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Chinese Journal of Chromatography
2022, Vol. 40, No. 2
Online: 08 February 2022

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Application of imine covalent organic frameworks in sample pretreatment YUAN Hongmei, LU Zeyi, LI Yuhuang, ZHANG Chengjiang, LI Gongke 2022, 40 (2):  109-122.  DOI: 10.3724/SP.J.1123.2021.04029 Abstract ( 324 )   HTML ( 237 )   PDF (1141KB) ( 166 )   Imine covalent organic frameworks (I-COFs), including imine-linked COFs and hydrazone-linked COFs, are a new type of crystalline porous organic materials constructed by the condensation of organic monomers by the Schiff-base reaction. Because they are composed of lightweight elements linked by strong covalent bonds, I-COF materials possess the advantages of low skeleton density, large surface area, high porosity, abundant monomer species, controllable pore size, functionalized structure, diverse synthetic methods, excellent adsorption performance, outstanding physical and chemical stabilities, etc. In recent years, interest in the field of I-COFs has increased tremendously because of their exceptional performance and broad applications in gas storage, gas separation, catalysis, sensing, photoelectric materials, sample pretreatment, drug delivery, and other fields. To date, imine bonds are one of the most widely used covalent bonds in COFs, and represent one of the most important ways to obtain I-COFs with excellent chemical stabilities. The synthesis methods for I-COFs include solvothermal synthesis, microwave synthesis, mechanochemical grinding synthesis, and room-temperature synthesis methods. Solvothermal synthesis is the most extensively used method for the production of I-COFs with high specific surface areas and good thermal stabilities. The microwave synthesis method is conducive to the rapid synthesis of COFs in industry, providing a more time-saving, simpler, and safer route for large-scale preparation of I-COFs. The mechanochemical grinding synthesis of porous solids has gained importance as an alternative to conventional solvothermal synthesis, because the process is quick, environment-friendly, and potentially scalable. The room-temperature method is characterized by mild reaction conditions and rapid reactions. It is an energy-saving, economic, safe, and green synthesis method, which has emerged as a hot spot in the preparation of I-COFs in recent years. Research progress over the past years on the application of I-COFs in the field of materials science has undoubtedly established the basis of its application in analytical chemistry. Owing to the excellent physical and chemical properties of I-COF materials, they are suitable for use as separation and enrichment media for trace target compounds in complex samples. The high specific surface area and porosity, extended conjugate network skeleton, and π-electron-rich nature of the materials endow it with a high adsorption capacity. These materials are highly enriched in target analytes by π-π interactions, acid-base interactions, donor-acceptor interactions, hydrogen bonding, hydrophobic interactions, and other intermolecular interactions. Precise control of the microporous structure of I-COFs was obtained by controlling the chain length, geometric structure, doping elements, and substituent groups of the organic monomers. Selective enrichment of target trace substances was achieved by modifying the groups of I-COFs based on the principle of host guest adaptation, molecular sieving, or microporous filling effect. At present, research on the synthesis of I-COF materials is in the stage of rapid development, and many I-COFs with excellent properties and great application potential have been synthesized, allowing widespread application of I-COFs in sample pretreatment medium. This review summarizes the current state-of-the-art on the main types and synthetic methods of I-COFs, as well as the applications of I-COFs in solid-phase extraction, magnetic solid-phase extraction, dispersive solid-phase extraction, and solid-phase microextraction. The prospects of I-COFs in sample pretreatment are also presented.

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Lipid metabolomic analysis in exosomes of osteonecrosis of the femoral head based on ultra performance liquid chromatography-tandem mass spectrometry GUO Minkang, ZHANG Jian 2022, 40 (2):  123-129.  DOI: 10.3724/SP.J.1123.2021.04016 Abstract ( 287 )   HTML ( 58 )   PDF (2806KB) ( 146 )   Supporting Information Osteonecrosis of the femoral head (ONFH) can lead to its collapse which requires total hip arthroplasty. Exosomes, which are important for intercellular communication are involved in a series of physiological and pathological processes, and therefore play a unique role in disease diagnosis and treatment. In this study, untargeted metabolomics was used to investigate the metabolic characteristics of lipids in exosomes of femoral head tissue with osteonecrosis and to explain the metabolic changes that occur in the body during this disease. Ultracentrifugation was used to separate and enrich exosomes from femoral head tissue with osteonecrosis. Exosomes were identified using dynamic light scattering (DLS), Western blotting, and transmission electron microscopy (TEM). Gradient elution was performed with ultrapure water and acetonitrile as mobile phases using a Kinetex XB-C18 column (100 mm×2.1 mm, 2.6 μm). The column oven temperature, flow rate of the mobile phase, and duration were 30 ℃, 300 μL/min, and 15 min, respectively. A triple TOF 4600 high resolution mass spectrometry system was used, and the mass scan range of m/z was set at 100 -1000. Other conditions were as follows: sheath gas, 380 kPa; auxiliary gas, 380 kPa; curtain gas, 170 kPa; and atomization temperature, 600 ℃. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with multivariate statistical analysis was used to identify the lipid metabolic profile of ONFH-derived exosomes. The exosome metabolites were characterized in detail, which enables their identification and provided a reliable method for quality evaluation. After transforming the obtained original data using MarkView software, peak identification, peak alignment, subtraction of solvent peak, impurity peak, noise filtering, and other treatments, a three-dimensional matrix was obtained from the exported data table. Principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA) in the SIMCA-P14.1 software were used for multivariate statistical analysis of differentially expressed exosome lipid metabolites. This strategy was validated using lipid metabolites from patients with ONFH and healthy controls. The correlation distribution was shown according to the point dispersion of the PCA score plot, and lipid metabolites from the same disease showed ideal clustering. This result indicates a small difference between the groups. A good clustering effect is also obtained using OPLS-DA, and the statistical model has high reliability. A total of 18 significantly altered lipid metabolites were detected in the exosomes, including acrylolipids, fatty acid esters, glycerides, and their derivatives. The pathway analysis was conducted with MetaboAnalyst (https://www.metaboanalyst.ca/) via database source including the HMDB (http://www.hmdb.ca/) and MMCD (http://mmcd.nmrfam.wisc.edu/) for confirming the impacted metabolic pathways and visualization. Metabolic pathway analysis showed that glycerophospholipid and sphingolipid metabolism were the most significantly altered in exosomes. An imbalance between sphingolipids and glycerophospholipids leads to lipotoxic damage, which is implicated in the pathophysiology of common metabolic diseases. Furthermore, glycerophospholipids are correlated with cell proliferation, differentiation, and apoptosis, and the change in glycerophospholipid ratio can reflect the disturbance in lipid metabolism. The metabolic changes in exosomes may reflect the metabolic changes in ONFH. In this study, lipid metabolomics analysis based on UPLC-MS/MS was used to determine metabolic differences between exosomes extracted from ONFN and femoral neck fracture (FNF). Metabolomic analysis of necrotic femoral head tissue-derived exosomes can help explore the most relevant pathways for assessing the changes in exosome metabolism that affect exosome metabolism in necrotic bone tissue.

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Determination of disulfoton and its metabolites in agricultural products by dispersive soild phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry SUN Qiang, LI Yubo, WEN Guangyue, WANG Weimin, DONG Maofeng, TANG Hongxia 2022, 40 (2):  130-138.  DOI: 10.3724/SP.J.1123.2021.04028 Abstract ( 238 )   HTML ( 62 )   PDF (4758KB) ( 141 )   Disulfoton, an organophosphorus pesticide, is used to control cotton, beet, potato, and other seedling period aphids, leaf moths, underground pests, etc., with internal absorption, killing, gastric poisoning, and fumigation. Disulfoton is a highly toxic organophosphate pesticide, which can inhibit cholinesterase activity, resulting in neurophysiological disorders by inhalation, feeding, and transdermal absorption. Disulfoton is difficult to degrade in the environment, which leads to enrichment in organisms and interference with endocrine. This compound is harmful to the ecological environment and human health. To ensure the quality and safety of food, it is important to develop a detection method for disulfoton and its metabolites in agricultural products. A reliable method based on dispersive solid phase extraction (d-SPE) with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed for the determination of disulfoton and its metabolites (disulfoton sulfone, disulfoton sulfoxide, demeton-S, demeton-S sulfone, and demeton-S sulfoxide) in agricultural products (pea, asparagus, wheat, coffee bean, and peanut). The optimal extraction method was as follows: 5.0 g the samples were extracted with acetonitrile (wheat, coffee bean, and peanut presoaked in 5 mL water) in a 50 mL centrifuge tube, followed by 10 min vortex. Before 30 s vortex, 4 g NaCl was added. After 5 min centrifugation, 1.5 mL of the supernatant was cleaned up with 50 mg octadecylsilane bonded silica (C18), 50 mg primary secondary amine (PSA), and 50 mg aminopropyl (NH2) adsorbents. The analytes were separated on a Thermo Syncronis C18 column (150 mm×2.1 mm, 5 μm) with gradient elution using water and acetonitrile at a column temperature of 40 ℃. The injection volume was 2 μL. Disulfoton and its metabolites were analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+) for the selective quantification. Qualitative and quantitative analyses were accorded to the retention times and characteristic ion pairs with one parent ion and two fragment ions. Quantitative analysis was performed by an external standard method using matrix-matched calibration curves. All the parameters that affected the extraction efficiencies were optimized. C18, PSA, and NH2 gave good recoveries of 87.9%-109.0%. Other adsorbents, multiwalled carbon nanotubes (MWCNTs), hydroxylated multiwalled carbon nanotubes (OH-MWCNTs), carboxylated multiwalled carbon nanotubes (COOH-MWCNTs), octylsilane bonded silica (C8), strong cation exchange (SCX) and neutral alumina (N-Al2O3), led to recoveries below 56.2%. The combination of adsorbents was also considered. Seven different combinations of 50 mg C18, 50 mg PSA, and 50 mg NH2 were chosen for the optimization experiments. There were no obvious differences in these combinations, and the target analytes recoveries ranged from 81.0% to 109.3% with relative standard deviations (RSDs) between 0.6% and 12.5%. The matrix effect could affect the extraction efficiency. The adsorbents of 50 mg C18, 50 mg PSA, and 50 mg NH2 showed weaker matrix effects as compared with other combinations of adsorbents in the instrument. The results for the matrix effect showed that peanuts and asparagus exceeded 20%, requiring matrix-matched calibration curves. Under the optimized conditions, disulfoton and its metabolites showed good linearities (R2≥0.9981) in the range of 2.0-200.0 μg/L. The average spiked recoveries of disulfoton and its metabolites in peas, asparagus, wheat, peanuts, and coffee beans ranged from 75.0% to 110.0%, with RSDs of 0.7% to 14.9%. The limits of detection (LODs) were between 0.02 and 2.0 μg/kg, and the limits of quantification (LOQs) were 5.0 μg/kg. The method was applied for the detection of 80 commercial productions, and neither disulfoton nor its metabolites were found. The proposed method is rapid, accurate, highly selective, and sensitive, and it is suitable for the simultaneous determination of disulfoton and its metabolites in grain, oil crops, vegetables, and other matrices.

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Dispersive solid-phase extraction combined with high-performance liquid chromatography for determination of seven anesthetics in aquatic products SHI Fang, SHOU Dan, JIN Micong, WANG Hongwei, CHEN Xuguang, ZHU Yan 2022, 40 (2):  139-147.  DOI: 10.3724/SP.J.1123.2021.08002 Abstract ( 252 )   HTML ( 58 )   PDF (3292KB) ( 141 )   Nowadays, anesthetics are widely used in fishery production processes, such as fish breeding, surgery, and fresh aquatic product transportation. Because of the widespread application of anesthetic drugs in aquatic products, there is an increasing demand for the rapid and sensitive detection of anesthetic drugs in aquatic products. The complex aquatic product matrix contains a variety of interfering substances, such as proteins, fats, and phospholipids, along with anesthetic drug residues at very low concentrations; therefore, it is necessary to adopt appropriate pretreatment methods for improving the sensitivity of detection. In this study, a dispersive solid-phase extraction (DSPE) method, combined with high-performance liquid chromatography, was established for the simultaneous detection of seven anesthetic drugs in aquatic products, viz. procaine, oxybuprocaine, tricaine, eugenol, methyl eugenol, isoeugenol, and methyl isoeugenol. For the DSPE step, pretreatment conditions, such as extraction solvent, extraction time, adsorbent amount, and DMSO dosage, were optimized. Sample pretreatment is a three-step process. First, in ultrasound-assisted extraction, 2.0 g samples were extracted using 10.0 mL 1.0% formic acid in acetonitrile under ultrasound conditions for 10 min. Then, DSPE was performed with mixed adsorbents: the solvent extracts were cleaned using 20 mg poly(styrene-glycidylmethacrylate) microspheres (PS-GMA), 50 mg primary secondary amines (PSA), and 10 mg C18, followed by separation by centrifugation. Finally, DMSO-assisted concentration was applied: the organic layer was collected and was dried at 40 ℃ in a N2 stream with 100 μL DMSO. Water was added to the residue to obtain a final volume of 1.0 mL for HPLC analysis. The seven anesthetic drugs were separated on a Welch welchrom C18 column (250 mm×4.6 mm, 5 μm) by gradient elution using methanol and 0.05% formic acid in 5 mmol/L ammonium acetate aqueous solution as mobile phases. The detection wavelengths were 235, 260, and 290 nm. Two matrix matching standard curves for fish and shrimp were applied for quantitative analysis. Under optimized conditions, the seven target anesthetics showed good linear relationships in their respective concentration ranges (R2>0.999), with the limit of detection (LOD) ranging from 0.011 to 0.043 mg/kg. In fish samples, the mean recoveries obtained at three concentration levels were between 79.7% and 109%, with relative standard deviations (RSDs) being less than 7.2%. In shrimp samples, mean recoveries were 78.0%-99.9%, with RSDs being less than 8.3%. This simple, rapid, accurate, and sensitive method can be applied to the detection of three kinds of aminobenzoic acid esters and four kinds of eugenol anesthetic drugs in aquatic products.

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Determination of nine food-borne stimulant drug residues in pork, egg, and milk by ultra-performance liquid chromatography-tandem mass spectrometry LIU Xuezhi, ZHAO Yinglian, MA Yue, DONG Shishi, WANG Bin, ZHANG Yang 2022, 40 (2):  148-155.  DOI: 10.3724/SP.J.1123.2021.04005 Abstract ( 235 )   HTML ( 58 )   PDF (1375KB) ( 119 )   β-Agonists, β-blockers, and protein assimilators are classified as stimulant drugs. Their illegal use during animal feeding and slaughtering leads to food-borne stimulant drug residues, which are harmful to human health. At present, methods for the detection of β-agonists and protein assimilators are prevalent, but those for the detection of β-blockers are rare. There is no national standard for the detection of β-blockers in food products of animal origin. A method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of nine food-borne stimulant drug residues, including β-agonists, β-blockers, and protein assimilators, in pork, egg, and milk. The optimal extraction conditions for this method were as follows: The samples were hydrolyzed with β-glucuronidase/aryl sulfate esterase in pH 5.2 ammonium acetate buffer. Enzymatic hydrolysis was carried out in a constant-temperature (37 ℃) water bath oscillator for 16 h. The enzymolyzed samples were cooled to room temperature and then extracted with acetonitrile, which was adjusted to pH 9.5 with NaOH solution. After extraction and homogeneous mixing, the extract was added to a salt package for salting out stratification. The clear supernatant was cleaned up using an enhanced lipid removal tube (EMR-lipid), which was pre-activated by water. Then, anhydrous magnesium sulfate was added to ensure dehydration of the extract and concentrated to near dryness under nitrogen flow. The residue was dissolved in 1 mL acetonitrile-0.1% formic acid aqueous solution (1∶9, v/v). Separation was performed on an ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) with gradient elution using methanol-0.1% formic acid aqueous solution as the mobile phase. The analytes were detected in the multiple reaction monitoring (MRM) mode after being ionized by an electrospray positive ion (ESI+). Quantitative analysis was performed by the internal standard method using matrix-matched calibration curves. The effects of the extraction solvent and pH on the extraction efficiency during pretreatment were optimized. The influence factors of different types of chromatographic column, mobile phase and dissolved solution in the process of instrumental analysis were discussed in detail. Under the optimal conditions, the method showed good linearity in the range of 0.5 to 20 μg/L, with correlation coefficients (r2) greater than 0.99. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.3-0.6 μg/kg and 1.0-2.0 μg/kg, respectively. The average recoveries of all the compounds ranged from 65.2% to 117.0% with relative standard deviations (RSDs) in range of 1.3%-14.4% at spiked levels of 1, 2, and 5 times the LOQs. The established method was used to determine the quality of animal-origin foods such as pork, eggs, and milk purchased from the market. The nine stimulant drug residues were not detected in these food samples. The analytical method is rapid, sensitive, accurate, and stable. It can be used for the determination of the nine food-borne stimulant drugs residue in pork, egg, and milk.

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Determination of seven Alternaria toxins in infant milk powder by solid phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry XING Jiali, ZHANG Zigeng, ZHENG Ruihang, XU Xiaorong, MAO Lingyan, CHENG Hai, SHEN Jian 2022, 40 (2):  156-164.  DOI: 10.3724/SP.J.1123.2021.05023 Abstract ( 156 )   HTML ( 30 )   PDF (3061KB) ( 105 )   Alternaria toxin is a general term for a class of toxic metabolites produced by Alternaria, which widely exists in soil, grain, vegetables, and fruits. This mycotoxin is extremely harmful to human health. It is well known that infant milk powder containing vegetable oil is easily contaminated by Alternaria alternata. Alternaria toxins have thus become an increasingly important focus in food. Rapid and accurate detection of Alternaria toxin residues in food is of great significance for food safety. This requires pretreatment to purify the target toxins and maximize the accuracy and precision of the analysis. In this study, a rapid method based on online solid phase extraction/purification and ultra-performance liquid chromatography-tandem mass spectrometry (online SPE UPLC MS/MS) was established to detect seven Alternaria toxins (alternariol monomethyl ether, altenuene, tenuazonic acid, alternariol, tentoxin, altenusin, and altertoxin Ⅰ) in infant milk powder. First, the mass spectrometry and chromatographic conditions were optimized. A BEH-C18 column (50 mm×2.1 mm, 1.7 μm) was selected, with 0.1% formic acid aqueous solution-acetonitrile as the mobile phase. Then, the extraction conditions (extraction agent ratio and extraction method) and the solid phase extraction process (extraction column, type and volume of the eluent, and pH of the sample loading solution) were optimized. One gram of milk powder (accurate to 0.01 g) was weighed into a 50 mL tip and bottom plug centrifuge tube. Acetonitrile-water (84∶16, v/v) was set as the extraction agent for the first two cycles, and acetonitrile-methanol-water (45∶10∶45, v/v/v) was set as the third extraction agent. Horizontal shaking for 30 min was the best extraction method. The sample was centrifuged at 9500 r/min for 10 min, and the supernatant extracted many times was mixed and blown with nitrogen at 40 ℃. The sample was redissolved in first-order water (pH 5.5), purified on an HLB column, and successively activated with 6 mL methanol and 6 mL first-order water (pH 5.5). The solution was then loaded onto the column, and the SPE was adjusted to ensure that the water sample flowed through the column at the rate of 1 mL/min so that the column did not dry up during the analysis process before the end of sample loading. The column was rinsed with 12 mL of first-order water. After leaching, the negative pressure filtration was continued for approximately 5 min, followed by elution with 10 mL methanol, and the eluted solution was directly tested after passing through a 0.22 μm filter membrane, without concentration. The analytes were determined by electrospray ionization (ESI) with alternating positive and negative ions. Under the optimal analysis conditions, the linear relationships of the seven Alternaria toxins were good in the mass concentration range of 0.5-200 μg/L, with coefficients of determination (R2)>0.9903. The limits of detection and limits of quantification were 0.15-0.64 μg/kg and 0.54-2.24 μg/kg, respectively. The recoveries of the seven Alternaria toxins were 79.1%-114.3%, and the relative standard deviations were less than 8.87% at different concentrations. The method was applied to the determination and analysis of 60 samples of infant milk powder, and the results showed that no toxin was found in stage one or stage two of the milk powder. Only one sample of the stage three of milk powder was detected, which was tentoxin, and the content was 4.97 μg/kg. The developed method is accurate, rapid, simple, sensitive, repeatable, and stable. It can be used for the practical determination of seven Alternaria toxins in infant milk powder.

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Simultaneous determination of 24 corticosteroids in sediments based on ultrasonic extraction, solid-phase extraction, liquid chromatography, and tandem mass spectrometry ZHOU Yongshun, GONG Jian, YANG Kexin, LIN Canyuan, WU Cuiqin, ZHANG Shuhan 2022, 40 (2):  165-174.  DOI: 10.3724/SP.J.1123.2021.03025 Abstract ( 206 )   HTML ( 46 )   PDF (2035KB) ( 110 )   Corticosteroids (CSs) are widely used to treat various inflammatory and immune diseases in humans and animals, such as arthritis and lupus. Thus far, CSs have been frequently detected in diverse pollution sources, such as in the influent and effluent of traditional wastewater treatment plants, livestock farms, and aquaculture. Owing to incomplete removal or limited treatment, CSs can enter the water environment and eventually be adsorbed in the sediment. Due to hydrodynamic effects, CSs can re-enter the surface water through the resuspension of sediments, and pose a hazard to the ecosystem and human health via the enrichment of aquatic organisms and transmission through the food chain. Therefore, trace analysis of CSs in sediments is significant for exploring their prevalence and behavior in multiple environments. However, existing research mainly focuses on the determination of glucocorticoids in water samples, and studies on the systematic quantitative analysis of CSs in environmental solid samples with more complex matrices are scarce. Moreover, majority of previous investigations focused on a limited number of glucocorticoids, making it important to widen the range of target compounds to be studied, including mineralocorticoids. In this study, the main factors which could influence the accuracy and sensitivity in the determination of 24 target CSs were systematically optimized in the sample pretreatment and instrument analysis. A novel method based on ultrasonic extraction coupled with solid phase extraction (SPE) for sample pretreatment was developed for the simultaneous determination of the 24 CSs in sediments using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The sediment sample was ground to homogenize the particle sizes after freeze-drying. The analytes from 2.0 g of the sample were ultrasonicated and extracted with methanol-acetone (1∶1, v/v). After concentrating and diluting each extract, SPE was performed. The water sample was extracted and purified using hydrophile-lipophile balance (HLB) cartridges, following which the extract was further purified with LC-NH2 cartridges. The extracts were concentrated using a rotary evaporator, dried under a gentle stream of nitrogen, and re-dissolved in methanol for instrumental analysis. Chromatographic separation was conducted on an Agilent ZORBAX Eclipse Plus C8 column (100 mm×2.1 mm, 1.8 μm), with a column flow rate of 0.3 mL/min and a gradient of mobile phases A (water with 0.1% acetic acid) and B (acetonitrile). The column temperature was set to 30 ℃ and the injection volume was fixed at 5 μL. Electrospray ionization MS in the dynamic multiple reaction monitoring (DMRM) and selected ion monitoring (SIM) modes were performed in the positive mode for the qualitative and quantitative analysis of the target compounds. Quantitation of the target compounds was carried out using the internal standard method. The effects of different extraction solvents, purification conditions, and MS conditions on the recoveries of the target compounds were investigated. The limits of detection (LODs) (S/N≥3) and limits of quantification (LOQs) (S/N≥10) of all 24 compounds were in the ranges of 0.14-1.25 μg/kg and 0.26-2.26 μg/kg, respectively. The correlation coefficients of linear calibration curves were higher than 0.995 in the range of 1.0-100 μg/L. The recoveries of the 24 CSs at 5, 20, and 50 μg/kg spiked levels ranged from 64.9% to 125.1% with relative standard deviations of 0.4%-12.6% (n=5). The developed method was applied to analyze the CSs in three sediment samples from the rivers of the Pearl River Delta. In all, 11 target compounds were detected in these samples, with contents in the range of 1.25-29.38 μg/kg. The characteristic of this method is efficient, sensitive, reliable, and suitable for the trace determination of varieties of natural and synthesized CSs in environmental sediments.

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Analysis of glycan ratio of Chinese hamster ovary cell-cetuximab antigen-binding segment via rapid enzyme digestion with endo-β-N-acetylglucosaminidase F CHENG Qian, JIA Daihui, ZHANG Bohui, XU Junyan, SHAO Zhe, HUANG Yingfeng, ZOU Xun 2022, 40 (2):  175-181.  DOI: 10.3724/SP.J.1123.2021.05008 Abstract ( 217 )   HTML ( 18 )   PDF (1656KB) ( 205 )   Supporting Information The N-glycosylation of proteins is a typical post-translational modification. Compared with other monoclonal antibodies, N-glycosylation modification in cetuximab is more complicated. Because cetuximab contains two N-glycosylation sites, one is located on the antigen-binding fragment (Fab) and the other is on the crystallizable fragment (Fc) of the heavy chain (HC). Among the two, the glycosylation of the Fab segment is more complicated. As this segment is located in the hypervariable region (VH), it may affect the affinity of the antibody antigen and cause other issues. Therefore, it is necessary to study glycosylation modification at this site. This modification is particularly challenging, necessitating the development of specific glycan cutting technology and a stable glycan ratio analysis method. In this study, cetuximab expressed in Chinese hamster ovary (CHO) cell was used as the experimental research object. Based on the digestion with endo-β-N-acetylglucosaminidase F2 (Endo F2), an experimental method was developed that can quickly release Fab glycans. Qualitative and glycan ratio analyses were carried out by ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The test was divided into two steps: in the first step, a non-denaturing (native state) glycosidase excision test was performed on the CHO-cetuximab drug substance. The drug substance was diluted to 1.0 mg/mL by adding ultrapure water, following which 1.0 μL of Endo F2 was directly added to 100 μL of the drug substance for enzyme digestion at 37 ℃. Through HRMS, the data were deconvoluted to obtain the accurate mass of the drug substance. The results showed that when the digestion time of Endo F2 was 5 min, the glycans in the Fab segment could be completely removed, whereas those in the Fc segment were not affected. Rapid enzyme cutting of the Fab glycans was realized; simultaneously, it was concluded that this method was also very specific for the removal of Fab glycans. In the second step, an accurate ratio analysis test was performed on Fab glycans excised from CHO-cetuximab. The released Fab glycans were precipitated with ice ethanol, the supernatant was centrifuged and spin-dried, and then labeled with para-aminobenzyl (2-AB). 2-AB labeling could make glycans have fluorescent detectable signals, and after reconstitution in 70% acetonitrile aqueous solution, was detected by UPLC coupled with a fluorescence detector (FLR). Good chromatographic peak separation was obtained using a hydrophilic interaction chromatography (HILIC) column. Thus, the test enabled stable glycan ratio analysis. The molecular weight results for three independent Endo F2 digestion cycles for 5 min showed that the masses after digestion were similar; subsequently, glycan ratio analysis was performed based on HILIC. The results of three independent glycan ratio analysis experiments were also similar, indicating that the rapid enzyme digestion of Endo F2 followed by glycan ratio analysis after 5 min of digestion yielded good stability and reliability. Data obtained by measuring the samples produced using two different processes employed by our company showed that there were distinct differences in the glycan profiles of the two processes, especially in terms of the sialic acid glycoforms. These results prove that the method developed in this study can accurately analyze the ratio of glycans. Monitoring the antibody production process is important and meaningful for the evaluation of the process.

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Simultaneous determination of 36 hypotensive drugs in fingerprints by ultra performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry DU Qiuyao, ZHANG Yunfeng, WANG Jifen, ZHAO Peng, WU Xiaojun, DONG Linpei, LI Jiayi, LIU Bingjie 2022, 40 (2):  182-189.  DOI: 10.3724/SP.J.1123.2021.05012 Abstract ( 208 )   HTML ( 40 )   PDF (989KB) ( 152 )   Fingerprints contain important information such as the ingredients ingested by the donor. By analyzing the characteristic components in fingerprints, the donor can be characterized, which would provide insights for investigation of a given case. This approach can also be used in the qualitative monitoring of drug intake. Therefore, the examination of hypotensive drugs in fingerprints has significant value in practical application. This study established a method based on ultra performance liquid chromatography-triple quadrupole composite linear ion trap mass spectrometry (UPLC-Q-TRAP/MS) for the simultaneous determination of 36 hypotensive drugs in fingerprints. The pre-treatment method was based on protein precipitation. A 3×3 cm filter paper was cut into pieces and placed in a 2 mL plastic centrifuge tube after fingerprint collection. Then, 0.50 mL methanol was added, followed by vortex mixing for 1 min and ultrasonic oscillation for 3 min. The filter paper was centrifuged at 12000 r/min for 5 min, and the supernatant was withdrawn for sample analysis. An ACQUITY UPLC® BEH C18 chromatographic column (100 mm×3.0 mm, 1.7 μm) was selected, with 0.01% aqueous formic acid and methanol as mobile phases for gradient elution. MS analysis involved scheduled multiple reaction monitoring-information dependent acquisition-enhanced product ion (SMRM-IDA-EPI) scanning. This method could be used to retrieve library researching during high-sensitivity analysis, which could increase the accuracy of qualitative results. The calibration curves showed good linearity in the range of 0.05-50.00 ng/fingerprint, with correlation coefficients (r) greater than 0.99 for all 36 analytes. The limits of detection and limits of quantification of the 36 hypotensive drugs were 0.001-0.045 ng/fingerprint and 0.002-0.050 ng/fingerprint, respectively. At spiked levels of 0.25, 2.50, 25.00 ng/fingerprint, the matrix effects, recoveries, intra-day precisions, and inter-day precisions of the 36 hypotensive drugs were 79.0%-119.2%, 79.3%-116.2%, 0.2%-18.3%, and 1.6%-19.1%, respectively. This method was used to detect hypotensive drugs in the fingerprints of 87 hypertensive patients, and hypotensive drug intakes were accurately detected in most cases. The established method is operationally simple, with high sensitivity and good selectivity, and it is suitable for screening and testing hypotensive drugs in fingerprints.

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Influence of chromatographic column selection on the determination of 23 preservatives LI Li, LI Shuo, WANG Haiyan, SUN Lei 2022, 40 (2):  190-197.  DOI: 10.3724/SP.J.1123.2021.05006 Abstract ( 307 )   HTML ( 44 )   PDF (877KB) ( 240 )   The influence of column selection on the determination results of 23 preservatives in cosmetics was studied by considering the corresponding detection methods for these preservatives. The test method for 23 preservatives specified in Safety and Technical Standards for Cosmetics was considered as the reference method. Twenty three preservatives were analyzed using 15 different brands and models of C18 chromatographic columns on two different high performance liquid chromatographs. The plate count and resolution of the chromatographic peaks were calculated to analyze the separation effect of the 23 components. The separation efficiency and equivalence of different chromatographic columns were evaluated and predicted using the database established by the United States Pharmacopeia (USP) and Product Quality Research Institute (PQRI). The experimental results showed that the separation effects of the 15 chromatographic columns on the 23 preservatives differed significantly. Further, only two chromatographic columns were able to completely separate the 23 components without changing the standard procedure for mobile phase elution and other conditions. The other 13 columns poorly separated some components. The retention times of phenyl 4-hydroxybenzoate, isobutyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate, and benzyl 4-hydroxybenzoate were similar. The separation of these substances was among the key outcomes of the chromatographic separation. The differences in the selectivity of the 4-hydroxybenzoates were distinct among the different chromatographic columns. The plate counts of the 4-hydroxybenzoates were similar when using the same chromatographic column on two high performance liquid chromatographs. However, the plate counts of 4-hydroxybenzoates using different chromatographic columns were considerably different. Control of the plate counts alone was not sufficient to achieve good separation efficiency of the 4-hydroxybenzoates. Moreover, the peak sequences of some compounds such as 4-hydroxybenzoic acid and methylisothiazolinone with different chromatographic columns were reversed. Some substances could be absorbed at multiple wavelengths. If separation from adjacent chromatographic peaks is not achieved at the baseline, large errors will occur when detecting the low-concentration components to be tested. The ZORBAX SB-C18 column was used as a reference column, and the similarity values provided by the USP and the PQRI databases were consulted. The actual separation efficiency of the typical column used in the experiment was compared with the predicted results of selectivity difference from the database. The results showed that the USP and the PQRI database could not predict a suitable equivalent column. There was no regularity in the experimental results; further, the USP and the PQRI datebase provided scarce reference values for the analysis of the 23 preservatives by liquid chromatography. The outcomes obtained using liquid chromatography methods are easily affected by factors such as the instrument model, mobile phase composition and pH, chromatographic column, column temperature, and flow rate. The chromatographic column is a key factor affecting the accuracy of determination by HPLC. The selectivity difference of the chromatographic column should be taken into account when the technique is performed in relevant laboratories. The standard technique will be employed by different laboratories at different times and under different test conditions, and many abnormalities or deviations may occur. The sequence of peaks should be confirmed using a single compound standard during the first standard method validation or during the replacement of columns. Based on existing research results, future research should focus on the development of a screening and prediction evaluation system for chromatographic columns, and thereby guide the separation of actual samples in complex cosmetic bases. Relevant laboratories should focus on the durability of chromatographic columns, and improve the system adaptability index when developing a detection method. They also should refine the column classification and enhance descriptive information, and thus, guide the rational selection of the column so as to mitigate the risk posed by inaccurate determination results due to improper selection of the chromatographic column.

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Simultaneous determination of monochloropropanediol esters and glycidyl esters in vegetable oils by acidic transesterification-gas chromatography-mass spectrometry WANG Xueting, LI Jingjing, JIANG Shan, SHEN Weijian, WANG Yiqian, GU Qiang 2022, 40 (2):  198-205.  DOI: 10.3724/SP.J.1123.2021.05009 Abstract ( 145 )   HTML ( 22 )   PDF (892KB) ( 87 )   A comprehensive analytical method based on gas chromatography-mass spectrometry (GC-MS) was developed for the determination of 3-monochloropropanediol esters, 2-monochloropropanediol esters, and glycidyl esters in vegetable oils. Different parameters, such as bromination reaction temperature, bromination reaction time, derivatization reagent dosage, and derivative reaction time, were studied. The optimal conditions were as follows: 0.25 g of oil was weighed in a 10-mL glass tube, followed by the addition of 2 mL tetrahydrofuran, 25 μL of internal working standard solutions, and 30 μL of acid aqueous solution of NaBr, homogenized, and the mixture was incubated at 50 ℃ for 15 min. The reaction was stopped by the addition of 3 mL of an aqueous solution of sodium hydrogen carbonate. To separate the oil from the water phase, n-heptane was added, and the upper layer was transferred to an empty test tube and evaporated to dryness under a nitrogen stream. The residue was dissolved in 1 mL of tetrahydrofuran. 1.8 mL of sulfuric acid solution in methanol was added to the sample, and the resulting mixture was incubated at 40 ℃ for 16 h. The reaction was stopped by the addition of 0.5 mL of an aqueous solution of sodium hydrogen carbonate. After purification by n-hexane and derivatization of phenylboric acid, the derivatives were extracted with n-hexane. After nitrogen blowing, the residue was dissolved in 1 mL of n-hexane, and then filtered through a 0.45-μm membrane filter unit prior to GC-MS analysis. Temperature programming was applied at an initial temperature of 80 ℃. After 0.5 min, the temperature was raised to 180 ℃ at a rate of 20 ℃/min, held for 0.5 min, raised to 200 ℃ at a rate of 5 ℃/min for 4 min, and finally raised to 300 ℃ at a rate of 40 ℃/min for 4 min. The target compounds were separated on a DB-5MS column (30 m×0.25 mm×1 μm). Identification and quantification were achieved using an electron impact (EI) ion source in the positive ion mode with the selected ion monitoring mode. The internal standard method was used to quantify the 3-chloropropanediol esters, 2-chloropropanediol esters, and glycidyl esters. Under the optimal conditions, the correlation coefficients of the standard calibration curves were greater than 0.999 in the mass concentration range of 0.01-0.80 mg/L. The limits of detection were 25, 25, and 20 μg/kg (S/N=3), and the limits of quantification were 75, 75, 60 μg/kg (S/N=10). Four samples of different matrix types were selected for scaling experiments. At spiked levels of 250, 500, and 750 μg/kg, the recoveries of 3-chloropropanediol esters, 2-chloropropanediol esters, and glycidyl esters in spiked samples ranged from 89.0% to 98.7%, with relative standard deviations between 2.05% and 7.81% (n=6). This method was used to determine 112 commercially available vegetable oil samples, among which 84 samples were detected with 3-chloropropanediol esters, 2-chloropropanediol esters, or glycidyl esters. The method developed in this study was remarkably different from the standard method, which are mentioned in the national standard method (GB 5009.191-2016) and industry standard method (SN/T 5220-2019), especially in the pretreatment step that involved acidic transesterification. Use of the acidic transesterification method can avoid side reactions, such as the conversion of 3-chloropropanediol, 2-chloropropanediol, and 3-bromopropanediol to free glycidol under alkaline conditions. The method developed in this study was more efficient, and the results were more accurate and reproducible. It has theoretical and practical significance for the control of 3-chloropropanediol esters, 2-chloropropanediol esters, and glycidyl esters residues in vegetable oils, establishment of detection standards, and optimization of the production process.

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Determination of fipronil and its metabolites in livestock and poultry liver by high performance liquid chromatography-tandem mass spectrometry with dispersive solid phase extraction WEI Yunji, BAO Huimei, HE Zhenghe, HUANG Gaoming, FENG Min, ZHU Zhenyi, HE Jian 2022, 40 (2):  206-211.  DOI: 10.3724/SP.J.1123.2021.04007 Abstract ( 209 )   HTML ( 33 )   PDF (1689KB) ( 112 )   The residues of fipronil and its metabolites, namely fipronil-desulfinyl, fipronil-sulfone, and fipronil-sulfide, have attracted increasing attention since the European egg contamination incident. In this study, by optimizing the pretreatment method and chromatographic conditions, a simple extraction method coupled with high performance liquid chromatography-tandem mass spectrometry was developed for the simultaneous determination of fipronil and its metabolites, including fipronil-desulfinyl, fipronil-sulfone, and fipronil-sulfide, in livestock and poultry liver. The optimal pretreatment method for fipronil and its metabolites was determined by comparing the recoveries obtained with different extraction solvents (methanol, acetonitrile, acetone, and ethyl acetate), and by purification with N-propyl ethylenediamine (PSA) and octadecylsilane (C18). The samples were extracted with 10 mL acetonitrile, then purified with 150 mg PSA and 100 mg C18, following which the extracted solutions were directly injected for analysis. Separation was performed on an Agilent ZORBAX SB-C18 column (150 mm×2.1 mm, 3.5 μm) with gradient elution using acetonitrile and water as the mobile phases. The target compounds were detected by electrospray ionization (ESI) in the negative mode under multiple reaction monitoring, and quantified by the external standard method with matrix-matched standard correction curves. The results indicated that the linear ranges for the four compounds ranged from 0.1 μg/L to 10.0 μg/L with correlation coefficients (r2) higher than 0.995. The limit of detection was 0.2 μg/kg and limit of quantitation was 0.5 μg/kg. The average recoveries were between 81.1% and 99.8% at three spiked levels of 0.5, 1.0, and 10 μg/kg, with relative standard deviations (RSDs) of 6.1%-11.7%. The matrix effect experiment showed that fipronil and its metabolites had matrix inhibition effects. Matrix-matched standard curve correction was performed to eliminate matrix inhibition effects. The proposed method was used for the analysis of 99 liver samples, where fipronil-sulfone was detected in four samples with values of 1.25-2.82 μg/kg. The method is simple, sensitive, and accurate, and is suitable for the determination of fipronil and its metabolites in livestock and poultry liver.