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Indian Journal of Pharmacology, Vol. 40, No. 4, July-August, 2008, pp. 164-169 Research Article Sample purification procedure for confirmation of 3'-hydroxy stanozolol by gas chromatography/high resolution mass spectrometry Ahi S, Reddy IM, Beotra A, Lal R, Jain S, Kaur T Dope Control Center, Sports Authority of India, J. N. Stadium, New Delhi Date of Submission: 07-Aug-2007 Code Number: ph08051 Abstract Objective: To improve the detection limit of 3'-hydroxy-stanozolol by using the double extraction procedure, specific for basic drugs.
Keywords: 3′-hydroxy-stanozolol, gas chromatography/high resolution mass spectrometry, purification, World Anti Doping Agency Introduction Since 1967, the use of performance enhancing drugs is banned in sports. Anabolic Steroids (AAS) have been included in the list banned by the International Olympic Committee (IOC) in 1976. Since the year 2002, the World Anti Doping Agency (WADA) has taken over and made more stringent guidelines for the testing of drugs by doping labs. [1] Stanozolol (17α-methyl-17β-hydroxy-5a-androstane- [3, 2, C] pyrazole), first synthesized in 1959, [2] is one of the most misused anabolic steroids in sports. [3] Stanozolol is metabolized to a large extent and the main metabolic product in urine is mono hydroxylated, 3′-hydroxy-stanozolol. Stanozolol and its metabolites are different from most anabolic steroids and are particularly difficult to detect in urine. These compounds have a poor gas chromatographic behavior and the measured concentrations are generally very low. In order to ensure that all doping control laboratories can detect the presence of prohibited substances uniformly, WADA has established a minimum detection capability for testing methods called minimum required performance limits (MRPL). The MRPL for anabolic steroids is 10 ng/mL, except clenbuterol, nandrolone, stanozolol, methyl testosterone and methandione, for which the detection limit is 2 ng/mL. In view of the above, it is very crucial to strengthen the testing protocol of the lab to improve the detection limit of these five drugs. We have already reported improvement in the detection limit of clenbuterol and epimetendiol with SPE cartridges. [4] The identification of stanozolol by various extraction procedures has been shown in literature. [5],[6] The sample preparation procedures vary from Solid phase extraction and Liquid-liquid extraction [7] to Immunoaffinity chromatography. [8] However, irrespective of the methods of sample preparation, the identification of stanozolol and its metabolites has proved to be problematic, particularly in cases of low concentrations, due to interfering matrix peak. Hence, it is required to improve sample extraction procedure, which would ensure removal of interfering matrix, thereby allowing the WADA criteria of confirmation by ion match to be fulfilled. The objective of the present paper is to develop a confirmation procedure for Stanozolol by comparing its efficacy with the existing procedure. Materials and Methods Reference standards, chemicals and reagents Instrumentation and conditions Mass spectrometric analyses were conducted using high-resolution mass spectrometer (JEOL JMS-700, Tokyo, Japan). The main working parameters of the mass spectrometer are summarized in [Table - 2]. The gas chromatographic system and high-resolution mass spectrometer were both controlled using JMS-700 M Station software (JEOL JMS-700, Tokyo, Japan). Sample extraction procedure Extraction procedure 1: Extraction procedure 1 is the existing procedure, which is being used in our lab for testing of anabolic androgenic steroids. [9],[10] Two/four mL of urine, based on the specific gravity, is applied on the pre-prepared XAD2 ® columns. The columns are washed with two mL of water to eliminate most of the water-soluble urinary constituents, which have not been absorbed on the solid support. The free and conjugated steroids are then eluted with 2.5 mL of methanol. The eluted effluent is evaporated under nitrogen stream at 60°C and the residue is dissolved in one mL of phosphate buffer (pH 7.0). Hydrolysis is performed with 50 μL of β-glucuronidase enzyme for one hour, at 60°C. The sample is alkalized by adding K 2 CO 3 and is extracted with 5 mL of distilled diethyl ether. After centrifugation, the organic phase is separated and dried with Na 2 SO 4 , 50 μL of internal standard 17α-methyl testosterone (Conc. 500 ng/mL) is added. It is then evaporated under Nitrogen stream at 60°C. Extraction procedure 2: The first step of elution of drug from XAD2 ® resin and the hydrolysis step is similar to that of the Procedure 1. Two/four mL of urine, based on the specific gravity, is applied on the pre-prepared XAD2 ® columns. The columns are washed and the drug is eluted with 2.5 mL of methanol. The eluted effluent is evaporated and hydrolysis is performed with 50 μL of β-glucuronidase enzyme for one hour, at 60°C. 10 μL of 5N HCl is added to hydrolysate to make it acidic. Oasis MCX cartridges are conditioned with 2 mL of methanol and water. The acidified effluent is applied on the Oasis MCX cartridges and washed with 2 mL HCl and methanol. The basic drug, 3′-hydroxy stanozolol is eluted with 2 mL methanol containing 1% ammonium hydroxide. Internal standard is added and evaporated as mentioned in Procedure 1. The dried residue is again re-dissolved in 200 μL of methanol and dried again followed by derivatization. Derivatization Results The identification criterion of stanozolol was as per the fragmentation ions 560.3650, 546.3493 and 545.3410. The detection of 3′-hydroxy stanozolol with Extraction Procedure 2 [Figure - 1] on the GC/HRMS showed improved recovery, as compared to Extraction procedure 1 [Figrue 2]. Hence, Extraction Procedure 2 was validated as per the WADA International Standard of Laboratories (ISL) [11] requirements. Method validation Calibration curve and linearity Limit of detection (LOD), signal to noise ratio Selectivity Precision and accuracy Total recovery The extraction recovery percentages were found to be within the limits and were acceptable. Internal standard recovery was also found to be good and agreeable, as it was consistent, precise and reproducible. Ion match criteria Doping control samples Discussion The objective of the present study was to improve extraction procedure of 3′-hydroxy stanozolol to achieve good recovery percentage and to achieve acceptable ion matching criteria laid down in WADA International Standard of Laboratories. The separation of drug was performed on gas chromatography and detection was done by high-resolution mass spectrometer. The lowest possible detection of 3′-hydroxy stanozolol is 1 ng/mL on GC/HRMS and was achieved with the present method. The total recovery percentages of 3′-hydroxy stanozolol were found to range from 53 to 71% with Extraction Procedure 1 and 56 to 97% with Extraction Procedure 2. The extraction recovery percentages were found to be within the limits and agreeable. Internal standard recovery is also found good and acceptable, as it was consistent, precise and reproducible. The first extraction procedure concentrates the drug in polystyrene divinyl benzene resin column and further extracts it through liquid-liquid extraction. However, the second Extraction Procedure involves the use of solid phase extraction with oasis cartridges. The elution of the drug was done with the alkaline organic solvent, which shows extraction recovery between 56 and 97%. The recovery percentages of the drug from both the extraction procedures were found to be in conformity at concentrations 5 and 10 ng/mL. However, recovery of drug at 1 and 2 ng/mL showed significant improvement by extraction Procedure 2. To declare a positive finding, it is required to detect a drug in athlete′s urine and also to fulfill ion match criteria wherein three characteristic ions of presumptive positive sample should match with reference standards. [11] The ion match criterion was not being fulfilled with the first extraction procedure at lower concentrations, which was the reason for planning the present work. However, the analysis of data revealed that the peak height and area for quantitation of ion 560.3650 was good with Extraction Procedure 1 but relative peak area and heights of 545.3410 and 546.3493 ions did not show conformance to WADA requirements, [11] which may be due to matrix interferences. The ion match criteria with Extraction Procedure 1 and 2 is shown in [Table - 4] and [Table - 5], which clearly shows removal of matrix interferences by Extraction Procedure 2, thereby fulfilling WADA criteria of ion match. This is essentially required to make it legally strong, which is required for doping labs. Conclusion The present study could successfully achieve detection, good percentage recovery and fulfillment of ion matching criteria of 3′-hydroxy stanozolol at MRPL as specified by WADA, using the second Extraction Procedure protocol, with detection level as low as 1 ng/mL. Excellent detection limits enable the long-term detection of stanozolol misuse in doping control. This procedure may be used for the confirmation of suspicious samples found in routine testing.References
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