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.