Simple Determination of 17a-Ethynylestradiol in Hair Restorer by High-Performance Liquid Chromatography Coupled with Fluorescence Detection After Pre-Column Derivatization 4-(N-Chloroformylmethyl-N Methylamino)-7-Nitro-2,1,3-Benzoxadiazole

The concentration of 17a-Ethynylestradiol (EE2) in hair restorer was analyzed by High-Performance Liquid Chromatography (HPLC) coupled with fluorescence detection (excitation wavelength, 470 nm; emission wavelength, 540 nm) after pre-column derivatization with 4-(N-chloroformylmethyl-Nmethylamino)- 7-nitro-2,1,3-benzoxadiazole (NBD-COCl). Derivatization with NBD-COCl was performed in borate buffer (pH 9.0) at room temperature for 3 min. The HPLC column was 150 mm×3.0 mm i.d., containing 5 μm particles of C22 packing material, and the mobile phase was prepared by addition of acetonitrile (580 mL) to 420 mL of Milli-Q water containing trifluoroacetic acid (0.1 v/v%). The sample was eluted from the column at room temperature at a flow rate of 0.5 mL/min, and the retention time of NBD-CO-EE2 was 10.7 min. The calibration plot was linear in the range of 0.05 to 1 μg/mL with an r2 value of 0.9952, and the lower limit of detection was 0.010 μg/mL (at a signal-to-noise ratio of 3:1, absolute amount of 0.050 ng/20 μL injection). The coefficient of variation was less than 9.5%. None of three tested estrogens (estriol, β-estradiol, estrone) interfered with the NBD-CO-EE2 peak. It was found that the content of EE2 in hair restorer was 14.3±0.9 μg/mL (range, 13.3 to 15.9 μg/mL). Recovery in addition-recovery tests was within the range of 70.5 to 98.4%.