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Chinese Journal of Chromatography
2022, Vol. 40, No. 4
Online: 08 April 2022

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Adulteration identification of wheat flour in chestnut flour based on differences in mycotoxin contamination by liquid chromatography-tandem mass spectrometry ZHOU Jian, CHEN Xiaohong, JIN Micong 2022, 40 (4):  303-312.  DOI: 10.3724/SP.J.1123.2021.10021 Abstract ( 220 )   HTML ( 198 )   PDF (870KB) ( 102 )   Supporting Information An analytical method based on dispersive solid-phase extraction (d-SPE) and ultrafast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was employed for the determination of 43 mycotoxins in chestnut flour and wheat flour. A total of 128 samples consisting of 48 chestnut samples and 80 wheat flour samples were collected randomly and subjected to analysis. Finally, five specific toxins were selected as markers to identify these two foodstuffs. Acetonitrile-water (84∶16, v/v) was used to extract mycotoxins from chestnut flour and wheat flour. After extraction, the supernatant was transferred to the d-SPE equipment, using which purification was performed with C18 and EMR-Lipid (lipid adsorbent). Chromatographic separation was carried out by gradient elution with eluent A (ESI+: 0.1% formic acid, ESI-: water) and eluent B (ESI+: methanol-acetonitrile (1∶1) containing 0.1% formic acid, ESI-: acetonitrile) on a BEH C18 column (100 mm×2.1 mm, 1.7 μm). Quantitative analysis was performed with the aid of matrix-matched curves. When establishing the method, the experimental matrix for optimization was designed by central-composite design based on the response surface methodology. Quadratic polynomial equations were deduced to describe the relationships between the responses and variables, and assess the interaction effects among the variables to acquire the true optimal conditions with less workload. Using the optimum experimental conditions, the accuracy of the proposed method was determined through three-level spiking tests, while the precision was evaluated in terms of the repeatability (six replications per level). Satisfactory precisions (RSDs≤7.5% in chestnut flour and RSDs≤9.3% in wheat flour) were achieved in all tested assays. The recoveries were also acceptable, and ranged from 72.4% to 109.4% for chestnut flour and from 70.7% to 112.9% for wheat flour. The matrix effects of mycotoxins were 48%-128% in wheat flour and 41%-112% in chestnut flour. The detectability of mycotoxins in the two matrices was assessed by spiking the blank extracts with various low concentrations, and determined as the lowest values that can produce chromatographic peaks at a signal-to-noise ratio (S/N) of 3∶1. The obtained limits of quantification varied from 0.10 μg/kg to 20 μg/kg (bongkrekic acid) in both investigated matrices. Satisfactory linearities were obtained, with correlation coefficients>0.9991 for all the analytes. After validation, the contamination status of the multiple mycotoxins was evaluated for various concentration ranges. Based on the obtained data, both wheat flour and chestnut flour were severely contaminated, with 17 mycotoxins detected in them. Particularly, chaetoglobosin A, ochratoxin B, and penicillic acid were only detected in chestnut flour, while 3-acetyl-deoxynivalenol, deoxynivalenol, and nivalenol were detected in wheat flour. Further, the positive rates and contamination concentrations of chaetoglobosin A, ochratoxin B, and penicillic acid were not significant; hence, they did not qualify as identification markers. On the other hand, the incidence of deoxynivalenol in wheat flour almost reached 100%, which is very significant. Finally, deoxynivalenol and its four derivatives (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, deepoxy-deoxynivalenol, and nivalenol) were treated as adulteration markers for the two foodstuffs. To improve the reliability of the conclusion, all samples were re-tested using the first method prescribed by the National Food Safety Standard, i. e., GB 5009.111-2016. Ten chestnut flour samples were also randomly selected to prepare moldy samples under suitable environmental conditions for the growth of Fusarium, to verify the production and release of deoxynivalenol and its derivative mycotoxins under the extreme conditions. The distribution data for these mycotoxins were consistent with those obtained by d-SPE, confirming that the adulteration criterion is trustworthy. The established method is simple, rapid, sensitive, and accurate, and can effectively meet the requirements for the simultaneous determination of multiple mycotoxins in chestnut flour and wheat flour. Moreover, the adulteration results, which were obtained for natural contaminants (deoxynivalenol and its four derivatives), are less affected by humans and hence, much more accurate and reliable.

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Simultaneous determination of 76 pesticide residues in the traditional Chinese medicine by magnetic hydrophilic-lipophilic-balanced materials assisted matrix solid phase dispersion extraction-high performance liquid chromatography-tandem mass spectrometry WEI Dan, GUO Ming 2022, 40 (4):  313-322.  DOI: 10.3724/SP.J.1123.2021.08014 Abstract ( 192 )   HTML ( 58 )   PDF (2737KB) ( 146 )   Supporting Information Traditional Chinese medicine (TCM) is gaining popularity worldwide, but its quality is often affected by excessive pesticide residues during cultivation and production. A sensitive and reliable method for the simultaneous determination of multi-residue pesticides in TCMs is the key to guarantee the quality and safety of TCMs. In this study, broad-spectrum hydrophilic-lipophilic balanced magnetic adsorbents were prepared for the magnetic matrix solid phase dispersion (MMSPD) extraction of 76 pesticides from three different TCMs before their detection by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Unlike the traditional matrix solid phase dispersion (MSPD), which required tedious SPE column packing, an external magnetic field was employed in our method for magnetic isolation and extraction from TCM samples, followed by grounding adsorption. First, broad-spectrum hydrophilic-lipophilic balanced magnetic adsorbents, Fe3O4@PLS, were successfully fabricated by the swelling polymerization of divinyl benzene (DVB) and N-vinyl pyrrolidone (NVP) on the surface of Fe3O4 magnetic particles. The prepared materials were systematically characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffractometry (XRD) for their morphologies, chemical structure, and crystalline structure. Then, the obtained magnetic particles (Fe3O4@PLS) were applied to MMSPD for the simultaneous extraction and analysis of the 76 pesticides in honeysuckle, chrysanthemum, and pseudo-ginseng root (dry) prior to detection by HPLC-MS/MS. In order to establish the optimal extraction conditions, the key parameters affecting the MMSPD extraction efficiency were optimized, including the amount of magnetic materials Fe3O4@PLS (5, 10, 12, 15, 20 mg), volume fractions of methanol in water for matrix purification (0%, 5%, 10%, 20%, 25%), grinding adsorption time for dispersion (2, 3, 4, 5, 6 min), type (methanol, methanol containing 0.1% (v/v) formic acid, acetonitrile, acetonitrile containing 0.1% (v/v) formic acid) and volume (0.5, 1, 2, 2.5, 5 mL) of elution solvent, and vortex time for desorption (1, 2, 3, 4, 5 min). Finally, the optimal conditions were set as follows: 10 mg of the magnetic adsorbent Fe3O4@PLS, 10 mL 20% (v/v) of methanol in water with a vortex time of 1 min for matrix purification, grinding time of 5 min for dispersion and adsorption, 0.5 mL acetonitrile containing 0.1% (v/v) formic acid as the elution solvent, and vortex time of 1 min for desorption. Then, the 76 pesticides 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 by positive electrospray ionization (ESI+). Under the optimal conditions, good linearities were obtained for the 76 pesticides in the concentration ranges of 10 to 200 μg/kg, with correlation coefficients (r 2)≥0.9965. The limits of detection (LODs, S/N=3) were in the range of 0.6-3.0 μg/kg, and the limits of quantification (LOQs, S/N=10) ranged from 2.0 to 10.0 μg/kg. The proposed method was successfully applied to 76 pesticide residue analysis in honeysuckle, chrysanthemum, and pseudo-ginseng root (dry). At three spiked levels, the recoveries were 69.1%-112.2%, 67.1%-102.8% and 70.1%-105.1%, with RSDs of 2.0%-12.4%, 2.1%-13.2% and 2.0%-13.5% for honeysuckle, chrysanthemum, and pseudo-ginseng root(dry), respectively. The prepared magnetic material Fe3O4@PLS has the characteristics of both strong magnetic responsibility and high broad-spectrum adsorption property for hydrophilic (e. g., carbendazim) and lipophilic (e. g., edifenphos) pesticides. The implementation of the MMSPD method before the analysis of the 76 pesticides by HPLC-MS/MS has the following advantages: less consumption of the magnetic materials, sample, and organic solvent; simple operation; high sensitivity; and satisfactory accuracy and precision. Hence, this is a potential method for the simultaneous determination of multiple pesticide residues in complex non-liquid TCM samples.

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Determination of five nonsteroidal anti-inflammatory drugs in water by dispersive solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry based on metal-organic framework composite aerogel LING Huijuan, WU Gege, LI Shuang, ZHOU Qian, LI Chunxin, MA Jiping 2022, 40 (4):  323-332.  DOI: 10.3724/SP.J.1123.2021.07014 Abstract ( 248 )   HTML ( 51 )   PDF (5834KB) ( 206 )   Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of synthetic drugs that do not contain glucocorticoids. NSAIDs are widely used for their analgesic, antipyretic, and anti-inflammatory effects. Due to their low adsorption coefficients and recalcitrance to biodegradation, NSAIDs readily enter environmental water through sewage discharge and exist stably for long periods. The long-term presence of trace amounts of NSAIDs in environmental water has adverse health effects on humans and animals. Therefore, it is important to establish an appropriately sensitive and reliable method for the determination of NSAIDs in environmental water, where their concentrations are low. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is highly selective and sensitive, and so is especially suitable for detection of NSAIDs. Solid phase extraction is one of the most commonly used pretreatment methods. The extraction efficiency depends mainly on the adsorbents used. Metal-organic framework (MOF) aerogel SPE materials combine the attributes of highly selective adsorption property and high affinity. Moreover, the monolithic structure of the MOF aerogel composite simplifies the solid-liquid separation process. In this work, a novel MOF/chitosan (CS) composite designated Co-UiO-67(bpy)/CS, was prepared as the adsorbent material to enrich ketoprofen (KPF), naproxen (NPX), flurbiprofen (FPN), diclofenac (DCF), and ibuprofen (IBF) in water. This facilitated the detection of these compounds by UPLC-MS/MS. Co-UiO-67(bpy) was synthesized by a solvothermal method by mixing zirconium chloride, cobalt chloride, and the organic ligand 2,2-bipyridine-5,5 dicarboxylic acid. A CS suspension was used to prepared the hydrogel, which was freeze-dried to obtain the Co-UiO-67(bpy)/CS aerogel. The prepared material was characterized by Fourier transform-infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). Co-UiO-67 (bpy) was embedded into chitosan. A layered porous MOF composite aerogel was observed. The extraction efficiency of the five NSAIDs was investigated and optimized by assessing type of extraction material, MOF amount, extraction time, sample pH, ionic strength, formic acid concentration in eluent, elution time, and elution volume. The optimized results showed that the target compounds could be completely adsorbed within 5 min. In the UPLC-MS/MS experiment, NSAIDs were analyzed in the negative ionization multiple radiation monitoring (MRM) mode. Gradient elution was carried out with 0.01% formic acid aqueous solution and methanol as the mobile phases. The analytical method was established in the optimized extraction conditions. The five NSAIDs displayed good linearity with linear correlation coefficients greater than 0.9937. The limits of detection (LODs) and limits of quantification (LOQs) of this developed method were 0.32-2.06 ng/L and 1.05-6.78 ng/L, respectively. Satisfactory recoveries of the five analytes were achieved within 74.5%-114.1% at three spiked concentrations of 40, 250, and 1500 ng/L, as well as good precision with relative standard deviations of 1.3%-12.3% (intra-day) and 1.3%-11.5% (inter-day). The method was then used to test real-world water samples. Trace amounts of ketoprofen and flurbiprofen were detected in municipal wastewater (14.52 ng/L and 10.05 ng/L, respectively). The method exhibited good sensitivity, accuracy, and precision, and the operation process was convenient. The present study thus presents a novel method for the detection of the trace NSAIDs in environmental waters.

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Determination of 42 antibiotic residues in seven categories in water using large volume direct injection by ultra high performance liquid chromatography-triple quadrupole mass spectrometry SUN Huijing, LI Peiwen, ZHANG Beibei, CHEN Huiming 2022, 40 (4):  333-342.  DOI: 10.3724/SP.J.1123.2021.08010 Abstract ( 480 )   HTML ( 60 )   PDF (1828KB) ( 272 )   Supporting Information Antibiotics are emerging contaminants that have recently attracted attention. They have been detected in natural water and pose health concerns owing to potential antibiotic resistance. Antibiotics are ubiquitous in aquatic environments, with a wide spectrum and trace levels. It is difficult to detect all types of antibiotics with completely different physicochemical properties. Solid phase extraction (SPE) is a common sample preparation procedure. For a fast and high-throughput continuous on-line analysis of these emerging contaminants, a method for the determination of 42 antibiotics (grouped into seven categories: sulfonamides, fluoroquinolones, lincosamides, macrolides, tetracyclines, cephalosporins, and chloramphenicols) in environmental water was developed based on ultra high performance liquid chromatography combined with tandem mass spectrometry (UHPLC-MS/MS) involving large volume direct injection without sample enrichment and cleanup. The collected water samples were filtered through a 0.22-μm filter membrane, their pH levels were adjusted to 6.0-8.0 after adding Na2EDTA, and then the solutions were mixed with an internal standard. The addition of Na2EDTA contributed to the release of tetracyclines and fluoroquinolones from the metal chelate. Improved recoveries were observed for all the compounds when the pH of the aqueous solution was set at 6.0-8.0. The optimized UHPLC conditions were as follows: chromatographic column, Phenomenex Kinetex C18 column (50 mm×30 mm, 2.6 μm); mobile phase, acetonitrile and 0.1% (v/v) formic acid aqueous solution; flow rate, 0.4 mL/min; injection volume, 100 μL. In the UHPLC-MS/MS experiment, chloramphenicol, thiamphenicol, and florfenicol were analyzed in the negative ionization scheduled multiple reaction monitoring mode (scheduled-MRM), while the other 39 antibiotics were analyzed in the positive scheduled-MRM mode. This acquisition method improved the response of each target compound by dividing the time of the analysis test cycle and scanning the ion channels of chromatographic peaks at different time periods. The ionspray voltage was set at 5500 and -4500 V in positive and negative modes, respectively. The source temperature for both ionization modes was set at 500 ℃, which was optimized to improve the sensitivity. Instrumental parameters like collision energy and declustering potential were also optimized. Good linearity was observed for all the tested antibiotics, with a correlation coefficient (r) greater than 0.995. The method detection limits (MDLs) were 0.015-3.561 ng/L. The average recoveries ranged from 80.1% to 125%, while the relative standard deviations (RSDs) were between 0.8% and 12.2%. The method was successfully applied to the determination of 10 source water samples and 5 tap water samples. Twelve antibiotics, viz. sulfachloropyridazine, sulfadiazine, sulfamethazine, sulfamethoxazole, sulfisomidine, clindamycin, lincomycin, roxithromycin, clarithromycin, erythromycin, thiamphenicol, and forfenicol, were detected in the 10 water samples with a detection frequency of 100%. The total antibiotic content in each sample ranged from not detected to 80.3 ng/L. Lincosamides and chloramphenicols were the predominant antibiotics in the water samples, with contents in the ranges of 3.83-13.7 and 4.23-33.6 ng/L, respectively. Therefore, the large volume direct injection method exhibited good performance in terms of MDL and recovery compared to standard methods and those reported previously. Compared with traditional pretreatment methods, the large volume direct injection method is simpler, more rapid, more precise, and more accurate. It is a viable alternative to SPE, and can be used for the determination of the 42 antibiotics at trace levels in cleaner water bodies, such as surface water, groundwater, and tap water.

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Determination of 19 illegally added chemical ingredients in hair loss prevention cosmetics by ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry DONG Yalei, NIU Shuijiao, QIAO Yasen, HUANG Chuanfeng, WANG Haiyan, SUN Lei 2022, 40 (4):  343-353.  DOI: 10.3724/SP.J.1123.2021.08019 Abstract ( 188 )   HTML ( 42 )   PDF (2188KB) ( 109 )   Cosmetic products for hair loss prevention are often mixed with prohibited substances such as hormones, antibiotics, and forbidden pharmacologically active substances. Although drugs increase the efficacy of cosmetic products, they cause skin irritation and allergic reactions, upon long-term exposure. Given the increasing number of hair loss prevention cosmetics on the market, the need to guarantee product safety calls for efficient and reliable methods to identify illegal ingredients in these products. Chromatography combined with high-resolution mass spectrometry offers the advantages of high resolution and high throughout, thus being a powerful technique for simultaneously detecting illegal ingredients in cosmetics. In this study, an ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) method for detecting 19 illegal chemical components was established. Combined with the scientific database, a screening platform for hair loss prevention cosmetics was constructed. The effect of extraction solvent was investigated. The chromatographic and mass spectrometry conditions were optimized. Under the optimal conditions, separation was achieved within 20 min on an ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm). Acetonitrile and 2 mmol/L ammonium formate solution containing 0.05% formic acid were used as mobile phases for gradient elution. The 19 compounds were detected by positive ion electrospray ionization (ESI) in the MSE mode. The chromatographic retention time, precursor ions, product ions of the target analytes, and abundance ratio were collected to construct a screening database with UNIFI software. The 19 compounds were well separated, with good linearity. The limits of detection (LODs) and limits of quantification (LOQs) were 0.025-0.05 μg/g and 0.075-0.15 μg/g, respectively. Hair lotion and shampoo, which are commonly marketed as hair loss prevention cosmetics, were selected as the respective matrices for the recovery experiment. The average recoveries of the 19 compounds ranged from 68.6% to 118.6%, and the relative standard deviations (RSDs) were 0.3%-10.3%. Then, 77 batches of cosmetic samples were detected and screened under the same conditions. The TOF-MS information, including the retention time, ion addition mode, mass-to-charge ratio of the parent ions and fragment ions, as well as the abundance ratio, were compared between the cosmetic samples and the standard MS information with UNIFI software. Finally, two batches of samples that were illegally adulterated with minoxidil and finasteride were identified. The ESI fragmentation pathway of the product ions from minoxidil was also proposed. The matrix matching external standard method was used to determine the amounts of minoxidil and finasteride in the two batches of hair lotion, and they were as high as 60 mg/g and 0.31 mg/g, respectively. This result revealed that multiple chemical components were simultaneously added to hair loss prevention cosmetics. Furthermore, the amount of the illegally added drug was very high, indicating high safety risk for consumers using such cosmetics. The present method has the advantages of simple operation, high sensitivity, and good reproducibility. It can be used for rapid screening and simultaneous quantitative analysis of various illegal chemicals in hair loss prevention cosmetics.

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Determination of polybrominated diphenyl ethers in serum using isotope internal standard-gas chromatography-high resolution dual-focus magnetic mass spectrometry WANG Mengmeng, XIE Linna, ZHU Ying, LU Yifu 2022, 40 (4):  354-363.  DOI: 10.3724/SP.J.1123.2021.10017 Abstract ( 152 )   HTML ( 29 )   PDF (2682KB) ( 115 )   A method for the determination of 14 polybrominated diphenyl ethers (PBDEs) in human serum using isotope internal standard-gas chromatography-high resolution dual-focus magnetic mass spectrometry (GC-HRMS) was developed. After thawed to room temperature, 0.5 mL serum samples were mixed with 13C-labeled isotopic internal standard. Subsequently, methanol was added to precipitate the proteins in the samples. The effects of three kinds of acids on the removal of cellulite from the serum samples and the corresponding recoveries were compared, and the results revealed that sulfuric acid was the most optimal. The target compounds were extracted by liquid-liquid extraction (LLE), and the effects of different extraction solvents on recoveries were compared. The results indicated that n-hexane (6 mL)-methyl tert-butyl ether (6 mL) was the best extraction solvent. The extracts were cleaned and eluted using solid phase extraction cartridges. Furthermore, the factors that influenced the cleanup effects and recoveries, including the solid phase extraction columns and elution solvents, were investigated in detail. The results indicated that the optimal conditions were cleanup with a silica gel column and elution with hexane-dichloromethane (1∶1, v/v). The eluate was re-dissolved in hexane after being blown to near dryness using nitrogen. The detection of PBDEs was performed using GC-HRMS. The instrument conditions were optimized, and the capillary column used was an Rtx-1614 column (30 m×0.25 mm×0.1 μm). Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The injector temperature was 290 ℃, and the oven temperature was programmed as follows: 150 ℃ for 2 min, 150 ℃ to 250 ℃ at 15 ℃/min, held for 1 min, 250 ℃ to 290 ℃ at 25 ℃/min, held for 3 min, and 290 ℃ to 320 ℃ at 25 ℃/min, held for 12.5 min. The injection volume was 1 μL in splitless mode. The samples were ionized in the positive electron ionization (EI) mode at 35 eV. Precursor ions and the production of each compound were identified using a voltage-selective ion detection (VSIR) program with a resolution of 10000. The ionization temperature was set at 280 ℃, and the transmission line temperature was set at 320 ℃. To ensure the integrity of the separation of low-brominated components, the column separation time was shortened, the response of high-boiling components was improved (BDE-190 and BDE-209), the decomposition of BDE-209 on the chromatographic column was effectively prevented, and the requirement of the simultaneous determination of multiple PBDEs was met. The method demonstrated good linearity in the range of 0.40 to 25 μg/L for BDE-209, and 0.08 to 5 μg/L for the other 13 PBDEs, with correlation coefficients greater than 0.995. The method detection limits (MDLs) were in the range of 0.01 to 0.51 μg/L, and the limits of quantification (LOQs) ranged from 0.04 to 1.70 μg/L. The recoveries of the 14 compounds ranged from 75.5% to 120.7%. The intra-day relative standard deviations (RSDs) were within 3.8%-10.9% (n=6) and the inter-day RSDs were within 4.2% to 12.4% (n=6). This method was successfully applied to the determination of 14 PBDEs in 15 serum samples from an adolescent population in an area. Notably, 1.86 to 4.66 ng/g lipid BDE-47 was detected in the serum samples with a detection frequency of 100%, and the other compounds were not detected. The results imply that the adolescent population in this region was exposed to some PBDE. Compared with the existing methods reported, this method has less sample demand and higher sensitivity and accuracy, can simultaneously determine 14 PBDEs, including BDE-209 in human serum, and effectively improve the efficiency of detection. This study offers a new method for studying the impact of polybrominated diphenyl ethers on population health in China.

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Preparation of cucurbitacin compounds in Siraitia grosvenorii roots by high speed countercurrent chromatography SUN Jiayi, SUN Jiaqi, LI Heping, YAN Xiaojie, LI Dianpeng, LU Fenglai 2022, 40 (4):  364-371.  DOI: 10.3724/SP.J.1123.2021.07010 Abstract ( 154 )   HTML ( 26 )   PDF (975KB) ( 61 )   Siraitia grosvenorii (Swingle) C. Jeffrey, belonging to the family Cucurbitaceae, is a natural sweetener. The roots of this plant are used in folk medicine for the treatment of rheumatoid arthritis. Cucurbitacins play an important role in the resistance of this plant to insects and adversity, and have anti-inflammatory, anti-tumor, and other biological activities. They usually exist as a variety of similar structures in Cucurbitaceae plants. Separation of a large amount of high-purity monomer compounds by the conventional separation method based on column chromatography is difficult, which limits the research and application of their activities. Therefore, we chose a new method for this separation. High-speed countercurrent chromatography (HSCCC) is a liquid-liquid chromatographic technique characterized by high recovery and reproducibility, and is considered a very effective method for the separation of natural compounds present in various plant extracts. An appropriate solvent system is the key for efficient separation, but its selection is tedious, which hampers the wider implementation of HSCCC in chemical research involving preparative separations. In this study, based on the general estimation strategy by using the TLC solvent system (GUESS), the corresponding relationship between the partition distribution coefficient (K value) and the TLC retention factor (Rf value) of the compounds was established by the partition experiment. The Rf value and separation coefficient α were calculated using the water-saturated organic phase as the expansion agent, which could minimize the number of countercurrent separation experiments required in solvent system selection. In this study, HSCCC was used to establish an efficient method for the extraction of cucurbitacins from the root extract of Siraitia grosvenorii. A fraction rich in cucurbitacins was obtained from the ethanol extract of Siraitia grosvenorii roots after separation by column chromatography on HPD-100, MCI, and C18 columns. Six types of solvent systems with different compositions were investigated using the GUESS method. The results showed that employing the solvent system of n-hexane-ethyl acetate-methanol-water (3∶7∶3∶7, v/v/v/v) to partition the cucurbitacin fraction could remove a large number of impurities. The components retained in the upper phase in the partition experiment were subsequently purified by HSCCC. The favorable solvent system for HSCCC was n-hexane-ethyl acetate-methanol-water (4∶6∶5∶5, v/v/v/v), while the upper and lower phases were selected as the stationary and mobile phases, respectively, with a flow rate of 2.0 mL/min, a rotation speed of 860 r/min, and an injected sample weight of 280 mg. Five cucurbitacin compounds were obtained by one-time separation. The weights of the five compounds were 14.73, 8.82, 30.74, 5.03, and 3.81 mg. The purities of these compounds were 97.0%, 95.4%, 96.3%, 91.6%, and 95.3%, respectively. Their structures were identified as cucurbitacin Q1, 23,24-dihydrocucurbitacin F-25-acetate, cucurbitacin B, 23,24-dihydrocucurbitacin B, and dihydroisocucurbitacin B-25-acetate by1H-NMR and 13C-NMR spectroscopies, along with comparison with the literature. This study demonstrated how GUESS guidance accelerates the selection of HSCCC solvent systems, simplifies the workflow, and it provides an efficient preparative method for the separation of chemical constituents from the Siraitia grosvenorii roots, which can also be used as a new method for the large-scale preparation of cucurbitacin compounds.

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Application of magnetic immunofluorescence assay based on microfluidic technology to detection of Epstein-Barr virus LI Junhao, HAN Guanhua, LIN Xiaotao, WU Liqiang, QIAN Chungen, XU Junfa 2022, 40 (4):  372-383.  DOI: 10.3724/SP.J.1123.2021.09005 Abstract ( 240 )   HTML ( 21 )   PDF (7219KB) ( 145 )   Early diagnosis of Epstein-Barr virus (EBV) can reduce the risk of major illnesses. Disadvantages of EBV antibody detection methods that are commonly used clinically include lengthy assay time, need for a lot of reagent, and low efficiency. Compared with traditional detection methods, microfluidics technology offers high throughput, low reagent consumption, less bio-contamination, and a higher degree of automation. Advantages of magnetic immunofluorescence technology include high detection efficiency and a strong signal. The combined advantages of the two methods can compensate for the shortcomings of traditional methods. In the present study, polymethyl methacrylate (PMMA) as the raw material was subjected to laser cutting and vacuum hot pressing to quickly obtain chips. Magnetic beads labeled with antigen and fluorescent microspheres labeled with anti-human antibody were then rapidly lyophilized into microspheres by freeze-drying and embedded into the chips. After incubation and cleaning, the last step was detection. Image J software was used to analyze the mean fluorescence intensity and obtain negative or positive test results. To determine the precision of the chip, high- and low-value samples of each item were retested 10 times. The mean values were calculated to obtain the relative standard deviation (RSD) for several common pathogens. Furthermore, the coincidence rate of clinical samples was tested using a chemiluminescence immunoassay (CLIA) to determine the potential clinical application value. The RSD of the precision test for each item was <10%, indicating good precision. The precision of the accelerated stability test was not verified. Specificity test results revealed no cross-reaction with some common pathogen antibodies, indicating good specificity. It remains to be verified whether the antibodies detected by this method cross-react with other herpes simplex viruses, such as types 1 and 2, Kaposi’s sarcoma-associated virus, and human herpes virus type 6 and 7. Of the 121 clinical samples tested, statistical analysis of the data indicated good agreement with the chemiluminescence immunoassay in clinical trials. EB viral capsid antigen (EB VCA) IgG positive coincidence rate was 95.77% (68/71), the negative coincidence rate was 86% (43/50) (Kappa=0.828, P<0.05), the limit of detection (LOD) was 1.92 U/mL, and the linear range was 1.92 to 200 U/mL. The EB VCA IgA positive coincidence rate was 92% (46/50), negative coincidence rate was 92.96% (66/71) (Kappa=0.847, P<0.05), LOD was 2.79 U/mL, and the linear range was 2.79 to 200 U/mL. The positive coincidence rate of EB nuclear antigen 1 (EB NA1) IgG was 92.96% (66/71), the negative coincidence rate was 92% (46/50) (Kappa=0.847, P<0.05), the LOD was 3.13 U/mL, and the linear range was 3.13 to 200 U/mL. The positive coincidence rate of EB NA1 IgA was 90% (45/50), the negative coincidence rate was 91.55% (65/71) (Kappa=0.813, P<0.05), the LOD was 1.53 U/mL, and the linear range was 1.53 to 200 U/mL. Compared with the traditional enzyme-linked immunosorbent assay, the novel method featured a shorter detection time, reduced use of reagent, high degree of automation, and less bio-contamination. Compared with CLIA, advantages of the novel method include multi-item combined detection, long luminescence time, and simple use as a basic health service. Compared with silicon and ceramic microfluidic chips, advantages of the selected PMMA material include low processing cost, short processing time, simple processing technology, and easy industrialization. A refinement that can still be made include the use of molding instead of laser cutting technology, which can further shorten the chip processing time. In summary, a microfluidic detection platform was initially built to provide a rapid, sensitive, simple, highly automated, and easy to be used by basic health service for the quantitative combined detection of EBV VCA and EB NA1 IgG and IgA.

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Multi-channel contactless conductivity detection device for online detection of free-flow electrophoresis separation LIANG Ziqi, ZHANG Qiang, JIANG Xiaoteng, LIU Xiaoping, CAO Chengxi, XIAO Hua, LIU Weiwen 2022, 40 (4):  384-390.  DOI: 10.3724/SP.J.1123.2021.11011 Abstract ( 161 )   HTML ( 20 )   PDF (2458KB) ( 55 )   Free-flow electrophoresis (FFE) is an all-liquid-phase electrophoresis technique without any supporting media, which has both analytical and preparative functions. Compared to other electrophoresis techniques, FFE has been used for the separation of peptides, proteins, cells, and microorganisms due to its advantages of mild separation environment, high recovery, and sustainable separation. Both the online detection of the characteristic parameters for each component solution and the real-time control of the progress of the separation experiment are of considerable importance for the study of FFE separation. Since the existing FFE devices do not have the online detection function, there are obvious deficiencies in their practicability. The absence of online detection function not only made it impossible to track the progress of the separation experiment in real time, but also made it difficult to detect the properties of the component solutions, which still require offline testing after separation. In this study, a multi-channel capacitively coupled contactless conductivity detection (MC-C4D) device has been developed to solve this problem, and an automatic measurement software has also been developed. The MC-C4D device used a parallel time-sharing contactless conductivity detection technique. It consisted of several contactless conductivity detection modules arranged in parallel, which in turn consisted of a number of contactless conductivity cells that were switched on/off by analog multiplexers for detecting the conductivity of the solution flowing through the cells in real time. The number of cells was equal to the number of components of the FFE. The components were connected to each of the FFE flow channels, such that the MC-C4D device could be used to measure the conductivity of the solution flowing through each channel in parallel online. To verify the performance of the MC-C4D device, calibration was conducted by using potassium chloride (KCl) standard solutions on MC-C4D device. The experimental data showed that the detection range of MC-C4D was 0.015-2.5 mS/cm, and the limit of detection (LOD) was 0.002 mS/cm. The intra-day relative standard deviation (RSD, n=3) was 2.31%, the measurement relative error (RE) was 3.03%, and the measurement difference between channels was 1.60%. All these data validated that the device had the advantages of wide detection range, low LOD, good repeatability, high accuracy, and low variation between channels. The MC-C 4D device was also applied to reciprocating free-flow isoelectric focusing (RFFIEF) electrophoresis for real-time online detection of the conductivity of each component solution during protein focusing. At the start of isoelectric focusing, when the ions had not reached equilibrium loading in the electric field and the pH gradient had not yet been fully developed, there was little difference in conductivity between the different channels and the channel conductivity curve was relatively flat. As the experiment progressed, the proteins gradually started to enrich the anodic end. As the proteins accumulated towards the isoelectric point, their own net charge gradually decreased, and thus, the conductivity of the solution in the channels near the anodic region also decreased. Under sufficient isoelectric focusing, protein enrichment was evident. In the focusing region, the conductivity of the solution in the corresponding channel decreased further. There was also an increase in the conductivity of the solution in the corresponding channel due to the accumulation of ions near the electrode ends. These results showed that the MC-C4D device not only enabled real-time online detection of the conductivity of each component solution in FFE, but also aided in mastering the progress of separation experiment in RFFIEF, thus improving the practicality of the FFE device. Thus, the MC-C4D device, which had the advantages of good performance, small size, simple circuit system, easy installation and commissioning, and low cost, could play an important role in multi-channel measurement, online inspection, and process monitoring.

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Preparation and application of porous organic cage capillary electrochromatographic chiral column JIA Wenyan, TANG Minghua, ZHANG Junhui, YUAN Liming 2022, 40 (4):  391-398.  DOI: 10.3724/SP.J.1123.2021.07004 Abstract ( 184 )   HTML ( 28 )   PDF (2719KB) ( 137 )   Capillary electrochromatography for enantioseparation has received considerable research attention in the past decades, because it integrates the advantages of classical electrophoresis and modern micro-column separation. Chirality is a fundamental feature of compounds found in nature and is also a major concern in the modern pharmaceutical industry. Porous organic cages (POCs) are defined as a class of porous materials with permanent ordered three-dimensional cavity structures that are different from those of porous materials, such as zeolite, metal-organic frameworks, covalent organic frameworks, and mesoporous silica. POCs have good solubility in general organic solvents and can be used as a chromatographic stationary phase conveniently coated inside a standard capillary column. Homochiral POCs with hydroxyl groups on the cage molecules were synthesized by imine-linked condensation of 2-hydroxy-1,3,5-triformylbenzene with (1R,2R)-1,2-diphenylethylenediamine. The thus-synthesized POCs were characterized by nuclear magnetic resonance (NMR) spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, X-ray powder diffraction (XRD) analysis, etc. In the FT-IR spectra, the absorption peaks at 1602, 1489, and 1458 cm-1 were attributed to the C=C-H and C=C tensile vibrations in the benzene ring. The strong characteristic absorption peak at 1636 cm-1 was attributed to the imine bond (C=N) stretching, the two peaks at about 2900 cm-1 were attributed to C-H bond vibration, and the absorption peak at 3420 cm-1 was attributed to the O-H pulling vibration. In the XRD patterns, the powder diffraction peaks of the POCs were consistent with the simulated data. These results indicated that POCs were successfully synthesized. Thermogravimetric analysis was performed in the temperature range of 25-800 ℃ (10 ℃/min), and the POCs were found to be stable up to 380 ℃. Dichloromethane was used as solvent to uniformly coat POCs on the capillary wall to prepare an electrochromatography column. Joule heat generated in electrophoresis was negligible under the experiment condition used for the open-tubular column. Four chiral compounds, viz. dihydroflavone, praziquantel, naproxen, and 3,5-dinitro-N-(1-phenylethyl)benzamide, were used as test compounds, and the electrochromatography separation conditions were optimized such that the best separations were obtained. The voltage was applied to separate the selected enantiomers in the range of 10-20 kV. Considering the good separation and appropriate migration time simultaneously, applied voltages of 13 kV and 12 kV were recommended for dihydroflavones and 3,5-dinitro-N-(1-phenylethyl)benzamide, respectively, as well as 14 kV for praziquantel and naproxen. The concentration of the buffer solution for dihydroflavonoids was 0.075 mol/L, and those for praziquantel, naproxen, and 3,5-dinitro-N-(1-phenylethyl)benzamide were 0.100 mol/L. The pH was 3.51 for all four substances. Resolutions of 2.99, 2.10, 2.58, and 3.59 were achieved on a POC chiral column for dihydroflavonoids, praziquantel, naproxen, and 3,5-dinitro-N-(1-phenylethyl)benzamide, respectively. Two positional isomers, viz. o,m,p-nitrophenol and o,m,p-nitrophenilamine, were also successfully separated with 0.100 mol/L Tris-H3PO4 at pH 3.51. Therefore, the chiral electrochromatography column showed good chiral recognition ability and the POC is an excellent separation material with excellent application prospect in chiral electrochromatography.