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Chinese Journal of Chromatography
2020, Vol. 38, No. 12
Online: 08 December 2020

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Volume 38, Number 12 Content 2020, 38 (12):  0-0.  Abstract ( 30 )   PDF (4672KB) ( 55 )

Communications

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Preparation of core-shell silica-carbon composite microspheres stationary phase and application in saccharide separation ZHAO Xingyun, ZHANG Hongyan, ZHOU Xiaoyu, WANG Li, WAN Lihong, WU Ren’an 2020, 38 (12):  1357-1362.  DOI: 10.3724/SP.J.1123.2020.08016 Abstract ( 155 )   HTML ( 12 )   PDF (3140KB) ( 149 )   In this study, core-shell mesoporous silica-carbon composite microspheres (Sil@MC) were prepared by one-step coating of the phenol formaldehyde polymer (PF) on SiO2 surface and by carbonizing the PF polymer under nitrogen atmosphere. The morphology observation of the Sil@MC stationary phase showed that it had good monodispersity. Surface area (302 m2/g), mean pore diameter (9.5 nm), and pore volume (0.63 cm3/g) of Sil@MC materials were also measured by pore structure analysis. The results showed that the Sil@MC was successfully immobilized on the silica particles via copolymerization and carbonization. As a stationary phase of HPLC, the Sil@MC column was filled by a slurry method. The Sil@MC materials formed after calcination of SiO2 coated with phenolic resin could be used for the separation of four polar sugar compounds (D-(+)-glucosamine hydrochloride, glucose, D-(+)-trehalose dihydrat and raffinose) with the mobile phase of acetonitrile-water (containing 0.1% (v/v) formic acid). However, the material formed by calcinating SiO2 without coating phenolic resin could not separate these polar sugar compounds by HPLC-MS. Finally, the representative oligosaccharide isomers of raffinose, melezitose and stachyose, nystose, and human milk oligosaccharide isomers, such as 3'-sialyllactose, 6'-sialyllactose and lacto-N-newtetraose, lacto-N-tetraose, were successfully separated by the Sil@MC column with good peak shapes. The results demonstrates that silica-carbon composites derived from phenolic resin have potential application in polar compounds chromatographic separation.

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Preparation of brazilein from Caesalpinia sappan by high performance countercurrent chromatography HE Wenqian, FAN Qingfei, ZHOU Lan, HUANG Fengmei, JIANG Xian, NA Zhi, HU Huabin, SONG Qishi 2020, 38 (12):  1363-1368.  DOI: 10.3724/SP.J.1123.2020.03016 Abstract ( 88 )   HTML ( 9 )   PDF (964KB) ( 63 )   Brazilein is among the main chemical constituents of Caesalpinia sappan. It has diverse pharmacological activities. Modern pharmacological studies have shown that the compound has antitumor, anti-inflammatory, antibacterial, antioxidant, immunomodulatory, and other pharmacological activities. Brazilein is often used as a stain in various industries. The separation of brazilein by traditional column chromatography will not only result in contamination of the chromatographic column materials, but also lead to loss of the active ingredient. Countercurrent chromatography is an advanced liquid-liquid chromatographic separation technique. It has been widely used for natural product separation and isolation as it offers several advantages, such as low solvent consumption, a highly selective solvent system, and high recoveries. Typical countercurrent chromatography techniques include centrifugal partition chromatography (CPC), high-speed countercurrent chromatography (HSCCC), and high performance countercurrent chromatography (HPCCC). It is well known that choosing a suitable solvent system is vital in countercurrent separation. Therefore, two methods were introduced for choosing a suitable solvent system. One is the generally useful estimation of solvent systems (GUESS) method, which employs thin-layer chromatography (TLC) to identify a suitable solvent system with minimal labor for the rapid purification of target compounds, and another is the Shake-Flash method. The solvent system could be determined by observing the distribution of the sample in the upper and lower phases. Two kinds of solvent systems were screened using the TLC-GUESS and Shake-Flash methods, and tested through the analysis mode of the HPCCC instrument. The results showed that chloroform-methanol-water (4∶3∶2, v/v/v) was the optimal solvent system for HPCCC separation. A total of 15.2 mg of brazilein and 5.7 mg of caesappanin C were obtained from an ethyl acetate extract with high purities (95.6% and 89.0%, analyzed by HPLC) in one step using the preparation mode of HPCCC, the reversed-phase liquid chromatography mode with the apparatus rotated at 1600 r/min, a flow rate of 10 mL/min, separation temperature of 25 ℃, and detection wavelength of 285 nm. Their structures were determined by spectroscopic and spectrometric analyses. Brazilein stained the solid packing material in the column and was difficult to elute. The results showed that the use of HPCCC for the separation of brazilein can not only prevent the loss of target active ingredients in Caesalpinia sappan, but also shorten the separation and purification times and improve the operating efficiency. Therefore, HPCCC can be used for the separation and preparation of other pigment compounds in Caesalpinia sappan and other dye plants.

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Advances in the development of detection techniques for organophosphate ester flame retardants in food YANG Jishuang, ZAHNG Qinghe, SU Liqiang 2020, 38 (12):  1369-1380.  DOI: 10.3724/SP.J.1123.2020.03026 Abstract ( 222 )   HTML ( 8 )   PDF (1727KB) ( 124 )   Organophosphate ester (OPE)-based flame retardants and plasticizers are widely utilized in various industrial products, and are being increasingly used as substitutes for gradually phased brominated flame retardants (BFRs). According to the different types of substituents used, OPEs are mainly divided into alkyls, halogenated compounds, and aromatics, which have widely varying physicochemical properties. OPEs can induce neurotoxicity, carcinogenicity, and damage in endocrine and reproductive systems in humans. Examples of halogenated OPE are tris(2-chloroisopropyl) phosphate (TCIPP) and tris(1,3-dichloropropyl) phosphate (TDCIPP), which are suspected to be carcinogenic. OPEs have emerged as pollutants in environmental and food matrices as a result of volatilization and abrasion processes. Due to its low content in the food matrix and serious background interference, there is a lack of reliable and sensitive analytical methods. Recently, there has been a focus on the detection of OPE flame retardants in food. In this paper, we have reviewed the current status and development trends of OPE detection methods in various foodstuffs. First, the physicochemical properties of more than 30 common OPEs were summarized. Even when using the same extraction solvent, there are obvious differences in extraction efficiency according to different compound properties. To simultaneously analyze multi-component OPE flame retardants in food, it is very important to choose the appropriate extraction solvent to meet the required extraction efficiency of compounds with a wide range of polarities. In addition, although OPE flame retardants are not easily hydrolyzed under neutral conditions, they will degrade to a certain extent under strong acidic and alkaline conditions. It is worth mentioning that avoiding the removal of lipids and other interferences in food matrices under strong acidic and alkaline conditions. Different pretreatment methods, such as accelerated solvent extraction, matrix solid-phase dispersion extraction, microwave-assisted extraction, ultrasonic-assisted extraction, QuEChERS, solid-phase extraction, gel permeation chromatography, and dispersive solid-phase extraction are also compared. Combining the advantages of ultrasonic assisted extraction (UAE) and QuEChERS pretreatment technology can reduce the waste of extraction solvent and internal standard solution. For lipid-rich matrices like biological samples, it is necessary to remove lipid interference by SPE columns or GPC purification. Furthermore, the characteristics of separation and detection techniques, such as GC, GC-MS/MS, and LC-MS/MS, are discussed. Comparing detection limits and recovery data with those reported in the literature, GC-MS/MS can provide improved selectivity, precision, and limits of detection in complex food matrices, but LC-MS often suffers from ion suppression, matrix interferences, and incomplete separation of some OPEs. Since electron impact (EI) has higher ionization efficiency, it produces many fragment ions, thus creating a more complete spectral library, which is conducive to structural identification. When using GC-MS/MS to determine OPE flame retardants, the EI mode was usually used. However, positive chemical ionization (PCI) and electron capture negative ionization (ECNI) modes were also used sometimes. In the section on quality control, the main sources of standards and internal standards, possible sources of blank contamination, and the research status of measures to reduce matrix effects have been reviewed. To avoid blank contamination, all the laboratory equipment should be carefully cleaned, heated at high temperatures, and rinsed with polar or non-polar organic solvents in order to remove all interfering organic residues. Isotopically labeled internal standard and isotopic dilution mass spectrum quantification methods are used to reduce matrix effects. Owing to the limited availability of commercial standards and the relatively high cost, alternative approaches, such as matrix-matched calibration or standard addition methods, are required. The screening and identification of unknown metabolites of OPEs and related analytical methods based on high resolution mass spectrometry could also be studied for precursor OPEs in foodstuffs in the future.

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Determination of four bisphenol environmental hormone residues in infant serum by liquid chromatography-tandem mass spectrometry LIU Fang, MENG Taoyu, CHEN Lian, WU Yajun, XIONG Shun, DING Li 2020, 38 (12):  1381-1387.  DOI: 10.3724/SP.J.1123.2020.03024 Abstract ( 500 )   HTML ( 10 )   PDF (1072KB) ( 96 )   Bisphenols are important industrial raw materials that are widely used to produce plastic bottles (feeding bottles), infant cups, and food and beverage (milk powder) cans. Because of the estrogen-like effect of bisphenols, even low-dose intake of these compounds by trace migration affects normal hormone levels in the human body. Therefore, it is imperative to develop a rapid, accurate, and highly sensitive method for the determination of bisphenols in serum. In this study, methyl tert-butyl ether (MTBE) was used as the extraction solvent, and the liquid-liquid extraction pretreatment method was used for sample processing. A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established for the simultaneous determination of bisphenol A (BPA), bisphenol B (BPB), bisphenol F (BPF), and bisphenol S (BPS) at trace levels in infant serum. The important parameters affecting the extraction efficiency, such as the extraction solvent, extraction time, and extraction solvent volume for the four bisphenol environmental hormones were optimized. Serum samples were extracted by MTBE at 40 ℃ for 15 min. The target compounds were separated on a Waters ACQUITY UPLC BEH C18 column (50 mm×2.1 mm, 1.7 μm) using ultrapure water and methanol solution containing 0.5 mmol/L ammonium acetate as the mobile phases, with gradient elution at a flow rate of 0.2 mL/min. Finally, the analytes were analyzed by HPLC-MS/MS in the negative ion mode. BPA, BPB, and BPS showed good linearity in the range of 0.25-100 μg/L, while BPF showed good linearity in the range of 1-100 μg/L. The correlation coefficients were 0.9929-0.9959, and the limits of detection for BPA, BPB, BPS, and BPF were 0.05, 0.05, 0.05, 0.5 μg/L, respectively. At the three spiked levels (5, 20, 100 μg/L), the average recoveries of BPA, BPB, BPS, and BPF ranged from 84.56% to 104.43%, and the relative standard deviations were less than 10%. The proposed method was successfully applied to the determination of bisphenol contents in 150 infant serum samples. BPA was detected in most serum samples, and the detection rates in the serum of boys and girls were 90.67% and 89.33%, respectively. The detection rates of BPF in the serum of boys and girls were 6.67% and 1.33%, respectively; the corresponding values for BPS were 5.33% and 16.00%. BPB was not detected. The proposed method has the advantages of simple operation, good recovery, and high precision, and it is suitable for the simultaneous determination of the four bisphenol environmental hormones in infant serum.

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Determination of 15 3-chloro-1,2-propanediol fatty acid esters in vegetable oils and fritters by ultra performance convergence chromatography-tandem mass spectrometry YANG Guangyong, GUO Cangting, XUE Guang, GUO Jinxi 2020, 38 (12):  1388-1395.  DOI: 10.3724/SP.J.1123.2020.03020 Abstract ( 118 )   HTML ( 8 )   PDF (899KB) ( 109 )   The presence of 3-chloro-1,2-propanediol fatty acid esters (3-MCPDE) in food and processed materials has recently become a topic of concern because of the toxicity of their metabolites. 3-MCPDE structurally similar to glyceride, which makes it difficult to separate or extract them from oils and fritters. A method based on ultra performance convergence chromatography-tandem mass spectrometry (UPC2-MS/MS) was established for the determination of 15 3-MCPDE in vegetable oils and fritters. Amino-packed columns were used to purify the samples. The analytical conditions were optimized, and the matrix effect was investigated. The sample was treated by column chromatography to remove glyceride and free fatty acids, which induce strong matrix effects. The amino-packed column was eluted with hexane and hexane-ethyl acetate (6∶4, v/v). Every 1 mL of the eluent was analyzed using a UPC2 and ACQUITY QDa detector. Elution curves were drawn based on the testing data and used to determine the collection volume. The collection volumes for 3-chloro-1,2-propanediol diesters and monoesters according to the elution curves were 7-14 mL and 3-9 mL. The collected eluent was mixed and dried under nitrogen flow at a temperature of 60 ℃. A hexane-isopropanol (98∶2, v/v, 1 mL) mixture was used to dissolve the residue. The resulting solution was separated on a Viridis HSS C18 SB column (150 mm×2.1 mm, 1.8 μm) under gradient elution. Supercritical carbon dioxide and methanol (containing 40% acetonitrile and 0.1% formic acid) were used as the mobile phases, and the flow rate was 1 mL/min. The separated compounds were analyzed by tandem MS with an electrospray ionization (ESI) source in positive and multiple reaction monitoring modes. Water (containing 97% isopropanol and 0.2% ammonia water) was used as the auxiliary pump mobile phase, and the flow rate was 0.2 mL/min. The method showed good linear relationships in the range of 0.5-100 μg/L (r2≥0.9973). The limits of detection (LODs) and limits of quantification (LOQs) were 0.01-0.68 μg/L (S/N=3) and 0.04-1.74 μg/L (S/N=10), respectively. The average recoveries (n=9) at the three spiked levels were in the range of 81.6%-98.5%. The relative standard deviations were in the range of 1.8%-6.4%. The matrix effects in the case of the oils and fritters were weak. The developed method was used to detect 44 oil samples and eight fritter samples. Meanwhile, some suspect 3-MCPDE compounds outside the scope of the investigation were analyzed based on their primary and secondary mass spectra. The detection rates of 3-MCPDE in oils and fritters were 84.1% and 87.5%, and their amounts were in the range of 0.024-4.481 mg/kg and 0.018-1.144 mg/kg, respectively. The detection rates of 3-MCPDE in rapeseed oil were higher compared to those for other kinds of oil. The method is specific, fast, simple, accurate, reliable, and environmentally friendly, in addition to being more sensitive than other methods and showing better matrix compatibility for oils. This method has been successfully used to determine the types and amounts of 3-MCPDE in vegetable oils and fritters. The research findings provided accurate data to assess the exposure risk of 3-MCPDE. The results of our experiment also provided valuable information for elucidating the formation mechanism of 3-MCPDE. The proposed method can be used to analyze waste edible oil based on large amounts of analysis data. However, this method has some limitations. The resolution ratio of the mass spectrometer used in this method is too low for the qualitative analysis of unknown compounds. The qualitative results for the suspect 3-MCPDE compounds are not particularly accurate, and a large variety of monomer standards are required for the quantitative determination of 3-MCPDE. The 3-MCPDE standards are expensive, and there is limited choice of these standards; moreover, they are difficult to synthesize. The poor ionization yield of 3-chloro-1,2-propanediol monoesters under the ESI conditions resulted in high LODs. Hence, it is necessary to develop a method for increasing the ionization of monoesters, for example, via derivatization.

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Determination of streptomycin and dihydrostreptomycin in grapes by liquid chromatography-tandem mass spectrometry LIU Zhenzhen, QI Peipei, HE Fuxiang, WANG Zhiwei, DI Shanshan, XU Hao, ZHAO Huiyu, WANG Qiang, WANG Xinquan 2020, 38 (12):  1396-1401.  DOI: 10.3724/SP.J.1123.2020.03034 Abstract ( 177 )   HTML ( 8 )   PDF (1362KB) ( 102 )   Supporting Information Streptomycin (STR) and dihydrostreptomycin (DSTR) are two of the most common aminoglycoside antibiotics used in veterinary medicine. STR is produced by some streptomyces griseus strains, and DSTR is a derivative of STR. In recent years, STR has been widely used in grapes to induce denuclearization. However, high levels of STR may have adverse effects like serious ototoxicity and nephrotoxicity. Therefore, to ensure the quality of grapes and the health of consumers, the regulation of STR and DSTR levels in grapes is required. An analytical method was developed for the identification and quantification of STR and DSTR in grapes by liquid chromatography-tandem mass spectrometry (LC-MS/MS). STR and DSTR are highly polar compounds due to the presence of various amino and hydroxyl groups in their structure. The determination of STR and DSTR poses a considerable analytical challenge, both during sample preparation and instrument analysis. In this study, the main factors governing the response, recovery, and sensitivity of these compounds, such as the type of chromatographic column, the type and proportion of the mobile phase and extraction solvent, the dosage of sodium 1-hexane sulfonate solution, and elution solvent and its volume, were investigated during sample pretreatment and instrument analysis. The STR and DSTR residues in the grape sample were extracted by ultrasonication with a phosphoric acid solution (pH 2), and cleanup and enrichment was performed using an Oasis HLB solid phase column. The analysis was performed using a UPLC Waters HSS T3 column (100 mm×2.1 mm, 1.8 μm) at the column temperature of 35 ℃. The injection volume was 2 μL. The mobile phase consisted of 0.1% formic acid aqueous solution and methanol with a volume ratio of 60∶40. ESI-MS/MS was operated in multiple reaction monitoring (MRM) mode. External standard calibration curves were used for quantification. Based on the optimized method, both analytes displayed good linearity between 2 and 400 μg/L. The correlation coefficients were 0.9991-0.9997. Recoveries in spiked blank grape samples (5, 10, 20, and 40 μg/kg) ranged from 76.8% to 91.9%, with the relative standard deviations (RSDs) less than 10.2%, in compliance with the current legislation. The limits of detection and the limits of quantification of both analytes were 1 μg/L and 5 μg/kg, respectively. To assess the feasibility and potential of the proposed approach for routine analyses of STR and DSTR in other kinds of grape samples, the developed method was applied to the analysis of these compounds in red grapes, xinyu grapes, and xiahei grapes. The recoveries of STR and DSTR in the three kinds of blank grape samples were 77.2%-83.9% and 70.8%-78.9%, respectively, and the RSDs ranged from 3.0% to 15.6%. The results showed that the optimized methods can yield satisfactory recoveries for the analytes in grapes. In this method, the combination of Waters HSS T3 column to overcome the difficulties of the retention and separation of these highly polar compounds in the reverse phase, avoids the use of an ion-pair additive in the mobile phase to increase their retention, which is known to cause severe contamination of the column and serious ion suppression with electrospray ionization detection. In addition, the ideal enrichment and purification effect can be achieved by adding a sodium 1-hexane sulfonate solution to the superstratum extract with the use of only Oasis HLB for sample treatment. The method described herein has the advantages of easy operation, accuracy, and selectivity, making it feasible for the identification and quantification of STR and DSTR residues in grapes.

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Establishment of screening and confirmation method for the analysis of pesticide residues in tea by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry QIAO Yongsheng, WANG Junhu, QIU Yajing, QIAN Zhongyi, HU Hui, CHEN Wei, WANG Ping 2020, 38 (12):  1402-1412.  DOI: 10.3724/SP.J.1123.2020.05005 Abstract ( 114 )   HTML ( 6 )   PDF (941KB) ( 111 )   Based on ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS), UNIFI 1.7.0 software was used to establish a screening and confirmation method for the analysis of 91 pesticide residues, which was qualitatively validated and applied to tea screening in the circulation market. By analyzing the collected pesticide certified reference materials (CRM), a mass spectral database of 91 pesticides was constructed. The database contains multiple types of information, including formulas, theoretical exact masses, retention times, characteristic fragment ions, and adduct types. The samples were extracted with acetonitrile, purified on a solid-phase extraction column, and separated on an Acquity BEH C18 column. All the data (ESI+) were acquired in MSE mode and analyzed using the UNIFI information system. Analyte detection was based on the retention time deviation ±0.1 min, accurate mass deviation ±5×10-6, and major adduct forms including [M+H]+, [M+Na]+, [M+K]+, and [M+NH4]+. Screening was performed by the software in an automated fashion. Compound identification was accomplished with retention time matching and accurate mass measurements of the primary diagnostic ions for each analyte. To ensure accuracy of the identification results, information from the MS/MS profiles must be compared with online mass spectral libraries such as PubChem and MassBank. The SANTE/11813/2017 protocol for the validation of the screening method was followed. A mixed standard solution was spiked to 21 tea samples at four levels (0.01 mg/kg, 0.05 mg/kg, 0.10 mg/kg, and 0.20 mg/kg) to determine the screening detection limit (SDL) of each pesticide, and a total of 1911 pesticide/sample combinations were evaluated. The results revealed 66 pesticides with an SDL of 0.01 mg/kg, eight pesticides with an SDL of 0.05 mg/kg, one pesticide with an SDL of 0.10 mg/kg, three pesticides with an SDL of 0.20 mg/kg, and 13 pesticides with an SDL greater than 0.20 mg/kg. One pesticide showed matrix-inhibitory effects in the screening tests. Finally, the established method was used to analyze the pesticide residues in 22 tea samples available on the market. Six pesticide compounds were found in the tea samples, all of which were confirmed to be positive after artificial identification. This method provides a reference for the high-throughput screening and detection of pesticide residues in tea as well as a research approach for the analysis of various chemical contaminants in other matrices.

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Determination of 10 fluoroquinolones residues in aquatic products by accelerated solvent extraction, magnetic solid-phase extraction, and high-performance liquid chromatography-tandem mass spectrometry WEI Dan, GUO Ming, ZHANG Ju 2020, 38 (12):  1413-1422.  DOI: 10.3724/SP.J.1123.2020.05002 Abstract ( 155 )   HTML ( 14 )   PDF (3327KB) ( 140 )   In the aquaculture industry, fluoroquinolones are widely used as effective therapeutic agents to prevent animal diseases. The wide bactericidal activity of fluoroquinolones strongly depends on their concentration. Abuse of fluoroquinolones is considered the main reason for the possible occurrence of residues in aquatic products. The increasing presence of residues in aquatic products may pose potential risks to human health. Therefore, it is important to develop an efficient, sensitive, and reliable method for the simultaneous determination of fluoroquinolones in aquatic products. In the analysis of fluoroquinolones, many HPLC methods with different detection techniques have been applied. Among the most common used techniques, HPLC-MS is possible for the determination of very low level analytes in matrix. For the determination of low concentrations of fluoroquinolone residues in aquatic products, preliminary extraction and purification steps are frequently needed to achieve low detection limits. Accelerated solvent extraction (ASE) is well suited for the determination of organic pollutants in solid samples. ASE has the advantages of a high degree of automation, sufficient extraction, high speed, and less solvent consumption, but it has the disadvantage of poor purification effects. Magnetic solid-phase extraction (MSPE) has attracted considerable attention on account of its benefits such as easy separation, less solvent consumption, and quick adsorption of antibiotic residues in liquid samples. The combination of ASE with MSPE makes it possible to sufficiently extract and further purify the target compounds from complex solid samples. Compared with the currently used purification methods of SPE and QuECHERS, MSPE has advantages such as no need of centrifugation and filtration, less solvent consumption, and low cost by appropriate choice of magnetic materials. In this study, a method based on ASE-MSPE-HPLC-MS/MS was developed for the simultaneous determination of sarafloxacin, ofloxacin, enrofloxacin, danofloxacin, lomefloxacin, pefloxacin, ciprofloxacin, enoxacin, norfloxacin, and difloxacin in yellow croaker, grass carp, black fish, prawn, and macrobrachium. As a magnetic purification sorbent, a graphene oxide nanoscale-coated zerovalent iron adsorbent composite (GO@nZVI), was facilely prepared at room temperature. GO and nZVI solutions were rapidly vortex-mixed at 25 ℃, and then, the magnetite precipitate was magnetically isolated to obtain GO@nZVI. Simpler than the usually used preparation methods, GO@nZVI can be fabricated without complicated multi-step synthesis, fussy operation and harsh conditions. nZVI nanomaterials have strong multiple interactions (hydrogen bonding, electrostatic interaction or their combination) with GO composite only with appropriate adjustment of pH values. The synthesized magnetic purification sorbents were characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD), indicating the successful formation of GO@nZVI. The magnetic material was used to purify and extract ten fluoroquinolone residues in aquatic products via MSPE, followed by ASE. In ASE step, the analytes were extracted from the aquatic products using methanol for 5 min at 70 ℃, under an extractive pressure of 10.34 MPa for three cycles. The extract was purified by MSPE using GO@nZVI. The target compounds were separated on an Agilent ZORBAX Eclipse Plus C18 column (100 mm×3.0 mm, 1.8 μm) with gradient elution, and analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+). Under the optimized conditions, good linearities were obtained for the ten fluoroquinolones in the range of 1-100 μg/kg, with correlation coefficients above 0.99. LODs (S/N=3) and LOQs (S/N=10) were 0.02-0.29 μg/kg and 0.07-0.98 μg/kg, respectively. At three spiked levels, the recoveries of the fluoroquinolones were between 81.6% and 105.8%, with RSDs between 4.2% and 13.6%. Overall, the major advantages of this combined ASE-MSPE-HPLC-MS/MS method are facile preparation of the magnetic purification material, automated and simple operation, high sensitivity, short extraction time, and less solvent consumption. This sensitive, repetitive method could be successfully employed for the determination of ten fluoroquinolone residues in aquatic products, with good recoveries.

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Screening and quantitative analysis of nine illicit antiallergics in emulsion cosmetics by ultra-high performance liquid chromatography-quadrupole-time-of-flight high-resolution mass spectrometry WANG Mengying, CHEN Yechao, TU Fengqin, HOU Jing, YANG Ming, LU Yuepeng, WANG Yuhong, YANG Zong, CHEN Dan 2020, 38 (12):  1423-1430.  DOI: 10.3724/SP.J.1123.2020.06017 Abstract ( 104 )   HTML ( 19 )   PDF (1661KB) ( 103 )   A rapid and accurate analysis method based on ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight high-resolution mass spectrometry (UPLC-Q-TOF-HRMS) was developed to screen and determine nine antiallergy drugs in emulsion cosmetics. First, a standard library of the target compounds was established. The library contained the TOF-MS information and secondary MS information such as retention time, ion addition mode, mass error, isotope distribution, mass-to-charge ratio of the parent ion, and fragment ion distribution. According to the European Union regulation (SANTE/11945/2015), the standard for the qualitative determination by HRMS was determined; that is, each compound was confirmed by two ions with a mass error below 5%, and the abundance ratio of the two ions was less than 30%. Second, the instrument conditions and sample pretreatment conditions for the determination of different compounds were optimized, and the influence of different levels of quantitative ions on the matrix effect was compared. The following observations were made: (1) the addition of 0.1% formic acid to the water phase improved the response of the chromatographic peaks; (2) among the various solvent amounts tested (4, 5, 6, 8 mL acetonitrile and 4, 5, 6, 8 mL methanol), 4 mL acetonitrile showed the best extraction efficiency; (3) PRiME HLB had a better purification effect than the other two purification columns (C18 and HLB solid-phase extraction cartridges), thus reducing the interference of impurities and ensuring good recovery of the target compounds; (4) the use of two pairs of secondary product ion quantification could significantly reduce the matrix effect of anti-allergic compounds and improve the quantification accuracy. Finally, based on the above findings, the experimental procedure was established. The lotion samples were first ultrasonically extracted with acetonitrile and purified on the PRiME HLB column. Chromatographic separation was performed on a Waters XBridge C18 column with gradient elution using 0.1% (v/v) formic acid in water and acetonitrile. Finally, the sequential window acquisition of all theoretical mass spectra (SWATH), which shows obvious advantages in continuous and high-throughput acquisition, was selected for MS data acquisition. The retention time, mass accuracy, isotope distribution, and fragment ion matching ratio were used for fast qualitative screening, while the peak areas of characteristic product ions were used for precise quantification. All the calibration curves showed good linearity (r2>0.99) within the tested ranges (5-100 μg/L) under the optimum conditions. The limits of quantification (LOQs) were in the range of 0.05-0.10 mg/kg. The recoveries were in the range of 65.3%-107% at three spiked levels (0.10, 0.20, and 0.60 mg/kg), with relative standard deviations (RSDs, n=6) below 20%. Compared with the existing ion exchange column methods, the proposed “one-step” purification method based on PRiME HLB is simpler and more rapid, where the extraction solution is filtered directly after allowing it to pass through the column, without any subsequent washing and elution procedures. In addition, the LOQs of this method are lower than those of other LC-MS/MS methods, indicating that the proposed method has higher sensitivity. The application of SWATH data acquisition makes it possible to achieve quantification with two pairs of product ions, thus reducing the matrix effect and ensuring accuracy of the quantitative results. Therefore, the proposed method is less time-consuming and operationally convenient, and it can be used for the rapid screening and accurate quantification of antiallergics in lotion samples.

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Separation and screening of antioxidant peptides from Scomberomorus niphonius based on nano flow liquid chromatography XUE Yaru, GUO Rui, ZHANG Bo 2020, 38 (12):  1431-1439.  DOI: 10.3724/SP.J.1123.2020.04019 Abstract ( 94 )   HTML ( 7 )   PDF (2846KB) ( 71 )   As a rich source of high activity antioxidant peptides, Scomberomorus niphonius is a key marine natural product with a high processing value. Due to the high complexity of fish tissues, high recovery extraction and high efficiency screening of the active antioxidant peptide species have become the major challenges for the research and development of preparation and separation techniques. Due to the high specificity of hydrolytic enzymes, when different types of enzymes are used for the hydrolysis of fish tissues, the resultant active peptides may have significant differences in chemical structures, biological functions, and physical activities. In this study, in order to obtain antioxidant peptides with high activity and functionality, defatted visceral powder of Scomberomorus niphonius was used as a raw material for sample preparation. Five hydrolytic enzymes (flavor protease, trypsin, acid protease, neutral protease, and alkaline protease) were selected and investigated for their hydrolyzing efficiencies in visceral solutions of Scomberomorus niphonius, according to their optimum hydrolyzing conditions. The diphenyl bitter hydrazine radical (DPPH·) scavenging rate, hydroxyl radical (·OH) scavenging rate, and degree of hydrolysis (DH) were adopted as indicators for hydrolytic enzyme selection and optimization. The data suggested that, among all the hydrolytic enzymes investigated, trypsin presented the best scavenging capabilities for both DPPH· and ·OH species, with scavenging rates of 88.93%±0.82% for DPPH· and 53.09%±0.73% for ·OH, respectively. Based on the single-factor test results, the DPPH· scavenging rate was further utilized as an indicator and was found to depend on the hydrolytic enzyme quantity, hydrolyzation temperature, and hydrolyzation time. According to the Box-Behnken center composed experimental design, a triple factor and triple level response surface method was adopted for further optimization of antioxidant peptide preparation from Scomberomorus niphonius viscera. The preparation method was systematically optimized, and as a result, a degree of hydrolysis of 23.66%, DPPH· scavenging rate of 93.78%, and ·OH scavenging rate of 62.59% were achieved. High performance nano flow liquid chromatography is a new generation micro scale liquid phase separation technique that has the advantages of low sample requirement, low solvent consumption, and high efficiency. In this study, we applied this method to marine natural product purification. In order to screen the most suitable chromatographic stationary phase for the separation and analysis of active antioxidant peptides from Scomberomorus niphonius viscera, a nano flow reversed-phase C18 column (15 cm×100 μm, 5 μm, 30 nm) and a strong cation exchange column (15 cm×100 μm, 5 μm, 100 nm) were investigated. A nano flow high performance liquid chromatography platform with a 1∶1000 splitting ratio was adopted for this study. Using the enzyme-hydrolyzed solution from Scomberomorus niphonius viscera as the sample, following filtration, the antioxidant peptides were separated, collected, freeze-dried, and finally tested for their antioxidant capability. The results showed that the strong cation exchange phase was more suitable for antioxidant peptide isolation and purification from Scomberomorus niphonius viscera, and an antioxidant peptide component with high activity was successfully screened. During the activity test, the 50% inhibition concentration (IC50) value of the DPPH· scavenging capability of this peptide was 0.672±0.051 mg/mL, which was 13.6 times higher than the activity value before the peptide species was purified. The current study for the first time reports the application of high performance nano flow liquid chromatography in the purification and analysis of antioxidant peptides from a marine natural product source and also demonstrates the effectiveness and future prospect of the use of nano flow ion exchange chromatography for high performance separation and high efficiency screening of antioxidant peptides.

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Establishment of chromatographic fingerprint of Squama Manis and its applications in animal source identification and quality grade discrimination QIAO Yali, LIU Zhe, SHEN Aijin, GUO Zhimou, LIU Yanfang, CHEN Xiangyin, XU Qing, LIANG Xinmiao 2020, 38 (12):  1440-1448.  DOI: 10.3724/SP.J.1123.2020.04007 Abstract ( 86 )   HTML ( 4 )   PDF (1023KB) ( 52 )   Squama Manis, or “Chuanshanjia” in Chinese, is a traditional Chinese medicine (TCM) for promoting blood circulation and reducing swelling and discharge; the only animal source used in TCM is the scales of Manis pentadactyla. However, in today’s pharmaceutical market, there are many scales from other species of the same genus that are difficult to distinguish from Squama Manis. High-quality and low-quality scales are also severely confused. To solve the above problems, various analytical methods have been developed, such as thin-layer chromatography, mass spectrometry and DNA detection. Owing to their low resolving ability, high equipment cost, and inconvenient operation, none of these methods are appropriate for routine identification of Squama Manis. A chromatographic fingerprint can comprehensively reflect the synergic action of multiple chemical compositions in TCM and has been widely used for the quality control of TCM. In the present study, we established a fingerprint of Squama Manis and explored its feasibility in identifying the origin and quality grade of scales. First, Squama Manis powder was hydrolyzed by hydrochloric acid (1 mol/L). Next, the extract was analyzed on a Symmetry 300 C18 column by linear gradient elution, using 0.1% trifluoroacetic acid (v/v) in water and 0.1% trifluoroacetic acid (v/v) in acetonitrile as the mobile phase and 280 nm as the detection wavelength. The established method was systematically validated, demonstrating good precision, repeatability and sample stability (relative standard deviation (RSD)<5%). Subsequently, samples of different sources and quality grades were distinguished by similarity evaluation and discrimination analysis based on the fingerprint data. In the similarity evaluation, the reference fingerprint was defined as the average fingerprint of twelve first-class samples, and seventeen chromatographic peaks were identified as common peaks. Similarities between the reference fingerprint and fingerprints with different base sources and quality grades were calculated using the absolute area of common peaks as original data. The similarities between Squama Manis and scales from other animals were all less than 0.776, while the similarities between Squama Manis of different grades overlapped significantly, varying from 0.988 to 0.996 for first-class samples and 0.950 to 0.995 for general samples. The results reflected the feasibility of similarity evaluation for discriminating base source and its limitation in the distinguishing between quality grades. Nonetheless, first-class scales showed higher average similarity and lower RSD than general scales, which indicates some level of revelation between fingerprint similarity and quality grade. Thus, a better algorithm or discriminant model is required to distinguish between quality grades. Therefore, a supervised chemometric technique, kernel-based support vector machine (SVM), was applied to construct predictive models. The SVM is a common discriminant model that classifies samples by constructing a separate hyperplane in n-dimensional space, maximizing the margin between classes. Combination with a kernel function can effectively avoid “dimension disaster” when dealing with nonlinear data. In the model, the quality grade was defined as a sample label, and the absolute peak areas constituted the data matrix. Verified by 10-fold cross-validation, the unbiased prediction accuracy was up to 95.83%. The predicted results were highly consistent with the actual classifications. The results indicate the high feasibility of the established model for determining quality grade, as it performed significantly better than the similarity evaluation. Samples from batches A and B were completely discriminated and only two samples from batch S were incorrectly classified. Given the batch bias, we believe that model error may have been caused by man-made tag errors rather than the model itself. In conclusion, we established a chromatographic fingerprint for Squama Manis quality analysis and demonstrated its feasibility in animal source identification and quality determination by combining different data analysis methods. The established strategy may provide a new method for improving the the validity and accuracy of Squama Manis in clinical use.

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Quantum chemical calculations for elucidating chiral resolution mechanism of chiral covalent organic frameworks 6 chromatographic stationary phase HU Yuanyuan, ZHANG Zhongjie, HUANG Lu 2020, 38 (12):  1449-1455.  DOI: 10.3724/SP.J.1123.2020.05009 Abstract ( 130 )   HTML ( 6 )   PDF (3651KB) ( 88 )   With the aim of exploring the chiral resolution mechanism of the chiral covalent organic frameworks 6 (CCOF6) chromatographic stationary phase, the ORCA program was used to optimize the structures of CCOF6 and four pairs of enantiomers. Then the molecular docking of CCOF6 with each enantiomer was performed by AutoDock program to obtain the initial interaction configurations of CCOF6 with four pairs of enantiomers. Energy calculations of the initial configurations were performed by ORCA program (B3LYP functional with DFT-D3 correction, def2-TZVP orbital basis set, def2/J auxiliary basis set, and RIJCOSX used to accelerate the calculation) to determine the interaction configurations of CCOF6 with four pairs of enantiomers and to obtain the corresponding binding free energy and binding free energy difference. Wave function analyses of ORCA calculation results were performed by Multiwfn program, and the weak interactions between CCOF6 and the four pairs of enantiomers were visualized by Visual Molecular Dynamics program. The results showed the following: ① for calculating the binding free energies of CCOF6 and the four pairs of enantiomers, the ORCA method with the solvation effect was more accurate than the AutoDock method as well as the ORCA method without the solvation effect; ② the greater the absolute value of the binding free energy difference between the CCOF6 stationary phase and enantiomers, the greater was the selectivity factor of the enantiomers but not the resolution of the enantiomers; ③ the hydroxyl group of S-1-phenyl-1-propanol interacted with the ether bond of CCOF6, but the hydroxyl groups of the other enantiomers all interacted with the carbonyl groups of CCOF6, and the binding force between S-1-phenyl-1-propanol and CCOF6 was the weakest; ④ from the peak time of the enantiomer and its binding free energy with CCOF6, it was confirmed that the elution ability of n-hexane/isopropanol for 1-phenyl-1-propanol was the weakest, followed by the elution ability for 1-phenyl-2-propanol; ⑤ the peak time of S-1-phenyl-1-propanol was longer than that of R-1-phenyl-1-propanol, while for the other enantiomers, the peak time of the R-enantiomers was longer than that of the S-enantiomers.

Technical Notes

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Determination of eight environmental phenols in human urine samples by high-throughput solid-phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry LIN Xiao, QIU Tian, ZHANG Xu, HU Xiaojian, YANG Yanwei, ZHU Ying 2020, 38 (12):  1456-1464.  DOI: 10.3724/SP.J.1123.2020.07021 Abstract ( 131 )   HTML ( 9 )   PDF (888KB) ( 224 )   A method combining 96-well plate solid-phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry (96-well SPE LC-MS/MS) was developed for the simultaneous determination of eight environmental phenols in urine samples. The samples included seven bisphenol compounds and triclosan. The urine samples were thawed to room temperature, and the target analytes were deconjugated by β-glucuronidase/aryl-sulfatase in ammonium acetate buffer solution at 37 ℃ overnight. Then, the effects of three kinds of 96-well solid-phase extraction plates and different elution conditions on the purification of the urine samples and the environmental phenol recoveries were compared. The best purification effect was achieved on Oasis HLB 96-well plate (60 mg) solid phase extraction, using 30% (v/v) acetonitrile aqueous solution as the rinse solution. The target analytes were then eluted by methanol solution and evaporated to dryness using a nitrogen blower. After reconstruction with 0.5 mL methanol/water (1∶1, v/v) solution, the target compounds were detected by UPLC-MS/MS. To achieve better chromatographic separation, two kinds of analytical columns (C18 and T3) and different types of mobile phases (methanol and acetonitrile as the organic phase) were also compared. The best chromatographic effect was achieved when the treated samples were separated on a C18 column (100 mm×2.1 mm, 1.7 μm) using acetonitrile/water as the mobile phase at a flow rate of 0.3 mL/min. Mass spectra were recorded by negative electrospray ionization under the multiple reaction monitoring (MRM) mode. The sample matrix effect was also evaluated. The absolute matrix effects of bisphenol A, bisphenol F, bisphenol S, bisphenol B, and bisphenol AF were in the range of 3.47% to 15.32%. Since the above mentioned matrix effect was weak, there was no need for compensation measures. On the contrary, tetrachlorobisphenol A, tetrabromobisphenol A, and triclosan showed an absolute matrix effect of 49.58% (moderate), 71.99% (strong), and 86.93% (strong), thus necessitating compensation measures. Therefore, this strategy uses a one-to-one corresponding isotope internal standard method to offset the matrix effect. Six different urine samples were used to evaluate the relative matrix effect. The relative standard deviations (RSDs) of the eight corresponding internal standard peak areas were 3.63%-9.06%, indicating that the relative matrix effect was stable. Under the optimized conditions, linearity ranges were 0.50-50 μg/L for bisphenol A and bisphenol AF; 0.05-50 μg/L for tetrachlorobisphenol A and bisphenol S; 0.01-50 μg/L for bisphenol F and tetrabromobisphenol A; 1.00-50 μg/L for bisphenol B; and 5.00-200 μg/L for triclosan. The correlation coefficients were all greater than 0.9995. At spiked levels of 2.5, 5, and 25 μg/L, the average recovery ratios of the eight target analytes were 81.01%-118.84%, while the intra-day and inter-day precisions were 0.38%-19.41% and 2.54%-17.83%, respectively. The limits of detection (LOD) were 0.002-1.09 μg/L, and the limits of quantitation (LOQ) were 0.007-3.63 μg/L. This method was successfully applied to the determination of the eight environmental phenols in 64 urine samples collected from Beijing area between 2019 and 2020. All the target environmental phenols were detected, except for bisphenol B and bisphenol AF. Bisphenol A and bisphenol S showed the highest detection rates of 100% and 96.9%, respectively. The detection rates of triclosan, tetrabromobisphenol A, tetrachlorobisphenol A, and bisphenol F were 57.8%, 46.9%, 23.4%, and 21.9%, respectively. The medium values of urinary concentration followed the order 1.44 μg/L(triclosan), 0.69 μg/L(bisphenol A), 0.086 μg/L (bisphenol S), 0.0032 μg/L (tetrabromobisphenol A), 0.00050 μg/L (tetrachlorobisphenol A), 0.00 μg/L (bisphenol F, bisphenol B, and bisphenol AF). The aforementioned results imply that the widespread environmental phenolic exposure of Beijing residents is worthy of attention. Compared with traditional solid-phase extraction methods, the method reported in this paper is time-saving, effective, and suitable for the simultaneous analysis of large quantities of samples; moreover, the small sample and organic solvent consumption make this method more environment- and operator-friendly.

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Determination of four antipyretic and analgesic drugs in water by solid-phase extraction coupled ultra-performance liquid chromatography-tandem mass spectrometry ZHU Feng, YAO Zhijian, HUO Zongli, JI Wenliang, LIU Hualiang, ZHOU Qing, LI Aimin, JIAO Wei, GU Jing 2020, 38 (12):  1465-1471.  DOI: 10.3724/SP.J.1123.2020.07002 Abstract ( 107 )   HTML ( 8 )   PDF (1327KB) ( 88 )   The widespread use of pharmaceutical and personal care products (PPCPs), including antipyretic and analgesic drugs, in the last two decades had led to the existence of PPCP residues in the environment, thus raising concerns about their pseudo-persistent nature and potential threat to human health. Generally, most of the detected contaminants are present at low levels (ranging from ng/L to μg/L) in environmental water. Therefore, advanced analytical methodologies are crucial to monitor the occurrence and distribution of antipyretic and analgesic drugs in environmental water. However, trace analysis of environmental pollutants is always challenging because it is necessary to extract analytes present in the sample at ultralow levels from complex environmental matrices. Therefore, an appropriate sample pretreatment is necessary to enrich the target compounds. Conventional solid-phase extraction materials show poor efficiency for the enrichment of antipyretic and analgesic drugs. We herein report a hydrophilic and lipophilic amphiphilic porous polymeric material GCHM (Guochuang hydrophilic material). GCHM was successfully prepared by a stepwise emulsification and micellization process using N-vinyl-2-pyrrolidone (NVP) and divinylbenzene (DVB) as raw materials. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of four antipyretic and analgesic drugs in water using our solid-phase extraction (SPE) column. The water samples were extracted and purified by the GCHM solid-phase extraction column, and then analyzed by UPLC-MS/MS. Gradient elution was carried out with 0.1% formic acid aqueous solution and acetonitrile as the mobile phase. The target analytes were separated on an ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm), and multiple reaction monitoring (MRM) was conducted in the positive electrospray ionization mode. The isotope internal standard method was used for quantitative correction. Comparison of the enrichment efficiencies of Oasis HLB, Bond Elut Plexa, and GCHM revealed that GCHM showed the best performance. Different pH values affecting the enrichment efficiency of the GCHM SPE column were optimized, and the matrix effect was evaluated. The results showed that the four target analytes gave the best enrichment effect on the SPE column at pH 7, and the matrix effect for each substance was between 82.8% and 102.2%, indicating obvious matrix removal after the water sample was purified by the GCHM SPE column. Good correlation coefficients (r) greater than 0.995 were observed for all the target compounds in the range of 1-100 μg/L. The method limits of quantitation (S/N=10) ranged from 1 ng/L to 5 ng/L. The corrected recoveries were 85.6% to 106.4%, and the relative standard deviations (RSD) were under 5.6%. The GCHM solid-phase extraction column is inexpensive and efficient, being suitable for the detection of the four antipyretic and analgesic drugs in water. Subsequently, the occurrence of these selected antipyretic and analgesic drugs in water samples from Shanghai, Jiangsu, and Guangdong provinces were studied. The GCHM column has potential advantages over the commercial imported SPE column and is worthy of widespread application. This column can also aid the enrichment and purification of other compounds with similar structures or properties in water.