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    Chinese Journal of Chromatography
    2021, Vol. 39, No. 12
    Online: 08 December 2021

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    Reviews
    Novel adsorption material for solid phase extraction in sample pretreatment of plant hormones
    LIN Shuting, DING Qingqing, ZHANG Wenmin, ZHANG Lan, LU Qiaomei
    2021, 39 (12):  1281-1290.  DOI: 10.3724/SP.J.1123.2021.03045
    Abstract ( 303 )   HTML ( 370 )   PDF (909KB) ( 283 )  

    Plant hormones (PHs) are of significance in plant growth, as they regulate the various processes related to plant growth, development, and resistance. Sensitive and precise quantitative analysis of PHs is a bottleneck in plant science research. Currently, liquid chromatography-tandem mass spectrometry is used for the accurate and efficient detection of PHs. Sample pretreatment is an indispensable step in the chromatography-mass spectrometry analysis of PHs because it directly affects the sensitivity and accuracy of subsequent detection methods. Among various pretreatment methods for PHs, solid phase extraction (SPE) is the most widely used. Various new types of SPE, such as dispersive SPE, magnetic SPE, and solid phase microextraction, have been developed by modifying the extraction cartridge. The choice of adsorption material is the key factor in the abovementioned SPE methods, which has a decisive effect on the extraction, purification, and enrichment effects of the target substance in the sample pretreatment process. Carbon-based materials, including carbon nanotubes, graphene, carbon and nitrogen compounds, as well as organic frameworks, including metal organic frameworks and covalent organic materials, are suitable adsorption materials because of their designable structure, large specific surface area, and good stability. Molecularly imprinted polymers and supramolecular compounds show specific molecular recognition based on host-guest interactions, which can significantly improve the selectivity of sample pretreatment methods. In this paper, SPE-related technology and the abovementioned types of functionalized adsorption materials in the pretreatment of PHs prevalent in the past five years have been reviewed. The related development trends are also summarized.

    Articles
    Pharmacokinetics and tissue distribution characteristics of the novel photosensitizer 32-(4-methoxyphenyl)-152-aspartyl-chlorin e6
    WANG Liu, DONG Yi, CAO Lei, SUN Yuming, LI Yueqing, ZHAO Weijie
    2021, 39 (12):  1291-1297.  DOI: 10.3724/SP.J.1123.2021.01010
    Abstract ( 273 )   HTML ( 202 )   PDF (839KB) ( 145 )  
    Supporting Information

    Photodynamic therapy (PDT) has garnered immense research interest. PDT can directly kill the cells via a combination of photosensitizer, light, and molecular oxygen. It has emerged as a promising therapeutic option for cancer treatment owing to its advantages such as minimized systemic toxicity, minimal invasiveness, high therapeutic efficacy, and potential for developing antitumor immunity. The novel photosensitizer 32-(4-methoxyphenyl)-152-aspartyl-chlorin e6 (DYSP-C34) was synthesized by introducing a 32-aryl substitution and amino acid substituent of the Chenghai chlorin (CHC). Briefly, 32-(4-methoxyphenyl) substitution was achieved via olefin metathesis reactions. The aspartic acid side chain was introduced regioselectively at C-152, followed by hydrolysis to yield the target DYSP-C34. CHC with the same chemical structure as chlorin e6 was prepared from chlorophyll a, which was extracted from Spirulina powders derived from Chenghai Lake in the Yunnan province of China. This strategy successfully endowed the resultant photosensitizer with better cellular permeability and increased water solubility. In addition, the photodynamic antitumor effects of PDT largely depend on the dose of photosensitizer used, time between photosensitizer administration and light exposure, and possibly other still poorly known variables. Determination of optimal conditions for PDT requires a coordinated interdisciplinary effort. Therefore, the pharmacokinetics and tissue distribution of DYSP-C34 in vivo are critical for the efficacy and safety of PDT. Herein, a high performance liquid chromatography-ultraviolet (HPLC-UV) detection method was established for the determination of the new photosensitizer DYSP-C34 in rat plasma. The sample preparation involved a protein-precipitation and liquid-liquid extraction method. Methanol was used to precipitate proteins and chloroform was used to extract chlorins. Then, DYSP-C34 was separated on a Unitary C18 column (250 mm×4.6 mm, 5 μm) with a mobile phase comprising methanol and 5 mmol/L tetrabutylammonium phosphate buffer solution (70∶30, v/v). The flow rate was 1.0 mL/min with UV detection using a wavelength of 400 nm at 40 ℃. Results showed that DYSP-C34 and chlorin e6 trimethyl ester (IS) were well separated under these conditions. The method was sensitive and sufficiently precise with a good linear relationship (determination coefficient (r2)=0.9941) over the range of 1-200 μg/mL in rat plasma. At three spiked levels (8, 40, and 120 μg/mL), the average recoveries were 74.39%, 69.71%, and 65.89%, respectively. The intra-day and inter-day relative standard deviations (RSDs) were lower than 5%. The precision met the requirements of biological sample determination. Furthermore, DYSP-C34 was stable in rat plasma under various storage conditions at room temperature, three freeze-thaw cycles, and long-term cryopreservation. The validated method was successfully applied to the pharmacokinetic study of DYSP-C34 after intravenous injection of a single dose in rat plasma. The pharmacokinetic parameters after intravenous injection of DYSP-C34 (16 mg/kg) were calculated. The plasma half-life (t1/2z) was 6.98 h, the area under the plasma concentration-time curve AUC(0-∞) was 1025.01 h·mg/L and the mean retention time MRT(0-∞) was 9.19 h. In addition, the results of DYSP-C34 distribution in tumor-bearing mice showed that DYSP-C34 could accumulate in tumor tissues, with higher concentrations in liver and kidney tissues, and lower concentrations in heart, spleen, and lung tissues. In summary, a specific, simple, and accurate HPLC-UV method was developed and validated for the determination of DYSP-C34 in rat plasma and tumor-bearing mouse tissues. The pharmacokinetics of DYSP-C34 after intravenous administration in rats and the tissue distribution characteristics of tumor-bearing mice were clarified for the first time. It is significant for clinical rational drug use and pharmacodynamic research. Therefore, choosing an appropriate time for light treatment time can achieve the best photodynamic effect. The results of pharmacokinetics and tissue distribution of DYSP-C34 provide vital guidance for subsequent pharmacodynamic research and further clinical trials in terms of dosage, light time, light toxicity and side effects.

    Determination of new carmine in beverages by one step rapid solid phase extraction based on metal organic framework extractant
    HUANG Rong, CHENG Lei, XIAO Yushi, CAO Qiang, LIU Na, CHEN Shiheng, WU Lidong
    2021, 39 (12):  1298-1305.  DOI: 10.3724/SP.J.1123.2021.01024
    Abstract ( 194 )   HTML ( 190 )   PDF (4108KB) ( 176 )  

    New coccine is an azo pigment that is widely used in food. To mitigate potential health issues arising from excessive consumption, China has issued provisions on the allowed addition limit of new coccine in food. Currently, there are certain difficulties with establishing detection methods for such trace pigments in foods; for example, preprocessing is complex and time-intensive. In addition, the low content of the target substance in the sample could be disturbed by food matrix, resulting in poor detection sensitivity. Metal organic frameworks (MOFs), as a novel class of highly efficient adsorbents, have attracted increasing attention because of their stability and large specific surface area. MOFs are porous coordination crystal structures that connect metal clusters with organic ligands via coordination. Owing to their molecule-sized pores, MOFs can be used in various fields such as adsorption, catalysis, and drug dispersion. However, at the same time, their ultra-high specific surface area also leads to ultra-low weight of the material itself; this makes it difficult to collect the material even under high-speed centrifugation. In this study, a MOF material (PCN-222) with a high specific surface area was prepared by the coordination of the carboxyl group in the porphyrin ring and metal zirconium ions. To simplify pretreatment, the nanomaterials were filled into an injection solid phase extraction device for the rapid extraction of new coccine pigments from beverages. The morphology, structure, and properties of the PCN-222 nanomaterials were studied by transmission electron microscopy, particle size analysis, X-ray single-crystal diffraction, infrared spectroscopy, and ultraviolet spectroscopy. The specific surface area of the synthesized material was 979 m2/g. A high specific surface area was conducive to the adsorption of trace target compounds. The surface charge of the material could be controlled by adjusting the pH value of the solution, which was beneficial to the selective adsorption and desorption of ionic pigments. The π-π interaction between the benzene ring of the porphyrin ring and the benzene ring of the azo pigment also promoted extraction. Thus, the extractant exhibited strong enrichment performance for the new coccine anionic pigment. The solid phase extraction conditions were optimized, and it was found that saturated adsorption capacity was achieved by filling 3 mg of extractant. The effect of pH on adsorption was also explored; the adsorption effect was the best at pH 3. In the desorption experiment, N,N-dimethylformamide at pH 11 was conducive to better elution of the target. Further elution volume studies showed that maximum recovery could be achieved by adding 3 mL of eluent. Subsequently, the sample pretreatment time was reduced to 5 min. The enriched sample was separated using a Zorbax eclipse XDB-C18 column (250 mm×4.6 mm, 5 μm), eluted with an ammonium acetate-methanol solvent system, and detected at 254 nm. Under the optimum conditions, the recoveries of the samples at high, medium, and low levels reached 99.5%-109.4%, and the relative standard deviation was less than 3%. The limit of detection (LOD, S/N=3) of this method was 0.1 μg/L and the limit of quantification (LOQ, S/N=10) was 0.3 μg/L. In the actual sample detection experiment, the detection signal of new coccine in the sample was amplified by solid phase extraction to achieve enrichment. In addition, the extraction capacity of PCN-222 remained higher than 90% after four uses, and the synthesized material could be recycled. The high precision and low detection limit indicate that the method is suitable for the enrichment and detection of trace carmine in beverages. The findings of this study will aid in the development of a new solid phase extraction technology for food safety evaluation.

    Determination of three urinary catecholamines and serotonin by on-line packed-fiber solid-phase extraction
    BI Yueling, XU Tong, CHEN Liqin
    2021, 39 (12):  1306-1313.  DOI: 10.3724/SP.J.1123.2021.07001
    Abstract ( 186 )   HTML ( 192 )   PDF (1838KB) ( 190 )  

    Biogenic monoamines, including catecholamines (CAs) and serotonin (5-HT), play critical roles in the central nervous system. They have recently been proven to be primarily useful as biomarkers for the diagnosis of CA-producing tumors. The highly polar properties of biogenic monoamines result in poor retention on conventional materials, making it challenging to simultaneously measure more biogenic monoamines from complex matrices. Moreover, the classical method of off-line pretreatment is relatively complex, labor-intensive, and incurs errors in repeatability among different operators. Therefore, the development of an on-line sample pretreatment method combined with the use of specific nanofiber adsorbents has been explored. An on-line procedure could avoid unnecessary and time-consuming steps, and enable full automation of the experimental process. In this study, an on-line packed-fiber solid-phase extraction (PFSPE) and determination method for urinary CAs (dopamine (DA), norepinephrine (NE), epinephrine (E)) and 5-HT was developed, using composite nanofibers of polycrown ether-polystyrene (PCE-PS). PCE-PS composite nanofibers prepared by electrospinning were used as adsorbents in the PFSPE column, which was connected to the on-line HPLC system. The PFSPE-HPLC equipment contained a dual ternary pump and a switching valve to enable enrichment, purification, and analysis directly in the system. The left pump was connected with the PFSPE column for sample enrichment and purification, while the right pump was attached to the analysis column for sample separation and testing. The switching valve was controlled such that after enrichment, the samples could be eluted to the analysis column for separation and detection.
    The current work expands on our previous research by analyzing more target substances, and developing an on-line sample pretreatment method to simultaneously analyze four biogenic monoamines. Gradient separation aided in the satisfactory separation of the biogenic monoamines within a short retention time. The running time was set at 16 min to enable thorough enrichment, elution, and analysis. The influence of the complexing reagent (diphenylborinic acid 2-aminoethyl ester, 2 mg/mL) was also investigated with this on-line PFSPE-HPLC system. The results showed that the intensity of most analytes was significantly higher when 50 μL of the complexing reagent was added. The influence of a buffer on the extraction of the biogenic monoamines was also tested. The optimum extraction condition for the target analytes was achieved when artificial urine (AU) samples were diluted in a volume ratio of 1∶1 by phosphate- buffered saline solution (PBS, pH 7.8). Under the optimum experimental conditions, the on-line PFSPE-HPLC procedure showed good linearity (in the range of 1 ng/mL to 200 ng/mL) with correlation coefficients above 0.996 for the quantitative detection of urinary CAs (DA, NE, E) and 5-HT. For the CAs, the limit of detection (LOD) was 1 ng/mL (S/N=3), while the limit of quantitation (LOQ) was 2.5 ng/mL (S/N=10). For 5-HT, the LOD was 2.5 ng/mL (S/N=3) and the LOQ was 5 ng/mL (S/N=10). Moreover, high recovery rates and good reproducibility were obtained. The recoveries of AU and real urine spiked with CAs and 5-HT were in the range of 83.5%-117.7%, and the intra-day precision was lower than 10%. Additionally, no significant changes in the nanofibers were observed after repeated extraction, which reflected the good stability and reusability of the nanofibers. The nanofibers could be reused for more than 95 times.
    The on-line PFSPE-HPLC system was successfully applied for the determination of urinary CAs and 5-HT with good precision and high sensitivity. This high level of integration and automation was significantly advantageous in terms of its repeatability, as well as reduction in the time and effort required. The proposed on-line pretreatment and determination method can provide strong technical support for the detection and diagnosis of, as well as research on related diseases in clinical practice.

    Simultaneous determination of three allergic proteins in rice and products by high performance liquid chromatography-tandem mass spectrometry combined with stable isotope-labeled peptides
    YANG Huan, CAO Zhaoyun, MA Youning, CHEN Mingxue
    2021, 39 (12):  1314-1323.  DOI: 10.3724/SP.J.1123.2021.06039
    Abstract ( 219 )   HTML ( 190 )   PDF (1498KB) ( 103 )  

    Rice is an important cereal that is consumed as both an energy and protein source by a large proportion of the population worldwide. However, clinical studies have found that rice grains are responsible for cases of severe asthma, eczema, and atopic dermatitis in some adult patients. Several allergenic proteins have been identified and biochemically and immunochemically characterized from rice grains. These include α-amylase/trypsin inhibitors, glyoxalase Ⅰ, and α-globulin. In this study, we proposed an approach for the simultaneous quantification of three allergenic proteins in rice and its products, based on a stable isotope-labeled signature peptide standard and liquid chromatography-tandem mass spectrometry. Samples of rice and products were extracted by a salt solution, hydrolyzed by Lys-C and Trypsin, and purified by C18-SD. The linear ion trap-high resolution mass spectrometry (LTQ-Orbitrap) and Protein Discovery software were used to acquire and identify allergenic proteins in rice samples. In present study, three proteins including seed allergenic protein RAG2, glyoxalase Ⅰ, and 19 kDa globulin were identified. To establish a stable quantitative detection method, the signature peptides selected from the identified enzymatic hydrolysis peptides must have greater abundance and higher specificity as characteristic peptides. Three corresponding signature peptides in rice were screened based on the principles of previous study, and were validated through comparisons of the basic local alignment search tool (BLAST) with the NCBI and UniProt databases. The three signature peptides were successively eluted by liquid chromatography and separated on a Poroshell column. They were then detected by positive electrospray ionization (ESI+) in multiple reaction monitoring mode and quantified by an isotope dilution method. To achieve an improvement in the detection sensitivity and specificity, mass spectrometry parameters, such as the collision energy of three ion pairs of each peptide, were optimized. Three recombinant allergenic proteins and the winged stable isotope-labeled signature peptide standard were synthesized. These were then used to compare the effects of different enzymatic conditions, including hydrolysis solvents containing sodium dodecyl sulfate (SDS) with different contents, as well as the enzymes and their amounts, on the digestion efficiency. The data showed that the digestion efficiency of the three proteins could be improved to 65.7%-97.3% when 1 g/L of the SDS-containing hydrolysis solvent, and the combined digestion strategy of Lys-C and Trypsin, were adopted in the enzymatic process. These results indicate the following inferences: a small amount of SDS (1 g/L) in the enzymatic hydrolysis system is beneficial to complete protein denaturation, a Lys-C and Trypsin combined digestion strategy can complement the shortcomings of the two enzymes and improve the digestion efficiency, and the recoveries of the three proteins was not significantly increased by increasing the amount of enzyme when the ratio of protein to enzyme reached more than 20∶1. The method displayed good linearity in the range of 1-200 nmol/L with the correlation coefficients greater than 0.9972. The limits of detection and limits of quantification of the three proteins were 3 mg/kg and 10 mg/kg, respectively. The average recoveries of the three proteins spiked at three levels in different matrices ranging between 80.6%-103.7%, with the intra-day and inter-day precision less than 11.5%. Due to its high stability, excellent sensitivity, and simple operation, this method presents a wide range of application prospects in the analysis of the three allergenic proteins in different rice and rice food products.

    Simultaneous determination of eight carbamate pesticide residues in tomato, rice, and cabbage by online solid phase extraction/purification-high performance liquid chromatography-tandem mass spectrometry
    LIU Xin, SUN Xiulan, CAO Jin
    2021, 39 (12):  1324-1330.  DOI: 10.3724/SP.J.1123.2021.01028
    Abstract ( 194 )   HTML ( 620 )   PDF (1537KB) ( 198 )  

    Carbamate pesticides are a class of synthetic pesticides having wide antimicrobial spectrum, good insecticidal efficacy, and a short residual period. These pesticides are used in agriculture, forestry, and animal husbandry. Their widespread use in the last two decades has led to the existence of drug residues in the environment, which are transferred to food, thereby raising concerns regarding the potential threat to human health. Rapid and accurate detection of carbamate pesticide residues in food is of great significance for food safety, and this requires pretreatment to purify the target components and maximize the accuracy and precision of the analysis. A rapid and accurate analytical method based on online solid phase extraction/purification-high performance liquid chromatography-tandem mass spectrometry (online SPE-HPLC-MS/MS) was established for the determination of eight carbamate pesticides in tomato, rice, and cabbage. About 5.0 g of tomato (without water), 2.0 g of cabbage, and 2.0 g of rice (mixed with 3 mL of water) were vortexed at 1000 r/min for 1 min. After adding 2 g of sodium chloride and 10 mL of acetonitrile containing 0.5% (v/v) formic acid, the samples were extracted and centrifuged. The supernatants were combined after the samples were extracted again. The reconstituted solutions were then purified on a CAPCELL PAK C18 column (50 mm×2.0 mm, 15 μm). When the volume ratio of 0.1% (v/v) formic acid aqueous solution and acetonitrile (used as the mobile phases) was 90∶10 and 35∶65, the eight carbamate pesticides could be completely adsorbed and eluted. The carbamate pesticides were separated on an ACQUITY UPLC CSH C18 column (100 mm×2.1 mm, 1.7 μm) under gradient elution and analyzed in the multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+). Under the optimum conditions, the calibration curves of the eight carbamate pesticide residues showed good linearity (r>0.995) within their respective linear ranges. The limits of quantification (LOQs) and limits of detection (LODs) were in the range of 0.05-1.0 ng/mL (S/N=10) and 0.01-0.3 ng/mL (S/N=3). The recoveries were in the range of 73.76%-112.32% at three spiked levels (2, 10, and 20 ng/mL), with relative standard deviations of 1.28%-13.14% (n=6). The online purification method showed better enrichment and purification ability for the target substances than did the offline purification method and greatly improved the pretreatment efficiency. The loading and purification could be completed within 12 min. The developed method has the advantages of high recovery rate, good reproducibility, accuracy, rapidness, sensitivity, and environment friendliness. It can be used for the determination of the eight carbamate pesticides in plant foods, such as tomato, rice, and cabbage.

    Simultaneous determination of beauvericin and four enniatins in eggs by ultra-performance liquid chromatography-tandem mass spectrometry coupled with cold-induced liquid-liquid extraction and dispersive solid phase extraction
    LIU Bolin, NI Man, SHAN Xiaomei, XIE Ji’an, DAI Yanyu, ZHANG Cheng
    2021, 39 (12):  1331-1339.  DOI: 10.3724/SP.J.1123.2021.02015
    Abstract ( 207 )   HTML ( 186 )   PDF (1762KB) ( 89 )  

    Enniatins (ENNs) and beauvericin (BEA), known as emerging mycotoxins, are the toxic secondary metabolites produced by various Fusarium species. Most grain and grain-based products are contaminated with ENNs and BEA. Animals have been exposed to ENNs and BEA primarily due to consumption of cereal grains and cereal by-products. ENNs and BEA have been detected in animal-derived food and human breast milk, and they pose significant threats to public health. Therefore, more contamination data are urgently needed for the risk assessment of ENNs and BEA present in animal-derived food. To ensure the quality of animal-derived food, a method has been developed for the simultaneous detection of five emerging mycotoxins (viz. enniatin B, enniatin B1, enniatin A, enniatin A1, and beauvericin) in eggs by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled with cold-induced liquid-liquid extraction (CI-LLE) and dispersive solid phase extraction (DSPE). The main factors governing the response, recovery, and sensitivity of the method, such as the type of extraction solvent, the temperature and duration of cold treatment in CI-LLE, the type and dosages of adsorbents, and apparatus conditions and the type of mobile phase used, were optimized during sample pretreatment and instrument analysis. The mycotoxin residues in eggs were extracted using 20 mL acetonitrile-water-acetic acid (79∶20∶1, v/v/v) mixture for 20 min by the vortex shock method. After mixing, the mixture was frozen for 30 min in a freezer at -40 ℃ and centrifuged for 10 min at 10000 r/min. A 2 mL aliquot of the upper acetonitrile layer was purified by using 70 mg of C18 adsorbents. After whirling, the mixtures were centrifuged at 10000 r/min for 5 min. The purified solution was then concentrated to nearly dry in nitrogen atmosphere at 40 ℃. The residues were dissolved in 1.0 mL 80%(v/v) acetonitrile aqueous solution. The target analytes were separated on an ACQUITY UPLC BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 μm) at a column temperature of 40 ℃, with a flow rate of 0.3 mL/min. The injection volume was 5 μL, and gradient elution was conducted using acetonitrile and 5 mmol/L ammonium formate solution as the mobile phases. Multiple reactions monitoring (MRM) was conducted in the positive electrospray ionization (ESI +) mode. The isotope internal standard method was used for quantification of BEA, and the matrix-matched external standard method was used for quantification of four ENNs. The results of the optimized method showed that the five analytes were completely separated by using the above-mentioned chromatographic column. Good linear relationships were obtained for the five mycotoxins in the concentration range of 0.1-50.0 μg/L; the correlation coefficient (r2) ranged from 0.9983 to 0.9997. The limits of detection (LODs) ranged from 0.05 to 0.15 μg/kg, while the limits of quantification (LOQs) ranged from 0.20 to 0.50 μg/kg. Accuracy and precision experiments were conducted by spiking egg samples with known amounts of analytes at three concentration levels (0.5, 5.0, and 25.0 μg/kg, in compliance with the current legislation) with six replicates. The average recoveries of the five analytes ranged from 81.1% to 106%, and the relative standard deviations (RSDs) were between 0.27% and 9.79%. The matrix effects of the analytes were between 2.70% and 45.1% in egg samples after pretreatment by CI-LLE coupled with DSPE. The developed method was applied to the determination of five mycotoxins in rural eggs and commercial eggs. BEA was detected in most rural egg samples, with detection rates of 30.4%. None of the four ENN residues were detected. Therefore, we can conclude that the method described herein has the advantages of sensitivity, stabilization, accuracy, good recovery, and easy operation, and is suitable for the simultaneous and rapid determination of BEA and ENN residues in eggs.

    Determination of fatty acids compositions and contents in Chinese mitten crabs by gas chromatography
    SHEN Zhaodong, HUANG Dongmei, FANG Changling, YE Hongli, TIAN Liangliang, WU Zi, ZHANG Jun
    2021, 39 (12):  1340-1346.  DOI: 10.3724/SP.J.1123.2021.01032
    Abstract ( 218 )   HTML ( 192 )   PDF (1062KB) ( 118 )  

    Determination of fatty acid compositions and contents in Chinese mitten crabs is of great significance to evaluate its nutritional value and quality. However, in the face of a wide range of fatty acid extraction and methyl esterification reagents, the measurement results are uneven, and it is difficult to accurately quantify the rich fatty acids in Chinese mitten crabs. In this paper, four kinds of oil extraction reagents and two kinds of methylating reagents, were investigated. Chloroform-methanol (1∶1, v/v) was used as the extraction solvent, and methanol containing 2% sulfuric acid was used as the derivatization reagent. A method for the determination of fatty acids in the muscle of Chinese mitten crabs by gas chromatography was established. The experiment was carried out under the condition of programmed temperature rise, 37 kinds of fatty acids were separated on a DM-2560 capillary column (100 m×0.25 mm×0.20 μm), detected by hydrogen flame ionization detector (FID) and quantified by external standard method. The linear relationships of the 37 fatty acids were good in the range of 0.5-100.0 μg/mL. The correlation coefficients (R2) were 0.9981-0.9999. The limits of detection (LODs) and limits of quantification (LOQs) were 0.01-0.02 mg/100 g and 0.04-0.06 mg/100 g, respectively. The methodology was validated by palmitic acid and stearic acid. The recoveries were 76.0%-97.5%, and the relative standard deviations (RSD, n=5) were 3.31%-7.90% at the spiked levels of 1, 2 and 10 mg/100 g. The method was applied to the determination of fatty acid compositions and contents in the muscle of Chinese mitten crabs. A total of 31 kinds of fatty acids were detected. The length of carbon chain ranged from 12 to 24, and the total content of fatty acids reached 281.03 mg/100 g. Oleic acid, docosahexaenoic acid and eicosapentaenoic acid were the main fatty acids in the muscle of Chinese mitten crabs. Thus, this method provided accurate and reliable theoretical data for the determination of fatty acids in Chinese mitten crabs. This method has the advantages of simple operation, small amount of reagent and sample, reliable qualitative, accurate quantitative, detection of more fatty acid types. It is suitable for the rapid detection of fatty acid compositions and contents in muscle tissue of Chinese mitten crabs.

    Separation and determination of clenbuterol enantiomers by ultra-performance convergence chromatography
    ZHANG Wenhua, HONG Deng, LEI Meikang, HU Xiaoli, HOU Jianbo, XIE Wen, XU Dunming, YI Xionghai, LI You
    2021, 39 (12):  1347-1354.  DOI: 10.3724/SP.J.1123.2021.06045
    Abstract ( 181 )   HTML ( 184 )   PDF (1006KB) ( 73 )  

    Clenbuterol enantiomers differ greatly in their bioactivities. By optimizing the conditions for chromatographic separation and method validation, ultra-performance convergence chromatography (UPC2) was adopted to separate the enantiomers of clenbuterol. Standard solutions of (+)-clenbuterol and (-)-clenbuterol were stored at -18 ℃ for 1, 3, 5, 7, 14, 30, and 60 d, and then, their stability was monitored. The impacts of different chromatographic columns, cosolvents, system backpressure, and chromatographic column temperature on the separation of the two enantiomers were investigated. Acquity Trefoil AMY1 (150 mm×3.0 mm, 2.5 μm) was used for separation, and CO2-0.5% (v/v) ammonium acetate was used as the mobile phase. Gradient elution at a flow rate of 2.0 mL/min was adopted. The detection wavelength was set to 241 nm, and the injection volume was set to 10 μL. The backpressure was set to 13.8 MPa, and the column temperature was maintained at 40 ℃. The two enantiomers showed good linear relationships in the range of 1.0 to 20.0 mg/L with correlation coefficients greater than 0.9997. The limits of detection (LODs, S/N=3) of (+)-clenbuterol and (-)-clenbuterol were both 0.5 mg/L. The relative standard deviation (RSD, n=6) for the peak area of the 10.0 mg/L mixed standard working solution with six replicate injections ranged from 0.65% to 0.76%. The effectiveness and practicability of this method were demonstrated by using it to detect standard clenbuterol racemate. The (+)-clenbuterol and (-)-clenbuterol contents were 5.6 mg/L and 5.5 mg/L, respectively, in the standard clenbuterol racemates, as determined by the external standard method of quantification. The detection results suggested that the content ratio of (+)-clenbuterol and (-)-clenbuterol was close to 1.02∶1.00, which is consistent with the literature data. The established method has the advantages of rapid analysis, good separation effect, and low consumption of organic solvents, and it is suitable for the separation of clenbuterol enantiomers. This method can also provide technical support for the separation of other chiral drugs, analysis of the effects of chiral drugs, and assessment of product quality.

    Determination of the enantiomers of salmeterol xinafoate in salmeterol fluticasone powder inhalant by chiral nonaqueous capillary electrophoresis
    ZHANG Xu, DONG Miaoxue, XU Yin, WANG Lijuan, QIAO Xiaoqiang
    2021, 39 (12):  1355-1361.  DOI: 10.3724/SP.J.1123.2021.06002
    Abstract ( 172 )   HTML ( 185 )   PDF (839KB) ( 130 )  

    Salmeterol xinafoate (SalX) is one of the ideal drugs used for the treatment of nocturnal asthma attacks and daily maintenance. The molecular structure of SalX contains a chiral carbon atom, and thus, SalX has two enantiomers, viz. (R)-SalX and (S)-SalX. It is clinically administered in the racemic form. Related studies have shown that the two enantiomers of SalX are quite different in pharmacology, toxicology, and other aspects. Therefore, it is of great significance to establish an analytical method for the chiral separation and determination of the SalX enantiomers to guarantee their quality and ensure their safety and effectiveness in clinical use. In this study, a chiral nonaqueous capillary electrophoresis (NACE) method, using a L(+)-tartaric acid-boric acid complex as the chiral selector, was established to determine the enantiomers of SalX in salmeterol fluticasone powder inhalant. The L(+)-tartaric acid-boric acid complex was synthesized in situ by the reaction of L(+)-tartaric acid and boric acid in methanol solution. The ion pair principle was considered the enantioseparation mechanism, and the non-aqueous system was found to be more favorable for ion pair formation, which is useful for chiral recognition. Chiral separation is based on the reversible formation of diastereomeric ion pairs between the negatively charged L(+)-tartaric acid-boric acid complex and the positively charged salmeterol enantiomers. Due to the difference in ion-pair binding ability between different enantiomers, the apparent electrophoretic mobilities of different enantiomers were also different, resulting in chiral separation in NACE. To achieve good resolution, the effects of L(+)-tartaric acid concentration, boric acid concentration, and apparent pH (pH* ) on the chiral separation were investigated. The optimized buffer solution (pH* 0.93) contained 120.0 mmol/L L(+)-tartaric acid and 120.0 mmol/L boric acid in methanol. Other experimental conditions were as follows: uncoated fused-silica capillary with an I. D. of 50.0 μm, a total length (Ltot) of 64.5 cm, and an effective length (Leff) of 55.5 cm, along with gravity injection of 17.5 cm×10.0 s, detection wavelength of 225 nm, room temperature, and operating voltage of 20.0 kV. Under these experimental conditions, the two enantiomers of SalX achieved a resolution of 2.18 within 18.0 min. Both enantiomers showed a good linear relationship of the peak area in the concentration range of 27.5-800.0 mg/L, the correlation coefficient (r) being greater than 0.9990. The detection limit (S/N=3) and quantitative limit (S/N=10) were 7.5 mg/L and 25.0 mg/L, respectively; the standard recovery was 98.1%-101.9%, with relative standard deviations (RSDs) of 1.2%-1.9%. The intra- and inter-day precisions were examined, and the RSDs of the peak area and migration time were found to be below 4.9% and 1.9%, respectively, indicating good repeatability (inter-day) and reproducibility (inter-day) of the method. The established chiral NACE method was used to determine the two SalX enantiomers in a random salmeterol fluticasone powder inhalant purchased from a local market. The results showed that the percentage of labeled quantities was 98.7% for both enantiomer 1 and enantiomer 2, with RSDs of 2.5% and 2.7%, respectively. Thus, this method is simple, feasible, accurate, and inexpensive, and can be applied for the determination of SalX enantiomers in commercially available salmeterol fluticasone powder inhalants.

    Determination of absolute mobility and dissociation constant of lovastatin using capillary electrophoresis and empirical equation of ion mobility
    LUO Fang, GUO Zehua, CAO Chengxi, FAN Liuyin, ZHANG Wei
    2021, 39 (12):  1362-1367.  DOI: 10.3724/SP.J.1123.2021.01014
    Abstract ( 165 )   HTML ( 178 )   PDF (809KB) ( 103 )  

    In capillary electrophoresis, determination of the basic physical and chemical properties of compounds, such as absolute mobility (m0) and dissociation constant (pKa), is of great practical significance. This is because the aforementioned properties are often used for the qualitative or quantitative analyses of the relevant compounds toward their application as potential drugs. Lovastatin is a potential drug candidate that can reduce the levels of cholesterol and low-density lipoprotein cholesterol in the blood, as well as prevent atherosclerosis and coronary heart disease. For a more convenient and rapid investigation of the properties and applications of lovastatin, it is necessary to determine its m0 and pKa values. However, existing research on capillary electrophoresis for lovastatin and other related drugs focus on their quantitative determination, and their action mechanism and functions. Unfortunately, there are very few studies aimed at the determination of the m0 and pKa values of lovastatin. Based on related studies, this paper herein proposed a novel method to determine m0 and pKa of lovastatin. The present study mainly included a calculation method and experimental verification. The calculation method was based on capillary zone electrophoresis (CZE) and the empirical formula of ion mobility. First, on the basis of the empirical formula, the calculation formula for m0 was derived from the relationship between the actual mobility (mact), effective mobility (meff) and m0. Second, for a monovalent acid (HA), according to the calculation formula for m0 part, considering the hydrogen ion concentration as the independent variable and the reciprocal of meff as the dependent variable, a straight line was obtained on the coordinate axis. From the slope of this straight line, the dissociation equilibrium constant Ka was obtained directly, and pKa was calculated easily. After the derivation of m0 and pKa in the theoretical part, the feasibility and reliability of this method were verified by using it to determine the m0 and pKa values of several organic acids and bases (barbituric acid, benzoic acid, benzylamine, phenol, and m-cresol) in the experimental part. Note that for the buffer system with pH<6.0, reverse capillary electrophoresis was used for the determination of pKa, because this technique helped shorten the migration time and facilitates the detection of analytes that could not reach the cathode. After obtaining m0 and pKa, the theoretical reference values for these parameters were obtained by PeakMaster 5.1. The experimental data were well consistent with the theoretical m0 and pKa values. The standard deviation (SDs) of m0 and pKa were less than 6.0% and 6.2%, respectively. From the correlation coefficient (R) of the linear regression equation, it was found that the linear regression lines of pKa fit well, indicating the excellent reliability of this method. Finally, with this simple and reliable method, dimethyl sulfoxide (DMSO) was used as a marker for electroosmotic flow to determine the m0 and pKa values of lovastatin (-1.70×10-8 m2/(V·s) and 9.00, respectively). This method is suitable for the determination of m0 and pKa of acidic and basic analytes. The method has high accuracy and is expected to play an indispensable role in drug analysis.

    Technical Notes
    Determination of bongkrekic acid in tremella and auricularia auricular by improved QuEChERS method combined with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry
    ZOU Pan, DUAN Shengxing, HU Xizhou, ZHENG Dan, XIA Zhenzhen, XIA Hong, PENG Xitian
    2021, 39 (12):  1368-1373.  DOI: 10.3724/SP.J.1123.2021.06013
    Abstract ( 281 )   HTML ( 192 )   PDF (926KB) ( 130 )  

    An improved QuEChERS (quick, easy, cheap, effective, rugged, safe) method, combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), was used to determine bongkrekic acid (BA) in tremella and auricularia auricular. BA is a fat-soluble, fatal bacterial toxin produced by the aerobic gram-negative bacteria Burkholderia gladioli pathovar cocovenenans. Tremella and auricularia auricular, which have rich nutritional values, are traditional edible fungi in China that are very popular among Chinese consumers. However, tremella and auricularia auricular are easily contaminated by BA during storage and foaming, and this poses a great threat to food safety and human health. Therefore, establishing a rapid and efficient analysis method for detecting BA in tremella and auricularia auricular is of considerable significance. However, the BA concentration in the actual sample is very low, and the matrices of tremella fuciformis and auricularia auricular are very complex. Thus, it is necessary to employ appropriate sample pretreatment technology to extract and purify BA from tremella and auricularia auricular samples prior to instrumental analysis. In this study, the QuEChERS method, combined with UHPLC-MS/MS, was used to detect BA in tremella and auricularia auricular. The key parameters, such as extraction solvent, extraction method, and adsorbent used for cleanup, were optimized to obtain high extraction efficiency. The content of acetic acid in the extraction solution strongly influenced the extraction efficiency of BA, and acetonitrile with 5%(v/v) acetic acid was determined to be the optimum extraction solvent. After salting out, the acetonitrile extract was purified by dispersive solid phase extraction using 200 mg C18 as a cleanup adsorbent. The sample was then separated on a Waters HSS T3 column (100 mm×2.1 mm, 1.8 μm), using a water solution containing 0.01% (v/v) formic acid and 0.05% (v/v) ammonia and methanol as mobile phases. MS analysis was performed using an electrospray ionization source in the negative and multiple reaction monitoring (MRM) modes. Under the optimized conditions, the matrix effects of UHPLC-MS/MS in tremella and auricularia auricular were -6.3% and -11.5%, respectively; this indicated that the method had a significant purification effect, and the sample matrix did not affect the MS detection of BA. Further study showed that in the concentration range of 1-200 μg/L, the square of the regression coefficient of the linear equation (R2) was greater than 0.999. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.15 μg/kg and 0.5 μg/kg, respectively. The average recoveries in samples spiked with 0.5, 10, and 50 μg/kg BA in tremella ranged from 92.4% to 102.6%, and the intra-day and inter-day relative standard deviations (RSDs) were 4.3%-4.9% and 3.2%-3.5%, respectively. For auricularia auricular, the average recoveries ranged from 89.6% to 102.3%, and the intra-day and inter-day RSDs were 2.4%-9.5% and 3.6%-4.1%, respectively. These results indicate that the proposed method has satisfactory sensitivity, accuracy, and precision. Finally, the method showed good performance when applied to the analysis of real samples. Compared with other reported methods, the LOD and LOQ of our proposed method were lower, with satisfactory recovery and precision. Taken together, this study provides an effective detection technology for the monitoring and risk control of BA in tremella and auricularia auricular.

    Determination of ten aminoglycoside residues in eggs by mixed-mode ion exchange liquid chromatography-tandem mass spectrometry
    WEI Lili, XUE Xia, WU Chuanxiang, DING Yi, LU Lanxiang, WANG Jun, LIU Yanming
    2021, 39 (12):  1374-1381.  DOI: 10.3724/SP.J.1123.2021.02027
    Abstract ( 248 )   HTML ( 201 )   PDF (2494KB) ( 186 )  

    Aminoglycosides (AGs) are a class of broad spectrum antibiotics that have bactericidal activity against some aerobic gram-positive and gram-negative organisms. AGs have been extensively employed in animal husbandry for the treatment of bacterial infections or growth promotion. Many countries have issued strict maximum residue levels (MRLs) for AGs in many animal-origin foods. Analysis of AGs is quite challenging due to their physicochemical properties. The lack of any notable chromophores or fluorophores makes direct detection using ultraviolet (UV) or fluorescence (FLR) spectroscopy unfeasible. Therefore, AGs must be derivatized before they can be analyzed by UV or FLR detection techniques. However, the sensitivity of such derivatization methods is relatively low. Methods based on chromatographic analysis coupled with tandem mass spectrometric detection are emerging as the most common way of identification and quantification. The retention of AGs on reversed-phase column is poor due to the presence of various amino and hydroxyl groups in their structures. Therefore, ion-pair chromatography has reportedly been used to improve the retention of AGs. However, electrospray ionization-mass spectrometric detection was hampered by using an ion pairing reagent due to the suppression of ionization. In this study, a method based on mixed-mode ion exchange liquid chromatography-tandem mass spectrometry was developed for the determination of ten AGs residues (streptomycin, dihydrostreptomycin, hygromycin B, kanamycin, amikacin, tobramycin, apramycin, spectinomycin, neomycin, and gentamycin) in eggs. The main factors governing the method, such as the type of chromatographic column used, the type and proportion of the mobile phase used, mass spectroscopy parameters, type and volume of the extraction solvent used, pH, and the type of solid phase extraction (SPE) column, were investigated during sample pretreatment and instrument analysis. The residues of AGs in the test samples were extracted by ultrasonication with 10 mmol/L ammonium acetate buffer solution (comprising 0.4 mmol/L EDTA and 50 g/L trichloroacetic acid). After adjusting the pH, the AG residues in the sample were purified and enriched using a PRiME HLB SPE column. The target analytes were separated on a SIELC Obelisc R column (150 mm×2.1mm, 5 μm), the column temperature being 40 ℃, the flow rate being 0.3 mL/min, and the injection volume being 5 μL. Gradient elution was carried out with acetonitrile and 1.0%(v/v) formic acid aqueous solution (including 1 mmol/L ammonium formate) as the mobile phases. The detection was performed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in multiple reaction monitoring (MRM) mode. The retention times and ionic ratios were used for qualitative analysis, and the peak areas were used for quantitative analysis by the external standard method. Good correlation coefficients exceeding 0.99 were observed for all the AGs in the concentration range of 5-200 μg/L under the optimum conditions. The limits of detection (LODs, S/N ≥ 3) and limits of quantification (LOQs, S/N≥10) for the ten AGs were 2-5 μg/kg and 5-10 μg/kg, respectively. The recoveries ranged from 68.1% to 111.3% (n=6) at three levels (LODs, 20 μg/kg, and 100 μg/kg) in spiked blank egg samples, and the relative standard deviations were 1.2%-12.3%. The matrix effects of the analytes were between 0.3% and 94.3% after purification on the PRiME HLB column. The applicability of the method was validated by analyzing egg samples purchased from local markets. Overall, the method of mixed-mode ion exchange liquid chromatography-tandem mass spectrometry has proven to be a reliable and powerful technique for the simultaneous quantification and confirmation of ten AGs without using an ion pair reagent. Moreover, the clean-up step only required a kind of PRiME HLB sorbent cartridge. The relative parameter data of established method were consistent with GB/T 27404-2008. With simple pretreatment, rapid determination and high sensitivity, the method can be used in the determination of AGs in eggs.