A validated LC-MS/MS method for determination of antiviral prodrug molnupiravir in human plasma and its application for a pharmacokinetic modeling study in healthy Egyptian volunteers.

A fully validated, simple, rapid and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine NHC (N-hydroxycytidine), the active metabolite of Molnupiravir (MOL) in human plasma; one of the limited treatment options for SARS-CoV-2 in plasma of healthy volunteers. The internal standard (IS) used was ribavirin. The extraction of analyte and IS from plasma was performed using acetonitrile as a solvent for protein precipitation. Agilent Zorbax Eclipse plus C18, 4.6 × 150 mm, (5 µm) was used for chromatographic separation using a mixture of methanol0.2 % acetic acid (5:95, v/v) as a mobile phase that was pumped at a flow rate of 0.9 mL/min. Detection was performed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) employing positive ESI interface using API4500 triple quadrupole tandem mass spectrometer system, with the transitions set at m/z 260.10 â†’ 128.10 and 245.10 â†’ 113.20 for NHC and IS respectively. Method validation was performed in accordance with United States FDA bioanalytical guidance. The concentration range of 20.0-10000.0 ng/mL was used to establish linearity via weighted linear regression approach (1/x2). Moreover, the analyzed pharmacokinetic data from twelve Egyptian healthy volunteers were used to develop a population pharmacokinetic model for NHC. The developed model was used to perform simulations and evaluate the current MOL dosing recommendations through calculating the maximum concentration (Cmax) "the safety metric" and area under the curve (AUC0-12 h) "the efficacy metric" for 1000 virtual subjects. Geometric mean ratios (GMR) with their associated 90% confidence intervals (CI) compared to literature values were computed. Geometric means of simulation-based Cmax and AUC0-12 were 3827 ng/mL (GMR = 1.05; 90% CI = 0.96-1.15) and 9320 ng.h/mL (GMR = 1.04; 90% CI = 0.97-1.11), respectively indicating that current MOL dosage can achieve the therapeutic targets and dose adjustment may not be required for the Egyptian population. The developed model could be used in the future to refine MOL dosage once further therapeutic targets are identified.

A novel LC-MS/MS method for determination of the potential antiviral candidate favipiravir for the emergency treatment of SARS-CoV-2 virus in human plasma: Application to a bioequivalence study in Egyptian human volunteers.

A novel, fast and sensitive LC-MS/MS method was developed and validated for the bioanalysis of the antiviral agent favipiravir (FAV); a promising candidate for treatment of SARS-CoV-2 (COVID-19) in human plasma using pyrazinamide as an internal standard (IS). Simple protein precipitation was adopted for plasma sample preparation using methanol. Chromatographic separation was accomplished on Eclipse plus C18 column (50 × 4.6 mm, 3.5 µm) using a mobile phase composed of methanol-0.2 % acetic acid (20:80, v/v) pumped at a flow rate 0.6 mL/min in an isocratic elution mode. The API4500 triple quadrupole tandem mass spectrometer was operated with multiple-reaction monitoring (MRM) in negative electrospray ionization interface for FAV and positive for IS. The MRM function was used for quantification, with the transitions set at m/z 156.00→ 113.00 and m/z 124.80→ 81.00 for FAV and IS. The method was optimized and fully validated in accordance to US-FDA guidelines. Linearity was acquired over a concentration range of 100.0-20000.0 ng/mL by computing using weighted linear regression strategy (1/x2). The proposed method was effectively applied for the pharmacokinetic evaluation of FAV and to demonstrate the bioequivalence of a new FAV formulation (test) and reference product in healthy Egyptian human volunteers.

Validated stability-indicating HPLC method for the determination of mesna in presence of its degradation products.

A rapid and simple stability-indicating liquid chromatographic method was developed and validated for analysis of mesna in the presence of its degradation products in drug substance and drug products in a run time not >5 min. The separation was achieved on a RP amide C16 column at room temperature using methanol-phosphate buffer (10:90, v/v, pH 3.0) as mobile phase at a flow rate of 1 mL min(-1) and UV detection at 210 nm. The detector response for mesna was linear over the selected concentration range from 50 to 1000 µg mL(-1) with a correlation coefficient 0.9998. The limit of detection and the limit of quantitation were 7.5 and 22.7 µg mL(-1), respectively. The solution was stable for at least 5 days. Baseline resolution between mesna and its degradation products was achieved. Diode array detection peak purity tests showed no peak interfered with mesna peak. Moreover, the method was successfully applied for the determination of mesna in two different commercially available drug products.