Analyst, January 1996, Vol. 121 (67-69) 67 Establishing the Cut-off Concentration for the Detection of Etorphine in Horse Urine Robert F. Smitha, Laurence S. Jackson4 and Andrew Moorec a Division of Biomedical Science and Health Research Institute, ShefSield Hallam University, Shefield UK S I 1 WB. E-mail: R.F.Smith@shu.ac.uk c Pain Relief Unit, The Churchill, Oxford UK OX3 7L.J Horse Racing Forensic Laboratory, Newmarket UK CB8 7DT An 1251 radioimmunoassay to determine the pattern of urinary excretion of etorphine (a semisynthetic opiate agonist) after its administration to horses is described. Three thoroughbred horses were each given 5,15,30 and 100 pg of etorphine intramuscularly. Urine was collected for up to 72 after administration. The maximum etorphine concentration after administration of a dose of 5 pg was 711 pg ml-1 (concentrations were greater than 100 pg ml-1 after 23 h in all three horses); a 15 yg gave 2661 pg ml-1 (levels remained above 100 pg ml-l for more than 44 h in each horse); a 30 yg dose gave a maximum of 3344 pg ml-l (levels were above 100 pg ml-l for 24,72 and 72 h); and 100 pg gave in excess of 10 000 pg ml-1 (levels were greater than 300 pg ml-1 for up to 70 h).Forty-eight urine samples from horses not given etorphine all had levels of etorphine less than 100 pg ml-1. There was no increase in apparent etorphine concentrations after hydrolysis of samples with p-glucuronidase and aryl sulfatase. The half-lives of etorphine equivalents (calculated with a mono-exponential equation after the 100 pg dose) in the urine of the three horses were 569,803 and 821 min, respectively.We conclude that radioimmunoassay can provide a useful first line screening procedure for the assessment of etorphine use in racing horses. Keywords: Etorphine; kinetics; equine; radioimmunoassay Introduction Etorphine (4,5-epoxy-3-hydroxy-6-methoxy-a,l7-dimethyl-a- propyl-6,14-ethenomorphinan-7-methanol), is a potent semi- synthetic opiate agonist, derived from thebaine, a phar- macologically inactive opium alkaloid. A characteristic of etorphine is its high potency, its rapid onset and short duration of action. In human beings the effects of etorphine are similar to those of morphine.' Etorphine is used in veterinary practice combined with a phenothiazine (Large Animal Immobilin) for use in restraining large animals, or with methotrimeprazine (Small Animal Immobilin) where it is used for sedation and analgesia in small-animal surgery.In horses, low doses of etorphine induce a locomotor stimulant response similar to that noted for other opiates such as morphine and fentanyl.2 This combination of locomotor effect and low dose has made etorphine eminently suitable for illegal and inappropriate use to influence the outcome of horse races. Etorphine has been successfully measured using a variety of techniques: GC-MS;3 HPLC;4 ELISA;5 and radioimmunoassay (RIA).6 The high sensitivity and ability to analyse large numbers of samples in a relatively short time has made immunoassay a useful tool for the screening of large numbers of samples for drugs before confirmation by other techniques.This paper describes the use of commercially available reagents to screen horse urine for the presence of etorphine and considers the setting of an appropriate cut-off point to determine positives from negatives. Experimental Etorphine Administration and Sample Collection Etorphine were administered to each of three thoroughbred horses on four separate occasions. Doses of 5,15,30 and 100 pg of etorphine were administered intramuscularly (IM) to each horse and urine was collected, whenever voided, for up to 72 h after the dose. In addition, 48 urine samples were collected, without preservative, from horses not given etorphine and stored frozen at -20 "C until analysis. Radioimmunoassay The RIA reagents were used as supplied by the manufacturer (EuroDPC, Glyn Rhonwy, Llanberis, UK).A volume of 25 yl of a standard solution of etorphine in urine (0, 100, 500, 1000, 2500,5000 pg ml-I), control urine (300 and 3000 pg ml-l) or sample urine were incubated, in duplicate, for 1 h at room temperature with 100 p1 of 1251 of etorphine solution and 100 yl of rabbit anti-etorphine solution. Following this primary incubation, 1 ml of cold precipitating solution (donkey anti- rabbit antiserum in 8% m/v polyethylene glycol-saline) was added, and the tubes incubated for a further 10 min before centrifugation at 3000 rpm for 20 min at 4 "C. After centrifuga- tion the supernatant fluid was decanted and the bound radioactivity in each tube was counted for 60 s in a gamma counter. The data were plotted using a 4-parameter logistic- curve fit of the percentage : bound by bound zero ratio of the response variable against the logarithm of the etorphine concentration.The etorphine concentration of the unknown samples was determined as etorphine equivalents by inter- polation from the standard curve. Urine Hydrolysis In order to assess whether the RIA cross-reacted with conjugated metabolites of etorphine, 45 samples found positive for etorphine were analysed before and after incubation with 8- glucuronidase and aryl sulfatase. Urine samples (100 pl) were diluted (1 + 1) with citrate-phosphate buffer (pH 5) containing 200 U ml-1 (1 U = 16.67 nkat) of P-glucuronidase (EC 3.2.1.3 1, Type HI, from Helix pomatia; Sigma, Poole, Dorset, UK) and 80 U ml-1 of aryl sulfatase (EC 3.1.6.1, type V, from Patella vulgata; Sigma).Samples were incubated for 1 h at 37°C in a water-bath before analysis for etorphine by using RIA. The results were corrected for dilution.68 Analyst, January 1996, Vol. 121 Results The etorphine RIA gave a standard curve between 100 and 5000 pg ml-*. Assessment of the assay precision was made by selecting three samples to give a range of values throughout the standard curve (220, 1008, and 2006 pg ml-1) and measuring the mean, s and relative standard deviation for 20 pairs of tubes in a single assay (intra-assay) and as a duplicate pair in 20 separate assays (inter-assay). The precision was found to be < 10% for both intra- and inter-assay. Sensitivity was assessed as the apparent etorphine concentration equivalent to the mean minus 2 s of the response (given by measurement of the zero calibrator 40 times) and was determined as 15.3 pg ml-l.The assay was shown to cross-react with N-dealkyl etorphine (77%) and diprenorphine (7 1 %). There was no significant increase in the apparent etorphine concentration after hydrolysis of samples with P-glucuronidase and aryl sulfatase. Assessment of Optimum Cut-off The forty-eight samples from horses not given etorphine all had levels c 100 pg ml-1. Etorphine was detectable in the urine of each animal given etorphine after each dose of drug (Figs. 1-4), although the duration of all positive tests varied with dose and with the choice of cut-off point. These findings are summarized in Table 1. 1000 Etorphine dose 5 pg 0 0 on v 100 Tirne/h Urinary etorphine concentrations against time for each of the three Fig.1 horses for a 5 yg dose. Etorphine dose 15 pg k‘ 0 0 0 A” 0 a 100 I I I I I I I 0 10 20 30 40 50 60 Time/h Urinary etorphine concentrations against time for each of the three Fig. 2 horses for a 15 yg dose. After administration of 5 pg of etorphine the maximum urinary etorphine concentration was 7 1 1 pg ml- and remained above 100 pg ml-l for more than 23 h in all three horses; after 15 pg of etorphine the maximum level was 2661 pg ml-1 and the etorphine remained above 100 pg ml-1 for more than 44 h in each horse; and after 30 pg of etorphine the maximum level was of 3344 pg ml-l and levels were above 100 pg ml-1 for 24, 72 and 72 h for each of the three horses, respectively. Early samples collected after the 100 pg dose contained etorphine concentrations greater than 5000 pg ml-1 and were still in excess of 300 pg ml-1 for up to 70 h.The half-lives of etorphine equivalents (calculated with a mono-exponential equation from the 100 pg dose) for the three horses were 569, 803 and 821 min. 10000 I - E a g 1000 e 5 cr, 1 .- c 1 I ) , I 100 I 0 10 20 30 40 50 60 70 80 Tirne/h Urinary etorphine concentrations against time for each of the three Fig. 3 horses for a 30 yg dose. 10000 - I E 0, Q -. .- r“ 1000 c P 5 100 Etorphine dose 100 I I I I I I I 0 10 20 30 40 50 60 70 80 Time/h Urinary etorphine concentrations against time for each of the three Fig. 4 horses for a 100 yg dose. Table 1 Range of times in the three horses given etorphine at each dose where urinary etorphine concentrations remained above the putative cut-off value Duration of positive test/h Dose/pg 500/pg ml-’ 250/pg ml-l lOO/pg ml-l 5 3 4 18-46+ 2446+ 15 10-18 2 w + 44-46+ 30 18-25 22-26 24-72+ 100 52-65 68+ 68+Analyst, January 1996, Vol.121 69 Discussion Etorphine has three important effects in the horse. It is a locomotor stimulant, an analgesic, and may extend the time to exha~stion,~ making it suitable for abuse in horse-racing. The low levels of drug found in the urine of horses administered etorphine and the large number of samples to be tested presents a considerable analytical challenge. Immunoassay combines the advantages of excellent sensitivity and high sample throughput; features which make the technique ideal for screening applica- tions before confirmation with other analytical techniques. The problem for any screening procedure lies in the establishment of an appropriate cut-off point which can accurately determine true positives from false positives.In the case of immunoassay this is not just a question of sensitivity but of the background noise or ‘matrix effect’ caused by use of undiluted, unextracted biological samples; a problem which is well known as a cause of false positives particularly affecting equine samples. The choice of cut-off point will always be a trade off between maximizing true positives and minimizing false positives, although in the case of drug abuse some false negatives may be tolerated in order to avoid the consequences which can ensue from false positives, even given that all positive samples will be further investigated. For etorphine to be effective a horse would need to receive a dose of drug very shortly before a race and sampling would be expected to take place as soon as possible after the race and in any case within 24 h.For these reasons a cut-off point of 100 pg ml-1 appears to be a level suitable for the screening of samples for the illegal use of etorphine. All 48 samples from horses not given etorphine had apparent etorphine concentrations below this level and, even at the lowest dose of etorphine, levels remained above 100 pg ml-1 for between 24 and 46 h. The incubation of etorphine positive samples with hydrolysing enzymes did not significantly increase the concen- tration of etorphine equivalents found in the urine.This is likely to be because the antiserum was equally cross-reactive to both conjugated and free etorphine. The long (> 10 h) half-life of etorphine and etorphine glucuronides in equine urine found in this study accords well with the long half-life of etorphine reported in human plasma,8 and with other potent opiates (such as buprenorphine) which are also known to be excreted into urine over many hours and days post-administration. We conclude that a simple radioimmunoassay using com- mercially available reagents is of value in the screening of urine samples for etorphine abuse in racing horses before confirma- tion with reference analytical techniques. References Jasinski, D., Griffith, D. R., and Cam, D. B., Clin. Pharmacol. Exp. Ther., 1979, 17, 267. Tobin, T., in Drugs and the Performance Horse, Charles C . Thomas, Springfield, IL, USA, 198 1. Bonnaire, Y., Plou, P., Pages, N., Boudene, C., and Jouany, J. M., J . Anal. Toxicol., 1989, 13(4), 193. Glasel, J. A., and Venn, R. F., J . Chromatogr., 1981, 213(2), 337. Stanley, S., Jeganathan, A., Wood, T., Henry, P., Turner, S., Woods, W. E., Green, M., Tai, H-H., Watt, D., Blake, J., and Tobin, T., J . Anal. Toxicol., 1991, 15, 305. Tai, C. L., Wang, C. J., Weckman, T. J., Popot, M. A., Woods, W. E., Yang, J. M., Blake, J., Tai, H. H., and Tobin, T., Am. J. Vet. Res., 1988,49(5), 622. Suann, C., Rose, R., Plummer, C., Knight, P., Proc. 8th Zntl. Con$ Racing Analyst Vet 1990. Friedrich, G., Braunstein, P., Friedrich, M., Vach, W., Beitr. Gerichtl. Med., 1991, 49, 111. Paper 5105353F Received August 10,1995 Accepted September 20, I995