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Optimization of Analytical Procedure for In-hospital Rapid Quantification of Serum Level of Favipiravir in the Pharmacological Treatment of COVID-19

An in-hospital rapid method for quantification of the serum level of favipiravir (FPV) in the pharmacological treatment of COVID-19 was developed by appropriate combination of a solid-phase extraction treatment and a reversed-phase HPLC/UV detection system. The quantification method was well-validated and applied to the measurement of serum FPV level in a clinical practice at a general hospital that accepts COVID-19 patients. Furthermore, analysis of data from our preliminary interaction analysis revealed, for the first time, that FPV selectively forms complexes with ferric (Fe 3+ ) and cupric (Cu 2+ ) ions.


Introduction
The current pandemic of coronavirus disease 2019 (COVID-19) has spread rapidly worldwide. Several drugs are currently being tested in clinical trials for their efficacy and safety in the treatment of COVID-19. An orally administered agent (tablet) that contains favipiravir (FPV) as the active substance is one of the candidate drugs for the treatment of COVID-19 in Japan and various countries. [1][2][3] FPV is an RNA-dependent RNA polymerase inhibitor that has been approved in Japan as a treatment for a new type of influenza. 1 FPV exhibits non-linear pharmacokinetics, and a blood level of FPV (i.e., a FPV concentration in plasma or serum) following the first and second doses of a tablet containing FPV increases considerably before gradually decreasing with time. 4,5 FPV inhibits replication by the target virus depending on FPV's plasma concentration 5 , and the dosage of FPV varies depending on the disease state of the patient. Furthermore, FPV has been reported to interact with other medicinal drugs. 6 Therefore, to provide pharmacological treatment for COVID-19 by the administration of FPV, therapeutic monitoring of the blood level of FPV is very important, and a rapid and accurate analytical method for quantification of FPV in human plasma (or serum) is urgently needed.
In this report, we propose a simple and rapid method for quantification of FPV in serum by an appropriate combination of a solid-phase extraction (SPE) treatment and a reversed-phase (RP) HPLC/UV detection system. The procedure of the method was well-optimized and simplified, and then applied for use by medical staff (such as a clinical laboratory technician or a pharmacist) in a general hospital that accepts COVID-19 patients.

UV-VIS spectroscopic analysis
A stock solution (1 mg mL -1 ) of FPV was prepared by dissolving 1 mg of FPV in 1 mL of water. A test solution of FPV (20 μg mL -1 ) for UV-VIS spectroscopy was prepared by diluting the stock solution 50-fold with an aqueous solution (0.1 % phosphate buffer, pH 2 or pH 7) containing 5 v/v% CH3CN. A UV-VIS absorption spectrum of the test solution was recorded on a UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan) in the range 200 to 450 nm.

Preparation of FPV test solutions for SPE and HPLC analyses
For use in optimization of SPE and HPLC conditions, a FPV test solution (50 μg mL -1 ) was prepared by diluting the stock solution (1 mg mL -1 ) 20-fold with an arbitrary aqueous solution or a normal human serum. In addition, solutions of FPV-spiked serum (100 μg mL -1 ; for preparing a calibration curve) and of three other concentrations of FPV-spiked sera (5, 20, and 80 μg mL -1 ; QC samples for validation of the present quantification method) were prepared by diluting the stock solution into normal human serum.

SPE cartridge and optimized protocol
A monolithic C18-silica disk built-in centrifugal spin-cartridge, MonoSpin C18 (GL Sciences, Inc., Tokyo, Japan), was chosen for the SPE treatment prior to HPLC analysis of FPV. The MonoSpin cartridge was pretreated by sequential passage of 500 μL each of CH3CN and water before use. Each solution was passed through the cartridge by centrifugation at 5,000 rpm (2,300 × g) for 1 min using a Centrifuge 5415R (Eppendorf, Hamburg, Germany). Detail of the SPE procedure using the pretreated cartridge were as follows. First, an aliquot (6 μL) of 5 M HCl was added to a FPV test solution or FPV spiked serum (150 μL) in a microfuge tube. After vortex mixing for 5 sec, one-third of the volume (52 μL) of the mixture was loaded onto the MonoSpin cartridge. Next, aliquots of water (500 μL; wash solution), and aqueous 10% CH3CN (150 μL; eluting solution for FPV) were sequentially passed through the cartridge, and the final eluate containing FPV was collected in a test tube for subsequent HPLC analysis.

HPLC apparatus and optimized conditions
All HPLC experiments were carried out with a Chromaster system (Hitachi High-Tech Science Corp., Tokyo, Japan) equipped with a 5310-column oven, a 5210 autosampler, a 5110 pump, and a 5430 diode-array detector. RP HPLC separation of FPV was performed at 30 °C on a Chromolith HighResolution RP-18 column (100 × 4.6 mm I. D., Merck, Darmstadt, Germany) equipped with a guard column (5 × 4.6 mm I. D.). An analyte containing FPV (injection volume: 20 μL) was eluted with a mobile-phase solvent (0.1% phosphoric acid and CH3CN, 95:5, v/v) at a flow rate of 2 mL/min, and FPV in the eluate was detected by UV absorption at 325 nm.

Validation method
A one-point calibration curve for HPLC quantitation of the serum level of FPV was prepared from the HPLC data of the SPE eluate of the FPV-spiked serum (100 μg mL -1 ) without use of an internal standard (I.S.) compound. The limit of detection (LOD) and the limit of quantitation (LOQ) of FPV were estimated from the chromatogram of the SPE eluate of the FPV-spiked serum (5 μg mL -1 ) at a signal-to-noise ratio 3:1 and 10:1, respectively.
An intra-day study (repeatability) was performed by analyzing the QC samples (serum spiked with FPV at 5, 20, and 80 μg mL -1 ) 4 times during the same day, while an inter-day study (intermediate precision) was performed by analyzing the QC samples once-per-day on 3 separate days. Accuracy was reported by calculating the bias, expressed as ((measured concentration)/(nominal concentration)) ×100 %; precision was reported as the coefficient of variation (CV), expressed as (SD/mean of measured concentration) ×100 %. Specificity was measured by analyzing blank serum and comparing its chromatograms with the corresponding FPV-spiked serum samples.

Medical application
To evaluate the applicability of the present method, human serum samples from four different patients with COVID-19 who had been treated with FPV were obtained from Hokushin General Hospital (Nagano, Japan). The study protocol was approved by the Ethics Committees of Hokushin General Hospital (Receipt No. 2020005). The patient sera were filtered with DISMIC 13HP syringe filters (0.45 μm, ADVANTEC), and each filtered sample (after addition of 5 mol/L HCl) was loaded onto a MonoSpin C18 cartridge by centrifugation for 3 min. The subsequent SPE protocol was the same as that described above.

Screening method for complexation between metal ions and FPV
For identifying metal ions capable of forming complexes with FPV, our recently developed screening method using MonoSpin cartridge 7 was applied for the analysis of complexation between FPV and each of twelve different metal ions. The general procedure of the method is described in Supporting Information.

Results and Discussion
Over the last decade, nine different papers bearing descriptions of procedures for HPLC quantification of plasma/serum levels of FPV have been published. 4-6, 8, 10-14 However, as listed in Table S1 (in Supporting Information, SI), detailed descriptions of the analytical procedures and chromatograms were lacking from most of these previous papers. Therefore, we performed our own research to develop a method for rapid quantification of serum FPV level in specimens from patients with COVID-19 administered pharmacological treatment with FPV.
In our preliminary HPLC experiment (see Fig. S1 in SI), FPV was well-retained on an octadecyl-silica (ODS; C18) column under acidic pH conditions below FPV's pKa value (5.1) conditions under which the compound exists in a hydrophobic non-ionized form. As shown in the UV spectra in Fig. 1, the λmax of FPV was 325 nm in the acidic (pH 2) mobile-phase solvent containing 5% CH3CN. Therefore, the UV detection wavelength of our LC system was fixed at the 325 nm. Under these conditions, a good RP-HPLC profile of FPV (eluted within 3 min) was obtained, as shown in Fig. S1-a (in SI).
We next sought to optimize the SPE conditions for FPV-spiked serum, prior to quantitation of the compound by HPLC. A SPE technique is commonly used for pretreatment of serum specimen before a drug analysis. 15 Table S2 in SI). Based on these data, we devised an optimized protocol for SPE of FPV-spiked serum, as described above in the Experimental section.
Ultimately, the analytical procedure for quantification of FPV in serum was established by using an appropriate combination with the SPE treatment and subsequent RP-HPLC/UV analysis as described above; the procedure of the proposed method is shown schematically in Fig. 2. Specifically, the HPLC chromatogram of the SPE eluate of the FPV-spiked serum (50 μg mL -1 ) is shown in Fig. 3b. Compared to the chromatogram of blank (FPV-free) serum (Fig. 3c), FPV-spiked serum exhibited no interfering peaks derived from serum constituents at retention times corresponding to those of FPV (Fig. 3b). Furthermore, comparison of the FPV peak areas in Fig. 3b and Based on these results, a quantification of FPV in serum was performed by the above method using a one-point calibration curve that was prepared from the FPV peak area of the SPE eluate of the FPV-spiked serum (100 μg mL -1 ) without use of an I.S.
compound. As shown by the results of the method validation using three levels of QC samples (sera spiked with FPV at 5, 20, and 80 μg mL -1 ), accuracy values ranged from 97.9 to 105.0 % for all QC samples (Table 1). Intra-day and inter-day precision (CV%) values were below 6 % for the three tested concentrations. Acceptance criteria for accuracy and precision were met in all QC samples. As a final step, we implemented a quantification of FPV levels in sera from four patients with COVID-19 who had been treated by administration of FPV; this experiment was intended to verify the utility of our method in clinical practice at a general hospital. The FPV concentrations in the serum specimens from the four patients were quantified as 2.7, 33.1, 77.6, and 93.5 μg mL -1 ; the HPLC chromatograms of the quantification assays are shown in Fig. S2 in SI. Interestingly, although all four patients had been administered FPV at the same dose level, their serum FPV levels (trough levels) varied over a greater-than-30-fold range. Although the actual cause of the observed variation remains unknown, we expected that each patient's serum contains distinct levels of certain intrinsic factors that may interact with FPV. Therefore, we additionally examined the sera for the presence of metal ions capable of forming a complex with FPV, given the fact that FPV has a typical multidentate ligand structure (see the chemical structure in Fig. 1). For this purpose, we used a metal ion screening method that we have recently developed. 7 As shown in Fig. 4, the screening data revealed that FPV selectively formed complexes with ferric (Fe 3+ ) and cupric (Cu 2+ ) ions among the twelve tested types of metal ions; more detailed data also are shown in  Accuracy (%) was calculated from the data of quadruplicate samples (n=4) at given concentration; Repeatability (CV %) was also from quadruplicate samples (n=4); Intermediate precision (CV %) was calculated over a series of three analyses on different days (3 days).