Immunoaffinity Chromatography Cleanup Research Articles

Survey and risk assessment of aflatoxins and sterigmatocystin in Japanese staple food items and the evaluation of an in-house ELISA technique for rapid screening

Aflatoxins and sterigmatocystin (STC) are carcinogenic mycotoxins frequently detected in many kinds of agricultural products. An analytical method for the simultaneous detection of these mycotoxins was developed using immunoaffinity chromatography clean-up and LC–MS/MS techniques to assess the health risks associated with the exposure to aflatoxins and STC present in staple Japanese food items. A survey targeting Japanese staple food items, including rice and wheat, was conducted after validating the analytical method by conducting a single-laboratory test. A total of 550 samples were analyzed. Aflatoxins were not detected in all samples, whereas STC was detected in 22.4% of the tested samples. The dietary exposure to STC was estimated based on the results of the survey, and the amount of food intake ranged from 0.11 to 0.67 ng/kg body weight/day. The results obtained following the margin of exposure (MOE) approach revealed that STC in staple food items posed little risk to the general Japanese population. In addition, an ELISA technique for detecting STC in brown rice and wheat samples was developed, as high levels of contamination were observed in these food items. The performance of the developed ELISA technique was evaluated using STC-spiked samples, and the average recoveries were in the ranges of 94.6–121.2% for brown rice and 91.5–117.9% for wheat. The developed two analytical methods based on LC-MS/MS and ELISA techniques were efficiently utilized for assessing the health risks associated with exposure to these carcinogenic mycotoxins.

Determination of gentamicins in meat by LC/FL accompanied with online immunoaffinity chromatography cleanup system

Determination of gentamicins in meat by LC/FL accompanied with online immunoaffinity chromatography cleanup system

Simultaneous determination of capsaicin and dihydrocapsaicin for vegetable oil adulteration by immunoaffinity chromatography cleanup coupled with LC-MS/MS.

Capsaicin and dihydrocapsaicin were selected as adulteration markers to authenticate vegetable oils. In this study, a method of immunoaffinity chromatography (IAC) combined with liquid chromatography-tandem mass spectrometry was established for the determination of capsaicin and dihydrocapsaicin in vegetable oils. In this method, immunosorbents were obtained by covalently coupling highly specific capsaicinoid polyclonal antibodieswith CNBr-activated Sepharose 4B, and then packed into a polyethylene column. In this paper, the major parameters affecting IAC extraction efficiency, including loading, washing and eluting conditions, were also investigated. The IAC column displayed high selectivity for capsaicin and dihydrocapsaicin with the maximum capacity of 240ng. The limit of detection (LOD) and limit of quantification (LOQ) for capsaicin were calculated as 0.02 and 0.08μgkg(-1), and for dihydrocapsaicin were 0.03 and 0.10μgkg(-1). The recoveries of capsaicin and dihydrocapsaicin in oil samples were in the range of 87.3-95.2% with the relative standard deviation (RSD) of less than 6.1%. The results indicated that capsaicinoid compounds could not be found in edible vegetable oils. Therefore, the proposed method is simple, reliable and adequate for routine monitoring of capsaicinoid compounds in vegetable oils and has an excellent potential for detection of adulteration with inedible waste oil.

Determination of T-2/HT-2 toxins in duplicate diets in the Netherlands by GC-MS/MS: method development and estimation of human exposure

T-2 and HT-2 toxins (T-2 and HT-2) are important trichothecenes. They have been subject of formal risk assessment by various organisations, including the European Food Safety Authority (EFSA). The EFSA CONTAM Panel recently established a group Tolerable Daily Intake (TDI) of 100 ng/kg body weight/day for the sum of T-2 and HT-2. To assess the actual dietary exposure of Dutch consumers to T-2 and HT-2 a study was conducted in the Netherlands, in which duplicate portions of 24-h diets collected in 2011 were investigated for these toxins. This collection comprised 128 duplicate diets of the adult segment of the Dutch population, divided over a spring and autumn collection period. The diets in the study were homogenised and processed to lyophilised powders. Aliquots of every two of the samples were pooled to test portions that were analysed with a method, based on immunoaffinity chromatography clean-up in combination with GC-MS/MS determination. The method had a limit of quantification of 0.01 ?g/kg original non-lyophilised diet for both T-2 and HT-2. Recoveries ranged from 92-114% for T-2 and from 71-106% for HT-2, determined at levels of addition ranging from 0.1-0.3 ?g/kg. In practically all samples investigated, numerical values for the concentrations of T-2 and HT-2 could be obtained. Exposure estimates of the sum of T-2 and HT-2 in the 2011 study ranged from non-detectable to 18.6 ng/kg body weight/day. In addition limited sets of pooled samples of duplicate diets retained from collections in the period 1976-2004 were analysed for T-2 and HT-2. In all samples the mean and individual intakes of the sum of T-2 and HT-2 of the respondents were below the group TDI of the EFSA CONTAM Panel. From this study it was concluded that no health risks are expected from current exposure of adult Dutch consumers to T-2 and HT-2.

Preparation of immunoaffinity chromatographic column specific to diniconazole and its applications to the pretreatment of samples

The purified anti-diniconazole antibody was polymerised to hydrolytic tetramethoxysilane (TMOS) to synthesize the immunosorbent for the immunoaffinity chromatographic (IAC) column specific to diniconazole. The optimized conditions of the IAC were as follows: water as equilibrium and adsorbent medium, 30% and 50% (v/v) methanol aqueous solutions as eluents. The results showed that the dynamic column capacity was up to 125.4 microg/g of bed volume. The river water and fruit samples spiked with diniconazole were cleaned up and enriched by the IAC, and the diniconazole in eluant was determined by high performance liquid chromatography. The average recoveries (n = 5) of diniconazole in river water sample were 90.36%-100.14% with relative standard deviations (RSDs) of 2.03%-6.08%, and the average recoveries in fruit samples were 85.55%-94.02% with RSDs of 3.38%-6.78%. The IAC cleanup procedure provided an effective pretreatment method for the determination of diniconazole in sample media such as river water and fruits.

Development of a Broad-Specificity Monoclonal Antibody-Based Immunoaffinity Chromatography Cleanup for Organophosphorus Pesticide Determination in Environmental Samples

An immunoaffinity chromatographic (IAC) method for the selective extraction and concentration of 13 organophosphorus pesticides (OPs, including coumaphos, parathion, phoxim, quinalphos, dichlofenthion, triazophos, azinphos-ethyl, phosalone, isochlorthion, parathion-methyl, cyanophos, disulfoton, and phorate) prior to analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The IAC column was prepared by covalently immobilizing a monoclonal antibody with broad specificity for OPs on CNBr-activated Sephrose 4B. The column capacity ranged from 884 to 2641 ng/mL of gel. The optimum elution solvent was 0.01 M phosphate-buffered saline containing 80% methanol. The breakthrough volume of the IAC column was found to be 400 mL. Recoveries of OPs from spiked environmental samples by IAC cleanup and HPLC-MS/MS analysis ranged from 60.2 to 107.1%, with a relative standard deviation below 11.1%. The limit of quantitation for 13 OPs ranged from 0.01 to 0.13 ng/mL (ng/g). The application of IAC cleanup coupled to HPLC-MS/MS in real environmental samples demonstrated the potential of this method for the determination of OP residues in environmental samples at trace levels.

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 a chitosan-supported immunoaffinity chromatography column for the selective extraction of methandrostenolone from food and feed samples

The development of a chitosan-supported immunoaffinity chromatography (IAC) column and its application to the selective extraction of methandrostenolone (MA) from food and feed samples were described in this paper. Using hybridoma technique, a monoclonal antibody (mAb) against MA was produced. The IAC column was prepared by coupling the produced antibody with crosslinked chitosan. Scanning electron microscopy and IR spectroscopy was used to characterize the chitosan crosslinking and antibody coupling. 2% and 90% methanol were respectively selected as loading and eluting solution by optimization. The maximum capacity of the column for MA was 1790 ng/mL gel. The extraction recoveries of the column for MA at three different spiked concentrations ranged from 83.7 to 98.5%. After 2 cycles of usage, the column capacity and extraction recovery still remained 84.6% and 80.5%. To further verify the effect of matrix on the IAC cleanup, MA-fortified food and feed samples were extracted using the prepared IAC column, and MA recovery rates were found to be 86.2% and 70.4%, respectively.

Immunoaffinity removal and immunoassay for rhodamine B in chilli powder

SummaryA rapid and simple immunoassay and a sol–gel‐based immunoaffinity chromatography (IAC) purification method for Rhodamine B (RB) were developed using spiked chilli powder. A polyclonal antibody against RB was generated by immunisation of rabbits with an immunogen hapten. An enzyme‐linked immunosorbent assay (ELISA) was developed that achieved a 50% inhibition (IC50) value of 0.94 ± 0.05 ng mL−1. The limit of detection of the ELISA method was 1 ng g−1 in chilli powder. For the IAC, recovery was 68.1–86.2% at 1 ng g−1 and 72.6–89.3% at 5 ng g−1 in spiked chilli powder. Fortified samples were analysed by high performance liquid chromatography–mass spectrometry (HPLC–MS) after IAC purification, and the results showed a good agreement between the two methods. The ELISA could be a convenient tool for screening RB residue in foods, and the IAC cleanup procedure coupled with HPLC–MS could be an effective alternative method for the determination of RB in various substances.

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.

Preparation of Acetochlor Antibody and Its Application on Immunoaffinity Chromatography Cleanup for Residue Determination in Peanuts

The preparation of an antibody against acetochlor and its application on immunoaffinity chromatography (IAC) cleanup was investigated. The hapten was synthesized by reacting acetochlor with 3-mercaptopropionic acid and then linked with a carrier protein by the carbodiimide method. After immunizing a rabbit with hapten-bovine serum albumin (BSA) conjugate, the obtained antiserum showed an IC(50) value of 0.2 ng/mL. By coupling the purified polyclonal antibody with CNBr-activated Sepharose 4B, an IAC column was prepared. The dynamic column capacity was 5560 ng/mL gel. The IAC was then used to clean up a peanut sample solution, followed by high-performance liquid chromatography (HPLC) separation and ultraviolet (UV) detection. The recoveries of acetochlor from spiked peanut samples at levels of 5-100 microg/kg ranged from 94.7 to 102.5%, with a variation coefficient of 2.6-8.0%, and the limits of detection and quantification were 1.5 and 5 microg/kg, respectively.

Immunoaffinity Chromatography Clean-Up and LC for Analysis of Salinomycin and Narasin in Chicken Muscle

A method has been developed for simultaneous analysis of salinomycin and narasin in chicken muscle. Muscle samples were extracted with acetonitrile. Clean-up of the extracts on an immunoaffinity chromatography column was followed by liquid chromatography with postcolumn derivatisation and UV–visible detection at 520 nm. The immunoaffinity columns were prepared by coupling the anti-salinomycin monoclonal antibody to CNBr-activated Sepharose 4B. When chicken muscle fortified at 5, 25, and 50 ng g−1 was analyzed, intra-assay mean recoveries of salinomycin and narasin were in the ranges 87.5–93.1 and 86.2–94.3%, respectively, with relative standard deviation (RSD) of 4.7–6.2 and 2.4–5.7%. Inter-assay mean recoveries were 86.0–93.0 and 86.0–92.1%, respectively, with RSD of 4.8–6.5 and 5.8–7.4%. The limit of detection of the method was 2.5 ng g−1 for both drugs in chicken muscle.

Occurrence of ochratoxin A in sweet wines produced in Spain and other countries

A survey for the presence of ochratoxin A (OTA) was undertaken from 2001 to 2005 in 188 samples of sweet wines produced in Spain and in 102 samples originating from other countries: France (n = 49), Austria (9), Chile (9), Portugal (9), Greece (6), Italy (5), Germany (3), Hungary (2), Slovenia (2), Switzerland (2), Canada (1), Japan (1), New Zealand (1), Ukraine (1), South Africa (1) and the USA (1). The analytical method was based on immunoaffinity chromatography clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection. The limit of detection (defined as a signal–noise ratio = 3) was estimated to be 0.01 µg l−1. The limit of quantification (0.02 µg l−1) was checked as being the lowest measurable concentration. OTA was detected in 281 out of 290 samples analysed (96.9% positive) at concentrations ranging from 0.01 to 4.63 µg l−1. The overall mean and median levels were estimated to be 0.50 and 0.14 µg l−1, respectively. In Spanish sweet wines OTA was found in 99% of the samples, with mean and median values of 0.65 and 0.19 µg l−1, respectively. The mean value obtained in this study for OTA in the Spanish sweet wines would result in an intake of about 37.5 and 3.2 ng day−1 of OTA for regular consumers and for the overall population, respectively. These figures represent a minor contribution to the provisional tolerable weekly intake (PTWI) or TWI established by the Joint Expert Committee on Food Additives (JECFA) and the European Food Safety Authority: 3.8 and 3.1% for regular consumers; and 0.4 and 0.3% for the whole adult population, respectively.

Simultaneous Analysis of Avermectins in Bovine Tissues by LC-MS-MS with Immunoaffinity Chromatography Cleanup

A rapid, simple and sensitive LC-MS-MS method was developed to simultaneously measure residues of avermectin, ivermectin, doramectin, and eprinomectin in bovine liver and muscle. The extracted samples were cleaned by an immunoaffinity column which was prepared by coupling anti-avermectin polyclonal antibodies with CNBr-activated Sepharose 4B. The residues eluted from the column were analyzed by high-performance liquid chromatography with tandem mass spectrometry. Recoveries fortified at levels 5−50 ng g−1 ranged from 62.93 to 84.03%, with relative standard deviations from 6.02 to 17.39% for all four compounds in liver tissue. The limits of quantification were 5 ng g−1, and limits of detection were 2.5 ng g−1 in liver for the four drugs. The procedure was also applied to bovine muscle, giving similar results.

Determination of Chloramphenicol Residue in Chicken Tissues by Immunoaffinity Chromatography Cleanup and Gas Chromatography with aMicrocell Electron Capture Detector

A rapid and sensitive gas chromatography (GC) method was developed to detect chloramphenicol in chicken tissues. The extracted samples were cleaned up using the immunoaffinity column prepared by coupling antichloramphenicol monoclonal antibody with cyanogen bromide-activated Sepharose 4B. The dynamic column capacity of chloramphenicol was 3265 ng/mL gel. The eluate was evaporated to dryness, and residues were derivatized and determined by GC with a microcell electron capture detector. Average recoveries were 86.6 to 96.9% for chicken muscle and 74.3 to 96.1% for chicken liver. The limit of quantitation of the method was 0.05 ng/g for chicken muscle and 0.1 ng/g for chicken liver.

Validation of Screening Method for Residues of Diethylstilbestrol, Dienestrol, Hexestrol, and Zeranol in Bovine Urine Using Immunoaffinity Chromatography and Gas Chromatography/Mass Spectrometry

A method was developed, using commercially available immunoaffinity chromatography cleanup cartridges, followed by detection by gas chromatography/mass spectrometry, to screen for residues of the hormone growth promotants diethylstilbestrol, dienestrol, hexestrol, and zeranol in bovine urine. The single-laboratory, in-house validation included assessment of recoveries, repeatability, linearity of response, detection capability, and specificity (cross-reactivity) with a suite of antibiotics and other hormonal growth promotants. The method was validated for screening at a target concentration of 2.0 microg/L in urine. The detection capabilities for the analytes were diethylstilbestrol, 0.24; dienestrol, 0.15; hexestrol, 0.84; and zeranol, 0.28 microg/L.

Deoxynivalenol in lebensmitteln: Ergebnisse einer Pilotstudie

Cereal food products (n=333) were purchased in retail stores from Germany in 2001 and analysed for deoxynivalenol (DON), either by enzyme immunoassay or by HPLC after immunoaffinity chromatographic cleanup. Detection limits were dependent of the sample matrix and varied from 20–100 μg/kg. The overall DON incidence was 53%, with mean and median levels for positives of 251 μg/kg and 142 μg/kg, respectively. The contamination with DON (mean/median value, μg/kg) as found for bread (90/87), wheat flour (161/124), and noodles (472/297) indicate that the levels of DON in cereal foods were significant in view of the tolerable daily intake (1 μg/kg body weight) as established by the European Union scientific committee on food.

Development of an enzyme-linked immunosorbent assay for the determination of maduramicin in broiler chicken tissues.

Maduramicin is one of the most widely used coccidiostats in the world. A rapid and accurate analytical method for this drug should provide producers and users with an effective management tool. The current chromatographic methods are sensitive but labor-intensive. This paper reports the development of an enzyme-linked immunosorbent assay (ELISA) based on an immunoaffinity chromatography cleanup procedure for the analysis of maduramicin in broiler chicken tissues (including muscle, liver, and fat). Recoveries from fortified tissue homogenates at levels of 30.0-120.0 microg kg(-)(1) ranged from 76.4 to 107.5% with coefficients of variation of 3.8-16.4%. The limits of detection were 1.0 ng g(-)(1) in muscle, 2.8 ng g(-)(1) in liver, and 1.5 ng g(-)(1) in fat. The ELISA results from the analysis of incurred residue in tissue samples showed the cleanup procedure is viable.

Immunoaffinity column clean-up for the determination of imazalil in citrus fruits

We have developed a new clean-up method with immunoaffinity purification using anti-imazalil monoclonal antibody (MAB). Immunoaffinity purification of residual imazalil in citrus fruits could lead to a more effective and specific clean-up technique for isolation and concentration. A column containing anti-imazalil MAB immobilized to CNBr-activated agarose gel was prepared, and utilized for immunoaffinity chromatographic (IAC) clean-up for imazalil in citrus fruit samples such as lemons, oranges, and grapefruits. The prepared IAC clean-up column had sufficient total column capacity, and could be reusable at least thirty times per one IAC clean-up column. The newly developed method was able to eliminate co-exiting sample matrices originated from citrus fruits more successfully than the conventional clean-up method in respect of the simplicity and the rapidity of the procedure. The recoveries from citrus fruit samples fortified with imazalil at 0.05, 0.5, and 5 ppm were in the range of 85.2 to 98.8% with coefficients of variation (CV) of 1.8–6.6%. It took only about 1 h to complete the whole procedure from extraction to HPLC analysis. Furthermore, a clean analysis was achieved by using the proposed IAC clean-up method. The applicability of this method was demonstrated by measuring the residual imazalil in commercial citrus fruits. The study suggested that the IAC clean-up column for imazalil will be able to overcome some difficulties the conventional analyses for pesticides residue in foods were not able to.

Ochratoxin A in dried vine fruit: method development and survey

A method is described for the determination of concentrations of the mycotoxin ochratoxin A in dried vine fruits (currants, raisins and sultanas) using acidic methanolic extraction,immunoaffinity chromatography clean-up and HPLC determination. The limit of detection was estimated as 0.2μg/kg, and recoveries of 63-77% were achieved at 5μg/kg. HPLC-mass spectrometric confirmation of the identity of ochratoxin was obtained. Ochratoxin A and aflatoxins were determined in 60 samples of retail dried vine fruits purchased in the United Kingdom. Ochratoxin A was found in excess of 0.2μg/kg in 19 of 20 currant, 17 of 20 sultana and 17 of 20 raisin samples examined, an overall incidence of 88%. The maximum level found was 53.67μg/kg. No aflatoxin was found in any sample analysed, using a method with a detection limit of 0.2μg/kg for each of aflatoxin B1, B2, G1 and G2.