Immunobiosensor--an alternative to enzyme immunoassay screening for residues of two sulfonamides in pigs.

A rapid immunoassay using an optical biosensor (BIAcore) for determining the presence of sulfamethazine (SMT) residues in pig bile was developed. The assay was used in a routine screening laboratory alongside a previously described biosensor method for sulfadiazine (SDZ). Sulfonamide bile concentrations, determined by enzyme immunoassay (EIA), have already been shown suitable for use in predicting the extent of sulfonamide accumulation in kidney. The ability of immunobiosensor based bile screening to predict violative tissue residues (greater than the maximum residue limit; MRL) was compared with results achieved using two conventional EIAs for two of these drug residues (SMT and SDZ). Analysis of 2081 samples for both sulphonamide residues, over an 8 month period, showed the false positive prediction rate of biosensor analysis to be 0.14% and 0.34% for SMT and SDZ, respectively, compared with false positive rates of 1.54% and 1.44% by EIA. Biosensor analysis showed no false negative predictions for either SMT or SDZ while EIA showed a false negative prediction rate of 0.14% for SMT and 0.24% for SDZ. The present study has clearly demonstrated that immunobiosensor assays can be developed for veterinary drug residue screening programmes. These methods have the potential for generating faster and more reliable results than conventional immunoassay methods.     

A fast immunoassay for the screening of beta-agonists in hair.

Hair has been shown to be an excellent site for the accumulation of clenbuterol residues. Compared with other matrices, hair sampling is very easy and this might result in large numbers of samples. In this study, a simple digestion-extraction procedure was combined with a sensitive clenbuterol ELISA, which resulted in an easy screening procedure suitable for the detection of at least four beta-agonists. Hair from untreated cows (n = 40) resulted in low blank levels (0.9 +/- 0.7 and 0.5 +/- 0.2 ng g-1 for black and white hair, respectively). The detection limits for clenbuterol, bromobuterol, mapenterol and mabuterol were determined as 1-1.5 ng g-1 for white and 3-4 ng g-1 for black hair. The accumulation of mabuterol and mapenterol in black and white hair from treated calves was demonstrated by GC-MS. The screening assay is not suitable for the detection of cimbuterol (owing to the low extraction efficiency) and for salbutamol and terbutaline (owing to the low cross-reactivity of the antibodies used for the ELISA and the low extraction efficiency). Black hair samples from cows treated with clenbuterol were still found to be positive (> 5 ng g-1) at 23 weeks after treatment. The fast screening procedure is a powerful means to detect and track the illegal use of clenbuterol, bromobuterol, mabuterol and mapenterol in animal production.     

Determination and quantification of sulfadiazine and trimethoprim in swine tissues using liquid chromatography with ultraviolet and mass spectrometric detection

High-performance liquid chromatographic procedures with ultraviolet detection were developed for the quantitative determination of sulfadiazine (SDA) and trimethoprim (TMP) in swine tissues (kidney, liver, muscle, fat and fat + skin). In addition, high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry was used for the confirmation of the identity of the analytes of interest. Chromatographic separation was achieved on a Spherisorb ODS-2 column (250 x 4.6 mm id, dp 5 microns). The mobile phase for SDA analysis consisted of 1% acetic acid in water-acetonitrile (85 + 15, v/v). For TMP analysis a 80 + 15 + 5 (v/v/v) mixture of 0.25% triethylammonium acetate in water, acetonitrile and methanol was used as the eluent. Sulfamerazine and ormethoprim were used as the internal standards for SDA and TMP analysis, respectively. For the isolation of the compounds of interest from biological samples, a liquid-liquid extraction with acetone and ethyl acetate, followed by a clean-up using a solid-phase extraction column (aminopropyl and benzenesulfonic acid for SDA, benzenesulfonic acid for TMP) was performed. Calibration graphs were prepared for all tissues and linearity was achieved over the concentration ranges tested (50-1000 ng g-1 for SDA, r > or = 0.9979; 25-500 ng g-1 for TMP, r > or = 0.9994). The method was validated at the maximum residue level (MRL, 100 ng g-1 for SDA and 50 ng g-1 for TMP), at half the MRL and at double the MRL for both SDA and TMP. The accuracy and precision (expressed as the within-day repeatability) were found to be within the required ranges for each specific concentration. The quantification limits were 50 ng g-1 for SDA and 25 ng g-1 for TMP. The limits of detection were below one half the MRLs. Both methods were selective for the determination of SDA and TMP. Biological samples (kidney, liver, muscle, fat and fat + skin) from pigs that received a commercial SDA-TMP preparation with the feed for five consecutive days (dose rate: 25 mg SDA and 5 mg TMP kg body weight-1 day-1) were analyzed using the described methods. The quantitative results were used to calculate a withdrawal time (12 days) to reach residue levels below the respective MRLs. This calculation was performed according to the recommendations of the European Agency for the Evaluation of Medicinal Products (EMEA/CVMP/036/95).     

Evaluation of an immunobiosensor for the on-site testing of veterinary drug residues at an abattoir. Screening for sulfamethazine in pigs

A study was conducted to determine the feasibility of performing "on-site" screening for sulfamethazine (SMT), at an abattoir, using a rapid immunobiosensor method. This involved transfer of the biosensor technology and an assay developed in the laboratory, to the cold, humid conditions of a modern pig-processing factory. A pre-determined threshold limit of 0.4 microgram ml-1 SMT in bile was used to identify the likelihood that corresponding tissue samples contained SMT concentrations in excess of the European maximum permissible residue limit of 0.1 mg kg-1. Bile samples containing SMT concentrations above the threshold limit were deemed positive and the corresponding kidney and muscle samples were sent to the laboratory for HPLC analysis. The robustness of the biosensor instrumentation in the harsh operating conditions was monitored throughout the project. The performance of the assay, on-site, was assessed by the regular inclusion of QA samples and by the submission of control 'SMT-positive' pigs to the abattoir. Sampling procedures, identification and traceability were also under scrutiny. During the project, 337 (9.35%) of the total kill were tested for SMT residues, representing 75% of all producers submitting pigs for slaughter. Twelve animals, including the ten controls, gave positive bile results. HPLC analysis confirmed SMT residues in all 12 kidneys (11 in excess of the permissible level). Ten muscle samples also contained violative SMT levels. Throughout the project, the biosensor performed reliably, with no adverse reaction of any mechanical or electrical components. The SMT assay also performed reliably. This is the first report of a biosensor being used for 'on-site' drug screening.     

Multi-residue analysis of penicillin residues in porcine tissue using matrix solid phase dispersion.

The aim of this study was to develop a multi-residue method for the analysis of penicillins in animal tissue. Matrix solid phase dispersion (MSPD) was employed to extract the residues and the extracts were then cleaned-up by C18 solid phase extraction (SPE). Pre-column derivatisation using acetic anhydride and 1,2,4-triazole in the presence of mercuric chloride was employed to allow detection in 325 nm. Gradient elution was required to elute amoxicillin, ampicillin, penicillin G, cloxacillin and dicloxacillin derivatives from a C18 reversed phase column using phosphate buffer-acetonitrile mobile phase. The developed method had a limit of detection of 20 ng g-1 and had recoveries in the range 40-90% for the 5 drugs in samples fortified at 40 and 200 ng g-1; the maximum residue limits (MRLs) for these drugs were in the range of 50-300 ng g-1 (ppb).     

Determination of low concentrations of potentially toxic elements in human liver from newborns and infants

One hundred and fifty-seven liver samples from newborns and infants who had died from sudden infant death syndrome (SIDS) or other known causes have been analysed by ICP-MS for Ag, Cd, Co, Pb and Sb. The median concentrations found were: 15.4 (Ag), 2.9 (Cd), 15.9 (Co), 65.2 (Pb) and 1.8 (Sb) ng g-1 wet mass. There was no measurable difference in the concentrations of any of these elements between the SIDS and non-SIDS groups. The validity of the results was assessed by analysis of appropriate reference materials, interlaboratory comparison and isotope dilution analysis. The instrumental limits of detection were 0.25 (Ag), 0.14 (Cd), 0.21 (Co), 3.8 (Pb) and 0.38 (Sb) ng g-1 wet mass. The limits of detection of the method depend on the reagent blank and the extent of background contamination.     

Biosensor analysis of beta-lactams in milk: comparison with microbiological, immunological, and receptor-based screening methods

Two recently developed surface plasmon resonance biosensor assays for detection of beta-lactams in milk were used to screen raw producer milk samples. Both assays use a beta-lactam receptor protein with carboxypeptidase activity for detection. The results of the biosensor assays were compared with those of various commercial screening tests, i.e., the Delvotest SP, Penzym S, Beta-STAR, SNAP, and Parallux. The results of the 2 biosensor assays showed good agreement with those of the other screening tests. Of 195 analyzed milk samples, the results of only 5 samples differed between the assays. Additionally, 30 milk samples with both negative and positive results in the screening assays were analyzed by liquid chromatography for identification and quantification of any beta-lactam residues. All screening tests showed 0% false-negative results with 15 incurred samples containing between 4.0 and 268 microg/kg penicillin G. The biosensor assays showed 27% positive results (false violatives) with 15 producer milk samples containing penicillin G concentrations between 0 and 3.6 microg/kg, i.e., below maximum residue limit. This figure varied between 27 and 53% for the other screening tests.     

Traceability of sulfonamide antibiotic treatment by immunochemical analysis of farm animal hair samples

The use of hair to trace use of unauthorized substances, therapeutic agents, or their misuse is becoming very attractive since residues can be detected for a long time after treatment. For this purpose, an indirect enzyme-linked immunosorbent assay (ELISA) has been evaluated for its capability to trace sulfonamide antibiotic treatment by analyzing cattle and pig hair samples. Pigmented and nonpigmented hair samples from control and sulfamethazine (SMZ)-treated pigs and calves were collected, extracted under different alkaline conditions, and analyzed by ELISA after just diluting the extracts with the assay buffer. Data analysis following the European recommendations for screening methods demonstrates that the ELISA can detect SMZ in hair samples with a limit of detection (90% of the zero dose (IC₉₀)) between 30 and 75 ng g⁻¹. The same samples have been analyzed by HPLC after a dual solid-phase extraction. The ELISA results matched very well those obtained by the chromatographic method, demonstrating that the immunochemical method can be used as a screening tool to trace animal treatments. Between the benefits of this method are the possibility to directly analyze hair extracts with sufficient detectability and its high-throughput capability. Preliminary validation data are reported using an experimental approach inspired on the Commission Decision 2002/657/EC criteria for screening methods.     

Determination of sulfadiazine and sulfamethoxazole by capillary electrophoresis with end-column electrochemical detection.

Capillary electrophoresis (CE) with end-column electrochemical detection (EC) of sulfadiazine (SDZ) and sulfamethoxazole (SMZ) is described. Under the optimum conditions, SDZ and SMZ were separated satisfactorily, and a highly sensitive and stable response was obtained at a potential of 1.1 V versus Ag/AgCl. Optimized end-column detection provides detection limits as low as 0.1 microM for both compounds, which corresponds to 0.024 and 0.021 fmol with peak efficiencies of 394,000 and 335,000 theoretical plates for SDZ and SMZ, respectively. The calibration graph was linear over three order of magnitude. The relative standard deviations (n = 12) of peak currents and migration times were 2.3 and 2.7%, and 0.8 and 1.3%, respectively, for the two compounds. The proposed method was applied to the analysis of tablets and human urine samples with satisfactory results.     

A novel fluorescent aptasensor based on mesoporous silica nanoparticles for the selective detection of sulfadiazine in edible tissue

Sulfadiazine (SDZ) is a broad-spectrum antibiotic used to treat bacterial infections in animals, and SDZ residues in food can be harmful to human health. As a result, an aptasensor based on silica nanoparticles was developed for the rapid detection of SDZ. An aptamer that specifically binds to SDZ was obtained using graphene oxide-SELEX and further truncated to a 13 nt sequence (SDZ30-1:5′-AACCCAATGGGAT-3′), which has a high affinity (K_d = 65.72 nM). In addition, it was found by molecular simulation that a steric hindrance could prevent the target molecule from entering the binding pocket formed by the key base “TGG”, which affects the total binding free energy of SDZ30-1 and the target molecule, thereby affecting the affinity of SDZ30-1 to the target. The SDZ30-1 was selected as the fluorescent probe to establish an aptasensor for the detection of SDZ residues in milk and honey. The aptasensor exhibited a wide dynamic linear range (3.125 – 100 ng/mL) and a limit of detection (LOD = 1.68 ng/mL). The aptasensor in spiked samples recovered at a rate of 95.12 – 105.47%, with a coefficient of variation of less than 13.18 %. The results of aptasensor were positively correlated with those of HPLC (R² > 0.8687). Based on the above results, it could be inferred that the aptasensor can be used sensitively and rapidly for the detection of SDZ residues in edible tissue.     

Optimized procedure for the determination of antimony in lipid-rich environmental matrices by flow injection hydride generation atomic absorption spectrometry

An analytical procedure for the reliable determination of Sb in digests of lipid-rich environmental matrices in the low ng l-1-range based on flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS) has been developed. Prior to HG-AAS, aliquots (250 to 320 mg) of dry samples were mineralized with 3 ml nitric acid and 0.5 ml of each sulfuric and perchloric acids in open digestion vessels made of glassy carbon in a heating block. Procedure detection and quantification limits of a previously developed procedure for the determination of Sb in plant materials by FI-HG-AAS were decreased with respect to the lower Sb concentrations in animal tissues, the sensitivity of the instrumental response was increased, and the composition of the acid digestion mixture was re-optimized for lipid-rich samples. The accuracy and precision of the developed procedure was evaluated by the analysis of the two reference materials Bovine Liver 1577a and Pig Kidney CRM 186. These reference materials have been additionally spiked with appropriate amounts of Sb to obtain recovery data. The solution detection limit (3 sigma) in digested samples was 0.021 microgram l-1, the detection limit for the whole procedure based on the dry powders was 7 pg g-1, the method quantification limit for a reliable determination of Sb was 23 pg g-1. The reproducibility of repetitive measurements was 6.0% at 0.1 microgram Sb l-1 and 2.2% at 0.5 microgram Sb l-1. Calibration curves were linear from 0.05 to 3 micrograms Sb l-1. To demonstrate the suitability of the developed method, concentrations of Sb have been determined in pigeon eggs (approximately 2 ng Sb g-1), as well as in bream livers (approximately 4 ng g-1) and in deer livers (approximately 5 to 8 ng g-1) from animals living in remote and urban-industrialized areas of Germany, respectively.     

Immunochemical detection of aminoglycosides in milk and kidney

In 1996, the European Union established provisional maximum residue limits (MRL) for gentamicin, neomycin, streptomycin and dihydrostreptomycin in milk and tissue (0.1-5 mg kg-1). For the detection of these four aminoglycosides, three enzyme linked immunosorbent assays (ELISA) for applications in milk and kidney were developed. The screening of defatted and diluted milk resulted in limits of determination (LDM) of < 0.01 mg l-1. Kidney samples were deproteinized with a trichloroacetic acid solution (3%) and after filtration and the addition of buffer, aliquots were used in the ELISA. The LDM of the four aminoglycosides in kidney were < 0.05 mg kg-1. The ELISA were found suitable for the semi-quantitative screening of milk and kidney for the presence of the four aminoglycosides far below the MRL levels. In randomly taken milk samples (n = 776) and in kidneys derived from healthy pigs (n = 124), the aminoglycoside residues found were far below their established MRL. In eight out of the 94 kidney samples obtained from diseased animals after emergency slaughter, aminoglycoside residues were above the MRL.     

Fully automated screening of veterinary drugs in milk by turbulent flow chromatography and tandem mass spectrometry

There is an increasing interest in screening methods for quick and sensitive analysis of various classes of veterinary drugs with limited sample pre-treatment. Turbulent flow chromatography in combination with tandem mass spectrometry has been applied for the first time as an efficient screening method in routine analysis of milk samples. Eight veterinary drugs, belonging to seven different classes were selected for this study. After developing and optimising the method, parameters such as linearity, repeatability, matrix effects and carry-over were studied. The screening method was then tested in the routine analysis of 12 raw milk samples. Even without internal standards, the linearity of the method was found to be good in the concentration range of 50 to 500 μg/L. Regarding repeatability, RSDs below 12% were obtained for all analytes, with only a few exceptions. The limits of detection were between 0.1 and 5.2 μg/L, far below the maximum residue levels for milk set by the EU regulations. While matrix effects—ion suppression or enhancement—are obtained for all the analytes the method has proved to be useful for screening purposes because of its sensitivity, linearity and repeatability. Furthermore, when performing the routine analysis of the raw milk samples, no false positive or negative results were obtained.     

Magnetic retrieval of graphene: extraction of sulfonamide antibiotics from environmental water samples

A new technique of retrieving graphene from aqueous dispersion was proposed in the present study. Two-dimensional planar graphene sheets were immobilized onto silica-coated magnetic microspheres by simple adsorption. The graphene sheets were used as adsorbent material to extract six sulfonamide antibiotics (SAs) from water samples. After extraction, they were conveniently separated from the aqueous dispersion by an external magnetic field. Under the optimal conditions, a rapid and effective determination of SAs in environmental water samples was achieved. The limits of detection for six SAs ranged from 0.09 to 0.16 ng/mL. Good reproducibility was obtained. The relative standard deviations of intra- and inter-day analysis were less than 10.7% and 9.8%, respectively.     

LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine

A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.     

Screening method for the detection of a range of nitrofurans in avian eyes by optical biosensor

An immunobiosensor assay was developed for the multi-residue screening of a range of nitrofuran compounds in avian eyes. A polyclonal antibody which binds at least 5 of the major parent nitrofurans was raised in a rabbit after inoculation with a nitrofuran mimic-protein conjugate. Sample homogenates were extracted into 0.1M hydrochloric acid and subjected to clean-up by solid phase extraction and micro-centrifugation prior to biosensor analysis. Validation data obtained from the analysis of 21 fortified samples has shown that the method has a detection capability (CCβ) of less than 1 ng eye(-1) for nitrofurazone (NFZ). In addition, cross-reactivity data and the analysis of a smaller number of fortified samples have shown that the method will also detect a range of other major parent nitrofurans including furazolidone (FZD), furaltadone (FTD), nitrofurantoin (NFA) and nifursol (NFS). Intra-assay variation (n=10) was calculated at 12.9% and 10.1% at concentrations of 1 ng eye(-1) and 2 ng eye(-1) NFZ respectively. Inter-assay variation (n=3) was determined to be 10.8% and 4.7% at the same NFZ concentrations respectively. The cross-reactivity profile and validation data for the detection of these nitrofurans are presented together with the results obtained following the analysis of a small number of incurred samples using the developed method.     

Determination of chlortetracycline residues in tissues using high-performance liquid chromatography with fluorescence detection

A method is described for the determination of chlortetracycline residues in tissue samples. The samples were extracted into a hydrochloric acid - glycine solution and the extracts concentrated and purified on cyclohexyl-bonded reversed-phase cartridges. Any chlortetracycline present was converted to iso-chlortetracycline at pH 12, which was then separated from interfering compounds on a reversed-phase polymeric column using high-performance liquid chromatography with fluorescence detection. The detection and determination limits of the assay were 20 and 50 ng g-1, respectively, making it suitable for statutory residue testing purposes.     

Determination of estrone and 17 beta-estradiol in human hair by gas chromatography-mass spectrometry

An efficient procedure is described for the determination of estrone and 17 beta-estradiol in hair by gas chromatography-mass spectrometry (GC-MS). The method involves alkyloxycarbonylation with isobutyl chloroformate (isoBCF) of phenolic hydroxy groups after alkaline digestion of hair samples. The resulting isobutyloxycarbonyl derivatives of estrone and 17 beta-estradiol are extracted with hexane and subjected to chlorodifluoroacetyl derivatization in order to protect the remaining alcoholic hydroxy groups. When GC-MS with selected ion monitoring (SIM) was used, the quantitative ions were at m/z 270 and 384 in the electron ionization mass spectra for estrone and 17 beta-estradiol, respectively. The detection limits for SIM of the steroids were 1 and 2 pg, respectively, and the SIM responses were linear with correlation coefficients varying from 0.991 to 0.994 in the concentration range 0.2-4.0 ng g-1 for the estrogens studied. The detection of estrone and 17 beta-estradiol in hair samples was possible in the concentration range of 0.24-1.30 ng g-1. The concentrations of the two estrogens detected were different in male and female hair samples.     

Automatic sample preparation of sulfonamide antibiotic residues in chicken breast muscle by using dynamic microwave-assisted extraction coupled with solid-phase extraction

In the work, a rapid, simple and high-throughput sample preparation method was developed for the determination of sulfonamide (SA) antibiotic residues in chicken breast muscle. The extraction and clean-up were online combined and up to 20 samples can be treated simultaneously in 6 min. The SAs were first extracted with acetonitrile under the action of microwave energy, and then the extract was directly introduced into the SPE column for on-line clean-up and concentration. Subsequently, the SAs eluted from the SPE column were determined by liquid chromatography-tandem mass spectrometry. The precisions of extraction results of 20 samples were in the range of 4.9-7.4%. The limits of detection and quantification obtained were in the range of 2.4-3.6 ng/g and 8.6-11.3 ng/g for SAs, respectively. The recoveries of SAs obtained by analyzing chicken muscles at three fortified levels (10, 50 and 500 ng/g) were in the range of 82.6-93.2%. The results of the validation process prove that the proposed method is suitable for treating numbers of complex samples simultaneously in a short time.     

Trace determination of sulfonamides residues in meat with a combination of solid-phase microextraction and liquid chromatography-mass spectrometry

An integrated method of combining solid-phase microextraction (SPME) with liquid chromatography-mass spectrometry (LC-MS) was evaluated for determination trace amount of sulfonamides in meat products. Eight commonly used sulfonamides, sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethazine (SMT), sulfamonomethoxine (SMMX), sulfamethoxazole (SMXZ), sulfaquinoxaline (SQX) and sulfadimethoxine (SDMX), were investigated in this study. Chromatography was performed on a C₁₈ reversed-phase column using an isocratic acetonitrile in water as the mobile phase. Fiber coated with a 65 μm thickness of polydimethylsiloxane/divinylbenzene (PDMS/DVB) was used to extract sulfonamides at optimum conditions. Analytes were desorbed with static desorption in an SPME-HPLC desorbed chamber for 15 min and then determined by LC-MS. The detection limits of these sulfonamides in pork were from 16 μg kg⁻¹ (SMT) to 39 μg kg⁻¹ (SMMX). According to the analysis, the linear range was from 50 to 2000 μg kg⁻¹ with relative standard deviation (R.S.D.s) value below 15% (intra-day) and 19% (inter-day). The proposed method was tested by analyzing meats from a local market for sulfonamides residues. Some sulfonamides in our study were detected in the meat samples. The concentration of these residual sulfonamides ranged from 66 μg kg⁻¹ (SDZ) to 157 μg kg⁻¹ (SQX) in a chicken sample. The results demonstrate that the SPME-LC-MS system is highly effective in analyzing trace sulfonamides in meat products.