A novel stacking method of repetitive large volume sample injection and sweeping MEKC for determination of androgenic steroids in urine.

In this research, a novel stacking capillary electrophoresis method, repetitive large volume sample injection and sweeping MEKC (rLVSI-sweeping MEKC) were developed to analyze the presence of three androgenic steroids considered as sport doping drugs, testosterone (T), epitestosterone (E) and epitestosterone glucuronide (EG) in urine. This method provides better sensitivity enhancement than the traditional large volume sample stacking-sweeping strategies due to sensitivity enhancement by repetitive injections. This multiple sampling method enhances sensitivity of monitoring of urine samples by UV detection (254 nm). Firstly, the phosphate buffer was filled into an uncoated fused silica capillary and the samples were injected into the capillary at 10 psi for 20s, and then stacked at -10 kV for 1 min using phosphate buffer containing SDS. The above injecting and stacking steps were repeated five times. Finally, separation was performed at -20 kV, using phosphate buffer containing methanol, SDS and (2-hydroxypropyl)-β-cyclodextrin. Method validation showed that calibration plots were linear (r≥0.997) over a range of 5-200 ng mL(-1) for T, 20-200 ng mL(-1) for E and 0.5-500 ng mL(-1) for EG. The limits of detection were 1.0 ng mL(-1) for T, 5.0 ng mL(-1) for E and 200.0 pg mL(-1) for EG. When evaluating precision and accuracy, values of RSD and RE in intra-day (n=3) and inter-day (n=5) analysis were found to be less than 10.0%. Compared with the simple LVSS-sweeping, which is also a stacking strategy, this method further improves sensitivity up to 25 folds (~2500 folds with MEKC without preconcentration). This method was applied to monitor 10 athletes' urine, and did not detect any analyte. The novel stacking method was feasible for monitoring of doping by sportsmen.