TY - JOUR
AU - , de Pinho, Paula G
AB - Abstract The development of accurate and reliable analytical methodologies to detect the abuse of doping agents in sport animals is crucial to ensure their welfare, as well as to support continuing social acceptance of these sports. The detection of doping agents in racing pigeons is difficult, especially owing to the disadvantages and limitations of obtaining samples from conventional matrices. The present study aimed to develop and validate an analytical methodology combining a two-step extraction procedure (liquid–liquid extraction and solid-phase extraction) in feathers from racing pigeons with analysis by liquid chromatography tandem mass spectrometry (LC–MS-MS) that enabled the simultaneous detection of a beta-agonist drug (clenbuterol) and three corticosteroids (prednisolone, betamethasone and budesonide). The method was validated concerning linearity (with coefficients of determination always higher than 0.99), accuracy (87.3–112.4%), precision (repeatability and intermediate precision coefficient of variation (CV%) always below 15%), recovery (71.6–98.2%), limits of detection (0.24–0.52 ng/g) and quantification (0.79 and 0. 1.74 ng/g) and specificity. The applicability of the method was performed using feathers from pigeons administered orally with a daily dose of 0.075 mg of betamethasone. The drug was administered during 60 days and successive analyses of feathers were performed, at the end of the administration protocol and also after ceasing the oral administration of the drug, for a three weeks period. Introduction There has been increasing evidence that drugs are being administered to racing animals for performance-improvement purposes. Fortunately, much has improved since the detection, control and discouragement of doping practices in horses has been extensively studied (1–6). However, for other animal species, these studies have been to some extent neglected. Regarding pigeons, despite the existence of races on a weekly basis all over the world, in the past few decades only three studies, dated 1996, 1997 and 2018, reporting doping control analytical methods, in this species, have been published (7–9). Furthermore, whereas newly developed methods to detect doping, not only in horseracing, but also in greyhounds and even camels competitions, have shifted to mass spectrometry detection approaches (10–14), two of the few studies about doping control in pigeons relied on immunoassays (7, 8). Considering that mass spectrometry is currently the gold standard in analytical chemistry and forensic toxicology (15–17), and that the promotion of animal welfare and fair-play is critical for continuing social acceptance of animal sports (18), the development of new and reliable analytical methods to detect the administration of performance-enhancing drugs to pigeons is required. Two classes of compounds generally flagged by pigeon sports associations are beta-adrenoceptor agonists and corticosteroids (19–22). Beta-adrenoceptor agonists, such as clenbuterol, are usually prescribed for their bronchodilator effects on smooth muscle in asthma. However, these drugs are frequently abused owing to their anabolic effects on skeletal muscle, resulting in muscular hypertrophy and also lipolysis (23–25). Beta-agonists are also particularly attractive since they provide a faster recovery after injury associated with increase in protein content and muscle fiber size (26, 27). The side effects of the repeated abuse of beta-agonists in humans include peripheral vasodilatation (28, 29) and tachycardia (28) and cardiac hypertrophy was previously demonstrated in rats (30, 31). Tragically, a fatal myocardial infarction in a 17-year-old body builder was attributed to the use of clenbuterol (32). Regarding corticosteroids, their administration in veterinary practice is mainly attributed to their anti-inflammatory and immunosuppressive effects (33). Still, it was already demonstrated that corticosteroids have the potential to increase muscle mass in cattle (34, 35). Additionally, as anti-inflammatory agents, corticosteroids suppress pain (36), an obvious advantage for doping purposes. Since exogenous glucocorticoids have been linked to inhibition of corticosterone deposition in feathers of greenfinches, it can be expected that the administration of these drugs may negatively impact the normal hormonal balance and health of birds (37). In order to ensure the welfare of the animals, and the protection of fair-play in the context of doping of birds involved in racings (mainly pigeons), the development of analytical methods capable of detecting drugs misuse in matrices from birds is crucial. Feathers represent an interesting alternative matrix to feces or blood, owing to the possible detection of drugs for longer periods, wide and permanent availability, and harmless collection. This study aimed to validate a method able to detect one beta-adrenoceptor agonist (clenbuterol) and three corticosteroid drugs (prednisolone, budesonide and betamethasone) in pigeon feathers. Using liquid chromatography tandem mass spectrometry (LC–MS-MS) as detection methodology, this approach afforded the sensitivity, selectivity, robustness and accuracy, required for legal purposes. Furthermore, this method can be applied to the control of the use of these drugs in poultry meat production in a consumer safety perspective. Experimental All reagents were of the highest grade available. All chemicals were from Merck (Darmstadt, Germany) except prednisolone, betamethasone, clenbuterol hydrochloride, budesonide, methyltestosterone (internal standard, IS) that were obtained from Sigma (St. Louis, MO, USA). HPLC-grade water was obtained with a MilliQ system (Millipore, Lisbon, Portugal). OASIS HLB 3 cc columns were purchased from Waters Technologies (Lisbon, Portugal). Apparatus The separation of the five analytes was achieved with a LC Waters 2695 XE separation module (Milford, MA). A core-shell Kinetex C18 column (2.6 μm; 100 × 2.1 mm) kept at 30°C was used. The mobile phase flow rate was 0.3 mL/min and the injection volume was 20 μL. The adopted gradient elution consisted of the following steps: (i) 0–2 min, 20% of B (methanol) in A (10 mM ammonium acetate/acetic acid, pH 4); (ii) from 2 to 5 min, 20–90% of B in A increase; (iii) 5–8 min, 90% of B in A; (iv) from 8 to 10 min; (v)90–98% B in A kept until 12 min. Afterwards, the column was rinsed and re-equilibrated (10 column volumes) for 10 min before the next injection. A Waters Micromass® Quattro micro™ API triple quadrupole mass analyzer equipped with an electrospray ionization source in the negative ion mode was used under the following conditions: (i) 3.00 kV capillary voltage; (ii) 120°C source temperature, and 200°C desolvation temperature; (iii) 500 L/h desolvation gas flow and (iv) 650 V multiplier. The cone and collision gases were nitrogen (≥99.999%) and argon (≥99.999%), respectively. The MRM transition, cone voltage and collision energy for each analyte can be depicted in Table I and were initially determined by flow injection analysis. The dwell time was set at 100 ms. Data acquisition was performed by the MassLynx V4.1 software. Table I. Chromatographic/mass spectrometry conditions: retention time, MRM transitions, and operating parameters Analyte Retention time (min) Parent Ion (m/z) MRM transitions (m/z) Cone voltage (V) Collision energy (eV) Clenbuterol 7.40 277 203a; 259b 25 15 Prednisolone 10.02 361 147a; 171b 20 20 Betamethasone 10.24 393 147a; 279b 20 33 Budesonide 11.01 431 147a; 173b 30 32 Methyltestosterone (IS) 11.03 303 109a; 97b 40 25 Analyte Retention time (min) Parent Ion (m/z) MRM transitions (m/z) Cone voltage (V) Collision energy (eV) Clenbuterol 7.40 277 203a; 259b 25 15 Prednisolone 10.02 361 147a; 171b 20 20 Betamethasone 10.24 393 147a; 279b 20 33 Budesonide 11.01 431 147a; 173b 30 32 Methyltestosterone (IS) 11.03 303 109a; 97b 40 25 aQuantifier ion. bQualifier ion. Table I. Chromatographic/mass spectrometry conditions: retention time, MRM transitions, and operating parameters Analyte Retention time (min) Parent Ion (m/z) MRM transitions (m/z) Cone voltage (V) Collision energy (eV) Clenbuterol 7.40 277 203a; 259b 25 15 Prednisolone 10.02 361 147a; 171b 20 20 Betamethasone 10.24 393 147a; 279b 20 33 Budesonide 11.01 431 147a; 173b 30 32 Methyltestosterone (IS) 11.03 303 109a; 97b 40 25 Analyte Retention time (min) Parent Ion (m/z) MRM transitions (m/z) Cone voltage (V) Collision energy (eV) Clenbuterol 7.40 277 203a; 259b 25 15 Prednisolone 10.02 361 147a; 171b 20 20 Betamethasone 10.24 393 147a; 279b 20 33 Budesonide 11.01 431 147a; 173b 30 32 Methyltestosterone (IS) 11.03 303 109a; 97b 40 25 aQuantifier ion. bQualifier ion. Methods Biological specimens Drug-free feathers that spontaneously fall out were collected from eight racing pigeons, weighing 300–350 g, maintained under a controlled drug-free diet, and kept in appropriate cages provided with a raised wire-mesh floor to avoid fecal contamination of the feathers. Tap water was available ad libitum. Fallen contour and flight feathers were collected during 60 days and kept at −20°C until analysis. All blank samples used for the quality control samples were previously checked for the absence of the analytes. For proof of concept, a racing pigeon was administered orally during 60 days with a daily dose of 0.075 mg of betamethasone, a very popular drug among pigeon fanciers (38). The dose was the same referenced in mammals and adjusted to the weight of the pigeon, as in previous studies (9, 38). Mature contour feathers of the dosed animal were collected at the end of the 60 days of administration. After the final administration, mature contour feathers were collected weekly for another 21 days, to determine if betamethasone would still be detected after drug administration had ceased. The Ethics Committee of the Faculty of Pharmacy of the University of Porto reviewed and approved all procedures involving animals that were performed in accordance with their guidance. Quality control samples One mg/mL methanol stock solutions of drug standards and IS were prepared and stored at −20°C, as well as all intermediate solutions. To obtain quality control samples, 5 mL of blank feather homogenates (prepared as described below) were spiked with the stock solutions of the different drugs to obtain the final concentrations of 0.122, 0.245, 0.491, 0.981, 1.96, 3.92, 7.85 μg/g of feather homogenates of betamethasone; 0.135, 0.269, 0.538, 1.08, 2.15, 4.31, 8.61 μg/g of feather homogenates of budesonide; 0.087, 0.173, 0.346, 0.693, 1.39, 2.77, 5.54 μg/g of feather homogenates of clenbuterol and 0.113, 0.225, 0.451, 0.901, 1.80, 3.60, 7.21 μg/g of feather homogenates of prednisolone. In all cases, the IS concentration was set at 0.756 μg/g. Extraction procedure The first section of the naturally fallen flight feathers (either from the wings or from tail) correspondent to the calamus was discarded. The remaining parts of the flight feathers (vanes, barbs and rachis) and entire contour feathers were used. Feathers were initially divided into 1-cm sections and samples of 100 mg were submitted to three cycles of 6 m/s for 45 s grinding in a Bead Ruptor 12 Homogenizer (Omni International, NW Kennesaw, USA), in 5.0- mL reinforced polypropylene tubes, each one previously filled with 17 ceramic beads of 1.4-mm diameter. To the processed powder obtained, 3 mL of 0.2 M sodium acetate solution pH 5.2 were added, the mixture was vortex mixed and finally transferred into 15 mL Falcon tubes. The tube was again vortex mixed with 1 mL of 0.2 M sodium acetate solution pH 5.2, and the obtained solution was combined in the Falcon tube. Afterwards, an extraction procedure previously validated for excreta was in this study adapted for feathers (9). Briefly, for the quality controls, blank homogenates were spiked with the appropriate amounts of the drug stock solutions. After spiking, 0.35 g of NaCl were added and the samples were continuously shaken for 10 min in a VV3 mixer (VWR, Lisbon, Portugal). The pH was then adjusted to 9.5 with 10 M NaOH, and the analytes were further extracted by vortex mixing with 5 mL of ethyl acetate, for 1 min, followed by 15 min of continuous shaking in the VV3 mixer. After extraction, the tubes were centrifuged for 15 min at 3220 g in a refrigerated centrifuge at 4°C. The organic phase was kept, and the aqueous phase was again submitted to a second extraction. The organic extracts were combined and evaporated to dryness under a gentle nitrogen stream, and the dry residue was dissolved with 4 mL of water/methanol (4:1) prior to the solid-phase extraction (SPE) extraction. Using a SupelcoTM Visiprep SPE vacuum manifold device (Sigma-Aldrich), the whole volume of the suspended sample was transferred onto the OASIS HLB 3 cc column previously conditioned with 3 mL methanol, followed by 3 mL water. Then, 3 mL of water were applied twice for washing. Finally, 4 mL of methanol were applied to elute the analytes into a clean glass tube. The obtained eluate was dried under nitrogen flow at 40°C and the dry residue was dissolved in 250 μL of acetonitrile (Figure 1). Figure 1. View largeDownload slide Summary of the sample preparation procedure, adapted from Moreira et al. (9). Figure 1. View largeDownload slide Summary of the sample preparation procedure, adapted from Moreira et al. (9). Method validation For the validation of the analytical procedure, parameters including specificity/selectivity, matrix effects, linearity, accuracy, precision (inter- and intra-day precision), recovery, limit of detection (LOD) and limit of quantification (LOQ), were evaluated. The validation of these parameters was performed according to the European Medicines Agency (EMA) guidelines (39). Evaluation of the specificity of the method was accomplished through the determination of matrix-related or other possible interferences resulting from the experimental procedure, with the target analytes, in extracted blank samples (no analytes) injected into the apparatus. For the investigation of the matrix effect, calibration curves prepared with methanolic solutions of the analytes were injected and compared to those obtained with the quality controls prepared in blank matrix. Since significant differences were observed in the respective calibration curves, the calibration curves as well as all other validation parameters, including the linearity, were determined with quality control samples that were always prepared with blank matrix spiked with the desired amounts of the analytes. For the linearity assessment, seven independent quality control samples containing all four analytes, with concentrations ranging from 0.122 to 7.85 μg/g of betamethasone (0.122, 0.245, 0.491, 0.981, 1.96, 3.92, 7.85 μg/g), 0.135 to 8.61 μg/g of budesonide (0.135, 0.269,0.538, 1.08, 2.15, 4.31, 8.61 μg/g), 0.087 to 5.54 μg/g of clenbuterol (0.087, 0.173, 0.346, 0.693, 1.39, 2.77, 5.54 μg/g) and 0.113 to 7.21 μg/g of prednisolone (0.113, 0.225, 0.451, 0.901, 1.80, 3.60, 7.21 μg/g) and a blank, were prepared and injected (20 μL) into the LC–MS-MS in six different days. The regression curves (ratio between analyte peak area and IS peak area vs. analyte concentration) and the coefficient of determination (r2) were calculated. The six independent calibration curves (Y = aX + b) that were obtained were compared using the GraphPad® 6.0 Software, and visually inspected to confirm linearity. Three different analyte concentrations representative of low, medium and high concentrations within the linear concentration range (0.245, 0.981 and 7.85 μg/g for betamethasone; 0.269, 1.08 and 8.61 μg/g for budesonide; 0.173, 0.693 and 5.54 μg/g for clenbuterol and 0.225, 0.451 and 7.21 μg/g for prednisolone), were chosen to evaluate accuracy and precision. For accuracy determination, the percentage deviation between the calculated value from the calibration curve and the nominal value was determined [Accuracy (%) = (mean experimental concentration)/(nominal concentration) × 100)] for five different replicates of quality control samples, containing all analytes, independently prepared and injected (20 μL), in triplicate, into the LC–MS-MS system. Precision was expressed as the coefficient of variation (%CV) of a series of measurements (30) that were determined both for the repeatability of the method (i.e., the intra-day precision, reflecting the same operating conditions over a short period of time) and for the intermediate precision of the method (i.e., the inter-day precision, expressing variations within different days). For this purpose, five blank matrix samples were spiked to obtain low, medium, and high concentrations within the linear concentration range (0.245, 0.981 and 7.85 μg/g for betamethasone; 0.269, 1.08 and 8.61 μg/g for budesonide; 0.173, 0.693 and 5.54 μg/g for clenbuterol and 0.225, 0.451 and 7.21 μg/g for prednisolone). These samples were extracted and injected (20 μL) in triplicates into the LC–MS-MS equipment, on the same day, in order to estimate the intra-day precision of the analytical method and instrumentation by calculating the CV % of the obtained values (ratio between analyte peak area and IS peak area obtained for each calibrator). A CV % value ≤15% was considered acceptable. To evaluate the inter-day precision of the analytical method and instrumentation, a similar procedure was adopted but this time, the samples were independently prepared and injected over five consecutive days. A CV% value ≤15% was considered satisfactory, also for the inter-day precision. The LOD and LOQ parameters were determined to estimate the sensitivity of the method. LOD and LOQ were calculated according to the following equations: LOD = 3.3σ/S and LOQ = 10σ/S, where, for each analyte, σ is the standard deviation of the response and S the slope of the calibration curve. For this purpose, at least 15 blank samples were extracted according to the protocol previously described, and injected (20 μL) into the LC–MS-MS apparatus, prior to the chromatographic analysis (the ratio between the analyte peak area and IS peak area were obtained for each blank sample, and the deviation of the analytical signal recorded was calculated). The recovery (percentage) was calculated by comparing the peak areas of the analytes for extracted and non-extracted samples. Therefore, a set of control samples was spiked with three different concentrations (low, medium and high concentrations: 0.245, 0.981 and 7.849 μg/g for betamethasone; 0.269, 1.076 and 8.611 μg/g for budesonide; 0.173, 0.693 and 5.544 μg/g for clenbuterol and 0.225, 0.451 and 7.209 μg/g for prednisolone). The peak areas were compared to those obtained with a second set of control samples that were spiked with the same concentrations, but only just before the last evaporation step. Statistical analysis The GraphPad Software 6.0 and Microsoft Excel 2016 were used for all statistical analysis. Results and discussion Method development It is widely recognized that prior to the study of depletion of drugs and residue analysis, the selection and validation of appropriate analytical methods are matters of special concern (40, 41). Some very recently published works dedicated to the detection and quantification of different compounds in biological samples from birds, have used feathers as analytical matrix, demonstrating its suitability for these purposes (42, 43). However, as far as the authors know, the detection of doping compounds in feathers of pigeons had never been addressed before and there is not a single method validated for this purpose. Therefore, it was aimed to implement and validate such a method, concerning major criteria that consisted of an extraction procedure combined with a LC–MS-MS analysis for corticosteroids and beta-agonists in pigeon feathers. It is mandatory that the method developed fulfilled the performance criteria required by international agencies (39). In pigeons, feathers might be a very interesting alternative to more conventional matrices. Since the volume of blood that would be available to be collected is insufficient for an anti-doping analysis and urine is combined with feces in the cloaca, the fecal matter would be the more obvious remaining alternative, being used in previous studies for the detection of doping compounds in pigeons (7–9). Nevertheless, several recently published studies evidenced the suitability of feathers as a matrix for the detection of endogenous corticoids in feathers that can be used as a biological marker to understand the impact of natural and anthropogenic stressors on the health, survival, and coping mechanisms of free-living animals (44). By measuring corticosterone in feathers as an endocrine marker of stress physiology, it has been possible to study the effects of group size in cuckoos (43), non-breading seasons in gannets (45) and common eiders (46), body size and age in kites (47), sex and migratory strategies in Cory’s shearwater (48), food availability in great tits (49, 50) and rhinoceros auklet (51) and body condition in tawny owls and kestrels (52). Although the non-invasiveness of fecal matter collection would be an apparent advantage, feathers are also a minimally invasive matrix. It is considered that the impact of collecting a few mature contour feathers is negligible (44). Nevertheless, in the present study, we ensured that the collection of partially grown feathers (that could result in bleeding) and flight feathers (that could handicap the bird) was avoided. The confirmation of feathers as a suitable matrix is not only important for doping control but also for the control of drugs misuse in meat poultry production. Malucelli et al. (53) demonstrated that beta-agonists (namely clenbuterol, salbutamol and terbutaline) might be found in several matrices from broiler chickens, including feathers. Among all analyzed matrices (liver, kidney, stomach, muscle, fat, eye and feathers), the feathers presented the highest concentrations of all three analytes (224 ng of clenbuterol per gram, 1.140 ng of salbutamol per gram and 1.159 ng of terbutaline per gram) and, at the end of the study (after 44 days of withdrawal), clenbuterol, salbutamol and terbutaline were still detectable in feathers (LOD = 0.08 ng/g). Despite the impressive results obtained in this mentioned study, these were obtained by enzyme immunoassays, often associated to questionable accuracy and reliability that compromise their standing in legal challenges. The method herein presented only used one-third of the amount of feathers per sample used by those authors (53) and also enabled the simultaneous detection of compounds of two different therapeutic groups. It is crucial to use the minimum amount of feathers in the case of pigeons, since these birds are very small and unlike broilers, feathers might be vital for flying. In the absence of those flying feathers, the pigeons are worthless for commercial and sport purposes. Some other previously reported methods for the detection of exogenous substances in feathers used amounts as high as 12 g per sample (40, 54, 55) and 5 g per sample (56) that would be unsuitable for pigeons. The extraction procedure adopted in the present study has been adapted from an extraction procedure developed and validated by our group for the extraction of the same compounds from pigeon feces (9). According to the EMA Guideline on bioanalytical method validation (39), full validation should be performed for each species and matrix concerned. Therefore, despite the fit-for-purpose demonstration of the method for feces of pigeons, its major criteria validation on feathers was deemed necessary. The first step in developing the method was to grind feathers’ samples. Using a silica-bead ruptor homogenizer, the processed samples were more effectively homogenized, granting access to the deposited drugs either in the rachis of the feather or in its inter locking barbs or vanes. Owing to the cleaner nature of this matrix as compared with feces, its homogeneous appearance and also to the fact that a relatively small amount of feathers was used, centrifugation and mixing times were spared. Such a faster execution method is more likely to be adopted for routine analysis. After a thorough investigation during method development, methyltestosterone was chosen as the internal standard, based on our own previous experience and on some recently published papers reporting analytical methods for doping detection in animals that also used methyltestosterone as internal standard (3, 9, 57). This substance presents several advantages that include, (i) its chemical stability and reliability; (ii) the unlikely presence in the test sample (doping agents preferred by pigeon fanciers are those that can be orally administered which is not the case of methyltestosterone that requires intramuscular administration); (iii) the absence of chromatographic interference with the analytes and (iv) similarities with the target analytes in terms of molecular weight and ionization response in electrospray ionization. Therefore, methyltestosterone was considered a viable alternative to deuterated analogs that nevertheless represent the first choice owing to their similar extraction recovery, ionization response in electrospray ionization mass spectrometry, and similar chromatographic retention time. Concerning the chromatographic conditions, other LC–MS methods have reported similar mobile phases (using ammonium acetate/acetic acid and methanol) for the detection of beta-agonists in human plasma and urine (58) and corticosteroids in bovine urine (59), bovine milk (60), pig fat (61) and bovine muscle, liver and kidney (62). Although we tested several other chromatographic conditions (namely a mobile phase composed of 0.1% formic acid in water and 0.1% acid formic in acetonitrile; and the same mobile phase herein described, but with different gradients), the best chromatographic profiles that could be obtained, without compromising the quality of the chromatographic resolution, were those presently reported—as solvent A, acetate buffer 10 mM at pH = 4 and, as solvent B, methanol, using a linear gradient program of 0–2 min, 20% of B in A; 2–5 min, increase 20–90% of B in A; 5–8 min, 90% of B in A; 8–10 min, 90–98% B in A and keep 98% of B until 12 min. The use of isocratic conditions was also considered, but resulted in excessively long chromatographic runs. Fixing the organic phase >90% at the end of the chromatographic run, enabled the elution of possible compounds that could damage the column. With these optimized extraction and chromatographic conditions well resolved peaks for all analytes, eluting in less than 12 min, were obtained. Although peak fronting can be observed in Figure 2, the reproducibility of the method was not compromised, since, as shown below, the CV% of the validated criteria were consistently low, ensuring an acceptable quantification. Figure 2. View largeDownload slide Representative chromatogram obtained after the injection of a blank sample spiked with a mixture of 0.245 μg/g of betamethasone, 0.269 μg/g of budesonide, 0.173 μg/g of clenbuterol, 0.225 μg/g of prednisolone and 0.756 μg/g of methyltestosterone, for the multiple reaction monitoring (MRM) transitions. Figure 2. View largeDownload slide Representative chromatogram obtained after the injection of a blank sample spiked with a mixture of 0.245 μg/g of betamethasone, 0.269 μg/g of budesonide, 0.173 μg/g of clenbuterol, 0.225 μg/g of prednisolone and 0.756 μg/g of methyltestosterone, for the multiple reaction monitoring (MRM) transitions. Method validation Figure 2 presents a typical chromatographic separation using the LC–MS-MS system method for the simultaneous detection of corticosteroid (clenbuterol) and beta-agonist drugs (prednisolone, betamethasone and budesonide). For this study, a total of 24 blank feather extracts were analyzed in triplicate to evaluate potential chromatographic interferences. It was confirmed that the method is specific for the simultaneous analysis and quantification of the analytes and IS, owing to the absence of interference peaks in their retention time. Previous to the method validation and post-validation, several other blank specimens were independently analyzed further confirming the absence of interferences in this analytical matrix. Still, the possibility of existence of other interfering substances that can be exogenously administrated cannot be ruled out. As suggested by EMA, as more data on the behavior of the analyte become available, the continuous evaluation of specificity must be conducted, even after the original validation is completed (39). After comparison of calibration curves obtained after the extraction of methanolic solutions of the analytes with calibration curves extracted from spiked blank matrix, it was possible to conclude that ion suppression occurs for prednisolone and budesonide while ion enhancement occurs for betamethasone and clenbuterol. Accordingly, calibration curves were constructed using feathers samples from racing pigeons free of corticosteroid and beta-agonist drugs (collected before the addition of any doping agents and analyzed to confirm the absence of these compounds). Linearity was determined with six independent curves, prepared in six different days, for which the coefficient of determination (r2) was always greater than 0.99, thus indicating a linear relationship between analyte/IS peak areas and analyte concentrations over the tested concentration range. The visual inspection of the calibration graphs also confirmed linearity. The linearity data, expressed in μg/g, can be depicted in Table II. Table II. Summary of calibration curves data for the test analytes 95% confidence intervals Analyte Equation* R square Slope Y-intercept X-intercept μg/g Clenbuterol Y = 4.53*X + 0.379 0.992 4.09–4.98 −0.0899–0.747 −0.196–0.0310 Prednisolone Y = 0.241*X + 0.0303 0.995 0.–0.269 0.0163–0.0443 −0.204–(−0.0627) Betamethasone Y = 0.487*X + 0.165 0.991 0.461–0.514 0.124–0.215 −0.458–(−0.249) Budesonide Y = 0.240*X + 0.00340 1.00 0.210–0.258 −0.0101–0.0175 −0.0759–0.0381 95% confidence intervals Analyte Equation* R square Slope Y-intercept X-intercept μg/g Clenbuterol Y = 4.53*X + 0.379 0.992 4.09–4.98 −0.0899–0.747 −0.196–0.0310 Prednisolone Y = 0.241*X + 0.0303 0.995 0.–0.269 0.0163–0.0443 −0.204–(−0.0627) Betamethasone Y = 0.487*X + 0.165 0.991 0.461–0.514 0.124–0.215 −0.458–(−0.249) Budesonide Y = 0.240*X + 0.00340 1.00 0.210–0.258 −0.0101–0.0175 −0.0759–0.0381 n = 5. Table II. Summary of calibration curves data for the test analytes 95% confidence intervals Analyte Equation* R square Slope Y-intercept X-intercept μg/g Clenbuterol Y = 4.53*X + 0.379 0.992 4.09–4.98 −0.0899–0.747 −0.196–0.0310 Prednisolone Y = 0.241*X + 0.0303 0.995 0.–0.269 0.0163–0.0443 −0.204–(−0.0627) Betamethasone Y = 0.487*X + 0.165 0.991 0.461–0.514 0.124–0.215 −0.458–(−0.249) Budesonide Y = 0.240*X + 0.00340 1.00 0.210–0.258 −0.0101–0.0175 −0.0759–0.0381 95% confidence intervals Analyte Equation* R square Slope Y-intercept X-intercept μg/g Clenbuterol Y = 4.53*X + 0.379 0.992 4.09–4.98 −0.0899–0.747 −0.196–0.0310 Prednisolone Y = 0.241*X + 0.0303 0.995 0.–0.269 0.0163–0.0443 −0.204–(−0.0627) Betamethasone Y = 0.487*X + 0.165 0.991 0.461–0.514 0.124–0.215 −0.458–(−0.249) Budesonide Y = 0.240*X + 0.00340 1.00 0.210–0.258 −0.0101–0.0175 −0.0759–0.0381 n = 5. The method’s accuracy and precision (both repeatability and intermediate precision), was demonstrated at three different concentrations within the linear concentration range. The accuracy was calculated as the percentage of target concentration and results ranging from 87% to 112% were obtained, which are within the accepted limits (100 ± 20%) (Table III). For the intra-day precision of both instrumentation and method, the obtained CV% was always below 10.4% (Table III). For the inter-day precision of the instrumentation and method, the obtained CV% values were always below 15%, indicating an acceptable inter-day precision for both instrumentation and method (Table III). Table III. Inter-assay and intra-day precision, accuracy, recovery, LOD and LOQ data for the test analytes Analyte Expected concentration Observed concentration Accuracy Precision Recovery LOD* LOQ* Inter-day Intra-day (μg/g) (μg/g) (%) (CV%) (CV%) (CV%) (ng/g) (ng/g) Clenbuterol 0.173 0.153 [18.7] 88.4 14.6 10.4 88.8 [10.3] 0.35 1.18 0.693 0.686 [15.4] 99.0 14.1 8.70 78.2 [13.3] 5.54 5.50 [13.3] 99.2 12.3 4.96 88.1 [7.1] Prednisolone 0.225 0.197 [21.6] 87.6 8.90 3.10 98.2 [6.37] 0.39 1.29 0.901 0.896 [6.60] 99.4 14.5 5.90 87.1 [11.0] 7.21 7.07 [3.60] 98.1 14.7 4.50 89.0 [12.3] Betamethasone 0.245 0.214 [34.8] 87.3 7.40 2.40 94.7 [10.5] 0.24 0.79 0.981 1.10 [7.30] 112.4 6.10 2.90 87.3 [9.9] 7.85 7.74 [4.60] 98.6 8.30 5.10 96.3 [4.4] Budesonide 0.269 0.279 [10.6] 103.7 10.7 4.90 79.0 [12.5] 0.52 1.74 1.08 1.10 [4.20] 101.8 14.1 4.10 71.6 [14.5] 8.61 8.61 [10.9] 100.0 12.2 6.40 74.6 [10.4] Analyte Expected concentration Observed concentration Accuracy Precision Recovery LOD* LOQ* Inter-day Intra-day (μg/g) (μg/g) (%) (CV%) (CV%) (CV%) (ng/g) (ng/g) Clenbuterol 0.173 0.153 [18.7] 88.4 14.6 10.4 88.8 [10.3] 0.35 1.18 0.693 0.686 [15.4] 99.0 14.1 8.70 78.2 [13.3] 5.54 5.50 [13.3] 99.2 12.3 4.96 88.1 [7.1] Prednisolone 0.225 0.197 [21.6] 87.6 8.90 3.10 98.2 [6.37] 0.39 1.29 0.901 0.896 [6.60] 99.4 14.5 5.90 87.1 [11.0] 7.21 7.07 [3.60] 98.1 14.7 4.50 89.0 [12.3] Betamethasone 0.245 0.214 [34.8] 87.3 7.40 2.40 94.7 [10.5] 0.24 0.79 0.981 1.10 [7.30] 112.4 6.10 2.90 87.3 [9.9] 7.85 7.74 [4.60] 98.6 8.30 5.10 96.3 [4.4] Budesonide 0.269 0.279 [10.6] 103.7 10.7 4.90 79.0 [12.5] 0.52 1.74 1.08 1.10 [4.20] 101.8 14.1 4.10 71.6 [14.5] 8.61 8.61 [10.9] 100.0 12.2 6.40 74.6 [10.4] n = 5 for all parameters except LOD and LOQ (*n = 24). Table III. Inter-assay and intra-day precision, accuracy, recovery, LOD and LOQ data for the test analytes Analyte Expected concentration Observed concentration Accuracy Precision Recovery LOD* LOQ* Inter-day Intra-day (μg/g) (μg/g) (%) (CV%) (CV%) (CV%) (ng/g) (ng/g) Clenbuterol 0.173 0.153 [18.7] 88.4 14.6 10.4 88.8 [10.3] 0.35 1.18 0.693 0.686 [15.4] 99.0 14.1 8.70 78.2 [13.3] 5.54 5.50 [13.3] 99.2 12.3 4.96 88.1 [7.1] Prednisolone 0.225 0.197 [21.6] 87.6 8.90 3.10 98.2 [6.37] 0.39 1.29 0.901 0.896 [6.60] 99.4 14.5 5.90 87.1 [11.0] 7.21 7.07 [3.60] 98.1 14.7 4.50 89.0 [12.3] Betamethasone 0.245 0.214 [34.8] 87.3 7.40 2.40 94.7 [10.5] 0.24 0.79 0.981 1.10 [7.30] 112.4 6.10 2.90 87.3 [9.9] 7.85 7.74 [4.60] 98.6 8.30 5.10 96.3 [4.4] Budesonide 0.269 0.279 [10.6] 103.7 10.7 4.90 79.0 [12.5] 0.52 1.74 1.08 1.10 [4.20] 101.8 14.1 4.10 71.6 [14.5] 8.61 8.61 [10.9] 100.0 12.2 6.40 74.6 [10.4] Analyte Expected concentration Observed concentration Accuracy Precision Recovery LOD* LOQ* Inter-day Intra-day (μg/g) (μg/g) (%) (CV%) (CV%) (CV%) (ng/g) (ng/g) Clenbuterol 0.173 0.153 [18.7] 88.4 14.6 10.4 88.8 [10.3] 0.35 1.18 0.693 0.686 [15.4] 99.0 14.1 8.70 78.2 [13.3] 5.54 5.50 [13.3] 99.2 12.3 4.96 88.1 [7.1] Prednisolone 0.225 0.197 [21.6] 87.6 8.90 3.10 98.2 [6.37] 0.39 1.29 0.901 0.896 [6.60] 99.4 14.5 5.90 87.1 [11.0] 7.21 7.07 [3.60] 98.1 14.7 4.50 89.0 [12.3] Betamethasone 0.245 0.214 [34.8] 87.3 7.40 2.40 94.7 [10.5] 0.24 0.79 0.981 1.10 [7.30] 112.4 6.10 2.90 87.3 [9.9] 7.85 7.74 [4.60] 98.6 8.30 5.10 96.3 [4.4] Budesonide 0.269 0.279 [10.6] 103.7 10.7 4.90 79.0 [12.5] 0.52 1.74 1.08 1.10 [4.20] 101.8 14.1 4.10 71.6 [14.5] 8.61 8.61 [10.9] 100.0 12.2 6.40 74.6 [10.4] n = 5 for all parameters except LOD and LOQ (*n = 24). Regarding recovery, the results ranged from 71.6% to 98.2%. The samples spiked with the high concentration quality controls resulted in recoveries ranging from 74.6% to 96.3%, whereas medium concentration quality controls resulted in a recovery between 71.6% and 87.3% and low concentration quality controls presented recoveries ranging from 79.0% to 98.2% (Table III). Method sensitivity was proved with the obtained LOD and LOQ of all analytes. The detection limits ranged from 0.24 ng/g to 0.52 ng/g and the quantification limits were set between 0.79 and 1.74 ng/g (Table III). The only method previously reporting the detection of clenbuterol in feathers of broiler chickens, presented a LOD of 0.08 ng/g (53). Still, as already mentioned, the authors used immunoassays, that usually ensures high sensitivity, but that do not stand in legal matters. Proof of applicability The applicability of this method in real samples was verified, following 60 days of daily oral administration of 0.214 mg/kg of betamethasone. The analysis of the feathers from a pigeon resulted in an unequivocal identification and successful quantification of this corticosteroid (Table IV). Additionally, after ceasing the drug administration, feathers were collected once a week, for three weeks. The concentration of betamethasone increased with time, for more than 20 days. Even at 21 days (3 weeks) after discontinuation of administration, the concentration of betamethasone was 7.96 μg/g. These results demonstrate that this method can be used as an alternative analytical methodology using feathers, owing to its probable wider time window for doping detection. These results are in accordance with other studies about the long detection window provided by feathers that targeted several antibiotics. In a study in which flumequine was repeatedly administered, this antibiotic remained at detectable concentrations in broiler chicken feathers 6 days after ceasing the administration (55), and in another study, the depletion time of tylosine also in broiler chicken feathers was of 27 days (56). The use of keratinized matrices of other species has also been highlighted in recent years. A matrix that has gained some popularity in doping control in horses is hair (63–66). Corroborating the previously mentioned studies on feathers, also hair enables a longer range of time for the detection of doping agents than urine, feces or blood (64, 67). Besides the long period of time for detection of drugs after administration, hair also has the advantage of being more easily obtained. Another important result of the present study was also the evidence that exogenous corticosteroids accumulate in bird feathers. Table IV. Betamethasone excretion in feather from a pigeon after the oral administration of a daily 0.075 mg dose, during 60 days Day Betamethasone concentration (μg/g) During daily drug administration 60 3,92 Post drug administration 67 4,70 74 5,43 81 7,96 Day Betamethasone concentration (μg/g) During daily drug administration 60 3,92 Post drug administration 67 4,70 74 5,43 81 7,96 Table IV. Betamethasone excretion in feather from a pigeon after the oral administration of a daily 0.075 mg dose, during 60 days Day Betamethasone concentration (μg/g) During daily drug administration 60 3,92 Post drug administration 67 4,70 74 5,43 81 7,96 Day Betamethasone concentration (μg/g) During daily drug administration 60 3,92 Post drug administration 67 4,70 74 5,43 81 7,96 Conclusion In conclusion, the present manuscript describes a development and validation of major criteria of a methodology for the efficient identification and quantification of drugs that are commonly used as doping agents in racing pigeons, using feathers as the analytical matrix. A proof of concept is additionally presented. The herein proposed method can be easily adapted to doping control laboratorial routine analysis. The use of feathers as matrix enables a fast, reliable and relatively simple sample pre-treatment, with a long detection window time, that can be useful for doping control purposes. Hereafter, this method might be adapted for other purposes involving the misuse of beta-agonists and corticosteroids, such as for the control of meat production in poultry industry. Acknowledgments The authors wish to express their sincere gratitude to the Laboratory of Bromatology/Hydrology, Department of Chemistry Sciences, Faculty of Pharmacy of the University of Porto, for providing the analytical conditions for the method validation. Funding This work was supported by the European Union (European Regional Development Fund funds POCI/01/0145/FEDER/007728); and National Funds (Fundação para a Ciência e a Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020UID/MULTI/04378/2013. The study is a result of the project NORTE-01-0145-FEDER-000024, supported by Norte Portugal Regional Operational Programme (NORTE, 2020), under the PORTUGAL 2020 Partnership Agreement (DESignBIOtecHealth— New Technologies for three Health Challenges of Modern Societies: Diabetes, Drug Abuse and Kidney Diseases), through the European Regional Development Fund (ERDF). A.M. wishes to thank the Fundação para a CiênciaTecnologia for grants SFRH/BPD/86898/2012. R.S. acknowledges Fundação para a Ciência e Tecnologia for her Post-doctoral Grant (SFRH/BPD/ 110201/2015). References 1 Liu , Y. , Uboh , C.E. , Soma , L.R. , Li , X. , Guan , F. , You , Y. , et al. . ( 2011 ) Detection and confirmation of 60 anabolic and androgenic steroids in equine plasma by liquid chromatography-tandem mass spectrometry with instant library searching . Drug Testing and Analysis , 3 , 54 – 67 . 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TI - The Use of Feathers from Racing Pigeons for Doping Control Purposes
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bky088
DA - 2019-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-use-of-feathers-from-racing-pigeons-for-doping-control-purposes-ozCBcylYB8
SP - 307
VL - 43
IS - 4
DP - DeepDyve
ER -