List of Issues

Chinese Journal of Chromatography
2022, Vol. 40, No. 9
Online: 08 September 2022

Previous Issue    Next Issue



For Selected:

Download Citations EndNote Ris BibTeX

Toggle Thumbnails

Perspective

Select

Recent advances in clustered regularly interspaced short palindromic repeats-based detection of severe acute respiratory syndrome coronavirus 2 ZHOU Wen, YANG Kaiguang, ZHANG Lihua, LIANG Zhen, ZHANG Yukui 2022, 40 (9):  773-781.  DOI: 10.3724/SP.J.1123.2022.08001 Abstract ( 123 )   HTML ( 57 )   PDF (5000KB) ( 83 )   The rapid global spread of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has introduced various challenges in global public health systems. The poor applicability and sensitivity of the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and antigen-based tests, as well as the persistent emergence of SARS-CoV-2 variants with different mutations hinder satisfactory epidemic prevention and control. Therefore, there is an urgent need for diagnostic technologies capable of distinguishing SARS-CoV-2 variants with high sensitivity and low (or no) equipment dependence. Diagnosis based on clustered regularly interspaced short palindromic repeats (CRISPR) has low equipment requirements and is programmable, sensitive, and easy to use. Various nucleic acid detection tools with great clinical potential have been developed for the diagnosis of infectious diseases. Therefore, this review focuses on the reported state-of-the-art CRISPR diagnostic technologies developed for the detection and differentiation of SARS-CoV-2 variants, summarizes their characteristics and provides an outlook for their development.

Review

Select

Applications of ion mobility-mass spectrometry in the chemical analysis in traditional Chinese medicines ZHAI Rongrong, GAO Wen, LI Mengning, YANG Hua 2022, 40 (9):  782-787.  DOI: 10.3724/SP.J.1123.2022.01028 Abstract ( 197 )   HTML ( 36 )   PDF (782KB) ( 116 )   Ion mobility-mass spectrometry (IM-MS) is a combination of ion mobility separation and mass spectrometry technologies. In IM-MS, analytes are ionized by the ion source to form gas-phase ions, which are then rapidly separated using ion mobility based on their mobility difference, under the influence of both neutral buffer gas and an electric field, and then traversed and detected using mass spectrometry, which can separate ions based on mass-to-charge ratio. Furthermore, IM-MS could provide not only mass-to-charge ratio parameters like MS1 and MS2 spectra but also new structural information for component identification like collision cross-section values, drift time, arrival time, compensation voltage, and so on, which can be employed to resolve complex chemical components, especially indistinguishable isomers using mass spectrometry alone. In recent years, with the development of IM-MS technologies, IM-MS has become more widely employed in chemical analysis in traditional Chinese medicines (TCMs). Firstly, IM-MS was been successfully used in the separation of complex TCMs complex extract from interfering isobaric species. Secondly, IM-MS also offers new types of MS/MS fragmentation modes, and the combination of IM separation and fragmentation modes enables the acquisition of more specific and detailed fragment ion spectra. Thirdly, the collision cross-section is introduced by IM-MS, which is a unique physicochemical property of a component. Related data post-processing strategies based on experimentally derived collision cross-section values have been continuously developed in recent years to make full use of the collision cross-section values, these data post-processing strategies include collision cross-section database matching, theoretical collision cross-section values matching, machine-learning-based collision cross-section values prediction matching, mass-to-charge ratio versus collision cross-section correlation trend lines and so on. In doing so, these diverse strategies can greatly enhance the reliability and accuracy of the structural annotation of TCM compounds. This review primarily briefly introduces the major types and basic principles of IM-MS. The applications of IM-MS in TCM chemical analysis are highlighted in this study. The current applications of IM-MS in improving TCM chemical component separation are summarized, followed by a discussion of several strategies for enhancing separation selectivity. This review also offers some new fragmentation modes, novel data acquisition approaches, and collision cross-section data post-processing strategies applied in TCM qualitative analysis. Finally, the prospect of IM-MS applied in TCM chemical analysis is also discussed. This review provides approaches and ideas for future IM-MS research on TCM’s chemical analysis.

Articles

Select

A novel method for efficient screening and annotation of important pathway-associated metabolites based on the modified metabolome and probe molecules LI Zaifang, ZHENG Fujian, XIA Yueyi, ZHANG Xiuqiong, WANG Xinxin, ZHAO Chunxia, ZHAO Xinjie, LU Xin, XU Guowang 2022, 40 (9):  788-796.  DOI: 10.3724/SP.J.1123.2022.03025 Abstract ( 131 )   HTML ( 40 )   PDF (4235KB) ( 73 )   Plants produce a wide variety of secondary metabolites in the process of evolution. Secondary metabolites have highly diverse structures due to the modification of the basic skeletons of metabolites. They are required for interaction with the environment and are produced in response to abiotic/biotic stress. Characterization of secondary metabolic pathways is significant to plant molecular breeding and natural product biosynthesis. The liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) is one of the major techniques for untargeted metabolomics study. The LC-HRMS/MS method could detect tens of thousands of metabolic features and provide abundant structural information. It has been widely used in the discovery and characterization of the secondary metabolome. However, due to the largely diverse structure and limited records in the mass spectral library, the annotation of the secondary metabolome is very difficult. To address the analytical challenges associated with the vast structural diversity and the large numbers of secondary metabolites, particularly those previously unknown structural metabolites, a novel method for the efficient characterization of pathway-associated metabolites was developed. Modification reactions and MS/MS spectral information were collected using the metabolic pathways database and mass spectral library. Screening and annotation of metabolites involved in phenylpropanoid metabolism in maize leaves were used as an example. First, a database of modified groups was established via pathway-associated modifications from open access metabolic pathway database and literature. Here, pathway databases included the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Plant Metabolic Pathways (PlantCyc). A total of 61 modification types were enrolled, including 10 generic and 51 pathway-specific modifications. Modified metabolomes were filtered from untargeted LC-HRMS/MS metabolomics data. Next, MS/MS spectra of the pathway-associated compounds (probe molecules) were collected in the Global Natural Products Social Molecular Networking (GNPS) MS/MS spectral library. The MS/MS of compounds assigned to chemical classes of phenylpropanoids were kept. An MS/MS spectral database of the probe molecules was constructed. It included 2677 spectra of 1542 phenylpropanoid compounds in the positive mode and 814 spectra of 661 phenylpropanoid compounds in the negative mode. Then, an MS/MS molecular network was generated by modified metabolome and probe molecules. The clusters comprising both probe molecules and modified metabolites were kept. To explore more previously unknown structural metabolites, the clusters with one more pathway-specific modified metabolite were retained even though they didn’t contain any probe molecule. A total of 392 and 417 phenylpropanoid pathway-related metabolic metabolites were obtained in positive and negative ion modes, respectively. The pathway-associated metabolites were annotated based on the propagation of the molecular network. For the metabolites within the co-cluster, annotations were performed using the probe molecules as the initial seed. The modification group’s substructure information was used for network propagation annotation. For the clusters containing only pathway-specific modified metabolites, the annotation is similar to the above process if identified nodes were present within the cluster. Otherwise, de novo annotation was manually executed based on substructure information. Finally, 129 unique metabolites were annotated after integration and removal of redundancy. Ten annotated metabolites were validated using commercially available or synthesized reference compounds. The other annotation results were validated using predicted chemical classes, in silico MS/MS, and predicted retention time. They are mainly involved in the downstream branch of phenylpropanoid pathways, including the flavonoid pathway (8 flavonoids, 19 flavonoid O-glycosides, 32 flavonoid C-glycosides), the hydroxycinnamic acid pathway (31 hydroxycinnamic acids and derivatives), and the lignan pathway (22 neo-lignans/lignan/lignan glycosides). All the annotated structures were searched against the PubChem and SciFinder databases. Among them, 26 metabolites were previously unreported in both the databases. In this study, the pathway-associated metabolites could be quickly discovered and annotated by the integration of probe molecules and modified metabolome. It provides a method for the in-depth study of the phenylpropanoid pathway.

Select

Determination of 32 oxidative dyes by high performance liquid chromatography and confirmation by high performance liquid chromatography-tandem mass spectrometry ZHOU Zhiming, LI Jing, CHEN Zhanghao, WU Yingying, LU Tuliu, XIAO Shuxiong 2022, 40 (9):  797-809.  DOI: 10.3724/SP.J.1123.2022.03003 Abstract ( 146 )   HTML ( 39 )   PDF (939KB) ( 77 )   Various types of oxidative dyes used in hair dye products possess poor stability and have varying frequency of use. Interference problems also frequently arise in actual sample measurements. Therefore, it is necessary to establish a simple, rapid, accurate, and specific method for the determination of common dyes in hair dye products for their effective regulation. In this study, dyes were grouped according to their frequency of use. Using a C18 column that minimizes the silanol effect and influence of metals, the quantitative high performance liquid chromatography (HPLC) method for 32 dyes listed in Safety and Technical Standards for Cosmetics (2015 edition) was optimized, and a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) confirmatory method for the dyes was established. The samples were extracted using a mixed solution of ethanol-water (1∶1, v/v) with 10 g/L sodium bisulfite solution as an antioxidant, vortexed and mixed, and then extracted by ultrasonication in an ice bath for 10 min. Methanol, acetonitrile, and phosphate buffer were used as the mobile phases in the HPLC analysis. Additionally, two different elution conditions (chromatographic gradient) were used for the separation of 32 oxidative dyes, which were detected at a wavelength of 280 nm. The HPLC separations were compared using columns of particle sizes 5 μm and 2.7 μm; 5 μm C18 columns with better anti-interference and antiblocking ability were selected. Satisfactory separation was achieved for all three commercial C18 columns with a particle size of 5 μm, and the method had good general usability. In condition 1, 17 commonly used dyes and three less commonly used dyes were assigned to group Ⅰ and separated by HPLC; in condition 2, eight banned dyes and four other less commonly used dyes were assigned to group Ⅱ and separated by HPLC. The HPLC-MS/MS method used 5 mmol/L ammonium acetate aqueous solution-acetonitrile and 5 mmol/L acetic acid aqueous solution-acetonitrile as mobile phases in the positive and negative ion modes, respectively. Multiple reaction monitoring (MRM) was performed for qualitative and quantitative analyses in the electrospray ionization mode. Under the examined conditions, six pairs of isomers were well resolved. For the HPLC and HPLC-MS/MS methods, the relative standard deviations (RSDs) of the intra-day precision and 48 h stability tests were less than 10%. The recoveries were between 82.6% and 114.9% (RSD<10%). In the HPLC method, 32 dyes showed good linearity in an approximate range of 10-500 mg/L (r2>0.99), and the limits of detection (LODs) were 9.7-40.1 μg/g. The linear range of hydroquinone in the HPLC-MS/MS method was 2.0-79.7 mg/L, and the LOD was 8.0 μg/g; the linear ranges of the other components were approximately 0.1-4 mg/L, and the LODs were 0.01-0.4 μg/g. The actual samples were simultaneously measured by HPLC, HPLC-MS/MS, and the standard method. Finally, 16 of the 32 dyes were detected, and the detected contents ranged from 58 to 25160 μg/g. The RSDs of the results obtained from the three detection methods were between 1.9% and 10.1%. All detected components were within the limits of group Ⅰ of this method. In comparison with methods reported in the literature and the standard method, this method covers all components for the routine regulatory inspection of oxidative dyes in cosmetics. The method can separate the commonly used dyes under the same HPLC conditions and avoid interference from 15 other commonly used dyes during the analysis of actual samples. A suitable HPLC-MS/MS confirmatory method was also established for the identification of currently unknown substances in the statutory inspection of cosmetics. The method is simple, rapid, accurate, and specific with general usability and good operability.

Special Column for ThermoFisher

Select

Determination of the species origin and thrombin-like enzyme content of Bothrops atrox venom by ultra-high performance liquid chromatography-tandem mass spectrometry based on marker peptide XIAN Ruiqing, HANG Baojian, GONG Liping, WANG Congcong, ZHANG Xunjie, PENG Li, SHI Feng 2022, 40 (9):  810-816.  DOI: 10.3724/SP.J.1123.2021.12020 Abstract ( 156 )   HTML ( 55 )   PDF (1307KB) ( 70 )   Snake venom thrombin drugs are hemostatic drugs prepared from Agkistrodon halys venom, and the main active ingredients are snake venom thrombin-like enzymes (svTLEs). The svTLEs derived from different snake species differ in their structures, hemostatic mechanisms, and pharmacological effects. Therefore, accurate identification of the source of snake venom species and determination of the svTLE content are essential to ensure the quality of these products. Based on proteomics technology, the marker peptides of svTLEs from Bothrops atrox were screened with species specificity for the first time in this study, and an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for species identification and determination of the svTLE content of Bothrops atrox was established. After reductive alkylation and trypsin enzymolysis of the purified svTLE from Bothrops atrox, enzymatic peptide fragments were obtained and determined by easy-nano liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (Nano LC-Q-Exactive-MS). The mass spectrum data were analyzed by Proteome Discoverer 2.2 software. The maker peptide “EAYNGLPAK”, which characterized the svTLE from Bothrops atrox, was finally screened and validated by comparison of the basic local alignment search tool (BLAST) with the NCBI and UniProt databases. For the marker peptide, the enzymolysis temperature, enzymolysis time and amount of enzyme for the sample preparation were optimized. The optimized enzymolysis conditions were as follows: enzymolysis temperature, 37 ℃; enzymolysis time, 4 h; and amount of enzyme, 10 μL. A qualitative and quantitative detection method based on UHPLC-MS/MS was established by optimizing the chromatographic and mass spectrometric conditions. Accordingly, 20 mg of the evenly mixed sample was weighed and placed in a 100 mL volumetric flask. Then, 25 mmol/L ammonium bicarbonate solution was added to dissolve the sample, and the solution was diluted to the scale. Precisely 1.00 mL of the solution was extracted; subsequently, addition of 10 μL trypsin solution was added, followed by shaking, and the mixture was placed in an incubator for 4 h to induce enzymolysis at a constant temperature of 37 ℃. The mixture was subsequently removed from the incubator, cooled to ambient temperature, centrifuged at 12000 r/min for 10 min, and analyzed by LC-MS. Separation was performed on the UPLC system with a Thermo Hypersil GOLD C18 column (100 mm×2.1 mm, 3.0 μm) under the gradient elution of acetonitrile containing 0.1% (v/v) acetic acid and water containing 0.1%(v/v) acetic acid, at a flow rate of 0.3 mL/min, column temperature of 30 ℃, and injection volume of 2 μL. The maker peptides were determined in the electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM) modes using the external standard curve method. The detection ions were m/z 481.9> 315.2 and 481.9> 485.2. There was a good linear relationship between the mass concentration of the marker peptide and the chromatographic peak area in the range of 2.5-30 ng/mL, and the correlation coefficient (r) was 0.9996, The limit of detection (S/N=3) and limit of quantification (S/N=10) were 2.5 mg/kg and 6.25 mg/kg, respectively. At spiked levels of 40, 80, and 120 mg/kg, the recoveries of the marker peptides were 95.5%-101.9%, while the relative standard deviations (RSDs) of the results for parallel analyses at various spiked levels were 1.1%-3.2%. The developed method is simple, rapid, sensitive, and specific, and it can be used for the identification of Bothrops atrox venom species and determination of the svTLE content. The findings of this study would help ensure the quality of hemocoagulase products from the relevant source and provide a reference for the quality control of other snake venom products.

Select

Rapid determination of 22 functional components in cosmetics using ultra-high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry XIONG Chensihui, DING Tianming, LIU Jie, YI Ou, DING Xiaoping, XIE Yun 2022, 40 (9):  817-824.  DOI: 10.3724/SP.J.1123.2022.03037 Abstract ( 224 )   HTML ( 48 )   PDF (1130KB) ( 117 )   Plant components from extracts of Sophora flavescens, rhodiola, ginseng, Centella asiatica, and tea play important roles in skin whitening, moisturizing, anti-aging, sun protection, anti-inflammation, antiseptic, bacteriostatic, and other effects of cosmetics. At present, no relevant standard methods have been established to detect the addition amounts of plant extracts in cosmetics. In addition, plant extracts listed in product labels may be undetectable due to their addition in trace quantities and the lack of technical support. Therefore, a quantitative method for the simultaneous determination of 22 functional components in cosmetics was established by ultra-high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UHPLC-LTQ/Orbitrap MS). Target compounds were extracted with methanol from samples using ultrasonic extraction, and then separated on a C18 column (100 mm × 2.1 mm, 1.8 μm) with gradient elution of 0.1% (v/v) formic acid aqueous solution (A) and acetonitrile (B). The gradient elution program were as follows: 0-5 min, 5%B-8%B; 5-25 min, 8%B-60%B; 25-35 min, 60%B-80%B; 35-36 min, 80%B-5%B; 36-45 min, 5%B. The flow rate was 0.3 mL/min and the injection volume was 5 μL. Accurate masses of precursor ions were used to detect cosmetic functional components in positive ionization mode. The fragment ions obtained by higher energy collisional dissociation were used for confirmation of the functional components. Each compound showed good linearity. The limits of detection (LODs) were in the range of 0.003-2.01 mg/kg, and the limits of quantification (LOQs) were in the range of 0.02-4.36 mg/kg. Recoveries at three levels were 63.2%-125.1%, and relative standard deviations (RSDs) were 0.18%-10.9%. Fifty-four batches of samples labeled with four monomer functional components and nine plant extracts were tested. In the 17 batches of samples labeled with nicotinamide, 4 batches labeled with caffeine, and 6 batches labeled with Sophora flavescens root extract, the labeled functional components were detected. One out of 11 batches of samples labeled with D-panthenol was not detected. Three of the seven batches of samples labeled with ascorbyl glucoside were not detected. In the 21 batches of samples labeled with licorice extracts, the corresponding functional components were not detected in 9 batches. In the 21 batches of samples labeled with Centella asiatica extract, the corresponding functional components were not detected in 11 batches. In the 13 batches of samples labeled with tea extract, the corresponding functional components were not detected in 8 batches. In 11 of the 12 batches containing ginseng root extract, the corresponding functional components were not detected. In five of the six batches of astragalus membranaceus root extract samples, the corresponding functional components were not detected. In samples labeled with Polygonum cuspidatum root extract, Rehmannia glutinosa root extract, and Ophiopogon japonicus root extract, the corresponding functional components were detected. The method is simple, rapid, reliable, accurate, and suitable for the determination of the 22 functional components in cosmetics.

Select

Determination of free amino acids in Eriocheir sinensis by ultra-high performance liquid chromatography-high resolution mass spectrometry GAO Ling, GU Qiang, WANG Hong, MA Xingkong, XUE Feng, ZHANG Xing, GE Jiachun, DING Tao, SHEN Weijian 2022, 40 (9):  825-832.  DOI: 10.3724/SP.J.1123.2022.03027 Abstract ( 143 )   HTML ( 42 )   PDF (1357KB) ( 71 )   Eriocheir sinensis is a unique freshwater crab found in China, which is well known for its rich nutrition and sweet and delicious taste. Free amino acids in Eriocheir sinensis are not only important nutrients but also are closely related to their unique taste and aroma. Therefore, the determination of the free amino acid contents in Eriocheir sinensis is of great significance for product quality evaluation, flavor research, authenticity, and origin identification. Herein we proposed an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based method for the determination of 17 free amino acids in Eriocheir sinensis. First, 5 g of the Eriocheir sinensis sample was weighed into a 50-mL polypropylene centrifuge tube. Then, 10 mL of extraction solvents was added to the centrifuge tube, and the resultant solution was mixed well using a vortex mixer. We compared a variety of solvents and finally selected 5%(v/v) perchloric acid aqueous solution as the optimum extraction solvent. The supernatant was transferred to another polypropylene centrifuge tube after centrifuging at 8000 r/min for 5 min. The extraction procedure was repeated according to the above-mentioned steps, and the extraction solution was combined with the supernatant. The extracts were then adjusted to pH 6.5 with 1 mol/L potassium hydroxide solution, and were diluted to 50 mL with water. After filtering by both qualitative filter paper and a 0.45-μm polyether sulfone syringe filter, the extracts were determined by UHPLC-HRMS. We compared three types of mobile phases and chose 0.1%(v/v) formic acid aqueous solution mixed with acetonitrile as the optimum one. Precise parent ion and ion source parameters were also optimized. The 17 analytes, viz. aspartic acid, threonine, serine, glutamic acid, proline, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, arginine, glycine, alanine, and histidine, were separated on an XDB-C18 column (100 mm×4.6 mm, 1.7 μm) with gradient elution. The amino acids were then detected by HRMS in electrospray ionization and selected ion monitoring modes, and the analytes were quantified using external standards. The instrumental detection limit (IDL) and the instrumental quantification limit (IQL) were 0.3 mg/L and 1.0 mg/L, respectively. The linear correlation coefficients were all above 0.9990 in the concentration range of 10.0-200.0 mg/kg. Three levels of free amino acid standards were spiked into the edible parts of Eriocheir sinensis. The recoveries of the amino acids were between 78.4% and 105.3%. The intra-sample, intra-day, and inter-day repeatabilities were below 4.2%, 5.2%, and 11.4%, respectively, which were within reasonable ranges. Twenty samples of Eriocheir sinensis were tested using the proposed method. Thus, in this study, we developed an alternative method for the determination of free amino acids in Eriocheir sinensis with simple pretreatment, good selectivity, and high accuracy.

Select

Determination of gypenoside XLVI and LVI in Gynostemma pentaphyllum from Fujian by ultra-high performance liquid chromatography-charged aerosol detector LU Pengxin, LI Gang, ZHENG Wei, LIANG Haizhen, ZHANG Jie, CHAI Ruiping, LUO Dingqiang, JIN Yan, GUO Baolin, MA Baiping 2022, 40 (9):  833-842.  DOI: 10.3724/SP.J.1123.2022.01024 Abstract ( 122 )   HTML ( 40 )   PDF (888KB) ( 112 )   Gynostemma pentaphyllum (Thunb.) Makino contains dammarane-type triterpenoid saponins, similar to ginseng, with a host of pharmacological activities. However, its planting resources and chemical composition are quite complex. The chemical constituents of Gynostemma pentaphyllum vary drastically among different origins and varieties. Thus, the corresponding quality control methods also need to be different. Currently, limited information is available about the quality control of Gynostemma pentaphyllum from Fujian. A new method based on ultra-high performance liquid chromatography-charged aerosol detection (UHPLC-CAD) was established for the determination of gypenoside XLVI and LVI in Gynostemma pentaphyllum. The major components of Gynostemma pentaphyllum were characterized using UHPLC-quadrupole time-of-flight-mass spectrometry (UHPLC-Q-TOF/MS) combined with UHPLC-CAD. The results revealed gypenoside XLVI, LVI, and their corresponding malonyl-containing acidic saponins as the main components. However, malonylgypenoside XLVI and LVI can easily remove their malonyl group and convert to gypenoside XLVI and LVI during the application of Gynostemma pentaphyllum. In this study, the samples were pretreated using alkali hydrolysis to transform the acid saponins completely, and the final contents of gypenoside XLVI and LVI were determined via UHPLC-CAD. The optimal alkaline hydrolysis, extraction, and liquid chromatography conditions were established. First, the alkaline hydrolysis conditions were optimized. The effects of the volume of ammonia and reaction time on the contents of gypenoside XLVI, LVI, malonylgypenoside XLVI, and LVI were examined. Malonylgypenoside XLVI and LVI could be transformed completely to gypenoside XLVI and LVI by standing for 24 h in an ethanol-water-ammonia (50∶46∶4, v/v/v) mixture. Furthermore, the extraction conditions were optimized. Next, effects of the different solvents, extraction time, and solid-liquid ratio on the extraction rates of gypenoside XLVI and LVI were investigated. The extraction method for Gynostemma pentaphyllum powder using the ethanol-water-ammonia (50∶46∶4, v/v/v) and a solid-liquid ratio of 1∶150 (g∶mL) for 30 min was established. Finally, a prepared test solution was separated on a Waters ACQUITY UPLC BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 μm). Acetonitrile and 0.1% (v/v) formic acid aqueous solution were used as the mobile phases for gradient elution. The flow rate was set to 0.5 mL/min and column temperature was maintained at 40 ℃. The separation was detected using a charged aerosol detector. Results indicated that the logarithm of the mass concentrations of gypenoside XLVI and LVI had a linear relationship with the logarithm of the peak area in the range of 9.94-318.00 μg/mL and 12.78-409.00 μg/mL, respectively. The correlation coefficients (r) were 0.9993 and 0.9995, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of gypenoside XLVI were 1.58 μg/mL and 6.36 μg/mL, respectively. The LOD and LOQ of gypenoside LVI were 2.05 μg/mL and 8.18 μg/mL, respectively. The relative standard deviations (RSDs) of precision, repeatability, and 24 h stability were less than 2.0% (n=6). The spiked recoveries of gypenoside XLVI were 100.2%-107.2% and the RSD value was 2.4%. The spiked recoveries of gypenoside LVI were 97.9%-104.2% and the RSD value was 2.6%. The results of 16 batches of Gynostemma pentaphyllum samples indicated that the gypenoside XLVI content was 0.57%-2.57%, and gypenoside LVI content was 0.66%-2.99%. Hence, this method has high sensitivity and good reproducibility. Therefore, it can be used for quality research and quality control of Gynostemma pentaphyllum from Fujian.

Technical Notes

Select

Simultaneous determination of 40 plant growth regulators, fungicides, insecticides, and antibiotics in bean sprouts by QuEChERS-high performance liquid chromatography-tandem mass spectrometry FENG Junjun, JIANG Haiyun, WANG Jing, JING Zhengyi, ZHANG Fan, TAN Tianyu, HE Feng, JIANG Lihua, LI Haiqin, CHANG Shimin, LI Tengfei 2022, 40 (9):  843-853.  DOI: 10.3724/SP.J.1123.2021.12028 Abstract ( 231 )   HTML ( 63 )   PDF (1152KB) ( 383 )   Chromatography combined with mass spectrometry is the most commonly used detection technology, and it offers the advantages of high sensitivity and high selectivity. The quick, easy, inexpensive, effective, rugged, and safe (QuEChERS) method is low-cost, effective, and time efficient. The application of the QuEChERS has now been extended to the analysis of contaminants in food samples. The aim of the study was to identify different concentration levels of multiple harmful drug residues in bean sprouts. In this study, QuEChERS coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the simultaneous determination of 40 plant growth regulators, fungicides, insecticides, and antibiotics in bean sprouts. In the HPLC-MS/MS experiment, gibberellic acid, 4-fluorophenoxyacetic acid, chloramphenicol, N6-(δ2-isopentenyl)-adenine, 6-benzylaminopurine, 4-chlorophenoxyacetic acid, and 2,4-dichlorophenoxyacetic acid (2,4-D) were analyzed by MS/MS with negative electrospray ionization (ESI-). The other 33 target analytes (chlormequat, ronidazole, metronidazole, pymetrozine, dimetridazole, methomyl, carbendazim, enoxacin, levofloxacin, pefloxacin mesylate, norfloxacin, ciprofloxacin, enrofloxacin, thiabendazole, lomefloxacin, chlorpyrifos, sarafloxacin, imidacloprid, etc.) were analyzed by MS/MS with positive electrospray ionization (ESI+). Sensitive MS conditions were realized by optimizing the instrumental parameters such as the desolvent temperature, collision energy, spraying needle position, precursor ions, and product ions. Then, the optimal pretreatment method was determined by comparing the recovery rates of the 40 drugs obtained with different extraction solvents (methanol, acetonitrile, acetonitrile containing 0.1% ammonia, acetonitrile with 1% acetic acid), different extraction methods (ultrasonic extraction, shaking extraction), and purification with primary secondary amine (PSA) and C18. In this study, the bean sprouts samples were extracted twice by 10 mL acetonitrile with 1% acetic acid, and extracted under ultrasonic conditions. Then, the extracting solution was only cleaned with 100 mg C18. The chromatographic separation of the 40 compounds was accomplished on a Waters ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) with gradient elution. Methanol and 0.01% formic acid aqueous solution were used as the mobile phases. The 40 compounds were analyzed in the multiple reaction monitoring (MRM) mode. The matrix matching external standard method was used for quantitative determination. The results showed that the 40 compounds could be analyzed within 15 min. Under the optimized conditions, the calibration curves showed good linearities for the 40 compounds, and the coefficients of determination (r2) were greater than 0.99 in the range of 2-200 μg/L. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.1-3 μg/kg and 0.3-9 μg/kg, respectively. Using negative bean sprouts as the substrates, the recovery tests were carried out at three spiked levels of 5, 10, and 50 μg/kg. The average recoveries of the 40 drugs were 78.5% to 115.3%, and the corresponding relative standard deviations (RSDs) were 1.3% to 9.7% (n=6). This method was successfully applied to the analysis of the 40 drug residues in 21 batches of local bean sprouts in Handan city. The results revealed the presence of extensive drug residues in the bean sprouts. The 26 batches were detected to varying degrees, among which 4-chlorophenoxyacetic acid, carbendazim, 6-benzyladenine, 2,4-D, enrofloxacin, and metronidazole were detected at high rates. The detection rates of 4-chlorophenoxyacetic acid, 6-benzyladenine, carbendazim, 2,4-D, gibberellic acid, and enrofloxacin were 28.6%, 19.0%, 9.5%, 9.5%, 4.8%, and 4.8%, respectively. The contents ranged from 37.5-352.4, 32.4-273.1, 28.8-38.7, 316.1-20.2, 19.9 and 13.6 μg/kg, respectively. Given its advantages of simplicity, rapidness, and high sensitivity, the developed method can be used for the rapid and accurate determination of trace levels of the 40 drug residues in large quantities of bean sprouts.

Select

Determination of dimethyl sulfate genotoxic impurities in tertiary amine drugs by ultra-high performance liquid chromatography-tandem mass spectrometry GONG Liping, HANG Baojian, XIAN Ruiqing, YANG Mingzheng, ZHANG Xunjie, WEI Xia 2022, 40 (9):  854-859.  DOI: 10.3724/SP.J.1123.2022.01008 Abstract ( 313 )   HTML ( 64 )   PDF (871KB) ( 127 )   Dimethyl sulfate is an important chemical raw material that is widely used in the synthesis of drugs, dyes, spices, and pesticides. The highly toxic and corrosive dimethyl sulfate residue in medicines is harmful to the human body, and hence, the residue level should be strictly controlled. Traditional detection methods use high-purity acetonitrile and anhydrous as the solvents, which limits the choice of detection solvents and degrades the versatility and accuracy of detection. Therefore, a simple and accurate method for the determination of dimethyl sulfate residues is urgently needed. Dimethyl sulfate is usually detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with pyridine as the methylation substrate. In this study, a new method for the detection of dimethyl sulfate was established using tertiary amines such as aminophenazone, which has many advantages over pyridine, as the methylation substrate. For example, the hybrid orbital and electron cloud of the N atom are different, resulting in stronger nucleophilicity of aminophenazone. High temperatures that are detrimental to the stability of dimethyl sulfate are not required when using aminophenazone, and the aliphatic quaternary ammonium salt product is more stable, with good stability, low interference, good ionization properties, and high response. The separation was performed on a Waters Atlantis HILIC C18 column (100 mm×2.1 mm, 3.0 μm) using a mobile phase consisting of 10 mmol/L ammonium acetate solution-0.1% formic acid methanol solution (50∶50, v/v) at a flow rate of 0.3 mL/min. The column temperature was set at 40 ℃, and the sample size was 1 μL. Dimethyl sulfate was determined in the electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM) modes. Dimethyl sulfate showed good linear relationships within the range of 0.9935 to 7.9480 ng/mL (r=0.9997). The limit of detection and limit of quantification for dimethyl sulfate were 0.50 ng/mL and 1.15 ng/mL, respectively. The recoveries (n=3)of dimethyl sulfate were 94.9% to 106.4%. The relative standard deviations (RSDs) were 1.44% to 5.51%. The RSD of the methylated aminophenazone peak area was 4.32%, indicating good stability of the reaction product. Dimethyl sulfate genotoxic impurities were not detected in 9 batches of aminophenazone, caffeine, and tegafur samples, which indicated that the drug manufacturers paid attention to the control of these impurities. The proposed method is advantageous over the existing techniques in terms of the better ion peak shape and higher molecular weight, without interference from other fragments. The method is specific, sensitive, simple, rapid, and accurate, and it can be used for the determination of dimethyl sulfate genotoxic impurities in aminophenazone and other medicines.