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Chinese Journal of Chromatography
2022, Vol. 40, No. 3
Online: 08 March 2022

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Recent advances in isolation and detection of circulating tumor cells with a microfluidic system CAO Rongkai, ZHANG Min, YU Hao, QIN Jianhua 2022, 40 (3):  213-223.  DOI: 10.3724/SP.J.1123.2021.07009 Abstract ( 416 )   HTML ( 184 )   PDF (4241KB) ( 210 )   The isolation and analysis of circulating tumor cells (CTCs) is an important issue in tumor research. CTCs in peripheral blood, which are important biomarkers of liquid biopsy, are closely related to the occurrence of cancer and are used to monitor the effect of treatment on cancer patients. However, the number of CTCs in the blood samples of cancer patients is very low, usually being present at only 0-10 CTCs/mL. Therefore, prior to the detection of CTCs, it is important to preprocess clinical blood samples for efficient separation and enrichment. With the advantages of low sample consumption, high separation efficiency, ease of automation and integration, microfluidic chips can be a suitable platform for the isolation of CTCs. In the last few years, CTC separation and detection using microfluidic chips have developed rapidly, and a variety of detection methods have been developed. According to the technical principle used, microfluidics for CTC separation can be divided into biological property-based methods and physical property-based methods. The biological property-based methods mainly depend on the interaction between the antigen and antibody, or the specific binding of the aptamer and target. These methods have high selectivity but low efficiency and recovery rates. Physical separation is based on the physical properties of CTCs such as their size, density, and dielectric properties. For example, CTCs can be blocked or captured by the microstructure in the channels of microfluidic chips, sorted by external physical fields (acoustic, electrical, magnetic), or screened by micro-scale hydrodynamics. Physical property-based methods generally have a higher flux but lower separation purity. However, the advantages of biological property-based methods and physical property-based methods can be integrated to provide microfluidic chips having better separation performance. In addition to the direct positive enrichment of CTCs, a negative enrichment strategy can also be adopted. The influence of direct screening on the activity of CTCs can be avoided by selectively removing white blood cells. In this paper, recent advances in microfluidics utilized in the isolation of CTCs, including physical and immune methods and positive and negative enrichment, are reviewed. We summarized the technical principles, detection methods, and research progress in CTC separation and detection using microfluidic chips. Developing trends in microfluidics for CTC separation and analysis are also discussed.

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Mirror cutting-assisted orthogonal digestion enabling large-scale and accurate protein complex characterization HAN Ruonan, ZHAO Lili, AN Yuxin, LIANG Zhen, ZHAO Qun, ZHANG Lihua, ZHANG Yukui 2022, 40 (3):  224-233.  DOI: 10.3724/SP.J.1123.2021.06010 Abstract ( 178 )   HTML ( 38 )   PDF (5007KB) ( 181 )   Supporting Information Protein complexes are involved in a variety of biological activities. Accurate and comprehensive characterization of the structures and interactions of protein complexes is crucial in determining their biological functions. Chemical cross-linking coupled with mass spectrometry (CXMS) is an emerging investigative technique for protein complexes. CXMS enables the sensitive high-throughput analysis of protein complexes without the requirements of molecular weight and purification. These attributes have spurred the increased use of CXMS for the structure and interaction characterization of purified protein complexes and complicated cell lysate samples. CXMS utilizes chemical cross-linking reagents to covalently connect two reactive amino acids in or between proteins that are spatially close to each other. Subsequently, the cross-linked proteins are digested into cross-linked peptides, followed by LC-MS/MS analysis, as well as database searching to provide cross-linking information for the composition, interaction, and structural site distance restrictions of protein complex identification. Therefore, identification of cross-linked sites has a decisive influence on the characterization of protein complexes. This identification is limited by the unsatisfactory quality of the cross-linked peptide spectrum. Insufficient b/y fragment ions and poor continuity of amino acid sequence matching lead to low coverage and accuracy of cross-linked site identification. Based on the complementary feature of mirror-cutting digestion, an orthogonal digestion strategy based on LysargiNase combined with trypsin was developed in this study. Trypsin is the most commonly utilized digestion enzyme in proteomics, with extremely high enzyme activity and specificity. Trypsin generates C-terminally charged peptides after lysine (K) and arginine (R). LysargiNase is a mirror protease complementary to trypsin that cleaves before the K and R residues. This generates peptides with an N-terminal positively-charged residue. Owing to the different physical and chemical micro-environments of the cross-linked peptides digested by LysargiNase and trypsin, the behavior of their detection ability in MS analysis is diverse. Using the orthogonal digestion strategy, both simple and complicated cross-linked samples were analyzed in this study. For the analysis of bovine serum albumin (BSA), 291 pairs of non-redundant cross-linked sites were obtained, of which 216 pairs of cross-linked sites were provided by trypsin digestion, whereas 75 pairs of cross-linked sites were exclusively supplied by LysargiNase digestion. Except for the 35% increase in the number of identified cross-linked sites, 32% of the spectra of the commonly identified cross-linked peptides have better quality with more b-type fragment ions and consecutive sequence matching. Furthermore, for the Escherichia coli sample, 726 pairs of cross-linked sites were obtained in total, among which, 624 and 274 pairs were identified from trypsin and LysargiNase digestion, respectively. LysargiNase digestion yielded 120 individual cross-linked sites, which resulted in a 16% increase in single trypsin digestion. Consistent with the BSA sample, the quality was improved in 35% of the spectra of commonly identified cross-linked peptides. Corresponding to the identified cross-linked peptides, 242 structural constraints with 607 pairs of intra-cross-linked sites and 29 sets of protein-protein interactions with 119 pairs of inter-cross-linked sites were obtained. The collective results demonstrated that, mirror cutting-assisted orthogonal digestion strategy could significantly increase the number of identified fragment ions and amino acid sequences matching the continuity of the spectra by contributing b-and y-type ions, respectively. This improved the accuracy and coverage of cross-linked peptide identification. The findings additionally demonstrate the superiority of our method in the accurate identification of the cross-linked peptide spectra and the increased number of identified cross-linked sites. In a word, this method is expected to provide new insights for the large-scale and highly accurate characterization of protein complexes.

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Preparation of modified rosin bonded silica high performance liquid chromatographic stationary phase and separation of Panax notoginseng saponins XIE Wenbo, XIA Lu, LI Hao, LI Wen, CAO Yu, HUANG Yun, LEI Fuhou 2022, 40 (3):  234-241.  DOI: 10.3724/SP.J.1123.2021.07008 Abstract ( 168 )   HTML ( 35 )   PDF (1793KB) ( 92 )   The sanqi is the dried root of Panax notoginseng (Burk.) F. H. Chen. The main components responsible for the drug actions of sanqi are notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd, which account for about 80% of the saponin content in sanqi. It is widely used in the treatment of anemia, coronary heart disease, hypertension, stroke sequelae, and other diseases. However, sanqi has many chemical components with complex and similar structures, which are difficult to separate. In this study, alkylated silica gel bonded with hydrogenated rosin hydroxyethyl acrylate (HRHA) was prepared via mercapto-ene click chemistry. A new type of modified rosin-bonded silica stationary phase (SiO2@HRHA) for high performance liquid chromatography was prepared for the separation of five saponins (R1, Rg1, Re, Rb1, and Rd). It was characterized by thermogravimetric analysis, Fourier-transform infrared spectroscopy, specific surface area and microporous physical adsorption and elemental analysis. The results showed that SiO2@HRHA had a regular spherical shape with porous surfaces, along with a specific surface area of 308.55 m2/g and an average pore diameter of 6.78 nm. Performance evaluation of the column revealed that the SiO2@HRHA column showed typical reversed-phase chromatographic behavior with better flowability and reproducibility. Results of the Tanaka test showed that SiO2@HRHA column had good stereoselectivity and hydrogen bond capacity. Compared to other stationary phases, e. g. silica modified with acrylopimaric acid (16-hydroxyethyl-34-hydroxyethyl acrylate) ester (AAE) and dihydroterpineol (DTP), which were prepared in our laboratory at the same time, the SiO2@HRHA column demonstrated better resolution (Rs) for the separation of the five saponins under optimal chromatographic conditions. The Rs values for R1, Rg1, Re, Rb1, and Rd were 3.33, 3.54, 20.17 and 9.72, respectively on the SiO2@HRHA column. Rs between Rg1 and Re was also better than that obtained on a C18 column. Panax notoginseng saponins were separated on the SiO2@HRHA column using acetonitrile and water as the mobile phases at the flow rate of 1.0 mL/min at 25 ℃. The optimal UV detection wavelength was 203 nm. It was found that the five saponins could be separated better using the SiO2@HRHA column than the SiO2@AAE and SiO2@DTP columns. Because the ternary phenanthrene skeleton of the rosin group in SiO2@HRHA had structural similarity and good stereoselectivity to the polycyclic compounds (Panax notoginseng saponins). In addition, according to the hydrophobicity evaluation, the SiO2@HRHA column showed the best hydrophobicity among the three columns, which may be conducive to the separation of the five saponins. Thus, this study can provide a new avenue for the separation and purification of Panax notoginseng saponins from actual samples.

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Comprehensive analysis of chemical constituents of tea flowers by ultra-performance liquid chromatography-high resolution mass spectrometry combined with integrated filtering strategy HUANG Sichen, ZHAO Hongpeng, HU Yongdan, REN Dabing, YI Lunzhao 2022, 40 (3):  242-252.  DOI: 10.3724/SP.J.1123.2021.07015 Abstract ( 299 )   HTML ( 37 )   PDF (4212KB) ( 146 )   Tea flowers and fresh tea leaves are biological products of tea, but tea flower is often regarded as waste during tea production, resulting in notable waste of tea flower resources. At present, analysis of the chemical components in tea flowers focuses on single types of chemical components such as amino acids and tea polyphenols, and there are only a few reports on the simultaneous analysis of numerous chemical components in tea flowers. Researchers are not completely clear about the types and amounts of the chemical components in tea flowers; this has hindered the in-depth development and effective utilization of tea flowers. In this study, ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was used to detect the chemical constituents of tea flowers. This technique was combined with the integrated filtering strategy (IFS) of nitrogen rule filtering (NRF), mass defect filtering (MDF), and diagnostic fragment ion filtering (DFIF) for screening the characteristic mass spectra of the target chemical components. Furthermore, the chemical constituents of tea flowers were annotated with information about the retention time, MS fragmentation, and MS/MS fragmentation. All the qualitative chemical components were divided into six categories with a total of 137 chemical constituents, including 3 alkaloids, 38 flavonoids, 31 phenolic acids and their derivatives, 37 catechins and their derivatives, 18 amino acids, and 10 other components. The internal standard method was used to quantify all the qualitative chemical components. The quantitative results showed that the amounts of the six kinds of chemical components in tea flowers were as follows: amino acids, 9371.42 μg/g; catechins and their derivatives, 9068.43 μg/g; phenolic acids and their derivatives, 8696.92 μg/g; alkaloids, 4392.52 μg/g; flavonoids, 1192.88 μg/g; and others, 139.94 μg/g. Quality control samples were used to evaluate the stability of the instrument and the repeatability of the tested data. Nine representative chemical components with high, medium, and low contents in tea were selected, and the relative standard deviation (RSD) of the results was used to evaluate the repeatability of the data. These nine chemical constituents are selected from amino acids, alkaloids, flavonoids, phenolic acids and their derivatives, catechins and their derivatives, and other components, and the response intensities were different. The relative standard deviations of the nine chemical components were in the range of 2.11% to 12.17%. The above results demonstrated the good stability of the instrument and excellent repeatability of the test data. Chlorogenic acid components (CGAs) and glycosylated quercetin components (GQs) were used as two representative components to explain the entire process of extracting the target compounds by IFS. CGAs comprise a class of special esters formed by the esterification of cinnamic acid derivatives with quinic acid as the parent structure. The most common cinnamic acid derivatives are p-coumaric acid, caffeic acid, and ferulic acid. On the one hand, according to the above information and the different positions and degree of quinic acid esterification, the CGAs were structurally classified as monosubstituted CGAs (Mono-CGAs), disubstituted CGAs (Di-CGAs), and trisubstituted CGAs (Tri-CGAs), and three different mass defect filtering windows were set. Therefore, 751 possible target components were selected from 3537 mass spectra in accordance with the nitrogen rule. On the other hand, 22 target components in accordance with the nitrogen rule were obtained by further screening the m/z 191.0551 ion as the diagnostic fragment ion of the CGAs. Combining the overall analytical data with the above mass defect filtering and diagnostic fragment ion filtering screening results, nine target CGAs were selected and characterized based on the MS information. This study reveals the types and amounts of the chemical components accumulated in tea flowers, thus providing valuable information and serving as data reference for the in-depth development and effective utilization of tea flowers.

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Rapid screening and identification of 167 illegally added medicines in herbal tea by ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry HE Jiawen, WEN Jiaxin, LIU Yaxiong, HU Jiazhe, CAO Yajing, LAI Yuhong 2022, 40 (3):  253-265.  DOI: 10.3724/SP.J.1123.2021.07006 Abstract ( 254 )   HTML ( 51 )   PDF (4924KB) ( 144 )   Supporting Information An accurate mass database and a method based on ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry (UHPLC-Orbitrap HRMS) were developed. These were applied in the screening and identification of illegally added medicines in herbal tea. Based on investigations, 167 medicines were selected to build an accurate MS database; these medicines included antipyretic analgesics, glucocorticoids, antibiotics, and antihistamines, among other categories. The database was established using Orbitrap HRMS and TraceFinder software. The database carried information on all selected compounds, including the molecular formula, accurate mass of precursor ions and fragment ions, retention time, and mass spectra. The samples were ultrasonically extracted with a 50% (v/v) methanol aqueous solution. The extracted solutions were separated using a Waters XBrigde BEH C18 column (100 mm×2.1 mm, 2.5 μm). As the mobile phases, 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid were used, with gradient elution. The sample solutions were analyzed by Orbitrap HRMS in the full-scan MS and data-dependent MS/MS acquisition modes (Full MS/dd-MS2). Positive and negative polarity data were simultaneously acquired. Some parameters were optimized to increase the peak intensity and sensitivity of all compounds. The resolutions in the full-MS scan and dd-MS2 scan were set to 70000 and 17500, respectively. In the full-MS mode, scanning was performed in the range of m/z 100 to 1000. In the MS/MS mode, the normalized collision energy (NEC) was set to 20%, 40%, and 60% for each compound. The inclusion list was not used during the measurement, and the dynamic exclusion time was set to 10.0 s. The loop count was set to 5. After acquiring the sample data with these conditions using Orbitrap MS, they were imported into TraceFinder software, through which the sample information was extracted and automatically matched with the information on compounds in the MS database. Screening and identification were conducted by comparing the retention times as well as the exact masses of precursor ions and fragment ions that were experimentally measured. If the errors between the experimentally and theoretically obtained masses of the precursor ions were below 5×10-6 and the deviations in retention times were less than 20 s, then suspicious positive compounds might be identified. Furthermore, if such compounds possess more than one similar fragment ion with a mass tolerance below 5×10-6, and exhibit similar ion distributions in the MS/MS profiles (compared to those in the database), they could be confirmed to be the same. The validation result showed that all compounds had good linear relationships, with correlation coefficients (r) greater than 0.99. Because pefloxacin, norfloxacin, desloratadine, astemizole and clindamycin had background interference, the method was not suitable for their quantification. Following experiments using three spiked concentrations, the recoveries of the rest 162 compounds were found to be in the range of 66.4%-118.1%, and the relative standard deviations (RSDs, n=6), in the range of 0.1%-16.1%. When the limit of detection (LOD) was 0.2 mg/kg, 83 compounds were detected, while when the LOD was 1.0 mg/kg, 167 compounds were detected. All compounds were matched successfully to the standard added sample with the MS database in TraceFinder software. To lower the likelihood of false positive and false negative results, a quality control method was recommended. The method was applied to analyze 245 herbal tea samples, among which 12 positive samples were detected. Thirteen positive compounds were found, including acetaminophen, diclofenac sodium, chlorpheniramine, brompheniramine, dexamethasone, dexamethasone 21-acetate, prednisone, prednisone 21-acetate, metronidazole, erythromycin, ciprofloxacin, amantadine, and dextromethorphan. In particular, amantadine, dextromethorphan, brompheniramine, and ciprofloxacin were newly detected, compared to standard methods. The developed method is rapid and accurate, and will be useful in the high-throughput screening of illegally added medicines in herbal tea.

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Determination of trace genotoxic impurities in nifedipine by ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry GUO Changchuan, TAN Huijie, LIU Qi, GONG Tengfei, WANG Xue, WANG Chenglin, XU Yuwen 2022, 40 (3):  266-272.  DOI: 10.3724/SP.J.1123.2021.06008 Abstract ( 221 )   HTML ( 38 )   PDF (1384KB) ( 276 )   A method based on ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry (UHPLC-Orbitrap HRMS) was established for the determination of genotoxic impurities 2, 6, and 12 in nifedipine. After extraction with methanol, the sample was injected into the UHPLC-Orbitrap HRMS system for analysis. An ACE EXCELTM 3 C18-AR column (150 mm×4.6 mm, 3 μm) was used for chromatographic separation. The mobile phase was methanol-0.1% formic acid aqueous solution (65∶35, v/v). The flow rate was 0.6 mL/min, while the column temperature and autosampler temperature were set as 35 ℃ and 8 ℃, respectively. The divert valve switching technique was used to protect the mass spectrometer. The six-way valve was set to divert the eluent of 7.5-11.6 min to waste and the rest of the eluent into the mass spectrometer. The Orbitrap mass spectrometer was coupled with the UHPLC system by an electrospray ion (ESI) source. The sheath gas and auxiliary gas flow rates were 60 and 20 arb (arbitrary units), respectively. The spray voltage was 3.5 kV, while the capillary temperature and auxiliary gas heater temperature were set as 350 ℃ and 400 ℃, respectively. The positive ion parallel reaction monitoring (PRM) scanning mode was adopted, and the mass spectral resolution was set to 35000 FWHM. The accurate masses of the [M+H]+ precursor ions of impurities 2, 6, and 12 were m/z 347.1230, 361.1026, and 347.1230, respectively. The accurate masses of the extracted [M+H]+ fragment ions of impurities 2, 6, and 12 were m/z 315.0968, 298.1069, and 315.0968, respectively. The normalized collision energies (NCEs) were optimized to 10%, 42%, and 10% for impurities 2, 6, and 12, respectively. The external standard method was utilized for quantitative analysis. The established method was validated in detail by investigating the specificity, linear range, limit of detection (LOD), limit of quantification (LOQ), recovery, precision, and stability. This method had good specificity, and the solvent did not interfere with the determination of impurities. The peak areas of impurities 2, 6, and 12 as well as their concentrations showed good linear relationships in the ranges of 0.2-100 ng/mL, with all correlation coefficients (r)≥0.9998. The recoveries of impurities 2, 6, and 12 at three levels (low, medium, and high) were in the range of 96.9%-105.0%, while the RSDs were between 1.21% and 5.12%. The LODs were 0.05 ng/mL and the LOQs were 0.2 ng/mL for all three impurities. This analytical method was used to determine impurities 2, 6, and 12 in three batches of nifedipine samples. Impurity 6 was not detected in the three batches, but impurities 2 and 12 were detected in all the three samples, and the detection amount was within the limit. The developed method is sensitive, fast, accurate, and easy to operate. It can provide a reference for the quality control of nifedipine by pharmaceutical companies and extend strong technical support for the supervision by drug regulatory authorities.

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Rapid and simultaneous determination of glyphosate, glufosinate, and their metabolites in soil by high performance liquid chromatography-tandem mass spectrometry PING Hua, ZHAO Fang, LI Cheng, WANG Beihong, KONG Hongling, LI Yang, MA Zhihong 2022, 40 (3):  273-280.  DOI: 10.3724/SP.J.1123.2021.08005 Abstract ( 626 )   HTML ( 70 )   PDF (1123KB) ( 270 )   Glyphosate (GLY) and glufosinate (GLUF) are non-selective translocated herbicides that are used in agricultural and non-agricultural land worldwide. The extensive use of GLY and GLUF may lead to their accumulation in soil, which causes soil pollution and affects the soil micro-ecological environment; the accumulated GLY and GLUF also migrate to groundwater via leaching. However, GLY, GLUF, and their metabolites are highly water-soluble and lack chromogenic and fluorescent groups, making them difficult to analyze. Currently, derivatization methods are mostly used to detect GLY, GLUF, and their metabolites. However, these methods also have some drawbacks, such as complex operation, long time consumption, and poor stability. In addition, these compounds are easily passivated and made inactive in soil; they also react with organic matter, humic acid, metal oxides, and heavy metal ions, making their extraction from soil difficult. To date, the method for the determination of GLY, GLUF, and their metabolites in soil is limited. Therefore, it is necessary to establish a quick and sensitive method to determine the residues of GLY, GLUF, and their metabolites in soil. In this study, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of GLY, GLUF, and their metabolites in soil. Parameters like extraction solvent, extraction temperature, extraction time, and adsorbents, which affected the extraction efficiencies, were optimized. Finally, the soil samples were extracted with 0.5 mol/L ammonia solution in a bath shaker at 50 ℃, and then centrifuged at 10000 r/min for 5 min. The supernatant was filtered through 0.2-μm syringe filters and then determined by HPLC-MS/MS. A Dikma Polyamino HILIC column (150 mm×2.0 mm, 5 μm) was used for chromatographic separation with good peak shape and high response of the target compounds. Ammonium acetate (0.2 mmol/L) with 0.07% ammonia aqueous solution and acetonitrile were used as the mobile phase. The flow rate of the elute was 0.25 mL/min. MS/MS was conducted under multiple reaction monitoring (MRM) mode using an electrospray ionization (ESI) source, and was quantified by the external standard method using matrix-matched calibration curves. All the target compounds were ionized in the negative ionization mode. The linear ranges of GLY and its metabolites were between 5.0 and 500 μg/L, and those of GLUF and its metabolites were between 2.0 and 500 μg/L. Linear correlation coefficients were greater than 0.99. The limit of detection (LOD) and limit of quantification (LOQ) were assessed using signal-to-noise (S/N) ratios of 3 and 10, respectively. The LOD and LOQ values of both GLY and (aminomethyl)phosphonic acid (AMPA) were 4.0 and 13.3 μg/kg, respectively. The LOD and LOQ values of GLUF, MPP, and N-acetyl glufosinate (NAG) were 2.0 and 6.7 μg/kg, respectively. Method accuracy was acquired by recovery test at three spiked levels (0.02, 0.05, 0.2 mg/kg). The average recoveries of five targets spiked in soil with low organic matter content were 74.2%-101%, and the relative standard deviation (RSD) was 0.93%-6.8%; the average recoveries of the five targets spiked in soil with high organic matter content were 90.8%-116%, and the RSD was 0.40%-7.1%. The established method was used to determine 20 soil samples in peach orchard, and the detection rates of AMPA, GLY, MPP, GLUF and NAG were 45%, 25%, 10%, 5% and 5%, respectively. The maximum residues were 147, 35.2, 154, 21.6 and 11.0 μg/kg, respectively. This method is simple, rapid, green, inexpensive, allows pretreatment without organic reagents, and affords high accuracy, high sensitivity, and good reproducibility. The method is suitable for testing a large number of soil samples with different organic matter contents. It can provide reliable technical support for the study of residue status and environmental behavior in soil.

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Simulation of gas chromatographic separation based on random diffusion SUN Yinlu, WANG Lin, YIN Zhiyu, ZHAO Jianwei 2022, 40 (3):  281-288.  DOI: 10.3724/SP.J.1123.2021.10011 Abstract ( 161 )   HTML ( 26 )   PDF (1164KB) ( 160 )   Understanding the diffusion behavior of particles during chromatographic analysis is critical for optimizing the operation conditions, improving the chromatographic performance, and designing a new separation device. Most of the existing simulations focus on chromatographic thermodynamics, while very few consider the overall diffusion and separation process. Herein, a new simulation method for gas chromatography separation was developed based on the random diffusion theory in microscale restricted space. This method retained the key information for controlling the diffusion behavior, simplified the interaction between the particles to be separated, and enlarged the time scale of each step one molecule walks. Thus, the operational efficiency could be significantly improved due to reduced calculation, and the entire diffusion process in the gas chromatography capillary column could be simulated. In this model, the capillary column was represented by a two-dimensional long and narrow rectangle where the particles to be separated randomly diffused. Besides, a directional velocity along the axis of the chromatographic column exerted on the particle represented the driving force of the mobile phase. If a particle collided with the inner wall of the column, it would remain at the collision position even after some time lapsed. When desorption occured, the particle would flow along with the mobile phase until its next adsorption on the surface. The interaction between the particle and the inner wall was expressed by the equivalent adsorption time. By dynamically tracking the trajectories of particles, the statistical distribution of time for the residence of the particles in the chromatographic column could be obtained, that is, the detection signal (retention time). Based on the previous simulation studies on the separation of n-alkane homologues, combined with the Kovats Retention Index, the functional relationships between adsorption steps and temperature as well as carbon number were established. As a result, the separation parameter systems for various homologues at different temperatures were set up. The separation of alcohol homologues at different temperatures was considered as an example to verify the reliability of the simulation method. The relative errors between the measured and simulated values were within 5% for the retention time and 0.75%-60% for the peak width. The reasons for the large relative errors in the peak width are summarized as follows. On the one hand, parameterization of alcohol homologues was performed on the basis of a previous study on the separation law of n-alkane. Given the limitations of the current computing capability, the insufficient iteration in the parameterized process led to large errors. In addition, the errors at different temperatures further accumulated in extrapolated approximations. On the other hand, the strong hydrogen bond force between the alcohol molecules was simplified with the elastic collision, which increased the magnitude of the errors. Although the simulation method proposed in this paper can accurately predict the retention time and reasonably describe the morphological characteristics of chromatographic peaks, there is still room for improvement. In particular, the description of the detailed interactions between molecules must be improved. For example, the method of molecular mechanics may be assistant with the investigation of the functional relationship between interaction potential and adsorption steps. The interaction potential calculated on the basis of molecular mechanics replaces the parameterized adsorption steps, yielding more accurate simulation results. In general, the simulation method proposed in this study is a valuable reference for the optimization of chromatographic operating conditions and for the development of new chromatographic techniques.

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Field-amplified sample injection and graphene quantum dot dual preconcentration in the analysis of melamine and dicyandiamide by capillary electrophoresis LI Chao, WANG Qi, ZHANG Zhaoxiang 2022, 40 (3):  289-295.  DOI: 10.3724/SP.J.1123.2021.08017 Abstract ( 150 )   HTML ( 20 )   PDF (1809KB) ( 56 )   Sulfur-doped graphene quantum dots (S-GQDs) were prepared by the pyrolysis of citric acid and mercaptopropionic acid. Compared with graphene quantum dots (GQDs), the S-GQDs have improved surface state and chemical reactivity, and thus, exhibited stronger interaction with cations. Based on its excellent affinity for cations, a dual preconcentration technique combining field-amplified sample injection (FASI) and S-GQDs as multianalyte carriers was developed for the determination of melamine and dicyandiamide by capillary electrophoresis (CE). During the FASI process, a large quantity of analytes was introduced into the capillary and accumulated at the capillary inlet. Concurrently, the S-GQDs migrated to the anode and captured the analytes on its surface at the boundary of the sample and buffer solution. The use of S-GQDs allows the capture of abundant analytes, which can amplify the detection signal. This new protocol was evaluated by the quantitative determination of melamine and dicyandiamide in metformin hydrochloride preparations. The effect of volume fraction of the S-GQDs in the buffer solution, the composition and pH of the buffer, and the sample injection time on the preconcentration and separation were investigated. By controlling the pH at 4.6, the sample injection time was prolonged to 450 s. A very large amount of melamine and dicyandiamide, bearing positive electric charges, were injected into the capillary and were captured by S-GQDs. The assay using FASI preconcentration and S-GQDs as enhancer resulted in a 1.6×105-fold improved sensitivity compared with that obtained with traditional 10-kV electrokinetic injection for 10 s. The calibration curves of melamine and dicyandiamide were obtained in the concentration range from 1.0×10-14 to 1.0×10-8mol/L, with correlation coefficients (r2) >0.999. The detection limits (S/N=3) were 2.6×10-15mol/L for melamine and 5.7×10-15mol/L for dicyandiamide. The recoveries of the two analytes were 95.9%-102.4% and 92.0%-106.0%, respectively, with relative standard deviations (RSDs) of no more than 5%. The RSD values of peak height, peak area, and migration time were all less than 5.6%. This method is reliable, easy, and exhibits a good separation effect. This proves that the S-GQD-enhanced CE method could be developed into a new and sensitive technique for the determination of melamine and dicyandiamide in different preparations of metformin hydrochloride.

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Separation of budesonide enantiomers with amylose-tris-[(S)-1-phenylethyl carbamate] chiral stationary phase and determination of its contents in pharmaceutical preparations HUANG Yongpeng, TANG Hui, MENG Xiangyan, CHEN Bo, ZHONG Hui, ZOU Zhiyun 2022, 40 (3):  296-301.  DOI: 10.3724/SP.J.1123.2021.06048 Abstract ( 173 )   HTML ( 28 )   PDF (794KB) ( 69 )   The drug budesonide exists as 22R and 22S enantiomers. However, the drug activity of 22R-budesonide is 2-3 times stronger than that of 22S-budesonide. The development of enantiomeric separation and quantitative analysis methods for budesonide can provide an important basis for its drug development and quality control. At present, the enantiomers of budesonide are separated on a reversed C18 solid phase column. However, chiral stationary phases are rarely reported for the separation of the enantiomers of budesonide. In this study, a high performance liquid chromatography (HPLC) method with a chiral stationary phase was developed for the rapid separation and determination of budesonide enantiomers. The effects of the type of chiral stationary phase, mobile phase additives, and column temperature on the resolution of the budesonide enantiomers were also investigated. The results showed that the chiral stationary phase amylose-tris-[(S)-1-phenylethyl carbamate] was more suitable for the separation of budesonide enantiomers. The mobile phase additives used in the experiment had no significant effect on the chromatographic parameters (peak height, peak width, and resolution) of the budesonide enantiomers. However, with an increase in the column temperature, the peak width of the budesonide enantiomers decreased, while the peak height and resolution increased. The optimized HPLC conditions were as follows: column, Chiralpak AS-RH (150 mm×4.6 mm, 5.0 μm); mobile phase, acetonitrile-water (45∶55, v/v); column temperature, 40 ℃; flow rate, 1.0 mL/min; detector, diode array detector (DAD); detection wavelength, 246 nm; injection volume, 10 μL. The external standard method was used to quantify the budesonide enantiomers. Under the optimized conditions, the enantiomers were well separated, and the retention times of 22R-budesonide and 22S-budesonide were 6.40 min and 7.77 min, respectively. The resolution of the enantiomers was 4.64. The linear ranges of 22R-budesonide and 22S-budesonide were 0.16-1000 μg/mL and 0.20-1000 μg/mL, respectively. The peak area of the enantiomers showed a good linear relationship with the corresponding concentration, and the correlation coefficients (R2) were 0.9999. The limits of detection (LODs) of 22R-budesonide and 22S-budesonide were 0.05 μg/mL and 0.07 μg/mL, respectively, based on a signal-to-noise ratio of 3. The limits of quantification (LOQs) were calculated to be 0.16 μg/mL and 0.20 μg/mL, respectively, based on a signal-to-noise ratio of 10. The recoveries at four spiked levels were in the range of 102.63% to 104.17%, with the relative standard deviations (RSDs) of 0.08% to 0.57% (n=6). The budesonide solution was stored in dark at 4 ℃ for 24 h, and no obvious degradation was observed. Finally, the method was applied to determine four actual samples of budesonide suspension for inhalation in a batch. The samples were dissolved in methanol, filtered through a 0.45 μm microporous membrane, and then analyzed. The amounts of 22R-budesonide and 22S-budesonide in the samples were in the ranges of 283.15-284.63 μg/mL and 259.86-261.51 μg/mL, respectively. This method is simple and rapid, in addition to having good repeatability and high accuracy. It can be used for the resolution of budesonide enantiomers and for quality control in budesonide preparations.