Preparation and characterization of an immunoaffinity column for the selective extraction of salbutamol from pork sample

A rapid, simple, and reliable determination method for salbutamol in pork was developed with immunoaffinity column (IAC) extraction followed by HPLC analysis. The salbutamol immunoaffinity column was prepared by coupling CNBr-activated Sepharose-4B with the anti-salbutamol polyclonal antibody which was purified by caprylic acid-ammonium sulfate. The coupling rate of the antibody and Sepharose-4B was 98.6%, and the dynamic column capacity of IAC was 400 ng/mL gel. The average recoveries of salbutamol from spiked pork samples at levels of 2, 10, 20, and 50 ng/g ranged from 83.3% to 92.2%, with the relative standard deviations of 2.8-7.0% (n=5), and the limits of detection and qualification were 0.25 ng/g and 0.5 ng/g, respectively.     

Determination of 1-nitropyrene in herbs after selective enrichment by a sol-gel-generated immunoaffinity column

Using the determination of 1-nitropyrene as an example the paper demonstrates the advantages of including a highly selective sol–gel-generated immunoaffinity column in the sequence of clean-up steps necessary to determine haptens in complex matrices. The sol–gel method to immobilise antibodies enlarges the variety of immunoaffinity columns available and leads to mechanically stable columns with constant retention characteristics. The sample preparation scheme proposed combines acetonitrile extraction, size-exclusion and immunoaffinity chromatography. 1-Nitropyrene is then separated by reversed-phase HPLC from interfering compounds and determined after catalytic on-line reduction to the corresponding amine by spectrofluorimetry. Concentrations in the range from 0.1 to 1.4 μg/kg 1-nitropyrene were detected in herbs.     

Preparation and characterization of an immunoaffinity chromatography column for the selective extraction of trace contraceptive drug levonorgestrel from water samples

The preparation and characterization of an immunoaffinity chromatography (IAC) column for the specific extraction and enrichment of trace contraceptive drug levonorgestrel (LNG) from water samples were described. The IAC column was constructed by covalently coupling specific polyclonal antibody against LNG to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions, as well as the effect of flow rate on the extraction were carefully optimized. Pure water, 5% of methanol and 50% of methanol were respectively selected as loading, washing and eluting solutions, while the flow rates in the loading, washing and eluting steps were selected to be 1.0, 2.0 and 0.5 mL min⁻¹, respectively. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, extraction recovery and stability. It was found that, for IAC column packed with 0.2 g of solid support immobilized with antibody, the maximum capacity for LNG was about 260 ng. The extraction recoveries of the column for LNG at three different spiked concentrations were within 95.3-106.9%. After more than 35 times repeated usage, there was not significant loss of specific recognition. Using high performance liquid chromatography (HPLC) as an analytical tool, trace amount of LNG in the range of ng L⁻¹ was found in river water and wastewater samples after 600-fold enrichment, demonstrated the feasibility of the prepared IAC column for LNG extraction.     

Determination of sulfonamides in swine meat by immunoaffinity chromatography

A procedure was developed for the preparation of anti-sulfonamide (SA) group-specific antibodies and immunosorbents. Sulfonamide haptens and conjugates were synthesized by building spacer arms on an N1 group of 4-aminobenzensulfonamide. The anti-SA group-specific antibodies and immunosorbents were prepared successfully. After extraction with methanol-water (8 + 2), sulfamonomethoxine, sulfadimethoxine, and sulfaquinoxaline were cleaned up on immunoaffinity columns and determined by reversed-phase liquid chromatography with UV detection at 270 nm. The recoveries from fortified swine meat (10-100 microg/kg) ranged from 70.8 to 94.1%, with coefficients of variation of 3.4-12.9%. Limits of detection were 1-2 microg/kg.     

Utilization of a mixed-bed column for the removal of iodine from radioactive process waste solutions

A boron carbide capsule was previously designed and tested by Pacific Northwest National Laboratory (PNNL) and Washington State University (WSU) for spectral-tailoring in mixed spectrum reactors. The presented work used this B₄C capsule to create a fission product sample from the irradiation of highly enriched uranium (HEU) with a fast fission neutron spectrum. An HEU foil was irradiated inside of the capsule in WSU’s 1 MW TRIGA reactor at full power for 200 min to produce 5.8 × 10¹³ fissions. After 3 days of cooling, the sample was shipped to PNNL for radiochemical separations and analysis by gamma and beta spectroscopy. Fission yields for products were calculated from the radiometric measurements and compared to measurements from thermal neutron induced fission (analyzed in parallel with the non-thermal sample at PNNL) and published evaluated fast-pooled and thermal nuclear data. Reactor dosimetry measurements were also completed to fully characterize the neutron spectrum and total fluence of the irradiation.     

Preparation and evaluation of a large-volume radial flow monolithic column

In this study, a 38 mL monolith with homogeneous porous structure was produced by a single polymerization from glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of porogens and an initiator. The uniform temperature distribution within the reaction system was achieved by adding reactant mixture continuously and enhancing the heat transfer ability of the polymerization system. Homogeneous porous structure in the monolith was proved by SEM and the pore size distribution profiles measured by mercury intrusion porosimetry. Experimental results from proteins separation indicated that the dynamic capacity and resolution of radial flow monolithic column were independent of flow rates. Furthermore, the pressure drop on the column was linearly dependent on the flow rate and did not exceed 1.7 MPa even at a flow rate of 50 mL/min, which proved that the prepared monolith could be used in the quick separation and preparation of biopolymers.     

一种聚甲基丙烯酸酯类亲水作用毛细管电色谱整体柱的制备与评价

以2-羟基乙基甲基丙烯酸酯(HEMA)为单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂,制备了亲水分离模式的聚HEMA-co-EDMA新型毛细管电色谱(CEC)整体柱。考察了整体柱的结构特征及其CEC性能,研究了极性物质的保留行为,并对其可能的保留机理进行了探讨。所制备的整体柱稳定性好,固定相表面带有极性羟基功能团,不仅能提供亲水相互作用位点,且能吸附流动相中的阴离子产生阳极电渗流(EOF)。在流动相中乙腈含量较高(＞62%,体积分数)的条件下,整体柱表现出典型的亲水作用,实现了对核苷、碱基和苯胺类带有碱性的强极性化合物的高效分离,并成功分离了苯酚类、苯甲酸类等中性或酸性的极性化合物。     

干凝胶无机整体柱的制备及评价

以硅酸钾为硅源,甲酰胺为催化剂在毛细管内原位聚合形成干凝胶柱,制备了一系列致密度不同的整体柱。该法在柱制备及高温干燥过程中不会发生柱床断裂和塌陷现象,此特点明显优于以烷氧基硅烷为前驱体制备无机整体柱的方法。考察了不同模数硅酸钾对整体柱柱床结构的影响,用扫描电镜(SEM)和氮吸附法对整体柱结构进行了表征,考察了整体柱柱压与流速的关系;对整体柱进行十八烷基修饰后,测定了反相整体柱(C18整体柱)对蒽的柱效,通过考察甲苯在该柱上的突破曲线,获得了其对甲苯的柱容量。结果显示该整体柱柱床刚性好,在高温、高柱压、高流速时柱床能保持其物理结构的稳定性,对蒽的柱效达到41400理论塔板/m,对甲苯的柱容量为61 ng。     

烯唑醇免疫亲和色谱柱的制备及其在样品前处理中的应用

将四甲氧基硅烷(TMOS)水解后与烯唑醇抗体聚合,采用溶胶-凝胶法合成了烯唑醇免疫亲和色谱(IAC)柱固定相,并用其制备了对烯唑醇具有特异性亲和力的IAC柱。对IAC柱条件进行了优化,选择超纯水作为吸附与平衡介质,30%～50%(体积分数)甲醇水溶液作为洗脱剂。结果表明: 在优化条件下,IAC柱对烯唑醇的动态柱容量达125.4 μg/g。在河水样品和水果样品中添加烯唑醇,经IAC柱净化富集,洗脱液采用高效液相色谱检测,河水中烯唑醇的平均回收率为90.36%～100.14%,相对标准偏差(RSD)为2.03%～6.08%;水果中烯唑醇的平均回收率为85.55%～94.02%, RSD为3.38%～6.78%。本研究为烯唑醇在河水、水果等样品中的残留分析提供了一种新的高效前处理手段。     

Preparation and evaluation of a novel surface-imprinted polymer for selective adsorption of dibenzothiophene

We have prepared a novel surface molecular imprinted polymer (S-MIP) using dibenzothiophene (DBT) as the template molecule, 4-vinylpyridine as the functional monomer, and potassium hexatitanate whisker as the carrier. The S-MIP was characterized by Fourier transform infrared spectroscopy and and scanning electron microscopy. The kinetics and isotherms for the adsorption of DBT on the S-MIP followed pseudo-second-order and Freundlich models, respectively. Thermodynamic parameters were determined. The overall adsorption process was endothermic and spontaneous. Selectivity studies showed that the S-MIP selectively adsorbs DBT over its structural analogs and can be regenerated. Its adsorption capacity was still at 84.8% at the sixth cycle.

Development and application of immunoaffinity column chromatography for atrazine in complex sample media

A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine was bound to the IA columns when the loading solvents were either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffered saline (PBS). Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating atrazine did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96 ± 2% (2.1%). Recoveries of the 500, 50, and 5 ng mL⁻¹ atrazine standard solutions from the four IA columns were 107 ± 7% (6.5%), 122 ± 14% (12%), and 114 ± 9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (∼0.16 μg of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and could be reused more than 50 times with no change in performance. The IA columns were challenged with soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) or ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.     

An immunoaffinity column for the determination of peanut protein in chocolate.

An immunoaffinity column was prepared from rabbit polyclonal antiserum for the determination of peanut protein from food matrixes. The anti-peanut immunoglobulin G was isolated from antiserum by affinity chromatography on a column coupled with peanut protein and then attached to an AminoLink gel. The column was applied to the determination of peanut protein in chocolate after extraction, immunoaffinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Overall recoveries from chocolate spiked with 0.2-3.2 micrograms/g of peanut protein averaged 77% (range, 72-84%), and the minimum detection limit was 0.1 microgram/g. Chromatography of extracts with the column improved detection limit and eliminated the matrix effect experienced with direct ELISA of chocolate extracts.     

伊瑞霉素键合手性毛细管整体柱的制备与对映体分离

以具有22个不同种类手性中心的新型大环抗生素伊瑞霉素为手性选择器,基于环氧基团高反应活性的特征,将伊瑞霉素用一步法键合到甲基丙烯酸酯整体柱表面制备伊瑞霉素键合手性毛细管整体柱。通过对制备条件进行优化,证实该制备方法可在较宽的pH范围(6.0～9.0)内进行,方法简单易行,反应条件温和。应用制备的手性毛细管整体柱在毛细管电色谱模式下,对5种手性氨基酸对映体和手性药物罗格列酮对映体进行拆分,均得到了基线分离,说明伊瑞霉素手性固定相具有较强的手性拆分能力。在优化的色谱条件下,6种对映体的分析时间均小于4 min,分析速度快。通过对有机调节剂、缓冲液pH值和缓冲盐浓度等分离条件进行系统考察,初步探讨了该手性毛细管整体柱对不同溶质的手性识别机理。     

两步液液萃取-固相萃取净化结合高效液相色谱-串联质谱法测定猪肉中11种磺胺类兽药残留

建立了猪肉中11种常见的磺胺类兽药残留的两步液液萃取-固相萃取净化-高效液相色谱-串联质谱（LC-MS/MS）检测方法。猪肉样品经乙酸乙酯（含2%（v/v）甲酸）及丙酮分两步液液萃取，正己烷脱脂，Oasis MCX混合阳离子固相萃取小柱净化，氮吹浓缩，定容，过滤膜后进行高效液相色谱-串联质谱分析。采用多反应监测正离子模式进行检测，以基质校准曲线外标法定量。结果表明，在20~400 μg/L范围内11种磺胺类药物均呈现良好的线性关系（相关系数（r²）≥ 0.99），检出限（LODs）（S/N=3）和定量限（LOQs）（S/N=10）分别为0.1~1.0 μg/kg和0.2~3.0 μg/kg。对阴性猪肉样品，在50、100、200 μg/kg 3个水平下分别进行加标回收试验，测得各待测物的平均回收率为79.3%~105.5%之间，相对标准偏差为1.3%~11.6%（n=6）。该方法比采用一步液液萃取法具有更高的提取效率，同时结合固相萃取净化方法进一步富集目标化合物，降低了基质干扰，提高了检测灵敏度。     

Quantification of deoxynivalenol in wheat using an immunoaffinity column and liquid chromatography

A simple and accurate method to quantify the mycotoxin deoxynivalenol (DON) in wheat is described. The method uses immunoaffinity chromatography for DON isolation and liquid chromatography (LC) for toxin detection and quantification. Wheat samples are extracted in water, filtered twice and applied to an immunoaffinity column. Following a water wash, DON is eluted from the column with methanol and injected onto an LC system with a UV detector for quantification. Test performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity and comparison with the GC-electron-capture detection (ECD) method of Tacke and Casper. Specificity of the immunoaffinity column cleanup procedure was confirmed with only DON (>80%) and its 15-C derivatives (40-50%) being recognized by the antibody while 3-C DON derivatives, nivalenol, T-2 and fusarenon-X did not bind. The limit of detection is at least 0.10 microg/g. Percentage recovery for the entire assay range averages 90% with an average relative standard deviation of 8.3%. Naturally contaminated samples showed comparable precision. Column capacity was determined to be 3.3 microg. The assay showed a high degree of linearity (r2=0.999) and an optimum assay range of 0.10 to 10.0 microg/g. Comparative analysis of 28 naturally or artificially contaminated wheat samples using DONtest-HPLC and the GC-ECD method of Tacke and Casper showed that DONtest-HPLC is a statistically significant predictor of the GC-ECD method (r2=0.982).     

Preparation of a fast-flow agarose-based chelating adsorbent with a novel dioxime derivative for selective column preconcentration of copper.

Fast-flow spherical homogeneous agarose beads were prepared by an emulsification method, and were cross-linked and activated by repeated treatment with allylbromide and bromine/water, followed by alkali. Bis(2-aminopyridyl)dioxime (APD) was synthesized by the reaction of 2-aminopyridine, and dichloroglyoxime and characterized by melting-point as well as IR, 1HNMR, 13CNMR and MS spectroscopies. APD was chemically linked to activated agarose beads to be employed for the column preconcentration of metal ions. Capacity measurements for eight metal ions indicated a high selectivity of the adsorbent towards Cu2+ with a capacity of 25.7 micromol per ml packed adsorbent. A factorial design was used for optimization of the effects of 5 different variables on the recovery of Cu2+. Under the optimized conditions, Cu2+ was quantitatively accumulated on a 0.25 ml packed column of the adsorbent in the pH range of 4 to 6, and simply eluted with 2 ml of a 1 mol 1(-1) hydrochloric acid solution. The column could tolerate salt concentrations up to 0.5 mol 1(-1), sample flow rates up to 15 ml min(-1), and sample volumes beyond 1000 ml. Matrix ions of Na+, Mg2+ and Ca2+ and potentially interfering ions of Ni2+, Cd2+, Zn2+, Fe3+ and Co2+ with relatively high concentrations did not show any significant effect on the analyte's signal. Preconcentration factors up to 500 and a detection limit of 0.16 microg 1(-1) were obtained for the determination of the analyte by flame AAS. Application of the method to the determination of natural and spiked copper in river water and seawater samples resulted in quantitative recoveries.     

Preparation and evaluation of a sulfoalkylbetaine‐based zwitterionic monolithic column for CEC of polar analytes

A novel polymethacrylate‐based monolithic column with covalently bonded zwitterionic functional groups was prepared by in situ copolymerization of N,N‐dimethyl‐N‐methacryloxyethyl N‐(3‐sulfopropyl) ammonium betaine (SPE), pentaerythritol triacrylate (PETA), and vinylsulfonic acid (VS) in a binary porogenic solvent consisting of cyclohexanol and ethylene glycol. This monolith was developed as a separation column for CEC. While SPE functioned as both an electrostatic interaction stationary phase and the polar ligand provider, VS was employed to generate EOF. PETA, which has much more hydrophilicity due to a hydroxyl sub‐layer, was used to replace ethylene dimethacrylate as a cross‐linker. The monolith provided an adequate EOF when VS level was maintained at 0.6% w/w. Different monolithic stationary phases were easily prepared by adjusting the ratio of PETA/SPE in the polymerization solution as well as the composition of the porogenic solvent. The observed RSD were ≤3.6, ≤4.3 and ≤5.6% for the EOF velocity, retention time, and column efficiency, respectively. The column efficiencies greater than 145 000 theoretical plates/m for thiourea and 132 000 theoretical plates/m for charged cytidine were obtained. The poly(SPE‐co‐PETA‐co‐VS) monolith showed good selectivity for neutral and charged polar analytes. It was found that the separation mechanism of charged polar solutes was attributed to a mixed mode of hydrophilic interaction and electrostatic interaction, as well as electrophoresis. No peak tailing was observed for the separation of basic compounds, such as basic nucleic acid bases and nucleoside on the monolith.     

Determination of vitamin B12 in infant formula and adult nutritionals using HPLC after purification on an immunoaffinity column: first action 2011.09

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," on June 29, 2011, the method "Determination of vitamin B12 in infant formula and adult nutritionals using HPLC after purification on an immunoaffinity column" was recommended by an Expert Review Panel and adopted as AOAC Official First Action status. The method is applicable for the determination of vitamin B12 in milk-based infant formula. Vitamin B12 is extracted from the sample in sodium acetate buffer in the presence of potassium cyanide. After purification and concentration with an immunoaffinity column (IAC), vitamin B12 is determined by LC with UV detection (361 nm). Data supplied by CLF demonstrated linear response over a wide range of concentrations (1.4-39 microg/100 mL). The analytical range is 0.2-10 microg/100 g, depending on the capacity of the IACs (0.01-0.5 microg), the input weight, and dilutions. Recovery rates were assessed using National Institute of Standards and Technology SRM 1849, and determined to be 95.1%, with SD of 0.34 and CV of 9.0. Measurement uncertainty (UE) was 0.8 microg/100 g, which was calculated from the validation data. It is an expanded measurement uncertainty and was obtained through multiplication with a coverage factor k. LOQ values were reported as 0.10 microg/100 g. The performance characteristics of the method met the standard method performance requirements set forth by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals; thus, the method was determined to be appropriate for First Action status.