Homogeneous liquid-liquid microextraction coupled with HPLC/DAD for determination of nirmatrelvir and ritonavir as COVID-19 combination therapy in human plasma

Abstract

The study reports the development of a high-performance liquid chromatography/diode array detection method to measure the levels of nirmatrelvir and ritonavir in human plasma. These two antiviral medications are used for the treatment of COVID-19 and are marketed as Paxlovid^®. The method employed sugaring-out induced homogeneous liquid-liquid microextraction to improve sensitivity. Optimization of the method was performed using the one variable at a time approach by adjusting several factors such as type of sugar, extractant, amount of sugar, volume of extractant, and pH of the aqueous sample to achieve the highest efficiency. The developed method was validated according to the Food and Drug Administration guidelines and demonstrated good linearity, accuracy, and precision. The range of linearity was from 1000 to 20,000 ng/mL for nirmatrelvir and 200 to 20,000 ng/mL for ritonavir with correlation coefficient values of 0.998 and 0.996, respectively. Selectivity studies revealed that no others peaks appeared in the retention times of the studied drugs. The stability of nirmatrelvir and ritonavir were also investigated through short term and three cycles of freeze-thaw, and both drugs were found stable. This analytical method could be useful for monitoring drug concentrations in patients undergoing treatment with these medications for COVID-19. In this work, for the first time, SULLME was used for the sensitive determination of nirmatrelvir and ritonavir in biological fluids. The developed method was able to determine both drugs in therapeutic levels with no need to sophisticated techniques like LC-MS. In addition to that, SULLME is considered a simple and green sample preparation in comparison with conventional sample preparation methods.

Keywords: COVID-19; HPLC; Nirmatrelvir; Ritonavir; SARS-CoV-2, Sugaring-out.

Fig. 1

Chemical structures of nirmatrelvir, ritonavir, and velpatasvir

Fig. 2

Procedures of the determination of NIRMA and RITONA by SULLME followed by HPLC/DAD

Fig. 3

Chromatographic separation of 1 velpatasvir (IS, 20 µg/mL), 2 nirmatrelvir (20 µg/mL) and 3 ritonavir (20 µg/mL) in an aqueous sample. Chromatographic conditions: Column: Thermo Hypersil ODS C₈ column (250 × 4.6 mm, 5 μm) at 35 °C, Mobile phase: phosphate buffer (50 mM, pH = 3): acetonitrile (35:65, v/v), Elution: Isocratic, Detection: DAD at 210 nm, Flow rate: 1 mL/min, Injection volume: 5 µL

Fig. 4

Effect of sugar and extracting solvent types on the microextraction efficiency of RITONA

Fig. 5

The effect of ACN volume on the extraction efficiency using 1 mL aqueous sample and 1000 mg of sucrose as a phase separating agent

Fig. 6

The effect of sucrose amount on the extraction efficiency using 1 mL aqueous sample and 500 µL of ACN

Fig. 7

The effect of pH of the aqueous sample on the extraction efficiency using 1 mL aqueous sample, 500 µL of ACN and 800 mg of sucrose

Fig. 8

Blank plasma sample spiked with velpatasvir (1, 20 µg/mL) as an internal standard. The arrows show the absence of peaks at the retention times of nirmatrelvir (2) and ritonavir (3). Chromatographic conditions: Column: Thermo Hypersil ODS C₈ column (250 × 4.6 mm, 5 μm) at 35 °C, Mobile phase: phosphate buffer (50 mM, pH = 3): acetonitrile (35:65, v/v), Elution: Isocratic, Detection: DAD at 210 nm, Flow rate: 1 mL/min, Injection volume: 5 µL

Fig. 9

Chromatograms of human plasma containing 1 velpatasvir (IS, 20 µg/mL), 2 nirmatrelvir (15 µg/mL) and 3 ritonavir (15 µg/mL) after sample preparation by protein precipitation (red) and SULLME (black). Chromatographic conditions: Column: Thermo Hypersil ODS C₈ column (250 × 4.6 mm, 5 μm) at 35 °C, Mobile phase: phosphate buffer (50 mM, pH = 3): acetonitrile (35:65, v/v), Elution: Isocratic, Detection: DAD at 210 nm, Flow rate: 1 mL/min, Injection volume: 5 µL