Chinese Journal of Chromatography

Enrichment strategy of cysteine-containing peptides based on covalent chromatography

MI Wei, WANG Jing, YING Wantao, JIA Wei, CAI Yun, QIAN Xiaohong

2010, 28 (2): 108-114.

Abstract ( 2391 ) [Full Text(HTML)] ()

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Automated multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely applied in large scale proteome profiling. However global proteome analysis remains a technical challenge due to the issues associated with sample complexity by tryptic digestion. The application of tag containing peptide enrichment approach for sample pre-separation could reduce the complexity of protein digest. Here, we demonstrated a simple and highly efficient cysteine-containing peptide enrichment method using a thiol specific covalent resin. The cysteine-containing peptides from the tryptic digests of the complex protein mixtures were selected by covalent chromatography based on thiol-disulfide exchange, identified by mass spectrometry. The strategy was firstly optimized and evaluated by using the tryptic peptides of bovine serum albumin (BSA). Then the method was applied with a relatively complicated sample from a five standard protein mixture. The results of these studies show that the enrichment method of cysteine-containing peptides is highly specific, efficient and reproducible. The effectiveness of this method in reducing the sample complexity and improving the identification of peptides by mass spectrometry has enabled high-throughput, automatic and large-scale qualitative and quantitative proteome analyses.

Analysis of mouse liver membrane proteins using multidimensional ion exchange chromatography and tandem mass spectrometry

WANG Zhuowei#, PENG Fuli#, WANG Yuan, TONG Wei, REN Yan, XU Ningzhi, LIU Siqi,

2010, 28 (2): 115-122.

Abstract ( 2622 ) [Full Text(HTML)] ()

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The analysis of membrane proteins is still a technical obstacle in proteomic investigation. A fundamental question is how to allow the hydrophobic proteins fully solubilizing in a proper solvent environment. We propose that the denatured membrane proteins in high denaturant solution are fully ionized and separated through ion exchange chromatography. The membrane proteins prepared from a mouse liver were dissolved in 4 mol/L urea, 20 mmol/L Tris-HCl buffer (pH 9.0), and loaded onto a tandem chromatography coupled with Q-Sepharose FF and Sephacryl S-200HR. With a linear NaCl gradient elution, the bound proteins were eluted and collected followed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to further separate the eluted proteins. The protein bound on SDS-PAGE were excised and in-gel digested by trypsin, while the digested peptides were delivered to reversed-phase high performance liquid chromatography (HPLC) and ion-trap mass spectrometry for the peptide identifications. Of a total of 392 proteins identified, 306 were membrane proteins or membrane associated proteins reported by literature. Based on the calculation of hydrophobicity, the GRAVY (grand average of hydropathicity) scores of 83 proteins are over or equal to 0.00. Taking all the evidence, we have established an effective approach which is feasible in the investigation towards mouse liver membrane proteomics.

Applications of Ti-SBA-15 mesoporous material in high performance enrichment of phosphopeptides

ZHANG Yu, QIN Hongqiang, WU Ren’an, ZOU Hanfa

2010, 28 (2): 123-127.

Abstract ( 2408 ) [Full Text(HTML)] ()

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A titanium-incorporated SBA-15mesoporous material (Ti-SBA-15) was synthesized via the co-condensation of tetraethyl orthosilicate (TEOS) and tetrabutyl titanate using surfactant P123 as the template. Due to the existence of Ti in the framework of SBA-15, the synthesized Ti-SBA-15 was applied as the selective adsorbent of phosphopeptides from the complex tryptic digest of β-casein. The matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis showed that the phosphopeptides of β-casein digest could be selectively enriched by the Ti-SBA-15 with Ti/Si molar ratio of 0.08, even under the interference of bovine serum albumin (BSA) with the molar ratio of β-casein to BSA up to 1:100. It can be concluded that the Ti-SBA-15 showed the specific adsorption toward the phosphorylated peptides, which provides great potential in the specific capture of phosphopeptides.

Application of peptide retention time in proteome research

SHAO Chen, GAO Youhe

2010, 28 (2): 128-134.

Abstract ( 2474 ) [Full Text(HTML)] ()

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Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been one of the most popular approaches in proteome analysis. As an independent parameter to mass spectrometry information, peptide retention time has been utilized to facilitate protein identification and quantification. In the field of peptide identification, the prediction of the retention time combined with routine tandem mass spectrometry database searching methods could help improve the confidence of identification. The sensitivity of identification could also be improved by matching peaks with both the accurate mass and retention time in multiple aligned LC-MS runs. Meanwhile, because small changes of liquid chromatography conditions lead to variability in retention times unavoidably, retention time alignment is crucial to label-free quantification. Additionally, post-translational modifications (PTM) could be identified by combining retention time shifts and mass deviation information.

Combination of matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry for the analysis of intact glycopeptides from horseradish peroxidase

CHEN Yaohan, YAN Guoquan, ZHOU Xinwen, YANG Pengyuan,

2010, 28 (2): 135-139.

Abstract ( 2255 ) [Full Text(HTML)] ()

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The mass spectrometric analysis of glycan composition and structure is a difficult but essential part in future study following the present glycoprotein identification. Intact glycopeptides analysis is an attracting field, in considering its capability to provide glycosite and corresponding glycan structure information at the same time. Mass spectrometry has been proven to be an important and a key tool for glycan analysis over the past few years. Making use of two soft ionization techniques—matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), through tandem mass spectrometry (MS/MS) analysis, the information for glycans of the glycopeptides can be obtained in the investigation. Meanwhile, by comparing the MALDI-MS/MS and ESI-MS/MS approaches using model glycoprotein (horseradish peroxidase, HRP), the complementary results have been verified experimentally. We believe that the combination of the two techniques is necessary and will provide useful information for the understanding of protein glycosylation.

Application of online two-dimensional separation system using monolithic columns for proteome analysis of human cartilage

XIE Shajie, WANG Fangjun, YAN Dan, ZHOU Guangdong, YE Mingliang, ZOU Hanfa

2010, 28 (2): 140-145.

Abstract ( 2203 ) [Full Text(HTML)] ()

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In Shotgun proteome analysis, where nano-flow is adopted to increase the sensitivity as well as extremely complicated samples such as proteolytic digest are inevitably confronted, monolithic capillary columns are widely used to improve the liquid chromatography separation performance. It is known that cartilage contains extensive amounts of extracellular matrix (ECM), in which collagens and aggrecans being the most abundant macromolecules. It is obvious that the high content of ECM components causes a challenge in the comprehensive proteome analysis of cartilage. In this study, a 7 cm×150 μm i.d. phosphate strong cation exchange (SCX) monolithic capillary column was coupled with an 85 cm×75 μm i.d. C12 reversed-phase monolithic capillary column for online two-dimensional separation of 20 μg tryptic digest of proteins extracted from human cartilage. After 14 salt steps fractionation and following gradient separation coupled with tandem mass spectrometry detection, finally 7434 unique peptides, corresponding to 1901 distinct proteins were positively identified. Then, the identified proteins were analyzed by Gene Ontology (GO), and it was found that most of the identified proteins were come from articular chondrocytes with low abundance, which is important for the researches of articular diseases.

Application of on-line two-dimensional liquid chromatography in comparative proteome analysis of antlers with different growing stages

GAO Liang, QIAO Xiaoqiang, LIANG Zhen, ZHANG Lihua, HUO Yushu, ZHANG Yukui

2010, 28 (2): 146-151.

Abstract ( 2329 ) [Full Text(HTML)] ()

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A method of on-line two-dimensional weak anion exchange chromatography-reversed-phase liquid chromatography (2D WAX-RPLC) was established, and applied for the comparative proteome study of antlers with four different growing stages. A five-protein-mixture was used as the sample to evaluate the reproducibility of the system. In addition, the capacity of such a system for the relative quantitative analysis was demonstrated by analyzing protein mixtures with different concentration ratios. Furthermore, five different lysis buffers were used to extract proteins from antlers with different growing stages. After the protein separation with 2D WAX-RPLC, the fractions with maximum peak height ratio larger than 2 were collected, digested, and further identified by micro-reversed-phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS). In total, 22 differential proteins were identified from 9 fractions. Our experimental results demonstrated that 2D WAX-RPLC based protein separation has the advantages of good reproducibility and automation, and might be a complementary method of two-dimensional gel electrophoresis for comparative proteome study.

Application of aspartic acid as a non-specific binding inhibitor in the enrichment of phosphopeptides with titanium dioxide

CHI Ming, BI Wei, LU Zhuang, SONG Lina, JIA Wei, ZHANG Yangjun, QIAN Xiaohong, CAI Yun

2010, 28 (2): 152-157.

Abstract ( 2374 ) [Full Text(HTML)] ()

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Titanium dioxide (TiO2) is one of metal oxides widely used for phosphopeptide enrichment in phosphoproteomic research nowadays. However it can bind to some non-phosphorylated peptides containing one or more aspartic acid residues and/or glutamic acid residues. These non-phosphorylated peptides can be eluted along with phosphorylated peptides and cause the reduction of the selectivity. Conventional inhibitors for the non-specific binding of non-phosphorylated peptides can often contaminate the ion source of mass spectrometry and therefore their applications are limited in liquid chromatography-mass spectrometry (LC-MS). In this study, aspartic acid was reported as a novel non-specific binding inhibitor for phosphopeptide enrichment by titanium dioxide. Firstly, the tryptic peptide mixtures of 3 and 9 standard proteins were used for the comparison of the enrichment efficiency of titanium dioxide. The effects with the presence of aspartic acid, glutamic acid and no-inhibitor in the enrichment systems were compared separately. The results showed that aspartic acid can greatly improve the selectivity of titanium dioxide for phosphopeptide enrichment. Then, aspartic acid was used for the enrichment of tryptic peptide mixture of C57BL/6J mouse liver lysate and good results were also obtained which demonstrated that aspartic acid was a promising non-specific binding inhibitor for complex biological samples. Besides, no contamination in the ion source occurred during the mass spectrometric analysis.

Optimization of two-dimensional high performance liquid chromatographic columns for highly efficient separation of intact proteins

HONG Guangfeng, GAO Mingxia, YAN Guoquan, GUAN Xia, TAO Qian, ZHANG Xiangmin,

2010, 28 (2): 158-162.

Abstract ( 2470 ) [Full Text(HTML)] ()

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In order to optimize two-dimensional liquid chromatographic (2D-LC) columns for highly efficient separation of proteins, several liquid chromatographic columns were investigated and evaluated. Weak anion-exchange (WAX) column was chosen as the first dimension because of its extensive protein separation power. By comparison of different WAX chromatographic columns for human liver protein separation, TSKgel DEAE-5PW column was selected as the first dimension of a 2D-LC system. For the second dimension, ten typical reversed-phase (RP) LC columns (250 mm×4.6 mm, 5 μm, 30 nm) were investigated and evaluated. Their silica based RP stationary phases were butyl (C4), octyl (C8) or octadecyl (C18). To evaluate the retention behavior and non-specific protein adsorption ability of these ten columns, four neutral compounds (uracil, nitrobenzene, naphthalene and fluorene) and three standard proteins (cytochrome C, myoglobin and albumin from chicken egg white) were adopted and separated by RPLC. Meantime, WAX fractions were used to investigate the separation ability of different alkyl-bonded silica stationary phase columns for complex protein samples. By comparison of column separation efficiency, adsorption of intact proteins and sample analysis, Jupiter 300 C4 column was finally employed for its excellent separation ability. Optimization of WAX and RPLC columns offers reliable foundation for the construction of 2D-LC protein separation systems.

Improvement of interface in comprehensive two-dimensional liquid chromatography and its application in the research of proteomics

LI Duxin, ZHANG Lingyi, LI Tong, DU Yiping, ZHANG Weibing

2010, 28 (2): 163-167.

Abstract ( 2162 ) [Full Text(HTML)] ()

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A two-dimensional liquid chromatography (2D-LC) system was constructed with improved trap column interface. Weak anion exchange (WAX) LC was used as the first dimension separation mode and reversed-phase (RP) LC as the second dimension. Two 35 mm long columns (column 1 and column 2) were used as trap columns to retain the fraction from the first dimension, and forward-flushing was employed to pre-separate the components when the trap column was connected to the second dimension. The interface greatly increases the efficiency of the second dimension column without losing the separation speed. Rat serum sample was separated on this system to evaluate the performance of the constructed system. The viscous fingering (VF) phenomenon was generated attribute to the difference in the velocities of the mobile phases of the two dimensions. The separation efficiency was theoretically increased by 70% when the 35 mm trap columns were used in the forward-flush mode.

Application of turbulent flow chromatography in the analysis of biological samples

LIU Peng, ZHOU Jianliang, AN Jingjing, LI Ping

2010, 28 (2): 168-174.

Abstract ( 2671 ) [Full Text(HTML)] ()

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The complexity of biological fluids makes the sample preparation necessary in the analytical process. The traditional techniques, such as liquid-liquid extraction and solid-phase extraction, are time-consuming and labor-intensive. Turbulent flow chromatography (TFC), as an on-line direct injection technique, possesses the advantages of reducing sample preparation steps and enabling the effective pre-concentration and clean-up of biological fluids. Therefore, it is a high-throughput and high-selectivity technique for biological sample pretreatment. In this review, the theory, advantages, characteristics and current status of TFC in bioanalysis are introduced and summarized.

Optimization of preparation of poly (glycidyl methacrylate- divinylbenzene) monolithic column with orthogonal experiments for separation of small molecules

MA Wei, XU Huanxin, LIU Zuozhen, NING Fanghong,

2010, 28 (2): 175-179.

Abstract ( 2394 ) [Full Text(HTML)] ()

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A monolithic column of macro porous poly(glycidyl methacrylate-divinylbenzene) (poly(GMA-DVB)) has been prepared by free radical polymerization within the confines of a chromatographic stainless steel tube (50 mm×4.6 mm). For the best separation and low back pressure, orthogonal experiments were carried out with V(cyclohexanol): V(dodecanol), V(GMA): V(DVB) and BPO dosage as the three main factors. The characteristic feature of the column, including specific surface area, pore volume as well as pore diameter distribution, was studied by scanning electron microscopy (SEM), mercury intrusion porosimetry analysis and BET analysis. The obtained optimum preparation conditions were that the volume ratio of GMA, DVB, cyclohexanol and dodecanol was 0.825:0.825:1.32:2.03 and the BPO dosage was 0.7%(mass percentage), then it was heated at 70 ℃ for 24 h. Using this monolithic column, benzene and ethylbenzene and a drug of oxiracetam can be well separated on a high performance liquid chromatographic (HPLC) system equipped with a ultraviolet (UV) detector at 254 nm. A solution of acetonitrile-water (50:50, v/v) for the separation of benzene and ethylbenzene, and acetonitrile-water (80:20, v/v) for the separation of oxiracetam were used as mobile phases at a flow rate of 1 mL/min. The theoretical plate number was 37000 plates/m and the resolution of peak width at half height (R1/2) was 7.14. The separation time was less than 10 min. The monolithic column prepared by this method is reproducible and has high column efficiency. It is an economical method to prepare monolithic column, which can be applied to separate small molecules.

Separation and identification of bovine lactoferricin by high performance liquid chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry

AN Meichen, LIU Ning

2010, 28 (2): 180-184.

Abstract ( 1980 ) [Full Text(HTML)] ()

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A high performance liquid chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (HPLC-MALDI-TOF/TOF MS) method was developed for the separation and identification of bovine lactoferricin (LfcinB). Bovine lactoferrin was hydrolyzed by pepsin and then separated by ion exchange chromatography and reversed-phase liquid chromatography (RP-LC). The antibacterial activities of the fractions from RP-LC separation were determined and the protein concentration of the fraction with the highest activity was measured, whose sequence was indentified by MALDI-TOF/TOF MS. The relative molecular mass of LfcinB was 3124.89 and the protein concentration was 18.20 μg/mL. The method of producing LfcinB proposed in this study has fast speed, high accuracy and high resolution.

Determination of perfluorooctane sulfonates in fire-fighting foam and other materials by high performance liquid chromatography-tandem mass spectrometry

CHEN Huiming, CHENG Yan, CHEN Wei, YU Wenlian, LI Xi, WANG Zheng

2010, 28 (2): 185-189.

Abstract ( 2395 ) [Full Text(HTML)] ()

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A novel method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the determination of perfluorooctane sulfonates (PFOS) in the fire-fighting foam, detergents and fabric finishing agents. The PFOS residue was extracted with water at first by ultrasonic, then separated by high-speed centrifugation. The supernatant was purified by pre-conditioned solid phase extraction (SPE) micro-column, and the extract was filtrated through a membrane with 0.2 μm diameter. The filtrated liquid was analyzed by HPLC using acetonitrile-10 mmol/L ammonium acetate solution (80:20, v/v) as mobile phase. The PFOS was detected by using negative electrospray ionization (ESI) on a tandem mass spectrometer in multiple reaction monitoring (MRM) mode. The qualitative analysis of the PFOS can be performed by using the relative abundance of two daughter ions of PFOS, and the quantitative analysis was performed by external standard method. The linear calibration curve was obtained in the range of 0.002~0.1 mg/L with a linear correlation coefficient (r2) of 0.998. The spiked recoveries for PFOS in the fire-fighting foam, detergents and fabric finishing agents were 93.4%~103%, 93.2%~102% and 91.8%~102% with the relative standard deviation of 0.48%~3.52%, 0.78%~1.79% and 0.47%~3.47%, respectively. And the detection limit for PFOS was 2 mg/kg (S/N≥10), which can meet the requirement for the PFOS restriction in fire-fighting foam, detergents and fabric finishing agents in the EU directives. With high accuracy and sensitivity, the method is simple and rapid, and can be used for PFOS inspection in fire-fighting foam, detergents and fabric finishing agents.

Simultaneous determination of seven sex hormones in fish products using ultra performance liquid chromatography-tandem mass spectrometry

ZHANG Aizhi, WANG Quanlin, SHEN Jian, ZHANG Shufen, CHEN Liren

2010, 28 (2): 190-196.

Abstract ( 2598 ) [Full Text(HTML)] ()

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A rapid, specific and highly sensitive method for the determination of seven sex hormones (norgestrel, methyltestosterone, testosterone propionate, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, and nandrolone) residues in fish products was developed using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with electrospray ionization (ESI) in positive mode. The target compounds were extracted with methanol after the enzyme hydrolysis of the fish products. ZnCl2 was added to the extract solution to remove lipids. Then target compounds were purified by an LC-C18 and an LC-NH2 solid phase extraction cartridges. The target compounds were separated on a Waters ACQUITYTM UPLC BEH-C18 column (100 mm×2.1 mm, 1.7 μm) and detected qualitatively and quantitatively in multi reaction monitoring (MRM) mode. For the seven sex hormones, the limits of detection (LOD) of the method were from 0.08 to 0.17 μg/kg and the limits of quantification (LOQ) were in the range of 0.24~0.58 μg/kg. At the spiked levels of 1 and 4 μg/kg, the average recoveries ranged from 76% to 118% with the relative standard deviations between 5.0% and 11.3% for the seven sex hormones using internal standard method; and the average recoveries ranged from 66% to 94% with the relative standard deviations between 4.5% and 10.7% using matrix matched external standard method. The results showed that both methods are able to meet the multi-residue detection of the seven sex hormone residues in fish products. The degreased large yellow croaker and roast fish fillet real samples from a local market were detected by the developed method, and the seven targets were not found.

Simultaneous determination of trace diphacinone and chlorophacinone in biological samples by high performance liquid chromatography coupled with ion trap mass spectrometry

JIN Micong, CHEN Xiaohong

2010, 28 (2): 197-203.

Abstract ( 2362 ) [Full Text(HTML)] ()

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A rapid qualitative and quantitative method for the simultaneous determination of trace diphacinone and chlorophacinone in biological samples has been established. The method mainly serves for the emergent poisoning detection. The whole blood was treated with methanol-acetonitrile (50/50, v/v) and the urine was cleaned-up by Waters Oasis HLB SPE cartridges. The samples were separated on an Extend C18 column (150 mm×4.6 mm, 5 μm) by using the mobile phase consisted of ammonium acetate-acetic acid (0.02 mol/L, pH 5.5)-methanol (15/85, v/v). The determination was performed by high performance liquid chromatography coupled with ion trap mass spectrometry (HPLC-IT-MS) using a negative electrospray ionization interface in the multiple reaction monitoring (MRM) mode. The transitions of m/z 339→167 for diphacinone and m/z 373→201 for chlorophacinone were selected for the quantificantions. For the whole blood samples, the calibration curves were linear within the ranges of 1.0~200.0 μg/L and 0.5~100.0 μg/L; the limits of quantification were 1.0 μg/L and 0.5 μg/L; the spike recoveries were 90.1%~92.2% and 87.6%~93.4%, the intra-day relative standard deviations (RSDs) were less than 6.8% and 7.4%, and the inter-day RSDs were less than 9.9% and 10.9% for diphacinone and chlorophacinone, respectively. For the urine samples, the calibration curves were linear within the ranges of 0.2~40.0 μg/L and 0.1~20.0 μg/L; the limits of quantification were 0.2 μg/L and 0.1 μg/L; the spike recoveries were 90.1%~94.5% and 90.0%~98.0%, the intra-day RSDs were less than 6.1% and 7.3%, and the inter-day RSDs were less than 8.9% and 11.2% for diphacinone and chlorophacinone, respectively. This method is simple and sensitive for the satisfactory determination of trace diphacinone and chlorophacinone residues in poisoned patients for the clinical diagnosis.

Determination of four components in Vitamin C Yinqiao Tablets using reversed-phase high performance liquid chromatography with dual wavelength detection

DONG Li, SUN Xiangde, LI Qin

2010, 28 (2): 204-208.

Abstract ( 2962 ) [Full Text(HTML)] ()

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A reversed-phase high performance liquid chromatographic (RP-HPLC) method with dual wavelength detection was developed for the determination of vitamin C, paracetamol, chlorphenamine maleate and chlorogenic acid in Vitamin C Yinqiao Tablets. The separation was performed on a Sinochrom ODS-BP column with the mobile phase consisting of 0.05 mol/L KH2PO4 (pH 3.0, containing 1% triethylamine and acetonitrile (75:25, v/v). The detection wavelength was set at 260 nm (λ1) and 326 nm (λ2). The method linearities were wide and the average recoveries were more than 99.4%. The relative standard deviations were less than 1.8% (n=5). This method is convenient, rapid and accurate. It is readily applied to the quality controls in the production process of Vitamin C Yinqiao Tablets.

Simultaneous determination of 7 active ingredients in Scutellaria barbata D. Don by capillary micellar electrokinetic chromatography

MI Xuan, ZHU Ruohua

2010, 28 (2): 209-214.

Abstract ( 2278 ) [Full Text(HTML)] ()

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A method of capillary micellar electrokinetic chromatography with a diode array detector was developed for the simultaneous determination of 7 active ingredients (baicalein, naringenin, wogonin, scutellarin, apigenin, luteolin, and protocatechuic acid) in Scutellaria barbata D. Don and its ointment. The 7 ingredients in samples were extracted with methanol in an ultrasonic bath. The important factors, such as pH, running buffer concentration, additive, detection wavelength, separation voltage and injection time, were optimized. Under the optimum conditions, the 7 analytes were separated within 12 min at a separation voltage of 25 kV in a 50 mmol/L borate-0.20 mol/L boric acid buffer (pH 8.4) containing 8.5 mmol/L sodium dodecyl sulfate (SDS). The detection was carried out at 260 nm and 335 nm. Notably, the linearity ranged from 8×10~6 to 3.2×10~4mol/L with the correlation coefficients (r2) at the range of 0.9965~0.9999. The detection limits (S/N=3) ranged from 7.0×10~8mol/L to 2.0×10~6mol/L for all the 7 analytes. The recoveries of the 7 active ingredients were all over 85%. This method is of good sensitivity, simple preparation, good reproducibility and reliability. It was successfully applied to the analysis of the 7 active ingredients in Scutellaria barbata D. Don and its ointment.

Enantiomeric separation of azelnidipine by high performance liquid chromatography with chiral stationary phase

ZHANG Kai, XUE Na, LI Lin, LI Fan, DU Yumin

2010, 28 (2): 215-217.

Abstract ( 2577 ) [Full Text(HTML)] ()

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A high performance liquid chromatographic (HPLC) method was established for the enantiomeric separation of azelnidipine. Baseline chiral separation of azelnidipine was achieved under normal-phase chromatographic mode using the Chiralpak AD-H chiral column (250 mm×4.6 mm, 5.0 μm, Daicel). The influences of the nature of chiral stationary phase, form of the mobile phase, and column temperature on the enantiomeric separation were studied. The optimized chromatographic conditions were hexane-isopropyl alcohol (90:10, v/v) as the mobile phase with a flow rate of 0.8 mL/min and detection at the wavelength of 254 nm. The column temperature was set at 20 ℃. The resolution of 3.3 for azelnidipine was achieved under the above chromatographic conditions. The method is simple, rapid and with good reproducibility.

Determination of arsanilic acid and sulfanilic acid as adulterant in feed additives by reversed-phase high performance liquid chromatography

XU Jinping, HE Heng, XU Mengyi, QU Yanhua

2010, 28 (2): 218-220.

Abstract ( 3057 ) [Full Text(HTML)] ()

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A reversed-phase high performance liquid chromatographic (RP-HPLC) method was established for the determination of arsanilic acid and sulfanilic acid as adulterant in the feed additives. The separation was carried out on a Waters Bondapak C18 column, and methanol-water (pH 2.9 adjusted by 0.01 mol/L phosphoric acid) (1:4, v/v) was used as the mobile phase with a flow rate of 1.0 mL/min. A diode array detector was used at 244 nm as the detection wavelength. Arsanilic acid and sulfanilic acid were separated within 3 min. The linear ranges all were 5~200 mg/L and the detection limits (S/N=3) were 0.20 and 0.15 mg/L for arsanilic acid and sulfanilic acid, respectively. This method is simple and rapid, and suitable for the simultaneous determination of arsanilic acid and sulfanilic acid in feed additives.

Research activities in chromatography

LIU Huwei

2010, 28 (2): 221-222.

Abstract ( 1689 ) [Full Text(HTML)] ()

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