Crazy Bulk Review – Detailed and HonestAnabolic androgenic 50my, such as stanozolol, are typically misused by athletes during preparation for competition. Out-of-competition testing presents a unique challenge stanozolol fm 30 ml 50mg the current anti-doping detection system owing to logistic reasons. Analysing hair for the presence of a prohibited drug offers a feasible solution for covering the wider window in out-of-competition testing. For method development, spiked drug free rat hair, blood and urine samples were used. The sustanon flu symptoms developed method was then applied to hair, urine and serum samples from five brown Norway rats after treatment intraperitoneal with stanozolol for six consecutive days at 5.
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Anabolic androgenic steroids, such as stanozolol, are typically misused by athletes during preparation for competition. Out-of-competition testing presents a unique challenge in the current anti-doping detection system owing to logistic reasons.
Analysing hair for the presence of a prohibited drug offers a feasible solution for covering the wider window in out-of-competition testing. For method development, spiked drug free rat hair, blood and urine samples were used.
The newly developed method was then applied to hair, urine and serum samples from five brown Norway rats after treatment intraperitoneal with stanozolol for six consecutive days at 5.
The assay for each matrix was linear within the quantification range with determination coefficient r 2 values above 0. The respective assay was capable of detecting 0. The accuracy, precision and extraction recoveries of the assays were satisfactory for the detection of both compounds in all three matrices. These methods can be used for in vivo studies further investigating stanozolol metabolism, but also could be extended for doping testing.
Owing to the complementary nature of these tests, with urine and serum giving information on recent drug use and hair providing retrospective information on habitual use, it is suggested that blood or urine tests could accompany hair analysis and thus avoid false doping results.
Among these, stanozolol is one of the most frequently identified AAS. Stanozolol is a synthetic derivative of the male sex hormone testosterone.
Depending on the dose administered, once in the body, stanozolol gets rapidly metabolised and the metabolites are generally detected in urine until ca. Furthermore, urinalysis also fails to distinguish between chronic use and single, accidental exposure of drugs [ 15 ]. The major elimination and deactivation pathway of AAS and their phase I metabolites is through glucuronide conjugation phase II metabolism , mainly catalysed by the enzyme UGT2B17, followed by excretion in urine [ 16 - 19 ].
However, inter-individual and inter-ethnic variations in the prevalence of deletion polymorphism in the gene coding of the UGT2B17 enzyme have been reported, which eventually influence the urinary excretion of AAS and potentially lead to false-negative doping results [ 20 , 21 ]. It has also been reported that the glucuronidation activity of UGT2B17 and other UGTs towards AAS is inhibited by commonly used anti-inflammatory drugs like diclofenac and ibuprofen, in vitro [ 22 - 26 ].
Common dietary substances such as red wine [ 27 ], white tea and green tea [ 28 ] have also shown similar inhibitory effects in in vitro studies. Although the inhibitory effect is yet to be examined and reported in vivo , these in vitro results indicate that concomitant use of such over-the-counter medication or common dietary products with AAS may lead to impaired urinary excretion of AAS and their metabolites. Considering that such genetic and metabolic variations may limit the efficacy of urinalysis in testing doping, it can be suggested that urinalysis, if used as a stand-alone test, is susceptible to confounding doping results [ 11 - 13 , 16 - 21 ].
Owing to the growing number of doping cases with AAS [ 1 - 6 ], there is an ever-increasing need to develop new methods to detect drug doping. The current anti-doping regime can be reinforced by employing additional biological samples like blood and hair analysed in tandem with urine.
Since impaired glucuronidation leads to reduction in the urinary excretion rate of AAS, it can be assumed that the levels of unconjugated AAS and their phase I metabolites in the systemic circulation will be elevated and thus higher levels of AAS and their phase I metabolites will be available to get incorporated into hair and other body tissues [ 21 ]. Hair analysis has been used in the past for detecting drug use [ 29 - 32 ] as it predominantly favours the direct detection of parent AAS and determines a retrospective history of drug use.
Thus, hair analysis and blood analysis [ 33 ] can provide complementary information to urinalysis to prevent false doping results. However, to investigate this option further, in vivo studies are required to establish a relationship between the drug levels detected in hair, urine and blood. Since, metabolites are generally difficult to detect in hair, it is reasonable to assume that a single-dose treatment may not be sufficient to investigate whether levels of metabolites can be determined in hair.
However, multiple doses of stanozolol along with sensitive analytical methods can provide this key information. Thus, in line with previous steroid-abuse rat studies [ 36 - 39 ], the present study was designed with a daily dose of 5.
Sodium hydrogen phosphate heptahydrate, sodium phosphate monobasic dihydrate, sodium hydroxide, formic acid, hydrochloric acid, LC-MS grade water, acetonitrile, methanol, HPLC grade dichloromethane, pentane, chloroform and ethylacetate were purchased from Sigma Aldrich Poole, UK. All chemicals were of analytical-reagent grade and were used without further purification. For the animal experiment, stanozolol, ketamine 2.
A SB C column 2. All animals were kept in an animal house located in Semmelweis University, Budapest, Hungary. Animals were housed in groups of three individuals in standard laboratory cages. Rats were kept in a constant room temperature environment with an alternating h light—dark cycle.
Food and water were available ad-libitum. The dose of stanozolol selected was in line with previous steroid studies using rat models [ 36 - 39 ] and considered equivalent to levels abused by humans on a milligram per kilogram of body weight basis [ 36 , 37 ]. Hair, urine and blood samples were collected on the 7 th day of the study, i.
The growth rate of rat hair was tested prior to the treatment regime by shaving the back of the experimental animals and the sampling protocol was adjusted accordingly.
Urine was collected by gently pressing the abdomen. Blood was taken from the tail vein. Before collecting blood and urine samples, the animals were anaesthetised with a mixture of ketamine and xylazine.
Two weeks before the experiment, the entire dorsal surface of the animal was shaved to the skin with an electric shaver and drug-free control hair was collected and preserved. Exactly the same dorsal surface was sampled on the 7 th day of the experiment to avoid any diluting effect of the hair grown before the stanazolol treatment period. Drug-free blood and urine samples were also collected before the experiment was initiated.
Hair samples were stored in sealed, clean envelopes at room temperature. Samples were analysed in Kingston University. After decontamination, hair samples were allowed to air dry and then pulverised using a ball mill. Serum and urine samples were thawed and vortex mixed. After cooling, the samples were purified by performing liquid—liquid extraction LLE.
The reconstituted solution was filtered through a 0. The LC system comprised of a binary pump, automatic degasser, column heater and infinity thermostated autosampler. The analytical column used was a SB C column 2. Mobile phase solvents comprised of water with 0. The mass spectrometer was equipped with an electrospray ionisation ESI source, which was operated in positive ion mode. The mass spectrometer was operated in multiple reaction monitoring MRM mode to monitor the precursor ions and the diagnostic product ions of each analyte and IS.
For the optimum ionisation of analytes, the following mass spectrometric conditions were applied: The mass spectrometric parameters were optimised using the Masshunter optimizer software version B. The validation of the analytical methods was performed according to the Food and Drug Administration FDA guidelines [ 40 ], by determining accuracy, precision, lower limits of quantification LLOQ , lower limits of detection LLOD , linearity, selectivity, and extraction recoveries [ 41 , 42 ]. Drug-free rat hair, urine and serum samples were used for method development and validation.
Quality control QC samples were prepared similarly at three concentration levels for each matrix distributed over the linear range. Calibration curves were prepared for each matrix by plotting the analyte to IS ratio against the known concentrations of analyte in each sample. The analyte to IS ratio for each analyte was obtained by dividing the peak area of analyte by the peak area of the IS.
Samples for calibration curves and quality controls were treated in a way similar to unknowns. The linearity of the method was investigated by using linear regression analysis.
Intra-day precision was determined by measuring 6 replicates per concentration level, on the same day. Inter-day precision was assessed by analysing 6 replicates per concentration level, on three consecutive days. LLOD was defined as the lowest concentration of the analyte which gave a peak response equivalent to three times the background noise [i. The analyte to internal standard peak area ratios obtained after extraction were then compared with analyte to internal standard peak area ratios of standard solutions prepared in methanol at the same final concentrations.
To determine matrix effects, blank hair, urine and serum samples from different animals were extracted as described above. In order to consider only the matrix effect and not losses during the extraction procedure, the blank extracts were spiked with known concentrations of analytes and ISs after the extraction step, followed by analysis. Operating the mass spectrometer in MRM mode enhanced the method selectivity, sensitivity and specificity. Internal standards were used to compensate for any: Use of different mobile phase solvents was investigated.
For instance, use of water as solvent A in combination with methanol or acetonitrile or a mixture of methanol and acetontrile Different gradient and isocratic mobile phase compositions were investigated.
Addition of formic acid 0. Optimum sensitivity and excellent peak shapes for all analytes and ISs were obtained when water with formic acid 0. However, when formic acid was added only to water solvent A at a concentration of 0. For hair analysis, alkali digestion was employed for the extraction of drugs from hair matrix. Alkali digestion ensures complete dissolution of the hair matrix and hence it is generally known to give good recoveries of drugs entrapped in the hair matrix.
However, a potential drawback of complete dissolution of hair is that the components of hair matrix in solution may interfere with the analysis. Thus, to reduce the unwanted matrices that may affect the analysis, sample purification was carried out using LLE. The extraction efficiencies of different solvents like pentane, hexane, chloroform, ethyl acetate and ethanol, and their combinations were investigated. It was found that a mixture of pentane, chloroform and ethyl acetate in the ratio 3: Also, owing to the hair decontamination step employed using dichloromethane , no external interferences were observed.
The validation results are within the limits set by the FDA guidelines [ 40 ]. Suppression or enhancement of analyte ionisation owing to co-eluting components of matrices was not observed. The assay for hair analysis was linear in the range 0.
The determination coefficient r 2 values were found to be higher than 0. The assay for urinalysis was linear in the range 0. The serum assay showed good linearity within the quantification range 0. Furthermore, difference in the amount of water consumed by animals can also lead to variations in the levels of drugs in their body. These findings are in agreement with previous reports [ 32 ]. Similarly, in another study carried out by Shen et al. The major disadvantage of such technique is that it requires a laborious and expensive sample derivatisation step.
Generally, the derivatives are unstable and susceptible to thermal decomposition during analysis, thus affecting the reproducibility of the method. Thus, suggesting that both compounds can be detected in serum at similar concentration levels and with equal ease.