Solid-phase extraction
In many racing chemistry laboratories throughout the world, SPE has replaced liquid–liquid extraction for the isolation of drugs from both urine and plasma. Based upon the studies of Shackleton and Whitney (1980), the early applications of SPE to racing chemistry were in the use of C18 bonded-phase cartridges to the isolation of anabolic steroids and their metabolites from equine urine (Dumasia et al. 1986). With the development of a range of more polar phases and, more importantly, ionic phases, the technique evolved to provide a simple, rapid, robust and efficient approach to the fractionation of drugs from biological fluids. Retention through a cation-exchange mechanism isolated the basic drugs, whereas an anion exchange mechanism isolated acidic drugs. Foster et al. (1990) have reported an automated SPE procedure for drugs in horse urine. Following initial extraction using Tox-Elut cartridges (cartridges that contain a specially modified form of diatomaceous earth), basic and acid–neutral drugs were separated using a cation exchange cartridge. Further purification of the acid–neutral fraction was achieved using silica SPE cartridges.
However, mixed-mode cartridges with reten tion mechanisms based upon both hydrophobic interactions and ion-exchange processes have found wide application in racing chemistry and many other disciplines associated with drug analyses. Dumasia and Houghton (1991) reported the application of the mixed mode cartridge for the extraction of beta-agonists, beta-antagonists and their metabolites in horse urine prior to their analysis by GC-MS. Westwood and Dumasia (1994) automated this approach for the general screening of basic drugs and their metabolites from horse urine. Wynne et al. (2001b) reported a multi-eluate approach to the SPE extraction of biological fluids to detect drug residues. The method employs the mixed-mode cartridge (C8–cation exchange) to separate the basic and acidic–neutral fractions. The acidic–neutral fraction was fractionated on a second mixed-mode cartridge (C18–anion exchange; Figure 1). Mixed-mode phases are also available in new poly(styrene–divinylbenzene) sorbents, produced by combining two classes of polymeric sorbents, copolymeric and functionalised polymers. The treatment of the functionalised polymer with sulfonic acid provides cation exchange characteristics. These cartridges have been shown to be effective in the extraction of a number of basic drugs from equine urine (Stanley et al. 2001). One of the major problems encountered in the SPE of horse urine, particularly if automated procedures are used, is cartridge blockage. This is primarily because of the viscous nature of horse urine and, to a lesser extent, suspended material. This problem has been addressed (Wynne et al. 2001a, 2001c) using non-conditioned SPE (NC SPE). The sorbents used in this technique are highly cross-linked with a unique combination of hydrophilic and lipophilic moieties and thus do not require conditioning. They have good flow characteristics and can be applied to the extraction of a wide range of drugs. Wynne et al. (2001c) suggested the application of NC-SPE for the pre-extraction of horse urine before application of the recovered extract to mixed-mode cartridges. The application of SPE to veterinary drug abuse has been reviewed by Wynne (2000).
Figure 1 Multi-eluate approach to SPE of biological fluids to detect drug residues.
