Analysis of Favipiravir in Human Plasma Using Fully Automated Sample Preparation LC/MS/MS System

Analytical Conditions and Pretreatment of Samples

Favipiravir (PN: C8720^*1), as the target compound, and[13C,15N]-favipiravir (PN: C8853^*1), as its stable isotope, were purchased from Alsachim, one of the companies of the Shimadzu Group. [¹³C,¹⁵N]-favipiravir was used as the internal standard (ISTD). Favipiravir was spiked with commercially available human plasma treated with EDTA 2K to prepare calibration curves and QC samples. Analysis was performed using the LC and MS analytical conditions shown in Table 1 and the MRM transition in Table 2. Shimpack Scepter^™ C18-120 (50 mm×2.1 mm I.D., 1.9 µm, P/N: 227-31012-03) was used as the analytical column. Fig. 3 shows the MS chromatograms and structural formulas of the compounds. A calibration curve was prepared using calibration points at plasma concentrations of 1, 2, 5, 10, 20, 50 and 100 µg/mL for favipiravir (n = 5 for each calibration point). [ ¹³C,¹⁵N]- favipiravir (20 µg/mL) solution was prepared using acetonitrile and used as ISTD. The pretreatment steps for the plasma sample spiked with favipiravir are shown in Fig. 2. Samples were automatically prepared through the following series of steps: mixing 20 μL of 75% isopropyl alcohol (IPA), 50 μL of plasma, 10 μL of ISTD and 200 μL of acetonitrile, shaking the mixture, and then filtration of the mixture using a PTFE membrane filter. Finally, the prepared sample was used for analysis. ^*1 Shimadzu GLC and Alsachim Product numbers

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