key: cord-0706540-dkjh7r0w authors: Mikhail, Ibraam E.; Elmansi, Heba; Belal, Fathalla; Ehab Ibrahim, Adel title: Green micellar solvent-free HPLC and Spectrofluorimetric determination of Favipiravir as one of COVID-19 antiviral regimens date: 2021-03-23 journal: Microchem J DOI: 10.1016/j.microc.2021.106189 sha: b1573f1d1d7a656ff435d6a157b3d2c143058cb2 doc_id: 706540 cord_uid: dkjh7r0w Quality control (QC) of pharmaceutical products requires fast, sensitive as well as economic methodologies in order to provide high throughput at low cost which are the main aspects considered by such economic facilities. Meanwhile, the ecological impacts must be considered by researchers to minimize the hazardous effects of research laboratories. Favipiravir (FAV) is an antiviral agent recently approved for treatment of COVID-19 infections during 2020 pandemic crisis, so the size of its production by international pharmaceutical corporations evolved dramatically within the past few months. Two novel simple, sensitive, and green methods were developed and validated for FAV determination based on solvent-free micellar LC and spectrofluorimetry techniques. To improve FAV native fluorescence, several factors were studied including solvent type, buffering, pH and added surfactants. The best sensitivity for FAV fluorescence was obtained in Britton-Robinson buffer (pH 4) at 436 nm after excitation at 323 nm within concentration range of 20-350 ng mL-1. Another HPLC method was validated using C18-RP (5µm, 250 x 4.6 mm) stationary phase and solvent-free mobile phase consisting of (0.02M Brij-35, 0.15M SDS, and 0.02M disodium hydrogen phosphate, pH 5.0) isocratically eluted at a flow rate of 1 mL min-1 and detection wavelength of 323nm. LC method was validated across concentration range of 10-100 µg mL-1 and FAV eluted in 3.8 min. The methods were validated according to the FDA guidelines and were applied successfully for determination of FAV in its marketed tablet dosage forms and in spiked human plasma samples. The proposed methods are eco-friendly since they are typically based on biodegradable reagents in aqueous solvent-free phases, which was proven by their assessment on two recent greenness metrics (GAPI and AGREE) to prove their eco-friendly properties. In December 2019, the WHO was alerted by the Chinese government of pneumonia 45 hospitalized cases with unidentified cause. These cases were identified later as COVID-19 in 46 which the causative virus was SARS-Cov-2. In March 2020, WHO declared Coronavirus 47 infection outbreak as pandemic. By the end of June 2020, the number of reported cases 48 worldwide exceeded 10 million cases with hundreds of thousands of deaths [1] . Symptoms in 49 mild cases were fatigue, fever and dry cough, however in severe infections failure of kidney and 50 respiratory system occurred [2] . The virus caused severe acute respiratory syndrome (SARS) for 51 about 16% of the infected cases after 5 days of exposure [3] . The high mortality rates caused by 52 COVID-19 (about 1-2%),endeavoured global scientists to discover antiviral agents that can 53 suppress the viral spread and enhance patients' recovery [3] . Since the process for approval of a 54 new drug for human use is complex and comprises multiple stages to establish safety data and 55 discover potential risks, the easiest and fastest way was to try FDA approved antivirals. Several 56 antivirals were clinically tried including lopinavir/ritonavir combination, favipiravir, umifenovir, 57 remdesivir and tocilizumab. infections and declared that FVP was found to enhance viral clearance and improve chest CT [3, 64 5, 6]. The side effects associated with the use of FAV were found mild and manageable. The use of fluorescence spectroscopy has several advantages including high selectivity, 75 specificity, sensitivity and fast outcome. Depending on 2 wavelengths for both excitation and 76 emission improves method's selectivity. The sensitivity of fluorescence is much higher than 77 absorption spectroscopy since it doesn't compare the intensity to a reference beam; it measures 78 the intensity directly against low background. The absence of mobile phase preparation and 79 conditioning steps required for chromatographic techniques increase the tool's output. Since claiming greenness of any analytical methodology is not enough, the proposed 106 methods were assessed against two recent greenness metrics, the green analytical procedure 107 index (GAPI) and AGREE tools [21, 22] . Another aim for this study is to provide a mini-review 108 about FAV determination methodologies, and a thorough comparison to some previously 109 reported methods to evaluate their greenness aspects and differences between used metrics. conditions. The effect of relative volume of BRB added per 10 mL of aqueous solvent was 215 tested. RFI of FAV increased with increasing the buffer volume up to 10 mL (Fig. 3) . So, the 216 validation study was performed by dilution of the drug solution under study with BRB. The use of organic solvents in normal phase chromatography is also less economic than aqueous 226 mobile phases used in RP-chromatography. So, C18 RP was the primary choice. Mobile phase pH: 228 The pH of the mobile phase affects the ionization state of the resolving analytes. pH of the 229 mobile phase was altered within range of 2.8 -6.5, since FAV has weak acidic pKa value of 5.1 230 in order to keep the molecule non-ionized and enhance its retention on RP-C18 phase. The best 231 retention was obtained on adjusting the pH of the mobile phase at pH 5.0. 257 LODs and LOQs were calculated from the slope (S) and standard deviation (σ) of the calibration 258 curve. LODs were calculated corresponding to values of (3.3σ/S) and LOQs were corresponding to values of (10σ/S). Results shown in (Table 1) indicate the sensitivities of the proposed 260 methods. 262 Accuracy was tested by calculating percentage recoveries for the prepared quality control 263 solutions prepared at low, medium and high concentration levels within the calibration ranges. Repeatability and Intermediate precisions were tested by repetition of analysis of the quality 265 control solutions within the same day (Intra-day) and at three different days (Inter-day). The 266 obtained results are presented in Table 2 , with acceptable percentage recoveries and standard forms and the results were statistically compared to each other to find any significant differences. However, the reported study was applied on a laboratory prepared mixture with the addition of 310 assumed excipients, instead of the commercial formulation, and the results obtained were 311 compared to a reference method which was missed from citing within the reported manuscript. Procedure Index (GAPI) [21] have been utilized extensively. AGREE assessment tool was 317 reported recently [22] . GAPI is composed of 15 pentagrams, each coloured (red, yellow or 318 green) to assess its environmental impact, where red represent bad impact, yellow for 319 intermediate and green for safe and low environmental hazards [30] . AGREE provides a clock-320 shaped graph with perimeter divided into 12 parts based on the 12 principles of Green Analytical evaluate the agreement of the analytical procedure to GAC principle. The heart of the AGREE 323 graph has an overall assessment colour together with an overall assessment figure into a scale of 324 0 to 1. 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