Immunochemical Approaches to the Analysis of Î² ... - ACS Publications

Chapter 5

Immunochemical Approaches to the Analysis of β-Agonistic Drugs Willem Haasnoot, Geert Cazemier, Piet Stouten, and Anniek Kemmers-Voncken

Downloaded by UNIV LAVAL on May 16, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch005

State Institute for Quality Control of Agriculture Products (RIKILT-DLO), Bornsesteeg 45, 6708 PD Wageningen, Netherlands

β-Agonistic drugs are the mostfrequentlyfound growth promoting agents in the European Union (E.U.). For tracing the illegal use of β -agonists, several types of sample materials (cattle feed, urine, feces, liver, kidney, blood, muscle, bile and retina) are used. For screening of the presence of a variety of β-agonists, three enzyme immunoassays (Clenbuterol-, Salbutamol- and Fenoterol-EIA) were developed. The Clenbuterol-EIA was found suitable for the detection of at leastfiveβ -agonists (clenbuterol, cimbuterol, bromobuterol, mapenterol and mabuterol) in all the sample materials applying the direct method for urine or a simple liquid-liquid (water-isobutanol) extraction. The detection limits depend on the β-agonist and the sample material, and varied between 0.1 ng/mL for plasma and 5 ng/g for feed samples. The detectability of β-agonists can be improved to at least eight compounds by applying the Salbutamol-EIA. The Fenoterol-ELA is specific to fenoterol and ractopamine; however, matrix effects prevent the applicati on for routine analysis.

The use of growth regulators (anabolic agents), i.e., naturally occurring steroid hormones, synthetic hormones, xenobiotic compounds and other growth-promoting com pounds, in food-producing ruminants (cattle, calves, sheep) causes a shift from fat to lean, an increase in growth rate, and reduced feed requirements for growth (7). The United States Food and Drug Administration (FDA) has maintained that hormones approved for use (estradiol, progesterone, testosterone, trenbolone-acetate and zeranol) are safe when used according to label directions (2). Within the European Union (EU) the use of all growth-promoters in livestock breeding is forbidden (3). For the last five years, β-agonistic drugs have become serious competitors of the anabolic steroids, βAgonistic drugs are chemically modified adrenalin (epinephrin) derivatives with a profound P -effect. They are originally used as bronchospasmoytics in humans, but administered to animals at a higher dosage, a positive effect on growth as well as the 2

0097-6156/96/0621-0060$15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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Immunochemical Approaches to β-Agonistic Drugs

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protein/fat tissue ratio also is observed. Several investigations have demonstrated that the β-agonists ractopamine (in pigs (4-7)), clenbuterol (in rats (8) and calves (9,70)) and cimaterol (in pigs (77) and lambs (72)) increase muscle mass and reduce fat deposition. In Europe, despite the E U ban on all growth-promoting agents, β -agonists are the most frequently found growth promoting agents. There have been reports of food poisoning related to the consumption of illicit β-agonists in liver in which clenbuterol exerts toxicity at a relatively high level (200-300 ng/g (13,14)). These reports and an increa sing misuse of β-agonists have led to increased control. The number of samples analyzed within the Dutch Ministry of Agriculture Nature Management and Fisheries for the presence of β-agonists increased from around 200 in 1988 to almost 20,000 in 1994. Urine is still die most frequently analyzed sample material, however, other materials are used for different reasons. In farmhouses, urine, feces and cattle feed can be sampled. Sampling of feces is much easier and faster than sampling urine, and the residue levels for β-agonists are comparable (75). At slaughter, edible tissues (liver, kidney and muscle) can be sampled next to body fluids (plasma, urine and bile) and eye samples. Bile is one of the most suitable sample material for the control on misuse of anabolic steroids and would, if possible, be preferred for the control of both steroids and β-agonists. Clenbuterol accumulates in the choroid/pigmented retinal epithelium tissue of the bovine eye (16,17), and even after a withdrawal period of 140 days clenbuterol can still be detected (18), which makes this material extreemly suitable for the control on misuse of clenbuterol. Besides the increased amount of different samples, other βagonists such as salbutamol, bromobuterol, mabuterol, mapenterol, cimaterol and terbutaline (see Table I) have been included in the control system. In order to detect all these β-agonists in different sample materials, three antisera (anti-clenbuterol, -salbutamol and -fenoterol) were raised in rabbits. These antisera were used to develop microtiter plate EIA's (19) and on-site tests (a strip test (20) and a tube test (27)). Immunoaffinity chromatography (IAC), using immobilized anti-clenbuterol, was used to simplify the sample preparation prior to confirmatory analysis (22,23). A l l these assays were mainly focussed on the analysis of urine samples. Applications of the microtiter plate EIA's in detecting β-agonists in different biological samples are descri bed. Experimental Materials. Helix Pomatia digestive juice (containing a minimum of 40 U/mL βglucuronidase and 20 U/mL arylsulphatase) and isobutanol were obtained from Merck (Darmstadt, Germany). Sheep anti-rabbit immunoglobulin G (IgG; whole molecule), Tween-20, bovine serum albumin (BSA), Pronase Ε (5.5 units/mg), clenbuterol hydro chloride and fenoterol hydrobromide were obtained from Sigma (St. Louis, M O , U.S.A.). Salbutamol sulfate and terbutaline sulfate were obtained from Bufa-Chemie (Castricum, The Netherlands). Robert Schilt (RDCILT-DLO, Wageningen, The Netherlands) supplied us with standards of mabuterol, mapenterol, bromobuterol, cimbuterol, tulobuterol, pirbuterol and cimaterol. Horseradish peroxidase grade I (Ε 1.11.1.7) was obtainedfromBoehringer (Mannheim, Germany). Flat bottom microtiter ELISA plates (96-well) were obtained from Greiner (Nurtingen, Germany). Tetramethylbenzidine

Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

62

IMMUNOASSAYS FOR RESIDUE ANALYSIS

Table L Structures of β-agonists 2

1

7

3 - H - j x U -


ÔH H

β-Agonist

Group at position / 2

Clenbuterol Mabuterol Bromobuterol Mapenterol Tulobuterol Cimaterol Cimbuterol Salbutamol Pirbuterol Carbuterol Isoproterenol Terbutaline Fenoterol Ractopamine

H H H H Cl H H H H H H H H H

CH

3

4

5

6

7

Cl CF Br CF, H H H H H H H

CHj

CHJ

CHJ

CHJ

CHJ CH2-CH3

H

OH

H

C C C c c c c c Ν C c c c c

CHj

OH

NH NH NH NH H NHj NH OH OH OH OH H H

Cl Cl Br Cl H CN CN CH OH CHjOH NH-CO-NH OH

3 2

2

2

2

2

2

OH

2

3

OH OH

CHJ CHJ

CHJ

CHj H CHj CHj

CHJ

CHJ

CHJ

CHJ

CHJ CHJ

H C H J CHj CHJ-CSHJ-OH H ΟΙ,-ΟΗ,-ΟΗτΟΗ H CHJ

(TMB) peroxidase substrate was obtained from Kirkegaard and Perry Labs (Gaithersburg, M D , USA). E L I S A Equipment. A Wellwash Model 4 microplate washer (Denley Instruments, Billinghurst, U.K.) and an Argus 400 microplate reader (Canberra Packard, Downers Grove, D, U.S.A.) were used. Antibody Preparation. Clenbuterol was conjugated to B S A (molar ratio 7:1) after diazotization, as described by Yamamoto and Iwata (24). Salbutamol-hemisuccinate was prepared according to Beaulieu (25). For the coupling of salbutamol-hemisuccinate to B S A (molar ratio 30:1), the procedure described by Kyrein (26) was used. Fenoterol was coupled to B S A (molar ratio 30:1) using 1,4-butane diglycidyl ether as a spacer (27). The B S A conjugates (ca. 0.4 mg) were mixed with Freund's complete adjuvant. The emulsions were subcutaneously injected into New Zealand white rabbits. After four weeks the rabbits were subcutaneously injected every six weeks with ca. 0.4 mg of conjugate in Freund's incomplete adjuvant and blood samples were taken one and two weeks after the respective immunizations. The collected sera were stored at -20 °C.


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Preparation of Horseradish Peroxidase Conjugates. For the coupling of salbutamolhemisuccinate and fenoterol to HRP (molar ratio 1:1), the procedures described by Kyrein (26) and Lommen (27) were used, respectively. Enzyme Immunoassays. Microtiter plates were coated overnight with 100 pL aliquots of sheep-anti rabbit IgG (10 pg/mL in 50 m M sodium-carbonate [pH 9.6]) at 4 °C. Plates were washed 3 times with PBST (5.4 m M Na-phosphate/1.3 m M K-phosphate/150 m M NaCl [pH 7.4], 0.05% Tween-20). In case of the Clenbuterol-EIA (results expressed as clenbuterol-equivalents), aliquots of 50 pL of diluted clenbuterol standard solutions (0.05-5 ng/mL) or 50 pL sample (extract) were added to the wells. In case of the Salbutamol-EIA (results expressed as salbutamol-equivalents), 50 pL of salbutamol standard solutions (0.05-5 ng/mL) were added. In case of the Fenoterol-EIA (results expressed as fenoterol-equivalents), 50 pL of fenoterol standard solutions (0.02-5 ng/mL) were added. Next, 25 pL of appropriately diluted salbutamol-HRP (Clenbuterol- and Salbutamol-EIA) or fenoterol-HRP (Fenoterol-EIA) and finally 25 pL of diluted antisera (all in PBST) were added. Raw sera were used in the three EIA's. The sera and enzyme-conjugates were diluted in glycerol (1:1; v/v) and stored at -20 °C. In case of the Clenbuterol-EIA, the serum and the salbutamol-HRP were used at final dilutions of 10,000 and 150,000, respectively. In case of the Salbutamol-EIA, the serum and the salbutamol-HRP were used at final dilutions of 1,000 and 60,000, respectively. In case of the Fenoterol-EIA, the serum and the fenoterol-HRP were used at final dilutions of40,000 and 20,000, respectively. The plates were incubated for 1 h at 4 °C and after washing (3 times with PBST), the bound peroxidase was assessed by adding 100 uL of a tetramethylbenzidine (TMB) peroxidase substrate system. After incubation in the dark for 20-30 min at 20 °C, the reaction was stopped by adding 100 pL aliquots of 1 M phosphoric acid and the colored product of the peroxidase reaction was measured at 450 nm. Origin of Samples. Lyophilized blank bovine urine samples (n = 20) were obtained from the bank of reference blank samples prepared by the European Community Reference Laboratory (CRL) the National Institute of Public Health and Environmental Protection (RTVM, Bilthoven, The Netherlands). Bovine urine (n = 1,730) and feed samples (n = 114) were taken by the General Inspection Service (Kerkrade, The Nether lands) in 1994 at 122 farm houses. Blank calf feces samples (n = 28) and calf feed samples (milk replacers; η = 28) were obtained from TNO Nutrition and Food Research (Zeist, The Netherlands). Urine, plasma, feces, liver, kidney and muscle samples from treated animals were obtained from an animal experiment in which two bulls were orally treated with low doses of clenbuterol (0.8 pg/kg b.w. for 28 days) and two bulls were treated with high doses of clenbuterol (8 pg/kg b.w. for 28 days). No withdrawal period was applied. This animal experiment was performed at the Research Institute for Livestock Feeding and Nutrition ( I W O - D L O , Lelystad, The Netherlands) in February 1993. Sample materialsfromtreated calves were obtained from an animal experiment in which calves (n = 20) were orally treated with a mixture of clenbuterol, mabuterol and mapenterol (each 0.4 pg/kg b.w.) for a period of 6 weeks. Urine, feces, liver, bile and retina samples were taken directly after treatment (no withdrawal period). This animal experiment was performed at IVVO-DLO in June 1993.



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IMMUNOASSAYS FOR RESIDUE ANALYSIS

Sample Preparation. Urine. Method I (direct method). The p H of the sample was adjusted to 7 ± 0.5 by adding a few drops of 1 M acetic acid and/or 1 M sodium hydroxide. The sample was dilutedfivetimes in PB ST and 50 μL were pipetted into the microtiter plate (0.2 mL of sample/mL). Method Π (hydrolysis). The pH of 1 mL of the urine sample was adjusted to 4.8 by adding a few drops of 1 M acetic acid. Afterwords, HelixPomatia Juice (25 μL) was added and the mixture was incubated for 2 h at 55 °C or overnight at 37 °C. To the hydrolyzed urine, PBST (4 mL) was added and the pH was adjusted to 7 ± 0.5 by adding a few drops of 1 M sodium hydroxide and 50 μL were pipetted into the microtiter plate (0.2 mL of sample/mL). Method ΠΙ (liquid-liquid extraction). The pH of the urine sample was adjusted to 9.5 ± 0.5 by adding a few drops of 1 M sodium hydroxide. To 1 mL of urine, isobutanol (2 mL) was added and after mixing (Vortex) for 1 min and centrifugation for 10 min at 1,500 X g, 1 mL of the isobutanol was evaporated at 50 °C under a stream of nitrogen. The residue was dissolved in PBST (0.5 mL) and 50 μL were pipetted into the microtiter plate (1 mL of sample/mL). Method I V (hydrolysis + liquid-liquid extraction). Urine was hydrolyzed according to method Π. The pH was re-adjusted to 9.5 ± 0.5 and 1 mL of the hydrolyzed sample was extracted with isobutanol according to method ΙΠ (1 mL of sample/mL). Feed To 5 g of feed, methanol (25 mL) and 0.2 M phosphoric acid (25 mL) were added. The extraction was performed using a mechanical shaker for 30 min. After centrifugation for 15 min at 3,000 X g at 4 °C, 0.25 mL of the supernatant was pipetted into 1 mL of PBST. The mixture was vortexed, centrifuged for 5 min at 3,000 X g and 50 pL were pipetted into the microtiter plate (0.02 g of feed/mL). Feces, Liver, Kidney, Bile and Plasma. To 1 g of sample, 0.1 M hydrochloric acid (5 mL) was added. The sample was homogenated with a Sorvall Omni-Mixer (Model 17106, Dupont, Newtown, USA) and placed in an ultrasonic bath for 15 min. After centrifugation for 10 min at 4,000 X g at 4 °C, the pH of the supernatant was re adjusted to 9.5 ± 0.5 by adding a few drops of 10 M sodium hydroxide. After centrifugation for 5 min at 1,500 X g, 2 mL of the supernatant was pipetted into 4 mL of isobutanol. The mixture was vortexed and centrifuged for 5 min at 1,500 X g. In case of feces, liver, kidney and bile, 1 mL of the supernatant was evaporated at 50 °C under a stream of nitrogen and the residue was dissolved in 500 pL of PBST (0.2 g of sample/mL). In case of plasma, 2.5 mL of the supernatant was evaporated and the residue dissolved in 250 pL PBST (1 mL of plasma/mL). 50 pL were pipetted into the microtiter plate. Choroid/Retina and Muscle. To 1 g of the sample, 4 mL of a pronase containing Trisbuffer (pH 8.0 + 1.25 mg pronase E) were added. After an overnight incubation at 55 °C and centrifugation for 10 min at 1,500 X g, 2 mL of the supernatant was pipetted into a test tube and the p H was adjusted to 9.5 ± 0.5 by the addition of a few drops of 10 M sodium hydroxide. To this mixture, isobutanol (5 mL) was added and after vortexing and centrifugation for 5 min, at 1,500 X g, the isobutanol was pipetted into a test tube. In case of the retina, 1 mL of the isobutanol was evaporated and the residue dissolved in 500 pL of PBST (0.2 g of sample/mL). In case of muscle, isobutanol (2.5


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Immunochemical Approaches to β-Agomstic Drugs

65

mL) was evaporated and the residue dissolved in 500 pL PBST (0.5 g of muscle/mL) and 50 pL were pipetted into the microtiter plate. G C - M S . The presence of β-agonists was confirmed by the procedure described before (28). Results and Discussion


Under the conditions described in the experimental, the dose response curves showed measuring ranges of 0.1 to 5 ng/mL for the Clenbuterol-EIA, 0.05-5 ng/mL for the Salbutamol-EIA and 0.02 to 5 ng/mL for the Fenoterol-EIA (see Figure 1). The Clenbuterol-EIA showed high cross-reactivity towards mapenterol, mabuterol, bromobuterol and cimbuterol (Table Π). Table Π: Cross-reactivity of the EIA's towards β-agonists and recovery (%) of the β-agonists after extraction in isobutanol β-agonist

Percentage of cross-reactivity Clenbuterol- Salbutamol- FenoterolEIA EIA EIA

clenbuterol cimbuterol salbutamol bromobuterol mapenterol mabuterol tulobuterol carbuterol terbutaline pirbuterol cimaterol fenoterol ractoparnine

100 60 6 100 80 70 2 5 4 4 6

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