Determination of epitestosterone in human urine by off-line immunoaffinity solid-phase extraction coupled with high performance liquid chromatography.
Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.
Xu
Li
L
Beijing National Laboratory for Molecular Sciences, BNLMS, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry, Peking University, Beijing 100871, China.
Qiu
Shuang
S
Sun
Cui-Jin
CJ
Deng
Qin-Pei
QP
Chen
Hong-Xu
HX
Zhou
Ying-Lin
YL
Zhang
Xin-Xiang
XX
D000328:Adult; D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004845:Epitestosterone / Q000302:isolation & purification / Q000652:urine; D006801:Humans; D007163:Immunosorbent Techniques; D007231:Infant, Newborn; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*
D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D004845:Epitestosterone