Investigations Into the Analysis of Intact Drug Conjugates In
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Ghent University Academic Bibliography Received: 30 October 2019 Revised: 7 February 2020 Accepted: 8 February 2020 DOI: 10.1002/dta.2779 RESEARCH ARTICLE Investigations into the analysis of intact drug conjugates in animal sport doping control – Development and assessment of a rapid and economical approach for screening greyhound urine Bob Gray1,2 | Lisa Tuckley1 | Charlotte Cutler1 | Simon Biddle1 | Simon Hudson1 | Simon Gower3 | Lynn Vanhaecke2,4 1Bob Gray, LGC Ltd, Newmarket Road, Fordham, Cambridgeshire, UK Abstract 2Laboratory of Chemical Analysis, Department Animal sport doping control laboratories are constantly reviewing ways in which they of Veterinary Public Health and Food Safety, can improve their service offering whilst ensuring that they remain economically via- Ghent University, Merelbeke, Belgium 3Greyhound Board of Great Britain, 6 New ble. This paper describes the development and assessment of a rapid and economical Bridge Street, London, UK method for the detection of intact glucuronide conjugates of three anabolic steroids 4 School of Biological Sciences, Institute for and their metabolites along with three corticosteroids in canine urine. The analysis of Global Food Security, Queen's University, Belfast, UK intact drug conjugates for animal sport doping control is generally not performed rou- tinely as it presents a number of analytical challenges, not least of which is the lack of Correspondence Bob Gray, LGC Ltd, Newmarket Road, availability of appropriate reference standards. Here, we report the development of a Fordham, Cambridgeshire, CB7 5WW, UK. UHPLC–MS/MS method using APCI in the negative ion mode for the detection of Email: [email protected] intact phase II conjugates, including the importance of in vitro incubations in order to Funding information provide appropriate reference materials. Cross-validation of the developed method Greyhound Board of Great Britain (GBGB) demonstrated that the detection capability of the intact phase II conjugates of stanozolol, boldenone, nandrolone, and their metabolites along with the corticoste- roids dexamethasone and methylprednisolone was equivalent to that achieved in routine race-day screens. The new process has been in operation for approximately 2 years and has been used to analyze in excess of 13500 canine urine samples, resulting in a number of positive screening findings. To the best of our knowledge, this is the first reported use of a routine screen for intact drug conjugates within ani- mal sport doping control. KEYWORDS anabolic steroids, doping control, glucuronide conjugates, greyhound urine, UPLC-MS/MS 1 | INTRODUCTION these animals would then be sold on to new owners prior to com- mencing a racing career. With the sale price being dependent upon In 2011, the Greyhound Board of Great Britain (GBGB) instigated the their performance whilst in training, it is clear to see how improved Point of Registration (POR) testing program.1 At that time there was a performance could result in an increased sale price. perception within the sport that some animals were being adminis- The POR test was introduced in order to provide a layer of pro- tered long acting prohibited substances, such as anabolic steroids, in tection against this practice, to increase buyer confidence when pur- order to gain a performance improvement whilst in training. Many of chasing racing greyhounds, and to demonstrate an on-going Drug Test Anal. 2020;1–12. wileyonlinelibrary.com/journal/dta © 2020 John Wiley & Sons, Ltd. 1 2 GRAY ET AL. commitment to the integrity of the sport. From May 2011 onwards, derivatization and subsequent analysis by gas chromatography mass all animals that were taken to a racecourse for microchipping and reg- spectrometry (GC–MS), which has traditionally been the analytical istration to race were required to have a urine sample collected at the technique of choice for the detection of anabolic steroids and their same time.1 This urine sample was collected in a tamper evident sam- metabolites in animal sport doping control.2-5 In addition, phase I ple kit, given a unique and anonymized sample identifier and subse- metabolites are usually more readily available as reference standards, quently transported to the laboratory at LGC for long-term frozen which greatly assists with method development. storage. Whilst relatively sparse, there are previous reports on the detec- If a racing greyhound was subsequently tested positive for a tion of intact phase II conjugates of anabolic steroids from animal prohibited substance in the first 6 months from the time of its regis- sport doping control laboratories. As early as 1983, Dumasia et al. tration then the POR sample would be removed from long-term stor- reported on the successful detection of intact sulfate conjugated age and analyzed in order to determine if the substance was present boldenone in equine urine6 using fast-atom bombardment MS, but at the time of registration. The results from both tests would then be the method was hampered by high levels of matrix interference and available to the GBGB for consideration at any subsequent enquiry. was not suitable for routine testing. In 2002, the detection of endoge- This approach allowed the GBGB to collect samples from all regis- nous intact boldenone sulfate in the urine of colts was reported by tered animals but was also relatively low cost with just a small storage the group of Ho et al.7 This work subsequently led to the establish- fee being applicable to all samples and an analysis fee only for those ment of an internationally agreed threshold for the detection of samples that required testing. boldenone in the urine of intact male horses. Grace et al.8 reported a In 2016, the GBGB proposed a change in the way in which the method for the detection of intact sulfate conjugates of both samples collected as part of the POR program were processed. The boldenone and nandrolone in the urine of male horses. The authors new approach would see all samples analyzed within 10 working days overcame an issue of interference from high concentrations of sulfate of their receipt in the laboratory for a selection of prohibited sub- conjugates of the female sex hormones estrone and estradiol by selec- stances known to be of long duration. As this test was to be applied tive hydrolysis of aryl sulfates using glucuronidase from Helix pomatia. to all animals newly registered with the GBGB, there was a require- This approach allowed for the targeted hydrolysis of the aryl sulfates ment to make the analytical procedure as cost effective as possible of estrone and estradiol without any significant hydrolysis of the alkyl and therefore the number of analytes covered was restricted to a sulfates of nandrolone and boldenone. By following up this highly number of key compounds of interest. The analytes selected for inclu- targeted hydrolysis approach with a targeted solid phase extraction, sion in the first iteration of the method were the anabolic steroids the newly liberated “free” phenolic steroids were removed to waste boldenone, nandrolone, and stanozolol along with the corticosteroids and the retained nandrolone and boldenone sulfates subsequently dexamethasone, betamethasone, and methylprednisolone. This group eluted. of analytes was based upon the results obtained from the routine The analysis of intact conjugated anabolic steroids has a number screening of race-day urine samples performed at LGC and following of potential advantages, not least of which is the elimination of the consultation with the GBGB. enzyme hydrolysis step, which is both timely and costly due to the The aim of the new method was to provide equivalent coverage, requirement for high quality purified enzyme solution. There are also in terms of detection capability, to that achieved in the routine concerns that variation in the hydrolysis step from one batch to screens in place at LGC for anabolic steroids and corticosteroids in another may lead to unreliable and unreproducible results.9,10 In addi- race-day greyhound urine. As such there was no requirement to quan- tion, when employing liquid chromatography tandem mass spectrome- titatively validate the method but rather to assess its performance try, the intact conjugates ionize readily in both the positive and against the current screening protocols, which we termed cross- negative mode without the need for a time consuming derivatization validation throughout the work. An adverse screening finding from step. As such, the analysis of intact phase II conjugates has been the POR test results in the dog being prevented from racing until such investigated in human urine samples, primarily for doping control pur- time as a negative result is obtained. poses.11-17 This approach has gained acceptance and is used to com- Urine has always been the matrix of choice for greyhound doping plement the more traditional approach to steroid analysis in urine control as it is easy to collect and generally contains higher analyte samples. Whilst the detection of intact drug conjugates, particularly of concentrations than blood. As such, the methods employed by animal anabolic steroids, is reported for human sport doping control, there is sport doping control laboratories are based upon the detection of very little information available on its routine use in animal sport dop- drugs in urine and in many cases this requires the extraction and anal- ing control. ysis of phase I and phase II metabolites. In the dog, conjugation with Whilst offering undoubted benefits, the successful development glucuronic acid is the most important phase II metabolic reaction of of methods for the detection of intact phase II conjugates is compli- anabolic steroids.2-4 Therefore, the analysis of anabolic steroids in cated by the lack of availability of appropriate reference standards. greyhound urine is usually undertaken following enzymatic hydrolysis For those compounds for which reference materials are not commer- using β-glucuronidase enzymes to cleave the glucuronic acid and to cially available the synthesis of phase II steroid conjugates by in vitro release the phase I metabolites.