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Chinese Journal of Chromatography
2023, Vol. 41, No. 12
Online: 08 December 2023

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Preparation of a block copolymer-based temperature-responsive affinity chromatography stationary phase for antibody separation and purification GUO Dongmei, XIA Yiran, Mujeeb ur RAHMAN, WANG Jianzhong, LIU Jiawei, BAI Quan 2023, 41 (12):  1045-1051.  DOI: 10.3724/SP.J.1123.2023.09028 Abstract ( 218 )   HTML ( 59 )   PDF (1049KB) ( 132 )   Antibodies play an essential role in cancer diagnosis and treatment because of the specificity for target biomolecules and reduction of side effects. However, antibodies separation and purification still face some challenges. Antibody elution from columns using a low-pH aqueous solution leads to aggregation or loss of activity of the antibody drugs. In this paper, a block copolymer-based temperature-responsive affinity chromatography (TRAC) stationary phase, SiO2-P[NIPAM-b-4VP]-MEP using the block temperature-responsive copolymer poly(N-isopropylacrylamide-b-4-vinylpyridine) (P[NIPAM-b-4VP]) as the space arms and 4-mercaptoethyl pyridine (MEP) as the ligand was prepared for antibody separation. The TRAC column was tested using bovine serum albumin (BSA) and γ-globulin as model proteins, and the effects of salt concentration in the mobile phase and temperature on their separation were studied in detail. At 40 ℃, the TRAC stationary phase only selectively retained γ-globulin due to the specific affinity interaction between antibodies and the ligand MEP. At 5 ℃, γ-globulin can be eluted from the column with a mass recovery of 92.7% using a Tris-HCl buffer (pH 8.0) solution containing 0.6 mol/L NaCl. The adsorption capacity of γ-globulin on this stationary phase was (71.5 ±2.1) mg/g (n=3), which was twice that of a traditional temperature-sensitive affinity chromatography stationary phase SiO2-PNIPAM-MEP. The stationary phase was also used to separate and purify immunoglobulin (IgG) in human serum in one step by altering the temperature and ion strength of the mobile phase, resulting in a purity of 97.4%±0.7%. Thus, this new technology has specific selectivity for antibodies, as well as mild and green elution conditions, ultimately resolving the problem of traditional affinity chromatography using acid elution, which can lead to the antibodies aggregation/inactivation. This technology has great application potential for the industrial production of antibody drugs.

Reviews

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Advances in synthesis methods and applications of microporous organic networks for sample preparation YU Tao, CHEN Li, ZHANG Wenmin, ZHANG Lan, LU Qiaomei 2023, 41 (12):  1052-1061.  DOI: 10.3724/SP.J.1123.2023.07003 Abstract ( 198 )   HTML ( 46 )   PDF (1340KB) ( 108 )   Sample pretreatment is an essential step in chromatographic analysis. Solid phase extraction (SPE) is a widely used sample pretreatment method. In SPE, the quality of the adsorbent directly affects the adsorption and enrichment efficiency of the target compounds as well as the sensitivity and selectivity of the pretreatment and subsequent analysis. Therefore, the selection and development of adsorbents has become a research hotspot. Microporous organic networks (MONs) are a novel type of covalent organic materials that are synthesized by the Sonogashira reaction of aromatic alkynes and aromatic halides. These networks have the advantages of modifiable structures, large specific surface areas, high porosity, and simple synthesis methods. This paper reviews the synthesis and functional modification methods of MONs, with an emphasis on their applications in sample pretreatment. Future development trends are also prospected. In terms of synthesis, the preparation methods for MON-based materials have progressed from reflux and solvothermal synthesis to room-temperature synthesis, the conditions of which tend to be milder and more efficient. In terms of functional modification, the introduction of macromolecules and active groups (including amino, hydroxyl, and carboxylic groups) can increase the selectivity and active sites of MON-based materials. The combination of MONs with Fe3O4, SiO2, and metal organic frameworks yields core-shell-structured MONs. Furtherly, they can be calcined and etched to form porous carbon structures or hollow multilayer materials. Functionalized MONs and their composite materials have multiple interaction mechanisms (e. g., hydrogen bonding, hydrophobic, electrostatic, and π-π interactions) with various target compounds, thereby realizing their efficient extraction. MONs can be used as adsorbent materials in SPE, Solid phase microextraction, dispersed solid phase extraction, magnetic solid phase extraction, and other pretreatment methods. When combined with chromatography and chromatography-mass spectrometry, MONs achieve good adsorption effects and high sensitivity, demonstrating the application potential of these materials in sample pretreatment.

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Application progress of on-line sample preparation techniques coupled with liquid chromatography-mass spectrometry system in the detection of food hazards ZHAI Hongwen, MA Hongyu, CAO Meirong, ZHANG Mingxing, MA Junmei, ZHANG Yan, LI Qiang 2023, 41 (12):  1062-1072.  DOI: 10.3724/SP.J.1123.2023.04026 Abstract ( 183 )   HTML ( 23 )   PDF (1291KB) ( 97 )   Food safety has received increased attention, and food detection is of great significance. The food matrix is complex, and diverse food hazards have been identified. Thus, the detection methods and sample preparation techniques for food matrices must be continuously optimized and updated. Several steps are usually required when a chromatographic system is used to determine food hazards: sample preparation, that is, the separation of targets from different substrates using a suitable preprocessing method and target-substance separation and purification, which is usually achieved using chromatographic separation. The selection of an appropriate detector for qualitative and quantitative analyses is usually based on the properties of the target compound. The sample preparation procedure is considered the most time-consuming aspect of the entire food-analysis process. It is also prone to analytical errors. Therefore, optimization of the sample preparation process is a key issue in the field of chemical analysis. Researchers have developed a series of new, efficient, and accurate sample preprocessing methods, and an on-line sample-preparation system has been found to be a feasible approach. On-line sample preparation coupled with liquid chromatography-mass spectrometry (LC-MS) presents many advantages. First, manual operation could reduce analytical errors to ensure good accuracy and repeatability. It could also reduce the consumption of chemical reagents and avoid cross-contamination between samples. Furthermore, an on-line sample-preparation system could shorten the sample-preparation time and improve the detection efficiency. On-line sample preparation coupled with LC-MS has been widely applied in the fields of environment, biology, and food. On-line sample preparation systems coupled with LC-MS are divided into two modules: the first modules involves sample preparation and the second module involves the LC system. The first module remove impurities and isolates the target compounds in preparation for their qualitative and quantitative detection. The coupling of these two modules depends mainly on valve switching. In this paper, we introduce the most frequently used on-line sample-preparation techniques, including on-line solid phase extraction (on-line SPE), in-tube solid phase microextraction (in-tube SPME), and turbulent chromatography (TFC). We then describe the basic principles and coupling equipment of these three on-line analytical technologies in detail. The coupling equipment establishes a physical connection between the two modules. Next, we discuss the properties of different purification fillers in an on-line sample-preparation column. The applications and research progress of on-line systems for pesticide residues, veterinary drug residues, and biotoxins are also discussed. Compared with offline sample preparation, on-line analytical systems present several advantages. On-line analytical systems can not only greatly reduce the analysis time and solvent consumption but also improve the detection sensitivity and accuracy. Such systems can be used to determine food hazards to ensure food safety. Finally, the existing problems and development trends of on-line analytical systems are discussed and prospected. To promote the applications of on-line analytical technology in food-safety detection, we suggest that the following three aspects be considered. First, more on-line purification columns with novel fillers, in addition to C18 or polymer fillers, should be developed. Second, compared with ordinary detectors, high-resolution MS detectors have better precision and accuracy. Coupling on-line analytical technologies with a high-resolution mass spectrometer may be beneficial for the further development of on-line analyses. Third, different food matrices should be compared and evaluated to continuously optimize the detection process and improve the efficiency of on-line analytical systems. As concerns regarding food safety issues have increased, the applications of on-line analytical technologies for food detection can be expected to become increasingly important.

Articles

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Tandem mass tag-based quantitative proteomics analysis of plasma and plasma exosomes in Parkinson’s disease ZHAO Yuan, LIU Xin, ZHANG Yidan, ZHANG Jian, LIU Xiang, YANG Guofeng 2023, 41 (12):  1073-1083.  DOI: 10.3724/SP.J.1123.2022.12022 Abstract ( 256 )   HTML ( 31 )   PDF (3512KB) ( 100 )   The cardinal clinical features of Parkinson’s disease (PD), a common neurodegenerative disease, include the irreversible impairment of movement coordination, such as tremors, gait rigidity, bradykinesia, and hypokinesia. Although various factors are associated with the pathological changes in PD, such as oxidative stress, mitochondrial dysfunction, and neuroinflammation, the availability of treatments to retard PD progression is limited. Therefore, novel biomarkers for PD diagnosis and therapeutic targets are urgently needed. The diagnosis of PD mainly depends on its clinical manifestations and has an error rate of approximately 20%. Studies have shown that α-synuclein (α-syn) levels are significantly increased in the cerebrospinal fluid of patients with PD; however, the invasive nature of lumbar puncture restricts further studies on its clinical applications. Hence, the development of novel peripheral blood markers would be helpful for the early diagnosis of PD. Exosomes are extracellular vesicles (EVs) released by various cell types under physiological and pathophysiological conditions. Because exosomes carry a variety of bioactive molecules, they play a key role in biological processes such as intercellular communication and the immune response. Central nervous system (CNS)-derived exosomes can be detected in the cerebrospinal and peripheral body fluids of patients with PD, and their contents are altered during the disease process, rendering them an attractive biomarker resource. Therefore, a comprehensive and high-throughput investigation of the plasma and its exosomes may enhance our understanding of PD. In this study, we isolated exosomes from plasma using standard differential centrifugation and performed tandem mass tag (TMT)-labeled quantitative proteomic analysis of plasma and plasma exosome samples from healthy individuals and patients with PD using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 724 proteins were quantified in the plasma samples, and 611 proteins were screened from the exosome samples. Among these 611 proteins, 413 were found in the Exosomal Protein Database (Exocarta). Using |log2FC|>0.26 and P-value (P)<0.05 as the cutoff, five upregulated and six downregulated proteins were identified in the plasma samples of the PD group compared with the healthy group. In the plasma exosome samples, compared with the healthy group, the PD group showed six upregulated and seven downregulated proteins. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted based on gene set enrichment analysis (GSEA). GO-cellular component (CC) analysis revealed that plasma-enriched proteins were mainly located in the nucleus whereas plasma exosome-enriched proteins were mainly located in the cytoplasm. According to the GO-molecular function (MF) analysis, the MFs of differentially expressed proteins in the plasma were mainly enriched in RNA, DNA binding, and complement binding. By contrast, the molecular functions of differentially expressed proteins derived from plasma exosomes were enriched in antioxidant activity, oxidoreductase activity, and peroxide acceptor activity. We then analyzed the enriched KEGG pathways of differentially expressed proteins derived from the plasma and plasma exosome samples. The enrichment pathways of differentially expressed proteins in the plasma samples included the lysosome pathway, cellular senescence, and protein processing in the endoplasmic reticulum. By contrast, the enrichment pathways of differentially expressed proteins in the plasma exosome samples included chemokine signaling and cytokine receptor interactions. Finally, we assessed the functions of some exosomal proteins in PD to elucidate their potential for PD diagnosis and treatment. Significant differences were observed between the plasma and plasma exosome protein profiles, and the functions of differentially expressed proteins in plasma exosomes were strongly related to the pathology of PD. Our study provides a reference for identifying the potential biomarkers and therapeutic targets of PD.

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Simultaneous determination of 83 glucocorticoids in cosmetics by ultra performance liquid chromatography-tandem mass spectrometry ZHAO Qianru, LIU Hua, MENG Yaping, LI Xiang, GAO Ruifang, LI Xiangsheng 2023, 41 (12):  1084-1094.  DOI: 10.3724/SP.J.1123.2023.04009 Abstract ( 239 )   HTML ( 40 )   PDF (1667KB) ( 191 )   Glucocorticoids, which are a class of steroidal hormones secreted by the adrenal cortex, have significant anti-inflammatory, immunosuppressive, and anti-allergic effects. Thus, these compounds are widely used in clinical practice. However, the long-term use of cosmetics containing glucocorticoids can lead to serious consequences, such as hormone-dependent dermatitis, hypertension, and other serious injuries. The Safety and Technical Specification for Cosmetics (2015 edition) and Regulation (EC) No. 1223/2009 of the European Parliament and Council on cosmetic products list glucocorticoids as prohibited raw materials. According to the National Medical Products Administration, reports on the illegal addition of glucocorticoids to cosmetics by manufacturers have increased in recent years. Therefore, establishing high-throughput screening methods to ensure the quality and safety of cosmetics is imperative. In this study, a comprehensive analytical method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the rapid screening of 83 glucocorticoids in cosmetics. A series of conditions were optimized using three matrices that are commonly used in cosmetics: water, lotion, and cream (o/w-type). Four mobile-phase systems and three chromatographic columns were then optimized to achieve the best separation effects. Various MS parameters, such as the capillary voltages, cone voltages, desolvation gas flow rates, and collision energies of the ion pairs of the target compounds, were also optimized. Furthermore, pretreatment was essential for glucocorticoid determination owing to the complex matrix effects of cosmetics. The analytes were divided into two groups, with lg Kow=4 as the limit, to compare the effects of the extraction solvent on recoveries. The extraction recoveries of target analytes with six extraction methods, namely, extraction with acetonitrile, extraction with acetone, extraction with ethyl acetate, dispersion in saturated sodium chloride solution followed by extraction with acetonitrile, dispersion in saturated sodium chloride solution followed by extraction with acetone, and dispersion in saturated sodium chloride solution followed by extraction with ethyl acetate, were compared. The recoveries from QuEChERS and solid-phase extraction (SPE) purification were also compared. Based on the experimental results, the final sample pretreatment method included acetonitrile vortex dispersion, ultrasonic extraction, and sample loading after filtration. The 83 target compounds were separated on a Thermo Accucore PFP column (100 mm×2.1 mm, 2.6 μm) with 0.1% (v/v) acetic acid in acetonitrile and 0.1% (v/v) acetic acid in water as the mobile phases. The analytes were determined by dynamic multiple-reaction monitoring (MRM) in electrospray positive ionization mode (ESI+) and quantified using the external standard method. Matrix standard curves were used to reduce matrix effects. The calibration curves of the 83 target compounds were linear in the mass concentration range of 2-200 μg/L (r>0.995). At three levels of addition, the recoveries were 74.5%-112.4%, and the relative standard deviations (RSDs, n=6) were 0.8%-9.9%. The limits of detection (LODs, S/N≥3) were 0.001-0.023 μg/g, and the limits of quantification (LOQs, S/N≥10) were 0.002-0.076 μg/g. The developed method was used to detect glucocorticoids in 41 cosmetic samples. Fluocinolone acetonide, beclomethasone dipropionate, desonide 21-acetate, and desonide were detected in four samples. The content range of glucocorticoids in the positive samples was 0.53-634.27 μg/g. Notably, desonide 21-acetate, which is not included in the scope of the statutory detection method, was detected in two batches of samples. In conclusion, the proposed method is simple, sensitive, reliable, and suitable for the high-throughput analysis of the 83 glucocorticoids in cosmetics with different matrices. This method could provide reliable technical support for the daily supervision of cosmetics and serve as a supplement to current glucocorticoid standards.

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Determination of 14 perfluoroalkyl substances in Chinese mitten crab by multi-plug filtration cleanup coupled with ultra-performance liquid chromatography-tandem mass spectrometry WANG Xianli, RAO Qinxiong, ZHANG Qicai, DU Penghui, SONG Weiguo 2023, 41 (12):  1095-1105.  DOI: 10.3724/SP.J.1123.2023.07017 Abstract ( 200 )   HTML ( 45 )   PDF (2522KB) ( 96 )   Perfluoroalkyl substances (PFASs) have become a new food-safety problem. Dietary intake is a major pathway of human exposure to PFASs. Chinese mitten crab (Eriocheir sinensis) is a high-end aquaculture product popular among consumers in China. Conventional extraction methods for PFASs are cumbersome and time consuming, and result in incomplete purification; thus, this technique does not meet the requirements for PFAS detection. Herein, an analytical strategy was established for the rapid detection of 14 PFASs in Chinese mitten crab based on multi-plug filtration cleanup (m-PFC) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The carbon-chain length of the 14 PFASs analyzed in this study ranged from 4 to 14, and they are perfluorobutanoic acid (PFBA), perfluoro-n-pentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), perfluorotetradecanoic acid (PFTeDA), perfluoro-1-butane sulfonic acid (PFBS), perfluoro-1-hexane sulfonic acid (PFHxS), perfluoro-1-octane sulfonic acid (PFOS), and perfluoro-1-decanesulfonate (PFDS). The m-PFC column was prepared using carboxy-based multiwalled carbon nanotubes, and used to reduce the interference of sample impurities. The samples were extracted with 5 mL of 0.1% formic acid aqueous solution, 15 mL of acetonitrile and extraction salt (2 g Na2SO4 and 2 g NaCl). The supernatant (10 mL) was purified using the m-PFC column, concentrated to near dryness under nitrogen, and then redissolved in 1 mL of methanol. Finally, the sample solution was filtered through a 0.22 μm polypropylene syringe filter for UPLC-MS/MS analysis. The target analytes were separated using a Shimadzu Shim-pack G1ST-C18 chromatographic column (100 mm×2.1 mm, 2 μm) using methanol (A) and 5 mmol/L ammonium acetate aqueous solution (B) as the mobile phases via gradient elution. The linear gradient program were as follows: 0-0.5 min, 10%A-35%A; 0.5-3 min, 35%A-60%A; 3-5 min, 60%A-100%A; 5-6.5 min, 100%A; 6.5-7 min, 100%A-10%A. The target analytes were analyzed using negative electrospray ionization in multiple-reaction monitoring mode, and quantitative analysis was performed using the internal standard method. In this study, we optimized the mobile-phase system as well as the extraction solvent, time, volume, and salt. The 14 PFASs exhibited good peak shapes and sensitivities when the 5 mmol/L ammonium acetate solution-methanol system was used as the mobile phase. Compared with acetonitrile or methanol alone, the extraction efficiencies of the 14 PFASs were significantly improved when 5 mL of 0.1% formic acid aqueous solution was added, followed by 15 mL of acetonitrile. The extraction efficiencies of the 14 PFASs did not differ significantly when the extraction time was within 3-15 min. The extraction salt (MgSO4, Na2SO4, NaCl, (NH4)2SO4, and Na2SO4+NaCl) significantly affected the extraction efficiencies of the 14 PFASs. The highest extraction efficiencies of the 14 PFASs, which ranged from 47.9% to 121.9%, were obtained when Na2SO4+NaCl was used as the extraction salt. Under the optimal experimental conditions, good linearities (R2=0.998-0.999) were obtained for seven PFASs (PFBS, PFHxA, PFHpA, PFHxS, PFDA, PFDoDA, PFTeDA) at 0.10-100 μg/L, and seven PFASs (PFBA, PFPeA, PFOA, PFOS, PFNA, PFUnDA, PFDS) at 0.50-100 μg/L. The average spiked recoveries for the 14 PFASs in Chinese mitten crabs at three levels ranged from 73.1% to 120%, with relative standard deviations (RSDs) in the range of 1.68%-19.5%(n=6). The limits of detection (LODs) and quantification (LOQs) of the 14 PFASs were in the range of 0.03-0.15 and 0.10-0.50 μg/kg, respectively. The developed method was applied to the analysis of crab samples collected from three farms in Shanghai, and PFASs with total concentrations of 3.52-37.77 μg/kg were detected in all samples. The detection frequencies for PFDA, PFUnDA, PFDoDA, PFTeDA, and PFOS were 100%. PFDA, PFUnDA, PFOS, and PFDoDA were the most abundant congeners, accounting for 31.2%, 30.6%, 15.0%, and 10.9%, respectively, of the 14 PFASs detected. The proposed method is simple, efficient, accurate, and suitable for the rapid analysis of 14 PFASs in Chinese mitten crabs.

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Determination of 14 β-agonists in animal meat by ultra high performance liquid chromatography-tandem mass spectrometry DONG Jieqiong, XIAO Jin, ZHOU Xin, LI Ning, WANG Xuesong, KANG Junjie 2023, 41 (12):  1106-1114.  DOI: 10.3724/SP.J.1123.2023.03008 Abstract ( 201 )   HTML ( 32 )   PDF (1292KB) ( 113 )   The addition of β-agonists to animal feed can significantly improve the lean-meat rate of pigs, cattle, sheep, and other animals. However, the food residues of β-agonists are harmful to human health. When meat with β-agonist residues is consumed, poisoning symptoms such as palpitation, dizziness, and muscle tremors may develop, and damage to the cardiovascular system, liver, and kidney may occur. In this study, a method based on ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established for the rapid detection of 14 β-agonists (clenbuterol, salbutamol, ractopamine, clorprenaline, terbutaline, tulobuterol, bromobuterol, bambuterol, zilpaterol, mabuterol, fenoterol, arformoterol, cimaterol, and cimbuterol) in animal food sources. The sample pretreatment method and chromatographic conditions were optimized. The samples were hydrolyzed with β-glucuronidase hydrochloride/aryl sulfate esterase in ammonium acetate buffer (pH 5.2). Enzymatic hydrolysis was performed in a constant-temperature water bath ((36±2) ℃) oscillator for 16 h. The samples were cooled to room temperature and extracted with 0.5% formic acid acetonitrile. NaCl was added to separate the organic and aqueous phases, and 5 mL of the upper organic layer was purified using a one-step purification solid-phase extraction column. After drying with nitrogen at 50 ℃, the residue was dissolved in 0.4 mL of 0.2% formic acid aqueous solution. The samples were passed through a 0.22 μm filter and detected by UHPLC-MS/MS with gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The analytes were separated on a Phenomenex Kinetex F5 column and detected by positive-ion scanning in multiple-reaction monitoring (MRM) mode. Internal and external standard methods were used for quantitative analysis. The effects of the extract pH, solid-phase extraction column, purification method, and dissolved solution on the extraction efficiency were optimized during pretreatment. UHPLC-quadrupole time-of-flight MS was used to verify the purification effect of the one-step purification solid-phase extraction column, and the results indicated that this type of column could remove most of the phospholipids, sphingolipids, and glycerides in the sample extract. The factors influencing the different chromatographic columns and mobile phases were investigated. MS scanning was conducted in positive-ion mode with needle pump injection in mass-only mode, and the two daughter ions with the highest responses for each target were selected as the quantitative and qualitative ions. The declustering potential (DP) and collision energy (CE) of each ion were separately optimized in MRM mode. The switching mode of the mass spectrum and waste liquid was used, and the mobile phase was switched to waste liquid after all the target peaks were removed. These steps ensured that impurities in the sample flowed out of the column in a timely manner and that the effects of excessive impurities on the mass spectra were avoided. The 14 β-agonists showed good linear relationships in the range of 1.0-50 μg/L, with correlation coefficients of >0.99. The limits of detection (LODs) and quantification (LOQs) were in the range of 0.1-0.2 and 0.3-0.6 μg/kg, respectively. The average recoveries of the 14 β-agonists ranged from 70.25% to 117.48%, with relative standard deviations (RSDs) in the range of 0.63%-14.29% at low, medium, and high spiked levels. Pork, beef, and mutton samples were selected and analyzed using the developed method. The results were close to those of the national standard method, indicating that the method is accurate and reliable. Moreover, the proposed method has good stability and high accuracy; thus, it is suitable for the qualitative and quantitative determination of β-agonists in animal meat.

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Off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography separation of Curcuma volatile oil TONG Xin, JIN Yang, JIN Jing, LIU Ping, WU Chunyan, TONG Shengqiang 2023, 41 (12):  1115-1120.  DOI: 10.3724/SP.J.1123.2023.04008 Abstract ( 102 )   HTML ( 14 )   PDF (1093KB) ( 38 )   The chemical constituents of volatile oils used in traditional Chinese medicine are highly complex. Thus, achieving the complete separation of volatile oils by one-dimensional chromatography is difficult owing to the low peak capacity of the technique. Although comprehensive two-dimensional gas chromatography provides an efficient means for separating volatile oils, it cannot be used to screen bioactive components because of its limitations. Therefore, developing a new method to separate volatile oils based on liquid chromatography is of great significance in efforts to obtain new approaches to screen bioactive components in volatile oil. The objectives of the present study are to establish an efficient method for separating the chemical constituents of Curcuma volatile oil using off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography (CCC-LC) and to investigate the two-dimensional peak capacity, orthogonality, and spatial coverage of this method. Both CCC and LC conditions were optimized. A biphasic n-hexane-methanol-water solvent system was selected via the colorimetric method, and the lower phase was used as the mobile phase in gradient-elution mode: 0-55 min, n-hexane-methanol-water (5∶2∶3 v/v/v); 55-170 min, n-hexane-methanol-water (5∶3∶2, v/v/v); 170-290 min, n-hexane-methanol-water (5∶4∶1, v/v/v). After gradient elution, elution-extrusion elution mode was applied within 290-375 min. Good resolution was achieved by the CCC separation process. The HPLC separation process was carried out with gradient elution using a mobile phase composed of acetonitrile (A)-water (B): 0-10 min, 50%A-65%A; 10-14 min, 65%A; 14-21 min, 65%A-85%A; 21-25 min, 85%A-95%A; 25-30 min, 95%A-55%A; 30-40 min, 55%A. Curcuma volatile oil was separated under the above optimized two-dimensional separation conditions, and the data obtained were drawn into a two-dimensional contour map using Matlab software. The calculated total peak capacity exceeded 954, which was 10 times more than that of one-dimensional chromatography. High orthogonality (r=0.17) and spatial coverage factor (68.1%) were also obtained. Our research provides a new methodology for separating volatile oils used in traditional Chinese medicine as well as an approach for evaluating the quality of traditional Chinese medicinal herbs using two-dimensional chromatographic fingerprints.

Technical Notes

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Determination of boric acid and silicic acid in mineral water by nonsuppressed ion chromatography YANG Zhanqiang, ZHANG Fangfang, HAN Chunxia, ZHENG Hongguo 2023, 41 (12):  1121-1126.  DOI: 10.3724/SP.J.1123.2023.09025 Abstract ( 148 )   HTML ( 33 )   PDF (823KB) ( 58 )   Boron and silicon are widely distributed in nature; in water, these compounds typically present in the forms of boric acid and silicic acid, respectively. The maximum allowable levels of silicic acid and boric acid in water are stipulated in relevant national and industry standards, such as GB 8538-2022. Quality changes in water, which are of great significance in water-quality evaluations, can be understood in terms of its silicic acid and boric acid contents. Boric acid content is usually determined by ion exclusion chromatography, whereas silicic acid content is usually determined by postcolumn derivatization. Therefore, traditional methods cannot achieve the simultaneous determination of silicic acid and boric acid contents in water. Modern ion chromatography has been widely used in the detection of ionic compounds, such as anions, cations, organic acids, organic amines, amino acids, and sugars. Boric (pKa=9.24) and silicic (pKa=9.77) acids are weak acids that dissociate into ionic states under alkaline conditions. Although these compounds cannot be tested using suppressed ion chromatography, they can be retained on ion chromatography columns. In this study, a method based on nonsuppressed conductance detection was established for the simultaneous determination of boric acid and silicic acid in water. The contents of boric acid and silicic acid were detected by nonsuppressed ion chromatography using a Dionex IonPacTM AS20 analytical column. The chromatographic conditions were as follows: flow rate, 1.0 mL/min; column temperature, 30 ℃; eluent, 6 mmol/L sodium hydroxide solution and 60 mmol/L mannitol; and sample injection volume, 50 μL. The effective separation of silicic acid and boric acid was achieved within 8 min. SiO32- and boric acid demonstrated good linear relationships in the concentration ranges of 0.25-100 and 0.5-100 mg/L (correlation coefficients, 0.9999), respectively. The method detection (MDL) and quantification (MQL) limits were 0.078 and 0.26 mg/L for SiO32-, and the MDL and MQL limits were 0.18 and 0.60 mg/L for boric acid. The average recoveries of boric acid and SiO32- (n=6) were 97.3%-105.3%. Moreover, the relative standard deviations were less than 0.9% for boric acid at four spiked levels and less than 0.30% for SiO32- at three spiked levels. Thus, the method meets detection requirements. The pretreatment method is very simple, and the sample can be directly injected through a 0.22 μm water filtration membrane and into the column. The boric acid and silicic acid contents in nine mineral drinking water samples were determined under the optimized analytical conditions. Boric acid was not detected in these nine samples, but silicic acid was detected in six samples. The silicic acid contents detected were between 18.70 and 62.08 mg/L, which was consistent with the concentration ranges marked on the manufacturers’ packaging. The proposed method can be used for the determination of boric acid and silicic acid in mineral drinking water and laboratory water, and provides a reference for the simultaneous detection of boric acid and silicic acid in ultrapure water used in the semiconductor industry.

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Determination of 14 aniline and benzidine compounds in soil by gas chromatography-mass spectrometry WU Lijuan, YANG Lili, HU Enyu, WANG Meifei, YANG Chao, YIN Mingming 2023, 41 (12):  1127-1134.  DOI: 10.3724/SP.J.1123.2023.01002 Abstract ( 187 )   HTML ( 29 )   PDF (870KB) ( 85 )   The complex matrix of soil samples and low extraction efficiency of aniline compounds limit many methods developed for detecting aniline and benzidine compounds in soil. In this study, a rapid and sensitive method based on gas chromatography-mass spectrometry was developed for the simultaneous determination of 14 aniline and benzidine compounds in soil. The collected soil samples were sealed with 5% sodium sulfite solution and refrigerated to inhibit the oxidation of the target compounds for up to 7 d. The extraction efficiencies of accelerated solvent extraction and oscillating dispersion extraction were compared, and the recovery of accelerated solvent extraction was found to be unsuitable. Hence, three-phase oscillating dispersion extraction was adopted. A certain amount of alkaline aqueous solution was added to the test system during extraction to improve the extraction efficiency because aniline and benzidine compounds are weakly alkaline substances. When the pH of the extracted water phase was greater than 12, relatively good recoveries were obtained. Next, a mixed solvent of ethyl acetate-methylene chloride (1∶4, v/v) was added to extract the target compounds via oscillation for 20 min. The solid phase was discarded via centrifugation, and the aqueous and organic phases were transferred to a liquid separation funnel for further separation. Finally, the organic phase was retained. This pretreatment process prevents the co-extraction of acidic compounds or other impurities, thereby enhancing the purification ability of the method. Solid phase extraction (SPE) is generally recommended for soil extraction and purification. A preliminary test showed that compared with other columns, the Florisil SPE column could better retain the target substances and exhibited higher elution efficiency. After purification, the organic phase was concentrated to 1 mL using a nitrogen blower. The analytes were analyzed by gas chromatography-mass spectrometry using a capillary column (DB-35MS, 30 m×0.25 mm×0.25 μm). The temperature program was optimized to separate the target compounds at the baseline. Specifically, the initial oven temperature was set to 60 ℃, held for 2 min, increased to 130 ℃ at a rate of 5 ℃/min, increased to 300 ℃ at a rate of 30 ℃/min, and held for 4 min. The injector and ion source temperatures were 250 and 300 ℃, respectively. Aniline-d5 and acenaphthene-d10 were used as the internal standards for quantification. The effects of antioxidant addition, extraction solvent type, salting out, and other factors on extraction efficiency were investigated. The results showed that the method performed well under the optimized experimental conditions when actual soils were used as real sample matrices. The accuracy and precision of the proposed method were verified. A total of 14 aniline and benzidine compounds demonstrated good linearities in the range of 0.5-100 mg/L. The method detection limits (MDLs) ranged from 0.02 to 0.07 mg/kg, and the limits of quantification (LOQs) ranged from 0.08 to 0.28 mg/kg. The target compounds were spiked at contents of 1 and 10 mg/kg. The spiked recoveries of the 14 targets in actual soils were 62.9%-101%, and the relative standard deviations (RSDs) of six precision tests were 3.8%-10.3%. The proposed method effectively inhibited the oxidation of aniline and benzidine compounds during extraction, and the target compounds exhibited high recoveries and good stabilities in the presence of three phases. Moreover, the operating procedure was simple and easy to implement. The proposed method was applied to the soil collected from an industrial enterprise in Jiangsu province that was suspected to be contaminated with aniline, and two aniline compounds were detected. The developed method requires a small sample size, and the preservation step is simple and effective. In addition, it can be applied to various types of actual soils. The method meets the requirements of current soil pollution risk control standards for aniline and benzidine compounds in soils.

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Chiral capillary gas chromatography for the separation of the enantiomers of 4-chloromethyl-2,2-dimethyl-1,3-dioxolane ZHANG Zhenyong 2023, 41 (12):  1135-1140.  DOI: 10.3724/SP.J.1123.2023.06010 Abstract ( 139 )   HTML ( 18 )   PDF (848KB) ( 52 )   Chiral compounds play an important role in the pharmaceutical industry owing to their unique biological activities. The enantiomers must be separated because they can exhibit different pharmacological activities. Thus, the development of chiral separation methods is essential to determine the purity of enantiomers. 4-Chloromethyl-2,2-dimethyl-1,3-dioxolane is an important chiral pharmaceutical intermediate. In this context, a method based on chiral capillary gas chromatography was established for the separation and determination of the enantiomers of 4-chloromethyl-2,2-dimethyl-1,3-dioxolane. The separation of (R)- and (S)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane was initially investigated using two conventional stationary-phase capillary columns: SH-I-5Sil MS and SH-WAX. The stationary phase of SH-I-5Sil MS consisted of 5% phenyl and 95% polymethylsiloxane, whereas the stationary phase of SH-WAX consisted of 100% crosslinked polyethylene glycol. Neither of the columns exhibited chiral selectivity, so they both were unable to separate the enantiomers of 4-chloromethyl-2,2-dimethyl-1,3-dioxolane. Subsequently, the separation of (R)- and (S)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane was investigated using four chiral columns: Rt-bDEXm, Rt-bDEXsm, Rt-bDEXse, and InertCap CHIRAMIX. Among the chiral columns, Rt-bDEXse, which used a stationary phase composed of 2,3-di-O-ethyl-6-O-tert-butyl dimethylsilyl β-cyclodextrin added to 14% cyanopropyl phenyl and 86% dimethyl polysiloxane, achieved the best separation of (R)- and (S)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane. Thus, this column was selected as the analytical column for further method optimization. Detection was performed using a hydrogen flame ionization detector. The effects of various gas chromatographic parameters, such as linear velocity, initial column temperature, column heating rate, and solvent type, on the separation of (R)- and (S)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane were investigated. The optimal chromatographic conditions included a linear velocity of 70 cm/s, an initial column temperature of 70 ℃, and a column heating rate of 2.0 ℃/min. The final column oven temperature was 150 ℃. Methanol, ethanol, ethyl acetate, n-hexane, dichloromethane, and dimethyl sulfoxide were selected as solvents. The results showed that dimethyl sulfoxide interfered with the peaks of the target compounds, whereas the other solvents had no significant effect on the peak shape and separation of (R)- and (S)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane. Methanol was finally selected as the solvent in this study. Further experiments revealed that (R)- and (S)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane could be rapidly separated within 10 min, with a resolution greater than 1.5. A good linear relationship was observed in the range of 0.5-50.0 mg/L, with a linear correlation coefficient greater than 0.998. The limits of detection for (R)- and (S)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane were 0.07 and 0.08 mg/L, respectively, and the corresponding limits of quantification were 0.22 and 0.25 mg/L, respectively. Spiked recovery tests were performed at three spiked levels of 0.5, 2.0, and 10.0 mg/L using methanol as the blank to determine the accuracy of the proposed method. The recoveries for (R)- and (S)-4-chloromethyl-2,2-dimethyl-1,3-dioxolane were 94.0%-99.1% and 96.0%-98.8%, respectively, with relative standard deviations (RSDs) of 1.26%-4.87% and 1.51%-4.46%, respectively. The established method is efficient and reliable; thus, it can serve as a reference for the separation of the enantiomers of 4-chloromethyl-2,2-dimethyl-1,3-dioxolane. It can also potentially be applied to evaluate the enantiomeric purity of other chiral compounds in the pharmaceutical industry and produce chiral drugs and other related compounds.

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Rapid detection of four amphetamine-type drugs in hair by pulsed direct current electrospray mass spectrometry MI Kun, ZHANG Wentian, WEN Luhong, WANG Jin 2023, 41 (12):  1141-1148.  DOI: 10.3724/SP.J.1123.2023.04002 Abstract ( 128 )   HTML ( 18 )   PDF (1274KB) ( 50 )   Amphetamine-type drugs are synthetic compounds with an amphetamine parent structure. These compounds cause addiction, central nervous system excitation, and hallucinations. The number of drug users worldwide has gradually increased because amphetamine-type drugs can be synthesized in a simple and artificial manner. The current methods for anti-drug screening and toxicant identification are limited by the large quantity and variety of the drug analytes and long detection times. Thus, the development of broad-spectrum, rapid, and high-throughput detection methods is an urgent necessity. In addition, conventional amphetamine-type drug test samples, such as blood and urine, are only suitable for short-term drug identification. Hair has the advantages of easy preservation, stability, and a long detection window, which can compensate for the deficiencies of body-fluid-based test materials. Hair samples can reflect long-term drug use, which is beneficial for tracing drug sources, and has become an important means of providing evidence in court. Because most laboratory instruments are unable to perform the rapid on-site detection of amphetamine-type drugs in hair, establishing a high-throughput, qualitative and quantitative rapid on-site detection method is necessary. In this study, pulsed direct current electrospray ionization (Pulsed-DC-ESI) coupled with mass spectrometry was used for the rapid detection of four amphetamine-type drugs (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethamphetamine) in hair. Methanol was used as the extraction solvent, and the grinding method was used for extraction. The pretreatment process included cutting, grinding, and centrifugation. The pretreatment time for each sample was about 10 min. Multiple samples could be processed in batches, greatly improving the efficiency of analysis. Pulsed-DC-ESI is an ambient ionization technology that can be conducted via direct injection without chromatographic separation. The tip of the spray capillary tube was immersed 1 cm below the surface of the sample solution to allow absorption via the capillary effect. When the spray capillary tube contained 1 μL of the sample solution, detection was performed. Pulsed-DC-ESI generates an electrospray at the same frequency as the mass spectrum, thereby avoiding the problem of sample wastage, which often occurs in traditional ESI. The portable mass spectrometer used for analysis is a linear ion trap mass spectrometer. The parameters of Pulsed-DC-ESI, such as the inner diameter of spray capillary tip, spray voltage, and distance between electrode and solution, were optimized based on the mass spectral responses of the amphetamine-type drugs. The optimized ion source conditions included a inner diameter of spray capillary tip of 25 μm, spray voltage of 2 kV, and the distance between electrode and solution of 20 mm. The optimal sample solvent was methanol. The optimized method can achieve simultaneous detection of the four amphetamine-type drugs within 20 s. The linear ranges of amphetamine, methamphetamine, and the two other drugs were 1-25, 1-100, and 1-50 ng/mg, respectively. The limits of detection and quantification of the four drugs in hair were 0.1-0.2 and 1 ng/mg, respectively. All linear correlation coefficients were greater than 0.99, and the average spiked recoveries were 86.6%-114.7%. The intra-day precisions were 4.14%-7.34%, and the inter-day precisions were 3.71%-8.43%. The proposed method was used to screen 2000 samples provided by various testing institutions. A total of five samples were positive for methamphetamine, which is consistent with the results of conventional forensic identification methods. Thus, the developed method can be used for the rapid detection of amphetamine-type drugs.