Metabolic Detection of Stanozolol Using GC Mass Spectrometry - Essay Example

?Metabolic Detection of Stanozolol Using GC – MS Metabolic Detection of Stanozolol Using GC – MS Different athletes usually misuse anabolic androgenic steroids especially Stanozolol for unfair competition. Nonetheless, different competition tests have currently provided unique challenges in the contemporary anti-doping detection of such drug misuse (Makin and Gower, 2010; pg. 799). Among the sensitive methods that should be adopted is the urinary metabolite using GC-MS. Numerous Properties of the Stanozolol make it suitably analysis with the GC analytical method as opposed to the HPLC method most of which are pegged on the properties of Stanozolol. It should be noted that Stanozolol is an anabolic – androgenic steroid easily extractable from urine not as an element but a metabolic mixture. Therefore, in this state, GC – Mass Spectrometry, the GC-MS provides a perfect illustration steps in extracting the element from the mixture (Makin and Gower, 2010; pg. 999). Additionally, this element usually melts at a temperature of 1550C and has a boiling point of 2350C (Behre and Nieschlag, 2004; pg. 725). Urine contain several impurities or elements and compounds that at these temperatures will still form part of the Stanozolol; therefore, separating it at gaseous state will provide proper and accurate determination since different element have different vaporization characteristics and the same cannot be determined using HPLC but GC. In other words, it should be noted that Stanozolol cannot be detected using the test methods for analyzing multi-residual steroids. Therefore, in this case, even the high performance thin layer chromatography (HPTLC) will limit Stanozolol’s detection since this analytical process makes it inferior to other steroids. Additionally, in the GC – MS, Stanozolol portrays different behaviors compared to the compounds. Moreover, the HPLC makes the drug to metabolize faster; thus, may interfere with its accurate detection. For these reasons, GC is usually a perfect analytical method for Stanozolol than HPLC and at the workshop, the UV was determined at the Stanozolol values of 20mg/ml. Stanozolol usually follow different GC- MS sample preparation stages (using solid Phase Extraction (SPE) Cartridge). The initial stage involves Rinsing the ODS (C18) SPE with MeOH followed with water. This procedure dissolves the residues. In this sense, the Stanozolol is extracted from the mixture. The second stage involved reduction of the sample’s pH values. The pH of the urine sample was adjusted to 10 and the passed through the cartridge, followed by water. The Ph values of the water used in the analysis are usually nearly neutral. Stanozolol sample is usually basic since it has pyrozole ring; thus, at neutral pH the element is usually slightly stable. Adjusting the pH to 10 will make the Stanozolol slightly basic to increase its stability. The next step involves passing Methanol through the cartridge and evaporating to dryness using nitrogen gas. The residue was then dissolved in acetate buffer at pH of 5.2 and then extracted with diethyl ether containing 5alpha-androstan- 17- one as an internal standard. The ether extract was the evaporated to dryness and the residue derivatised by silylation for the GC-MS. This procedure was carried out as a means of sample purification. It is only with pure samples that the desired results can be obtained. It should be noted from the above step, the sample contains only Stanozolol mixed with some elements and water. Evaporation will help in eradicated all the volatile elements including water from the sample mixture (Behre and Nieschlag, 2004; pg. 771). The acetate buffer extract remaining from step 3 above was exposed to a stream of nitrogen gas to remove any trace of diethyl ether and then a crude beta-D-glucuronidase or sulfatase enzymic prepared from H. pomatia was added. The buffered urinary extract was afterwards incubated for 16 hour at 370C to clean up the sample towards obtaining a clean sample for analysis. The analytical instruments and procedure to be deployed in this analysis is quite sensitive and selective; thus, it is vital to provide only clean and dry sample for analysis that will not interfere with separation in gas phase. Finally, the incubated extract was cooled to 200C and potassium carbonate added and dissolved. The mixture was then extracted with ether containing 5alpha-androstan-17-one as an internal standard. The extracted ether was dried over anhydrous sodium sulfate then filtered and evaporated to dryness. The residue was derivatised (BSTFA) for the GC-MS analysis. This extract was labeled B in the chromatographic results. This method was applied to allow effective ion selection process. It should be note that at high temperatures, the ion selection is usually difficult and inaccurate results may be obtained. This is because numerous ions and elements in the sample are in their excited states. Therefore, cooling of the same and drying the same will reduce excitation and interference of vapor in any chromatograph phase. Stanozolol requires derivatisation for to metabolize successfully. It should be noted that the gas chromatography coupled with fluorescence and UV detectors are usually employed to analyze steroids. Nonetheless, fluorescence coupled with HPLC detections usually involves elaborate preparation steps while the GC-MS needs a complicated derivatisation sample step (International Mass Spectrometry Conference, 1985; pg. 888). This step is usually required since it makes the analytical process feasible and accurate; however, it makes the entire process elaborate and expensive. Additionally, the derivtisation steps may be required in the analysis since they make the sample unstable; thus, susceptible to thermal decomposition. The commonly used derivative for the metabolisms is the male sex hormone or the testosterone. Effective GC – MS analysis usually deploy different chemical and environmental conditions. The temperature required for this analysis is expected to range between 130 °C and 300 °C. Notably, the sample may last differently in any given temperature range depending of the aims of a particular step of analysis. Therefore, the Temperature Ramp for the analysis will appear as in the table below. Other than the chemical and environmental factors, the Stanozolol analysis is also affected by the sensitivity of equipments used and it should be noted that the use of MS compares different in sensitivity as GS full scan analysis. The amount of information collected and fed into the GC usually affects the degree of the sensitivity of the analytical data. However, as the GC peak increases the sensitivity of the analysis usually decreases. For the SIM analysis, the smaller quantities of the analytical sample usually determine the degree of sensitivity; however, the certainty degree in the SIM analysis reduces with the analytical sample size. On the other hand, a full scan MS targets numerous mass fragment range; the larger the scan range, the lower or the reduced the sensitivity ((International Mass Spectrometry Conference, 1985; pg. 982). GC –MS Results The amount of Stanozolol as per diagrams A and B (provided in the lab manual) is relatively the same in both samples. Notably, both samples provide same degree resonance and absorption; therefore, they have nearly the same and equal amount of the element sample. However, depending on the molecules surrounding C-17, this carbon shows different range and degree of resonance or absorption of the UV rays. From the table, it is apparent that di-hydroxylation have high retention levels compared to the mono – hydroxylation. This difference may be pegged on the amount of energy each requires to ionize (International Mass Spectrometry Conference, 1985; pg. 1012). The same results show that the cis- and trans formed about the C- 16 affect the retention time. Notably, the trans has high retention time than cis. Different athletes usually misuse anabolic androgenic steroids including Stanozolol for unfair competition. Nonetheless, different competition tests have currently provided unique challenges in the contemporary anti-doping detection of such drug misuse (Makin and Gower, 2010; pg. 799). Additionally, such detection mechanisms have solved the problems since they currently provide systematic logistic reasoning towards challenging unfair competition in sporting activities. These techniques, though in vivo, have helped in determining the drug levels in the urine or blood of an athlete. Nonetheless, it would be prudent to have sensitive methods that provide quick results of Stanozolol levels in the users’ systems. Such quick results will reduce anxieties among athletes in cases of suspicion. Among the sensitive methods that should be adopted is the urinary metabolite using GC-MS. Reference BEHRE, H. M., & NIESCHLAG, E. (2004). Testosterone: action, deficiency, substitution. Cambridge [u.a.], Cambridge Univ. Press. INTERNATIONAL MASS SPECTROMETRY CONFERENCE. (1985). Advances in mass spectrometry. Chichester, WileyMAKIN, H. L. J., & GOWER, D. B. (2010). Steroid analysis. Dordrecht, Springer. Read More