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Determination of carazolol residues in porcine tissue by radioreceptor assay
Analytica Chimica Acta 462 (2002) 149–156
Determination of carazolol residues in porcine tissue by radioreceptor assay Susanne A. Meenagh∗ , John D.G. McEvoy, Christopher T. Elliott Department of Agriculture and Rural Development, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK Received 8 January 2002; received in revised form 13 March 2002; accepted 18 April 2002
Abstract A radioreceptor assay was developed for the determination of the -blocker carazolol in porcine muscle and kidney. The method involves a simple alkaline extraction procedure using diethyl ether followed by a competitive assay between carazolol residues and [3 H]-dihydroalprenolol ([3 H]-DHA) using solubilised 2-adrenoceptors isolated from a transfected cell line. The limit of detection (LOD) was determined using 20 reference blank samples of pig kidney and pig muscle. LODs for muscle (0.93 g kg−1 ) and kidney (1.47 g kg−1 ) were well below their respective European community maximum residue limits, (MRLs 5 and 25 g kg−1 , respectively). The assay was used to investigate if carazolol residues persisted in pig tissues for up to 30 h post-intramuscular injection at the recommended dose rate (10 g carazolol/kg body weight). The highest mean ±S.D. concentrations were detected at 1 h post-injection in kidney (10.84 ± 1.3 g kg−1 ) and muscle (3.59 ± 0.2 g kg−1 ) which were less than the respective MRLs. It is concluded that this method offers a robust and rapid alternative to other methods for the screening of carazolol residues in pig meat. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Carazolol; Radio-receptor assay; Porcine muscle; Porcine kidney; Incurred tissues
1. Introduction In veterinary medicine carazolol 4-(2-hydroxy-3isopropylaminopropoxy)carbazole (Fig. 1) is a -blocking agent prescribed for use in pigs to relieve the stress of parturition, reduce the incidence of the mastitis, metritis, agalactia syndrome, prevent frenzy during mating and alleviate tachycardia. However, it is most widely used to reduce stress during transportation from farm to slaughterhouse and the subsequent incidence of pale, soft and exudative (PSE) meat caused by stress-induced accelerated glycogen
∗ Corresponding author. Tel.: +44-28-90-525-636; fax: +44-28-90-525-626. E-mail address: [email protected] (S.A. Meenagh).
metabolism in muscle. Such meat is less marketable and the farmer may be financially penalised. Carazolol is not licensed for the treatment of food animals in the UK, however, it is available in Spain as a licensed veterinary medicine (SuacronTM , Bayer). In the EU, maximum residue limits (MRLs) have been elaborated for both cattle and pigs. The compound is listed in Annex I of [1] and the MRLs for pig kidney and muscle are 25 and 5 g kg−1 , respectively [2]. The marker residue is the parent substance. Under Council Directive 96/23/EC, member states are mandated to examine animals and animal products for carazolol residues [3]. Although residues have not been detected in the UK, the Belgian monitoring programme has frequently detected residues of this compound and other tranquillisers in pig meat throughout the 1990s [4,5].
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Fig. 1. Chemical structure of carazolol 4-(2-hydroxy-3-isopropylamino-propoxy)carbazole.
Even though the licensed product has a zero meat withholding time (and is indicated for the reduction of stress prior to slaughter) there are unconfirmed reports of black market preparations being used which may explain the regular findings of carazolol residues. This is of concern since carazolol is also used in human medicine to treat conditions including tachycardia due to hyperactivity of the sympathetic nervous system. Residues of the drug may pose a hazard to human health. Several methods have been described detailing the detection of carazolol residues in animal tissues including high performance thin layer chromatography (HPTLC) [6], fluorescence spectrometry [7], liquid chromatography (LC) [8–10] and mass spectrometry [11]. These methods often require extensive sample clean up before application to the assay and have limited sample throughput. A radioimmunoassay (RIA) for porcine urine and an enzyme immunoassay (EIA) for porcine tissue have also been described for carazolol [12,13]. However, such methods tend to be compound specific and unsuitable for multi-analyte screening. The current study describes the use of a rapid and sensitive radioreceptor assay which exploits the affinity of -agonist and -blocker residues for a solubilised 2-adrenoceptor isolated from a transfected cell line [14]. The transfected cell line expresses a high concentration of 2-adrenoceptors and has previously shown affinity for a number of -agonist compounds including clenbuterol and cimaterol [15]. This receptor has been shown to bind to several -blockers in this laboratory including labetalol, nadolol, pindolol and propranolol. In this assay, carazolol competes with a radiolabelled
antagonist [3 H]-dihydroalprenolol ([3 H]-DHA) for the receptor binding site. The extraction procedure for tissue involves homogenisation with sodium hydroxide followed by extraction with diethyl ether before application to the assay. Both fortified and incurred tissue samples were analysed using the method and a short study was conducted in pigs to establish whether carazolol residues were detectable for up to 30 h post-injection following the use of a commercially available veterinary formulation (SuacronTM , Bayer).
2. Experimental 2.1. Chemicals Carazolol (analytical standard) and SuacronTM (a veterinary preparation of Carazolol) were gifts from Bayer (Barcelona, Spain). Sodium acetate, anhydrous disodium hydrogenorthophosphate hydrate, sodium chloride, potassium hydrogen carbonate and magnesium chloride 6-hydrate were all Analar grade material and obtained from BDH Ltd. (Lutterworth UK). [3 H]-DHA, specific activity 97 Ci mmol−1 was obtained from Amersham Pharmacia Biotech. UK Ltd. (Buckinghamshire, UK). Optiphase hi-safe scintillation fluid was obtained from Wallac (Turku, Finland). Protein estimation kit, aprotinin, digitonin, isoproterenol, leupeptin, polyethylenamine and trizma hydrochloride (tris(hydroxymethyl)aminomethane hydroenamine) were obtained from Sigma (Poole, UK). 2.2. Cell line and growth media A transfected cell line NCB20-D1 (murine neuroblastoma × embryonic Chinese hamster brain cells) with a high expression of human 2-adrenoceptors was a gift from Professor Graeme Milligan, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland. Dulbecco’s modified Eagle Medium (DMEM) was obtained from Life Technologies (Paisley, UK). The media for cell growth was supplemented with 10% foetal bovine serum (Labtek Int. Ltd., East Sussex, UK) and gentamicin sulphate which (50 l ml−1 ) obtained from Biowhittaker UK
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Ltd. (Wokingham, UK). Geneticin sulphate was obtained from Sigma. 2.3. Preparation of buffers Phosphate buffered saline (PBS) pH 7.2 was prepared by dissolving anhydrous disodium hydrogenorthophosphate (10.61 g), sodium dihydrogenorthophospate (3.44 g) and sodium chloride (85 g) in 1 l of deionised water. The stock solution was diluted 1:10 in deionised water to give a 0.01 M solution. Lysis buffer was prepared by dissolving Tris–HCl (0.3 g), potassium hydrogencarbonate (0.1 g) and magnesium chloride 6-hydrate (0.5 g) in 1 l of deionised water. Leupeptin (10.0 g) and aprotinin solution (200 l) were added to the buffer immediately prior to use.
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they were group housed and acclimatised for 3 days. Immediately prior to the experiment they were individually identified, weighed. One animal was removed from the group and housed in an adjacent pen. The remaining 12 animals were injected intramuscularly behind the right ear with Suacron at the recommended dose rate of 10 g carazolol/kg−1 body weight. The single animal functioned as an unmedicated (negative) control, and was slaughtered prior to the first batch of medicated animals. The treated animals were slaughtered by captive blot stunning and exsanguination in pairs at 1, 2, 4, 8, 24 and 30 h post-injection. Samples of gluteal muscle, diaphragm, and both kidneys were removed and individually bagged for analysis. Diaphragm samples were extensively trimmed prior to analysis to remove excess fat, which may interfere with the assay. All samples were stored at −20 ◦ C prior to analysis.
2.4. Preparation of standards 2.7. Methodology 1 mg ml−1
A stock solution of of carazolol was prepared in methanol. From 1 mg ml−1 standard dilutions were prepared in 1 mM sodium acetate solution to obtain standard solutions of 125, 250, 500, 625, 1250, 2500, and 5000 ng ml−1 . Standards were stored at 4 ◦ C and kept for 1 month. 2.5. Apparatus Standard laboratory apparatus was used throughout for the preparation of media and chemical solutions. A Sigma 3K30C high speed centrifuge (Wishart Scientific, Ballyclare, Northern Ireland) was used in the solubilisation of 2-adrenoceptors from cell membranes. A 1225 sampling manifold was obtained from Millipore UK Ltd. (Watford, UK). GF/B glass fibre filters (25 mm diameter) were obtained from Whatman (Maidstone, UK). Filter papers were pre-treated with 0.3% polyethylenamine and were used to filter reaction mixtures. Radioactivity was counted using an LKB 1219 scintillation counter (Wallac) with on-line data analysis using Multicalc V2.4 software (Wallac). 2.6. Production of carazolol incurred pig tissues Thirteen Landrace pigs aged 4 months were used for a small scale depletion study. Upon arrival at the unit
2.7.1. Preparation of cell membranes NCB20-D1 cells were routinely cultured as described previously [16] in DMEM and incubated under an atmosphere of 95% air and 5% CO2 in a humidified incubator at a temperature of 37 ◦ C. When the culture flasks were 90–95% confluent, cells were scraped from the flask surface using a cell scraper and collected in lysis buffer. The cell suspension was homogenised with 10–12 strokes of a PTFE hand held homogeniser, before centrifuging for 5 min at 4 ◦ C at 1000 × g. The resulting supernatants were collected and further centrifuged at 28,000 × g for 10 min at 4 ◦ C. The membrane pellet was resuspended in ice cold lysis buffer and assayed for protein concentration using a commercial assay kit. The membrane suspension was used immediately in solubilisation procedures or stored frozen at −70 ◦ C pending future use. 2.7.2. Solubilisation of β2-adrenoceptors The cell membrane suspension was mixed with a 1% (m/v) aqueous solution of digitonin at a final protein concentration of ca. 5 mg ml−1 . The suspension was vortexed for 10 s using a Whirlmix and kept on ice. Vortexing was repeated every 15 min. After four vortexing cycles the suspension was centrifuged at 50,000 × g for 1 h at 4 ◦ C. The resulting supernatant
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was assayed for protein concentration and either stored at −70 ◦ C or used immediately in the kidney and muscle binding assays. 2.7.3. Extraction procedure (kidney and muscle) Finely chopped tissue (6 × 5 g) was weighed into glass universals (25 ml) and spiked with carazolol standards prepared previously, to give a standard range of 0–50 g kg−1 for muscle and 0–100 g kg−1 for kidney. Spiked tissues were allowed to stand for 10 min. Sample tissue (5 g) was also weighed into a glass universal. Following the addition of 10 ml of sodium hydroxide solution (1 M) to the standards and samples, the tissues were homogenised for 30 s and a further 5 ml of sodium hydroxide solution was added. Samples were mixed on an end-over-end mixer for 30 min, before centrifugation at 2000 × g for 10 min. The supernatant was decanted into clean universals excluding floating fat particles that aggregated on the surface of the solution. Supernatant (1 ml) was added to glass test tubes and diethyl ether (1 ml) was added. The tubes were shaken for 10 min on a test tube shaker, before centrifugation at 1000 × g for a further 10 min. Sample tubes were frozen using an aluminium block, which had been pre-cooled in liquid nitrogen. The diethyl ether layer was transferred to a clean test tube, and the sample tubes were allowed to thaw before the extraction process was repeated by addition of a further 1 ml of diethyl ether. The ether extracts were combined and evaporated to dryness under a gentle stream of nitrogen on a dry-block sample concentrator (TechneDB-3A) at 60 ◦ C. The resulting sample residue was reconstituted in 1 ml of PBS and vortexed gently before application to the receptor assay. 2.7.4. Radioreceptor assay Tissue extracts were analysed in triplicate by incubating 100 l of extract with 200 g of solubilised receptor protein and 0.23 nM 3 H-DHA in a final incubation volume of 500 l PBS. Non-specific binding was determined in the presence of an excess of the -agonist drug (500 M isoproterenol). Incubations were carried out for 45 min at 30 ◦ C. Reactions were terminated by adding 3 ml of ice cold PBS and filtering over GF/B glass fibre filters pre-treated with 0.3% polyethylenamine. Filters were washed three times with 1 ml of ice cold PBS before soaking for 2 h in
3 ml of Optiphase scintillation fluid. Radioactivity was quantified by liquid scintillation counting for 1 min. 2.7.5. Data analysis Raw data obtained (dpm) for each of the points of the five standard curves generated during the reproducibility experiments for matrix standard curves (kidney and muscle), were normalised against the response of the zero standard (0 ng/g carazolol) of each corresponding standard curve. Normalised data sets were fitted using non-linear regression analysis (Unistat, London, UK). 2.7.6. Assay validation The limit of detection (LOD) was determined by assaying a panel of kidney and muscle samples taken from known untreated pigs (n = 20). The mean and the standard deviation were calculated from the assay data. The LOD was calculated from the assay data as the mean value plus three times the standard deviation (s). Assay reproducibility was determined by performing repeated analysis (within and between assays) of blank samples fortified with carazolol at concentrations ranging from 2.5 to 10 g kg−1 for muscle and from 5.0 to 25 g kg−1 for kidney. 3. Results and discussion 3.1. Assay performance A typical calibration curve (n = 5) obtained for the radioreceptor muscle assay is shown (Fig. 2). The LOD for this matrix was 0.93 g kg−1 . The assay exhibited good repeatability data for both intra and inter-assay variations (Table 1). The intra-assay coefficient of variation was 3.5, 6.1 and 6.7% for samples fortified with carazolol at levels of 2.5, 5.0, and 10 g kg−1 , respectively, while inter-assay variation at these fortification levels was 9.9, 6.2 and 4.0%, respectively. Calibration curves (n = 5) for the radioreceptor kidney assay are shown (Fig. 3). The LOD for this matrix was 1.47 g kg−1 . This is comparable to reported LODs for LC methods available for the determination of carazolol [8–10]. Intra-assay variation was determined as 7.8, 10.8 and 4.1% at concentrations of 5, 12.5 and 25 g kg−1 with inter assay variations
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Fig. 2. Calibration curve (n = 5) for the radioreceptor muscle assay. Error bars represent standard deviation at each calibration point.
determined as 5.2, 4.4 and 6.5%. Inter-assay variations were 5.2, 4.4 and 6.5% at these fortification concentrations (Table 2). 3.2. Results of incurred residue study The results from the incurred residue study are shown in Table 3. At each of the sampling points, there was considerable variation in the concentrations of carazolol observed in individual animals. However,
the highest concentrations of carazolol residues were consistently detected in kidney medulla and cortex at 1 h post-injection. The concentration of the compound had decreased in the medulla by 2 h post-injection with only a minor decrease observed in the kidney cortex. As carazolol is a lipophilic -blocker, this explains why residues persisted in the cortex. At the three remaining time points (8, 24 and 30 h) carazolol residues were not detected in the left kidney (medulla and cortex), or in the right kidney medulla. However,
Table 1 Assay validation data recorded for carazolol muscle No. of assays
Spiking concentration of carazolol (g kg−1 )
Repeatability Mean ± S.D. concentration of carazolol measured (g kg−1 )
Sr (%)
Intra-assay 5 5 5
2.5 5.0 10.0
2.61 ± 0.09 4.94 ± 0.30 9.24 ± 0.62
3.5 6.1 6.7
Inter-assay 5 5 5
2.5 5.0 10.0
2.43 ± 0.24 5.51 ± 0.34 9.56 ± 0.38
9.9 6.2 4.0
No. of blank (negative) tissue samples tested
Mean ± S.D. concentration of carazolol measured in tissue blanks (g kg−1 )
Limit of detection (mean ± 3S.D.)
20
0.69 ± 0.08
0.93
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Fig. 3. Calibration curve (n = 5) for the radioreceptor kidney assay. Error bars represent standard deviation at each calibration point.
concentrations in excess of the LOD were detected in the right kidney cortex. These findings illustrate that when scheduling kidney for carazolol residue analysis both kidneys should be sampled and cortex should preferentially be screened. Carazolol was only detectable in diaphragmatic muscle at 1 and 2 h post-injection. Corresponding gluteal muscle samples were negative. If the results of the present small scale study are true, i.e. that carazolol concentrations are higher in diaphragm than in other muscle groups, this has implications for the
choice of muscle tissue for routine monitoring. It is reassuring that in most monitoring programmes, diaphragmatic muscle is the muscle of choice for sampling since it is easy to remove and does not devalue the carcass. This phenomenon has also been reported for tilmicosin [17], where widely differing concentrations were observed in different muscle groups within individual animals. Overall, the results from the depletion study demonstrated that, when dosed at the recommended dose rate, kidneys and muscle from treated pigs did not contain
Table 2 Assay validation data recorded for carazolol kidney No. of assays
Spiking concentration of carazolol (g kg−1 )
Repeatability Mean ± S.D. concentration of carazolol measured (g kg−1 )
Sr (%)
Intra-assay 5 5 5
5.0 12.5 25.0
4.75 ± 0.37 12.94 ± 1.40 23.40 ± 0.96
7.8 10.8 4.1
Inter-assay 5 5 5
5.0 12.5 25.0
5.49 ± 0.29 13.04 ± 0.58 26.12 ± 1.71
5.2 4.4 6.5
No. of blank (negative) tissue samples tested
Mean ± S.D. concentration of carazolol measured in tissue blanks (g kg−1 )
Limit of detection (mean ± 3S.D.)
20
0.42 ± 0.35
1.47
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Table 3 Concentrations of carazolol (g kg−1 ) residues in pig muscle and kidney samples taken from pigs treated with carazolol at 10 g kg−1 body weight by single intramuscular injection. With the exception of the unmedicated control animal (n = 1), two pigs were slaughtered at each of the time points. Values shown are mean ± S.D. concentrations of three replicate determinations per tissue Time (h)
Kidney medulla
Kidney cortex
Muscle
Left
Right
Left
Right
Diaphragm
Gluteal
2
Pig 3 Pig 4
3.13 ± 1.93
4.02 ± 1.56
6.06 ± 1.50 5.32 ± 1.30
6.10 ± 1.88 7.72 ± 0.63
2.22 ± 0.08 2.45 ± 0.16
4
Pig 5 Pig 6
7.11 ± 2.6
3.69 ± 0.55 6.28 ± 1.02
8
Pig 7 Pig 8
2.59 ± 1.20 3.27 ± 0.30
24
Pig 9 Pig 10
2.00 ± 0.34 1.91 ± 1.04
30
Pig 11 Pig 12
2.37 ± 0.19 1.79 ± 0.83
Negative control 1
LOD, limit of detection; 1.47 and 0.93 g kg−1 for kidney and muscle, respectively.
carazolol residues in excess of the MRL. This finding supports the label claim of zero meat withholding for the drug. Our results are comparable to those reported from pharmacokinetic studies performed using radiolabelled carazolol (10 g kg−1 body weight) with analysis performed using HPLC [18]. Mean ± S.D. concentrations of carazolol detected in porcine muscle (n = 6) were 1.77 ± 0.78 and 2 ± 0.57 g kg−1 at 2 h post-injection. Concentrations measured in two pigs at 16 h post-injection had decreased to 0.14 and 0.02 g kg−1 , respectively. The concentrations in muscle detected at the 16 h time point were less than the LOD for our muscle radioreceptor assay. In this earlier study [18], concentrations of radiolabelled carazolol detected in kidney (n = 6) ranged from 20.9 ± 6.2 g kg−1 after 2 h, to 4.7 g kg−1 after 16 h. These concentrations were higher than those detected in the incurred residue study reported in this paper. As carazolol is a lipophilic -blocker which undergoes extensive metabolism [19], the higher kidney concentrations of radiolabelled analyte measured by LC may be attributed to the sum of the parent drug plus (radioactive) metabolites. Although there is little published data on carazolol metabolites in the pig, Rudolph and Steinhart [20] detected three conjugated carazolol metabolites in pig urine—monoglucuronide, lactate and acetate.
Such conjugations facilitate elimination from the body and common sense dictates that these metabolites are less likely to have biological activity (and affinity for) the 2-receptor. This assumption may explain the discrepancies observed between the two studies.
4. Conclusions The present study, has led to the development of a rapid, sensitive radioreceptor assay for the detection of carazolol residues in porcine muscle and kidney. Its major advantage over other published methods is the minimal sample clean up required before application to the receptor assay. The LOD is comparable to those reported for existing methods used for screening for carazolol residues. As this receptor assay may have the potential for being used as a multi-residue screening method for a range of agonistic [15] and antagonistic compounds acting via the 2-receptor, further investigations will be performed to assess the suitability of this receptor for other -blocking drugs. It is concluded that this assay is suitable for routine monitoring of residues of carazolol in pig meat and may have a future potential application for screening residues of other -blockers and -agonists.
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Acknowledgements The authors wish to thank Mildred Wylie and Deborah Snodden for their assistance with tissue cultures during this study, Ciaran Mulligan for the handling and post-mortem of animals and Peter Watson and Angeles Fornells from Bayer for supplying the carazolol. References [1] Council regulation (EEC) 2377/90 of 26 June 1990, Official J. Eur. Commun. No. L224 (1990) 1–6. [2] Commission regulation (EC) No. 1442/95 of 26 June 1995, Official J. Eur. Commun. L143 (1995) 26–30. [3] Council directive 96/23/EC of 29 April 1996, Official J. Eur. Commun. No. L125 (1996) 10–32. [4] Annual Report of the Institute of Veterinary Inspection (Belgium) for 1999 http://cemu10.fmv.ulg.ac.be/OSTC/ MonitoringIEV/99iev.htm. [5] Comission Européene, Direction Générale XXIV. XXIV/ 1516/98-MR-final (04/03/99 http://europa.eu.int/comm/food/ fs/inspections/vi/reports/belgium/vi rep belg 1516-1998 fr. pdf. [6] N. Haagsma, E.R. Bathelt, J.W. Engelsma, J. Chromatogr. 436 (1988) 73–76.
[7] J.W. Engelsma, J. Simons, Vet. Quart. 7 (1985) 73–76. [8] M. Rudolph, H. Steinhart, J. Chromatogr. 392 (1987) 371– 378. [9] M. Rose, G. Shearer, J. Chromatogr. 624 (1992) 471– 477. [10] H. Keukens, M.M.L. Aerts, J. Chromatogr. 464 (1989) 149–161. [11] M. Dubois, D. Fluchard, S. Kiebooms, Y. Colemonts, P. Delahaut, in: L.A. van Ginkel, A. Ruiter (Eds.), Proceedings of the Euroresidue IV Conference, Euroresidue Foundation, Utrecht, The Netherlands, 2000, pp. 398–405. [12] E. Rattenberger, Arch. Lebensmittel. Hygiene 40 (1989) 118. [13] E. Rattenberger, O Herr, Arch. Lebensmittel Hygiene 44 (1993) 135–137. [14] I. Mullaney, B.H. Shah, A. Wise, G. Milligan, J. Neurochem. 65 (1995) 545–553. [15] S. Meenagh, C. Elliott, R. Buick, Analyst 126 (2001) 491–494. [16] E. Kelly, M. Keen, P. Nobbs, J. MacDermot, Br. J. Pharmacol. 99 (1990) 309–316. [17] J.G. Beechinor, F.J. Bloomfield, Vet. Rec. 149 (2001) 182–183. [18] FAO Food and Nutrition Paper 41/4, 1991, pp. 23–37. [19] W. Riess, S. Brechbuhler, L. Brunner, P. Imhof, D.B. Jack, Therapiewoche 25 (1975) 4259. [20] M. Rudolph, H. Steinhart, J. Chromatogr. 392 (1987) 371–378.
Determination of carazolol residues in porcine tissue by radioreceptor assay Susanne A. Meenagh∗ , John D.G. McEvoy, Christopher T. Elliott Department of Agriculture and Rural Development, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK Received 8 January 2002; received in revised form 13 March 2002; accepted 18 April 2002
Abstract A radioreceptor assay was developed for the determination of the -blocker carazolol in porcine muscle and kidney. The method involves a simple alkaline extraction procedure using diethyl ether followed by a competitive assay between carazolol residues and [3 H]-dihydroalprenolol ([3 H]-DHA) using solubilised 2-adrenoceptors isolated from a transfected cell line. The limit of detection (LOD) was determined using 20 reference blank samples of pig kidney and pig muscle. LODs for muscle (0.93 g kg−1 ) and kidney (1.47 g kg−1 ) were well below their respective European community maximum residue limits, (MRLs 5 and 25 g kg−1 , respectively). The assay was used to investigate if carazolol residues persisted in pig tissues for up to 30 h post-intramuscular injection at the recommended dose rate (10 g carazolol/kg body weight). The highest mean ±S.D. concentrations were detected at 1 h post-injection in kidney (10.84 ± 1.3 g kg−1 ) and muscle (3.59 ± 0.2 g kg−1 ) which were less than the respective MRLs. It is concluded that this method offers a robust and rapid alternative to other methods for the screening of carazolol residues in pig meat. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Carazolol; Radio-receptor assay; Porcine muscle; Porcine kidney; Incurred tissues
1. Introduction In veterinary medicine carazolol 4-(2-hydroxy-3isopropylaminopropoxy)carbazole (Fig. 1) is a -blocking agent prescribed for use in pigs to relieve the stress of parturition, reduce the incidence of the mastitis, metritis, agalactia syndrome, prevent frenzy during mating and alleviate tachycardia. However, it is most widely used to reduce stress during transportation from farm to slaughterhouse and the subsequent incidence of pale, soft and exudative (PSE) meat caused by stress-induced accelerated glycogen
∗ Corresponding author. Tel.: +44-28-90-525-636; fax: +44-28-90-525-626. E-mail address: [email protected] (S.A. Meenagh).
metabolism in muscle. Such meat is less marketable and the farmer may be financially penalised. Carazolol is not licensed for the treatment of food animals in the UK, however, it is available in Spain as a licensed veterinary medicine (SuacronTM , Bayer). In the EU, maximum residue limits (MRLs) have been elaborated for both cattle and pigs. The compound is listed in Annex I of [1] and the MRLs for pig kidney and muscle are 25 and 5 g kg−1 , respectively [2]. The marker residue is the parent substance. Under Council Directive 96/23/EC, member states are mandated to examine animals and animal products for carazolol residues [3]. Although residues have not been detected in the UK, the Belgian monitoring programme has frequently detected residues of this compound and other tranquillisers in pig meat throughout the 1990s [4,5].
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 3 3 8 - 0
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Fig. 1. Chemical structure of carazolol 4-(2-hydroxy-3-isopropylamino-propoxy)carbazole.
Even though the licensed product has a zero meat withholding time (and is indicated for the reduction of stress prior to slaughter) there are unconfirmed reports of black market preparations being used which may explain the regular findings of carazolol residues. This is of concern since carazolol is also used in human medicine to treat conditions including tachycardia due to hyperactivity of the sympathetic nervous system. Residues of the drug may pose a hazard to human health. Several methods have been described detailing the detection of carazolol residues in animal tissues including high performance thin layer chromatography (HPTLC) [6], fluorescence spectrometry [7], liquid chromatography (LC) [8–10] and mass spectrometry [11]. These methods often require extensive sample clean up before application to the assay and have limited sample throughput. A radioimmunoassay (RIA) for porcine urine and an enzyme immunoassay (EIA) for porcine tissue have also been described for carazolol [12,13]. However, such methods tend to be compound specific and unsuitable for multi-analyte screening. The current study describes the use of a rapid and sensitive radioreceptor assay which exploits the affinity of -agonist and -blocker residues for a solubilised 2-adrenoceptor isolated from a transfected cell line [14]. The transfected cell line expresses a high concentration of 2-adrenoceptors and has previously shown affinity for a number of -agonist compounds including clenbuterol and cimaterol [15]. This receptor has been shown to bind to several -blockers in this laboratory including labetalol, nadolol, pindolol and propranolol. In this assay, carazolol competes with a radiolabelled
antagonist [3 H]-dihydroalprenolol ([3 H]-DHA) for the receptor binding site. The extraction procedure for tissue involves homogenisation with sodium hydroxide followed by extraction with diethyl ether before application to the assay. Both fortified and incurred tissue samples were analysed using the method and a short study was conducted in pigs to establish whether carazolol residues were detectable for up to 30 h post-injection following the use of a commercially available veterinary formulation (SuacronTM , Bayer).
2. Experimental 2.1. Chemicals Carazolol (analytical standard) and SuacronTM (a veterinary preparation of Carazolol) were gifts from Bayer (Barcelona, Spain). Sodium acetate, anhydrous disodium hydrogenorthophosphate hydrate, sodium chloride, potassium hydrogen carbonate and magnesium chloride 6-hydrate were all Analar grade material and obtained from BDH Ltd. (Lutterworth UK). [3 H]-DHA, specific activity 97 Ci mmol−1 was obtained from Amersham Pharmacia Biotech. UK Ltd. (Buckinghamshire, UK). Optiphase hi-safe scintillation fluid was obtained from Wallac (Turku, Finland). Protein estimation kit, aprotinin, digitonin, isoproterenol, leupeptin, polyethylenamine and trizma hydrochloride (tris(hydroxymethyl)aminomethane hydroenamine) were obtained from Sigma (Poole, UK). 2.2. Cell line and growth media A transfected cell line NCB20-D1 (murine neuroblastoma × embryonic Chinese hamster brain cells) with a high expression of human 2-adrenoceptors was a gift from Professor Graeme Milligan, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland. Dulbecco’s modified Eagle Medium (DMEM) was obtained from Life Technologies (Paisley, UK). The media for cell growth was supplemented with 10% foetal bovine serum (Labtek Int. Ltd., East Sussex, UK) and gentamicin sulphate which (50 l ml−1 ) obtained from Biowhittaker UK
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Ltd. (Wokingham, UK). Geneticin sulphate was obtained from Sigma. 2.3. Preparation of buffers Phosphate buffered saline (PBS) pH 7.2 was prepared by dissolving anhydrous disodium hydrogenorthophosphate (10.61 g), sodium dihydrogenorthophospate (3.44 g) and sodium chloride (85 g) in 1 l of deionised water. The stock solution was diluted 1:10 in deionised water to give a 0.01 M solution. Lysis buffer was prepared by dissolving Tris–HCl (0.3 g), potassium hydrogencarbonate (0.1 g) and magnesium chloride 6-hydrate (0.5 g) in 1 l of deionised water. Leupeptin (10.0 g) and aprotinin solution (200 l) were added to the buffer immediately prior to use.
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they were group housed and acclimatised for 3 days. Immediately prior to the experiment they were individually identified, weighed. One animal was removed from the group and housed in an adjacent pen. The remaining 12 animals were injected intramuscularly behind the right ear with Suacron at the recommended dose rate of 10 g carazolol/kg−1 body weight. The single animal functioned as an unmedicated (negative) control, and was slaughtered prior to the first batch of medicated animals. The treated animals were slaughtered by captive blot stunning and exsanguination in pairs at 1, 2, 4, 8, 24 and 30 h post-injection. Samples of gluteal muscle, diaphragm, and both kidneys were removed and individually bagged for analysis. Diaphragm samples were extensively trimmed prior to analysis to remove excess fat, which may interfere with the assay. All samples were stored at −20 ◦ C prior to analysis.
2.4. Preparation of standards 2.7. Methodology 1 mg ml−1
A stock solution of of carazolol was prepared in methanol. From 1 mg ml−1 standard dilutions were prepared in 1 mM sodium acetate solution to obtain standard solutions of 125, 250, 500, 625, 1250, 2500, and 5000 ng ml−1 . Standards were stored at 4 ◦ C and kept for 1 month. 2.5. Apparatus Standard laboratory apparatus was used throughout for the preparation of media and chemical solutions. A Sigma 3K30C high speed centrifuge (Wishart Scientific, Ballyclare, Northern Ireland) was used in the solubilisation of 2-adrenoceptors from cell membranes. A 1225 sampling manifold was obtained from Millipore UK Ltd. (Watford, UK). GF/B glass fibre filters (25 mm diameter) were obtained from Whatman (Maidstone, UK). Filter papers were pre-treated with 0.3% polyethylenamine and were used to filter reaction mixtures. Radioactivity was counted using an LKB 1219 scintillation counter (Wallac) with on-line data analysis using Multicalc V2.4 software (Wallac). 2.6. Production of carazolol incurred pig tissues Thirteen Landrace pigs aged 4 months were used for a small scale depletion study. Upon arrival at the unit
2.7.1. Preparation of cell membranes NCB20-D1 cells were routinely cultured as described previously [16] in DMEM and incubated under an atmosphere of 95% air and 5% CO2 in a humidified incubator at a temperature of 37 ◦ C. When the culture flasks were 90–95% confluent, cells were scraped from the flask surface using a cell scraper and collected in lysis buffer. The cell suspension was homogenised with 10–12 strokes of a PTFE hand held homogeniser, before centrifuging for 5 min at 4 ◦ C at 1000 × g. The resulting supernatants were collected and further centrifuged at 28,000 × g for 10 min at 4 ◦ C. The membrane pellet was resuspended in ice cold lysis buffer and assayed for protein concentration using a commercial assay kit. The membrane suspension was used immediately in solubilisation procedures or stored frozen at −70 ◦ C pending future use. 2.7.2. Solubilisation of β2-adrenoceptors The cell membrane suspension was mixed with a 1% (m/v) aqueous solution of digitonin at a final protein concentration of ca. 5 mg ml−1 . The suspension was vortexed for 10 s using a Whirlmix and kept on ice. Vortexing was repeated every 15 min. After four vortexing cycles the suspension was centrifuged at 50,000 × g for 1 h at 4 ◦ C. The resulting supernatant
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was assayed for protein concentration and either stored at −70 ◦ C or used immediately in the kidney and muscle binding assays. 2.7.3. Extraction procedure (kidney and muscle) Finely chopped tissue (6 × 5 g) was weighed into glass universals (25 ml) and spiked with carazolol standards prepared previously, to give a standard range of 0–50 g kg−1 for muscle and 0–100 g kg−1 for kidney. Spiked tissues were allowed to stand for 10 min. Sample tissue (5 g) was also weighed into a glass universal. Following the addition of 10 ml of sodium hydroxide solution (1 M) to the standards and samples, the tissues were homogenised for 30 s and a further 5 ml of sodium hydroxide solution was added. Samples were mixed on an end-over-end mixer for 30 min, before centrifugation at 2000 × g for 10 min. The supernatant was decanted into clean universals excluding floating fat particles that aggregated on the surface of the solution. Supernatant (1 ml) was added to glass test tubes and diethyl ether (1 ml) was added. The tubes were shaken for 10 min on a test tube shaker, before centrifugation at 1000 × g for a further 10 min. Sample tubes were frozen using an aluminium block, which had been pre-cooled in liquid nitrogen. The diethyl ether layer was transferred to a clean test tube, and the sample tubes were allowed to thaw before the extraction process was repeated by addition of a further 1 ml of diethyl ether. The ether extracts were combined and evaporated to dryness under a gentle stream of nitrogen on a dry-block sample concentrator (TechneDB-3A) at 60 ◦ C. The resulting sample residue was reconstituted in 1 ml of PBS and vortexed gently before application to the receptor assay. 2.7.4. Radioreceptor assay Tissue extracts were analysed in triplicate by incubating 100 l of extract with 200 g of solubilised receptor protein and 0.23 nM 3 H-DHA in a final incubation volume of 500 l PBS. Non-specific binding was determined in the presence of an excess of the -agonist drug (500 M isoproterenol). Incubations were carried out for 45 min at 30 ◦ C. Reactions were terminated by adding 3 ml of ice cold PBS and filtering over GF/B glass fibre filters pre-treated with 0.3% polyethylenamine. Filters were washed three times with 1 ml of ice cold PBS before soaking for 2 h in
3 ml of Optiphase scintillation fluid. Radioactivity was quantified by liquid scintillation counting for 1 min. 2.7.5. Data analysis Raw data obtained (dpm) for each of the points of the five standard curves generated during the reproducibility experiments for matrix standard curves (kidney and muscle), were normalised against the response of the zero standard (0 ng/g carazolol) of each corresponding standard curve. Normalised data sets were fitted using non-linear regression analysis (Unistat, London, UK). 2.7.6. Assay validation The limit of detection (LOD) was determined by assaying a panel of kidney and muscle samples taken from known untreated pigs (n = 20). The mean and the standard deviation were calculated from the assay data. The LOD was calculated from the assay data as the mean value plus three times the standard deviation (s). Assay reproducibility was determined by performing repeated analysis (within and between assays) of blank samples fortified with carazolol at concentrations ranging from 2.5 to 10 g kg−1 for muscle and from 5.0 to 25 g kg−1 for kidney. 3. Results and discussion 3.1. Assay performance A typical calibration curve (n = 5) obtained for the radioreceptor muscle assay is shown (Fig. 2). The LOD for this matrix was 0.93 g kg−1 . The assay exhibited good repeatability data for both intra and inter-assay variations (Table 1). The intra-assay coefficient of variation was 3.5, 6.1 and 6.7% for samples fortified with carazolol at levels of 2.5, 5.0, and 10 g kg−1 , respectively, while inter-assay variation at these fortification levels was 9.9, 6.2 and 4.0%, respectively. Calibration curves (n = 5) for the radioreceptor kidney assay are shown (Fig. 3). The LOD for this matrix was 1.47 g kg−1 . This is comparable to reported LODs for LC methods available for the determination of carazolol [8–10]. Intra-assay variation was determined as 7.8, 10.8 and 4.1% at concentrations of 5, 12.5 and 25 g kg−1 with inter assay variations
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Fig. 2. Calibration curve (n = 5) for the radioreceptor muscle assay. Error bars represent standard deviation at each calibration point.
determined as 5.2, 4.4 and 6.5%. Inter-assay variations were 5.2, 4.4 and 6.5% at these fortification concentrations (Table 2). 3.2. Results of incurred residue study The results from the incurred residue study are shown in Table 3. At each of the sampling points, there was considerable variation in the concentrations of carazolol observed in individual animals. However,
the highest concentrations of carazolol residues were consistently detected in kidney medulla and cortex at 1 h post-injection. The concentration of the compound had decreased in the medulla by 2 h post-injection with only a minor decrease observed in the kidney cortex. As carazolol is a lipophilic -blocker, this explains why residues persisted in the cortex. At the three remaining time points (8, 24 and 30 h) carazolol residues were not detected in the left kidney (medulla and cortex), or in the right kidney medulla. However,
Table 1 Assay validation data recorded for carazolol muscle No. of assays
Spiking concentration of carazolol (g kg−1 )
Repeatability Mean ± S.D. concentration of carazolol measured (g kg−1 )
Sr (%)
Intra-assay 5 5 5
2.5 5.0 10.0
2.61 ± 0.09 4.94 ± 0.30 9.24 ± 0.62
3.5 6.1 6.7
Inter-assay 5 5 5
2.5 5.0 10.0
2.43 ± 0.24 5.51 ± 0.34 9.56 ± 0.38
9.9 6.2 4.0
No. of blank (negative) tissue samples tested
Mean ± S.D. concentration of carazolol measured in tissue blanks (g kg−1 )
Limit of detection (mean ± 3S.D.)
20
0.69 ± 0.08
0.93
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Fig. 3. Calibration curve (n = 5) for the radioreceptor kidney assay. Error bars represent standard deviation at each calibration point.
concentrations in excess of the LOD were detected in the right kidney cortex. These findings illustrate that when scheduling kidney for carazolol residue analysis both kidneys should be sampled and cortex should preferentially be screened. Carazolol was only detectable in diaphragmatic muscle at 1 and 2 h post-injection. Corresponding gluteal muscle samples were negative. If the results of the present small scale study are true, i.e. that carazolol concentrations are higher in diaphragm than in other muscle groups, this has implications for the
choice of muscle tissue for routine monitoring. It is reassuring that in most monitoring programmes, diaphragmatic muscle is the muscle of choice for sampling since it is easy to remove and does not devalue the carcass. This phenomenon has also been reported for tilmicosin [17], where widely differing concentrations were observed in different muscle groups within individual animals. Overall, the results from the depletion study demonstrated that, when dosed at the recommended dose rate, kidneys and muscle from treated pigs did not contain
Table 2 Assay validation data recorded for carazolol kidney No. of assays
Spiking concentration of carazolol (g kg−1 )
Repeatability Mean ± S.D. concentration of carazolol measured (g kg−1 )
Sr (%)
Intra-assay 5 5 5
5.0 12.5 25.0
4.75 ± 0.37 12.94 ± 1.40 23.40 ± 0.96
7.8 10.8 4.1
Inter-assay 5 5 5
5.0 12.5 25.0
5.49 ± 0.29 13.04 ± 0.58 26.12 ± 1.71
5.2 4.4 6.5
No. of blank (negative) tissue samples tested
Mean ± S.D. concentration of carazolol measured in tissue blanks (g kg−1 )
Limit of detection (mean ± 3S.D.)
20
0.42 ± 0.35
1.47
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Table 3 Concentrations of carazolol (g kg−1 ) residues in pig muscle and kidney samples taken from pigs treated with carazolol at 10 g kg−1 body weight by single intramuscular injection. With the exception of the unmedicated control animal (n = 1), two pigs were slaughtered at each of the time points. Values shown are mean ± S.D. concentrations of three replicate determinations per tissue Time (h)
Kidney medulla
Kidney cortex
Muscle
Left
Right
Left
Right
Diaphragm
Gluteal
2
Pig 3 Pig 4
3.13 ± 1.93
4.02 ± 1.56
6.06 ± 1.50 5.32 ± 1.30
6.10 ± 1.88 7.72 ± 0.63
2.22 ± 0.08 2.45 ± 0.16
4
Pig 5 Pig 6
7.11 ± 2.6
3.69 ± 0.55 6.28 ± 1.02
8
Pig 7 Pig 8
2.59 ± 1.20 3.27 ± 0.30
24
Pig 9 Pig 10
2.00 ± 0.34 1.91 ± 1.04
30
Pig 11 Pig 12
2.37 ± 0.19 1.79 ± 0.83
Negative control 1
LOD, limit of detection; 1.47 and 0.93 g kg−1 for kidney and muscle, respectively.
carazolol residues in excess of the MRL. This finding supports the label claim of zero meat withholding for the drug. Our results are comparable to those reported from pharmacokinetic studies performed using radiolabelled carazolol (10 g kg−1 body weight) with analysis performed using HPLC [18]. Mean ± S.D. concentrations of carazolol detected in porcine muscle (n = 6) were 1.77 ± 0.78 and 2 ± 0.57 g kg−1 at 2 h post-injection. Concentrations measured in two pigs at 16 h post-injection had decreased to 0.14 and 0.02 g kg−1 , respectively. The concentrations in muscle detected at the 16 h time point were less than the LOD for our muscle radioreceptor assay. In this earlier study [18], concentrations of radiolabelled carazolol detected in kidney (n = 6) ranged from 20.9 ± 6.2 g kg−1 after 2 h, to 4.7 g kg−1 after 16 h. These concentrations were higher than those detected in the incurred residue study reported in this paper. As carazolol is a lipophilic -blocker which undergoes extensive metabolism [19], the higher kidney concentrations of radiolabelled analyte measured by LC may be attributed to the sum of the parent drug plus (radioactive) metabolites. Although there is little published data on carazolol metabolites in the pig, Rudolph and Steinhart [20] detected three conjugated carazolol metabolites in pig urine—monoglucuronide, lactate and acetate.
Such conjugations facilitate elimination from the body and common sense dictates that these metabolites are less likely to have biological activity (and affinity for) the 2-receptor. This assumption may explain the discrepancies observed between the two studies.
4. Conclusions The present study, has led to the development of a rapid, sensitive radioreceptor assay for the detection of carazolol residues in porcine muscle and kidney. Its major advantage over other published methods is the minimal sample clean up required before application to the receptor assay. The LOD is comparable to those reported for existing methods used for screening for carazolol residues. As this receptor assay may have the potential for being used as a multi-residue screening method for a range of agonistic [15] and antagonistic compounds acting via the 2-receptor, further investigations will be performed to assess the suitability of this receptor for other -blocking drugs. It is concluded that this assay is suitable for routine monitoring of residues of carazolol in pig meat and may have a future potential application for screening residues of other -blockers and -agonists.
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Acknowledgements The authors wish to thank Mildred Wylie and Deborah Snodden for their assistance with tissue cultures during this study, Ciaran Mulligan for the handling and post-mortem of animals and Peter Watson and Angeles Fornells from Bayer for supplying the carazolol. References [1] Council regulation (EEC) 2377/90 of 26 June 1990, Official J. Eur. Commun. No. L224 (1990) 1–6. [2] Commission regulation (EC) No. 1442/95 of 26 June 1995, Official J. Eur. Commun. L143 (1995) 26–30. [3] Council directive 96/23/EC of 29 April 1996, Official J. Eur. Commun. No. L125 (1996) 10–32. [4] Annual Report of the Institute of Veterinary Inspection (Belgium) for 1999 http://cemu10.fmv.ulg.ac.be/OSTC/ MonitoringIEV/99iev.htm. [5] Comission Européene, Direction Générale XXIV. XXIV/ 1516/98-MR-final (04/03/99 http://europa.eu.int/comm/food/ fs/inspections/vi/reports/belgium/vi rep belg 1516-1998 fr. pdf. [6] N. Haagsma, E.R. Bathelt, J.W. Engelsma, J. Chromatogr. 436 (1988) 73–76.
[7] J.W. Engelsma, J. Simons, Vet. Quart. 7 (1985) 73–76. [8] M. Rudolph, H. Steinhart, J. Chromatogr. 392 (1987) 371– 378. [9] M. Rose, G. Shearer, J. Chromatogr. 624 (1992) 471– 477. [10] H. Keukens, M.M.L. Aerts, J. Chromatogr. 464 (1989) 149–161. [11] M. Dubois, D. Fluchard, S. Kiebooms, Y. Colemonts, P. Delahaut, in: L.A. van Ginkel, A. Ruiter (Eds.), Proceedings of the Euroresidue IV Conference, Euroresidue Foundation, Utrecht, The Netherlands, 2000, pp. 398–405. [12] E. Rattenberger, Arch. Lebensmittel. Hygiene 40 (1989) 118. [13] E. Rattenberger, O Herr, Arch. Lebensmittel Hygiene 44 (1993) 135–137. [14] I. Mullaney, B.H. Shah, A. Wise, G. Milligan, J. Neurochem. 65 (1995) 545–553. [15] S. Meenagh, C. Elliott, R. Buick, Analyst 126 (2001) 491–494. [16] E. Kelly, M. Keen, P. Nobbs, J. MacDermot, Br. J. Pharmacol. 99 (1990) 309–316. [17] J.G. Beechinor, F.J. Bloomfield, Vet. Rec. 149 (2001) 182–183. [18] FAO Food and Nutrition Paper 41/4, 1991, pp. 23–37. [19] W. Riess, S. Brechbuhler, L. Brunner, P. Imhof, D.B. Jack, Therapiewoche 25 (1975) 4259. [20] M. Rudolph, H. Steinhart, J. Chromatogr. 392 (1987) 371–378.