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.     

Preparation and evaluation of a mixed-bed immunoaffinity column for selective purification of sixteen sulfonamides in pork muscle

This paper describes the preparation of a novel mixed-bed immunoaffinity chromatography (IAC) column by coupling four monoclonal antibodies against different sulfonamides (SAs) to Sepharose 4B. The IAC column can be used to simultaneously extract and purify 16 SAs in pork muscle. The dynamic column capacities for all SAs in mixed standard solution were between 312 and 479 ng/mL gel. After simple extraction and IAC cleanup, the sample solution can be directly injected for liquid chromatography-ultraviolet analysis. The recoveries of SAs from spiked samples at levels of 25, 50 and 100 μg/kg ranged from 83.3 to 103.1% with variation coefficient less than 8.6%. The comparison of IAC with liquid-liquid extraction and solid phase extraction indicated that IAC has better purification effect and needs less organic solution than conventional methods, thus it would be an ideal method for selective purification of SAs in pork muscle.     

Development and characterization of an immunoaffinity monolith for selective on-line extraction of bisphenol A from environmental water samples

Matrix interference and contamination from analytical procedures are two major issues in the detection of trace level of bisphenol A (BPA) in environmental water. In this paper, we report a highly selective and efficient method for on-line extraction of BPA from water samples followed by quantification with liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI/MS/MS). Poly(ethylene dimethacrylate-glycidyl methacrylate) monolith was synthesized in 50 mm × 4.6 mm i.d. stainless steel cartridges and the epoxy-groups on the surface of the monolith were hydrolyzed and oxidized to aldehyde functions. Antibodies against BPA were covalently immobilized onto the monolithic column via Schiff base reaction. The optimum application buffer and elution buffer were found to be pH 5.5 phosphate buffered saline (PBS) and methanol–water (70:30, v/v), respectively. The obtained immunoaffinity monolithic columns were on-line coupled to LC–MS/MS using column-switching valves and the system was applied to analyze BPA in real environmental water samples. The method achieved a detection limit of 0.3 ng L⁻¹ using a sample volume of 100 mL. The linear calibration range was 1.0–160 ng L⁻¹. Samples including tap water, lake water and effluent from municipal sewage treatment plant were all measured with satisfactory results.     

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.     

Preparation and evaluation of molecularly imprinted solid-phase micro-extraction fibers for selective extraction of phthalates in an aqueous sample

A novel molecularly imprinted polymer (MIP) that was applied to a solid-phase micro-extraction (SPME) device, which could be coupled directly to gas chromatograph and mass spectrometer (GC/MS), was prepared using dibutyl phthalate (DBP) as the template molecule. The characteristics and application of this fiber were investigated. Electron microscope images indicated that the MIP-coated solid-phase micro-extraction (MI-SPME) fibers were homogeneous and porous. The extraction yield of DBP with the MI-SPME fibers was higher than that of the non-imprinted polymer (NIP)-coated SPME (NI-SPME) fibers. The MI-SPME fibers had a higher selectivity to other phthalates that had similar structures as DBP. A method was developed for the determination of phthalates using MI-SPME fibers coupled with GC/MS. The extraction conditions were optimized. Detection limits for the phthalate samples were within the range of 2.17-20.84 ng L⁻¹. The method was applied to five kinds of phthalates dissolved in spiked aqueous samples and resulted in recoveries of up to 94.54-105.34%, respectively. Thus, the MI-SPME fibers are suitable for the extraction of trace phthalates in complicated samples.     

Preparation and characterization of molecularly imprinted microspheres for selective extraction of trace melamine from milk samples

We describe molecularly imprinted microspheres (MIMs) for the selective extraction of melamine from milk. The MIMs were made from melamine as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the linking agent. The MIMs were synthesized by suspension polymerization and characterized by rebinding experiments. They displayed high adsorption capacity, fast rebinding kinetics, and highly specific rebinding of melamine. The imprinting factor is 4.1. Scatchard analysis revealed a one-type rebinding behavior, the dissociation constant and maximum rebinding capacity being 37.59 g L^?1 and 30.85 μmol g^?1, respectively. The MIMs exhibited a 25% cross-reactivity towards atrazine, but less than 3.0% towards prometryn, clenbuterol and metronidazole. In addition, a MIM-based solid phase extraction (MISPE) column for melamine was prepared by packing MIMs into a common SPE cartridge. The MISPE extraction gave recoveries of 89.8 to 100.6% of melamine, with relative standard deviations of 5.9 to 7.5%. There was no significant loss of rebinding capacity after more than 60 repeated uses, thus demonstrating the high stability of the MISPE column. The MSPE column also was applied to the extraction of melamine from spiked liquid and powdered milk with satisfying accuracy and precision.

Establishment of an immunoaffinity chromatography for simultaneously selective extraction of Sudan I, II, III and IV from food samples

The establishment of an immunoaffinity chromatography (IAC) for simultaneously selective extraction of four illegal colorants Sudan dyes (Sudan I, II, II and IV) from food samples was described. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against Sudan I to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. It was observed that IAC column was able to separately capture Sudan I, II, III and IV with maximum capacity of 295, 156, 184 and 173ng, respectively. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the extraction recoveries of the IAC column for Sudan I-IV at two different spiked concentrations were within 95.3-106.9%. After 50 times repeated usage, 64% of the maximum capacity was still remained. Six food samples randomly collected from local supermarket without spiking Sudan dyes were extracted with IAC column and detected by high performance liquid chromatography (HPLC). It was found that there was no detectable Sudan II, III and IV in all six food samples, but Sudan I with the content of 2.7-134.5ngg(-1) was detected in three food samples. To further verify the extraction efficiency, other three negative samples were spiked with Sudan I-IV at the concentrations of 20ngg(-1) and 50ngg(-1), which were then extracted with IAC column. The extraction recoveries and relative standard deviation (RSD) were 68.6-96.0% and 4.8-15.2%, respectively, demonstrating the feasibility of the prepared IAC column for Sudan dyes extraction.     

Preparation and characterization of an extraction chromatography column for technetium separation based on Aliquat-336 and silica gel support

An extraction chromatographic material based on Aliquat-336 anchored on hydrophobized silica gel support was prepared as an ion exchanger. The prepared material appeared to be suitable for the separation of ⁹⁹Tc from environmental matrices in column application. The properties of the material, sorption characteristic and distribution coefficient of ^99mTcO₄ ^-in various media were studied. The prepared sorbent was conditioned by washing with nitric acid. The solution containing ^99mTcO₄ ^- in 0.1M HNO₃ was passed through the column. Tc was eluted from the column by 8M HNO₃. The flow rate was 0.4 ml/min. The chemical yield of technetium during the separation process was determined using ^99mTc tracer and gamma measurement. The sorption recovery of Tc from the prepared sorbent with 0.1M HNO₃ solution was more than 98%, and the desorption recovery from the column using 8M HNO₃ varied between 92-96%. It was found that the prepared sorbent is suitable for the separation of technetium from environmental matrices and radioactive wastes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of an immunoaffinity extraction column for enrichment of adducts between human serum albumin and hexahydrophthalic anhydride in plasma

An immunoaffinity extraction (IAE) column was prepared for extraction of adducts between human serum albumin (HSA) and hexahydrophthalic anhydride (HHPA). HHPA is a strong sensitizer inducing immunoglobulin E antibodies in vivo. Polyclonal antibodies from a rabbit immunized with keyhole limpet hemocyananin-HHPA conjugate were purified using a Protein A Sepharose gel. To obtain antibodies with optimal affinity towards HHPA-protein adducts, HHPA-specific antibodies were selected using an N-hydroxysuccinimide-Sepharose column coupled with albumin-HHPA conjugate. Antibodies eluted from this column at pH 2.2 were selected to prepare the IAE column. The column was evaluated using 2 mL plasma spiked with HSA-HHPA conjugate. The column was eluted with glycine buffer at pH 2.0. The conjugates in the eluate were hydrolyzed to the corresponding HHP acid and quantified by mass spectrometry. The average recovery of HHPA adducts in 11 experiments was 68% with a coefficient of variation (CV) of 7%. The column's capacity to bind protein-HHPA adducts was found to be linear in the range of 0.15-1.2 nmol conjugate. The evaluation showed that the IAE column had adequate affinity towards the HHPA adducts and that the adducts could be extracted with good recovery and precision from a large volume of plasma.     

Novel molecularly imprinted monolithic column for selective on-line extraction of ciprofloxacin from human urine

Water-compatible ciprofloxacin-imprinted monolithic columns were synthesized in water-containing systems for selective extraction of ciprofloxacin from human urine samples. Methanol-water (10:3, v/v) was used as a porogenic solvent and the obtained monolithic imprinted polymers reveal high selectivity to ciprofloxacin in an aqueous environment; the affinity can be easily controlled by adjusting the pH of the mobile phase. Owing to the unique porous structure and flow-through channels existing in the network skeleton of the monolithic MIP, urine samples could be directly injected into the column, proteins and other biological matrix were quickly washed out and ciprofloxacin was selectively retained and enriched. Good linearity was obtained from 0.08 to 400 mg/L (r=0.998) with the relative standard deviations less than 3.6%. The limit of detection of the method was 0.04 mg/L and the recoveries were more than 94.5% at three different concentrations. Moreover, by increasing the injection volume to 2.0 mL, the sensitivity of the method could be improved 100-fold according to the peak height of ciprofloxacin. This expedient greatly simplified the overall procedure, resulting in a rapid and efficient sample analysis while maintaining precision and accuracy.     

Development and characterisation of an immunoaffinity chromatographic column for the on-line determination of the pesticide triclopyr

An immunoaffinity column for the selective extraction and concentration of the herbicide triclopyr from water samples and quantification on line by HPLC has been developed. The immunoaffinity device was prepared by immobilising triclopyr antibodies to hydrazide derivatized azlactona beds. Efficient desorption of bound triclopyr was achieved with 70% ethanol/water solution. The column was evaluated regarding selectivity, recovery, capacity, saturation volume and reusability. Obtained results show that immunoaffinity chromatography (IAC) can be used for quantitative extraction, concentration and determination of triclopyr from water samples.     

Novel immunoassay and rapid immunoaffinity chromatography method for the detection and selective extraction of naringin in Citrus aurantium

In this work, a novel monoclonal antibody specific for naringin was prepared and characterized. Subsequently, an indirect competitive enzyme‐linked immunosorbent assay for naringin was developed, with an effective range from 4.8 to 156 ng/mL naringin. Next, an immunoaffinity column was obtained by coupling anti‐naringin monoclonal antibodies to CNBr‐activated Sepharose 4B and a rapid immunoaffinity chromatography assay for naringin was developed. The immunoaffinity column was used to separate naringin from Citrus aurantium. The results showed that 1 g of the dry Sepharose 4B can couple 10 mg of immunoglobulin G. And the immunoaffinity column can efficiently and specifically capture approximately 250 μg of naringin without cross reacting with its structurally similar compounds. Moreover, our results indicate that the application of immunoaffinity chromatography can simplify the pretreatment and the isolation process greatly compared to conventional methods, providing a potential method for extracting the target component from structurally similar compounds in natural products.     

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.     

Utilization of fat retainers in supercritical fluid extraction for the selective extraction of polychlorinated biphenyls from a model fat sample

Two common fat retainers used in supercritical fluid extraction--basic alumina and the silica based adsorbent Florisil--were investigated using lard fat as model material. With a fat retainer in the extraction cell it was possible to obtain fat-free time windows. Activation by heating did not influence the length of the time windows, while deactivation of the retainers with 10% water (w/w) drastically decreased the fat retaining capabilities. The influence of modifier addition was also investigated. Finally, a method was developed, where basic alumina was utilized to selectively extract polychlorinated biphenyls (PCBs) from a model fat sample, containing PCBs, triglycerides and phospholipids. The PCBs could be quantitatively extracted in a totally fat-free time window.     

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).     

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。     

Removal of poliovirus type 1 from a protein mixture using an immunoaffinity chromatography column.

An immunoaffinity matrix was prepared using human polyclonal antibody (Intragam) attached to Sepharose 4B activated with CNBr. The immunoaffinity matrix was then assessed with regard to its capacity to remove viruses. The challenge virus, poliovirus type 1 was loaded in high titre in either PBS or a preparation derived from human plasma known as supernatant II + III. This fraction is depleted of IgG and is used to prepare human albumin. It was shown that an average greater than 5 logs of spiked virus were removed in one passage through the column. This type of approach may prove useful as a viral removal method in biopharmaceutical manufacturing.     

一种新型固相萃取柱结合超高效液相色谱-串联质谱法选择性富集测定猪肉中的盐酸克伦特罗

发展了一种测定猪肉中盐酸克伦特罗残留的简便、高效、准确的固相萃取-超高效液相色谱-串联质谱(SPE-UPLC-MS/MS)的方法。将搅碎的猪肉样品用5%(v/v)高氯酸超声提取,再以10000 r/min离心15 min后,上清液用SMCX固相萃取柱进行富集和净化。因SMCX是以硅胶为基质兼有反相/强阳离子交换的混合作用模式,因此可以有效地去除复杂基质干扰,达到目标样品的选择性富集和净化的目的。方法学结果表明,该方法在0.25～50 μg/kg范围内具有良好的线性关系,相关系数r为0.9982; 3个添加水平(1.25、12.5、50 μg/kg)的平均回收率为62.2%～ 72.0%,相对标准偏差(RSDs)为4.2%～ 6.1%;检出限(S/N=3)为0.05 μg/kg。所发展的样品前处理和检测方法简单、快速,可用于瘦肉精类成分的选择性富集和分离检测。     

On-line immunoaffinity sample pre-treatment for column liquid chromatography: evaluation of desorption techniques and operating conditions using an anti-estrogen immuno-precolumn as a model system.

An immunoaffinity precolumn (immuno-precolumn) containing an immobilized antibody directed against estrogen steroids, was used as a model system for the evaluation of different desorbing techniques, suitable for on-line coupling to column liquid chromatography (LC). Desorption of estrogen analytes from the immuno-precolumn proved to be impossible with the buffers and chaotropic solutions frequently used in affinity desorption. Micellar solutions are effective in obstructing the antibody-antigen reaction, but their use as desorbing solutions was not found to be practical because of the large interferences introduced into the chromatograms. Desorption with aqueous solutions at elevated temperature, created by microwave action or conventional heating, although effective is not practical in this instance, because the agarose used in this study as the stationary phase for the immuno-precolumn is prone to heat decomposition. The most effective and practical approach is desorption with a methanol-water mixture (95 + 5 v/v). On-line dilution of the eluate allows the concentration of the desorbed analytes using a reversed-phase LC system with subsequent separation and ultraviolet detection. The performance of the system with spiked urine and plasma samples, which were introduced directly into the system, was evaluated and the results were compared with immunoselective desorption.