Chinese Journal of Chromatography

Recent advances in the development and application of effervescence-assisted microextraction techniques

YE Hanzhang, LIU Tingting, DING Yongli, GU Jingjing, LI Yuhao, WANG Qi, ZHANG Zhan’en, WANG Xuedong

2023, 41 (4): 289-301. DOI: 10.3724/SP.J.1123.2022.06001

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Effervescence-assisted microextraction (EAM) is a novel sample pretreatment method based on the reaction of CO₂ and H⁺ donors to generate CO₂ bubbles and promote rapid dispersion of the extractant. During this process, the unique dispersion method increases the contact area between the target molecule and the extraction solvent, and the adsorption/extraction efficiency of the adsorbent/extractant toward the target molecule is also enhanced. The EAM technique is of particular interest due its convenient application, low running costs, reduced solvent consumption, high extraction efficiency, and environmental friendliness. Benefiting from the rapid development of extractants, the evolution and application of the EAM technology is becoming more tuned and diversified. Indeed, the synthesis of new extractants, such as nanomaterials with multi-pore structures, large specific surface areas, and rich active sites, has attracted extensive attention, as has the development of ionic liquids with strong extraction abilities and high selectivities. As a result, the EAM technology has been widely applied to the pretreatment of target compounds in various samples, such as food, plant, biological, and environmental samples. However, since these samples often contain polysaccharides, peptides, proteins, inorganic salts, and other interfering substrates, it is necessary to remove some of these substances prior to extraction by EAM. This is commonly achieved using methods such as vortexing, centrifugation, and dilution, among others. The treated samples can then be extracted using the EAM method prior to detection using high performance liquid chromatography (HPLC), gas chromatography (GC), and atomic absorption spectroscopy (AAS) to detect substances such as heavy metal ions, pesticide residues, endocrine-disrupting compounds (EDCs), and antibiotics. Using effervescence as a novel assisted method for the dispersion of solvents or adsorbents, the concentrations of Pb²⁺, Cd²⁺, Ni²⁺, Cu²⁺, bisphenol, estrogen, and the pyrethyl pesticides have previously been successfully determined. Moreover, many influencing factors have been evaluated during method development, including the composition of the effervescent tablet, the solution pH, the extraction temperature, the type and mass/volume of extractant, the type of eluent, the eluent concentration, the elution time, and the regeneration performance. Generally, the cumbersome single factor optimization and multi-factor optimization methods are also required to determine the optimal experimental conditions. Following determination of the optimal experimental conditions, the EAM method was validated by a series of experimental parameters including the linear range, the correlation coefficient (R²), the enrichment factor (EF), the limit of detection (LOD), and the limit of quantification (LOQ). In addition, the use of this method has been demonstrated in actual sample testing, and the obtained results have compared with those achieved using similar detection systems and methods to ultimately determine the accuracy, feasibility, and superiority of the developed method. In this paper, the construction of an EAM method based on nanomaterials, ionic liquids, and other emerging extractants is reviewed, wherein the preparation method, application range, and comparison of similar extractants were evaluated for the same extraction system. In addition, the current state-of-the-art in relation to EAM research and application when combined with HPLC, cold flame AAS, and other analytical techniques is summarized in terms of the detection of harmful substances in complex matrices. More specifically, the samples evaluated herein include dairy products, honey, beverages, surface water, vegetables, blood, urine, liver, and complex botanicals. Furthermore, issues related to the application of this technology are analyzed, and its future development trend in the field of microextraction is forecasted. Finally, the application prospects of EAM in the analysis of various pollutants and components are proposed to provide reference for monitoring pollutants in food, environmental, and biological samples.

Applications of functional materials-based solid phase microextraction technique in forensic science

XIE Weiya, ZHU Xiaohan, MEI Hongcheng, GUO Hongling, LI Yajun, HUANG Yang, QIN Hao, ZHU Jun, HU Can

2023, 41 (4): 302-311. DOI: 10.3724/SP.J.1123.2022.06018

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Sample extraction is a crucial step in forensic analysis, especially when dealing with trace and ultra-trace levels of target analytes present in various complex matrices (e. g., soil, biological samples, and fire debris). Conventional sample preparation techniques include Soxhlet extraction and liquid-liquid extraction. However, these techniques are tedious, time-consuming, labor-intensive and require large amounts of solvents, which poses a threat to the environment and health of researchers. Moreover, sample loss and secondary pollution can easily occur during the preparation procedure. Conversely, the solid phase microextraction (SPME) technique either requires a small amount of solvent or no solvent at all. Its small and portable size, simple and fast operation, easy-to-realize automation, and other characteristics thus make it a widely used sample pretreatment technique. More attention was given to the preparation of SPME coatings by using various functional materials, as commercialized SPME devices used in early studies were expensive, fragile, and lacked selectivity. Examples of those functional materials include metal-organic frameworks, covalent organic frameworks, carbon-based materials, molecularly imprinted polymers, ionic liquids, and conducting polymers, all widely used in environmental monitoring, food analysis, and drug detection. However, these SPME coating materials have relatively few applications in forensics. Given the high potential of SPME technology for the in situ and efficient extraction of samples from crime scenes, this study briefly introduces functional coating materials and summarizes the applications of SPME coating materials for the analysis of explosives, ignitable liquids, illicit drugs, poisons, paints, and human odors. Compared to commercial coatings, functional material-based SPME coatings exhibit higher selectivity, sensitivity, and stability. These advantages are mainly achieved through the following approaches: First, the selectivity can be improved by increasing the π-π, hydrogen bonds, and hydrophilic/hydrophobic interactions between the materials and analytes. Second, the sensitivity can be improved by using porous materials or by increasing their porosity. Third, thermal, chemical, and mechanical stability can be improved by using robust materials or fixing the chemical bonding between the coating and substrate. In addition, composite materials with multiple advantages are gradually replacing the single materials. In terms of the substrate, the silica support was gradually replaced by the metal support. This study also outlines the existing shortcomings in forensic science analysis of functional material-based SPME techniques. First, the application of functional material-based SPME techniques in forensic science remains limited. On one hand, the analytes are narrow in scope. As far as explosive analysis is concerned, functional material-based SPME coatings are mainly applied to nitrobenzene explosives, while other categories, such as nitroamine and peroxides, are rarely or never involved. Research and development of coatings is insufficient and the application of COFs in forensic science has not yet been reported. Second, functional material-based SPME coatings have not been commercialized as they don’t yet have inter-laboratory validation tests or established official standard analytical methods. Therefore, some suggestions are proposed for the future development of forensic science analyses of functional material-based SPME coatings. First, research and development of functional material-based SPME coatings, especially fiber coatings with broad-spectrum applicability and high sensitivity, or outstanding selectivity for some compounds, is still an important direction for SPME future research. Second, a theoretical calculation of the binding energy between the analyte and coating was introduced to guide the design of functional coatings and improve the screening efficiency of new coatings. Third, we expand its application in forensic science by expanding the number of analytes. Fourth, we focused on the promotion of functional material-based SPME coatings in conventional laboratories and established performance evaluation protocols for the commercialization of functional material-based SPME coatings. This study is expected to serve as a reference for peers engaged in related research.

Determination of 12 typical personal care products in human urine samples by ultra performance liquid chromatography-tandem mass spectrometry

HAN Linxue, ZHANG Xu, HU Xiaojian, ZHANG Haijing, QIU Tian, LIN Xiao, ZHU Ying

2023, 41 (4): 312-322. DOI: 10.3724/SP.J.1123.2022.05032

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A rapid and sensitive method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 12 typical personal care products (PCPs) in human urine. These PCPs included five paraben preservatives (PBs), five benzophenone UV absorbers (BPs), and two antibacterial agents. Accordingly, 1 mL of the urine sample was mixed with 500 μL of β-glucuronidase-ammonium acetate buffer solution (enzymatic activities are 500 units/mL) and 75 μL of a mixed internal standard working solution (internal standard contents are 7.5 ng), followed by enzymatic hydrolysis overnight (≥16 h) at 37 ℃ in a water bath. The 12 targeted analytes were enriched and cleaned up using an Oasis HLB solid phase extraction column. Separation was performed on an Acquity BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm) using an acetonitrile-water system as the mobile phase, in negative electrospray ionization (ESI^-) multiple reaction monitoring (MRM) mode, for target detection and stable isotope internal standard quantification. The optimal MS conditions were established by optimizing the instrument parameters and comparing two analytical columns (Acquity BEH C₁₈ and Acquity UPLC HSS T₃) as well as different types of mobile phases (methanol or acetonitrile as the organic phase) to achieve better chromatographic separation. In order to obtain higher enzymatic and extraction efficiency, different enzymatic conditions, solid phase extraction columns, and elution conditions were investigated. The final results showed that methyl parabens (MeP), benzophenone-3 (BP-3), and triclosan (TCS) showed good linearities in the ranges of 4.00-800, 4.00-800 and 5.00-200 μg/L, respectively, the other targeted compounds showed good linearities in the ranges of 1.00-200 μg/L. The correlation coefficients were all greater than 0.999. The method detection limits (MDLs) were in the range of 0.06-1.09 μg/L, and the method quantification limits (MQLs) ranged from 0.08 to 3.63 μg/L. At three spiked levels, the average recoveries of the 12 targeted analytes ranged from 89.5% to 111.8%. The intra-day and inter-day precisions were 3.7%-8.9% and 2.0%-10.6%, respectively. The results of the matrix effect assessment showed that MeP, ethyl paraben (EtP), and benzophenone-2 (BP-2) exhibited strong matrix effects (26.7%-103.8%); propyl paraben (PrP) exhibited moderate matrix effects (79.2%-112.0%); and the other eight target analytes exhibited weak matrix effects (83.3%-113.8%). The matrix effects of the 12 targeted analytes after correction using the stable isotopic internal standard method ranged from 91.9% to 110.1%. The developed method was successfully applied to the determination of the 12 PCPs in 127 urine samples. Ten typical PCPs were detected, with the overall detection rates ranging from 1.7% to 99.7%, except for benzyl paraben (BzP) and benzophenone-8 (BP-8). The results revealed that the population in this area was widely exposed to PCPs, especially MeP, EtP and PrP; the detection rates and concentrations of these PCPs were found to be very high. Our analytical method is simple and sensitive, and it is expected to be an effective tool for biomonitoring PCPs in human urine samples as part of environmental health studies.

Determination of complexation ability of rhubarb with copper ions by ultra-high performance liquid chromatography

LIU Yapeng, ZHANG Wang, LIU Xinjie, WU Huan, ZHOU An

2023, 41 (4): 323-329. DOI: 10.3724/SP.J.1123.2022.06020

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Gandou decoction (GDD) is a traditional Chinese medicine prescription that has been widely used to treat copper metabolism disorders in China with remarkable clinical effect and lower toxicity. However, evaluation of the complexation ability of copper ions is challenging, which hinders screening and discovery of coordinate active ingredients in GDD. An analytical method is needed to determinate the complexation ability of chemical constituents with copper ions. In this study, a rapid and accurate method based on ultra-high performance liquid chromatography (UHPLC) was developed to determine the complexing ability of rhubarb with copper ions. First, the optimal coordination reaction conditions between active ingredients of rhubarb and copper ions were determined. The samples were separated using an Agilent Eclipse Plus C18 column (50 mm×2.1 mm, 1.8 μm) with 5 μL injection volumes. The mobile phase was gradient eluted with methanol and water containing 0.1% (v/v) phosphoric acid at a flow rate of 0.3 mL/min. The detection wavelength was 254 nm and the column temperature was 30 ℃. Under the optimized chromatographic conditions, the rhubarb constituents were effectively separated. Next, peak areas of rhubarb were calculated before and after the coordination reaction between copper ions. The complexing ability of active ingredients in rhubarb with copper ions was evaluated by calculating the rate of changes of their chromatographic peak areas. Finally, ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to identify the coordination active ingredients in rhubarb extract. Focusing on the coordination reaction conditions between active ingredients of rhubarb and copper ions revealed that the active ingredients of rhubarb and copper ions reached equilibrium by coordination reaction at pH 9 for 12 h. Methodological evaluation revealed the good stability and repeatability of the method. Under these conditions, 20 major components of rhubarb were identified by UPLC-Q-TOF-MS. According to the coordination rate of each component and copper ions, eight components with strong coordination were screened out (gallic acid 3-O-β-D-(6'-O-galloyl)-glucopyranoside, aloe emodin-8-O-β-D-glucoside, sennoside B, l-O-galloyl-2-O-cinnamoyl-glucoside, chysophanol-8-O-β-D-(6″-O-acetyl)-glucoside, aloe-emodin, rhein and emodin). The respective complexation rates of the components were 62.50%, 29.94%, 70.58%, 32.77%, 34.61%, 26.07%, 28.73% and 31.78%. Compared with other reported methods, the presently developed method can be used to screen the active ingredients of traditional Chinese medicines that have complexing ability with copper ions, especially in complex mixture systems. This study describes an effective detection technology for evaluating and screening the complexing ability of other traditional Chinese medicines with metal ions.

QuEChERS-liquid chromatography-tandem mass spectrometry for determination of 22 triazole pesticide residues in Chinese herbal medicines

WANG Jiao, WU Tong, WANG Xinquan, LIU Zhenzhen, XU Hao, WANG Zhiwei, DI Shanshan, ZHAO Huiyu, QI Peipei

2023, 41 (4): 330-338. DOI: 10.3724/SP.J.1123.2022.08005

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Eight well-known herbals in Zhejiang Province, Zhebawei, are commonly used as traditional Chinese herbal medicines owing to their rich active ingredients. However, the unavoidable use of pesticides during agricultural production has led to pesticide residue problems in these herbs. In this study, a simple, rapid, and accurate method was established to determine 22 triazole pesticide residues in Zhebawei. An improved QuEChERS method was used for sample pretreatment, and Rhizoma Atractylodis Macrocephalae was used as a representative sample. The sample was extracted with acetonitrile to eliminate some polar and nonpolar compounds, pigments, and other impurities, and the purification effects of multiwalled carbon nanotubes (MWCNTs), amino-modified multiwalled carbon nanotubes (MWCNTs-NH₂), carboxylated multiwalled carbon nanotubes (MWCNTs-COOH), crosslinked polyvinylpyrrolidone (PVPP), zirconium dioxide (ZrO₂), 3-(N,N-diethylamino)-propyltrimethoxysilane (PSA), octadecyl (C18), and graphitized carbon black (GCB) were compared. MWCNTs-COOH and C18 were selected as the purification adsorbents, and their dosages were systematically optimized. The combination of 10 mg of MWCNTs-COOH and 20 mg of C18 was eventually selected as the purification adsorbents. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for analysis, and box graphs were plotted to present the dispersion of each group of recoveries, thus enabling the identification of the data outliers, dispersion distribution, and data symmetry. The established method was systematically verified and showed good linearity over the concentration range of 1-200 μg/L (except for bromuconazole, epoxiconazole, and etaconazole) with correlation coefficients >0.99. The average recoveries of the 22 pesticides at spiked levels of 10, 20, 100, and 200 μg/kg were in the range of 77.0%-115% with relative standard deviations (RSDs) <9.4%. The limits of detection and quantification were 1-2.5 μg/kg and 10-20 μg/kg, respectively. The applicability of the developed method to other herbals was investigated at 100 μg/kg, and the average recoveries of the target pesticides in different matrices ranged from 76.4% to 123% with RSDs <12.2%. Finally, the method established was used to detect triazole pesticide residues in 30 actual Zhebawei samples. The results showed that triazole pesticides were present in Bulbus Fritillariae Thunbergii and Dendranthema Morifolium. Difenoconazole was detected in Bulbus Fritillariae Thunbergii at contents ranging from 41.4 μg/kg to 110 μg/kg, while difenoconazole, myclobutanil, triadimenol and propiconazole were detected in Dendranthema Morifolium at contents ranging from 16.1 μg/kg to 250 μg/kg. The established method can meet the requirements for the accurate quantitative analysis of triazole fungicides in Zhebawei.

Direct determination of five xanthic acids in water by ultra performance liquid chromatography-tandem mass spectrometry

ZHU Weihong, WANG Chao, ZHANG Linlin, YUAN Mao

2023, 41 (4): 339-347. DOI: 10.3724/SP.J.1123.2022.09002

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Xanthates with different alkyl groups, such as ethyl, propyl, butyl, and amyl groups, are widely used in large quantities in the mining flotation of metallic minerals. Xanthates enter environmental waters through mineral processing wastewater discharge and are ionized or hydrolyzed into ions or molecules of xanthic acids (XAs) in water. XAs endanger aquatic plants and animals, as well as human health. To the best of our knowledge, XA analysis is mainly limited to butyl xanthate. Moreover, the isomers and congeners of XAs cannot be determined separately using the existing methods. Herein, a novel method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established to separate and analyze five XAs, namely, ethyl-, isopropyl-, n-butyl-, isobutyl-, and amyl-XAs, in water. Water samples were filtered through a 0.22 μm hydrophilic polytetrafluoroethylene (PTFE) membrane and directly injected into the UPLC-MS/MS instrument. Separation was performed using a Waters Acquity UPLC BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm) with ammonia solution (pH 11)-acetonitrile (9∶1, v/v) as the mobile phase for isocratic elution. The five XAs were detected in the negative electrospray ionization (ESI^-) and multiple reaction monitoring (MRM) modes. An internal standard method was used for quantification. The pretreatment and UPLC-MS/MS conditions were comprehensively optimized to achieve the separation and analysis of the five XAs via direct injection. The XAs showed negligible adsorption on hydrophobic PTFE, hydrophilic PTFE, hydrophilic polypropylene, and polypropylene membranes during filtration. However, the amyl-XA showed obvious adsorption on nylon and polyether sulfone membranes. The five XAs mainly formed [M-H]^- parent ions in the ESI^- mode and the main daughter ions obtained following collisional fragmentation depended on the alkyl groups of the XAs. Increasing the pH of the ammonia solution in the mobile phase to 11 led to the isomeric separation of n-butyl- and isobutyl-XAs. The optimized mobile phase inhibited the tailing of the chromatographic peak of amyl-XA and effectively improved all the chromatographic peak shapes of XAs. The BEH C₁₈ column was selected as the chromatographic column owing to its better compatibility with high-pH solutions compared with the T3 C₁₈ column. Preservation experiments conducted over 8 d showed that the concentration of all five XAs decreased over time at room temperature; among the XAs analyzed, the concentration of ethyl-XA revealed the most significant decrease. However, the recoveries of the five XAs at 4 and -20 ℃ remained high, ranging from 101% to 105% and from 100% to 106%, respectively, on the 8th day. The preservation observed with a high concentration of XAs was similar to that found with a low concentration. The preservation time was extended to 8 days at pH 11 and 4 ℃ away from the light. No significant matrix effects were observed for the five XA samples in surface water and groundwater, but industrial sewage exerted obvious matrix inhibitory effects on ethyl- and isopropyl-XAs. Owing to the short retention times of ethyl- and isopropyl-XAs, the co-fluxed interferents in the industrial sewage depressed the MS signals. The five XAs showed good linearity in the range of 0.25-100 μg/L, with correlation coefficients greater than 0.9996. The method detection limits were as low as 0.03-0.04 μg/L, and the intra- and inter-day precisions were 1.3%-2.1% and 3.3%-4.1%, respectively. The recoveries obtained under low, medium, and high spiked levels (1.00, 20.0, 80.0 μg/L) were 96.9%-133%, 100%-107%, and 104%-112%, respectively. The corresponding RSDs were 2.1%-3.0%, 0.4%-1.9%, and 0.4%-1.6%, respectively. The optimized method was successfully applied to the analysis of XAs in surface water, groundwater, and industrial sewage. The method could separate and detect various congeners and isomers of XAs without the need for cumbersome pretreatment processes, and its advantages include smaller sample requirements, simpler operation, higher sensitivity, and longer preservation time. The proposed technique presents excellent application potential in XA environmental monitoring and water evaluation, and mineral flotation studies.

Rapid screening of Chemical Weapons Convention-related chemicals in oil matrix by headspace solid-phase microextraction and gas chromatography-mass spectrometry

CHEN Jia, LIU Yulong, XU Bin, LIU Qin, XIE Jianwei

2023, 41 (4): 348-358. DOI: 10.3724/SP.J.1123.2022.07007

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The Chemical Weapons Convention (CWC) requires verification of a large number of compounds with different types and properties. The results of the verification are of great political and military sensitivity. However, the sources of verification samples are complex and diverse, and the contents of the target compounds in these samples are usually very low. These issues increase the likelihood of missed or false detection. Thus, establishing rapid and effective screening methods for the accurate identification of CWC-related compounds in complex environmental samples are of great importance. In this study, a fast and simple procedure based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-electron ionization mass spectrometry (GC-EI/MS) in full-scan mode was developed to determine CWC-related chemicals in oil matrix. A total of 24 CWC-related chemicals with different chemical characteristics were selected to simulate the screening procedure. The selected compounds were divided into three groups based on their properties. The first group included volatile and semi-volatile CWC-related compounds with relatively low polarity, which could be extracted by HS-SPME and directly analyzed by GC-MS. The second group included moderately polar compounds with hydroxyl or amino groups; such compounds are related to nerve, blister, and incapacitating agents. The compounds in the third group included non-volatile CWC-related chemicals with relatively strong polarity, such as alkyl methylphosphonic acids and diphenyl hydroxyacetic acid. These compounds must be derivatized into vaporizable derivatives prior to extraction by HS-SPME and analysis by GC-MS. Variables that influence the SPME process, such as fiber type, extraction temperature and time, desorption time, and derivatization protocol, were optimized to improve the sensitivity of the method. The screening procedure for CWC-related compounds in the oil matrix samples included two main steps. First, low-polarity volatile and semi-volatile compounds (i. e. the first group) were extracted by HS-SPME with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fibers and analyzed in split-injection mode (split ratio, 10∶1) using GC-MS. The use of a large split ratio can reduce the solvent effect, which is conducive to the detection of low-boiling-point compounds. If necessary, the sample could be extracted once more and analyzed in splitless mode. The derivatization agent bis(trimethylsilyl)trifluoroacetamide (BSTFA) was then added to the sample. Mid- and high-polarity compounds (i. e. the second and third groups) were extracted with polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibers after derivatization and analyzed in splitless mode using GC-MS. The established method exhibited good repeatability and sensitivity. The detection limits for the compounds in the first group ranged from 0.5 ng/mL to 100 ng/mL, whereas the detection limits for the compounds in the second and third groups ranged from 20 ng/mL to 300 ng/mL. Except for compounds with extremely high boiling points and a few compounds that are not suitable for derivatization with BSTFA, the method can be used to analyze most CWC-related compounds in oil matrix samples. In particular, it greatly shortened the preparation time of the oil matrix samples and reduced the loss of low-boiling-point compounds owing to the sample concentration process, thereby avoiding missed detection. The method was successfully applied to the Organization for the Prohibition of Chemical Weapons (OPCW) proficiency tests and proved to be a useful technique for the rapid screening of trace levels of CWC-related chemicals in oil matrix.

Determination of four fatty acid ethyl esters in olive oil by solid phase extraction-gas chromatography

LU Huiyuan, WANG Lijuan, ZHANG Jiongkai, ZHANG Chizhong, LI Tianjuan, JI Ruixue, SHEN Weijian

2023, 41 (4): 359-365. DOI: 10.3724/SP.J.1123.2022.09011

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The fatty acid ethyl ester (FAEE) content of olive oil is an important indicator of its quality. At present, the international standard method used to detect FAEEs in olive oil is silica gel (Si) column chromatography-gas chromatography (GC); however, this technique presents a number of disadvantages, including complex operation, long analysis times, and high reagent consumption. In this study, a method based on Si solid phase extraction (SPE)-GC was established to determine four FAEEs in olive oil, namely, ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate. First, the effects of the carrier gas were investigated, and He gas was ultimately selected as the carrier gas. Next, several internal standards were screened, and ethyl heptadecenoate (cis-10) was determined as the optimal internal standard. The SPE conditions were also optimized, and the effects of different brands of Si SPE columns on the recoveries of analytes were compared. Finally, a pretreatment method in which 0.05 g of olive oil was extracted with n-hexane and purified through a Si SPE column (1 g/6 mL) was developed. A sample could be processed within approximately 2 h using a total reagent volume of about 23 mL. Validation of the optimized method revealed that the four FAEEs have good linearities within the range of 0.1-5.0 mg/L (coefficients of determination (R²)>0.999). The limits of detection (LODs) of the method were within 0.78-1.11 mg/kg, and its limits of quantification (LOQs) were in the range of 2.35-3.33 mg/kg. The recoveries ranged from 93.8% to 104.0% at all spiked levels tested (4, 8, and 20 mg/kg), and the relative standard deviations were 2.2%-7.6%. Fifteen olive oil samples were tested using the established method, and the total FAEEs of three extra-virgin olive oil samples were found to exceed 35 mg/kg. Compared with the international standard method, the proposed method has the advantages of simpler pretreatment process, shorter operation time, lower reagent consumption and detection cost, high precision, and good accuracy. The findings provide an effective theoretical and practical reference for improving olive oil detection standards.

Determination of seven monoaromatic hydrocarbon metabolites by ultra performance liquid chromatography-tandem mass spectrometry

QIU Tian, ZHANG Xu, YANG Yanwei, HU Xiaojian, LUO Song, ZHU Ying

2023, 41 (4): 366-375. DOI: 10.3724/SP.J.1123.2022.05016

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Monoaromatic hydrocarbons (MAHs) such as benzene, toluene, and xylene are important anthropogenic pollutants in the urban atmosphere. The detection of urinary MAH metabolites are included in human biomonitoring programs in several countries, including Canada, the United States, Italy, and Germany, because their evaluation is vital to monitor the exposure of humans to MAHs. To this end, herein, a method was developed for the determination of seven MAH metabolites through ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). An aliquot of 0.5 mL urine was fortified with an isotopic labeled internal standard solution before being hydrolyzed by 40 μL of 6 mol/L HCl solution, followed by extraction using a 96-well EVOLUTE^®EXPRESS ABN solid-phase extraction plate. The samples were washed with 1.0 mL of methanol-water (10∶90, v/v) and eluted with 1.0 mL methanol. The eluate was diluted four times with water prior to use in instrumental analysis. Chromatographic separation was achieved using an ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm), with gradient elution using 0.1% formic acid as mobile phase A and methanol as mobile phase B. The detection of seven analytes was performed using a triple-quadrupole mass spectrometer equipped with a negative electrospray ionization source in the multiple reaction monitoring mode. The linear ranges of the seven analytes varied from 0.1-20 μg/L to 2.5-500 mg/L, with correlation coefficients greater than 0.995. The method detection limits were 1.5, 0.02, 0.1, 900, 0.6, and 4 μg/L for trans,trans-muconic acid (MU), S-phenylmercapturic acid (PMA), S-benzylmercapturic acid (BMA), hippuric acid (HA), 2-methyl hippuric acid (2MHA), and 3-methyl hippuric acid (3MHA)+4-methyl hippuric acid (4MHA), respectively. The limits of quantification were 5, 0.05, 0.4, 3000, 2, and 12 μg/L for MU, PMA, BMA, HA, 2MHA, and 3MHA+4MHA, respectively. The method was verified by spiking urine samples at three different concentration levels, with recovery rates ranging from 84% to 123%. The intra- and inter-day precisions were 1.8%-8.6% and 1.9%-21.4%, respectively. The extraction efficiencies were 68%-99%, and the matrix effects ranged from -11% to -87%. The urine samples obtained from the German external quality assessment scheme (round 65) were used to assess the accuracy of this method. Both high and low concentrations of MU, PMA, HA, and methyl hippuric acid were within the tolerance range. All analytes in the urine samples were found to be stable for up to seven days at room temperature (20 ℃, absence of light), with less than 15% change in concentration. Analytes in urine samples were found to be stable for at least 42 d at 4 ℃ and -20 ℃, or for six freeze-thaw cycles and up to 72 h in an autosampler (8 ℃). The method was applied to the analysis of 16 non-smokers’ and 16 smokers’ urine samples. The detection rates of MU, BMA, HA, and 2MHA were 100% in both non-smokers’ and smokers’ urine samples. PMA was detected in 75% non-smokers’ and 100% smokers’ urine samples. 3MHA+4MHA was detected in 81% non-smokers’ urine and in all smokers’ urine samples. Statistical differences were found for MU, PMA, 2MHA, and 3MHA+4MHA between the two groups (p<0.001). The established method has good robustness and can provide reliable results. The experiments were carried out in a high-throughput manner with large sample sizes, owing to the small sample volume, and allowed the successful detection of the seven MAH metabolites in human urine.

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