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Application Notes: Anabolic Steroids
General
Recently, the anabolic steroids have come into public prominence. These steroids are used both in meat production and sport. In human sport, they are sometimes used in large quantities for increasing the body bulk of athletes. In horse racing they are used to improve a horse's appetite and produce behavioral changes of the masculinizing type. Because of their long term deleterious effects on physiologic processes and unfair use in sport, control agencies have begun to assess their use.
Testesterone is the principal hormone of the testes. In humans the major metabolites are androstanediol, androsterone, and androstenedione. In horses the major metabolite is androstanediol which is excreted in the urine as the glucuronide or sulfate conjugate.
Analysis of the conjugated steroids requires hydrolysis of the sample. An enzymatic and an acid hydrolysis procedure are given. There is evidence that the enzymatic procedure will hydrolize only the glucuronide. The sulfate conjugates require the acid hydrolysis.
The procedure described here may be used for the total or conjugated steroid content of urine, plasma, or amniotic fluid. Total steroid content is obtained by hydrolyzing the sample. Free steroid content can be determined by extraction of the unhydrolized.sample. The conjugated portion is then the difference between the hydrolized and unhydrolized amounts.
Sample Preparation
A. Buffer Preparation
1. pH 5.0 acetate buffer - 14.8 ml 0.2 M acetic acid plus 35.2 ml 0.2 M sodium acetate diluted to 100 ml with water.
B. Hydrolysis Procedures
1. Acid hydrolysis - Add 0.75 ml HCl per 5 ml sample (urine, plasma, amniotic fluid ) in a screw capped test tube. Screw the tube top on loosely and heat in a boiling water bath for 60 minutes. Adjust to pH 7 with 1.0 N NaOH.
2. Enzymatic hydrolysis - Adjust 10 ml urine to pH 4.5 with HCl. Add 1 ml acetate buffer (pH 5) containing 1000 units glucuronidase. Incubate at 37°C for 24 hours. (NOTE: An expedited procedure is to heat at 55°C for 60 minutes.)
C. Extraction Procedure
1. Condition a SPICE C18 cartridge in sequence with: 2 ml water / acetone (8 : 2), 2 ml methanol, 2 ml water. Allow each solvent to pass through before adding the next.
2. Add the sample from the hydrolysis procedure ( or unhydrolyzed sample ) to the cartridge. Allow to completely flow through under vacuum, and discard the eluent.
3. Wash the cartridge in sequence with: 2 ml water, 2 ml water / acetone (8 : 2). Discard all eluents.
4. Elute the steroids with 1 ml methanol. Collect the eluent, evaporate, and reconstitute in 100 µl methanol.
D. Recovery
1. Recovery of androsterone and androstenedione from SPICE sample extraction of urine spiked with radiolabeled steroid was 99% and 72%, respectively.
Thin Layer Chromatography
A. Separation
1. Apply 10 and 20 µl of the reconstituted sample to an Analtech silica gel G layer. Apply standard androstanediol ( 1 µg / µl methanol ) to the reference lane.
2. Develop the chromatogram in a mobile phase of isopropyl ether / acetone (8 : 2).
B. Visualization
1. Examine the plates under UV254. Most of the steroids quench at the short UV wavelength.
2. Spray with 10% sulfuric acid in ethanol. Heat at 150°C for 2 - 3 minutes.
3. Examine the plate under UV366. The sulfuric acid spray gives fluorescent spots with colors leading to some specificity.
| Compound | Rf |
|---|---|
| dihydroepiandrosterone | 0.59 |
| androstenedione | 0.55 |
| epitestosterone | 0.48 |
| testosterone | 0.47 |
| androsterone | 0.35 |
Quantitation
Below is a scan of some androgens for fluorescence at 366nm. The mobile phase for this separation was isopropyl ether / acetone (95 : 5).
