Nanoscale affinity chip interface for coupling inhibition SPR immunosensor screening with Nano-LC TOF MS.

The on-line nanoscale coupling of a surface plasmon resonance (SPR)-based inhibition biosensor immunoassay (iBIA) for the screening of low molecular weight molecules with nano-liquid-chromatography electrospray ionization time-of-flight mass spectrometry (nano-LC ESI TOF MS) for identification is described. The interface is based on a reusable recovery chip (RC) that contains a nanoscale biosorbent composed of a hydrogel layer modified with antibodies raised against the analyte featuring the unique possibility of performance characterization using the SPR biosensor. Various hydrogel chemistries were evaluated, and the standard Biacore CM5 chip showed the highest capture capacity in combination with affinity-purified polyclonal antibodies. The procedure has four stages: the samples are prepared (1) and screened using a screening chip (SC) in the iBIA (2). Suspected noncompliant samples as being noncompliant are reinjected over the RC, and the analyte is captured at subnanogram level (3). The captured analyte is released, and the eluate is analyzed with nano-LC ESI TOF MS via a loop-type interface (4). The coupling of the technologies proved effective for screening enrofloxacin, a model compound, in incurred chicken muscle samples followed by identity confirmation in suspected noncompliant samples. Ciprofloxacin, a known metabolite of enrofloxacin, was identified as well in incurred chicken samples. This demonstrates the potential of the technologies coupled by means of a RC for the rapid screening and identification of known as well as unknown compounds. Finally, we demonstrate the feasibility of combining the two biosensor chips (SC and RC) with a robust chip-based nano-LC chip TOF MS system, thus providing a robust alternative triple-chip system.

Spreeta-based biosensor assays for endocrine disruptors.

The construction and performance of an automated low-cost Spreeta-based prototype biosensor system for the detection of endocrine disrupting chemicals (EDCs) is described. The system consists primarily of a Spreeta miniature liquid sensor incorporated into an aluminum flow cell holder, dedicated to support a Biacore chip frame, in combination with a simple pressurized air-driven fluid system. During the optimization, a monoclonal antibody (MAb)-based immunoassay for the estrogenic compound bisphenol A (BPA) was used as a model. After the optimization two thyroxine transport protein inhibition assays for thyroid endocrine disruptors were implemented. The average noise of the system for 1 min of baseline was 1.1 microRIU (refractive index units) and it could be operated in the range of 18-22 degrees C with a minimum baseline drift (5-10 microRIU/100 min). Optimum signal to noise ratio (S/N R) was obtained using a flow cell height of 100 microm and a flow rate of 180 microl/min. The sensitivity of the Spreeta-based biosensor inhibition assays implemented (50% inhibition concentration (IC50) of 30.2 nM for BPA using MAb12 and 12.3 and 11.6 nM for thyroxine (T4) using thyroxine-binding globulin (TBG) and recombinant transthyretin (rTTR), respectively) was comparable to the sensitivity previously obtained using a Biacore 3000 (IC50 of 39.9 nM for BPA and 8.6 and 13.7 nM, respectively, for T4). The results indicate that the alternative prototype system can be used in combination with ready-to-use biosensor chip surfaces and it is potentially a useful tool for the bioeffect-related screening of EDCs.

Immunoaffinity chromatography for the sample pretreatment of Taxus plant and cell extracts prior to analysis of taxanes by high-performance liquid chromatography.

The application of immunoaffinity chromatography for the purification of Taxus plant and cell extracts prior to the HPLC analysis is described. Polyclonal antibodies raised against 10-deacetylbaccatin III (10-DAB III), paclitaxel's main precursor in plant, were characterised by enzymed-linked immunosorbent assay. Immunoglobulins from selected antisera were immobilised on CNBr-activated Sepharose 4B. The immunoaffinity column was used for the purification of plant and plant cell culture extracts prior to their analysis by HPLC. Immunoaffinity chromatography enabled the selective concentration of taxoids and enhanced sample clean-up.

Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder.

The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders. Affinity-purified polyclonal antibodies raised against the three protein sources were immobilized in different flow channels (Fcs) on the biosensor chip (CM5). Dissolved milk powders were injected (20 microL injections at 20 microL min(-1)) through the serially connected Fcs, and the antibody-bound plant proteins were detected directly. The total run time between samples, including a regeneration step with 5 microL of 10 mM HCl, was 5 min. The limits of detection in milk powder were below 0.1% of plant protein in the total milk protein content. The antibodies also recognized some proteins from other plant sources, which made this BIA even more suitable as a broad screening assay for nonmilk proteins.

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.

Production and characterization of polyclonal antibodies to sulfamethazine and their potential use in immunoaffinity chromatography for urine sample pre-treatment.

An immunoaffinity chromatographic (IAC) method for isolating sulfamethazine (SMZ) from incurred urine samples was developed. This was achieved by (i) generating polyclonal antibodies that recognize equally well SMZ and its major urinary metabolites, (ii) evaluating in an ELISA procedure the influence of methanol, salt and pH on the antigen-antibody interaction in order to determine the optimum conditions for IAC and (iii) covalent coupling of the IgG fractions of anti-SMZ to CNBr activated Sepharose for the preparation of re-usable immunoaffinity columns, having a high capacity for SMZ (1900 ng SMZ mL-1 gel). For desorbing SMZ from the immunoaffinity column, different elution modes were evaluated, with 40% MeOH-0.1 mol L-1 HOAc-0.5 mol L-1 NaCl being the most efficient combination. Using the IAC column for processing SMZ spiked urine samples resulted in high recoveries, ranging from 92 to 100%. Because of the high cross-reactivity with the major metabolites of SMZ present in urine of treated animals, the antibodies show excellent properties for use in both IAC and ELISA. For the isolation and concentration of the parent drug and its major metabolites, the urine could be applied directly to the IAC column, without the time-consuming step of deconjugation. Moreover, the use of IAC prior to ELISA for the analysis of incurred urine samples showed good efficiency for the elimination of matrix interferences. Owing to the urine-tissue relationship, the urine concentrations can be used to predict the presence of the parent drug in tissues and so possible violations of the maximum residue limit (MRL) can be controlled.

Combinations of growth promoters in veal calves: consequences for screening and confirmation methods.

This study investigates the influence of low dosages of beta-agonists combined with other growth promoters on screening and confirmation methods in male veal calves. Five groups of four calves were treated for 6 weeks with combinations of low dosages of beta-agonists (beta AG, clenbuterol, mabuterol and mapenterol, 0.4 microgram/kg each twice daily) in combination with 17 beta-estradiol (E2, 5 mg per animal per 14 days), methylthiouracil (MTU, 2.857 mg/kg bw twice daily) and dexamethasone (DEX, 4 mg per animal per 10 days during the last 20 days of treatment). Another group of four untreated animals served as controls. The weight and size of the thymus was reduced in the DEX group, the MTU group showed enlarged thyroids. Histologically the prostates showed vacuolar degeneration in the beta AG animals and metaplasia in the E2 group. Some vacuolization was also observed in the controls. The testis showed impaired development in all treatment groups, E2 leading to the most severe changes. DEX led to cortical atrophy of the thymus. MTU induced hyperplastic changes in the thyroid. These results indicate that comedication does not markedly affect the histological changes induced in the hormonal target tissues. For screening purposes histology of the prostate can be used as a marker for oestrogens, whereas the weight of the thymus and thyroid can be used as an indication for the use of corticosteroids and thyreostatics, respectively. Vacuolization in the prostate appeared no reliable indication for beta-agonists. Samples of urine, faeces, liver and eye (retina/choroid) were analysed for beta-agonists. E2 significantly increased the levels of all beta-agonists in the liver and faeces, whereas DEX significantly reduced these levels. These observations show that additional medication with different groups of growth promoters can markedly alter the excretion of beta-agonists and thus influence (regulatory) control.

Development of a one step strip test for the detection of sulfadimidine residues.

The one step strip test described is a competitive immunoassay in which the detector reagent consists of colloidal gold particles coated with affinity purified polyclonal anti-sulfadimidine (SDD) antibodies. The capture reagent in the assay is an SDD-ovalbumin conjugate which is immobilised on the lateral flow membrane of the test device. In the test procedure, 150 microliters (four drops) of a liquid sample (buffer, urine or milk) are brought into the sample well of the test device and allowed to migrate over the membrane. The more analyte present in the sample, the more effectively it will compete with the SDD immobilised on the membrane for binding to the limited amount of antibodies of the detector reagent. A sufficient amount of SDD in the sample will therefore prevent the binding of the detector reagent to the SDD immobilised on the membrane. Therefore, a positive sample will not show a test line in the read-out zone. With spiked buffer or calf urine this was obtained at a level of > 10 ng ml-1 of SDD and with spiked (diluted) fresh cow milk at a level > 20 ng ml-1 of SDD. At these levels, the test is applicable only as a qualitative assay. The presence or absence of a test line indicates lower or higher levels of SDD, respectively. The major advantages of the one step strip test are that results can be obtained within 10 min and that all reagents are included in the test device.

Pour on application of growth promoters in veal calves: analytical and histological results.

To investigate the possibilities for screening and confirmation methods when the 'pour on' method of application is used for administration of growth promoters, an animal experiment was performed using a cocktail of a combination of growth promoters derived from (illegal) practice. Two cocktails were used, cocktail A consisting of stanozolol and estradiol benzoate and cocktail B consisting of stanozolol, estradiol benzoate and beclomethasone dipropionate. The intended dose per animal was 110 mg stanozolol, 25 mg estradiol and 10 mg beclomethasone. The experiment was performed on 20 male veal calves, 16 treated and 4 vehicle treated controls and 3 female veal calves, 2 treated and 1 vehicle treated control. Half of the animals were shaven prior to the application of the drugs. The cocktails were administered using two types of vehicles: vehicle A; Miglyol 840 with butylated hydroxytoluene and vehicle B; di(ethyleneglycol) monobutylether. During a 28 day treatment period, one group of animals was treated once a week, another group of animals was treated once every two weeks and slaughtered. Preliminary results showed that pour on application of anabolic steroids markedly increased growth performance of veal calves, the animals treated with cocktail A performed better than the animals treated with cocktail B. Macroscopically, the thymus was reduced in weight and size in the B animals. The bulbo-urethral glands were enlarged in all treated animals. Histologically all treated animals showed squamous metaplasia in the prostate, bulbo-urethral gland and Bartholins glands. Moreover, a changed secretion pattern was observed in both the prostate and the bulbo-urethral gland. Severe cortical atrophy was observed in the thymus and to a lesser extent the adrenals of the beclomethasone treated animals. The recently discovered 16 beta-hydroxy-metabolite of stanozolol was detected in urine, in relatively high concentrations. This is the first report of the excretion of this metabolite in urine after pour on administration showing the prospect for detection of dermal treatment. Estradiol levels were remarkably elevated (up to 200 micrograms l-1) exceeding the endogenous levels (< 1 microgram l-1).

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.

Application of an enzyme immunoassay for the determination of sulphamethazine (sulphadimidine) residues in swine urine and plasma and their use as predictors of the level in edible tissue.

The potential of an enzyme immunoassay (EIA) with high cross-reactivity towards the major metabolite (N4-acetyl-sulphamethazine) of sulphamethazine was tested for screening fluids and tissues. Healthy pigs were given 20 mg sulphamethazine per kg body weight per day in their drinking water for 2 days. Groups of four pigs were slaughtered after 3, 4 and 7 days withdrawal. The results were compared with liquid chromatographic analysis for urine, plasma, kidney, liver, gluteal muscle and diaphragm. In general, concentrations found by the EIA were higher than those found by liquid chromatography (LC) because sulphamethazine metabolites were detected by the EIA and not by LC. Using the EIA for the detection of sulphamethazine and the major metabolite in urine and plasma, predictive relationships (tissue-fluid ratios) for the concentration of the parent drug in tissue, determined by LC, were calculated. The tissue-plasma ratios for muscle, liver and kidney were 0.1, 0.2 and 0.1, respectively. The tissue-urine ratios for muscle, liver and kidney were 0.02, 0.03 and 0.03, respectively. Owing to the higher concentration of the parent drug in both fluids, the presence of the major metabolite in urine and the sensitivity of the EIA, tissue can be screened for low concentrations of sulphamethazine.

Development of a tube enzyme immunoassay for "on-site' screening of urine samples in the presence of beta-agonists.

An on-site screening test for the detection of beta-agonistic drugs in urine was developed. The test is based on the principle of an enzyme immunoassay in polystyrene tubes. Results can be obtained by visual interpretation or by measurement with a differential photometer. The total time required to perform the test for a set of samples (five samples+one cut-off standard) is about 20 min (visual interpretation) with an additional 2 min for an instrumental interpretation. Owing to its speed and simplicity, the test can be performed in slaughter- and farmhouses. In the tube test, a mixture of antibodies raised against clenbuterol and salbutamol is used, which makes this test sensitive towards a range of beta-agonists (multi-test). In this study, the test was applied to the screening of 269 bovine urine samples. Bovine urine samples with a level of 3 ng ml-1 of clenbuterol and higher were found positive with this on-site test. Owing to fewer matrix effects, a lower level (1 ng ml-1) could be detected in calf urine. The detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.

Expression of CD46, CD55, and CD59 on renal tumor cell lines and their role in preventing complement-mediated tumor cell lysis.

Nucleated cells are protected from complement-mediated injury by the expression of membrane-bound regulators of complement activation (mRCA) CD46, CD55, and CD59. Increased expression of these mRCA may be a mechanism by which tumor cells protect themselves from complement-mediated injury and prevent an inflammatory response. In the present study, we have investigated whether human renal tumor cell lines and cultured proximal tubular epithelial cells express CD46, CD55, and CD59 and whether these mRCA influence complement-mediated lysis of these cells. The expression of CD46, CD55, and CD59 was measured by flow cytometry. To determine the effect of mRCA on lysis, tumor cells were opsonized with complement activating anti-HLA class l mAb. Lysis was measured in the presence or absence of anti-CD46, anti-CD55 or anti-CD59 mAb and serum as a source of complement, using a 51Cr release assay. Flow cytometric analysis revealed that renal tumor cell lines and proximal tubular epithelial cells all express CD46, CD55, and CD59. Lysis of renal tumor cell lines in the presence of rabbit serum depended on the number of HLA class I molecules expressed by the tumor cells. Using human serum, complement-mediated lysis was decreased by at least one-third as compared with rabbit serum. The susceptibility of renal tumor cells for complement-mediated lysis could be increased up to the level observed with rabbit serum by inhibiting the function of CD59. Inhibition of the function of CD46 or CD55 with mAb directed against these mRCA had no substantial effect on lysis. We conclude from this work that renal tumor cells and proximal tubular epithelial cells express CD46, CD55, and CD59. Of these mRCA, CD59 is most efficient in preventing complement-mediated lysis of these cells. Expression of mRCA on tumor cells may influence the effectiveness of immunotherapy with tumor-associated mAb.

Determination of fenoterol and ractopamine in urine by enzyme immunoassay.

Fenoterol and ractopamine are phenethanolamines with beta-adrenergic agonist activity. An enzyme immunoassay (EIA) for these compounds was developed using antibodies raised in a New Zealand white rabbit against fenoterol-bovine serum albumin and fenoterol coupled to horseradish peroxidase (HRP). The calibration graphs of fenoterol and ractopamine showed linearity over the concentration ranges 0.1-5 and 0.2-25 ng ml-1, respectively. Isoxsuprine showed a cross-reactivity of 0.7% while the cross-reactivity of other beta-agonists was < 0.1%. The screening assay was used to detect fenoterol in urine samples obtained from an animal experiment in which male calves were treated with fenoterol (100 micrograms of fenoterol per kg of bodymass per meal for a period of four weeks). Using a direct method, without sample preparation, fenoterol concentrations ranged from 22 to 210 ng ml-1. The mean concentration of fenoterol after extraction in isobutanol was 3.5 times lower compared with the direct method. On applying enzymic hydrolysis in combination with isobutanol extraction, the mean concentration was eight times higher than that obtained when using extraction only. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed the presence of fenoterol in most of these samples. However, probably owing to the absence of a proper GC-MS internal standard, the correlation between GC-MS and EIA concentrations was low (r = 0.7976). In general, the concentrations found by the EIA are much higher than those found by GC-MS, which might be caused by the presence of metabolites detected with the EIA. Fenoterol is excreted in urine mostly (about 85%) as glucuronidated-sulfated conjugate. The antibodies partly recognize the conjugated fenoterol, which makes it possible to use a direct screening assay. In blank calf urine the detection limits, mean background +3 times the standard deviation, are 1.3 (fenoterol) and 2.6 ng ml-1 (ractopamine). In bovine urine, however, owing to matrix effects, the detection limits are 20 times higher.

Production of bi-specific monoclonal antibodies in a hollow-fibre bioreactor.

In the present study we have measured the feasibility of producing high amounts of bi-specific monoclonal antibodies (MAb) for therapeutic purposes using a hollow-fibre bioreactor. We have studied the isotype composition and functional capacity of an OKT3/OV-TL 3 quadroma (IgG2a/IgG1) and we have compared the production of bi-isotypic MAb in this system with tissue culture flasks or mouse ascites. Both culture supernatant and purified bi-isotypic MAb were able to enhance the cytolytic activity of a CD8+ T cell clone against ovarian tumour cell lines, demonstrating the presence of functional bi-isotypic MAb (bi-specific MAb). The total amount of immunoglobulin produced after 38 days was 375 mg. After purification by protein A affinity chromatography, 79 mg of bi-isotypic MAb (IgG1/IgG2a) was obtained. This amount of bi-isotypic MAb was similar to the amount obtained from ascitic fluid produced by 200 mice or 38 l of tissue culture flask supernatant.

Test strip enzyme immunoassays and the fast screening of nortestosterone and clenbuterol residues in urine samples at the parts per billion level.

The preliminary results of an investigation into the development of "on-site" test strip enzyme immunoassays for the screening of urine samples for the presence of growth promoters, such as 17 beta, 19-nortestosterone and clenbuterol at the parts per billion level are described. Urine samples, enzyme-labelled analyte and a nitrocellulose test strip, containing immobilized antibodies, are incubated together, after which the strip is placed in a chromogen-containing substrate solution for colour reaction. Using prefabricated strips, the tests can be performed in 45-60 min. A similar assay was worked out using a dot-blotting device, allowing the test to be performed in 20-50 min. The tests are simple and easy to perform outside the laboratory. Urine samples identified positive by gas chromatography mass spectrometry were also found to be positive with these test strips and, so far, no false-positive results have been encountered. With standard additions to blank urine samples, positive samples could be distinguished above the 5 ng ml level. However, samples from treated calves contain one or more metabolites of the parent compound, which increase the sensitivity of the assays. Although the tests described can be improved and still have to be evaluated further by analysing more urine samples, the preliminary results are very promising and give a lead to further research into the applicability of such "on-site" tests in residue analysis.

Determination of clenbuterol in urine of calves by high-performance liquid chromatography with in series ultraviolet and electrochemical detection.

A method for the determination of clenbuterol in calf urine is described. After a simple two-step sample pretreatment, involving an Extrelut-3 column and a solid-phase extraction column (C2), the separation of clenbuterol from interfering compounds present in urine samples was performed with ion-pair chromatography on a LiChrospher RP-Select-B column with a mixture of acetonitrile and sodium dodecyl sulphate/acetate buffer (pH 3.5) as mobile phase. To obtain a higher specificity, two different physico-chemical detection techniques, i.e. UV-absorption (244 nm) and electrochemical detection (+1250 mV), were applied in series. The lowest limit of determination was 0.5 ng ml-1 and the mean recovery of clenbuterol spiked at 10 ng ml-1 level was 79.9% (RSD = 6.3%; n = 9). The analysis of one urine sample, including sample preparation, took less than 2 h. Results obtained with this method correlated well with GC-MS analysis. With the described method about 400 urine samples were analysed. In a pilot experiment, in which a calf received orally 4 micrograms clenbuterol.HCl per kilogram body weight twice a day (five times the therapeutic dose for oral application) for 5 days, the highest concentration of clenbuterol found in urine was 73 ng ml-1. In a second experiment, in which two calves received the therapeutic dose of clenbuterol.HCl twice a day over a period of 2 weeks, the highest concentration of clenbuterol was 75 ng ml-1 of urine. Eight days after the final application, concentrations of clenbuterol were lower than 0.5 ng ml-1. From this excretion study for clenbuterol a half-life value of approximately 1.5 days was calculated.

Rapid determination of clenbuterol residues in urine by high-performance liquid chromatography with on-line automated sample processing using immunoaffinity chromatography.

A liquid chromatographic column-switching system for automated sample pretreatment and determination of clenbuterol in calf urine, using an immunoaffinity precolumn with Sepharose-immobilized polyclonal antibodies against clenbuterol, is described. A second precolumn packed with C18-bonded silica was used for the reconcentration of desorbed clenbuterol prior to the analytical separation. Urine, after 2-fold dilution with buffer (pH 7.4), was loaded directly onto the immuno precolumn, where clenbuterol was trapped by the immobilized antibodies. This immuno precolumn has been used for more than 200 runs with standard solutions and samples. Bound analyte was desorbed with 0.01 M acetic acid and transferred, via the second precolumn, to the analytical column. The total runtime per sample was 35 min. Using a sample load of 27 ml of dilute urine and UV detection at 244 nm, the detection limit was 0.5 ng/ml. The mean recovery of clenbuterol added to a blank urine sample at the 5 ng/ml level was 82 +/- 2% (n = 5) as determined with standard solutions loaded onto the same system. Urine samples from treated animals were analysed and the clenbuterol concentrations were comparable to those obtained by high-performance liquid chromatography using solid-phase extraction for sample clean-up.

Screening and confirmation of thyreostatics in urine by gas chromatography with nitrogen-phosphorus detection and gas chromatography-mass spectrometry after sample clean-up with a mercurated affinity column.

Methods are described for the screening and confirmation of residues of the thyreostatics thiouracil, methylthiouracil and propylthiouracil in urine samples of cattle at levels down to 25 micrograms/l. After a selective preconcentration of the thiol-containing thyreostatics on a mercurated affinity column, the analytes are derivatized by extractive alkylation and analysed by gas chromatography with nitrogen-phosphorus or mass spectrometric detection.

Determination of beta-19-nortestosterone and its metabolite alpha-19-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid chromatography with on-line immunoaffinity sample pretreatment.

An immunoaffinity precolumn (immuno precolumn) packed with Sepharose-immobilized polyclonal antibodies against the anabolic hormone 17 beta-19-nortestosterone (beta-19-NT) was used for the selective on-line pretreatment of raw extracts of urine, bile and tissue samples by high-performance liquid chromatography. Using UV detection (247 nm), beta-19-NT and its metabolite 17 alpha-19-nortestosterone (alpha-19-NT) can be determined in biological samples with a detection limit of 0.05 microgram/kg. Owing to the high clean-up efficiency of the immuno precolumn and the large sample volumes used, confirmation by gas chromatography-mass spectrometry is possible at this level. In urine samples from a calf treated with 19-nortestosterone 17 beta-laurate, the maximum concentrations of beta-19-NT (1.3 micrograms/l) and alpha-19-NT (3.1 micrograms/l) were found seven days after intramuscular administration. In a bile sample from this calf only alpha-19-NT (55 microgram/l) was detected. In meat samples from three treated calves, the concentration of beta-19-NT varied from 0.1 to 1.6 micrograms/kg and no alpha-19-NT could be detected. In liver samples from these calves, the concentrations of beta-19-NT and alpha-19-NT were less than 0.05-0.1 and 0.5-0.9 micrograms/kg, respectively. In the corresponding kidney samples, the concentrations of beta-19-NT and alpha-19-NT were 0.4-0.5 and 0.5-1.6 micrograms/kg, respectively. The application of the same immuno precolumn to the determination of 17 beta- and 17 alpha-trenbolone, two structurally related steroids, is also demonstrated.