2.3. Preparation of standards and quality control samples
By dissolving 10 mg of A1 or RA in 100 ml of methanol, the standard stock solution was prepared. Then, individual solutions were diluted with methanol to achieve ideal concentrations and used as standard solutions. We also dissolved and diluted PPD (10.0 mg) in methanol to obtain a stock solution with a concentration of 1.0 mg/ml, which was subsequently diluted to obtain an IS working solution at a concentration of 5.0 μg/ml. These solutions were kept at 4 °C and subsequently exposed to normal temperature prior to usage. One hundred microliters of blank plasma was spiked with 50 μl of appropriate RA and A1 standard solutions to obtain RA or A1 concentrations of 5, 25, 50, 100, 250, 500 and 750 ng/ml, and calibration standards were used every day. In a similar manner, quality control (QC) samples were prepared at low, medium, and high concentrations to obtain samples containing 10, 100, and 500 ng/ml RA and A1, respectively.