Introduction

Glecaprevir mainly acts as hepatitis C Virus nonstructural protein 3/4A protease inhibitor (Babu, C.M.Madhusudhana, & Mastanamma, 2018; GampaVijayKumar & Reddy, 2018; Kumar & Raja, 2018) It is used for the treatment of chronic hepatitis C Infection along Pibrentasvir.

Pibrentasvir acts as NS5A inhibitor anti-viral agent (Sangameshwar, 2019; Saradhi, 2018; Sreeram & Venkateswarlu, 2018). The combination of Glecaprevir and Pibrentasvir was being approved by US Food and Drug Administration for hepatitis treatment (Issarani, 2013). The chemical structures of Glecaprevir and Pibrentasvir are shown in Figure 1 and Figure 2.

Materials and Methods

Chemicals and Reagents

Glecaprevir and Pibrentasvir were kindly gifted by Pharmatrain Labs Pvt Ltd, Hyderabad certified to contain 99.9% and 99.8% purity respectively. Purification was not performed for the gifted samples and are used directly for Analysis. All the solvents utilized in analysis were of HPLC grade. Mavyret Tablets (label claim 100 mg of Glecaprevir and 40 mg of Pibrentasvir) of Abbvie pharma was used in analysis.

Instrument

LC system used consists of Waters HPLC having Empower Software with 2695 separation module having PDA detector with 20 μLinjection capacity. The column used was Xterra C₁₈ Column, 5μ (250× 4.6 mm) at controlled temperature. Various solvents were tested in order to sort out the simplest conditions for drug separation at a particular time.

Optimized Chromatographic conditions

The mobile phase having phosphate buffer (pH 4.0) and methyl alcohol in the proportions of 30:70 v/v was selected because it was found that it ideally resolve the peaks with retention time (RT) 2.205 min and 4.996 min for Glecaprevir and Pibrentasvir respectively. Isobestic point was determined byperforming the spectroscopic analysis in the range of 200 nm to 390nm. Both the components showed reasonably good response at 251 nm with characteristic peak as shown in the Figure 3.

Preparation of Phosphate Buffer

Precisely Weigh 1.732g of monobasic potassium phosphate was taken in a 500ml volumetric flask, dissolved and diluted to 500ml with HPLC water and the volume was adjusted to pH 4.0 with Ortho Phosphoric Acid.

Preparation of Mobile Phase

Accurately measured 300 milli litre (30%) of phosphate Buffer and 700 milli litre of Methanol(70%) were mixed and degassed in inaudible water tub for 10 minutes then filtered through 0.45 µ filter under vacuum filtration. Diluent was mobile phase.

Standard Solution Preparation

Weigh precisely a quantity which is equivalent to 10 mg of Pibrentasvir and 25 mg of Glecaprevir and dissolve in 25 ml of mobile phase which comprises of Phosphate buffer and methanol. Sonicate the above solution for few minutes until the drug samples dissolves completely and pipette out 3 ml of PibrentasvirandGlecaprevir from standard stock solution into two separate 10ml volumetric flasks and make up the volume to the mark with diluents. Inject 20 µL of the standard, sample into the chromatographic system and measure the areas for glecaprevir and pibrentasvir peaks and calculate the % Assay by using the formulae. The Chromatogram of standard solution was shown in Figure 4.

Sample Solution Preparation

Weigh precisely a quantity which is equivalent to 5 mg of Pibrentasvir and 12.5 mg of Glecaprevir and dissolve in 25 ml of mobile phase which comprises of Phosphate buffer and methanol. Sonicate the above solution for few minutes until the drug samples dissolve completely and pipette out 3 ml of Pibrentasvir and Glecaprevir from standard stock solution into two separate 10ml volumetric flasks and make up the volume to the mark with diluents. The Chromatogram of sample solution was shown in Figure 5.

Recovery studies

To analyze the correctness of the sample by the process developed and to study the levels of interference of formulation additives recovery experiments were carried out by standard addition method at various levels. From the overall amount of drug found, recovery was estimated. The results were reported in Table 1.

Results and Discussion

Preparation of Calibration Curves by HPLC

Serial dilutions of Glecaprevir (100-500 μg/ml) and Pibrentasvir (40-200 μg/ml) were prepared using a mobile phase as solvent using 10 ml volumetric flasks. Each drug solution was analyzed by HPLC system, and the chromatograms were recorded. The peak spaces of each drug were calculated and also the individual activity curves were planned against magnitude relation of the area underneath curve and concentration of the drug. The chromatogram reports are shown in Table 2.

HPLC Method Validation

Method validation was assessed by different parameters, which include linearity, accuracy, Specificity, precision, the limit of detection, the limit of quantitation in accordance with International Council for Harmonisation guidelines.

Linearity and Range

The one-dimensionality of a technique is its ability to get take a look at results that area unit directly proportional to the sample concentration over a given vary. The peak area and concentration were aforethought to induce atypical standardization plot. The linearity range for Pibrentasvir and Glecaprevir was found to be 40 to 200 µg/ml and 100-500 µg/ml, respectively. The Correlation coefficient value for the calibration plot of Pibrentasvir and Glecaprevir was 0.999; it shows good linearity for both the drugs. The calibration curves were given in Figure 6 and Figure 7.

Accuracy

Accuracy was determined by sneakiness of the measured worth to actuality worth for the sample. The percentage recovery of Pibrentasvir was found to be 100.23%, 100.50% and 100.02% for accuracy 50%, 100% and 150% samples respectively. The mean recovery for Pibrentasvir is 100.25. The %RSD of the samples was found to be less than 2, and Glecaprevir was found to be 100.54%, 100.73% and 99.94% for accuracy 50%, 100% and 150% samples respectively. The mean recovery for Glecaprevir is 100.40. The %RSD of the samples was found to be less than 2. Reports of accuracy were given in Table 3.

Precision

Repeatability studies were studied by a series of measurements are done with pibrentasvir and glecaprevir. Peak area was measured for six replicate injections of both the drugs. % Relative Standard Deviation for the area measured of repeated injections was found to be at intervals the desired range. The reports for precision were given in Table 4.

Limit of Detection and Limit of Quantification

The Limit of Detection and Limit of Quantification for Pibrentasvir was 3.00 and 9.98. The Limit of Detection and Limit of Quantification for Glecaprevirwas2.98 and 10.00, respectively. The results obtained are within the limits.

Robustness and Ruggedness

Robustness and Ruggedness studies were studied by five replicate injections of Pibrentasvir and Glecaprevir on different days. The acceptance criteria for robustness and ruggedness can be assessed by measuring the percentage relative standard deviation, which should not be of not more than 2%. The reports were tabulated Table 5 in and Table 6.

Degradation Studies

The International Council for Harmonization (ICH) guideline entitled stability testing of recent pharmaceutical compounds and merchandise needs that stress sampling is dispensed to explain the stability of active pharmaceutical ingredients. The aim of this work was to perform the degradation studies on the Pibrentasvir and Glecaprevir using the developed method.

Hydrolytic degradation under acidic condition

Accurately measure 3 ml of the above solution and add 3 ml of 0.1N HCL. Transfer the above contents into 10 ml volumetric flask. Keep the Volumetric flask at a temperature of 60ºC for one day and neutralization was carried out by 0.1N NaOH. Make up the volume up to 10 ml with diluent and filtration was performed by using whatmann grade filter paper and the filtrate was stored in vials.

Hydrolytic degradation under alkaline condition

Accurately measure 3 ml of the above solution and add 3 ml of 0.1N NaOH. Transfer the above contents into 10 ml volumetric flask. Keep the Volumetric flask at a temperature of 60ºC for one day and neutralization was carried out by 0.1 N HCL. Make up the volume up to 10 ml with diluent and filtration was performed by using whatmann grade filter paper and the filtrate was stored in vials.

Oxidative degradation

Accurately measure 3 ml of the above solution and add 1 ml of 30% w/v of Hydrogen peroxide. Transfer the above contents into 10 ml volumetric flask. Keep the Volumetric flask at a temperature of 60ºC for 15 minutes. Make up the volume up to 10 ml with diluent and filtration was performed by using whatmann grade filter paper and the filtrate was stored in vials.

Thermal-induced degradation

Glecaprevir and Pibrentasvir sample was taken in Petri dish and placed in Hot air oven at 110⁰ C for 3 hours. Dilution was performed by adding diluents to the sample and was analyzed by injecting into HPLC. All the results of stability testing were tabulated in Table 7.

Conclusion

Several trials were carried out to achieve optimized chromatographic conditions. The initial attempt was to use as much low proportion of organic solvents for the chromatographic separation of the compounds. Usage of binary compound solvents results in enhancement of elution time of each the compounds. The proposed method can be used for acceptable results. The proposed technique is simple, speedy and statistically valid for its accuracy. Hence no interfering peaks were found in the chromatograms shows that anyone excipient is not interfered during the chromatographic separation of both the drugs.