Metabolic Detection of Stanozolol Using GC MS - Essay Example

Metabolic Detection of Stanozolol Using GC – MS Metabolic Detection of Stanozolol Using GC – MS Properties of the Stanozolol that make it suitably analysis with the GC analytical method as opposed to the HPLC method The same suitability is mainly pegged on the properties of Stanozolol. It should be noted that Stan is an anabolic – androgenic steroid that is 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. 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 inferior to other steroids. Additionally, in the GC – GS, 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. Form the workshop values; the UV was obtained at 20mg/ml. The sample preparation stages (using solid Phase Extraction (SPE) Cartridge) 1. Rinsing the ODS (C18) SPE with MeOH followed with water This procedure is taken to dissolve the residues. In this sense, the Stanozolol was being extracted from the mixture. 2. 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 solution, the Stanozolol slightly basic to increase its stability. 3. Methanol passed through the cartridge, collected, and evaporated to dryness using nitrogen gas. The residue 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 evaporated to dryness and the residue derivatised by silylation for the GC-MS. This procedure was carried out as 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. 4. The acetate buffer extract remaining from step 3 above is exposed to a stream of nitrogen gas to remove any trace of diethyl ether and then a crude beta-D-glucuronidase or sulfatase enzymic preparation from H. pomatia was added. The buffered urinary extract was then incubated for 16 hour at 370C This is still a cleanup procedure 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 only provide clean and dry sample for analysis. 5. 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 usually becomes 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. Why is the required derivatisation for Stanozolol and its metabolites and to which group or groups is the derivatised required in the Stanozolol? 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. 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 unstable; thus, susceptible to thermal decomposition. The commonly used derivative for the metabolisms is the male sex hormone or the testosterone. Part C: The GC – MS Conditions C1. The temperature required for this analysis ranges between 130 °C and 300 °C. Notably, the sample may last within a given temperature range depending of the aims of a particular step of analysis. C2. Temperature Ramp for the analysis C3. The sensitivity of MS compared to the sensitivity of 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. D. GC –MS Results D1. the amount of Stanozolol as per diagrams A and B is relatively the same in both sample. Notably, both samples provide same degree resonance and absorption; therefore, they have nearly the same and equal amount of the element sample. D2. 1. Depending on the molecules surrounding C-17, this carbon shows different range and degree of resonance or absorption of the UV rays. 2. 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. 3. The cis- and trans formed about the C- 16 affect the retention time. Notably, the trans has high retention time than cis. Summary Different athletes for unfair competition usually misuse anabolic androgenic steroids including Stanozolol. 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. Nonetheless, sensitive methods that provide quick results for Stanozolol levels in the users should be adopted to reduce anxieties among athletes in cases of suspicion. Among the sensitive methods that should be adopted is the urinary metabolite using GC-MS. Reference MAKIN, H. L. J., & GOWER, D. B. (2010). Steroid analysis. Dordrecht, Springer. Read More