Development and method validation for testosterone and epitestosterone in human urine samples by liquid...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Endocrinology

Background:

• Accurate measurement of testosterone (T) and epitestosterone (ET) is crucial for clinical diagnostics and doping control.
• Existing methods for T and ET determination in urine often involve complex extraction procedures.
• Development of a robust and efficient chromatographic method is needed to simplify T and ET analysis.

Purpose of the Study:

• To develop and validate an isocratic high-performance liquid chromatographic (HPLC) method for quantifying testosterone (T) and epitestosterone (ET) in human urine.
• To compare the efficacy of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) for sample preparation.
• To establish the method's suitability for evaluating urinary T, ET, and the T/ET ratio in a healthy male population.

Main Methods:

• Isocratic high-performance liquid chromatography (HPLC) with a Hypersil C18 column.
• Mobile phase: water-acetonitrile (57:43, v/v).
• Detection: UV absorbance at 245 nm.
• Sample preparation: Liquid-liquid extraction (LLE) or solid-phase extraction (SPE).

Main Results:

• The HPLC method achieved optimal separation of T and ET.
• Recoveries for T and ET were comparable between LLE and SPE, indicating procedural interchangeability.
• Quantitation limits were 8.6/5.4 ng/mL (LLE) and 7.3/5.7 ng/mL (SPE) for T/ET, respectively.
• The method was successfully applied to assess urinary T, ET, and T/ET ratios in healthy males.

Conclusions:

• A validated isocratic HPLC method provides reliable determination of urinary testosterone and epitestosterone.
• Both LLE and SPE are effective and interchangeable sample preparation techniques for this assay.
• The developed method is suitable for epidemiological studies and clinical monitoring of androgen status.