMA-NTA-Ti4+ nanoparticles were used for labelling the phosphorylated proteins in Western Blot experiment. The electrophoretic band of α-casein could be clearly labeled with the FITC@SiO2-MPS-GMA-NTA-Ti 4+ nanoparticles, while the bovine albumin band could not be labelled. This indicates that the luminescent FITC@SiO2-MPS-GMA-NTA-Ti4+nanoparticles can be used to label the phosphorylated proteins in Western Blot experiments.

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Urine metabolomics analysis based on ultra performance liquid chromatography-high resolution mass spectrometry combined with osmolality calibration sample concentration variability HE Zhian, LIN Houwei, GUI Juan, ZHU Weichao, HE Jianhua, WANG Hang, FENG Lei 2021, 39 (4):  391-398.  DOI: 10.3724/SP.J.1123.2020.06018 Abstract ( 170 )   HTML ( 35 )   PDF (2894KB) ( 96 )   Urine is an important source of biomolecular information for metabolomic studies. However, the acquisition of high-quality metabolomic datasets or reliable biomarkers from urine is difficult owing to the large variations in the concentrations of endogenous metabolites in the biofluid, which are caused by diverse factors such as water consumption, drugs, and diseases. Thus, normalization or calibration is essential in urine metabolomics for eliminating such deviations. The urine osmolality (Π), which is a direct measure of the total urinary solute concentration and is not affected by circadian rhythms, diet, gender, and age, is often considered the gold standard for estimation of the urine concentration. In this study, a pre-data acquisition calibration strategy based on osmolality was investigated for its feasibility to overcome sample concentration variability. Before data acquisition, the product of the osmolality×injection volume of all samples was set to be equivalent through the uses of a customized injection volume or dilution. After ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) analysis of the sample, the raw dataset was normalized to the total ion abundance or total useful MS signals (MSTUS) to achieve further calibration. The osmolality of each urine sample was determined with a freezing-point depression osmometer. For the instrumental analysis, a Vanquish UPLC system coupled to a Q-Exactive Plus HRMS device was used for metabolite analysis and accurate mass measurement. Full-scan mass spectra were acquired in the range of m/z 60-900, and the MS/MS experiments were conducted in “Top5” data-dependent mode. A Waters UPLC column (100 mm×2.1 mm, 1.8 μm) was used for chromatography separation. The raw data were imported into Progenesis QI software for peak picking, alignment, deconvolution, and normalization. SIMCA-P software was used for the principal component analysis (PCA) and orthogonal partial least-squares discrimination analysis (OPLS-DA). This strategy was first applied to sequentially diluted urine samples, where three frequently used normalization methods were compared. In the identical injection volume experiment, the points were scattered and showed relevant distribution according to the dilution multiple in the plot of PCA scores. There was little improvement after normalization to either the total ion abundance or MSTUS. In the customized injection volume experiment, the urine samples derived from the same source showed ideal clustering. With total ion abundance and MSTUS normalization, the dataset was further improved in the PCA model fitting and prediction. As a result, there were more peaks with a peak area RSD of <30%, which indicated better parallelism. The diluted urine solutions had higher Spearman’s coefficient values with their sample source than those without calibration, which suggested less intra-group differences. The strategy was further validated using data from a metabolomic study of children with congenital hydronephrosis and healthy controls. As a concentration estimator, osmolality showed better linear correlation with the mass signal and was less influenced by physiological or pathological factors, thus obtaining broader application and more accurate results than creatinine. The concentration variability was effectively eliminated after customized dilution calibration and showed a more obvious clustering effect in the PCA score plot. The OPLS-DA-based statistical model used to identify discriminate metabolites was improved, with less chance of overfitting. In conclusion, the calibration strategy based on osmolality combined with total ion abundance or MSTUS normalization significantly overcame the problem of urine concentration variability, eliminated intra-group differences, and possessed better parallelism, thus giving better clustering effects in PCA or OPLS-DA and higher reliability of the statistical model. The results of this study provide guidance and a reference for future metabolomic studies on urine.

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Simultaneous determination of 12 lipophilic shellfish toxins in plasma and urine by ultra-high performance liquid chromatography-tandem mass spectrometry LIN Qiang, YANG Chao, LI Meili, WANG Jia, HOU Hanran, SHAO Bing, NIU Yumin 2021, 39 (4):  399-405.  DOI: 10.3724/SP.J.1123.2020.11001 Abstract ( 175 )   HTML ( 34 )   PDF (1614KB) ( 117 )   Lipophilic shellfish toxins pose significant threats to the health of seafood consumers and public health. The symptoms of these kinds of toxins include severe diarrhea, abdominal cramps, nausea and gastrointestinal disorders. These symptoms could be hardly distinguished with many other symptoms of food poisoning and diseases. Therefore, a fast and accurate determination method in human biological samples is urgently needed for the accurate judgement of food poisoning incident, which is important for the investigation of public health emergencies and clinical treatment of poisoned patients. However, there were several flaws of the previous studies reported on the analysis of lipophilic shellfish toxins: (1) limited target compounds were covered; (2) the pre-treatment process was complex; (3) the sensitivity of the compound was low. In this study, a simple extraction method coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 12 lipophilic shellfish toxins, including azaspir acid 1 (AZA1), azaspir acid 2 (AZA2), azaspir acid 3 (AZA3), dinophysistoxin 1 (DTX1), dinophysistoxin 2 (DTX2), gymnodimine (GYM), hyessotoxin (HYTX), okadaic acid (OA), pinnatoxin (Pntx), pectenotoxins 2 (PTX2), spirolides 1 (SPX1), yessotoxin (YTX), in plasma and urine. Firstly, the instrument conditions were optimized. Different additions in mobile phase were compared and 0.05% (v/v) ammonia solution was selected since it can improve the peak shape of YTX and HYTX, and increase the respondence by four times. Secondly, the volume of acetonitrile (0.2, 0.4, 0.6, 0.8, 1.0 mL) use for the extraction of the target compounds in plasma was optimized. Satisfactory recoveries were obtained when 0.6 mL of acetonitrile was used. At the same time, satisfactory recoveries were obtained when 0.9 mL of acetonitrile was used in urine samples. Finally, under the optimized conditions, the 12 compounds in plasma and urine samples were ultrasonically extracted with acetonitrile. Chromatographic separation was performed on a Phenomenex Kinetex C18 column (50 mm×3 mm, 2.6 μm) with 90% (v/v) acetonitrile aqueous solution and water containing 0.05% (v/v) ammonia as mobile phases. Gradient elution with a flow rate of 0.40 mL/min was employed. The 12 compounds were monitored in the multiple reactions monitoring (MRM) mode with electrospray ionization (ESI) under both positive and negative conditions. The matrix effects of the 12 compounds ranged from 0.8 to 1.1. Therefore, external standard calibration curves were used for the quantification. The 12 shellfish toxins showed good linear relationship in the range of 0.03-36.25 μg/L with the correlation coefficients greater than 0.995. The limits of detection (LODs, S/N=3) were 0.08-0.21 ng/mL for the urine samples and 0.10-0.28 μg/L for the plasma samples, respectively. The limit of quantitations (LOQs, S/N=10) were 0.23-0.63 μg/L for the urine samples and 0.31-0.84 μg/L for the plasma samples, respectively. The recoveries of the 12 compounds were in the range of 72.7%-124.1% at three spiked levels (i. e., LOQ, three times LOQ, and ten times LOQ). The intra-day and inter-day precisions were 2.1%-20.0% and 2.1%-15.3%, respectively. The method was applied in the detection of the 12 lipophilic shellfish toxins in the urine and plasma samples of healthy humans and mice previously injected with the 12 shellfish toxins intraperitoneally. None of the 12 toxins were found in the samples from healthy human, while all of the 12 lipophilic shellfish toxins were found in the urine and plasma samples collected from the poisoned mice in the range of 1.14-2.35 μg/L and 1.01-1.17 μg/L, respectively. The established method has the advantages of sensitive, quick, easy to operate, and of low sample volume. It can be used for the simultaneous determination of 12 lipophilic shellfish toxins in urine and plasma samples.

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Simultaneous determination of 11 prohibited and restricted veterinary drugs and their metabolites in animal-derived foods by ultra performance liquid chromatography-tandem mass spectrometry coupled with solid phase extraction LIU Bolin, XIE Jian, ZHAO Ziwei, WANG Xiuli, SHAN Xiaomei 2021, 39 (4):  406-414.  DOI: 10.3724/SP.J.1123.2020.05012 Abstract ( 189 )   HTML ( 50 )   PDF (1771KB) ( 154 )   Chloramphenicols, nitroimidazoles, lincosamides, and macrolides are common antibiotics used in veterinary medicine. Overdoses of these drugs will lead to residual substances in animal-derived foods and accumulate in the body through the food chain, thereby exerting adverse effects on human health. Therefore, regulation of veterinary drug levels is imperative to ensure the quality of animal-derived foods and safeguard the health of consumers. In this study, a method based on ultra performance liquid chromatography-tandem mass spectrometry coupled with solid phase extraction (SPE-UPLC-MS/MS) was developed for the simultaneous determination of eight prohibited and restricted veterinary drugs and three metabolite residues across four categories (chloramphenicols, nitroimidazoles, lincosamides, and macrolides) in eggs, liquid milk, chicken, and freshwater fish. The main factors affecting the response, recovery, and sensitivity of the method, such as the type and pH values of the extraction solvent, dilution solution for the analytes, type of chromatographic column, and type and proportion of the mobile phase, were optimized during sample pretreatment and instrument analysis. The samples were hydrolyzed and dispersed in 0.1 mol/L phosphate buffer solutions (pH 9.0) and extracted with acetonitrile. The extract was further extracted using ethyl acetate. After centrifugation, the supernatant ethyl acetate was concentrated to near dryness in nitrogen below 40 ℃. The residue was dissolved in 0.3 mL methanol, followed by the addition of 5.7 mL phosphate buffer solution. After shaking, the solutions were purified and enriched on an Oasis HLB SPE column. The target analytes were separated on an ACQUITY UPLC BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 μm) at a column temperature of 40 ℃ with a flow rate of 0.4 mL/min. The injection volume was 10 μL. Gradient elution was carried out with methanol and 0.1% formic acid aqueous solution as the mobile phases. Multiple reaction monitoring (MRM) was conducted in the positive and negative electrospray ionization modes. The isotope internal standard method was used for quantitative analysis. Under optimal conditions, each analyte showed a good linear relationship in each range, and the correlation coefficient (R2) was greater than 0.99. The limits of detection (LODs) ranged from 0.050 to 0.50 μg/kg, and the limits of quantification (LOQs) ranged from 0.20 to 1.5 μg/kg. With eggs, freshwater fish, chicken, and liquid milk as the matrix samples, the recoveries in spiked blank samples were determined at different addition levels in compliance with the current legislation. The average recoveries of the 11 analytes were 65.3% to 108%. The relative standard deviations (RSDs) were between 0.40% and 21%. The matrix effects of the analytes were between 0.0124% and 46.80% in four different samples after purification on the Oasis HLB column. The practicality of the proposed approach for routine analyses of the eight prohibited and restricted veterinary drugs, and three metabolite residuals was evaluated by applying it to the determination of these compounds in animal-derived food samples. The samples, including 80 eggs, 80 chicken, 40 liquid milk, and 32 freshwater fish, were procured from a supermarket and a farm product market. The results of the positive samples were consistent with those observed with the standard methods. The method described herein is easy to operate, sensitive, and accurate. It is suitable for the simultaneous and rapid determination of various prohibited and restricted veterinary drug residues and metabolites in animal-derived foods.

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Simultaneous determination of 22 sunscreen agents in cosmetics by ultra-high-performance liquid chromatography using diode array detector ZHANG Keming, DENG Ming, SU Yuanyuan, XIE Dong, XU Youcheng, LIU Xianghong 2021, 39 (4):  415-423.  DOI: 10.3724/SP.J.1123.2020.12001 Abstract ( 211 )   HTML ( 30 )   PDF (1085KB) ( 142 )   A fast and simple ultra-high-performance liquid chromatography using diode array detector (UHPLC-DAD) technique has been developed for the simultaneous determination of 22 sunscreen agents (UV filters), viz. phenylbenzimidazole sulfonic acid (PBS), terephthalydene dicamphor sulfonic acid (TDS), benzophenone-4 (BZ4), camphor benzalkonium methosulfate (CBM), benzophenone-2 (BZ2), benzylidene camphor sulfonic acid (BCS), benzophenone-3 (BZ3), 3-benzylidene camphor (3BC), isoamyl p-methoxycinnamate (IMC), 4-methylbenzylidene camphor (MBC), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), octocrylene (OCR), butyl methoxydibenzoyl methane (BDM), ethylhexyl dimethyl PABA (EDP), ethylhexyl methoxycinnamate (EMC), homosalate (HS), ethylhexyl salicylate (ES), diethylhexyl butamido triazone (DBT), ethylhexyl triazone (ET), drometrizole trisiloxane (DRT), methylene bis-benzotriazolyl tetramethylbutylphenol (MBP), and bis-ethylhexyloxyphenol methoxyphenyl triazine (EMT) in cosmetic products. Different parameters, such as column type, oven temperature, mobile phase composition, and detection wavelength, were studied. The best chromatographic separation was obtained under the following conditions: Poroshell 120 EC-C18 (100 mm×4.6 mm, 2.7 μm) column set at 25 ℃ and gradient acetonitrile-isopropanol-water (containing 50 mmol/L ammonium acetate and 0.05% (v/v) formic acid) as the mobile phase, pumped at a flow rate of 0.5 mL/min, with a wavelength of 311 nm. The proposed UHPLC-DAD technique provided separation of the 22 target sunscreen agents within 35 min, with the optimized sample pretreatment procedure below. First, samples were mixed thoroughly by adding 2 mL or 5 mL tetrahydrofuran, followed by vortex and dispersal. If the wax samples were still not homogenized completely, an ultrasonic dispersal protocol with heating to 50 ℃ was adopted. Second, the homogeneous samples were ultrasonically extracted with ethanol containing 0.1% (v/v) formic acid. The developed method showed good linear relationships, with correlation coefficients of no less than 0.998. Two kinds of samples with different matrix types were fortified at three levels. The average spiked recoveries of 22 UV filters ranged from 85.2% to 112.3%, with the relative standard deviations (RSDs) ranging from 0.5% to 6.5%. The limits of detection were between 0.7 and 64 mg/kg, and the limits of quantification ranged from 2.4 mg/kg to 215 mg/kg. Moreover, the stabilities of the mixed standard solutions at the levels of 2, 10, and 50 mg/L were tested. The stability results showed that drometrizole trisiloxane was stable for 12 h, while the others were stable for 36 h. The reliability of the developed method was demonstrated by applying it to 16 commercial sunscreen-containing cosmetic samples obtained from the Chinese market. The levels determined in this study agreed well with those of five commercial samples (such as emulsion and cream). The method developed was remarkably different from the standard method, which is mentioned in the Safety and Technical Standards for Cosmetics (2015 edition), especially in terms of mobile phase composition and extraction solvent. Compared to the standard method, this method bypassed the use of large amounts of corrosive solvents like tetrahydrofuran and perchloric acid, thus improving the extraction efficiency of low-polarity components like drometrizole trisiloxane, methylene bis-benzotriazolyl tetramethylbutylphenol, and bis-ethylhexyloxyphenol methoxyphenyl triazine, and the analytes were well separated with better stability. Benzophenone-2 was added to this method as another detection component. The good analytical features, as well as their environment-friendly characteristics, make the presented method suitable not only for routine analysis in cosmetics industries, but also as a candidate reference method for sunscreen analysis.

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Quantitative analysis of nine types of virus-like particles in human papilloma virus bulk by size-exclusion chromatography LONG Zhen, LI Xiaoyu, LI Xiuling, LIU Junkai, NIE Jianhui, LI Changkun, LI Yueqi, HUANG Taohong, HUANG Weijin 2021, 39 (4):  424-429.  DOI: 10.3724/SP.J.1123.2020.06032 Abstract ( 124 )   HTML ( 28 )   PDF (835KB) ( 111 )   Cervical cancer is the fourth most common cancer among women. Human papilloma virus (HPV) is the most common cause of cervical cancer which accounts for 5% of all human cancers and results in about 528000 cases and 266000 deaths every year. HPV vaccines are considered the most effective strategy for the prevention of HPV infection and cervical carcinoma. Since 2006, three prophylactic vaccines against HPV have been available on the market, including bivalent vaccines, quadrivalent vaccines, and nine-valent vaccines. Among them, nine-valent vaccines have been reported to be the most effective. They can prevent 97% of the high-grade pre-cancer lesions. Virus-like particles (VLPs), which are arranged as 360 copies of capsid proteins L1, are the only antigens of the HPV vaccine. Nine-valent HPV vaccines are prepared by mixing nine types of VLPs with adjuvants. Thus, the quality of the VLPs, including their stability and content in the HPV bulk, is very important for developing HPV vaccines. In this study, a method was developed for the determination of the nine types of VLPs (HPV6/11/16/18/31/33/45/52/58) in HPV bulk by size exclusion chromatography (SEC). The parameters of this method were optimized in terms of column brand, pore size of stationary phase particles, buffer concentration, and pH value. SHIMSEN Ankylo SEC-300 column (300 mm×7.8 mm, 3 μm) combined with a buffer aqueous solution containing 300 mmol/L NaCl and 50 mmol/L phosphate (pH 7.0) was utilized to separate the VLPs from the matrix since a narrow peak shape and good repeatability for VLPs could be obtained with this column and mobile phase. The optimized method had a wide linear range, good repeatability (RSDs of peak area were not more than 5.0%), and a satisfactory sensitivity (LOQs in the range of 4.58-15.24 μg/mL). The optimized method was used to determine the VLPs in the HPV bulk. The LOQs of the current method were much lower than the content of the nine types of VLPs in the HPV bulk, indicating that this method was sensitive enough for the determination of the nine types of VLPs in the HPV bulk. The method was also used to determine the VLPs in an HPV bulk that had been stored at 4 ℃ for one week. A decrease in the nine types of VLPs in the range of 10.0%-62.6% was observed after they were stored at 4 ℃ for one week. An HPV vaccine was prepared by mixing the VLPs with an adjuvant. Thereafter, the VLPs were adsorbed on the surface of the adjuvant. The developed method was applied to determine the free VLPs in twelve batches of HPV vaccines from three different manufacturers. No obvious free protein was detected in the twelve batches of the HPV vaccines from the three manufacturers, indicating that VLPs from these manufactures react well with their aluminum adjuvant. Folin-phenol (Lowry assay) is commonly used for the determination of proteins in vaccines. It is based on the reduction of phosphomolybdotungstic mixed acid chromogen in the phosphomolybdotungstic reagent, which results in an absorbance maximum at 650 nm. The Lowry method was sensitive to interfering substances. Most interfering substances caused a lower color yield, while some detergents caused a slight increase in color. To reduce the effect of the interfering substances, a procedure for precipitating the proteins was usually required before the sample was tested. Thus, the Lowry assay is complex, time-consuming, and of low selectivity. Compared to the Lowry method, the method we developed is simpler and more automatic. It is a high-throughput method of determining VLPs. It can be used to determine VLPs in HPV bulk and free VLPs in HPV vaccines.

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Separation and identification of impurities from intermediates of istradefylline WANG Yiyun, LÜ Xiaofang, XU Haojie, MENG Zihu, LI Jiarong, XU Zhibin, XUE Min 2021, 39 (4):  430-436.  DOI: 10.3724/SP.J.1123.2020.10013 Abstract ( 127 )   HTML ( 24 )   PDF (1719KB) ( 85 )   Istradefylline is a novel selective adenosine A2A receptor antagonist that is used to treat Parkinson’s disease and improve motor dysfunction in the early stage of this disease. During the synthesis of intermediate A1 (6-amino-1,3-diethyl-2,4-(1H,3H)-pyrimidinedione), at least two by-products were formed under alkaline or high-temperature conditions. In a previous study, one of the by-products in the synthesis of the intermediate was studied, and its structure was identified as (E)-N-ethyl-2-cyano-3-ethylamino-2-butene amide. In this study, we used high performance liquid chromatography (HPLC) to analyze another impurity formed during the synthesis of A1, and the following steps were executed: 0.4 g of intermediate was weighed and added to a 50 mL beaker, followed by the sequential addition of 8 mL water and 8 mL acetonitrile, and then, ultrasonic dissolution was performed. Finally, the solution was filtered through a 0.45-μm organic membrane and the test sample solution was obtained. We used the Agilent zorbax C18 chromatography column, with acetonitrile (A)/water(B) as the mobile phase under gradient elution ((tmin/A∶B)=t0/20∶80, t15/60∶40, t20-t50/90∶10). The detector wavelength is 268 nm. In order to separate the impurity from A1, we used a Ceres B preparative column, with acetonitrile-water (30/70, v/v) as the mobile phase. The flow rate was set at 30 mL/min, and the detection wavelength was 268 nm. The structure of the impurity was confirmed by high-resolution mass spectrometry (HRMS), one-dimensional nuclear magnetic resonance (NMR), and two-dimensional nuclear magnetic resonance (2D NMR), and characterized by single-crystal X-ray diffraction (XRD). In MS experiments, an electrospray ionization (ESI) source was used with positive ion scanning. In the NMR experiments, we used tetramethylsilane (TMS) as the internal standard and deuterated dimethyl sulfoxide (DMSO-d6) as the solvent to obtain the spectra. The results of preparative high performance liquid chromatography (Prep-HPLC) showed that good separation effect could be achieved by isocratic elution, and the impurity was perfectly separated. The1H-NMR spectral data are as follows:1H-NMR (600 MHz, DMSO): δ 1.01 (q, J=6.9 Hz, 3H), 1.02 (q, J=6.9 Hz, 3H), 1.07 (t, J=6.9 Hz, 3H), 3.04 (p, J=6.8 Hz, 2H), 3.74 (q, J=7.0 Hz, 2H), 3.94 (q, J=7.1 Hz, 2H), 5.85 (s, 1H). The 13C-NMR spectral data are summarized as follows: 13C-NMR (150 MHz, DMSO): δ13.9, 14.1, 15.9, 34.6, 34.9, 36.9, 81.9, 152.2, 153.3, 159.3, 162.0. The impurity was characterized by single-crystal XRD, and its spatial structure was further verified and determined as 1-(1,3-diethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-3-ethylurea. Based on the chemical structure of the impurity, we propose the following mechanism for the impurity: when A1 is synthesized under alkaline conditions or at high temperature, excessive diethylurea continues to undergo amidation with A1 to obtain this by-product. Although the formation mechanism of the impurity studied in this paper is different from that of the intermediate A1 impurity (E)-N-ethyl-2-cyano-3-ethylamino-2-butene amide, both the impurities are formed at high temperatures. Both will be accompanied by A1 in the subsequent reaction of istradefylline synthesis. The relationship between drug impurities and drug safety is a complex relationship that is affected by many factors. Generally, most impurities in drugs have potential biological activities, and some even interact with the drugs, thus affecting their efficacy and safety and inducing toxic effects. Therefore, to ensure the quality of istradefylline, it is necessary to control the impurity content during the production. The findings of this paper may provide guidelines for controlling the impurity content in istradefylline.

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Evaluation of thermal adsorption and desorption properties of dioxins on 11 adsorbents WANG Lin, WANG Longxing, NI Yuwen, ZHANG Haijun, CHEN Jiping 2021, 39 (4):  437-443.  DOI: 10.3724/SP.J.1123.2020.10009 Abstract ( 129 )   HTML ( 21 )   PDF (1535KB) ( 73 )   The key to the online thermal capture of dioxins is the appropriate choice of adsorbent for efficient capture at low temperatures and rapid desorption at high temperatures. Efficient adsorbents can allow for capture and separation during the online monitoring of dioxins or during offline dioxin tests. In this study, 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) and pentachlorodibenzofuran (1,2,3,8,9-PCDF) were used as model compounds for dioxin monomers, and an electron capture detector (ECD) was used to detect the dioxin signals. The retention volumes of 1,2,3,4-TCDD and 1,2,3,8,9-PCDF on 11 types of adsorbents were determined using a packed-column gas chromatography system. Then, the corresponding van der Hoff equation was established, and these adsorbents were evaluated for the thermal trapping of dioxin. The linear coefficients of determination (R2) of these adsorbents were greater than 0.96, which indicated a strong correlation between the adsorption capacity and 1/T (T: temperature). The gas-solid partition coefficients (KSA) of the adsorbent at 120, 150 and 180 ℃ were predicted according to the van der Hoff equation, which was obtained in an earlier study. Among the 11 adsorbents, florite had the largest adsorption capacity at 120, 150, and 180 ℃, especially at 120 ℃; the KSA values for 1,2,3,4-TCDD and 1,2,3,8,9-PCDF on florite were as high as 1.82×108 m3/g and 1.46×1013 m3/g, respectively. Carbon-based adsorbents of the Chinese stilbene polymer porous microspheres GDX series, GDX-101, GDX-102, GDX-103, GDX-105, and GDX-203, can facilitate thermal desorption below 270 ℃, which is the maximum tolerance temperature for series 1 and 2, thus providing evidence for the feasibility of using these adsorbents for the thermal adsorption/desorption of dioxins. When the detection temperature is less than 310 ℃, 1,2,3,4-TCDD is thermally desorbed from mordenite, but 1,2,3,8,9-PCDF is not; this indicates the selective adsorption of dioxin monomers on zeolite. However, diatomite and montmorillonite have poor adsorption capacity for dioxins in the gas phase, thus being unsuitable for the thermal trapping of dioxins. Florite, silica gel, alumina, GDX-102, GDX-103, and GDX-203, which have strong adsorption capacities, were selected as possible absorbents for the next evaluation. Comparison of the lnKSA values of dioxin monomers on the same adsorbent at 120 ℃ and 270 ℃ revealed that the retention volume of florite was the largest at both temperatures. When the thermal trapping performance of dioxin at low temperatures is considered, florite is thought to be the best among the 11 adsorbents for capturing dioxins. However, when the desorption performance at high temperatures is considered, GDX-102 is the best adsorbent for the thermal desorption of dioxins, and its lnKSA,270 ℃ is the smallest among those for the aforementioned six adsorbents. The lnKSA,120 ℃ and lnKSA,270 ℃ values of silica gel, GDX-103, and GDX-203 are similar to those of GDX-102, and hence, they can also be used as rapid thermal adsorption/desorption materials. In this study, the thermal adsorption/desorption properties of 1,2,3,4-TCDD and 1,2,3,7,8-PCDF on 11 adsorbents were systematically evaluated to obtain a new solution for the sampling and preparation of dioxins and to provide technical support for the thermal capture of dioxins. It should be noted that these results were obtained under ideal conditions of nitrogen, without considering the influence of the complex conditions of flue gas (such as moisture and CO2) on the thermal capture. To achieve the thermal capture of dioxins in incineration flue gas, it is necessary to carry out the relevant evaluation and test research in a flue gas atmosphere.

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Hydrophilic interaction liquid chromatography for removal of pesticide residues in ginseng extracts SUN Lingli, LIU Jia, GUO Xiujie, WU Lidong, DUAN Zhengchao, WANG Chaoran, WANG Lianzhi 2021, 39 (4):  444-452.  DOI: 10.3724/SP.J.1123.2020.08017 Abstract ( 145 )   HTML ( 38 )   PDF (1463KB) ( 105 )   Ginseng extracts are rich in a variety of ginseng monomer saponins, which have pharmacological functions of retarding aging, enhancing immunity, stimulating blood circulation, and lowering blood pressure. Ginseng is widely used in health products and dietary supplements in the domestic and foreign market. However, the amount of pesticide residues is an important index for measuring the quality of ginseng and ginseng extracts. Therefore, studies focused on methods for the removal of pesticide residues in ginseng extract are of great significance. Hydrophilic interaction liquid chromatography (HILIC) is used to improve the retention and separation selectivity of strongly polar substances, and it is widely employed in drug analysis, metabolomics, proteomics, etc. In this study, a method for the removal of pesticide residues was developed based on the difference in the retention behavior of pesticide residues and ginsenosides on the HILIC column. Using commercially available ginsenoside extracts, the retention behaviors of pesticide residues and ginsenosides on reverse chromatography and hydrophilic chromatographic columns were evaluated by high performance liquid chromatography. The results proved that on the reversed-phase liquid chromatography (RPLC) stationary phase, in addition to the strong retentions of quintozene and pentachloroaniline, which could be clearly separated from the saponins, the retentions of the other five pesticide residues including carbendazim, azoxystrobin, procymidone, iprodione and propiconazole were similar to total ginsenosides. The seven ginsenosides showed strong retention due to the formation of hydrogen bonds between the hydroxyl groups on the sugar chain and the carboxyl groups on the HILIC stationary phase. However, the pesticide residues were not well retained because of their poor hydrophilicity and small molecular weights. For this reason, the pesticide residues and ginsenosides could be completely separated on the HILIC column. Thus, enrichment of the seven ginsenosides and removal of the 14 pesticide residues was realized in one step on the HILIC column. In addition, the effects of loading amount, loading volume, and washing volume on the removal of pesticide residues in ginsenosides were investigated using the Click XIon SPE column. Then, taking the ginsenoside recoveries and pesticide residue removal rates into account, we confirmed the following: the ratio of the maximum sample loading mass to the filler mass was 1∶10; the optimal elution volume was twice the column volume; and the optimal loading volume was twice the column volume. The ginseng extracts were solvated with a 95% ethanol solution and loaded onto an HILIC column. The sample was subjected to pesticide residue removal, and ginsenoside purification and enrichment under the optimum removal conditions. Gradient elution was carried out using ethanol and water as the mobile phases. The total ginsenoside content in the final extracts was increased to 69.61%. The recovery of the total ginsenosides was 94.4%. The pesticide residues in the samples were quantitatively detected by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) in the multiple reaction monitoring (MRM) mode. The 14 pesticide residues in the original ginsenoside extracts were effectively removed. The amounts of five residues were reduced to below 0.05 mg/kg, while the other nine residues were completely eliminated. This study demonstrates the application of HILIC to pesticide residue removal in traditional Chinese medicine extracts and reveals a new technique for the purification of natural products. The proposed method shows a high removal rate of pesticide residues and a high recovery of total ginsenosides. It is safe, efficient, and environment-friendly, and can aid the development of high-quality ginsenoside extracts.