Doran et al.: Journal of aoaC InternatIonal Vol. 96, no. 6, 2013 1487 The Analysis of Pergolide Residues in Horse Plasma by LC with Fluorescence Detection GreGory Doran Charles Sturt University, School of Agricultural and Wine Sciences, EH Graham Centre for Agricultural Innovation, Wagga Wagga 2678, NSW, Australia Kristopher huGhes Charles Sturt University, School of Animal and Veterinary Sciences, Wagga Wagga 2678, NSW, Australia DaviD renDle Charles Sturt University, School of Animal and Veterinary Sciences, Wagga Wagga 2678, NSW, Australia; The Liphook Equine Hospital, Forest Mere, Liphook, Hampshire, GU30 7JG, United Kingdom scott eDwarDs Charles Sturt University, School of Animal and Veterinary Sciences, Wagga Wagga 2678, NSW, Australia Received July 9, 2012. Accepted by JB December 30, 2012. Corresponding author’s e-mail: gdoran@csu.edu.au DOI: 10.5740/jaoacint.12-282 VETERINARY DRUG RESIDUES Pergolide is used to treat pituitary pars intermedia dysfunction (equine Cushing’s Disease), a neurodegenerative condition associated with loss of dopaminergic inhibition of the pituitary in horses. After oral administration, only low concentrations of the drug are achieved in plasma, making drug detection and quantifcation diffcult. While direct analysis of plasma using sensitive MS/MS techniques is possible, dirty plasma samples and mobile phase buffers can cause instrumentation to become rapidly incapacitated. A method using LC with fuorescence detection was developed for pergolide analysis. LOQ for the instrumentation was 2 ng/mL when using direct injection of horse plasma samples, while interferences from the matrix were nominal. The use of SPE provided cleaner extracts and increased the LOQ in plasma samples to 0.15 ng/mL. The LC method developed allowed high sample throughput before pre-columns required replacement, which was extended when SPE cleanup was used. The effectiveness of SPE for the cleanup and preconcentration of plasma samples containing pergolide was demonstrated with spiked and naturally incurred samples; LC-MS/MS was used to validate the SPE method against direct injection samples. P ergolide [8-beta-((methylthio)methyl)-6-propylergoline- methanesulfonate] is a dopamine receptor agonist originally released by Eli Lily and Co. in 1988 for use in humans suffering from Parkinson’s disease. The drug was subsequently removed from sale by the U.S. Food and Drug Administration in 2007 due to its association with the hardening of heart valves (cardiac fbrosis; 1). However, in recent years, it has been reborn as an equine pharmaceutical used to treat pituitary pars intermedia dysfunction, a condition that may result in a range of clinical signs, most notably laminitis, as a result of increased production of pituitary-derived peptides. Pergolide is an ergoline derivative that has low solubility in most solvents, resulting in it being supplied as pergolide mesylate salt. Its low solubility coupled with its low volatility can make its analysis by liquid and gas chromatographic methods more challenging than many other organic chemicals. Consequently, only a handful of publications report its analysis. With a boiling point of approximately 490°C, direct analysis by GC is not possible, but derivatization may permit GC analysis by improving its volatility. The remaining literature sources only report methods focused on LC. While normal phase LC with acetonitrile mobile phase has been reported (2), most work tends to rely on the preferable RP-LC (3–7) methodology using methanol or acetonitrile mobile phases that have been acidifed with formic acid (4), ammonium carbonate (3, 5) or ammonium acetate (6, 7). Traditionally, detection relied upon nondestructive fow-through cells in UV-Vis spectrophotometers, which resulted in a LOQ of approximately 10 µg/mL (3, 5, 6). This LOQ was inadequate for studying therapeutic levels of pergolide in blood and would require extensive preconcentration with liquid–liquid extraction or SPE to increase pergolide concentrations prior to LC analysis. More recently, LC-MS (7) and LC-MS/MS (2, 4) have been applied to pergolide analysis, particularly in serum, resulting in LOQs of approximately 50 ng/mL and 10 pg/mL, respectively. While these destructive detection methods provide an enhanced LOQ over UV-Vis techniques, they suffer from several problems including a need for a high proportion of organic solvent in the mobile phase to improve desolvation for MS detection, and a tendency for instrumentation to become blocked by buffer salt and dirty samples. Pergolide’s interconnected aromatic and aliphatic ring structure lends itself to detection by fuorescence spectrophotometry (Figure 1). Additionally, the fow-through cell design of an LC fuorescence detector eliminates the concerns arising from the analysis of dirty samples and the use of mobile phase buffers that occur in MS instrumentation, while providing enhanced sensitivity over UV-Vis detectors. While fuorescence provides similar sensitivity to LC-MS (7), it is still several orders of magnitudes less sensitive than LC-MS/MS (2, 4). However, this problem may be addressed by the inclusion of preconcentration of plasma samples using SPE, with the added benefts of a cleaner sample being applied to the chromatography column and fewer interferences passing Downloaded from https://academic.oup.com/jaoac/article/96/6/1487/5655093 by guest on 23 September 2022