Development and validation of a simple reversed-phase high-performance liquid chromatography method for the determination of testosterone in serum of males.
An isocratic high-performance liquid chromatographic method with detection at 234 nm was developed, optimized and validated for the determination of testosterone in human serum. Propylparaben was used as internal standard. A Hypersil BDS RP-C18 column (150 mmx4.6 mm, 5 microm), was equilibrated with a mobile phase composed of acetonitrile and water (35:65, v/v) and having a flow rate of 1 ml/min. The elution time for testosterone and internal standard was approximately 11.6 and 9.9 min, respectively. Calibration curves of testosterone in serum were linear in the concentration range of 1-20 ng/ml. Limits of detection and quantification in serum were 0.4 and 1.1 ng/ml, respectively. Recovery was greater than 92%. Intra- and inter-day relative standard deviation for testosterone in serum was less than 2.1 and 3.9%, respectively. This method was applied to the determination of testosterone serum levels of 12 healthy males and data were correlated with data obtained using a radioimmunoassay method.
Loi
V
V
Department of Endocrinology, Diabetes and Metabolism, Red Cross Hospital, Athens, Greece.
Vertzoni
M
M
Vryonidou
A
A
Phenekos
C
C
D000097:Acetonitriles / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008297:Male; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D013739:Testosterone / Q000097:blood*; D013997:Time Factors; D014867:Water / Q000737:chemistry
D000097:Acetonitriles; D014867:Water; D013739:Testosterone; C032159:acetonitrile