Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 39, Num. 1, 2007, pp. 39-42

Indian Journal of Pharmacology, Vol. 39, No. 1, January-February, 2007, pp. 39-42

Research Paper

Purification of urine samples to improve detection limit of anabolic agents

Reddy MadhusudhanaI, Beotra A, Jain S, Kaur T, Lal Ranjit

Dope Control Centre, Sports Authority of India, J. N. Stadium, New Delhi - 110 003
Correspondence Address:Dope Control Centre, Sports Authority of India, J. N. Stadium, New Delhi - 110 003, drabeotra@rediffmail.com

Date of Submission: 31-Mar-2006
Date of Decision: 08-Aug-2006
Date of Acceptance: 30-Aug-2006

Code Number: ph07009

Abstract

Keywords: Anabolic steroid, detection limit, mass spectrometry, SPE column, urine test, XAD2 extraction.

Introduction

Materials and Methods

Reference standards and chemicals

The reference standards of clostebol and clenbuterol were purchased from Sigma-Aldrich (St Louis, MO, USA) and of nandrolone, stanozolol, epimetendiol from National Analytical Research Laboratory (National Measurement Institute, Sydney, Australia). The derivatizing reagent N-methyl-N-Trimethysilyltrifluoroacetamide (MSTFA), iodo-TMS and Dithioerythritol were purchased from Sigma-Aldrich (St Louis, MO, USA). The hydrolyzing enzyme b-glucuronidase, E. coli is purchased from Roche, Germany. All other reagents and chemicals such as methanol, diethyl ether, and anhydrous sodium sulphate were of analytical grade or HPLC grade.

Two types of solid phase extraction columns were used. The Amberlite XAD2 was purchased from Sigma-Aldrich (St Louis, MO, USA) and C-18 Samprep; 100 mg/3 mL from Rankem, India was purchased.

Extraction procedure

The samples were processed by XAD2 extraction[12] and SPE column.

XAD2 extraction: About 2 mL of centrifuged urine was passed into pre-prepared XAD2 column and 50 mL of clostebol (conc. 2.5 ng/mL) was added as the internal standard. The column was eluted with 2.5 mL of methanol and the eluent dried under nitrogen evaporator at 60°C. To this was added 1 mL of 0.2 M-phosphate buffer pH-7.0 and 50 µL of b-glucuronidase ( E. coli ) and the mixture was incubated at 60°C for 60 minutes. The hydrolysate was alkalinized by adding 250 µL of 7% potassium carbonate solution to adjust the pH of the mixture between 9 and 10. About 5 mL of distilled diethyl ether was added and mixed by moderate horizontal mixing in the shaker for 10 minutes. The ether layer was transferred to a labeled, glass test tube and the ether evaporated at 60°C under the nitrogen evaporator. After evaporation of diethyl ether the tube was placed for 10-15 minutes in a dessicator containing P ₂ O ₅, to remove traces of moisture if any.

SPE extraction: SPE columns were conditioned with 2 mL of methanol and equal amount of distilled water. The procedure followed was similar to that of XAD2 extraction.

Derivatization procedure

The dried residue was dissolved in 50 µL of MSTFA/iodo-TMS/dithioerythritol (1000/2/2:v/v/w) mixture. The mixture was incubated at 60°C for 15 min and then transferred into 200 µL conical glass vials. Nearly 2 mL was injected into HRMS for analysis.

Instrumentation and conditions

HRMS was used for evaluation of concentration of the samples. The parameters of gas chromatograph [Table - 1] and Mass Spectrometer [Table - 2] are mentioned.

Results

Limit of detection (LOD)

Limit of detection was defined as the lowest level at which a compound could be identified in urine samples, with diagnostic ions present with a signal to noise (S/N) ratio greater than 3. A single diagnostic ion 405.2640, 546.3493, 335.0690, and 448.3193 was used to determine the LOD of nandrolone, stanozolol, clenbuterol, and epimetendiol in urine sample, respectively, by HRMS. The LODs of clenbuterol and epimetendiol as required by WADA is 2 ng/mL.[2]

Recovery, accuracy, and precision

The preliminary work was done with six spiked urine samples of each of clenbuterol, nandrolone, and epimetendiol at 1, 2, 5, and 10 ng/mL and stanozolol at 5, 10, 20, and 40 ng/mL and were analyzed with HRMS. The results showed that for nandrolone and stanozolol the drug recovery at various concentrations (upto 10 ng/mL) was not different between two columns [Table - 3],[Table - 4]. At 20 and 40 ng/mL, recovery of stanozolol was significantly more ( P < 0.00) with XAD2 columns.

In a later study the extraction accuracy of the sample preparation procedure with three concentrations of clenbuterol and epimetendiol was undertaken ( n =10) and processed with XAD2 and SPE columns.

Clenbuterol . The percentage recovery was 30.45, 46.63, and 41.60% with XAD2 and 72.09, 72.16, and 89.24% with SPE columns [Table - 5]. The recovery percentage was significantly higher with SPE column extraction. The significance levels were P < 0.002 (2 ng/mL), P < 0.001 (5 and 10 ng/mL).

Epimetendiol . The recovery percentage for 2, 5, and 10 ng/mL with XAD2 were 105, 69.69, 66.63, and 136.87, 104.69, and 99.65% with SPE [Table - 5]. The recovery percentage was significantly higher with SPE column extraction. The significance level were, P < 0.015 (2 ng/mL), P < 0.010 (5 ng/mL) and P < 0.014 (10 ng/mL).

The recovery of clenbuterol and epimetendiol at 2, 5, and 10 ng/mL were calculated individually for XAD2 and SPE columns [Table - 6]. The total ion chromatogram of clenbuterol and epimetendiol with both XAD2 and SPE columns is shown in [Figure - 1],[Figure - 2].

Discussion

The results of the present study reveal that the extraction procedure for nandrolone involving XAD2 column, is good and there is no additional advantage using SPE column. With both types of columns nandrolone recovery was comparable. However, there is no available literature on comparison of these two types of columns for nandrolone recovery, though both types of columns have been studied separately.[4],[5]

The recovery of stanozolol at 5 and 10 ng/mL was comparable in both types of columns, whereas recovery was significantly more ( P < 0.000) at 20 and 40 ng/mL with XAD2 extraction. However, improvement in recovery of Stanozolol at higher concentration (20 and 40 ng/mL) is not of much value because it is required to improve detection at lower concentration (2 and 5 ng/mL). The purification techniques for Stanozolol reported are Immunoaffinity chromatography (IAC)[9] and use of Oasis cartridges.

There was a significant increase in the recovery of clenbuterol and epimetendiol at different concentrations, which necessitates the need to do confirmation testing with C 18 SPE columns. Though use of C-18 Sep-pak columns for extraction of clenbuterol has been reported.[13] There is no study comparing recovery percentage of two types of extraction. Further use of SPE columns for epimetendiol extraction has not been reported in literature so far. The purification techniques used for epimetendiol are based on HPLC clean up[6] and IAC gels.[6]

Conclusion

References

Copyright 2007 - Indian Journal of Pharmacology

The following images related to this document are available:

Photo images