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Chinese Journal of Chromatography
2008, Vol. 26, No. 3
Online: 30 May 2008

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Developments of microfluidic chip-based capillary electrophoresis for protein separation DONG Yani, FANG Qun 2008, 26 (3):  269-273.  Abstract ( 3166 )   [Full Text(HTML)] () PDF (146KB) ( 1740 )   In this paper, the developments of microfluidic chip-based capillary electrophoresis (CE) for protein separation in recent years are reviewed. Various chip-based CE systems for protein separation based on different CE separation modes are introduced. The approaches to suppress the adsorption of proteins on the surface of microchannel on chips are discussed. The application prospect for chip-based CE in protein separation is also proposed. Forty-seven references are cited.

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Free flow electrophoresis and its application development QU Feng, HAN Bin, DENG Yulin, ZHANG Lihua, ZHANG Yukui 2008, 26 (3):  274-279.  Abstract ( 2742 )   [Full Text(HTML)] () PDF (363KB) ( 1356 )   The principal, separation modes, influence factors and the equipment development of free flow electrophoresis (FFE) are introduced. Its latest applications on ion, small molecule and particles separation, peptides and proteins separation, cells and organelles separation, enantiomers separation, microchip device and proteomics are reviewed with cited 73 papers.

Articles

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Simultaneous determination of 14 phthalate ester residues in animal innards by gas chromatography-mass spectrometry with electron impact ionization LIN Zhuguang, SUN Ruonan, ZHANG Lili, ZOU Ximei, CHEN Meiyu, TU Fengzhang, MA Yu, JIANG Wenjing 2008, 26 (3):  280-284.  Abstract ( 2927 )   [Full Text(HTML)] () PDF (229KB) ( 1240 )   An analytical multiresidue method was developed for the simultaneous determination of 14 phthalate esters (PAEs) in animal innards by gas chromatography-mass spectrometry with electron impact ionization (GC-EI/MS). After the optimization of different parameters such as the extraction solvent, PAEs were extracted from animal innards with hexane-dichloromethane (11, v/v) in an ultrasonic bath and cleaned up on a Florisil column, then were determined by GC-EI/MS in selected ion monitoring mode with diphenyl phthalate (DPhP) as internal standard. The recovery studies were performed at 100, 200 and 400 μg/kg levels for each PAE, and the recoveries ranged from 60%-110% with the relative standard deviations between 0.8% and 10.3% for different PAEs. The detection limit of the method was less than 1.74 μg/kg for most of PAEs except dimethoxyethyl phthalate (DMEP) and di(2-ethoxyethyl) phthalate (DEEP). The method was linear over the range of 50.0-800.0 μg/kg with the correlation coefficients larger than 0.9994. The method has been successfully applied to the determination of 14 PAEs in six animal innards.

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Rapid determination of volatile flavor compounds in soy sauce using head space solid-phase microextraction and gas chromatography-mass spectrometry YAN Liujun, ZHANG Yanfang, TAO Wenyi, WANG Liping, WU Shengfang 2008, 26 (3):  285-291.  Abstract ( 3881 )   [Full Text(HTML)] () PDF (309KB) ( 1955 )   A rapid and simple method was developed for the determination of volatile flavor compounds (VFCs) in soy sauce by head space solid-phase microextraction (HS-SPME) coupled to capillary gas chromatography-mass spectrometry (GC-MS). Five types of SPME fibers, including 85 μm PA, 100 μm PDMS, 75 μm CAR/PDMS, 65 μm PDMS/DVB, 50 μm DVB/CAR/PDMS were investigated. Three parameters for HS-SPME in terms of adsorption time, salt concentration, and extraction temperature were optimized. Adsorption time tested in this study were 20, 40 and 60 minutes; the salt concentrations were 180, 210, 250, 270 and 300 g/L; and extraction temperatures were 25, 35, 45, 55 and 65 ℃. The concentrations of the compounds were calculated based on their relative peak areas to the internal standard of 2-octanol. An 85 μm PA fiber, adsorption time of 40 min, a temperature of 45 ℃ and NaCl concentration of 250 g/L were selected as th optimum conditions. This optimized method was applied to evaluate a real sample. As a result, 97 compounds in a soy sauce sample were isolated and identified successfully. The results showed that alcohols, carboxylic acids, esters and phenols were the major VFCs of soy sauce. The most important groups of volatile compounds in the soy sauce sample were ethanol, hexadecanoic acid, phenylethyl alcohol and 2,3-butanediol. In addition, some oxo-compounds and heterocyclic compounds were also found. The average relative standard deviation of the relative peak area was 12.1%, and the recoveries were 79.9%-109.6%. The method is simple, fast and accurate with high reproducibility, high sensitivity and low cost.

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Two sample pretreatment methods and their applications in the determination of 111 pesticides and related chemicals in aquatic products SU Jianfeng, LIN Guyuan, LIAN Wenhao, ZHANG Jinhu, CHEN Donghua 2008, 26 (3):  292-300.  Abstract ( 3670 )   [Full Text(HTML)] () PDF (420KB) ( 1697 )   Two sample pretreatment methods were developed and applied to determine 111 residues of pesticides in aquatic products. For Method Ⅰ, the residues were extracted from homogenized tissue with acetone-ethylacetate-n-hexane (1∶1∶1, v/v/v), and then were redissolved in acetonitrile; for Method Ⅱ, the residues were extracted from homogenized tissue with acetonitrile-water under ultrasonication, and then were separated by liquid-liquid partition. The supernatants were purified using solid phase extraction (Envi-18 and PSA columns) prior to the GC-MS analysis. The determination was performed in selected ion monitoring (SIM) mode with the external calibration for quantitative analysis. Under the optimal conditions, the detection limits (S/N=3) for residues of pesticides were in the ranges of 0.001-0.026 mg/kg. The recoveries of Method Ⅰ for the spiked standards at concentration of 0.25 mg/kg were 72%-113% with the relative standard deviations (RSDs) of 3.4%-12.1%. The recoveries of Method Ⅱ were 51%-127%, among which the recoveries for 94% pesticides were between 70% and 120% with the RSDs of 3.2%-13.8%.

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Analysis of volatile components in Qingshanlvshui Tea using solid-phase microextraction/accelerated solvent extraction-gas chromatography-mass spectrometry ZHAN Jiafen, LU Sheming ， MENG Zhaoyu， XIANG Nengjun，CAO Qiu’e， MIAO Mingming 2008, 26 (3):  301-305.  Abstract ( 3452 )   [Full Text(HTML)] () PDF (270KB) ( 1771 )   The volatile components of Qingshanlvshui Tea were extracted using solid phase micro-extraction (SPME) and accelerated solvent extraction (ASE), and then were identified by gas chromatography-mass spectrometry (GC-MS). It showed that ninety-one compounds were identified, including forty-nine by SPME, fifty-six by ASE, and fourteen by both of them. The main constituents were β-myrcene, 3,5,5-trimethyl-1,5-heptadiene, L-limonene, α-ocimene, β-ocimene, β-pinene, 2-methylbenzaldehyde, 5-(hydroxymethyl)-2-furfural. Both SPME and ASE have their advantages. SPME is excellent at simplicity, rapidity, solvent-free, high enrichment, low detection limit, environment friendly etc. ASE has characteristics of time and solvent saving, automation, simplicity, as well as high efficiency.

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Application of single drop microextraction in the determination of phthalate esters and parabens in drugs by gas chromatography-mass spectrometry QI Aiming, LI Meigui, MAO Liqiu 2008, 26 (3):  306-309.  Abstract ( 3048 )   [Full Text(HTML)] () PDF (1511KB) ( 1340 )   Single drop microextraction (SDME) was used for the determination of phthalate esters and parabens in drugs by gas chromatography-ion trap mass spectrometry (GC-IT/MS). The effects of the nature of extraction solvents, microdrop volume, the depth of microdrop in sample solution, extraction time and stirring rate on the extraction efficiency were investigated separately. The optimal SDME conditions, 1.5 μL of toluene, 0.8 cm of the depth of microdrop, 1000 r/min of stirring rate and 20 min of extraction time, were obtained and used for the analysis of methylparaben (MP), ethylparaben (EP), propylparaben (PP), iso-propylparaben (IPP), butylparaben (BP), dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP) in drugs. The results showed that the working curves for 8 phthalate esters and parabens were linear in the range of 0.032-80 mg/L by GC-MS on selective ion storage mode. The limits of detection (LOD) were between 0.6 μg/L and 1.28 mg/L, the overall recoveries were 95.85%-148.85% with the relative standard deviations of 3.9%-14.9%.

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Determination of monoethylglycinexylidide concentration in serum using solid phase extraction and capillary gas chromatography-mass spectrometry WANG Zhenxia, ZHANG Ruiming, CAO Wei, OUYANG Xiaohui, ZHAO Haiping, DONG Peide, MENG Xingkai 2008, 26 (3):  310-313.  Abstract ( 2988 )   [Full Text(HTML)] () PDF (219KB) ( 1305 )   A novel method for the determination of monoethylglycinexylidide (MEGX) （lidocaine metabolin） in serum using solid phase extraction (SPE) and capillary gas chromatography-mass spectrometry (GC-MS) was established. The serum sample was extracted with a CN-SPE column. An HP-5MS capillary column (15 m×0.25 mm×0.1 μm) was used. The initial temperature of the column was set at 100 ℃, held for 1 min, then raised to 200 ℃ at 40 ℃/min, and held at 200 ℃ for 0.5 min. The sample size was 2 μL, and the split ratio was set at 1∶1. The carrier gas was high purity helium with a flow rate of 1.0 mL/min. The monitoring ions for the determination were m/z 58 for MEGX and m/z 86 for procaine （internal standard）. The calibration curve of MEGX had good linear relationship in the range of 1.562-25 ng/mL (r=0.9981). The limit of detection was 0.5 ng/mL. The extraction recovery ranged from 80.1% to 85.7%. The method advanced the quantitative analysis of MEGX in serum by combining rapid and efficient SPE with specific and sensitive quantitation by GC-MS.

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Rapid determination of alkaloids in tobacco using gas chromatography-mass spectrometry with full scan-selected ion monitoring mode WANG Baoxing; YANG Shihua;HOU Ying;ZENG Xiaoying;WU Yi;XU Guowang 2008, 26 (3):  314-317.  Abstract ( 4309 )   [Full Text(HTML)] () PDF (262KB) ( 1404 )   Simultaneous Scan-Selected ion monitoring mode (Scan-SIM) is a new improvement method of data acquisition of gas chromatography mass spectrometry (GC/MS). The determination method of alkaloids in tobacco was established by Scan-SIM of GC/MS. Nicotine, nornicotine, anabasine and anatabine were determined with Scan mode, and myosmine, nicotyrine, 2,3'-Bipyridyl and cotinine were determined with SIM mode. The average recoveries of alkaloids ranged from 94.8% to 98.8％ and relative standard deviations (RSDs) were less than 6.0% (n=5). Therefore, it is a simple, rapid, accurate method. Tobacco samples in different years were determined with this method, and the results were satisfactory.

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Determination of flumioxazin residue in foods using gas chromatography-mass spectrometry LIN Li, YE Gang, XIE Liqi, LAN Fang, WU Weidong, JIN Baohui 2008, 26 (3):  318-321.  Abstract ( 3044 )   [Full Text(HTML)] () PDF (325KB) ( 1380 )   A method for the determination of flumioxazin residue in foods by gas chromatography-mass spectrometry (GC-MS) has been developed. The food sample was extracted with acetonitrile or ethyl acetate, concentrated in a rotary evaporator, then dissolved in acetonitrile-methyl benzene （3∶1, v/v） and purified with an NH2-solid phase extraction （SPE） column. The final extract was analyzed by gas chromatography-mass spectrometry with selected ion monitoring mode (GC-MS-SIM). External standard method was used for the quantification. The mean recoveries of flumioxazin spiked in foods were 79.4%-101%, and the relative standard deviations were 0.242%-7.15%(n=10). The detection limit for each was 0.01 mg/kg. This method has high sensitivity, veracity and is suitable for the determination of pesticide residues in foods.

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Determination of ifenprodil in human plasma using liquid chromatography with electrospray tandem mass pectrometry YANG Bo, CHEN Yongchuan, XIANG Rongfeng, YANG Dan, XIA Peiyuan, LIU Songqing 2008, 26 (3):  322-326.  Abstract ( 2382 )   [Full Text(HTML)] () PDF (494KB) ( 1012 )   A method for the determination of ifenprodil levels in human plasma was established. Ifenprodil and the internal standard (IS), ketoconazole, were extracted from the plasma with ethyl acetate using liquid-liquid extraction. The extracts were separated by high performance liquid chromatography (HPLC) using methanol-6 mmol/L ammonium acetate (pH value was adjusted to 7.40) (90∶10, v/v) as the mobile phase, and were then detected using mass spectrometry (MS). Electrospray source was applied and operated in positive ion mode. Selected reaction monitoring (SRM) mode with the transition of m/z 326.1→308.2 was used to quantify ifenprodil, and m/z 531.0→82.1 for IS. The excellent sensitivity and selectivity of the HPLC-MS/MS method allowed quantitation and identification of ifenprodil at low levels with a run time of 6.0 min. The assay was linear over the range from 0.25 to 50 μg/L. The intra-day and inter-day precisions measured as relative standard deviations (RSDs) were less than 2.7% and less than 6.5%, respectively. The average recoveries varied between 101.3% and 105.0%, and the detection limit was 0.08 μg/L. Due to its simplicity and accuracy, the established method is suitable for the application in a pharmaceutical study of the intervenous drop infusion of ifenprodil tartrate.

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Determination of simvastatin in human plasma using ultra-performance liquid chromatography- tandem mass pectrometry WANG Dong, QIN Feng, CHEN Lingyun, HAO Yu, ZHANG Yi, LI Famei 2008, 26 (3):  327-330.  Abstract ( 2754 )   [Full Text(HTML)] () PDF (369KB) ( 1242 )   A rapid, sensitive and specific method has been developed for the determination of simvastatin in human plasma with ultra-performance liquid chromatography-tandem mass spectrometry. Simvastatin and the internal standard （lovastatin） were extracted from human plasma with diethyl ether-n-hexane-isopropanol (80∶20∶3, v/v/v), then separated on a Waters ACQUITY UPLCTM BEH C18 column (50 mm×2.1 mm, 1.7 μm) with isocratic elution at a flow rate of 0.25 mL/min. The mobile phase was composed of 85% acetonitrile and 15% water (containing 10 mmol/L ammonium acetate). Electrospray ionization (ESI) source was applied and operated in positive ion mode. Multiple reaction monitoring (MRM) mode with the transitions of m/z 419.2 →m/z 199.0 and m/z 405.0→m/z 199.0 was used to quantify simvastatin and the internal standard, respectively. The linear calibration curve was obtained in the concentration range of 0.051-20.4 ng/mL. The lower limit of quantification was 0.051 ng/mL. The inter-day and intra-day precision (relative standard deviation) were less than 10%, and the accuracy (relative error) was within -2.7%-0% calculated from quality control samples. The mean extraction recovery of simvastatin was 91.6%. The method was proved to be selective, sensitive, rapid and suitable for the pharmacokinetic study of simvastatin.

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Fast separation and preparation of proteomics samples of human serum using high performance hydrophobic interaction chromatographic cake LI Ying, BAI Quan, CHEN Gang, WANG Lili 2008, 26 (3):  331-334.  Abstract ( 2361 )   [Full Text(HTML)] () PDF (349KB) ( 994 )   A method for the fast separation of human serum and enrichment of low abundance proteins was developed using offline 2D liquid chromatography (2D-LC) consisted of chromatographic cake (10 mm×20 mm i.d.) and reversed-phase liquid chromatography (RPLC). The protein after separation and enrichment was detected using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). This method was validated by four standard proteins at very low concentration. It was found that the detection limits were 1 pmol/μL for the enriched cytochrome-c and myoglobin, and 0.1 pmol/μL for enriched lysozyme and insulin. This method has been applied to the proteomic research of human serum, and it was found that the signal intensity and the number of detected proteins/peptides in MALDI-TOF MS increased with the increase of the loading sample volume of the human serum on chromatographic cake. A total of 285 fractions (Mr<15000) were found when 1.0 mL serum sample was loaded on the chromatographic cake. In addition, cytochrome-c in low abundance was also separated and enriched successfully when 1 μg cytochrome-c was added into 0.5 mL original serum. The results showed that 2D-LC consisting of the chromatographic cake and RPLC was successfully applied to not only the fast separation and preparation of human serum sample with large loading volume in one cycle of analysis, but also the efficient isolation and enrichment of the lower abundance proteins/peptides in human serum. Moreover, it successfully increased the detection efficiency of the low abundance proteins/peptides in human serum with MALDI-TOF MS.

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Analysis of chlorogenic acids in Helianthus tuberosus Linn leaves using high performance liquid chromatography-mass spectrometry YUAN Xiaoyan, GAO Mingzhe, WANG Kai, XIAO Hongbin, TAN Chengyu, DU Yuguang 2008, 26 (3):  335-338.  Abstract ( 2736 )   [Full Text(HTML)] () PDF (324KB) ( 1346 )   To identify chlorogenic acids in Helianthus tuberosus Linn leaves, a method of high performance liquid chromatography-ultraviolet-mass spectrometry (HPLC-UV-MS) was developed. HPLC analysis was performed on an Inertsil ODS-3 column (250mm×4.6 mm, 5 μm). The mobile phase consisted of 1% acetic acid (A) and methanol (B). A gradient program was adopted as follows: 0-10 min, 20%B to 35%B; 10-25 min, 35%B to 50%B; 25-35 min, 50%B to 80%B. The flow rate was set at 1.0 mL/min and the column temperature was 35 ℃. HPLC chromatogram was extracted at 327 nm. The mass spectrometer used was a TSQ triple quadrupole MS equipped with an electrospray ionization （ESI） interface. Initially, the mass spectrometer was programmed to perform full scan ranging from m/z 250-1200. For MS/MS, negative ion monitoring mode was used and the collision energy was set at 10-45 eV. By analyzing UV characteristics and MS fragmentation patterns, 7 chlorogenic acids were assigned to be three categories, i.e., three caffeoylquinic acids, one feruloylquinic acid and three dicaffeoylquinic acids. As the results of the method development efforts, an effective and fast method for the qualitative identification of the chlorogenic acids in Helianthus tuberosus Linn leaves was established.

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Determination of melamine residue in feeds by ultra performance liquid chromatography coupled with electrospray　tandem mass spectrometry CAI Qinren, OUYANG Yingyu, QIAN Zhenjie, PENG Yufen 2008, 26 (3):  339-342.  Abstract ( 2813 )   [Full Text(HTML)] () PDF (282KB) ( 1906 )   A method for fast determination melamine residue in feeds was established. An ultra performance liquid chromatography coupled with electrospray ionization quadrupole tandem mass spectrometry (UPLC-ESI-MS/MS) was used. The residue was quantified with multiple reaction monitoring (MRM) mode. The method was validated and good results were obtained with respect to precision, repeatability and spiked recovery. The limit of detection was 10 μg/kg for melamine, and the linear range was from 10 to 5000 μg/kg. The average recoveries were between 83% and 94% in the spiked range of 10-100 μg/kg, and the relative standard deviations (RSDs) were between 4.2% and 6.5%. The method has good repeatability and high sensitivity, and can be applied for the determination of melamine residue in feeds.

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Isolation and identification of lipopeptides produced by Bacillus subtilis using high performance liquid chromatography and electrospray ionization mass spectrometry CHEN Hua, WANG Li, YUAN Chengling, ZHENG Zhiming, YU Zengliang 2008, 26 (3):  343-347.  Abstract ( 2480 )   [Full Text(HTML)] () PDF (549KB) ( 1169 )   Bacillus subtilis JA produces a broad spectrum of bioactive lipopeptides with great potential applications in agriculture and industry. Crude lipopeptides were extracted with 100% methanol from the precipitate which was obtained by adding 6 mol/L HCl to the cell-free culture broth. The crude extract was separated on a Diamonsil C18 column (5 μm, 250 mm×4.6 mm) in reversed-phase high performance liquid chromatography （HPLC） system to separate the lipopeptide homologues. Five peaks were eluted from HPLC. Electrospray ionization mass spectrometry (ESI-MS) was used to analyze each HPLC fraction. The results showed three series of ion peaks. According to the values of m/z, the three series of ions were classified into surfactin, iturin and fengycin homologues, which were well-known biosurfactants produced by B. subtilis strains. The major ions were structurally characterized using tandem mass spectrometry. This study suggested a reasonable method for the isolation and identification of lipopeptides produced by B. subtilis strains.

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Simultaneous determination of sulfonamides and fluoroquinolones residues in chicken by high performance liquid chromatography-electrospray tandem mass spectrometry LIU Pengyan, JIANG Ning, WANG Yingfeng, YAN Lizhi 2008, 26 (3):  348-352.  Abstract ( 2242 )   [Full Text(HTML)] () PDF (577KB) ( 1259 )   1.College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China; 2.The Department of Chemistry, Capital Normal University, Beijing 100037, China; 3.Langfang Entry-Exit Inspection and Quarantine Bureau, Langfang 065000, China

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Determination of Sudan Residues in Eggs by Liquid Chromatography Tandem Mass Spectrometry WANG Peng, GUO Shaofei, JING Tao, HU Xiaozhong, LIN Yanfei, LUO Jing, SONG Qi, ZHOU Yikai, MEI Surong 2008, 26 (3):  353-357.  Abstract ( 2716 )   [Full Text(HTML)] () PDF (570KB) ( 1123 )   A new method was established for determining Sudan Ⅰ- Ⅳ in eggs by liquid chromatography tandem mass spectrometry (LC-MS/MS). The targeted analytes in eggs were extracted by solid-phase disperse technique with a mixture of chloroform and acetonitrile (9:1,v:v). Chromatographic separation was achieved by using ZORBAX SB-C18 column. Identification and quantification were performed using multiple- reaction monitoring (MRM) and electrospray ionization in positive mode. The linearity range of Sudan Ⅰ- Ⅳ was 0.5～100ng/g, 5.0～100ng/g，1.0～100ng/g and 2.0～100ng/g, respectively, and r>0.99. The recovery of determination of Sudan residues in eggs was between 87.3 %-113 % with RSD less than 9.1 %. The detection limit of this method was 0.1μg/kg (Ⅰ), 2.0μg/kg (II), 0.2μg/kg (III) and 0.4μg/kg (Ⅳ), respectively, which could meet the requirement of the Sudan residues monitoring both abroad and home.

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Simultaneous determination of gatifloxacin and ambroxol hydrochloride from tablet dosage form using reversed-phase high performance liquid chromatography SHAHED Mirza, NANDA Rabindra, DEHGHAN Muhammad Hassan, NASREEN Huda, FEROZ Shaikh 2008, 26 (3):  358-361.  Abstract ( 2159 )   [Full Text(HTML)] () PDF (208KB) ( 900 )   A reversed-phase high performance liquid chromatography (HPLC) method was developed, validated, and used for the quantitative determination of gatifloxacin (GA) and ambroxol hydrochloride (AM), from its tablet dosage form. Chromatographic separation was performed on a HiQ Sil C18 column (250 mm×4.6 mm, 5 μm), with a mobile phase comprising of a mixture of 0.01 mol/L potassium dihydrogen orthophosphate buffer and acetonitrile (70∶30, v/v), and pH adjusted to 3 with orthophosphoric acid, at a flow rate of 1 mL/min, with detection at 247 nm. Separation was completed in less than 10 min. As per International Conference on Harmonisation (ICH) guidelines the method was validated for linearity, accuracy, precision, limit of quantitation, limit of detection, and robustness. Linearity of GA was found to be in the range of 10-60 μg/mL and that for AM was found to be 5-30 μg/mL. The correlation coefficients were 0.9996 and 0.9993 for GA and AM respectively. The results of the tablet analysis (n=5) were found to be 99.94% with ±0.25% standard deviation (SD) and 99.98% with±0.36% SD for GA and AM respectively. Percent recovery of GA was found to be 99.92%-100.02% and that of AM was 99.86%-100.16%. The assay experiment shows that the method is free from interference of excipients. This demonstrates that the developed HPLC method is simple, linear, precise, and accurate, and can be conveniently adopted for the routine quality control analysis of the tablet.

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Preparation of asiaticoside and madecassoside from the extract of Centella asiatica (L.) Urb using preparative high performance liquid chromatography GAO Mingzhe, YUAN Xiaoyan, XIAO Hongbin 2008, 26 (3):  362-365.  Abstract ( 2783 )   [Full Text(HTML)] () PDF (235KB) ( 1433 )   A high performance liquid chromatographic (HPLC) method was successfully used to separate asiaticoside and madecassoside from the extract of Centella asiatica (L.) Urb. The raw material containing 20% asiaticoside and 45% madecassoside was dissolved in methanol and used for the preparation of the pure compounds. The flow rate of the mobile phase, injection volume and detection wavelength were optimized. The optimum operation parameters were selected as follows: a C18 column (50 mm×200 mm, 5 μm) with methanol-water (60∶40, v/v) as the mobile phase at a flow rate of 100 mL/min, the detection wavelength of 220 nm, and the injection volume of 1.5 mL at the concentration of 40 mg/mL. Asiaticoside and madecassoside can be obtained in a 20 min runtime, and the purities of the products were detected by melting point, thin layer chromatography (TLC) and HPLC method. The results showed that the purities of products were above 98%. The effective and rapid method has been successfully applied to the preparation of asiaticoside and madecassoside from Centella asiatica (L.) Urb.

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Preparation of three capsaicinoid components using preparative high performance liquid chromatography DONG Xinrong, LIU Zhonghua, LI Benxiang, WANG Kunbo 2008, 26 (3):  366-369.  Abstract ( 2458 )   [Full Text(HTML)] () PDF (283KB) ( 1203 )   A method was established for the isolation of three capsaicinoid components from capsaicinoids using preparative high performance liquid chromatography (Pre-HPLC). A preparative PRC-ODS column (250 mm×21.5 mm, 13 μm) was used in the preparation process. By isocratic elution with methanol-water (70∶30, v/v) as the mobile phase at a flow rate of 15 mL/min followed by concentration, three capsaicinoid components were obtained from 80% capsaicinoids, and subsequently identified by 1H NMR （nuclear magnetic resonance） and EI-MS （electron impact-mass spectrometry） as nordihydrocapsaicin, capsaicin and dihydrocapsaicin, and the purities of the three components analyzed by HPLC were 98.12%, 99.93% and 100.7% with yields of 60.1%, 58.9% and 72.3%, respectively.

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Relationship of resistance to diseases and water-soluble amino acids in Konjac leaves CHEN Yongbo, JIANG Qiaolong 2008, 26 (3):  370-373.  Abstract ( 2787 )   [Full Text(HTML)] () PDF (295KB) ( 1150 )   Reversed-phase high performance liquid chromatography was used to analyze water-soluble amino acids in the normal Amorphophallus Konjac, Amorphophallus albus, Amorphophallus bulbifer, and the soft rot Amorphophallus Konjac, to determine the relationship of the different soft-rot resistant Konjac varieties and the proportion and content of the multiple water-soluble amino acids. The results showed that there are remarkable differences in the content and proportion of water-soluble amino acids in different resistant varieties and the same variety of normal and diseased leaves of Amorphophallus. In this study, the bank of fingerprint chromatogram was established and can be used to analyze the related characteristic peaks and the resistance of Amorphophallus.

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Establishment and Application of Two-dimensional Liquid Chromatography System(NPLC／RPLC) LAN Tao, JIAO Fenglong, TANG Tao, WANG Fengyun, LI Tong, ZHANG Weibing 2008, 26 (3):  374-377.  Abstract ( 2465 )   [Full Text(HTML)] () PDF (1856KB) ( 990 )   A two-dimensional liquid chromatography (NPLC×RPLC) was developed. Normal phase liquid chromatography (NPLC) with a Hypersil SiO2 column (4.6mm×50 mm i. d.) was used as the first dimension, and reversed phase liquid chromatography (RPLC) with a Kromasil C18 column (4.6mm×250 mm i. d.) was used as the second dimension. The interface was based on a ten-port, two-position valve with a storage loop. As to improve the incompatibility of the two dimensional mobile phase and be good for adjusting the selectivity of separation, 1,4-Dioxane was used in the mobile phase of the first dimension, and isopropyl alcohol was used in the mobile phase of the second dimension based on the character of organic solvent. By rising the temperature of the second dimension, the system avoid the incompatibility of the two dimensional mobile phase. Zhengtian Wan was analyzed by this system, the total peak capacity reached 1120.

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Preparation of affinity chromatographic packings by immobilization of Cibacron Blue F3GA based on monodispersed non-porous hydrophilic poly (glycidylmethacry-co-ethylenedimethacrylate) particles and their application BO Chunmiao, WANG Youxian, GONG Bolin, YAN Chao 2008, 26 (3):  378-383.  Abstract ( 2393 )   [Full Text(HTML)] () PDF (789KB) ( 959 )   An affinity chromatographic packing was prepared by the immobilization of Cibacron Blue F3GA as dye ligands on the base of monodispersed, 3.0 μm non-porous hydrophilic poly (glycidylmethacry-co- ethylenedimethacrylate) particles. The effects of the ionic strength, organic solvent in the mobile phase and flow rate on protein retention were investigated on the chromatographic stationary phase. The apparent dissociation constant between the immobilized Cibacron Blue F3GA and lysozyme (Lys) was 5.26×10-5 mol/L which was evaluated by frontal chromatography. Purifications of Lys from egg white and bovine serum albumin (BSA) from calf blood were performed on the dye-ligand chromatographic column, separately, and the purities of the obtained fractions were determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purities of the purified Lys and BSA were determined to be 95% and 92%, respectively.

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Separation and purification of porcine hemoglobin from porcine blood ZHOU Bo, BIAN Liujiao 2008, 26 (3):  384-387.  Abstract ( 2868 )   [Full Text(HTML)] () PDF (510KB) ( 1150 )   In order to perform the isolation and purification of porcine hemoglobin from porcine blood. A three-step method, utilizing ultrafiltration, DEAE-Sepharose Fast Flow anion-exchange chromatography and Sephadex G-75 gel exclusion chromatography in sequence, for the separation and purification of porcine hemoglobin was developed. The obtained hemoglobin was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high- performance size-exclusion chromatography and reversed-phase high performance liquid chromatography. Through the three-step purification process, the purity of the obtained hemoglobin was more than 99% with a concentration of 1.328 g/L.

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Analysis of computational chemistry for the coordinationcharacter of Cu2+ and quercetin and kaempferol PAN Jian, WANG Jia, GAN Changsheng, ZHANG Chenguang 2008, 26 (3):  388-391.  Abstract ( 2809 )   [Full Text(HTML)] () PDF (2269KB) ( 2043 )   A model based on computational chemistry was established to simulate the coordination character of Cu2+and flavone compounds. Grid program package in MVD2007 was used in calculating the simulation of the coordination effect between Cu2+and two flavone compounds, kaempferol and quercetin, both of which have the lowest-energy conformation. Then the potential energy surface of interactional force field and relative binding energy were obtained. By comparing the relative binding energy of the two different compounds and analyzing the result of high performance liquid chromatographic (HPLC) method, it is indicated that the link between Cu2+and kaempferol is stronger than that between Cu2+and quercetin. A good correlation between the values of relative binding energies and the HPLC result suggests that the calculation method is reliable, and hence provides a theoretical basis for the separation in the area of coordination chemistry.

Technical Notes

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Simultaneous determination of canthaxanthin and astaxanthin in feedstuffs using solid phase extraction-reversed- phase high performance liquid chromatography ZHANG Hua, YANG Xin, MA Ying, DONG Aijun, ZHANG Yingchun 2008, 26 (3):  392-394.  Abstract ( 3318 )   [Full Text(HTML)] () PDF (180KB) ( 1355 )   A method was developed for the simultaneous determination of canthaxanthin and astaxanthin in feedstuffs using reversed-phase high performance liquid chromatography （RP-HPLC）. The sample was extracted by acetonitrile, and cleaned up by an LC-NH2 column. An Agilent ZORBAX Eclipse XDB-C18 analytical column (150 mm×4.6 mm, 5 μm) was used and kept at 25 ℃. Acetonitrile-methanol (95∶5, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. The detection was performed by a diode array detector at 474 nm. The quantitive analysis of external standard calibration curves was used. The linear ranges of the method for canthaxanthin and astaxanthin were 1.0- 30.0 mg/L (r=0.9990) and 1.0-20.0 mg/L (r=0.9991), respectively. The average recoveries were 90%-101% with the relative standard deviations of 0.62%-3.68%. The detection limits were 0.84 and 0.60 mg/L for canthaxanthin and astaxanthin, respectively. The method is simple, precise, sensitive and reproductive. It can be used to determine the contents of canthaxanthin and astaxanthin in feedstuffs.

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Determination of streptomycin residue in royal jelly by high performance liquid chromatography with post-column derivatization ZHANG Xiaoyan, XU Jinzhong, SHEN Chongyu, CHEN Huilan, WU Bin 2008, 26 (3):  395-397.  Abstract ( 2723 )   [Full Text(HTML)] () PDF (193KB) ( 1105 )   A reliable and sensitive method was introduced for the analysis of streptomycin in royal jelly. The method was based on a separation by a C8 analytical column with β-naphthoquinone-4-sulfonate post-column derivatization and fluorescence detection. The clean-up of the extract was done by solid-phase extraction, firstly with an octadecyl cartridge and secondly with a cation-exchange cartridge. The linear range was 0.02-0.5 mg/L and the correlation coefficient was 0.9958. The limits of detection (S/N=3) and quantitation (S/N=10) of streptomycin were 0.005 mg/kg and 0.01 mg/kg, respectively. The recoveries ranged from 84.0%to 104.0% with the relative standard deviations not larger than 7.9%. The method reduced the possibility of the false positive, and it could meet the need of the current work.

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Determination of triazolam in human urine by gas chromatograph-micro electron capture detector QIN Huajing 2008, 26 (3):  398-399.  Abstract ( 2297 )   [Full Text(HTML)] () PDF (148KB) ( 903 )   Triazolam is often abused in criminal activities. It is necessary to develop a simple, fast and sensitive method for determining triazolam in human urine. After the triazolam in urine was extracted with ether at pH 9, the analysis was performed on an HP-5 capillary column with gas chromatograph-micro electron capture detector (GC- μECD). The calibration curve of triazolam showed good linearity in the range of 0.2-50 ng/mL with the correlation coefficient of 0.9995. The inter-day relative standard deviation (RSD) and intra-day RSD were 4.17% and 5.31%, respectively. The average recovery was 93.9%. This simple, sensitive and accurate method can be easily implemented for monitoring triazolam in urine.