Preparation of an immunoaffinity column and its application in sample cleanup for methandrostenolone residues detection

Methandrostenolone (MA) is a steroid used as veterinary medicine on stockbreeding to promote animal growth. The use of MA has been strictly regulated because of its harmful effect on consumers. This paper describes the production of polyclonal antibody (pAb) against MA, the preparation of immunoaffinity column (IAC) and its potential application to the selective extraction of MA residues from animal tissue and feed samples. The produced pAb exhibited good sensitivity to MA with an IC(50) value of 5.6 ng/mL. The cross-reactivity values of the antibody with MA structurally related compounds of testosterone propionate (TP) and trenbolone (TR) were lower than 0.6%. By coupling the produced antibody with CNBr-activated Sepharose 4B, an IAC was prepared. 2% methanol and 80% methanol were selected as loading and eluting solution by optimization. The maximum capacity of the column for MA was approximately 334 ng/mL gel. The average recovery of 20, 40 and 60 ng/mL MA standard solutions from IACs was 97.9% with the relative standard deviation (RSD) among columns of 6.7%. After 3 times of repeated usage, the column capacity and recovery rate still remained 82.0% and 92.6% respectively. The IACs were then challenged with MA-fortified animal tissue and feed samples, recoveries of MA were found to be in the range of 83.5-99.7%.     

Selective sample cleanup by immunoaffinity chromatography for determination of fenvalerate in vegetables

This paper describes the establishment of an immunoaffinity chromatography (IAC) for selective extraction of fenvalerate from vegetable samples. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against fenvalerate to CNBr-activated Sepharose 4B and packed into a cartridge. The extraction conditions were carefully optimized, including loading, washing and eluting solutions. Under the optimal conditions, the IAC column was able to capture fenvalerate with the maximum capacity of 4000 ng. An average recovery of 94.5% and a RSD of 8.8% were obtained with six IAC columns prepared on six different days. Three vegetable samples spiked with fenvalerate at four different concentrations were extracted with IAC column and determined by gas chromatography with electron capture detection (GC-ECD). Chromatograms of final extracts were clean and fenvalerate could be easily detected without the interferences. The extraction recoveries and RSD were 74.7-96.5% and 2.5-5.2%, respectively, and the calculated limit of detection of the whole method was 0.008-0.012 ng g(-1).     

Development and application of a multi-target immunoaffinity column for the selective extraction of natural estrogens from pregnant women's urine samples by capillary electrophoresis

In this paper, a methodology for the determination of three naturally occurring estrogens (estradiol, estrone and estriol) in pregnant women's urine has been described. The procedure included immunoaffinity column (IAC) extraction of 4 mL of urine sample and subsequent analysis of the extraction by micellar electrokinetic chromatography (MEKC). A multi-target polyclonal antibody that has high affinity to three estrogens was produced. Then the IAC was developed by coupling polyclonal antibody to CNBr-activated Sepharose 4B. The IAC showed high affinity for these estrogens. Recoveries of three estrogens from human serum matrix were greater than 92% with R.S.D. less than 4.5%. The final elute of urine sample was diluted with running buffer and then quantitated with MEKC. The experimental results demonstrated that IAC was a useful technique for extraction and concentration of estrogens from biological samples. Three estrogens levels in six pregnant women's urine were measured by both the present method and enzyme-linked immunoadsorbent assay (ELISA). The results of this method have been found to correlate well with those of ELISA.     

Determination of deoxynivalenol in cereals by immunoaffinity clean-up and ultra-high performance liquid chromatography tandem mass spectrometry

An immunoaffinity column (IAC) was prepared with a new deoxynivalenol (DON) monoclonal antibody and used as a clean-up tool before ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis of DON in cereals. The developed IAC clean-up method showed high recoveries for DON. They ranged from 61% to 103% in wheat, rice, and millet with intra-day and inter-day variations below 19% and 17%, respectively. The column capacity was 2.86μg DON per mL of gel, and it maintained above 0.68μg/mL of gel after 10 cycles of usage at 2 days intervals. The limit of detection (LOD) and limit of quantification (LOQ) were 0.3 and 0.8μg/kg, respectively. Twenty-one out of 40 analyzed commercial cereal samples were positive at DON concentrations from 7 to 534μg/kg.     

Purification of antibody against Ara h 2 by a homemade immunoaffinity chromatography column

Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody, a homemade immunoaffinity chromatography (IAC) column method was established. The properties of homemade column were compared with those of the mAb affinity protein G (MPG) agarose high flow, a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits’ antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG. The homemade IAC could separate the corresponding antibody with higher specificity and lower cost but with lower recovery and column capacity than those of MPG. Thus, the homemade IAC is a specific, inexpensive, and suitable method that can be used for various laboratory purifications.     

Improved clean-up method for the enkephalins in plasma using immunoaffinity chromatography

A rapid and selective method of sample clean-up using immunoaffinity chromatography (IAC) was developed to isolate enkephalins from plasma. The enkephalin antibodies were produced utilizing novel protein carriers. Two antibodies, LE4H8 and 33FC6, were selected because of their moderate binding affinity and different epitopes. Enkephalin-spiked plasma was loaded onto the immunoaffinity column and eluted with acidic pH buffer. The eluate was derivatized with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA-CN), and the enkephalins were separated using reversed-phase liquid chromatography (RPLC). IAC sample clean-up of enkephalin-spiked plasma was compared to the existing solid-phase extraction method. The limit of detection for IAC was 30 pmol. The recovery of the enkephalins from plasma was 90% with a variance ranging from 2 to 9%. The immunoaffinity column was used for approximately 70 samples without any deterioration in performance.     

Comparison and evaluation of the specificity and binding capacity of commercial and in house affinity columns used in sample preparation for analysis of growth-promoting drugs

Immunoaffinity chromatography (IAC) and affinity chromatography (AC) are widely used for extraction of drugs from biological samples. Fifteen column types were purchased from five different manufacturers and their ability to bind specific drugs including β-agonists and anabolic steroids over a range of analyte concentrations in fortified bovine urine samples was assessed. The performance data obtained from these columns were compared with columns produced in this laboratory (in house columns). The in house columns gave the highest recoveries, ranging from 92 to 100% at the 1 ng spiking concentration, for five of the seven analytes assessed. Forty percent (11 of 27) of all the commercial column assessments recorded recoveries of less than 50% even when the lowest spiking concentration was applied (1 ng). For one manufacturer, only one of seven different columns purchased delivered extraction efficiencies greater than 50%. The extraction efficiencies of the clenbuterol columns were the highest with all commercially prepared columns showing at least 50% binding of radiolabelled tracer. Recoveries of -nortestosterone were the lowest. The variability of these products with respect to quality control requires constant monitoring.     

Development and characterization of an immunoaffinity column for the selective extraction of bisphenol A from serum samples

An immunoaffinity column (IAC) was developed by covalently coupling polyclonal antibodies against estrogenic bisphenols to CNBr-activated Sepharose 4B. The IAC showed high affinity for bisphenol A, while phenol was barely retained. Proteins in the sample matrix showed little nonspecific adsorption on the column. The best binding solvent for bisphenol A was found to be 0.01 mol l(-1) phosphate-buffered saline (PBS) and the optimal operating temperature was 4 degrees C. The bound bisphenol A could be quantitatively recovered by 1 ml of methanol-water (80:20) with an average recovery of 91.8% and a relative standard deviation of 7.1% (n=6). The immunoaffinity column has been successfully used for the isolation and purification of bisphenol A from serum samples.     

Efficient purification and preconcentration of erythropoietin in human urine by reusable immunoaffinity column

In this paper, an efficient method is proposed for purification and preconcentration of erythropoietin (EPO) in human urine samples. The EPO-specific immunoaffinity column (IAC) was generated by covalent immobilization of anti-EPO polyclonal antibodies on Sepharose 4B support. The EPO-binding capacity of the IAC was found to be about 2.0 microg (6.6IU) per 1.5 mL of gel and the activity recoveries of EPO at low concentrations of 7.8, 10 and 120 m IU/mL by the IAC were between 78 and 86%. Substantial cleanup effect was observed when the IAC was applied to human urine samples.     

HPLC determination of fumonisin mycotoxins in maize: a comparative study of naphthalene-2,3-dicarboxaldehyde and o-phthaldialdehyde derivatization reagents for fluorescence and diode array detection

Fumonisins are mycotoxins produced by various species of Fusarium and occur naturally in contaminated maize and maize-based foods. Ingestion of fumonisins has considerable health implications for humans and animals. Since fumonisins lack a useful chromophore or fluorophore, their determination in maize is routinely achieved via HPLC with fluorescence detection (FLD) after precolumn derivatization. This study optimized naphthalene-2,3-dicarboxaldehyde (NDA) derivatization of fumonisins in naturally contaminated maize following strong anion exchange (SAX) solid phase extraction (SPE) clean-up and utilizing diode array detection (DAD) as a practical alternative simultaneously to FLD. The limit of detection (LOD) for fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) with FLD was 0.11 ng, 0.50 ng and 0.27 ng, respectively, and with DAD it was 13.8 ng, 12.5 ng and 6.6 ng, respectively injected on column. The coefficient of variation (CV, n = 6) for FB(1), FB(2) and FB(3) in a naturally contaminated samples obtained with FLD was 2.6%, 1.8% and 5.3%, respectively, compared to 6.0%, 3.4% and 9.5%, respectively, obtained with DAD. Subsequently the optimized NDA derivatization was compared to the widely used o-phthaldialdehyde (OPA) derivatization agent as well as alternative sample clean-up with immunoaffinity column (IAC) by analyzing naturally contaminated maize samples (n = 15) ranging in total fumonisin (TFB = FB(1)+FB(2)+FB(3)) levels from 106 to 6000 μg/kg. After immunoaffinity column clean-up of extracted samples, the recoveries of spiked maize samples for NDA-FLD of FB(1), FB(2) and FB(3) were 62%, 94% and 64%, respectively. NDA proved to be an effective derivatization reagent of fumonisin in naturally contaminated maize samples following IAC clean-up, except for DAD at TFB levels below 1000 μg/kg. In contrast NDA derivatization following SAX clean-up produced results comparable to OPA only for levels below 1000 μg/kg. Aside from the difference in detection limits, FLD and DAD produced comparable results irrespective of the clean-up method or the derivatization agent.     

Simultaneous determination of thiamphenicol,florfenicol and florfenicol amine in swine muscle by liquid chromatography–tandem mass spectrometry with immunoaffinity chromatography clean-up

A rapid and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512 ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4–50 ng/g, the average recoveries were 85.2–98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0 μg/kg.     

Carry-over of deoxynivalenol into eggs of laying hens — Preliminary results

Carry-over of deoxynivalenol (DON) into eggs was investigated within the scope of a 16-week experiment with laying hens, in which the birds were fed a maize-based diet containing DON at 11.9 mg/kg dry matter. Eggs were collected during weeks 2, 4, 8, and 16. DON and its metabolite deepoxy-DON were analysed separately in freeze-dried yolk and albumen. Yolk was extracted with water and the extract was purified using an immunoaffinity column (IAC). Albumen was extracted with acetonitrile-water and the extract was pre-cleaned before applying an IAC. All albumen and some yolk samples were incubated with β-glucuronidase prior to extraction. DON and de-epoxy-DON were determined by high performance liquid chromatography (HPLC) with diode array detection (DAD). The detection limits of both toxins were 20 ng/g and 15 ng/g in freezedried yolk and albumen, respectively, corresponding to approximately 10 ng/g and 2 ng/g in fresh samples. The recovery of DON/de-epoxy-DON in spiked samples (50–200 ng/g) was 87/83% (yolk) and 87/77% (albumen) with coefficients of variation of 4–15%. Neither DON nor de-epoxy-DON were detected in any of the samples. In order to achieve lower detection limits, the methods are currently optimized. However, these preliminary results indicate that eggs do not contribute significantly to the dietary DON intake of the consumer. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004     

On the isolation of polychlorinated dibenzo-p-dioxins and furans from serum samples using immunoaffinity chromatography prior to high-resolution gas chromatography–mass spectrometry

Immunoaffinity chromatography (IAC) for the purification of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) from biological samples was explored as a means to simplify the cleanup procedure and thereby decrease the time and cost of dioxin analysis. A monoclonal antibody (DD3) was used to produce IAC columns and to isolate the PCDD/Fs from serum. Native and ¹³C-labeled PCDD/Fs were spiked at the ppq to ppt range into serum. Quantitation of the PCDD/Fs was performed by a standard dioxin analytical method, i.e. high-resolution gas chromatography–mass spectrometry (GC–MS), which was easily compatible with IAC. Five of the most toxic PCDD/Fs consistently showed acceptable recoveries (>25%) and were reliably quantitated. The congeners specifically recovered by this method represent almost 80% of the toxic equivalency of dioxins and furans present in the serum samples. Dioxin-like polychlorinated biphenyls (PCBs) were not recognized by this antibody column. Compared to conventional dioxin cleanup methods, IAC decreased solvent usage by 1.5 l/sample and took only 2 h to process a sample for analysis.     

Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC-APCI-MS

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.     

Development of a reference material using methamphetamine abusers' hair samples for the determination of methamphetamine and amphetamine in hair

In the present study, we developed a reference material (RM) using authentic hair samples for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) in human hair. MA abusers' hair samples were collected, homogenized and finally bottled. The concentration of each bottle was determined using two extraction methods, agitation with 1% HCl in methanol at 38 degrees C and ultrasonication with methanol/5M HCl (20:1), followed by gas chromatography/mass spectrometry (GC-MS) after derivatization with trifluoroacetic anhydride (TFAA). Both analytical procedures were fully validated and their extraction efficiency was compared. The homogeneity of analytes was evaluated and their property values were determined with their uncertainties. The two methods were acceptable to analyze MA and AP in human hair through the validation and comparative studies using spiked and authentic hair samples as well as NIST SRM 2379 certified reference material. Satisfying homogeneity was reached for MA and AP in the prepared RM. Finally, a human hair RM containing MA and AP is prepared at the level of 7.64+/-1.24 and 0.54+/-0.07 ng/mg, respectively. This material can be useful in forensic laboratories for internal quality control and external quality assurance.     

Determination of epitestosterone in human urine by off-line immunoaffinity solid-phase extraction coupled with high performance liquid chromatography

Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.     

Evaluation of different primary recovery methods for E. coli-derived recombinant human growth hormone and compatibility with further down-stream purification

Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.     

A simple progesterone radioimmunoassay without column chromatography

A radioimmunoassay for plasma progesterone without Chromatographie purification was developed. The 11-hemi-succinate of 11 -hydroxy-progesterone conjugated to bovine serum albumin was injected into rabbits to stimulate antibody production. The resulting antisera was used at a final dilution of 1:3500. The mean recovery of labeled progesterone added to 100 samples after ether extraction (88.9 ± 9.1%) was higher than the recovery obtained when column chromatography followed ether extraction (84.8 ± 7.5%). For comparison, plasma pools were assayed for progesterone with and without the use of columns. A female plasma pool (luteal phase) gave a mean of 546.3 ± 26.5 (SD) ng/100 mls (n = 5) without column chromatography and 557.2 ± 20.8 (SD) ng/100 mls (n = 5) with column chromatography. Another female plasma pool (follicular phase) gave a mean of 87.9 ± 9.6 (SD) ng/100 mls (n = 24) with column chromatography and 93.3 ± 8.6 (SD) ng/100 mls (n = 7) without column chromatography. A male plasma pool gave a mean of 22.8 ± 4.4 (SD) ng/100 mls (n = 13) with column chromatography and 21.8 ± 7.7 (SD) ng/100 mls (n = 3) without column chromatography. The intra assay and inter assay precision gave a coefficient of variation of 3.7 for six samples and 10.9 for 24 samples, respectively. The specificity of the antibody was determined by checking cross reactivity with 26 steroids. The sensitivity (25 pg) and accuracy were proven to be highly satisfactory.     

Validation of an HPLC Analytical Method Coupled to a Multifunctional Clean-up Column for the Determination of Deoxynivalenol

To evaluate a method using a multifunctional clean-up column coupled with high performance liquid chromatography as an official analytical method for the determination of deoxynivalenol in wheat used as food or feed, an inter-laboratory study was performed in 12 laboratories using four naturally contaminated wheat samples and one spiked sample. The relative standard deviations for repeatability (RSD_r) and reproducibility (RSD_R) of naturally contaminated wheat were in the range 5.8–11.3% and 12.0–20.7%, respectively. The HORRAT was less than 1.0 in each sample. From the spiking test, the recovery rate, RSD_r, RSD_R and HORRAT value were 100.0%, 11.2%, 10.3% and 0.5, respectively. The limit of quantification is 0.10 mg/kg from the range obtained in a linear calibration. Thus, it should be useful as a sensitive and validated analytical method for the determination of deoxynivalenol in wheat intended for use in food and feed.     

Determination of Diniconazole in Agricultural Samples by Sol-Gel Immunoaffinity Extraction Procedure Coupled with HPLC and ELISA

Background

In the European Union (EU), the use of diniconazole-M is no longer authorized. However, residues of diniconazole-M occur in various plant commodities.

Methodology/Principal Findings

A selective and simple analytical method for the trace level determination of diniconazole in soil, fruit, vegetables and water samples was developed based on immunoaffinity extraction followed by Enzyme-linked immunosorbent assay (ELISA) and the high-performance liquid chromatography (HPLC) analysis. The ELISA was based on monoclonal antibodies highly specific to diniconazole and was a fast, cost-effective, and selective screening method for the detection of diniconazole. The results of the ELISA correlated well with gas chromatography (GC) results, with the correlation coefficient of 0.9879 (n = 19). A simple gel permeation chromato- graphy clean-up method was developed to purify extracts from matrices containing high amounts of fat and natural pigments, without the need for a large dilution of the sample. The immunoaffinity column (IAC) capacity was 0.180 mg g⁻¹. The columns could be re-used approximately 20 times with no significant alteration in capacity. The recoveries from complex samples were in the range of 89.2% to 96.1% with a relative standard deviation (RSD) of 0.770%–6.11% by ELISA. The results were in good agreement with those obtained by HPLC method.

Conclusion/Significance

The IAC extraction procedure coupled with HPLC and ELISA analysis could be also used as alternative effective analytical methods for the determination of diniconazole concentrations in complex samples.