Novel dummy molecularly imprinted polymer for simultaneous solid-phase extraction of stanozolol metabolites from urine.

Stanozolol, a synthetic derivative of testosterone, is one of the common doping drugs among athletes and bodybuilders. It is metabolized to a large extent and metabolites are detected in urine for a longer duration than the parent compound. In this study, a novel dummy molecularly imprinted polymer (DMIP) is developed as a sorbent for solid-phase extraction of stanozolol metabolites from spiked human urine samples. The optimized DMIP is composed of stanozolol as the dummy template, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker in a ratio of 1:10:80. The extracted analytes were quantitively determined using a newly developed and validated ultrahigh-performance liquid chromatography tandem mass spectrometry method, where the limits of detection and quantitation were 0.91 and 1.81 ng mL-1, respectively, fulfilling the minimum required performance limit decided on by the World Anti-Doping Agency. The mean percentage extraction recoveries for 3'-hydroxystanozolol, 4ß-hydroxystanozolol, and 16ß-hydroxystanozolol are 97.80% ± 13.80, 83.16% ± 7.50, and 69.98% ± 2.02, respectively. As such, the developed DMISPE can serve as an efficient cost-effective tool for doping and regulatory agencies for simultaneous clean-up of the stanozolol metabolites prior to their quantification.

Inhibitory Effects of Diclofenac on Steroid Glucuronidation In Vivo Do Not Affect Hair-Based Doping Tests for Stanozolol.

In vitro studies show that diclofenac inhibits enzymatic steroid glucuronidation. This study was designed to investigate the influence of diclofenac on the excretion of stanozolol and 3'-hydroxystanozolol via analyses in hair, blood and urine in vivo in a rat study. Brown Norway rats were administered with stanozolol (weeks 1-3) and diclofenac (weeks 1-6). Weekly assessment of steroid levels in hair was complemented with spot urine and serum tests. Levels of both stanozolol and 3'-hydroxystanozolol steadily increased in hair during stanozolol treatment and decreased post-treatment, but remained readily detectable for 6 weeks. In contrast, compared to control rats, diclofenac significantly reduced urinary excretion of 3'-hydroxystanozolol which was undetectable in most samples. This is the first report of diclofenac altering steroid metabolism in vivo, detrimentally affecting detection in urine, but not in hair, which holds considerable advantages over urinalysis for anti-doping tests.

In Vivo Administration of Stanozolol in Cattle: Depletion and Stability Studies Using UHPLC-Q-Orbitrap.

An in vivo study was performed in order to evaluate the depletion time of stanozolol and its main metabolites using naturally incurred urine sample collected after the administration of intramuscular injections in 12 steers. A stability study was also carried out to investigate the influence of the storage period and the freeze-thaw cycles. A fast parent drug metabolization was observed, because within 6 h after drug administration, the signal of the metabolite 16ß-hydroxystanozolol was predominant. After the second drug administration, a detection window of 17 days was obtained. The stability was studied using ANOVA, in which a storage condition of -20 °C proved stable during 240 days, which was also confirmed after 5 freeze-thaw cycles.

Stanozolol-N-glucuronide metabolites in human urine samples as suitable targets in terms of routine anti-doping analysis.

The exogenous anabolic-androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis, and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17-epistanozolol-1'N-glucuronide and 17-epistanozolol-2'N-glucuronide in stanozolol-positive human urine samples due to the access to high-quality reference standards. Examination of excretion study samples shows large detection windows for the phase-II metabolites stanozolol-1'N-glucuronide and 17-epistanozolol-1'N-glucuronide up to 12 days and respectively up to almost 28 days. In addition, we present appropriate validation parameters for the analysis of these metabolites using a fully automatic method online solid-phase extraction (SPE) method already published before. Limits of identification (LOIs) as low as 100 pg/ml and other validation parameters like accuracy, precision, sensitivity, robustness, and linearity are given.

High-throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction.

In the recent years, a lot of effort was put into the development of multiclass initial testing procedures (ITP) to streamline analytical workflow in antidoping laboratories. Here, a high-throughput assay based on liquid chromatography-triple quadrupole mass spectrometry suitable for use as initial testing procedure covering multiple classes of compounds prohibited in sports is described. Employing a 96-well plate packed with 10 mg of weak cation exchange polymeric sorbent, up to 94 urine samples and their associated positive and negative controls can be processed in less than 3 h with minimal labor. The assay requires a 0.5-ml urine aliquot, which is subjected to enzymatic hydrolysis followed by solid phase extraction, evaporation, and reconstitution in a 96-well collection plate. With a 10-min run time, more than 100 analytes can be detected using electrospray ionization with polarity switching. The assay can be run nearly 24/7 with minimal downtime for instrument maintenance while detecting picogram amounts for the majority of analytes. Having analyzed approximately 28,000 samples, nearly 400 adverse analytical findings were found of which only one tenth were at or above 50% of the minimum required performance level established by the World Anti-Doping Agency. Compounds most often identified were stanozolol, GW1516, ostarine, LGD4033, and clomiphene, with median estimated concentrations in the range of 0.02-0.09 ng/ml (either as parent drug or a metabolite). Our data demonstrate the importance of using a highly sensitive ITP to ensure efficient antidoping testing.

Development and validation of a simple online-SPE method coupled to high-resolution mass spectrometry for the analysis of stanozolol-N-glucuronides in urine samples.

Stanozolol is still the most commonly used illicit anabolic-androgenic steroid (AAS) in professional sports. Therefore, accurate and fast analysis and long detection windows are of great interest in the field of antidoping analysis. In this work, a very simple, fast, and highly sensitive online solid-phase extraction method coupled with liquid chromatography-high-resolution tandem mass spectrometry (HPLC-HRMSMS) for the analysis of stanozolol-N-glucuronides was developed. This fully validated procedure is characterized by only a few manual steps (dilution and addition of internal standard) in the sample preparation. A limit of identification (LOI) of 75 pg/mL, high accuracy (87.1%-102.1%), precision (3.1%-7.8%), and sensitivity was achieved. Furthermore, good linearity (> 0.99) and robustness, as well as no carry-over effects, could be observed. In addition to excellent confirmation analysis performance, this method shows sufficient potential for the identification and characterization of unknown metabolites. Using this method, it was possible to unambiguously confirm the presence of 1'N- and 2'N-stanozolol-glucuronide in human urine for the first time due to the access to reference material.

Simultaneous immunochemical detection of stanozolol and the main human metabolite, 3'-hydroxy-stanozolol, in urine and serum samples.

Two enzyme-linked immunosorbent assays (ELISAs) have been established for the analysis of stanozolol (St) and 3'-hydroxy-stanozolol (3'OH-St), the main metabolite found in humans. The immunizing hapten N2'-(5-valeric acid)-androst-2-eno[3,2-c]-pyrazol-17a-methyl-17b-ol (hapten 8) has been designed with the aid of molecular modeling and theoretical tools to allow immunochemical detection of both compounds. Using an ELISA based on a homologous antisera/coating antigen combination, St can be selectively quantified without significant interference of the St metabolites or other steroids potentially present in the biological samples. On the other hand, St immunoreactivity equivalents due to the additional presence of 3'OH-St can also be quantified using an ELISA based on a heterologous antisera/coating antigen combination, in which the metabolite can be detected with 51% cross-reactivity. Thus, As147/5BSA detects 3'OH-St and St in buffer with IC(50) values of 1.46 and 0.68 microg L(-1), respectively. In contrast, As147/8BSA is highly specific for St with an IC(50) of 0.16 microg L(-1) and a limit of dection of just 0.022 microg L(-1). Performance of both assays in urine and serum samples has been evaluated and demonstrate that inappropriate use of stanozolol by athletes or young people can be detected in these matrices after simple cleanup methods, with IC(50) values below the minimum performance required levels established by the World Antidoping Agency.

Is zebrafish (Danio rerio) a tool for human-like metabolism study?

One of the greatest challenges in anti-doping science is the large number of substances available and the difficulty in finding the best analytical targets to detect their misuse. Therefore, metabolism studies involving prohibited substances are fundamental. However, metabolism studies in humans could face an important ethical bottleneck, especially for non-approved substances. An emerging model for metabolism assessment is the zebrafish, due to its genetic similarities with humans. In the present study, the ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with a well-known metabolism that are widely used as doping agents in sports, was evaluated. They represent 2 of the most abused classes of doping agents, namely, stimulants and anabolic steroids. These are classes that have been receiving attention because of the upsurge of synthetic analogues, for which the side effects in humans have not been assessed. The samples collected from the zebrafish tank water were hydrolysed, extracted by solid-phase extraction, and analysed by liquid chromatography with high resolution mass spectrometry (LC-HRMS). Adult zebrafish could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidise, and excrete several metabolites in a manner similar to humans. Therefore, adult zebrafish seem to be a very promising tool to study human-like metabolism when aiming to find analytical targets for doping control. Copyright © 2017 John Wiley & Sons, Ltd.

A novel HPLC-MRM strategy to discover unknown and long-term metabolites of stanozolol for expanding analytical possibilities in doping-control.

Stanozolol is one of the most commonly abused anabolic androgenic steroids (AAS) by athletes and usually detected by its parent drug and major metabolites. However, its metabolic pathway is complex, varied and individually different, it is important to characterize its overall metabolic profiles and discover new and long-term metabolites for the aims of expanding detection windows. High performance liquid chromatography coupled with triple quadrupole mass spectrometer (HPLC-MS/MS) was used to analyze the human urine after oral administration of stanozolol. Multiple reaction monitoring (MRM), one of the scan modes of triple quadrupole mass spectrometer showing extremely high sensitivity was well used to develop a strategy for metabolic profiles characterization and long-term metabolites detection based on typical precursor to product ion transitions of parent drug and its major metabolites. Utilizing the characteristic fragment ions of stanozolol and its major metabolites as the product ions, and speculating unknown precursor ions based on the possible phase I and phase II metabolic reactions in human body, the metabolite profiles of stanozolol could be comprehensively discovered, especially for those unknown and low concentration metabolites in human urine. Then these metabolites were further well structure identified by targeted high resolution MS/MS scan of quadrupole-time of flight mass spectrometry (Q-TOF). Applying this strategy, 27 phase I and 21 phase II metabolites of stanozolol were identified, in which 13 phase I and 14 phase II metabolites have not been reported previously. The 9 out of 48 metabolites could be detected over 15days post drug administration. This strategy could be employed effectively to characterize AAS metabolic profiles and discover unknown and long-term metabolites in sports drug testing.

Detection of stanozolol O- and N-sulfate metabolites and their evaluation as additional markers in doping control.

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC-MS/MS to establish potential long-term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M-I to M-XI) were detected and characterized by LC-MS/MS. This paper provides valuable data on the ionization and fragmentation of O-sulfates and N-sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long-term metabolite (epistanozolol-N-glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.

Use of ion trap gas chromatography-multiple mass spectrometry for the detection and confirmation of 3'hydroxystanozolol at trace levels in urine for doping control.

Stanozolol, a synthetic anabolic androgenic steroid, is often abused in sports to enhance performance. Consequently, the anti-doping laboratories daily screen for its metabolites (3'hydroxystanozolol and 4beta hydroxystanozolol) in all urines, mainly by GC-MS, after enzymatic hydrolysis and TMS derivatization. A sensitive and specific method by GC-MS(3) has been developed for the identification in urine of 3'hydroxystanozolol at trace levels. Full mass spectra and diagnostic ions are presented and a case report is commented. In this case, it was possible to attest the presence of a low concentration of stanozolol metabolite in a sample obtained from a competition test. This would have not been possible with other analytical techniques used in the laboratory. Through this case report, it was also possible to demonstrate the importance of sampling and the difficulties that has to face the laboratory when the pre-analytical step is not correctly performed.

Complementing the characterization of in vivo generated N-glucuronic acid conjugates of stanozolol by collision cross section computation and analysis.

Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 Å(2) , thus largely covering the observed and computed CCSs for stanozolol-N1'-, stanozolol-N2'-, and stanozolol-O-glucuronide found at values between 195.1 and 212.4 Å(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 Å(2) an excellent correlation (R(2) = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves.

Studies on anabolic steroids--12. Epimerization and degradation of anabolic 17 beta-sulfate-17 alpha-methyl steroids in human: qualitative and quantitative GC/MS analysis.

The epimerization and dehydration reactions of the 17 beta-hydroxy group of anabolic 17 beta-hydroxy-17 alpha-methyl steroids have been investigated using the pyridinium salts of 17 beta-sulfate derivatives of methandienone 1, methyltestosterone 4, oxandrolone 7, mestanolone 10 and stanozolol 11 as model compounds. Rearrangement of the sulfate conjugates in buffered urine (pH 5.2) afforded the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes in a ratio of 0.8:1. These data indicated that both epimerization and dehydration of the 17 beta-sulfate derivatives were not dependent upon the respective chemical features of the steroids studied, but were instead inherent to the chemistry of the tertiary 17 beta-hydroxy group of these steroids. Interestingly, in vivo studies carried out with human male volunteers showed that only methandienone 1, methyltestosterone 4 and oxandrolone 7 yielded the corresponding 17-epimers 2, 5 and 8 and the 18-nor-17,17-dimethyl-13(14)-enes 3, 6 and 9 in ratios of 0.5:1, 2:1 and 2.7:1, respectively. No trace of the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of mestanolone 10 and stanozolol 11 was detected in urine samples collected after administration of these steroids. These data suggested that the in vivo formation of the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of 17 beta-hydroxy-17 alpha-methyl steroids is also dependent upon phase I and phase II metabolic reactions other than sulfation of the tertiary 17 beta-hydroxyl group, which are probably modulated by the respective chemical features of the steroidal substrates. The data reported in this study demonstrate that the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes are not artifacts resulting from the acidic or microbial degradation of the parent steroids in the gut as previously suggested by other authors, but arise from the rearrangement of their 17 beta-sulfate derivatives. Unchanged oxandrolone 7 was solely detected in the unconjugated steroid fraction whereas unchanged steroids 1, 4 and 11 were recovered from the glucuronide fraction. These data are indirect evidences suggesting that the glucuronide conjugates of compounds 1 and 4 are probably enol glucuronides and that of compound 11 is excreted in urine as a N-glucuronide involving its pyrazole moiety. The urinary excretion profiles of the epimeric and 18-nor-17,17-dimethyl-13(14)-ene steroids are presented and discussed on the basis of their structural features.

Determination of 16beta-hydroxystanozolol in urine and faeces by liquid chromatography-multiple mass spectrometry.

This paper describes the optimisation of the detection of stanozolol and its major metabolite 16beta-hydroxystanozolol in faeces and urine from cattle. Faeces are extracted directly with diisopropyl ether. Urine is first submitted to an enzymatic hydrolysis and then extracted over a modified diatomaceous earth column (Chem-Elut) with a mixture of diisopropyl ether-isooctane. In a final step an acidic back extraction is performed. For the LC-MS-MS detection two approaches are discussed. In a first approach the final extract is detected without derivatization, while the second approach makes use of a derivatization step for 16beta-hydroxystanozolol. While the MS-MS spectrum without derivatization exhibits extensive fragmentation, the spectrum of the derivative shows two abundant diagnostic ions with much more reproducible ion ratios. The derivatization method and the method without derivatization enable the detection of 16beta-hydroxystanozolol up to 0.03 microg l(-1) in urine and 0.07 microg kg(-1) in faeces. Until now there is no literature available for the detection of 16beta-hydroxystanozolol in faeces and urine at the ppt level.

Confirmatory analysis of residues of stanozolol and its major metabolite in bovine urine by liquid chromatography-tandem mass spectrometry.

A reliable method for the confirmation of the synthetic hormone stanozolol and its major metabolite, 16beta-hydroxystanozolol, in bovine urine by liquid chromatography coupled with tandem mass spectrometry has been developed. [2H3]Stanozolol was used as internal standard. Sample preparation involved enzymatic hydrolysis, liquid-liquid extraction and purification on an amino solid-phase extraction column. The analytes were ionized using atmospheric pressure chemical ionization with a heated nebulizer interface operating in the positive ion mode, where only the protonated molecules, [M+H]+, at m/z 329 and m/z 345, for stanozolol and 16beta-hydroxystanozolol, respectively, were generated. These served as precursor ions for collision-induced dissociation and three diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring liquid chromatography-tandem mass spectrometry. The accuracy ranged from 19.7 to 14.9% and from 18.9 to 13.2% for stanozolol and 16beta-hydroxystanozolol, respectively. The precision ranged from 12.4 to 2.4% and from 13.1 to 1.8% for stanozolol and 16beta-hydroxystanozolol, respectively. The limit of quantification of the method was 1 ng/ml in the bovine urine for both stanozolol and 16beta-hydroxystanozolol. The developed method fulfils the European Union requirements for confirmatory methods.

Detection of stanozolol and its metabolites in equine urine by liquid chromatography-electrospray ionization ion trap mass spectrometry.

The equine phase I and phase II metabolism of the synthetic anabolic steroid stanozolol was investigated following its administration by intramuscular injection to a thoroughbred gelding. The major phase I biotransformations were hydroxylation at C16 and one other site, while phase II metabolism in the form of sulfate and beta-glucuronide conjugation was extensive. An analytical procedure was developed for the detection of stanozolol and its metabolites in equine urine using solid phase extraction, acid solvolysis of phase II conjugates and analysis by positive ion electrospray ionization ion trap LC-MS.

Anabolic steroid abuse and cardiac sudden death: a pathologic study.

CONTEXT: Androgenic anabolic steroids (AAS) used for improving physical performance have been considered responsible for acute myocardial infarction and sudden cardiac death. OBJECTIVE: To establish the relationship between AAS and cardiac death. DESIGN: Case report. PATIENTS: Two young, healthy, male bodybuilders using AAS. MAIN OUTCOME MEASURES: Pathologic cardiac findings associated with AAS ingestion. RESULTS: The autopsy revealed normal coronary arteries. In one case, we documented a typical infarct with a histologic age of 2 weeks. A segmentation of myocardial cells at the intercalated disc level was observed in the noninfarcted region. This segmentation was the only anomaly detected in the second case. No other pathologic findings in the heart or other organs were found. Urine in both subjects contained the metabolites of nortestosterone and stanozolol. COMMENT: A myocardial infarct without vascular lesions is rare. To our knowledge, its association with AAS use, bodybuilding, or both lacks any evidence of a cause-effect relationship. The histologic findings in our 2 cases and in the few others reported in medical literature are nonspecific and do not prove the cardiac toxicity of AAS. A better understanding of AAS action on the neurogenic control of the cardiac function in relation to regional myocardial contraction and vascular regulation is required.

Testing for anabolic steroids in hair from two bodybuilders.

Two male bodybuilders were recently arrested by the French customs in Strasbourg (France) in possession of 2050 tablets and 251 ampoules of various anabolic steroids. It was claimed that the steroids were for personal use and not for trafficing as suggested by the police. Urine and hair specimens were collected from both suspects to clarify the claims. Nandrolone, stanozolol, testosterone and their corresponding metabolites were identified in the urine of both subjects. After decontamination, the hair was hydrolyzed by sodium hydroxide in presence of deuterated internal standards. After extraction with ethyl acetate and silylation, the drugs were identified by GC-MS in the electron impact mode. Hair from both males were positive for nandrolone (196 and 260 pg/mg), testosterone (46 and 71 pg/mg) and stanozolol (135 and 156 pg/mg), clearly indicating steroids abuse. Although not yet recognized by the International Olympic Committee, hair analysis may be a useful adjunct to conventional drug testing in urine from athletes.

Quantitative determination of stanozolol and its metabolite in urine by gas chromatography/mass spectrometry.

The amount of stanozolol and 3'-hydroxystanozolol in urine was determined after oral administration of stanozolol. A gas chromatograph/mass selective detector equipped with a capillary column was used for these determinations. The GC/MS was operated in the SIM mode, and m/z 581, 669, and 315 were monitored for stanozolol, 3'-hydroxystanozolol, and calusterone (internal standard), respectively. The detection limit was approximately 1 ng/mL for both steroids. The maximum excretion rate of stanozolol was reached in 8 h and the maximum of 3'-hydroxystanozolol in 19 h. However, only 3% of the administered amount was recovered in urine.

Confirmatory analysis of stanozolol metabolites in bovine, pig and sheep urines using an optimized clean-up and liquid chromatography-tandem mass spectrometry.

This paper describes a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the analysis of three stanozolol metabolites (16ß-hydroxystanozolol, 3'-hydroxystanozolol, and 4ß-hydroxystanozolol) in urines of animal origins. The solid-phase extraction (SPE) clean-up procedure was optimized to reduce the matrix effects in the LC-MS/MS analysis and to enhance recovery. Four different approaches were tested to prepare the sample, which include anion, and cation mixed-mode ion exchange, reversed-phase and normal-phase SPE cartridges. Mixed-mode anion exchange Strata-XL-A SPE column with diethyl ether elution yielded the best values. The separation of metabolites was optimized on Kinetex XB column using isocratic elution. The best mobile phase composition was achieved at the acidic pH with 0.1% (v/v) formic acid in water and methanol composition. The main advantages of the approach applied in the present study over other known methods include the single step SPE clean-up, relatively fast separation on HPLC column packed with core-shell particles, and lowering the limit of detection of target metabolites to the range between 0.05 and 0.15µg/l. Additionally, the developed method was successfully validated for the first time for three species in accordance with the European Union (EU) 2002/657/EC decision. Finally, the efficiency of method was demonstrated by analyzing incurred samples.