A TLC plate was cut to approximately 7 cm by 10 cm. A 10 µl sample of the each solutions prepared for the calibration curve was loaded onto the first 4 lanes of the TLC plate. A 10 µl plasma extract was applied to the 1X lane. For the 3X lane, three 10 µl samples were sequentially loaded, allowing each to dry before the next. Loading three samples in this sequential manner increased the signal which was important for low concentration samples. Loading more than 3 spots caused observed distortions to the bands that could interfere with quantification.