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Chinese Journal of Chromatography
2021, Vol. 39, No. 7
Online: 08 July 2021

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Applications of separation technology in novel coronavirus research, epidemic prevention and detection LI Linsen, ZHU Chao, ZHAO Xinying, QU Feng 2021, 39 (7):  679-685.  DOI: 10.3724/SP.J.1123.2021.03022 Abstract ( 215 )   HTML ( 217 )   PDF (1125KB) ( 195 )   The novel coronavirus disease 2019 (COVID-19) outbreak has brought to light unprecedented challenges to global public health security. Researchers have devoted their efforts to in-depth research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bring the epidemic under control as rapidly as possible. Among the many areas of burgeoning SARS-CoV-2 related research, various analytical technologies have been applied to the advancement of virus detection, and development of vaccines and innovative therapies. Separation technologies with the merits of simple operation, high separation efficiency, and high selectivity, have become widely used and are key to progress in life science, medicine, pharmaceutical discovery and development, and other fields. Separation technologies have played an irreplaceable role in the isolation, detection, diagnosis, treatment, and prevention of this novel coronavirus. In this review, an overview of the relevant literature is presented from ISI Web of Science spanning Jan. 1st, 2020-Dec. 31, 2020, using “SARS-CoV-2” or “COVID-19” as keywords. The top 20 research directions are summarized, based on papers published in high impact international journals (e. g. Nature, Science, and Cell). Incorporating the impact of published papers, this review summarizes the primary separation technologies applied in these coronavirus studies, and discusses contributions of the following six technologies: affinity chromatography and size exclusion chromatography, liquid chromatography, magnetic bead separation technology, centrifugal technology, micro/nano-separation technology, and electrophoresis. First, affinity chromatography and size exclusion chromatography are discussed, which are the most frequently used protein purification techniques in Nature, Science, and Cell. The SARS-CoV-2 related proteins purified by affinity chromatography and size exclusion chromatography are summarized, and their applications in coronavirus transmission, infection mechanisms, and drug screening are introduced. Subsequently, high performance liquid chromatography (HPLC) is introduced, which is mainly employed for assessing the purity of candidate drugs. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) incorporates the strengths of HPLC and MS, offering both high separation efficiency and structural analysis capabilities with extended applications. LC-MS/MS has been applied to characterization of the binding of SARS-CoV-2 related proteins to potential inhibitors, and to metabolic analyses of candidate drugs. In SARS-CoV-2 nucleic acid tests, magnetic bead separation technology plays a crucial role in the separation of novel coronaviruses. In combination with other analytical techniques, magnetic bead separation technology can be applied to cytological analyses and immunological detection by functionalization of bead surfaces. Centrifugal technology is undoubtedly the most basic separation technology. It has been employed in almost all SARS-CoV-2 related researches. By controlling centrifugation speed, centrifugal technology can rapidly isolate virus particles or cultured cells from complex samples. Micro-nano separation technologies, such as microfluidics, offer advantages including small size, low sample consumption, rapid diffusion, and large surface area. In general, microfluidic technologies are often used in combination with other technologies to realize highly sensitive detection of SARS-CoV-2 related proteins. Finally, the applications of electrophoresis are introduced, which commonly engages in the analysis of polymerase chain reaction (PCR) products. In novel coronavirus studies, the application of electrophoresis has been relatively limited but has potential with further development to contribute significantly to future research. In conclusion, this review summarizes the contributions of six primary separation technologies to novel coronavirus studies, including epidemic detection and prevention, analyzes the main problems facing coronavirus detection efforts, and discusses the role of separation technologies in addressing these problems, with the aim of providing references for broader application of separation technologies.

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Large-scale enrichment and identification of human urinary N-glycoproteins/N-glycopeptides SHANG Shiting, DONG Hangyan, LI Yuanyuan, ZHANG Wanjun, LI Hang, QIN Weijie, QIAN Xiaohong 2021, 39 (7):  686-694.  DOI: 10.3724/SP.J.1123.2021.01039 Abstract ( 199 )   HTML ( 43 )   PDF (2615KB) ( 234 )   N-Glycosylation of proteins, an important post-translational modification in eukaryotic cells, plays an essential role in the regulation of cell adhesion, migration, signal transduction, and apoptosis. Abnormal changes in protein glycosylation are closely related to the occurrence of many critical diseases, including diabetes, tumors, and neurological, kidney, and inflammatory diseases. A non-invasive type of liquid biopsy, urine sampling has the advantage of reducing the complexity of proteomic analysis. This facilitates the design of large-scale and continuous or multi-time point sampling strategies. However, the dynamic range of urinary protein abundance is relatively large, owing to individual differences and physiological conditions. Currently, there is a lack of specialized research on individual differences, physiological fluctuations, and physiological abundance ranges of urinary N-glycoproteins in large healthy populations. Therefore, it is difficult to accurately distinguish individual differences and normal physiological fluctuations from changes caused by disease; this poses a great challenge in disease marker research. Liquid chromatography-mass spectrometry (LC-MS) is an analytical technique widely used for the large-scale profiling of proteomes in biological systems, and the enrichment of N-glycopeptides is a prerequisite for their detection by MS. In this study, we established an approach based on hydrophilic interaction chromatography (HILIC) by optimizing the activation, cleaning, and elution processes of the enrichment method, for instance through the optimization of particle size and solvent composition, and investigated the identification number, selectivity, and stability of N-glycoprotein/N-glycopeptide enrichment under different experimental conditions. We found that N-glycoproteins and N-glycopeptides were highly enriched in a trifluoroacetic acid system with 5-μm filling particles in the HILIC column. On this basis, we analyzed the levels of N-glycoproteins/N-glycopeptides in urine samples. The consistency of N-glycoprotein/N-glycopeptide levels in urine samples taken from the same healthy person for five consecutive days was investigated by correlation analysis. This analysis revealed that the urinary N-glycoproteome of the same healthy person was relatively stable over a short period of time. Next, urinary samples from 20 healthy male volunteers and 20 healthy female volunteers were enriched for N-glycoproteins/N-glycopeptides, which were profiled by MS through qualitative and quantitative analyses. Screening and functional analysis of differential proteins were then carried out. A total of 1016 N-glycoproteins and 2192 N-glycopeptides were identified in the mid-morning urine samples of the 40 healthy volunteers. A label-free quantitation strategy was used to investigate the fluctuation range of the physiologically abundant urinary N-glycopeptides. The abundance of urinary N-glycopeptides spanned across approximately five orders of magnitude. Subsequently, gender differences in the N-glycosylation levels of urinary proteins were also explored in healthy people. Functional analysis of the N-glycoproteins that exhibited gender differences in abundance was performed. Based on multivariate statistical analysis, 206 differentially expressed proteins (p<0.05, fold change (FC)> 4) were identified. In females, we found 175 significantly down-regulated N-glycoproteins and 31 significantly up-regulated N-glycoproteins with respect to males. The expression levels of N-glycopeptides between the two groups suggested a clear gender difference. To investigate the biological processes and functions of these proteins, gene ontology (GO) analysis was performed on the N-glycoproteins/N-glycopeptides differentially expressed between males and females. Metabolic pathway analysis was also carried out based on the kyoto encyclopedia of genes and genomes (KEGG). Differentially expressed N-glycoproteins were mostly associated with platelet degranulation, extracellular region, and ossification. The top three relevant pathways were glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, and lipid metabolism. Overall, sex may be an important factor for urinary N-glycoproteome differences among normal individuals and should be considered in clinical applications. This study provides relevant information regarding the function and mechanisms of the urinary glycoproteome and the screening of clinical biomarkers.

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Simultaneous determination of three quaternary ammonium muscle relaxants in blood by high performance liquid chromatography-tandem mass spectrometry HUANG Yongpeng, TANG Hui, SONG Yunyang, CHEN Bo, ZHONG Hui 2021, 39 (7):  695-701.  DOI: 10.3724/SP.J.1123.2020.09020 Abstract ( 185 )   HTML ( 36 )   PDF (840KB) ( 152 )   Vecuronium, rocuronium, and pancuronium are widely used as non-depolarizing muscle relaxants. There have been occasional cases of allergic reactions and even death when using such muscle relaxants. Rapid determination of the concentration of these muscle relaxants in blood can provide valuable information for early clinical diagnosis. As quaternary ammonium compounds, these muscle relaxants are highly polar. Hence, they cannot be retained effectively on reversed-phase chromatographic columns with conventional mobile phases. These quaternary ammonium muscle relaxants are mainly separated by ion-pair chromatography. Using an ion-pairing reagent can help improve the retention capabilities of quaternary ammonium muscle relaxants. Nevertheless, the sensitivity of MS detection is significantly decreased because of ionic inhibition caused by the ion-pairing reagent in the mobile phase. Furthermore, ion-pairing reagents can pollute the MS system. A method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the simultaneous determination of the three quaternary ammonium muscle relaxants in blood. The blood samples were diluted and subjected to high-speed centrifugation. The supernatant was purified on a Bond Elut AL-N solid phase extraction column and then filtered through a 0.45 μm microporous membrane. The quaternary ammonium muscle relaxants were separated on a ZIC-cHILIC analytical column (50 mm×2.1 mm, 3.0 μm) with gradient elution. Acetonitrile and 0.1% formic acid aqueous solution were used as mobile phases. The separated compounds were analyzed by tandem MS with an electrospray ionization (ESI) source in positive and multiple reaction monitoring (MRM) modes. The matrix effects of vecuronium, rocuronium, and pancuronium in blood were 88.1% to 95.4%. The calibration curves for vecuronium, rocuronium, and pancuronium showed good linear relationships in each range, and all correlation coefficients (R2) were > 0.996. The limits of detection of vecuronium, rocuronium, and pancuronium were 0.2-0.8 ng/mL, with the corresponding limits of quantification being 0.5-2.0 ng/mL. The recoveries of vecuronium, rocuronium, and pancuronium were 92.8% to 110.6%, with relative standard deviations (RSDs) of 3.2%-9.4%. This method is sensitive, accurate, and easy to operate, and it can be used to rapidly determine vecuronium, rocuronium, and pancuronium in blood.

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Determination of myclobutanil enantiomers in wheat and its processed products by ultraperformance liquid chromatography-tandem mass spectrometry based on a chiral stationary phase QI Yanli, GAO Jing, WANG Weirong, JIN Jing, LÜ Ying, QIN Shu 2021, 39 (7):  702-707.  DOI: 10.3724/SP.J.1123.2021.03001 Abstract ( 135 )   HTML ( 31 )   PDF (885KB) ( 78 )   A valid method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a chiral stationary phase was established for the determination of myclobutanil enantiomer residue in wheat grain and its processed products (flour, bran, pasta, steamed bun, noodle, and cooking water). The wheat grain and processed product samples were extracted with acetonitrile and purified with primary secondary amine (PSA) and C18. The enantiomers of myclobutanil were separated by Chiral column Lux Cellulose-1 (150 mm×2.0 mm, 3 μm, Phenomenex). The column temperature, sample volume injected, and flow rate were 30 ℃, 5 μL, and 0.25 mL/min, respectively. The mobile phase consisted of phase A (25%), water with 0.1% formic acid and 4 mM ammonium acetate, and phase B (75%), methanol with 0.1% formic acid and 4 mM ammonium acetate. A Waters Xevo TQ-S Micro MS/MS system (Waters, USA) was used for mass spectrometric analysis. An electrospray ionization (ESI) source operating in the positive ionization mode. MS analyses were performed in the multiple reaction monitoring (MRM) mode. The qualitative ions of myclobutanil were m/z 288.9/69.9 and 288.9/124.9, and the quantitative ion of myclobutanil was m/z 288.9/69.9. The source voltage was 3000 V, and the desolvation temperature was 400 ℃. The desolvation gas flow was 800 L/h, and the source temperature was 150 ℃. The matrix effect of wheat grains and their processed products on the determination of myclobutanil enantiomers by UPLC-MS/MS was investigated. S-(+)-myclobutanil and R-(-)-myclobutanil had a mid signal suppression effect on wheat grain, bran, pasta, steamed bun, and noodle, while S-(+)-myclobutanil and R-(-)-myclobutanil had a mid signal enhancement effect on flour and cooking water. Finally, the matrix-matched calibration method was effective in all matrices and was selected for the quantification of the myclobutanil enantiomer residue in the samples. The results showed that the two enantiomers of myclobutanil were well separated by this method. The first and second eluted enantiomers were S-(+)-myclobutanil and R-(-)-myclobutanil, respectively, with the corresponding retention times being 4.34 min and 5.13 min. The limits of detection (LOD) and limits of quantification (LOQ) of S-(+)-myclobutanil and R-(-)-myclobutanil in wheat and its processed products were 0.2 μg/kg and 0.5 μg/kg, respectively. In the linear range of 0.5-25 μg/L, the peak areas of the myclobutanil enantiomers showed a good linear relationship with the concentration, and the R2 values were all greater than 0.99. At fortification levels of 5, 50, and 100 μg/kg (enantiomer concentration), the average recoveries of S-(+)-myclobutanil in wheat grain and its processed products ranged from 82% to 110%, with RSDs between 0.9% and 6.8%. The average recoveries of R-(-)-myclobutanil in wheat grain and its processed products ranged from 80% to 109%, with RSDs between 0.9% and 6.8%. This method fulfils the requirements for pesticide residue analysis. The established method was applied to analyze five flour samples, two noodle samples, and two steamed bread samples. The results showed that S-(+)-myclobutanil and R-(-)-myclobutanil enantiomers were not detected in the samples. In this study, methods for the enantiomeric separation and residue analysis of myclobutanil in wheat were evaluated at the enantiomeric level, which enriched the methods of enantiomeric separation and residue analysis of chiral pesticide myclobutanil enantiomers in raw agricultural product (wheat grain) and its processed foods. This method is effective for the residue analysis of chiral pesticide myclobutanil enantiomers in raw agricultural commodities and its processed products.

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High performance liquid chromatographic method for determination of active components in lithospermum oil and its application to process optimization of lithospermum oil prepared by supercritical fluid extraction SHEN Jie, SHEN Wei, CAI Xue, WANG Jingxia, ZHENG Minxia 2021, 39 (7):  708-714.  DOI: 10.3724/SP.J.1123.2020.12009 Abstract ( 127 )   HTML ( 30 )   PDF (827KB) ( 69 )   Lithospermum erythrorhizon has the functions of cooling blood, activating blood, as well as detoxifying and penetrating rash. Lithospermum oil extracted from Lithospermum erythrorhizon can prevent and treat diaper rash, skin ulceration, eczema, and other skin diseases. Supercritical fluid extraction is the optimal method for the extraction of active components from lithospermum. In this study, an analytical method was established for simultaneously determination of six active components in lithospermum oil with high performance liquid chromatography (HPLC), and the contents of the active components as the evaluation index were used to investigate several important factors in the preparation of lithospermum oil by supercritical fluid extraction. The optimized HPLC conditions were as follows: separation column, Diamonsil C18 (250 mm×4.6 mm, 5 μm); mobile phases, acetonitrile containing 0.1% (v/v) formic acid-0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium formate (75∶25, v/v); flow rate, 1 mL/min; injection volume, 15 μL; room temperature; photodiode array detector (PAD); detection wavelength, 275 nm. The supercritical fluid extraction was optimized for ensuring stability of the amounts of effective components and the reliability of the quality of lithospermum oil. This will serve as the basis for preparation and quality control processes. Three factors and three levels orthogonal tests were adopted to investigate the important factors, viz. the pressure, temperature and CO2 flow rate in the preparation of lithospermum oil. The results showed that the developed HPLC-PAD method can be used for the simultaneous determination of shikonin, acetylshikonin, β-acetoxyisovaleryl akanin, isobutyryl shikonin, β,β-dimethylacryl shikonin, and 2-methylbutyryl shikonin in 30 min. The method has good precision, accuracy and repeatability. The contents of the active components were the highest when the extraction pressure, extraction temperature, and CO2 flow rate were 23 MPa, 40 ℃, and 27 L/h, respectively. The optimized conditions are suitable for the preparation and actual production of lithospermum oil. The HPLC-PAD method is simple, feasible, accurate, and reliable. It can be used for the preparation and quality control of lithospermum oil by supercritical fluid extraction. Thus, with this method, the stability of the contents of active ingredients and the reliability of the quality of lithospermum oil can be ensured; moreover, safe and effective drug use can be realized. The established method has obvious advantages over the traditional process and is a good candidate for widespread use.

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Determination of silanol group content on the surface of fumed silica by chemical reaction-headspace gas chromatography BAI Yun, DUAN Xianjian, WANG Dahai, HU Guanghui, WU Chunlei, ZHANG Mei, LIU Weili 2021, 39 (7):  715-720.  DOI: 10.3724/SP.J.1123.2020.11007 Abstract ( 169 )   HTML ( 25 )   PDF (793KB) ( 82 )   Fumed silica is prepared by flame pyrolysis, where silicon halide is combusted in an oxygen-hydrogen flame, resulting in finely dispersed and thermally stable silicon dioxide. Because of its unique physical and chemical properties, including high porosity, large pore volumes, large specific area, and high chemical activity, fumed silica is widely used in rubbers, plastics, adhesives, paints, and printing inks for reinforcement, as well as in thixotropy, anti-setting, and anti-sagging applications. These functional properties of fumed silica are related to the silanol group on its surface. However, there is no accurate and convenient test method to determine the silanol group content on the surface of fumed silica. This work explores a novel method to determine the silanol group content on the surface of fumed silica by chemical reaction-headspace gas chromatography (HS-GC). Theoretically, by this method, the silanol group can rapidly react with the Grignard reagent and generate methane, the amount of which can be determined accurately by GC analysis. GC analysis was conducted using a headspace flask as a closed reactor to transform the silanol group into a volatile component through a chemical reaction, so as to realize the accurate determination of silica hydroxyl. The amount of methane produced in the reaction was directly proportional to the content of silanol groups on the surface of fumed silica. Therefore, the silanol group content was calculated using the chemical reaction equation. Before the experiment, fumed silica was dried for 2 h in an oven at 105 ℃ to remove adsorbed moisture. The dried fumed silica sample was then reacted with the Grignard reagent dispersed in toluene in an airtight reaction bottle. Toluene was used as a dispersion agent to promote contact and reaction between the fumed silica sample and Grignard reagent. The methane produced by the reaction was injected into a gas chromatograph for separation and further detected using a flame ionization detector (FID). Methane was quantified from the peak areas of the GC signals using the external standard method, and the silanol content in the sample was obtained. Simultaneously, factors influencing the outcome of the method, such as the dosage of the Grignard reagent and reaction time with it, were optimized by a comparison test. Accordingly, 2.0 mL of 0.3 mol/L Grignard reagent and a reaction time of 15 min were found to be optimal for testing. The test results showed that there was good linear correlation between the content of the silanol group and the GC signals, with a correlation coefficient of 0.9990. The limit of detection was 0.30 mg/g, and the limit of quantification was 1.00 mg/g. The relative standard deviation of reproducibility was less than 3%. Based on an interlaboratory test conducted by four laboratories on five samples with different silanol group contents, the repeatability limit (r) was less than 2.5%, and the reproducibility limit (R) was less than 6.5%. Compared with the traditional chemical method, the method involving HS-GC presents distinct advantages in terms of lower reagent consumption, high sensitivity, good stability, and reliability. It is suitable for the rapid detection of the silanol group content on the surface of fumed silica, and can aid in the quality control of fumed silica during its production and application. This method has important theoretical and practical significance for developing accurate methods to determine silica hydroxyl in the silicon industry for standards and the optimization of industrial technology. This study serves as a foundation to standardize and promote the rapid development of silicon material-related industries.

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Efficient screening for 8-oxoguanine DNA glycosylase binding aptamers via capillary electrophoresis HAN Shimiao, ZHAO Liping, YANG Ge, QU Feng 2021, 39 (7):  721-729.  DOI: 10.3724/SP.J.1123.2020.12017 Abstract ( 121 )   HTML ( 23 )   PDF (3394KB) ( 61 )   8-Oxoguanine DNA glycosylase (OGG1) is an important enzyme that plays a key role in oxidative DNA damage repair. OGG1 can specifically recognize and excise 8-oxoG (a product of oxidative damage found in double-stranded DNA) through base excision repair (BER). OGG1 is expressed in normal tissues, and in most tumor tissues. Oxidative cellular damage can produce an inflammatory reaction, alleviating some measure of constitutive OGG1 inhibition. OGG1 inhibition in cancer cells shows some promise as a new method of cancer treatment. Most current OGG1 research focuses on regulating OGG1 with targeted small molecules. To date, no aptamer screen for OGG1 has been reported. Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides that can bind to a target with high affinity and specificity in vitro, that can be identified by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers can be used as chemical ligands to regulate intermolecular interactions. In this study, a screen for aptamers with OGG1 affinity was performed for the first time. Capillary electrophoresis (CE) is a microanalytical technique that offers speed and high separation efficiency. In this work, two screening methods based on CE-SELEX technology were established: a one-round pressure controllable selection, and a multi-round selection. The most important criterion for successful one-round pressure controllable selection is to select a competitive target with a different CE migration time than that of the target of interest. We mixed OGG1 with a competitive target and a nucleic acid library for CE analysis. Two proteins competitively bind sequences in the library, forming independent complexes. The concentration of the competitive target is continuously increased until complexes with the target stop decreasing, indicating that the target and the ssDNA library have formed a stable complex. Complexes were collected for PCR amplification, purification, and high-throughput sequencing to obtain high affinity aptamers. This method greatly improves screening efficiency, and reduces non-specific binding to the target, which is helpful for obtaining aptamers with high affinity and specificity. One-round pressure controllable selection for high affinity OGG1 selective aptamers was performed using single strand binding protein (SSB) to competitively and tightly bind nucleic acids in the library. The competitive screening pressure was increased by increasing the SSB concentration to eliminate sequences with low affinity for OGG1 from the random oligonucleotide library. Nucleic acid sequences with high OGG1 affinity were obtainable in one step, and OGG1-ssDNA complexes were collected by creating a timed program on Beckman P/ACE MDQ capillary electrophoresis. Collection occurred from 2.2 to 2.8 min. Under identical incubation and electrophoresis conditions, multiple round selections were conducted by injecting samples of co-incubated nucleic acid library and target into the capillary. After separation under a high-voltage electric field, nucleic acid target complexes were collected, amplified by PCR, purified, and used as an enriched secondary library in the next round of screening. High affinity aptamers were generally obtained within three rounds. Comparing results of the two screening methods, the three candidate aptamer sequences found with the highest frequency were consistent, and displayed KD values ranging from 1.71 to 2.64 μmol/L. Molecular docking analysis suggests that Apt 1 may bind to the OGG1 active pocket, which functions to repair oxidative damage. Comparison of the two screening methods indicates that one-round pressure controllable selection is more rapid and efficient, providing guidance for the design of other protein aptamer screening methods. The obtained aptamer is expected to be function effectively as an OGG1-mediated DNA repair inhibitor.

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Rapid identification of chemical components in Qi-Yu-San-Long decoction by ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry HUANG Mengwen, WU Huan, YU Wei, WANG Ying, WANG Fengcan, ZHANG Chunchun, ZHOU Longsheng, LI Zegeng 2021, 39 (7):  730-743.  DOI: 10.3724/SP.J.1123.2020.10016 Abstract ( 245 )   HTML ( 39 )   PDF (4266KB) ( 113 )   Qi-Yu-San-Long decoction (QYSLD) is a classic traditional Chinese medicine prescription consisting of ten types of herbal medicines, including Astragali Radix, Polygonati Odorati Rhizoma, Scolopendra, Pheretima, Solanum nigrum L., Hedyotis diffusa Willd., Coicis Semen, Euphorbia helioscopia L., Curcumae Rhizoma, and Fritillariae Cirrhosae Bulbus, combined in a ratio of 15∶5∶3∶3∶10∶10∶10∶3∶5∶3 by weight. QYSLD has been used to treat non-small cell lung cancer (NSCLC) for over 20 years in clinical practice, and its curative effect is considered credible. However, the chemical constituents of QYSLD have not been revealed because of their complexity, which has significantly hindered the systematic clarification of the efficacy of the materials and quality evaluation. In this study, a reliable strategy based on the data-independent acquisition (DIA) technology of ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) combined with a targeted screening method was established to investigate the chemical components of QYSLD. A 2-μL aliquot from each vial was injected into a Waters ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) to separate complex components. The temperature of the column was 35 ℃, and the flow rate was set at 0.2 mL/min. The mobile phase consisted of 0.1% formic acid aqueous solution and acetonitrile. Detection was conducted using an Xevo G2-XS QTOF-MS with a LockSpray capable-electrospray interface. The data for complex components in QYSLD were collected by full-information tandem mass spectrometry (MS E) in the positive and negative ion modes. In the MSE mode, data acquisition was performed using a mass spectrometer by rapidly switching from a low-collision-energy (CE) scan to a high-CE scan during a single LC run. Thus, accurate precursor and fragment ions were collected in a single run, which was helpful for the structural elucidation of multiple components in QYSLD. In addition, systematic information on isolated chemical compounds was collected and distinguished from the ten individual herbs in QYSLD using databases such as China Academic Journals Full-text database (CNKI), PubMed, Web of Science, Medline, and ChemSpider. Accordingly, a self-building library of QYSLD, including the component name, molecular formula, and structure of the components from the herbs, was established. Subsequently, the raw MSE data of the collected samples and the self-building chemical composition library were imported into a natural product post-processing screening (UNIFI) platform for targeted screening of the chemical components in QYSLD. The parameters for UNIFI platform were as follows: the retention time deviation was ±0.1 min; an error margin of no more than 5×10 -6 for the identified compounds was allowed; positive adducts, including [M+H]+and [M+Na]+, were selected; and negative adducts, including [M-H]- and [M+HCOO]-, were selected. The results showed that a total of 166 compounds were initially identified, including 22 saponins, 13 alkaloids, 27 flavonoids, 32 terpenes, 20 amino acids, 16 phenylpropanoids, 9 organic acids, 6 sterols, 6 anthraquinones, and 15 other components. Among them, sixteen components were confirmed unambiguously with the reference substances. To better understand the chemical contribution of individual herbs to the entire decoction, the attributes of each component were summarized. This study provides a foundation for exploring the pharmacodynamic substances of QYSLD.

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Simultaneous determination of pentostatin and 2'-amino-2'-deoxyadenosine in fermentation broth by high performance liquid chromatography-tandem mass spectrometry ZHAO Minmin, ZHANG Hongyu, LOU Tingting, ZHAO Kongxiang, WANG Suying 2021, 39 (7):  744-749.  DOI: 10.3724/SP.J.1123.2020.09018 Abstract ( 127 )   HTML ( 28 )   PDF (802KB) ( 73 )   An analytical method was established for the simultaneously determination the pentostatin and 2'-amino-2'-deoxyadenosine contents in fermentation broth by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After high-speed centrifugation, aqueous solution dilution, vortex shock, and microfiltration, the fermentation broth samples were analyzed by HPLC-MS/MS. The samples were separated on a Waters Atlantis® T3 column (100 mm×2.1 mm, 5 μm) using a gradient elution program with 10 mmol/L ammonium formate (containing 0.1% formic acid) and methanol (containing 0.02% formic acid) as the mobile phases. Moreover, a chromatographic protection column (5 mm×2.1 mm, 5 μm) was added to preserve the column efficiency. The flow rate, column temperature, and injection volume were set at 0.3 mL/min, 25 ℃, and 10 μL, respectively. Qualitative and quantitative analyses of the target compounds were performed using an ESI+ source. MS parameters such as the collision energies and tube lens offsets of pentostatin and 2'-amino-2'-deoxyadenosine were optimized. The quantitative ion pairs of pentostatin and 2'-amino-2'-deoxyadenosine were m/z 269.17>153.20 and m/z 267.00>136.10, respectively; the corresponding collision energies were 11 V and 18 V. The external standard method was used for quantitative analysis. The established method was verified rigorously in terms of the linear range, limit of detection, limit of quantification, recovery rate, and precision. Pentostatin and 2'-amino-2'-deoxyadenosine showed good linear relationships in the range of 1.0-250 μg/L. The correlation coefficients ranged from 0.9969 to 0.9996, and the relative standard deviations (RSDs) ranged from 6.51% to 8.35% (n=8). This result indicated good accuracy and exactitude in the detection of the pentostatin and 2'-amino-2'-deoxyadenosine. The recoveries (n=6) at three spiked levels (1.0, 5.0, and 25 μg/L) were in the ranges of 97.94%-104.46% and 89.96%-107.21% for the pentostatin and 2'-amino-2'-deoxyadenosine, respectively; the corresponding RSDs were in the ranges of 3.74%-4.88% and 4.81%-13.29%. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥10) of the 2'-amino-2'-deoxyadenosine and pentostatin in the fermentation broth were 0.003-0.060 μg/L and 0.010-0.200 μg/L, respectively. The validated experimental method was used for the detection of actual samples, viz. the stored multiple pentostatin-producing mutagenic strains in our laboratory. The HPLC-MS/MS method for the determination of the pentostatin and 2'-amino-2'-deoxyadenosine in fermentation broth offered the advantages of small sampling volume, strong maneuverability, good stability, and high sensitivity. Compared with previously published methods, this systematically established and optimized method significantly reduced the detection time, and matrix effects were well suppressed. Moreover, the peak shape and stability of the target compounds were greatly improved. This method provides a methodological basis and meaningful reference for the detection of the pentostatin and 2'-amino-2'-deoxyadenosine in fermentation broth.

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Determination of hydrazine in prednisolone by derivatization-gas chromatography-triple quadrupole mass spectrometry QIAN Chong, ZHANG Mei, LIU Shanshan, GOU Xinlei, WANG Wei, HU Guanghui 2021, 39 (7):  750-757.  DOI: 10.3724/SP.J.1123.2021.03002 Abstract ( 108 )   HTML ( 21 )   PDF (1551KB) ( 73 )   Prednisolone is an adrenal glucocorticoid drug with immunosuppressive, anti-inflammatory, anti-allergic, and antiviral effects that are widely exploited in clinical treatment. The hydrazine residue to prednisolone directly affects medication safety and threatens the patient’s health. At present, there are no relevant laws, regulations, and standards to control the residual limit of hydrazine in drugs at home or abroad. Therefore, a simple, rapid, accurate, reliable, sensitive, and selective method is urgently needed for the determination of trace hydrazine in prednisolone. Hydrazine has strong polarity and reductivity, with unstable physical and chemical properties, thus being easily oxidized. In addition, because of the lack of chromophores and low molecular weight, the detection of hydrazine is very difficult. Therefore, a derivative reagent should be introduced to reduce its polarity and generate a derivative product with a high molecular weight as well as stable physical and chemical properties. Acetone, as a common laboratory reagent, is inexpensive and can rapidly react with hydrazine; therefore, it is an ideal derivative reagent for the determination of hydrazine. In this study, a method based on precolumn derivatization with gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) was developed for the determination of hydrazine in prednisolone by optimizing the derivatization reagent, GC and MS conditions, solvent system, and derivatization conditions. Method validation was then carried out using the established method, and the results were satisfactory. In this study, 1 g of prednisolone sample was weighed and placed in a 10 mL centrifuge tube with a plug; then, a methanol-dichloromethane dilution solvent (14∶23, v/v) was added to the scale line, and the sample was vortexed until completely dissolved. About 100 μL of the test solution prepared above was pipetted into the sample vial, followed by the addition of 900 μL acetone. The resulting solution was vortexed and mixed well. The sample was diluted and derivatized simultaneously in acetone solution, acetone/methanol-dichloromethane dilution solvent (9∶1, v/v), and then detected and analyzed by GC-MS/MS. In this study, the derivatization reaction between hydrazine and acetone did not require the addition of acetic acid and ultrasound conditions, or the use of other reagents for the extraction operation. The reaction was instantaneous, and rapid determination of hydrazine in prednisolone could be achieved. The standard curve was obtained with a good correlation coefficient (r2=0.9999) in the range 1-12 μg/L. The limits of detection and quantitation were 0.03 mg/kg and 0.10 mg/kg, respectively. The relative standard deviation (RSD) of injection precision was 1.10%. The recoveries and repeatability were good; the recoveries of low-, medium-, and high-concentration spiked samples were 96.15%-96.46% at spiked concentrations of 1, 6, and 12 μg/L, respectively, and the corresponding RSDs were 1.77%-2.12%. The intermediate precision was good, and the RSD of the determination results obtained on the same instrument by different laboratory technicians at different times was 1.77%. The durability was good, and the degree of influence of the detection results was studied by changing the chromatographic conditions. Under the original condition or conditions with initial column temperature ±5 ℃, heating rate ±2 ℃/min, or column flow rate ±0.1 mL/min, the hydrazine content in the sample solution at a spiked concentration of 6 μg/L was detected, and the RSD of the detection results was 2.58%. The established method was applied to detect hydrazine in a prednisolone standard substance procured from the market and nine batches of prednisolone samples provided by a pharmaceutical company. No hydrazine was detected in any of these samples. The established method is simple, reliable, highly sensitive, and highly selective, and it can be applied for the detection of hydrazine in prednisolone.

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Simultaneous determination of six haloacetonitriles in finished water for drinking by purge and trap-gas chromatography-triple quadrupole mass spectrometry ZHAN Wei, HAN Zhiyu, LI Yong, LIU Fei, ZHANG Yong 2021, 39 (7):  758-763.  DOI: 10.3724/SP.J.1123.2020.08026 Abstract ( 226 )   HTML ( 29 )   PDF (944KB) ( 133 )   Haloacetonitriles (HANs) are widely used in finished water as unregulated disinfection by-products. HANs may pose much threat to human health, and there is no relevant standard examination method for these compounds. A method was established for the simultaneous determination of six HANs (chloroacetonitrile (CAN), dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), bromoacetonitrile (BAN), bromochloroacetonitrile (BCAN), and dibromoacetonitrile (DBAN)) in finished water by using purge and trap-gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). The purge and trap technology helps realize automatic determination of samples after collection, without using any harmful reagent. The cost and analytical efficiency of this method were superior to those of solid phase microextraction (SPME). Considering the instability of HANs, the analysis must be carried out as soon as possible after sampling, in order to avoid significant changes in their concentration during storage. In particular, the use of an appropriate quenching agent was critical to sample collection. In this study, ascorbic acid was chosen as the quenching agent. The stabilities of the spiked samples at the levels of 0.1 (TCAN), 0.2 (CAN), 1.0 (DCAN), 1.0 (BAN), 1.0 (BCAN), 4.0 (DBAN) μg/L were tested. The effect of sample storage time (0, 0.5, 1, 2, 3, 4, and 6 h) on the responses of the target component was evaluated. The stability results showed that within 6 h, the relative standard deviations of the responses for the six HANs ranged from 2.32% to 6.98%. To validate the method, first, different traps, viz. 7# (Tenax), 10# (Teanx/silica gel/cms), 11# (VOCARB), and 12# (BTEXTRAP) were optimized. Second, various chromatographic columns (VF-5, Rxi-624, DB-VRX, and HP-INNOWAX) were compared to investigate their influence on the peak shape. Under the optimal detection conditions, the six HANs in finished water were extracted with the 10# trap. The volume of the water sample was used 25 mL, with purging at 35 ℃ for 11 min, and desorbed at 190 ℃ for 1 min. Chromatographic separation was performed on a Rxi-624Sil MS chromatographic column (60 m×0.25 mm×1.40 μm). Gas chromatographic conditions were obtained under the following conditions: split ratio, 1∶10; linear velocity, 30 cm/s. The triple quadrupole mass spectrometer was operated in the electron impact (EI) mode. The target compounds were detected in the multiple reaction monitoring (MRM) mode. Quantitation was carried out using the external standard method. The results showed that the matrix effects of the six HANs ranged from 0.85 to 1.09. Good linearities were obtained in the range of the standard curves. The correlation coefficients (r) were greater than 0.9991. The limits of detection (LODs, S/N=3) were 0.8-120.0 ng/L. The limits of quantification (LOQs, S/N=10) were 1.5-300.0 ng/L. The average recoveries of the six HANs ranged from 84.2% to 106%, and the RSDs were in the range of 1.81%-10.7%. In August 2020, 38 samples of finished water were tested. All of the six HANs were found in the finished water. The concentrations of the HANs were in the range of 0.0101-1.28 μg/L, and the total detection rate was 92.1%. The detection rates of the individual components followed the order DCAN>BCAN>CAN>TCAN>BAN>DBAN. The developed method is efficient, sensitive, and environmentally friendly. It provides a high-quality technical choice for monitoring and health risk assessment of the emerging disinfection by-products of HANs.

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Determination of eight organic residues in ion exchange resins by headspace gas chromatography ZHU Hewen, ZANG Yingchao, ZHANG Guangsheng, LU Lixin, XIA Haifeng 2021, 39 (7):  764-770.  DOI: 10.3724/SP.J.1123.2020.12021 Abstract ( 132 )   HTML ( 31 )   PDF (831KB) ( 97 )   An analytical method based on headspace gas chromatography was developed for the determination of eight organic residues in ion exchange resins, methyl isopropyl ketone, methyl butyrate, 3-pentanone, 1,3-diethyl benzene, 1,4-diethyl benzene, dichloroethane, 1,3-dichlorobenzene, and methyl methacrylate. The organic residues in different types of resins were studied to provide a basis for the safe use of ion-exchange resins in food and medicine. The main factors (chromatographic column, equilibrium temperature, equilibrium time, flow rate, etc.) that affect the accuracy and sensitivity of the eight organic residues were investigated during instrument analysis. The extraction solvent and chromatographic conditions for the samples were optimized. According to the extraction efficiencies of methyl benzene, methyl alcohol and dimethyl sulfoxide, 2.0 g of the sample was extracted with dimethyl sulfoxide under ultrasonic conditions at 20 ℃. A DB-23 chromatographic column (60 m×0.32 mm×0.25 μm) and hydrogen flame ionization detector (FID) were selected for the GC method, and good separation and quantitative results were obtained for the eight organic residues. The process and conditions are summarized as follows. The equilibration time of the headspace sampler was 30 min, and the equilibrium temperature was 80 ℃. The temperature of the sampler was 240 ℃, while that of the FID detector was 300 ℃, with nitrogen being used as the carrier gas. The programmed temperature of the column was maintained at 60 ℃ for 16 min, then increased to 200 ℃ at a heating rate of 20 ℃/min, and maintained at this level for 2 min. The flow rate was 1.2 mL/min for detection. The external standard method was utilized for quantitative analysis. Good linear relationships were observed for the eight organic residues, and the correlation coefficients (R2) were all above 0.999 in the mass concentration range of 0.02-200 mg/L. The limits of detection (LODs) were 0.0050-0.0375 ng/g. The average recoveries for the eight organic residues were in the range of 82.3% to 109.2% at three spiked levels, and the relative standard deviation (RSD, n=6) was 1.06% to 4.16%. Eleven types of resin samples were detected by this method, and a certain amount of organic compounds were observed in the resin samples. The methyl methacrylate content in the methacrylate resin XAD761 was 414.4 μg/g, while that in the styrene resin LX-69B was as high as 470.8 μg/g. As opposed to traditional analytical methods, the present method has high sensitivity, good accuracy, and precision, with simple operation without derivatization or the need for acid-base treatment of the sample to reduce contamination. This method can be used to simultaneously detect a variety of organic residues in ion-exchange resins, so that the detection efficiency is significantly improved.