EQUINE VETERINARY JOURNAL Vol. 7. No. 3. July 1975
173
Recent Advances in the Field of Doping Detection MICHAEL S . MOSS Racecourse Security Services' Laboratories, Soharn House, Newrnarket, Suffolk
PROGRESS in chemical toxicology, of which dopetesting is a specialised field, has been primarily due to improvements in the chemist's ability to resolve complex mixtures into their component parts, and secondarily to his ability to identify the components unequivocally even when present in only small quantity. Priorities for development of dope detection methods are dictated by the supposed patterns of abuse. The earliest workers were concerned principally with the suppression of doping by plant alkaloids, mainly morphine and its derivatives, cocaine, strychnine and caffeine. With the successful development of detection techniques, these compounds have been much less abused, and latterly have almost ceased to be detected except in cases of accidental administration. Such administrations have been confined mainly to the methyl xanthines, often due to admixture of food supplements containing cocoa husk, or to the administration of a therapeutic substance at a time which did not allow a sufficient period for clearance of the drug from the horse before racing. Phenylbutazone and procaine have occurred frequently for this reason. Sound detection methods do, on the whole, appear to influence the patterns of doping. This being so, the nature of contemporary drug abuse must be to some extent spxulative but it would be facile to suppose that the disappearance from the scene of detectable drugs heralded a suppression of all forms of doping. Deliberate administration of drugs affecting the CNS and autonomic nervous system seems to have declined in Britain. There has been evidence of an increase in the use of anti-inflammatory drugs, internationally, notably phenylbutazone, but this too is on the decline in this country following its detection at low concentrations after quite long periods. The firm line taken by the Jockey Club condemning its use, and the recommendations of the Royal College of Veterinary Surgeons concerning the period that should be allowed prior to a race have probably been the principal causes of its virtual disappearance as a drug of abuse. Much speculation now centres on the abuse of steroids, and these fall into two main classes, the corticosteroids (or more correctly synthetic glucocorticoids, being Paper presented at The British Equine Veterinary Association Annual General Meeting, London, 1975.
variations upon the structure of natural glucocorticoids), and the anabolic steroids (being variations upon the structure of testosterone). The former are used principally in the treatment of inflammatory conditions, and thus occupy a position as doping agents with drugs such as phenylbutazone. The anabolic steroids are a relatively new class of drug in which the androgenic effects associated with testosterone have reputedly been suppressed by modification of the molecular structure. Androgenic side-effects appear, nevertheless, to be a feature of many such compounds. Because of the similarities in structure between the members of the glucocorticoids, and also between the members of the anabolic steroids, the possibility exists that radioimmunoassay might be applicable to group screening techniques. The requirement for such methods is to some extent mutually exclusive-namely that a suitable antibody will recognise a wide range of synthetic steroids but should not recognise endogenous compounds. Whether these two conflicting requirements can be effectively resolved in all cases must await the outcome of experiments, but early work at R.S.S. Laboratories with certain glucocorticoids is promising. A second approach to the problem of routine screening may lie in the use of gas-liquid chromatography making use of mass spectrometry with multiple peak monitoring. This system makes use of the mass spectrometer as a detector. Samples from the gas chromatograph are bled into a mass spectrometer, and the instrument pre-focussed to detect selected peaks of the mass spectrum of the compound to be detected. The use of capillary gas chromatography columns can be expected to increase the resolution of the system. There are, however, certain serious practical limitations in the use of such techniques routinely, and the more conventional forms of gas chromatography with mass spectrometry may prove to be more practicable. One other technique deserving special mention is that of high performance liquid chromatography. This technique (a development of conventional column chromatography systems) uses very fine column packings affording a much higher resolution of components. The very low flow rates that would thus be obtained under gravity are greatly increased by the use of high pressure pumps. This system of chromatography has many
EQUl N E VETERl N A R Y J O U R N A L
174
advantages over gas systems, especially for large molecules such as steroids, and may be expected to supplement or to supplant such systems in many future applications. SUMMARY Patterns of doping in Britain have on the whole changed from the use of CNS stimulants and depressants to therapeutic substances, or to accidental doping by feeding contaminated food or food supplements. Progress in detection and identification is largely a result of improvements in separation and isolation, especially by gas chromatography, together with the introduction of mass spectrometry. Radioimmunoassay and high performance liquid chromatography promise to be important techniques in the future, especially in detection of anabolic steroids and corticosteroids.
Les progrits obtenus dam la detection et dans I’identification sont diis largement a I’amelioration des techniques de separation et d’isolement, notamment A la chromatographie gazeuse et ii la spectromttrie de masse. Ulttrieurement les techniques employant les radio elements et la chromatographie liquide devraient se revtler d’importance notamment pour la detection des corticosteroids et des anabolisants steroidiens.
ZUSAMMENFASSUNG Die Dopingmethoden haben in England im allgemeinen vom Gebrauch ZNS-stimulierender oder-deprimierender Mittel gewechselt zu therapeutischen Substanzen oder zu zufalligem Doping durch die Verfutterung kontaminierten Futters oder von Supplementen. Fortschritte in Nachweis und ldentifizierung sind vor allem das Resultat verbesserter analytischer Methoden fur die Trennung und lsolierung der Substanzen besonders durch GasRESUME Les modalites du doping en Grande Bretagne ont dans chromatographie zusammen mit der Einfuhrung der I’ensemble change, delaissant les stimulants et les Massenspektrometrie. Radioimmunmethoden und hochdkpresseurs du systitme nerveux au profit des substances leistungsfahige flussige Chromatographie versprechen in therapeutiques, une autre forme pouvant Ctre I’emploi Zukunft Bedeutung zu erlangen besonders fur den d’aliments ou de supplements alimentaires “contaminis”. Nachweis anaboler Steroide und von Corticosteroiden.
Hardback Binders for Equine Veterinary Journal Hardback binders for Equine Veterinary Journal are obtainable from the Publishers. Copies of the Journal are inserted into detachable vertical wires. The title of the Journal and the Association’s emblem are in gold block lettering on the spine. Each binder holds twelve copies. Price is f2.30 each including postage and packing (US $7.OO-postage
by
sea mail) supplied in cardboard carton. Orders with remittance should be sent to :
BRITISH EQUINE VETERINARY ASSOCIATION 93 Mill Lane, Fordham, Ely Cambridgeshire CB7 5NH
173
Recent Advances in the Field of Doping Detection MICHAEL S . MOSS Racecourse Security Services' Laboratories, Soharn House, Newrnarket, Suffolk
PROGRESS in chemical toxicology, of which dopetesting is a specialised field, has been primarily due to improvements in the chemist's ability to resolve complex mixtures into their component parts, and secondarily to his ability to identify the components unequivocally even when present in only small quantity. Priorities for development of dope detection methods are dictated by the supposed patterns of abuse. The earliest workers were concerned principally with the suppression of doping by plant alkaloids, mainly morphine and its derivatives, cocaine, strychnine and caffeine. With the successful development of detection techniques, these compounds have been much less abused, and latterly have almost ceased to be detected except in cases of accidental administration. Such administrations have been confined mainly to the methyl xanthines, often due to admixture of food supplements containing cocoa husk, or to the administration of a therapeutic substance at a time which did not allow a sufficient period for clearance of the drug from the horse before racing. Phenylbutazone and procaine have occurred frequently for this reason. Sound detection methods do, on the whole, appear to influence the patterns of doping. This being so, the nature of contemporary drug abuse must be to some extent spxulative but it would be facile to suppose that the disappearance from the scene of detectable drugs heralded a suppression of all forms of doping. Deliberate administration of drugs affecting the CNS and autonomic nervous system seems to have declined in Britain. There has been evidence of an increase in the use of anti-inflammatory drugs, internationally, notably phenylbutazone, but this too is on the decline in this country following its detection at low concentrations after quite long periods. The firm line taken by the Jockey Club condemning its use, and the recommendations of the Royal College of Veterinary Surgeons concerning the period that should be allowed prior to a race have probably been the principal causes of its virtual disappearance as a drug of abuse. Much speculation now centres on the abuse of steroids, and these fall into two main classes, the corticosteroids (or more correctly synthetic glucocorticoids, being Paper presented at The British Equine Veterinary Association Annual General Meeting, London, 1975.
variations upon the structure of natural glucocorticoids), and the anabolic steroids (being variations upon the structure of testosterone). The former are used principally in the treatment of inflammatory conditions, and thus occupy a position as doping agents with drugs such as phenylbutazone. The anabolic steroids are a relatively new class of drug in which the androgenic effects associated with testosterone have reputedly been suppressed by modification of the molecular structure. Androgenic side-effects appear, nevertheless, to be a feature of many such compounds. Because of the similarities in structure between the members of the glucocorticoids, and also between the members of the anabolic steroids, the possibility exists that radioimmunoassay might be applicable to group screening techniques. The requirement for such methods is to some extent mutually exclusive-namely that a suitable antibody will recognise a wide range of synthetic steroids but should not recognise endogenous compounds. Whether these two conflicting requirements can be effectively resolved in all cases must await the outcome of experiments, but early work at R.S.S. Laboratories with certain glucocorticoids is promising. A second approach to the problem of routine screening may lie in the use of gas-liquid chromatography making use of mass spectrometry with multiple peak monitoring. This system makes use of the mass spectrometer as a detector. Samples from the gas chromatograph are bled into a mass spectrometer, and the instrument pre-focussed to detect selected peaks of the mass spectrum of the compound to be detected. The use of capillary gas chromatography columns can be expected to increase the resolution of the system. There are, however, certain serious practical limitations in the use of such techniques routinely, and the more conventional forms of gas chromatography with mass spectrometry may prove to be more practicable. One other technique deserving special mention is that of high performance liquid chromatography. This technique (a development of conventional column chromatography systems) uses very fine column packings affording a much higher resolution of components. The very low flow rates that would thus be obtained under gravity are greatly increased by the use of high pressure pumps. This system of chromatography has many
EQUl N E VETERl N A R Y J O U R N A L
174
advantages over gas systems, especially for large molecules such as steroids, and may be expected to supplement or to supplant such systems in many future applications. SUMMARY Patterns of doping in Britain have on the whole changed from the use of CNS stimulants and depressants to therapeutic substances, or to accidental doping by feeding contaminated food or food supplements. Progress in detection and identification is largely a result of improvements in separation and isolation, especially by gas chromatography, together with the introduction of mass spectrometry. Radioimmunoassay and high performance liquid chromatography promise to be important techniques in the future, especially in detection of anabolic steroids and corticosteroids.
Les progrits obtenus dam la detection et dans I’identification sont diis largement a I’amelioration des techniques de separation et d’isolement, notamment A la chromatographie gazeuse et ii la spectromttrie de masse. Ulttrieurement les techniques employant les radio elements et la chromatographie liquide devraient se revtler d’importance notamment pour la detection des corticosteroids et des anabolisants steroidiens.
ZUSAMMENFASSUNG Die Dopingmethoden haben in England im allgemeinen vom Gebrauch ZNS-stimulierender oder-deprimierender Mittel gewechselt zu therapeutischen Substanzen oder zu zufalligem Doping durch die Verfutterung kontaminierten Futters oder von Supplementen. Fortschritte in Nachweis und ldentifizierung sind vor allem das Resultat verbesserter analytischer Methoden fur die Trennung und lsolierung der Substanzen besonders durch GasRESUME Les modalites du doping en Grande Bretagne ont dans chromatographie zusammen mit der Einfuhrung der I’ensemble change, delaissant les stimulants et les Massenspektrometrie. Radioimmunmethoden und hochdkpresseurs du systitme nerveux au profit des substances leistungsfahige flussige Chromatographie versprechen in therapeutiques, une autre forme pouvant Ctre I’emploi Zukunft Bedeutung zu erlangen besonders fur den d’aliments ou de supplements alimentaires “contaminis”. Nachweis anaboler Steroide und von Corticosteroiden.
Hardback Binders for Equine Veterinary Journal Hardback binders for Equine Veterinary Journal are obtainable from the Publishers. Copies of the Journal are inserted into detachable vertical wires. The title of the Journal and the Association’s emblem are in gold block lettering on the spine. Each binder holds twelve copies. Price is f2.30 each including postage and packing (US $7.OO-postage
by
sea mail) supplied in cardboard carton. Orders with remittance should be sent to :
BRITISH EQUINE VETERINARY ASSOCIATION 93 Mill Lane, Fordham, Ely Cambridgeshire CB7 5NH
