4.5. Preparation of Standard Solution, Calibration Standards, and Quality Control Samples

Each standard stock solution of T4C, T3C, T2C, C4C, TCT, PIC, and SAF (1.0 mg/mL) with IS1and IS2 (1.0 mg/mL) was prepared separately in the methanol to prepare calibration standards and quality control samples. The primary stock solutions were made up of methanol to prepare mixed working solutions (WSa). All the standard stock solutions were stored at 4 °C. The calibration curve (CC) stock solutions (WSa) were diluted with methanol to prepared working solutions with a range of 100 to 32,000 ng/mL (WSb). The stock solutions of IS1 and IS2 were diluted to prepare 0.1 mg/mL (WSc).

The blank plasma samples of volume 45 µL were spiked with each of 5 µL standards (WSa) and IS (WSc) solutions, respectively. With further 200 µL, methanol was added and vortex for 2 min, followed by centrifugation for 10 min at 2000 RPM. The supernatant was collected in HPLC vials for analysis. A nine-point CC (10, 30, 100, 200, 400, 800, 1600, 2400, and 3200 ng/mL) was prepared for analysis. Quality control samples were prepared separately by spiking respective working standard solutions to achieved LLOQ (10 ng/mL), LOQ (30 ng/mL), MOQ (1600 ng/mL), and HOQ (2400 ng/mL).

An aliquot of 45 µL of rat plasma was spiked with each 5 µL of IS1 and IS2, and 200 µL of methanol was added, vortexed for 1–2 min, then centrifuged for 10 min at 2000 RPM. Collect the supernatant into HPLC vials and injected it into the UFLC-MS/MS system for analysis.

The linearity of T4C, T3C, T2C, C4C TCT, PIC, and SAF over nine points (10, 30, 100, 200, 400, 800, 1600, 2400, and 3200 ng/mL) were prepared by spiking of 45 µL drug-free rat plasma with the appropriate amount of analyte and IS1 and IS2. The linearity was assessed with three different calibration curves with maintaining the standard internal concentration at 10 ng/mL. A nine-point CC was set up by plotting T4C, T3C, T2C, C4C, TCT, PIC, and SAF peak area ratio against the control matrix’s nominal concentration of calibration standards. Calculation of linear regression data with 1/X² weighting factor.