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Chinese Journal of Chromatography
2020, Vol. 38, No. 11
Online: 08 November 2020

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Volume 38,Number 11 Content 2020, 38 (11):  0-0.  Abstract ( 20 )   PDF (4489KB) ( 31 )

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Preparation and application of urushiol methacrylate-bonded silica liquid chromatographic stationary phase Lei ZENG, Yu CAO, Xingdong YAO, Guoxiang LI, Fuhou LEI, Boan SHI 2020, 38 (11):  1257-1262.  DOI: 10.3724/SP.J.1123.2020.07039 Abstract ( 80 )   HTML ( 33 )   PDF (5433KB) ( 59 )   A novel stationary phase for high performance liquid chromatography was prepared using urushiol methacrylate as the chromatographic ligand. The mixed urushiol methacrylate was prepared using urushiol and methacryloyl chloride via a substitution reaction and then coated onto the surface of spherical silica by physical adsorption. The spherical silica was chemically modified with 3-methacryloyloxypropyl trimethoxysilane. Then, the urushiol methacrylate-bonded silica stationary phase (USP) was synthesized via the surface radical polymerization of urushiol methacrylate and the pendant vinyl groups onto the surface of the spherical silica. The stationary phase was characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, and elemental analysis. The results revealed that the urushiol methacrylate was successfully immobilized on the spherical silica surface after the surface polymerization reaction, and that it had excellent monodispersity. The stationary phases were packed in a stainless-steel hollow column by the slurry packing method, with methanol as the slurry solvent and absolute ethanol as the propelling solvent. The chromatographic performance of the stationary phases were investigated for the separation of Gastrodia elata extract. Acetonitrile-0.05% phosphoric acid solution (3:97, v/v) was employed as the mobile phase at a flow rate of 0.4 mL/min, with the detection wavelength of 220 nm. The separation performance for Fructus evodia extract was also studied, using acetonitrile-water (50:50, v/v) as the mobile phase at a flow rate of 0.5 mL/min, and the detection wavelength was 290 nm. This column showed good separation performance for both these extracts. Out of the five peaks observed for the Gastrodia elata extract, one was attributed to gastrodin, but the other four peaks need to be further verified. Two peaks assignable to evodiamine and rutaecarpine were observed for the Fructus evodia extract. Compared with C18 column, the USP column allowed for more effective separation of the components from the Gastrodia elata extract, with baseline separation; on the other hand, the chromatographic conditions for the separation of the components of the Fructus evodia extract were more environmentally friendly and safer. Because of the low flow rates adopted for the separation of the Gastrodia elata and Fructus evodia extracts, the amount of mobile phase used could be reduced. This study provides not only a new method for the separation and purification of gastrodin and evodiamine in real samples, but also a new strategy for the preparation of chromatographic stationary phases. It expanded the application of raw lacquer in chromatographic separation materials.

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Preparation and chromatographic properties of 1-vinyl-3-dodecylimidazole bromide silica-bonded stationary phase Xinting LI, Peng LIANG, Yufeng ZHOU, Xiaoqiang QIAO 2020, 38 (11):  1263-1269.  DOI: 10.3724/SP.J.1123.2020.02012 Abstract ( 98 )   HTML ( 22 )   PDF (898KB) ( 74 )   As the main components of cell membranes, lipids play important roles in organisms. Lipids have been proved to be closely associated with the occurrence and development of serious diseases, such as cancers and metabolic diseases. The development of novel separation materials for use in high-performance liquid chromatography (HPLC) is essential for high-efficiency lipid separation. Such materials can promote further in-depth research of the structure and biological functions of lipids. In this study, we focused on the preparation of an ionic-liquid-modified silica-bonded HPLC stationary phase and on its chromatographic retention mechanisms and separation performances for lipids. An imidazolium-based ionic liquid with C12 side chain, viz. 1-vinyl-3-dodecylimidazole bromide (VDI), has shown good biocompatibility and has previously been used for the solubilization of membrane proteins. Thus, VDI was first exploited as the functionalized monomer for the HPLC stationary phase. It was grafted onto the surface of thiol-functionalized silica spheres by a one-step click reaction to afford a new VDI silica-bonded stationary phase (Sil-VDI). Fourier-transform infrared (FT-IR)spectroscopy and thermogravimetric analysis were used to prove the successful preparation of Sil-VDI and characterize its structure. The chromatographic retention properties of the column packed with Sil-VDI was first studied using hydrophobic alkylbenzenes. The results showed that the Sil-VDI column was a typical reversed-phase liquid chromatography retention column. Since Sil-VDI has a permanent cationic imidazole structure, it should demonstrate anion exchange retention. Thus, inorganic anions BrO3 - , NO3 - , and IO3 - were selected to further investigate the retention mechanism of the Sil-VDI column. The results demonstrate that the Sil-VDI column also possesses ion-exchange retention mode. Thus, the Sil-VDI column has typical reversed-phase and ion-exchange mixed-mode retention characteristics. Based on the reversed-phase retention characteristics of the Sil-VDI column, hydrophobic alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), basic anilines, and benzene derivatives were selected for testing the HPLC separation performances of the Sil-VDI column. The results demonstrate that this new column gave good separation selectivity with good peak shapes. For example, PAHs like diphenyl, o -terphenyl, m -terphenyl, and triphenylene were used to investigate the chromatographic performances of the Sil-VDI column. These four PAHs were baseline separated within 7 min with good peak shapes. In addition, the positional isomers o -terphenyl and m -terphenyl show good separation efficiency, with resolution as high as 3.26. Based on the ion-exchange retention characteristics of the Sil-VDI column, inorganic anions BrO3 - , NO3 - , and IO3 - were selected to test the separation performance of the column for ionic compounds. Using 250 mmol/L KCl solution (pH 4.1) as the mobile phase, baseline separation of the three anions was achieved within 6 min. These results demonstrate that the Sil-VDI column has good potential for separation of ionic compounds. The separation performance of the Sil-VDI column was further verified based on the separation of lipids extracted from egg yolk and lung adenocarcinoma cells. Six main chromatographic peaks could be recognized within 7 and 5 min for the lipids extracted from lung adenocarcinoma cells and egg yolk, respectively. These results primarily demonstrate that the Sil-VDI column has good potential for the separation of lipid samples. In conclusion, a new ionic-liquid-based Sil-VDI stationary phase material was successfully fabricated via a simple synthesis method. The Sil-VDI column shows good separation performances for versatile samples. In future, further research will be performed on the separation ability of the Sil-VDI column for different biological samples.

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Click preparation and application of chiral stationary phase based on intrinsic recognition ability of cyclodextrin Ming CHEN, Xiaoning JIN, Xiaofei MA, Yong WANG 2020, 38 (11):  1270-1280.  DOI: 10.3724/SP.J.1123.2020.02011 Abstract ( 120 )   HTML ( 29 )   PDF (2665KB) ( 129 )   Most of the studies on cyclodextrin (CD)-based chiral stationary phase (CSP) have focused on the functional derivatization of CD or the bridging arms to introduce more interaction sites and thus improve the chiral resolution ability. At present, there are only a few reports on CSP that can reflect the intrinsic recognition ability of natural CD. In this study, a mono(6-mercapto-6-deoxy)-β -CD CSP (CSP1) with a clear and controllable structure was synthesized by the "thiol-ene" click reaction. CSP1 retained the intrinsic structure of natural CD to the maximum extent, and the bridge arm had no recognition site. The results of 13 C solid-state nuclear magnetic resonance (SSNMR) and Fourier transform infrared (FTIR) analyses confirmed the successful preparation of CSP1. Elemental analysis results showed that compared with double-bond functionalized silica, the percentages of C, H, and N in CSP1 increased, and the calculated CD loading of CSP1 was 0.82 μmol/m2 . Reversed-phase high performance liquid chromatography was performed for the chiral resolution of more than 50 chiral enantiomers, including isoxazoline, chiral lactide, chiral ketone, flavone, and dansyl amino acid. This fully demonstrated the intrinsic chiral recognition ability of natural CD, and the results showed that the intrinsic recognition ability of cyclodextrin was more conducive to the separation of Ph-Ph samples containing two hydrophobic benzene ring groups in the isoxazoline samples. For the Ph-Py and Ph-OPr samples, the separation effect was not satisfactory. The separation results for the Ph-Py samples were not ideal because the outer hydroxyl group of cyclodextrin could form a hydrogen bond with the pyridine nitrogen, thus hindering the inclusion and the separation effect. This eventually led to poor separation of the enantiomers. While the pyrrolidone group in the Ph-OPr sample could also form a good inclusion complex with cyclodextrin, its higher polarity weakened the inclusion effect compared to that for benzene rings, thus leading to poor chirality separation results. For chiral lactides, the intrinsic recognition ability of CD was good only for the separation of some samples. In the separation of chiral ketones, large steric hindrance effect inhibited the intrinsic recognition ability of CD, and the separation effect of such samples on CSP1 was not ideal. External functional groups were required in some cases to further regulate the chiral recognition performance. The molecular structure of dansyl amino acids played an important role in the separation effect, in addition to the intrinsic recognition ability of CD. The number of side chains in the substituent also affected the quality of separation. Lengthening the side chain or increasing the hydrophobicity could effectively improve the separation efficiency. The separation effect of flavanone samples on CSP1 was ordinary. The substituent positions also affected the separation effect. In order to further explore the intrinsic recognition ability of CD, the functional triazole-bridged CD-CSP (CSP2) and imidazole-bridged CD-CSP (CSP3) (the surface CD loadings of CSP2 and CSP3 were 0.51 μmol/m2 and 0.46 μmol/m2 , respectively) prepared earlier were selected and compared under the same chromatographic conditions. The results showed that the separation of the sample was related not only to the structure of the chiral medium but also to the structure of the sample molecules. Functional modification of the bridge arm could improve the selectivity of some enantiomers, but would also cause partial loss of the intrinsic chiral recognition ability of CD. For samples with the intrinsic recognition ability of CD to facilitate separation, no functional group had to be added to the bridge arm when designing a chiral medium. This study provides a useful reference for the design of CD-based CSPs.

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Determination of trace α -amanitin in urine of mushroom poisoning patient by online solid phase extraction-liquid chromatography-tandem mass spectrometry Xiaomin XU, Jingshun ZHANG, Zengxuan CAI, Zhen MENG, Baifen HUANG, Qing CHEN 2020, 38 (11):  1281-1287.  DOI: 10.3724/SP.J.1123.2020.03010 Abstract ( 78 )   HTML ( 18 )   PDF (1297KB) ( 71 )   An analytical method was established for the determination of trace α -amanitin in the urine of patients suffering from mushroom poisoning by online solid phase extraction-liquid chromatography-tandem mass spectrometry (online SPE-LC-MS/MS). The sample was protein precipitated with formic acid acidified acetonitrile-methanol (5:1, v/v). Reversed-phase liquid-liquid microextraction was used to remove the organic solvent from the sample extract. The toxin was purified by online SPE using an ODS micro column (5 mm×2.1 mm, 5 μm), and separated on an XBridgeTM BEH C18 column (150 mm×3.0 mm, 2.5 μm). Finally, the toxin was measured by MS/MS in the negative electrospray ionization (ESI- ) mode. Multiple reaction monitoring (MRM) was used, and the conditions were m /z 917.4>205.1 (quantitative ion transition) and m /z 917.4>257.1. Collision energy for both transitions was 55 eV. A fast valve-switching technique with a quantitative loop was used as an interface between the online SPE and LC-MS/MS modules. The two modules were independent, neither the mobile phase nor the pressure would interfere with each other, thus ensuring the stability of the system. Precise purification by the online system could effectively eliminate the matrix effects in the subsequent MS detection. Weak matrix suppression effects were found, with results of 88.7%-96.5%. The linear range of α -amanitin in urine was 0.1-50 μg/L with a correlation coefficient (r 2 ) of 0.9983. The limit of detection (LOD) and limit of quantification (LOQ) in the sample matrix were 0.03 μg/L and 0.1 μg/L, respectively. The average recoveries at three spiked levels (0.1, 2.0 and 20 μg/L) were 84.3%-91.7% with relative standard deviations (RSDs) of 3.8%-7.2%. The accuracy and precision were evaluated using quality control samples with toxin contents of 0.1 μg/L (LOQ), 0.2 μg/L (2-fold LOQ), 2.0 μg/L (medium level), and 20 μg/L (high level). The calculated average intra-day accuracy was 85.1%-96.0% with the precision of 4.1%-7.8%. The inter-day accuracy was 82.9%-94.8% with the precision of 5.0%-9.5%. The specificity of the method was verified by negative samples derived from patients who suffered only gastroenteritis poisoning, without hepatotoxic symptoms. α -Amanitin was found in urine samples from nine mushroom poisoning patients with hepatotoxic symptoms. The sampling time ranged from 19 h to 92 h. The toxin contents were 0.11-53.1 μg/L. For patients with a high intake of poisonous mushrooms, the toxin content was 53.1 μg/L in a patient's urine sampled 19 h after accidental consumption and 0.19 μg/L in another patient's urine sampled 92 h after poisoning. The content of α -amanitin was only 0.53 μg/L in the urine sample obtained 23 h after consumption for a patient with low intake and 0.11 μg/L in the urine sampled from another patient 40 h after poisoning. Amatoxins can metabolize rapidly in vivo. The laboratory identification of amatoxin poisoning requires a method for trace-level analysis in the biological matrix. It is proved that this method is simple, accurate and sensitive by the application to the analysis of actual samples. The protein precipitation and reversed-phase liquid-liquid microextraction steps are fast and simple. Hence, they can be used as a rapid and effective pre-treatment method for online SPE-LC-MS/MS analysis of water-soluble toxins in biomaterial matrix. Highly sensitive analysis of α -amanitin in urine can be obtained using a precise purification technology via online SPE in this study. The problem of qualitative confirmation of the toxin at trace levels (0.03 μg/L) after poisoning can be solved. The laboratory identification time for amatoxin poisoning in some patients exceeds 90 h. The developed analytical method at trace level (0.1 μg/L of LOQ) can provide reliable technical support for establishing the dose-response relationship of α -amanitin in vivo. It can satisfy for the determination of trace α -amanitin in urine samples from patients with hepatotoxic mushroom poisoning.

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Determination of N -nitrosodimethylamine in metformin hydrochloride and its preparations by high performance liquid chromatography-tandem mass spectrometry Changchuan GUO, Qi LIU, Lei ZHANG, Jing ZHENG, Yong WANG, Shujuan YANG, Zhijie CHU, Chong NIU, Yuwen XU 2020, 38 (11):  1288-1293.  DOI: 10.3724/SP.J.1123.2020.03008 Abstract ( 195 )   HTML ( 29 )   PDF (1075KB) ( 114 )   A method was established for the determination of N -nitrosodimethylamine (NDMA) in metformin hydrochloride active pharmaceutical ingredient (API) and preparation samples by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Water was used as the extraction solvent for the metformin hydrochloride API and preparation samples. The samples were analyzed by HPLC-MS/MS after vortex mixing, constant temperature shaking, high speed centrifugation and microfiltration. An ACE EXCEL 3 C18-AR column (150 mm×4.6 mm, 3 μm) was used for chromatographic separation. The mobile phases were water and methanol both containing 0.1% formic acid with gradient elution. The flow rate, column temperature, and autosampler temperature were set as 0.8 mL/min, 40℃, and 10℃, respectively. The valve switching technique was used to protect the mass spectrometer, while six-way valve switching was adopted to allow the mobile phase with a retention time of 2.85-7.00 min to enter the mass spectrometer and the mobile phase with other retention times to enter the waste liquid. For the mass spectrometer, an atmospheric pressure chemical ionization (APCI) ion source was used in positive ion MRM scanning mode. The other conditions were as follows:atomizer flow, 3 L/min; heater flow, 10 L/min, interface temperature, 300℃; desolvation line (DL) temperature, 250℃; heating block temperature, 400℃; and dryer flow, 10 L/min. The quantitative transition of NDMA was m /z 75.0→43.1 with a collision energy of-17.0 eV, while the qualitative transition was m /z 75.0→58.2 with a collision energy of-16.0 eV. 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, limit of quantification, recovery, precision, and stability. This method showed good specificity, since the solvents and excipients did not interfere with the determination of NDMA. A good linear relationship was observed the NDMA peak area and the mass concentrations in the range of 1.00-100.00 ng/mL with an excellent correlation coefficient (r >0.9999). The limit of detection and limit of quantification in solution were 0.20 ng/mL and 1.00 ng/mL, respectively. The recoveries of NDMA at low, medium, and high spiked levels ranged from 94.55% to 114.67%, and the RSDs ranged from 4.73% to 13.46%, indicating good accuracy and precision for the quantification of NDMA. Stability tests showed that NDMA was stable when placed in the autosampler for 0, 8, 24 h, since the RSD of the peak area was as low as 2.08%. The validated method was then applied to the determination of NDMA in metformin hydrochloride raw materials and preparations (tablets, capsules or enteric tablets). The detected amount of NDMA in the API did not exceed the limit in 113 batches of samples, but NDMA was detected and exceeded the limit in eight batches of preparations. This method is sensitive, accurate, and easy to operate, and it can be used for the determination of NDMA in metformin hydrochloride raw materials and preparation samples.

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Simultaneous determination of paraquat and diquat in plasma and urine by ion chromatography-triple quadrupole mass spectrometry Xiuyao ZHANG, Xinxin CAI, Xiaoyi ZHANG, Ruifen LI 2020, 38 (11):  1294-1301.  DOI: 10.3724/SP.J.1123.2020.02008 Abstract ( 187 )   HTML ( 24 )   PDF (899KB) ( 124 )   Paraquat (PQ) and diquat (DQ) are widely used as non-selective contact herbicides. Several cases involving accidents, suicide, and homicide by PQ or DQ poisoning have been reported. Poising by PQ, which is mainly concentrated in the lungs, causes acute respiratory distress syndrome and leads to multiple organ toxicity. The toxic effects of DQ are similar to those of PQ but relatively less intense. The mortality rates in PQ and DQ poisoning are high. Simultaneous monitoring of the PQ and DQ concentrations in plasma and urine can provide valuable information for early clinical diagnosis and prognosis. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is the main analytical method used to detect PQ and DQ in plasma and urine. As both these compounds are highly polar and water soluble, they cannot be retained effectively on a reversed-phase column with conventional mobile phases. The separation of PQ and DQ by ion-pair chromatography or hydrophilic chromatography has been reported. The use of an ion-pairing reagent helps in improving the retention capabilities of PQ and DQ. However, the sensitivity of MS detection is noticeably decreased because of ion suppression caused by the ion-pairing reagent in the mobile phase; furthermore, ion-pairing reagents may contaminate the MS system. The separation of PQ and DQ by hydrophilic chromatography is easily affected by matrix components in the sample, and their retention times are not stable. Considering PQ and DQ are bicharged cation species in solution, they are more suitable for separation by cation-exchange chromatography. A method based on ion chromatography-triple quadrupole mass spectrometry was established for the determination of PQ and DQ in plasma and urine. The plasma and urine samples were diluted with water, and then purified on a solid-phase extraction column containing a polymer-reversed phase and weak ion-exchange mixed-mode adsorbent (Oasis WCX). PQ and DQ were separated on an IonPac CS 18 analytical column (250 mm×2.0 mm, 6.0 μm) with gradient elution using a methylsulfonic acid solution electrolytically generated from an on-line eluent generation cartridge. An in-line suppressor was used to remove methylsulfonate and other anions from the eluent before the eluent entered the mass spectrometer. Between the suppressor and the ion source in MS, the addition of 3% (v/v) formic acid in acetonitrile as an organic modifier (using an auxiliary pump and a T-piece) aided desolvation in the ion source, resulted in a one-or two-fold improvement of the response, and eliminated the residual effects of the adsorption of PQ and DQ caused by ion source. The analytes were detected by triple quadrupole tandem mass spectrometry using positive electrospray ionization in the multiple reaction monitoring (MRM) mode. PQ-d8 and DQ-d4 were used as internal standards. The calibration curves for PQ and DQ showed good linear relationships in the ranges of 1.0-150 μg/L and 0.5-75 μg/L, respectively, and the correlation coefficients were > 0.999. The average matrix effects of PQ and DQ in plasma were 84.2%-89.3% and 84.7%-91.1%, while the average matrix effects of PQ and DQ in urine were 50.3%-58.4% and 51.9%-59.4%. The average recoveries of PQ and DQ in plasma were 93.5%-117% and 91.7%-112%, respectively, with relative standard deviations (RSDs) of 3.4-16.7% and 2.8%-13.2%, and that in urine were 90.0%-118% and 99.2%-116%, with relative standard deviations of 5.6%-14.9% and 2.4%-17.3% (n =6). The limits of detection of PQ and DQ in plasma and urine were 0.3 μg/L and 0.2 μg/L, respectively, with the corresponding limits of quantification being 1.0 μg/L and 0.5 μg/L. This method is sensitive and accurate, and it can be used to determine PQ and DQ for clinical diagnosis and prognosis in patients.

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Determination of dacarbazine in the urine of mice with melanoma by high performance liquid chromatography Yuhua YUE, Bingjun ZHOU, Jiayuan AI, Shun FENG 2020, 38 (11):  1302-1307.  DOI: 10.3724/SP.J.1123.2020.01003 Abstract ( 133 )   HTML ( 16 )   PDF (1275KB) ( 48 )   Dacarbazine (DTIC) is a first-line chemotherapy drug that is widely used in clinical practice for malignant melanoma. DTIC is metabolized by the liver in vivo . Some drugs are excreted in urine in the form of a prototype. Hence, DTIC in urine can be monitored to evaluate its utilization and conversion rate in the human body, and then to determine its therapeutic effect. Urine is the only body fluid that can be obtained in large quantities without damage, and it plays an important role in the analysis of body functions. However, the composition of urine is complex and there is large matrix interference, because of which trace analysis or trace component analysis is difficult. At present, the main analytical methods for DTIC are high performance liquid chromatography (HPLC) with/without mass spectrometry (MS). HPLC and HPLC-MS have the advantages of good separation effect, good selectivity, high detection sensitivity, automatic operation, and wide application range. Unfortunately, DTIC is a strongly polar and weakly basic compound; thus, it is difficult to achieve good separation and obtain good peak shapes by conventional reversed-phase chromatography. To overcome these defects, it is necessary to develop a novel method for the analysis of DTIC. In this study, mice were subjected to 12 h of fasting; then, blueberry anthocyanin was administered by gavage, and DTIC was administered by intraperitoneal injection. Then, morning urine was collected in a metabolic cage. Urine collection was continued every 4 days for a total of 5 times. Within 2 h, the collected urine was centrifuged (3000 g, 4℃) for 10 min to remove solids. The supernatant was stored in a refrigerator at-80℃. Before analysis, the urine samples were removed from the refrigerator and thawed naturally at room temperature. Then, the samples were treated by the acetone-sediment method, freeze-dried, dissolved in the mobile phase, and subjected to HPLC analysis with isocratic elution. The separation was performed on a Shimadzu-GL ODS column (250 mm×4.6 mm, 5 μm). The mobile phase was methanol/acetonitrile (1:1, v/v)-0.01 mol/L NaH2 PO4 (pH 6.5; 20:80, v/v) at a flow rate of 1 mL/min. The detection wavelength, column temperature, and running time were 280 nm, 30℃, and 15 min, respectively. Under the optimized conditions, the retention time of DTIC was 5.3 min, and a good peak shape was obtained. The linearity ranged from 0.25 to 1000 μg/mL (r 2 =0.999). The limits of detection and quantification were calculated to be 0.12 μg/mL and 0.25 μg/mL based on signal-to-noise ratios of 3 and 10, respectively. At three spiked levels (50.0, 375, and 500 μg/mL), the average recoveries were 98.9%, 102%, and 99.1% with relative standard deviations (RSDs) of 3.2%, 1.3%, and 1.2% (n =5), respectively. The RSDs of the interday and intraday measurements were lower than 3.8% and 4.4%, respectively. The proposed method allowed for the accurate determination of DTIC in urine using a mixed organic solvent/phosphate buffer solution as the mobile phase, with equivalent elution for 15 min. This method was successfully applied to monitor the change in DTIC concentration in the urine of C57BL/6 mice in various stages of melanoma. The results demonstrate that the method is simple, reliable, and easy to apply.

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Simultaneous determination of polycyclic aromatic hydrocarbons and phthalate esters in surface water by dispersive liquid-liquid microextraction based on solidification of floating organic drop followed by high performance liquid chromatography Jiwei YUAN, Jincheng WANG, Weili XU, Fangxi XU, Xianbo LU 2020, 38 (11):  1308-1315.  DOI: 10.3724/SP.J.1123.2020.01020 Abstract ( 82 )   HTML ( 16 )   PDF (1640KB) ( 61 )   Polycyclic aromatic hydrocarbons (PAHs) and phthalate esters (PAEs) are internationally recognized as priority pollutants; hence, it is important to monitor their concentrations in the environment. However, the low concentrations of PAHs and PAEs in surface water make the direct and sensitive determination of these compounds by instrumental methods difficult. Therefore, the development of an accurate and rapid sample pretreating method for the determination of PAHs and PAEs in water has always been the goal of environmental scientists. Dispersive liquid-liquid microextraction based on solidification of floating organic droplet (DLLME-SFO) is a simple, rapid, low-cost, sensitive, and environmentally friendly method. Methods based on DLLME-SFO for the simultaneous determination of PAHs and PAEs in surface water have rarely been reported. In this study, a novel DLLME-SFO method was developed for the simultaneous determination of 16 PAHs and 6 PAEs in surface water samples. To optimize the extraction efficiency for the target compounds, various parameters, including the types and volumes of extractants and dispersants, ionic strength, and extraction time, were investigated. First, 1-undecanol (melting point:19℃) and 1-dodecanol (melting point:24℃) were selected as extractive solvents, and their extraction efficiency was investigated. The results showed that 1-dodecanol had better extraction efficiency. The melting point of 1-undecanol was relatively low, and the droplets that solidified during the experiment were easy to melt and break, which led to the low recovery rate of extraction. Then, the effect of the volume (10, 20, 30, 40, 50 μL) of 1-dodecanol was investigated, and the extraction efficiency of the target compounds was found to decrease with increasing volume of 1-dodecanol. Second, the effect of four dispersive solvents (methanol, ethanol, acetonitrile, and acetone) on the extraction efficiencies was studied. The extraction efficiencies of the target compounds were the highest when methanol was used as the dispersant; hence, the effect of different volumes of methanol on the extraction efficiency was further examined. When the volume of methanol was less than 500 μL, the contact area between the extraction solvent and the water phase increased with increasing methanol volume, and the extraction efficiency increased. However, when the volume of methanol was more than 500 μL, the excessive dispersant increased the solubility of the target compound in the water phase, which led to a decrease in the extraction efficiency. Finally, the effects of salt addition and vortex oscillation time on the extraction efficiency were probed. The experimental results indicated that the extraction efficiency increased with an increase in the quantity of NaCl. When the NaCl quantity was greater than 0.2 g, there was no notable change in the extraction efficiency. Vortex oscillation could accelerate the establishment of the extraction equilibrium, and the extraction efficiency reached a stable state when the vortex oscillation time was more than 2 min. According to the abovementioned results, the optimized DLLME-SFO conditions were established as follows:for 5.0 mL water samples, 10 μL of 1-dodecanol was chosen as the extraction solvent, 500 μL of methanol was used as the dispersive solvent, the vortex oscillation extraction time was 2 min, and the NaCl quantity was 0.2 g. The target compounds were analyzed by high-performance liquid chromatography. Separation of the PAHs and PAEs was achieved on a SUPELCOSILTM LC-PAH column (150 mm×4.6 mm, 5 μm) with acetonitrile-water as the mobile phase using a gradient elution program. Fifteen PAHs were detected using a fluorescence detector, and six PAEs and acenaphthylene were detected by an ultraviolet detector. Quantitative determination was achieved by the external standard method. This method was successfully validated for the analyses of the 16 PAHs and 6 PAEs in two types of water samples (tap water and river water). The average recoveries of the target compounds were 60.2%-113.5%, and the corresponding relative standard deviations (RSDs, n =3) were 1.9%-14.3%. The limits of detection (LODs, S /N =3) ranged from 0.002 μg/L to 0.07 μg/L for the PAHs and from 0.2 μg/L to 2.2 μg/L for the PAEs. The limits of quantification (LOQs, S /N =10) ranged from 0.006 μg/L to 0.23 μg/L for the PAHs and from 0.8 μg/L to 7.4 μg/L for PAEs. The proposed method is simple, fast, low-cost, and environmentally friendly, and it is suitable for the rapid determination of trace PAHs and PAEs in surface water samples.

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3D printed portable gel electrophoresis device for rapid detection of proteins Yingying LI, Dingyi WANG, Qiying NONG, Lihong LIU, Meng ZHANG, Yong LIANG, Ligang HU, Bin HE, Guibin JIANG 2020, 38 (11):  1316-1322.  DOI: 10.3724/SP.J.1123.2020.02018 Abstract ( 97 )   HTML ( 13 )   PDF (2252KB) ( 65 )   The growing demand for rapid, portable, and economical detection methods for environmental analysis has resulted in increasing demands on the portability and miniaturization of analytical instruments. The miniaturization of scientific instruments facilitates analysis in the field of medicine, food, and environment, especially for the under-resourced areas. The gel electrophoresis devices currently available for protein separation are primarily used in laboratories. Miniaturized instruments that can be used for on-site and rapid separation of protein have not yet been reported. In this study, a portable gel electrophoresis device for rapid separation and detection of proteins was developed and manufactured by 3D printing in a laboratory, which was economical, convenient, and quick. First, four kinds of portable gel electrophoresis devices that included three kinds of columnar gel and one slab gel electrophoresis device were designed with computer-aided design software SolidWorks 2017 (Dassault Systemes SE, France); the components including gel tubes, gel plates, and gel electrophoresis tanks were then printed using a 3D printer after optimization of the printing parameters. Then, the performance of the four kinds of gel electrophoresis devices was investigated using prestained protein molecular weight standards. The results showed that the single-channel slab gel electrophoresis design can quickly separate proteins with the best separation efficiency. Moreover, the effect of different separation gel lengths (5, 10, 15, and 20 mm) on protein separation was studied and it was found that 10% separation gels with a length of 5 mm could effectively separate prestained protein molecular weight standards (in the range of 15-250 kD) in 20 minutes. Next, the battery was optimized for the portable GE device and a 25 V lithium battery (70 mm×60 mm×40 mm) was used as the power supply, which could provide a constant voltage of 25 V for 100 hours during gel electrophoresis. Then, the One-Step BlueTM reagent (Biotium, USA) was used to color the separation results of the five standard proteins (carbonic anhydrase, ovalbumin, bovine serum albumin, conalbumin, ribonuclease A), and the results were recorded by mobile phone. Finally, the proposed gel electrophoresis device was compared with the commercial device. The results showed that the two devices are comparable; however, the slab gel electrophoresis was faster, portable, and economical. In summary, this research designed and manufactured a portable gel electrophoresis device using 3D printing technique, which can be used for on-site analysis and detection of proteins. The device presents the following advantages compared with the commercial devices:1) small and portable:the size of the electrophoresis tank of the device is only 15 mm×20 mm×17 mm and the 25 V lithium battery has a working time of approximately 100 hours; 2) low cost:it can be processed in 5 hours using 3D printing technology, with 10 mL of printing material while the total cost is less than 400 RMB; 3) fast separation:this device can quickly achieve protein separation compared with commercial devices and can further use multiple electrophoresis tanks in parallel to analyze more samples at the same time. Besides, this research also highlights the advantages of 3D printing for the development of miniaturized analytical equipment. Though this study has achieved preliminary results for rapid separation of proteins using gel electrophoresis devices, the quantitative analysis of proteins following protein detection and the application of more samples need further research. Meanwhile, the continued application of 3D printing technology will promote the development of miniaturized and portable experimental equipment.

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High-efficiency separation and analysis of monosaccharides in Pueraria polysaccharides by pressurized capillary electrochromatography Xinyao XU, Soumia CHEDDAH, Yan WANG, Chao YAN 2020, 38 (11):  1323-1331.  DOI: 10.3724/SP.J.1123.2020.01005 Abstract ( 95 )   HTML ( 14 )   PDF (1892KB) ( 43 )   Pueraria polysaccharides have been proven to possess biological activities such as bacteriostasis, anti-oxidative, anti-tumor, and immunity boosting activities. The variation in the structure, composition, and amount of monosaccharides in these polysaccharides may lead to different spatial structures and biological activities. Therefore, extraction of Pueraria polysaccharides and determination of the monosaccharide composition are of great significance for activity analysis and quality control. Direct detection of saccharides is difficult because they are strongly polar and do not show absorption in the ultraviolet region. At present, the commonly used methods for saccharide detection are liquid chromatography-ultraviolet detection, gas chromatography-ultraviolet detection, and mass spectrometry. Pressurized capillary electrochromatography is a high-efficiency microseparation technology. In this study, two kinds of Pueraria polysaccharides were extracted by an ultrasonic-assisted method, and response surface methodology was performed to explore the conditions for ultrasonic-assisted extraction of polysaccharides from Pueraria. The interaction effects of four factors, the liquid-solid ratio, ultrasonic extraction time, ultrasonic extraction temperature, and ultrasonic power, on the extraction rate of the polysaccharides were analyzed. By combining the optimal conditions predicted by the software and the actual equipment conditions, the optimal extraction conditions for Pueraria polysaccharides were chosen as follows:ultrasonic extraction temperature, 90℃; liquid-solid ratio of Pueraria thomsonii Benth , 20 mL/g; liquid-solid ratio of Pueraria lobata Ohwi, 40 mL/g; ultrasonic extraction time, 30 min; ultrasonic power, 180 W. Through data fitting, the multiple quadratic regression equation of the four factors on the extraction rate of Pueraria polysaccharides was established. A novel method based on pressurized capillary electrochromatography for the separation and analysis of eight neutral monosaccharides has been established. The monosaccharides were derivatized by the 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization method. The separation conditions for these monosaccharides were explored, and the buffer concentration, buffer pH, applied voltage, type of chromatographic column, and mobile phase ratio were optimized. Finally, the established pressurized capillary electrochromatography-ultraviolet detection method was applied to the detection and identification of two kinds of actual Pueraria polysaccharide samples. The results of response surface analysis showed that among the four experimental factors, ultrasonic extraction temperature had the greatest influence on the extraction rate of polysaccharides from the two kinds of Pueraria, followed by the liquid-solid ratio; the influence of the ultrasonic extraction time and ultrasonic power was relatively weak. The experimental conditions were determined as follows:the separation of eight neutral monosaccharide derivatives could be realized within 24 min on a Halo-2.7 μm core-shell C18 capillary column with acetonitrile-50 mmol/L ammonium acetate aqueous solution (18:82, v/v, pH 4.1) as the mobile phase, by detection at 250 nm under an applied voltage of-20 kV. The separation and detection speeds and the column efficiency achieved with this method were much better than those obtained with the traditional liquid chromatography method. The results show that the proposed method has a good linear relationship and good repeatability. The separation and identification results for the actual samples showed that the polysaccharides of Pueraria thomsonii Benth were mainly composed of glucose, mannose, rhamnose, and fucose in the molar ratio 1.00:0.16:0.14:0.07. The polysaccharides of Pueraria lobata Ohwi were mainly composed of glucose and mannose in the molar ratio 1.00:0.70. This study provides a novel method for the rapid and efficient separation and detection of neutral monosaccharides, and serves as a reference for analyzing the monosaccharide composition of Pueraria polysaccharides.

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Simultaneous and rapid determination of malachite green and leucomalachite green by a label-free colorimetric aptasensor Wenwei WU, Yi WANG, Kexin LIU, Tiansong LI, Yongjie YANG 2020, 38 (11):  1332-1339.  DOI: 10.3724/SP.J.1123.2020.04010 Abstract ( 109 )   HTML ( 13 )   PDF (3743KB) ( 61 )   A label-free colorimetric aptasensor, using a bispecific aptamer (A3) as a sensing probe, gold nanoparticles (AuNPs) as an indicator, and NaCl solution as an aggregation inducer, was successfully developed for the simultaneous, rapid and visual detection of malachite green (MG) and leucomalachite green (LMG) in aquatic products. This method is based on the aptamer A3 having bispecific binding ability with MG and LMG, making it an ideal recognition receptor for MG and LMG. It can adsorb on the AuNPs and protect AuNPs against salt-induced aggregation, maintaining the red color of the solution. When MG or LMG was added to a solution, aggregation of AuNPs was specifically induced by desorption of aptamer from the AuNPs surface upon formation of the aptamer-target complex. Therefore, the salt could trigger aggregation of AuNPs and the solution color was changed from red to blue. This color change allowed the qualitative determination of MG and LMG visually, and quantitative determination by measuring the ratio of the absorbances at 520 nm and 650 nm. In this study, 50 μL of the nucleic acid aptamer A3 (final concentrations 150 nmol/L) and 150 μL of AuNPs (final concentrations 1.25 nmol/L) were incubated at room temperature (RT) for 6 min, then 50 μL of the sample was added and incubated at RT for 30 min, and finally 50 μL NaCl solution (final concentrations 150 mmol/L) was added. After 4 min, the solution color change was observed, and the absorbances at 520 nm and 650 nm were measured. Under the optimal conditions, MG and LMG could be detected specifically without any cross-reactivity with sulfadiazine (SDZ) and nitrofurantoin (NFT). The absorbance were related to the concentrations of MG and LMG, and a good linear relationship was obtained in the range of 0-17.5 μmol/L. The correlation coefficients (R 2 ) were 0.9938 and 0.9715, respectively. The limits of detection of MG and LMG were 6.93 nmol/L and 6.38 nmol/L, respectively. The spiked recoveries of MG and LMG ranged from 88.60% to 93.30% and 101.80% to 107.00%, respectively. The relative standard deviations (RSDs) of MG and LMG ranged from 2.27% to 3.55% and 2.62% to 3.75%, respectively. This colorimetric method is simple, rapid, sensitive, and allows visual, and it can provide a new method for the simultaneous and rapid determination of the MG and LMG in aquatic products.

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Determination of cholesterol and vitamin E isomers in milk powder by gas chromatography-tandem mass spectrometry coupled with rapid saponification Lu YING, Shufen ZHANG, Jiali XING, Yang LI, Xiaobao WANG, Xiaoli BI, Xinda ZHOU 2020, 38 (11):  1340-1347.  DOI: 10.3724/SP.J.1123.2020.04013 Abstract ( 124 )   HTML ( 21 )   PDF (1328KB) ( 53 )   Cholesterol and tocopherols, which are important quality indicators in milk powder, are essential nutrients for the human body. Current pretreatment methods for the detection of cholesterol and four isomers of vitamin E (α -tocopherol, β -tocopherol, γ -tocopherol, and δ -tocopherol) are based on national food safety standards, which are complicated, time-consuming, and unsuited for simultaneous measurements. Thus, developing a simple, fast, and simultaneous detection method for cholesterol and the four kinds of tocopherols is of practical significance. In this study, gas chromatography-tandem mass spectrometry (GC-MS/MS) was used to establish qualitative and quantitative methods for the determination of cholesterol and the above mentioned four isomers of vitamin E. The sample was digested with lipase and then saponified rapidly using a potassium carbonate-ethanol system. The optimal pretreatment method was established by optimizing the enzymolysis time, saponification temperature, type and volume of the extraction solvent, and extraction time. Then, cholesterol and the four tocopherols in milk power were simultaneously determined. The results revealed a good linear relationship for cholesterol and the tocopherols in the range of 0.5-50.0 mg/L and 0.25-25.0 mg/L, respectively. The correlation coefficients (r 2 ) were greater than 0.99; the recoveries were 76.6%-93.1%; and the relative standard deviations were 0.9%-3.3%. The limits of quantification for cholesterol and the tocopherols were 10.0 μg/100 g and 5.0 μg/100 g, respectively. The recoveries of the added standards did not fully reflect the ability of the method to decompose and extract the actual sample, especially given that the five compounds considered in this study were fat-soluble. Thus, the added standard recovery could not verify the enzymatic hydrolysis effect. In order to investigate the effectiveness of this method for actual milk powder samples, the amounts of cholesterol and the four tocopherols in infant milk powder were determined according to the national standard methods (GB 5009.82-2016, GB 5009.128-2016) and the proposed method. For each method, six sets of measurements were carried out in parallel. The cholesterol content measured by this method was slightly lower than that measured by the national standard method, while the amounts of the four tocopherols were slightly higher. There was no significant difference (p > 0.05) between the national standard method and our method on the amounts of cholesterol and the four tocopherols in milk powder. Twenty kinds of infant formula milk powder and four kinds of low-fat milk powder were randomly selected from the market, and the amounts of cholesterol and the four tocopherols were analyzed. The results showed that the amounts of cholesterol and the four tocopherols in the infant formula milk powder were higher than those in the low-fat milk powder. This method is simple, fast, sensitive, and accurate, thus meeting the detection requirements for cholesterol and tocopherols in milk powder. The findings of the study would provide a theoretical foundation for the rapid estimation of milk powder quality.

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Simultaneous determination of four opioids in urine by solid-phase extraction and derivatization coupled with gas chromatography-mass spectrometry Hang GAO, Yaxun LIU, Wei KE, Kai LIU, Liuyang NI, Tao TAO 2020, 38 (11):  1348-1354.  DOI: 10.3724/SP.J.1123.2020.06002 Abstract ( 89 )   HTML ( 18 )   PDF (2670KB) ( 49 )   Police officers currently use the colloidal gold rapid testing method to detect heroin in the urine of drug abusers, but the results are often rendered erroneous due to the presence of antitussive drugs, which contain opioids. The traditional manual liquid-liquid extraction method for urine testing has low efficiency and poor sensitivity, and hence, it fails to meet the requirements of the public security department to crack down on drug abusers. Therefore, to avoid punishment, most rapid-test-positive people make false claims about intaking cough suppressants. It is imperative to establish a highly efficient automatic method for the simultaneous determination of multiple opioids in urine, to rule out the use of heroin. A method based on solid-phase extraction and derivatization coupled with gas chromatography-mass spectrometry (GC-MS) has been developed for the simultaneous detection of morphine, O 6 -acetylmorphine, codeine, and acetyl codeine in urine. Since these four opioids exists as cations in acidic aqueous solution, the urine samples collected from dead bodies or drug addicts were adjusted to pH 6 by using phosphate buffer, enriched, and purified by MCX-SPE columns. Then, morphine, O 6 -acetylmorphine, and codeine were derivatized by N -methyl-N -(trimethylsilyl) trifluoroacetamide (MSTFA) for GC-MS testing. The effects of sample loading and elution flow rate, percentage of formic acid in the wash solvent (methanol), percentage of ammonia in the eluent (methanol), volume of the wash solvent, and drying time of the cartridge on the extraction efficiency were investigated in detail. The best results were obtained under the following conditions:sample loading and elution flow rate, 1.0 mL/min; volume fraction of formic acid in the wash solvent, 3%; volume fraction of ammonia in the eluent solvent, 5%; volume of 3% (v/v) formic acid in methanol (eluent), 1 mL; and drying time of the cartridge, 1 min. The GC-MS results showed good linearity in the range of 0.02-0.8 μg/mL with correlation coefficients (r 2 ) ≥ 0.998. The limits of detection (LODs) and limits of quantification (LOQs) were 0.0016-0.0039 μg/mL and 0.0054-0.0128 μg/mL, respectively. The recoveries of the target analytes were between 93.0% and 110.3% at spiked levels of 0.02, 0.1, and 0.2 μg/mL. As opposed to similar reported methods, our method showed high sensitivity and recovery; furthermore, the matrix interference was eliminated, and the chromatographic peaks of the analytes were completely separated from the impurity peaks at the level of 0.2 μg/mL. The automatic solid-phase extraction equipment is convenient to operate and allows one to process samples in batches. The conditions for solid-phase extraction can be precisely controlled, and the detection accuracy is greatly improved. In addition, a large number of sample tests can be performed by a few experimenters. Hence, this method facilitates simple and rapid forensic toxicology testing and drug abuse monitoring on a large scale.