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Chinese Journal of Chromatography
2012, Vol. 30, No. 11
Online: 28 November 2012

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A novel method for the identification of illegal cooking oil (1): detection of three capsaicinoids with liquid chromatography-mass spectrometry 2012, 30 (11):  1094-1099.  DOI: 10.3724/SP.J.1123.2012.08051 Abstract ( 1950 )   [Full Text(HTML)] () PDF (836KB) ( 686 )   Illegal cooking oil (ICO, also named swill-cooked dirty oil) has recently become a serious food safety problem in China. Now, the identification method of ICO is also a hot research area. Owning to the special eating habits of Chinese people, cayenne is widely used in catering business. Capsaicinoids are main spicy compounds in cayenne. So, they are potential evaluation indices for the identification of ICO. In this study, a solid phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS) method has been developed to detect the trace residues of three capsaicinoids (capsaicin, dihydrocapsaicin and nonylic acid vanillylamide) in cooking oil. The oil sample was first extracted with 20 g/L sodium hydroxide, the C18 SPE cartridge was then used to clean-up the sample and enrich the analytes before the liquid chromatography-mass spectrometry (LC-MS) detection. With this method, sixty seven blind samples provided by China National Center for Food Safety Risk Assessment were analyzed. The results showed that the capsaicinoids are good evaluation indices for the identification of ICO. In all the 48 ICO samples, 36 samples were successfully recognized. All the 19 normal oil samples were accurately identified. This method has been chosen and authorized as one of the four standard instrumental identification methods for ICO by the National Ministry of Health of China.

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A novel method for the identification of illegal cooking oil (2): determination of special odd-chain fatty acids by multidimensional gas chromatography-mass spectrometry JIN Jing, WANG Longxing, CHEN Jiping*, TIAN Yuzeng, ZOU Lili, ZHANG Baoqin, WANG Shuqiu, WANG Xingfu 2012, 30 (11):  1100-1107.  DOI: 10.3724/SP.J.1123.2012.08052 Abstract ( 1763 )   [Full Text(HTML)] () PDF (1251KB) ( 681 )   Endogenesis referents from illegal cooking oil (ICO), namely, 13-methyl-tetradecanoic acid and undecanoic acid have been confirmed via non-target and target screening of fatty acids. The former is mainly originated from animal oil, and the later from heated vegetable oil. Based on the retention interactions between gas chromatographic columns with different polarities and alkyl acids, the alkyl acids with various carbon chain or their isomers with the same carbon chains can be separated effectively using multidimensional gas chromatography-mass spectrometry. And the accurate quantification of 13-methyl-tetradecanoic acid and undecanoic acid was obtained. The target compounds can be cleaned up online and enriched with the developed method. Subsequently, the fourth and fifth assessments organized by China National Center for Food Safety Risk Assessment were performed in our laboratory. After continuous improvement, the precision of the method was increased. In detail, 100% vegetable oils, 71% ICO (the 4th batch) and 75% ICO (the 5th batch) have been identified. In combination with the capsaicinoid contents, edible oils can be identified in a comprehensive way, and the precision for ICO samples was increased to 89% and 100% for the 4th batch and the 5th batch samples, respectively. Owing to the aforementioned advantages, the developed method has been chosen as one of the four instrumental methods for the identification of ICO by China Ministry of Public Health, waiting for the validation from authoritative departments.

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Determination of capsaicinoids and eugenol in waste-edible-oil by liquid-liquid extraction and liquid chromatography-tandem mass spectrometry ZHANG Zhong*, REN Fei, ZHANG Pan 2012, 30 (11):  1108-1112.  DOI: 10.3724/SP.J.1123.2012.08054 Abstract ( 2013 )   [Full Text(HTML)] () PDF (818KB) ( 660 )   A method was developed for the determination of capsaicinoids (capsaicin, dihydrocapsaicin and synthetic capsaicin) and eugenol in waste-edible-oil extracted by liquid-liquid extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The capsaicinoids and eugenol in waste-edible-oil were extracted by methanol, and then separated by a SUPEL COSIL ABZ+Plus dC18 column (150 mm×4.6 mm, 5 μm). The analysis was performed by MS/MS with electrospray ionization in positive and negative ion modes with multiple reaction monitoring (MRM). The limits of detection for capsaicin, dihydrocapsaicin, synthetic capsaicin and eugenol were 0.02, 0.03, 0.03 and 0.6 μg/L, respectively. The good linear relationships were obtained in certain concentration ranges of capsaicinoids and eugenol. The relative standard deviations (RSDs, n=5) of same-worker and different-worker were less than 5%. The method is exclusive, sensitive and accurate, and can be used in waste-edible-oil determination.

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Determination of sodium and chloride in hogwash oil and their molar ratio by ion chromatography ZHANG Zhong1*, WANG Lichun1, LU Yuntian2 2012, 30 (11):  1113-1116.  DOI: 10.3724/SP.J.1123.2012.08053 Abstract ( 1745 )   [Full Text(HTML)] () PDF (810KB) ( 526 )   By reference to edible oil using process as well as hogwash oil refining technology, a method is presented to determine the contents of the sodium and chloride in hogwash oil based on ion chromatography. The molar ratio of the sodium and chloride was analyzed in order to determine whether the sample contained hogwash oil. A hogwash oil sample was extracted by deionized water before analysis. The ion chromatographic separation of the chloride was carried out on an AS19 column (250 mm×4 mm) at 30 ℃, using 20 nmol/L KOH solution as mobile phase at a flow rate of 1 mL/min and suppressor current of 112 mA. The ion chromatographic separation of the sodium was carried out on a CS12 column (250 mm×4 mm) at 30 ℃, using 20 nmol/L methanesulfonic acid (MSA) as mobile phase at a flow rate of 1 mL/min and suppressor current of 59 mA. The injection volume was 25 μL and the detector was an electron capture detector (ECD). The external standard method was used to quantify chloride and sodium. The detection limits of this method were 0.005 mg/L for chloride and 0.001 mg/L for sodium. The linear range was from 0 to 5 mg/L with r2=0.999988 for chloride and r2=0.999926 for sodium. The average recoveries and relative standard deviations were 94.2% and 2.4% for chloride and 92.5% and 2.7% for sodium, respectively. The molar ratio of sodium and chloride in edible oil was approximately 1, while that in hogwash oil was more than 4. The determination of the contents and molar ratio of the chloride and sodium in hogwash oil can be used as an important basis for the judgment of hogwash oil.

Reviews

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Recent advances in the application of high performance capillary electrophoresis for food safety DONG Yalei, CHEN Xiaojiao, HU Jing, CHEN Xingguo* 2012, 30 (11):  1117-1126.  DOI: 10.3724/SP.J.1123.2012.07009 Abstract ( 1653 )   [Full Text(HTML)] () PDF (1931KB) ( 605 )   In recent years, food safety incidents become a serious social problem. Foods are usually complex mixtures consisting of a large diversity of molecules. Analysis of foods is a topic that demands the development of rapid, robust, efficient, sensitive and cost-effective analytical methodologies. Therefore, new techniques for food safety purpose are required by analytical chemists. Capillary electrophoresis (CE) is a popular separation technique that possesses fast and efficient performances in an automated way with minimum consumption of sample and reagents. Nowadays, CE represents a desired strategy for the determination of many compounds or molecules in various kinds of food. In this paper, the review intends to provide the recent innovative developments reported in food safety analysis using CE methods for a full overview. As a fundamental review, it focuses on the introduction and detection of several common hazardous materials existing in food such as non-food additives, pesticide residues, veterinary drug residues, heavy metal ion contaminants, toxins, biphenol A and phthalates in packaging materials and so on. Furthermore, this review prospects the main development direction of CE in this field for the future. A total of 63 papers published during the period of Jan 2009~Jun 2012 are included in the present review.

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An enzyme reactor based on aptamer modified microfluidic chip for protein analysis XIAO Peng, LI Dalei, MAN Yan, GENG Lina, L Xuefei, DENG Yulin* 2012, 30 (11):  1127-1132.  DOI: 10.3724/SP.J.1123.2012.07019 Abstract ( 1850 )   [Full Text(HTML)] () PDF (1674KB) ( 428 )   As a kind of recognition molecule, aptamer has been studied and applied widely in numerous science fields in recent years. Immobilized enzymatic reactor has drawn much attention because of its striking advantages, such as high digestion efficiency and ease in coupling with the separation and detection systems. In this study, a novel microfluidic enzymatic chip, which immobilized trypsin based on aptamer, was prepared and proposed. An online analysis platform, which consisted of an aptamer-based chip and high performance liquid chromatography tandem mass spectrometry, was established by using a 6-port valve and applied to protein analysis. The enzymatic capacity and stability performance of chip reactor were characterized by using mixed protein sample, which consisted of bovine serum albumin (BSA), myoglobin (Mb) and cytochrome c (Cyt.c). The sample digestion time of the chip reactor was about 5.76 s while 1 μL/min of flow rate was adopted; and moreover, 5 ng of Mb was identified successfully with the sequence coverage of 37%. Furthermore, the sequence coverages and the relative standard deviations were 44.3% and 6.5% for BSA, 65.0% and 2.7% for Mb, 62.0% and 5.6% for Cyt.c respectively when 500 ng digest of mixed proteins were analyzed in three runs. According to experimental results, the online analysis platform possesses the ability of high sensitivity and good stability, which can provide a promising tool for rapid and high-throughput proteomics study in the near future.

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Preparation of a stir bar sorptive extraction coating based on molecularly imprinted polymer and its application in the extraction of dienestrol and hexestrol in complicated samples NONG Shuyu, LIN Fuhua, HUANG Xiaojia*, YUAN Dongxing 2012, 30 (11):  1133-1142.  DOI: 10.3724/SP.J.1123.2012.08034 Abstract ( 1752 )   [Full Text(HTML)] () PDF (2510KB) ( 437 )   A new stir bar sorptive extraction (SBSE) coating based on molecularly imprinted polymer (MIP) with diethylstilbestrol as replaced template molecule was prepared. The influences of the contents of template molecule and monomer in the polymerization mixture on the extraction performance of MIP-SBSE were investigated thoroughly. The MIP was characterized by elemental analysis, scanning electron microscopy and infrared spectroscopy. In order to evaluate the usability of the new coating, the MIP-SBSE was combined with high performance liquid chromatography (HPLC) and diode array detector (DAD) with dienestrol (DS) and hexestrol (HS) as detected solutes. To achieve optimally selective extraction performance for DS and HS, several parameters, including extraction and desorption times, desorption solvent, ionic strength and pH value in sample matrix were investigated. The results showed that under the optimized experimental conditions, the present method has high selectivity and sensitivity. When drying-redissolving procedure was taken during sample preparation, the limits of detection for DS and HS were as low as 0.04 μg/L and 0.14 μg/L, respectively. Good linearities were obtained for analytes with the correlation coefficients (R2) above 0.99. Finally, the proposed method was successfully applied to the determination of DS and HS in wastewater, honey and cow urine samples. The recoveries of spiked target compounds in real samples ranged from 61.3% to 120%. The developed method is simple, selective, sensitive and applicable for the analysis of trace DS and HS in complicated samples.

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Determination of ten aminoglycoside residues in milk and dairy products using high performance liquid chromatography-tandem mass spectrometry GONG Qiang, DING Li, ZHU Shaohua, JIAO Yanna, CHENG Jing, FU Shanliang, WANG Libing* 2012, 30 (11):  1143-1147.  DOI: 10.3724/SP.J.1123.2012.06024 Abstract ( 1896 )   [Full Text(HTML)] () PDF (830KB) ( 672 )   A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analytical method was developed for the simultaneous determination of ten aminoglycoside residues (streptomycin, dihydrostrepmycin, neomycin, kanamycin, tobramycin, gentamycin, apramycin, hygromycin B, paromomycin, and amkacin) in milk and dairy products. The sample was extracted with 5% trichloroacetic acid aqueous solution, then the extract was purified by a hydrophilic-lipophilic balance (HLB) cartridge. The ten aminoglycoside residues were separated by ion-pair reversed phase high performance liquid chromatography. Heptafluorobutyric acid was used as ion pair agent due to its volatility. Then the analytes were detected by electrospray ionization tandem mass spectrometry. The pretreatment condition of the sample, the HPLC condition and the MS operation parameters were optimized. The results showed that the linearities of the ten aminoglycoside residues in 20~1000 μg/L had the correlation coefficient between 0.9946~0.9997. The recoveries ranged from 71.2% and 101.7% with the relative standard deviations of 3.4%~13.8%. The proposed method was successfully applied to the determination of the mass concentrations of the analytes in related samples, which provides a simple, and convenient method for the quality control of milk and dairy products. Furthermore, this method is effective for the safety monitoring of aminoglycoside residues in milk and dairy products.

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Determination of three drugs and their metabolites in saliva by ultra performance liquid chromatography-tandem mass spectrometry CHEN Yue1, ZHU Jun2, YU Zhongshan2, ZHANG Yunfeng2, LIU Yao2* 2012, 30 (11):  1148-1152.  DOI: 10.3724/SP.J.1123.2012.07015 Abstract ( 1542 )   [Full Text(HTML)] () PDF (837KB) ( 480 )   An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the methamphetamine, morphine and O6-acetylmorphine in saliva. The sample was extracted and the protein was precipitated with acetonitrile. The matrix-matched standard solutions were used to prepare the curve of quantitative analysis. The drugs were separated on a BEH HILIC UPLC column. The mass spectrometric acquisition was carried out by means of electrospray ionization in positive mode (ESI+) with multiple reaction monitoring (MRM) method. The isotope internal standards were used to check the drugs. The average recoveries at four levels of 10, 20, 50 and 100 μg/L ranged from (68.7±6.5)% to (110.8±4.6)%. The intraday precisions were lower than 16.5% and interday precisions were lower than 16.3%. The detection limits (LOD, S/N＞3) and the quantification limits (LOQ, S/N＞10) of the three drugs were 0.02~0.05 μg/L and 0.1~0.2 μg/L, respectively. The saliva matrix effect was investigated. The method is rapid, simple, accurate and highly sensitive. The qualitative analysis and quantitative analysis of the collected saliva samples for the drugs can be finished within one hour, which is conducive to the rapid identification of the suspected drug addicts.

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Simultaneous determination of nine effective components in Jiaweizuojin Pills by ultra performance liquid chromatography with tandem mass spectrometry QIN Sha1,2, WANG Jin2, XU Yuanjin1,2* 2012, 30 (11):  1153-1158.  DOI: 10.3724/SP.J.1123.2012.06040 Abstract ( 1391 )   [Full Text(HTML)] () PDF (857KB) ( 402 )   A method for the simultaneous determination of paeoniflorin, tetrahydropalmatine, jatrorrhizine, berberine, palmatine, evodiamine, saikosaponin C, saikosaponin A and saikosaponin D in Jiaweizuojin Pills was established by ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The UPLC separation was performed on a Zorbax RRHD Eclipse Plus C18 column (50 mm×2.1 mm, 1.8 μm) by using 0.2% (v/v) formic acid aqueous solution and methanol as mobile phases with the gradient elution at a flow rate of 0.4 mL/min. The analytes were detected by tandem mass spectrometry under the positive ion mode with the electrospray ionization (ESI) source and in the multiple reaction monitoring (MRM) mode. Under the optimized conditions, the calibration curves were linear in the ranges of 0.025~5.0 mg/L for paeoniflorin, 0.0010~2.0 mg/L for tetrahydropalmatine, 0.0023~7.2 mg/L for jatrorrhizine, 0.0027~28.9 mg/L for berberine, 0.0023~9.1 mg/L for palmatine, 0.0050~1.0 mg/L for evodiamine, 0.050~10 mg/L for saikosaponin C, 0.0050~1.0 mg/L for saikosaponin A and 0.0075~1.5 mg/L for saikosaponin D with the detection limits of 5.0, 0.20, 0.45, 0.54, 0.45, 1.0, 10, 1.0, 1.5 μg/L, respectively. The average recoveries of the nine effective components were between 99.3% and 105% with the relative standard deviations not more than 2.6%. The developed method is simple, rapid and accurate, and suitable for the quality control of the nine effective components in Jiaweizuojin Pills.

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Determination of multi-pesticides in black tea by subcritical water extraction and gas chromatography-tandem mass spectrometry PAN Yuchen1, YI Xionghai2*, DENG Xiaojun2, ZHAO Shanzhen2, CHEN Shunsheng1, YANG Huiqin2, HAN Li2, ZHU Jian2 2012, 30 (11):  1159-1165.  DOI: 10.3724/SP.J.1123.2012.06033 Abstract ( 1508 )   [Full Text(HTML)] () PDF (878KB) ( 458 )   A gas chromatography-tandem mass spectrometry (GC-MS/MS) method with subcritical water extraction was developed for the determination of 21 organochlorine and pyrethroid pesticides in black tea. Under the extraction pressure of 5 MPa, the target compounds were extracted with subcritical water at the temperature of 150 ℃ for 15 min, transferred into acetone-n-hexane (1:1, v/v), and cleaned-up by an ENVI-Carb solid phase extraction (SPE) column. The GC separation was performed on a DB-5 capillary column. The pesticides were determined by MS/MS in multiple reaction monitoring (MRM) mode and quantified by matrix-matched internal standard method. The calibration curves showed good linearities in the range of 5.0~320.0 μg/L with the correlation coefficients greater than 0.99. The limit of quantification (S/N＞10) was 50 ng/g, and the limit of detection (S/N＞3) was 10 ng/g. The recoveries of pesticides spiked in the tea at three levels of 50, 100 and 200 ng/g were ranged from 70.18% to 119.98% with the relative standard deviations (RSDs) of 5.01%~11.76%. The sensitivity, accuracy and precision of the method meet the technical standard of the pesticide determination. The method can be applied to the determination of organochlorine and pyrethroid pesticides in black tea.

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Determination of fatty acids in vegetable oils using comprehensive two-dimensional gas chromatography coupled to quadropole mass spectrometry ZHENG Yueming1,2, FENG Feng2, GUO Wei2, CHU Xiaogang2, PAN Jiarong1,3*, JIA Wei2 2012, 30 (11):  1166-1171.  DOI: 10.3724/SP.J.1123.2012.06041 Abstract ( 1554 )   [Full Text(HTML)] () PDF (1315KB) ( 406 )   Comprehensive two-dimensional gas chromatography with quadropole mass spectrometry (GC×GC-qMS) was applied to the detection of 31 fatty acids in vegetable oils. The sets of columns, modulation period, scan range of quadropole mass spectrometer were optimized. The results demonstrated that the separation was achieved in 50 min with the column set of DB-1 (30 m×0.25 mm×0.25 μm) as the 1st column and DB-Wax (3.2 m×0.1 mm×0.1 μm) as the 2nd column. All fatty acids were accurately and sensitively determined while the modulation period was 3.5 s and the scan range of quadropole MS was m/z 40~350. Most of the fatty acids were identified by NIST library spectra search, the other fatty acid isomers were identified by single standard injection analysis. When applying this method to the real vegetable oil samples, not only the sensitivities were 100 times higher than those obtained with GC-qMS methods, but also some minor fatty acids were identified. This work suggested a new technical approach in analyzing fatty acid components in vegetable oils, which is meaningful to prohibit adulteration and ensuring the quality safety of edible vegetable oils.

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Determination of 17 pyrethroid pesticide residues in vegetables by gas chromatography-mass spectrometry with negative chemical ionization SHEN Weijian1*, CAO Xiaowen2, LIU Yijun1, ZHANG Rui1, FAN Xin1, ZHAO Zengyun1, SHEN Chongyu1, WU Bin1 2012, 30 (11):  1172-1177.  DOI: 10.3724/SP.J.1123.2012.07044 Abstract ( 2322 )   [Full Text(HTML)] () PDF (858KB) ( 663 )   A method was established for the determination of 17 pyrethroid pesticide residues in vegetables using QuEChERS (quick, easy, cheap, effective, rugged and safe) clean-up method and gas chromatography-mass spectrometry (GC-MS) with negative chemical ionization (NCI). The pyrethroid pesticides in the sample were extracted with acetonitrile. After QuEChERS clean-up with a mixture of primary secondary amine and graphitized carbon black packings, the extract was analyzed by GC-NCI-MS in selected ion monitoring (SIM) mode. An isotope internal standard of cypermethrin was employed to the quantification. The limits of quantification ranged from 0.02 to 5 μg/kg. The recoveries of the pyrethroid pesticides spiked in three different matrixes (peas, broccoli and Chinese onion green) at four spiked levels of 10, 20, 30 and 100 μg/kg were from 71.0% to 139.0%, and the relative standard deviations were less than 12.8%. This method can be used as a conclusive evidence method of the 17 pyrethroid pesticide residues in vegetables.

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Determination of synthetic nitro-musks in cosmetics by gas chromatography coupled with negative chemical ionization-triple quadrupole mass spectrometry WANG Zheng* 2012, 30 (11):  1178-1182.  DOI: 10.3724/SP.J.1123.2012.07003 Abstract ( 1504 )   [Full Text(HTML)] () PDF (833KB) ( 438 )   A confirmatory method is presented for the determination of five nitro-musks (musk ambrette, musk xylene, musk moskene, musk tibeten and musk ketone) in different cosmetics by gas chromatography coupled with negative chemical ionization (NCI)-triple quadrupole mass spectrometry (GC-MS/MS). The samples were extracted under ultrasonication using a mixture of acetone and hexane. The extract was concentrated and then cleaned up by CNWBOND Si solid phase extraction cartridge. Five different instrument parameters such as the temperature programmed, ion source temperature, reagent gas pressure, collision energy, monitoring ion pairs were optimized for higher sensitivity. Then the analytes were qualitatively and quantitatively analyzed under the multiple reaction monitoring (MRM) mode after the chromatographic separation on an HP-5MS capillary column (30 m×0.25 mm, 0.25 μm), and employing d15-musk xylene as internal standard. The mixed standards were spiked in the blank cosmetics samples (each nitro-musk was about 500 ng/kg), and the recoveries were in the range of 85.81%~103.77% with the relative standard deviations (RSDs) not more than 5.32%. The limits of quantification of the method were about 50.0~500 ng/kg. The method is accurate, rapid, sensitive and can be used in the inspection of the five nitro-musks in cosmetics.

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Simultaneous determination of eight derivatives of propranolol in cornea perfusate in vitro by high performance liquid chromatography WU Haitao1*, CHEN Chuanbing2, WANG Ningsheng1, MI Suiqing1, LIAO Nanying1 2012, 30 (11):  1183-1187.  DOI: 10.3724/SP.J.1123.2012.06039 Abstract ( 1151 )   [Full Text(HTML)] () PDF (1123KB) ( 332 )   A high performance liquid chromatographic method was developed for the simultaneous determination of eight derivatives of propranolol. Cassette dosing method was used in the epithelium side of cornea in vitro to get the effect of penetrant, and the perfusate was collected in the side of endothelium. The protein in the sample was precipitated and discarded by high speed centrifugation before injection. An Agilent Zorbax Extend column (150 mm×3 mm, 5 μm) was used at 30 ℃. The mobile phase system contained acetonitrile and 0.03% (v/v) phosphoric acid aqueous solution and the percentage of acetonitrile changed between 3% and 20% (v/v) in a linear gradient elution. The samples were detected by an ultraviolet (UV) detector at 205 nm. The results showed that the eight derivatives of propranolol were completely separated and determined in 31 min. The correlation coefficients were above 0.9970 and good linear relationships were obtained in the range of 0.2(0.1)~40.0 μmol/L. Under the optimized conditions, the recoveries of the derivatives were in the range of 91.12%~105.73%. The intra-day relative standard deviations (RSDs) were in the range of 1.00%~11.63%, and the inter-day RSDs were in the range of 1.18%~18.58%. The sample showed stability under room temperature, freeze and three cycles of freeze-thaw conditions. This method is fast and accurate for the quantitative analysis of the derivatives of propranolol in transmembrane absorption such as cornea perfusion in vitro or transwell cell system.

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Separation of benzoxazine enantiomers on β-cyclodextrin bonded chiral stationary phases XU Xuefeng, GUO Zhimou*, LIANG Xinmiao 2012, 30 (11):  1188-1193.  DOI: 10.3724/SP.J.1123.2012.07011 Abstract ( 1606 )   [Full Text(HTML)] () PDF (869KB) ( 393 )   Chiral separation of benzoxazine enantiomers was studied on click chemistry based beta-cyclodextrin stationary phases under reversed-phase high performance liquid chromatographic mode. The effects of the type and percentage of the organic modifier, the concentration of triethylammonium acetate buffer (TEAA), and pH on enantioselective separation were examined and studied. It was demonstrated that acetonitrile was better for the chiral separation of benzoxazine enantiomers than methanol. It was observed that the retention time and the resolution of benzoxazine enantiomers decreased with the increase of the volume ratio of TEAA from 0.1% to 1.0%. The separation of benzoxazine enantiomers was of the maximum resolution at pH 4.1. With the optimized mobile phase of the mixture of acetonitrile and 0.1% TEAA (pH 4.1), all the enantiomers were separated at the baseline. The chiral recognition mechanism is also discussed. The separation was probably based on the inclusion complex interaction and the hydrogen bonding between enantiomers and chiral stationary phases. This work provided the experience for the intensive study of click beta-cyclodextrin bonded stationary phases, and it also illustrated the potential of click chemistry in the preparation of beta-cyclodextrin based chiral stationary phase.

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Adsorption of endotoxins on Ca2+ iminodiacetic acid by metal ion affinity chromatography André Moreni LOPES1*, Jorge Sánchez ROMEU2#, Rolando Páez MEIRELES2, Gabriel Marquez PERERA2, Rolando Perdomo MORALES3, Adalberto PESSOA Jr1, Lourdes Zumalacárregui CáRDENAS4 2012, 30 (11):  1194-1202.  DOI: 10.3724/SP.J.1123.2012.06007 Abstract ( 1464 )   [Full Text(HTML)] () PDF (729KB) ( 393 )   Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin’ concentration values less than 100 EU/mL and 100000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.

Technical Notes

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Determination of deoxynivalenol in grain and its products by solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry HUANG Juan1*, CHEN Guosong2, ZHANG Xiaoyan1, SHEN Chongyu1, LV Chen2, WU Bin1, LIU Yan1, CHEN Huilan1, DING Tao1 2012, 30 (11):  1203-1207.  DOI: 10.3724/SP.J.1123.2012.06007 Abstract ( 1558 )   [Full Text(HTML)] () PDF (823KB) ( 428 )   A method was established for the determination of deoxynivalenol (vomitoxin) in grain and its products based on solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample was firstly extracted by acetonitrile-water (84:16, v/v). The extract was then cleaned-up by an HLB solid phase extraction cartridge. The separation was carried out on a Phenomenex Kinetex C18 column (100 mm×4.6 mm, 2.6 μm) with a gradient elution using 0.3‰ ammonia solution-acetonitrile as mobile phases. The analysis of deoxynivalenol was performed under electrospray negative ionization mode. The limit of detection (LOD, S/N=3) and the limit of quantification (LOQ, S/N=10) were 20 μg/kg and 50 μg/kg, respectively. A good linearity (r ＞0.99) was achieved for the target compound over the range of 20~1000 μg/L. The recoveries at the three spiked levels (50, 100, 500 μg/kg) in the blank matrices such as flour, barley, soybean, rice, cornmeal, cassava and wheat, were varied from 75.6% to 111.0% with the relative standard deviations no more than 13.0%. The method is accurate, efficient, sensitive and practical. The cost of pretreatment is obviously reduced by replacing immunoaffinity columns and Mycosep columns with HLB columns which have the same purification effect.