The quantitative determination of TH was performed
by HPLC. The HPLC method used was newly developed.
The mobile phase was methanol-water-acetic
acid-triethylamine (90:10:0.05: 0.14, v/v/v/v) at a flow
rate of 1.0 mL/min. The HPLC method was done
using a Shimadzu LC-20A HPLC system, which consisted
of a binary gradient pump (model LC-20A), a
SPD-20MA diode array detector at 283 nm, a SiL-20A
auto sampler, a DGU-20A3 degasser and CTO-20A
column oven. The apparatus was interfaced to a Dell
PC compatible computer using LC solution software.
Twenty μL of injection volume was eluted in a reverse
phase column (Waters-ODS C18, 5 μm, 4.6 × 150 mm).
TH peaks were separated with a retention time of 6.5
min. The area under the peak was used to calculate
the concentration of TH. The method was validated in
terms of specificity, linearity and reproducibility. The
result showed linearity over the concentration range
of 0.1-100 μg/mL (R2 = 0.9999). The intra- and interday
variations were less than 1.5%.