INTRODUCTION:

Hepatitis C is treated with a fixed dose combination of Glecaprevir/Pibrentasvir¹, marketed under Mavyret and Maviret. It combats all six forms of hepatitis C.Glecaprevir is an antiviral agent and a macro-cyclic HCV protease inhibitor that inhibits the non-structural (NS) 3/4A protein of the hepatitis C virus (HCV). The molecular weight of Glecaprevir is 838.87 g/ mole (Figure 1A). pKa of the drug is 3.74.

Pibrentasvir^1,2 is an antiviral agent and NS5A inhibitor of HCV that aims to inhibit viral RNA replication and the entire virion. The molecular weight of Pibrentasvir is 1,113.20 g·mol⁻¹ (Figure 1B). pKa of the drug is 5.3.

A review of the literature reveals that many methods, such as HPLC^3-9 have been established to determine Glecaprevir and Pibrentasvir simultaneously in dosage forms and bulk. However, the UPLC method was developed since it is more advanced than the HPLC method. Sri Datla V.V.S.S.N¹⁰ et al developed a method with higher LOD and LOQ. However, another method was developed by Naga Venkata Indira¹¹ et al and Deepthi R¹² et al with 1 mL/min flow rate which requires a higher amount of mobile phase than the current method. Marakada Sridevi¹³ et al, Pushpa Kumari¹⁴ et al and Geetha Sushmitha¹⁵ et al developed a method utilizing a 50 mm column which is more costly than the current column used. Therefore, I can state that the created method is novel to the previously established methods. As such, during the development of the method, we encountered many straightforward procedures and some challenging methods. The goal of the project is to provide a quick UPLC analysis of Glecaprevir and Pibrentasvir in tablets and API with a forced degradation study.

Fig. 1: A) Glecaprevir structure B) Pibrentasvir structure.

MATERIALS AND METHODS:

Materials:

UPLC system (Waters Acquity UPLC with TUV detector system) has been used in the development of a method for estimating Pibrentasvir and Glecaprevir using Empower 2.0 version software for data acquisition. During method development, various chemicals and solvents such as Ammonium acetate and Acetonitrile are used. We acquired Ammonium acetate, HPLC-grade water, and Acetonitrile solvents from Merck. Spectrum Pharma Solutions provided pure samples of both drugs. A tablet formulation containing Pibrentasvir and Glecaprevir is available on the market under the brand name Mavyret. According to the tablet's label, it has 40 mg of Pibrentasvir and 100 mg of Glecaprevir.

Preparation of buffer:

Transferred 0.77gm of Ammonium acetate into a 1000 mL container with about 3/4^th volume of HPLC grade water, mixed and diluted with water, filtered using 0.22 µm membrane filters, and degassed.

Mobile phase Preparation: Degassed the mixture of buffer and Acetonitrile. (30:70%v/v).

Preparation of diluent: A 50:50% v/v mixture of Acetonitrile and HPLC grade water was sonicated to degas.

Procedure for standard solutions preparation:

Glecaprevir 25 mg and Pibrentasvir 10 mg Standards were weighed and then transferred into a 50 ml volumetric flask. Diluent of 3/4^th volume was then added, mixed, and sonicated and added diluent to the volume. After adding 1 mL of the solution into a 10 mL volumetric flask, diluted to solution to volume with diluent. (50µg/mL of Glecaprevir, 20µg/mL of Pibrentasvir).

Preparation of sample solution:

The mean weight of 10 tablets was found by weighing them. By using a mortar and pestle the tablets were then ground into powder. Weighed about 40 mg of Pibrentasvir and 100 mg of Glecaprevir and added to a 100 mL volumetric flask. Diluent of ¾ th volume was added and mixed and after sonicating for about 25 minutes, added diluent to the final volume mixed and filtered using 0.22 µm PVDF filters. 0.5 mL was pipetted to a 10 mL volumetric flask and was diluted to volume. (50µg/mL of Glecaprevir, 20µg/mL of Pibrentasvir).

METHOD DEVELOPMENT:

Initially tried with HPLC grade water and HPLC grade methanol in 50:50 %v/v on Acquity CHS 100 x 2.1mm, 1.7m with peak absorption maxima at 248 nm. In trial 1 SST was not within the limits, so further trial was carried out. Trail -2 was with Acetonitrile and OPA in 50:50% v/v on the same column. In trial 2 also SST was not within the criteria, so further trial was carried out. Trail-3 was with Acetonitrile and Ammonium acetate in 50:50% v/v on the same column. In trial 3 also SST was not meeting the limit. Trail -4 was carried with Acetonitrile and Ammonium acetate in 65:35% v/v on the same column, Glecaprevir was found at 1.155 min and Pibrentasvir at 1.422 min and SST was within the acceptance criteria. So Trail 4 was optimized but for better resolution, we carried out another trial. Trail-5 was carried with Acetonitrile and Ammonium acetate in 70:30 % v/v on the same column, Glecaprevir was eluted at 1.166 minutes, and Pibrentasvir at 1.502 min. The resolution, USP plate count, % RSD, and USP tailing factor were found to be satisfactory and the optimized parameters were reported in Table No.1.

Table No. 1: Optimized Chromatographic conditions

Chromatographic conditions	UPLC
Column	ACQUITY CHS C18 100x 2.1mm, 1.7 µm
Flow rate	0.3 mL/min
Detector wavelength	248.0 nm
Column temperature	30°C
Injection volume	1.0 µL
Run time	3.0 minutes
Diluent	Acetonitrile: Water (50:50%v/v)
Mobile phase	0.01 M Ammonium acetate and Acetonitrile (30:70 %v/v)

Fig. 2: Chromatogram Under Optimized Conditions By UPLC

METHOD VALIDATION:^16,17

1) System suitability:

It was determined with one injection of blank, and six injections of standard solution. % RSD, plate count, tailing, and resolution have been assessed to establish system suitability.

2) Specificity:

Specificity is ascertained by injecting one injection of blank and a placebo and observing the blank interference and placebo interference at the main peaks of Glecaprevir and Pibrentasavir.

3) Precision:

Prepared six samples and injected 1 µl of each sample preparation into UPLC. The method precision is measured by calculating the average and % RSD for six assay results.

4) Linearity:

Three sets of six linear solutions were calculated each with concentrations ranging from 25% to 150% of standard ppm were prepared and then injected. Calibration curves were generated using concentration representing the abscissa (X-axis) and an area representing the ordinate (Y-axis) for Glecaprevir and Pibrentasvir separately and the square of the correlation coefficient (r²) was calculated.

5) Accuracy:

Accuracy refers to the proportion of the analyte that is successfully recovered. Recovery trials were conducted in triplicate. The standard amount of drug added to the sample solution at 50%, 100%, and 150% in triplicate were done. % recovery is determined by dividing the % drug recovered to the % drug added and the average % recovery is also calculated.

6) LOD and LOQ:

LOD values and LOQ values were derived from the Standard Deviation and the slope obtained from the linearity curves.

7) Robustness:

By analysing Standards under modified method-specified conditions, system suitability parameters were ascertained including Resolution, %RSD of areas, USP plate count, and USP tailing were determined. Variations of Flow rate (±0.1 mL/min), mobile phase, and temperature (± 2 ˚C) were measured in robustness.

8) Forced degradation studies:

9) After subjecting 1 mL stock solution to the aforementioned conditions, diluent was added to the resulting solution, which was then diluted to 10 mL, before being injected.

Table No. 2: Forced degradation conditions

S. No	Condition	Method applied
1	Oxidation	1 mL of 20% H₂O₂/60⁰C/30 minutes
2	Acid	1mL of 2N HCl/60⁰C/30 minutes
3	Alkali	1mL of 2N NaOH/60⁰C/30 minutes
4	Dry Heat	105⁰C/6 hours
5	Photo Stability	UV Chamber for 7 days
6	Neutral	5 mL of water /6 hrs/60ºc

RESULTS AND DISCUSSION:

The method was validated for System suitability, Specificity, Precision, linearity, Accuracy, LOD and LOQ, Robustness, and Forced degradation parameters.

1) System suitability:

% RSD for six standard injections for Glecaprevir is 0.6 and for Pibrentasvir is 0.6. The plate count for Glecaprevir is 4652 and for Pibrentasvir is 5105, tailing factor for Glecaprevir is 1.17, and for Pibrentasvir is 1.22 respectively. Resolution between the two peaks was 5.2

Table No. 3: System suitability data

Parameter	Glecaprevir	Pibrentasvir
% RSD	0.6	0.6
Plate count	4652	5105
Peak tailing	1.17	1.22
Resolution	NA	5.2

2) Specificity: Blank and Placebo interference was not seen at the main peaks indicating the method is specific.

(A)

(B)

Fig. 3: Chromatograms of A) Blank B) Placebo

3) Precision: % Assay of Glecaprevir and Pibrentasvir were found to be between 98%-101% and the %RSD for Glecaprevir is 0.5, %RSD for Pibrentasvir is 1.0. It was found that %RSD was below 2%, demonstrating that the procedure was precise.

Table No. 4: Precision data

S. No	% Assay of Glecaprevir	% Assay of Pibrentasvir
1	99.8	99.2
2	99.5	100.9
3	99.9	99.5
4	100.3	100.6
5	98.8	98.3
6	99.8	99.1
Average	99.7	99.6
STD	0.5036	0.9797
%RSD	0.5	1.0

4) Linearity: The square of the Correlation coefficient is 0.9998 for Glecaprevir and 0.9998 for Pibrentasvir indicating that the method is linear between 25% to 150%.

Table No. 5: Linearity table

Percentage (%)	Glecaprevir		Pibrentasvir
Percentage (%)	Concentration (PPM)	Peak area	Concentration (PPM)	Peak area
25	12.5	63730	5	37810
50	25	130453	10	77816
75	37.5	193872	15	115707
100	50	257797	20	153368
125	62.5	317751	25	189376
150	75	381269	30	227140
Regression equation	y = 5083.4x + 1496.7		y = 7572.2x + 876.94
r2	0.9998		0.9998

5) Accuracy: Recovery was found to be between 98%-101% reflecting that the method is accurate.

Table No. 6: Recovery studies

Glecaprevir				Pibrentasvir
Spiked Level	µg/ml added	µg/ml found	% Recovery	µg/ml added	µg/ml found	% Recovery
50%	25	24.9	99.8	10.0	9.95	99.5
50%	25	24.9	99.5	10.0	9.85	98.5
50%	25	25.2	100.7	10.0	9.96	99.6
100%	50	49.5	99.0	20.0	20.20	101.0
100%	50	50.2	100.4	20.0	20.16	100.8
100%	50	49.2	98.4	20.0	20.10	100.5
150%	75	75.3	100.3	30.0	29.46	98.2
150%	75	73.8	98.4	30.0	29.59	98.6
150%	75	74.9	99.9	30.0	29.88	99.6
Average			99.6	Average		99.6
%RSD			0.8	%RSD		1.0

6) Robustness: The % RSD for six injections of the standard solutions for Glecaprevir and Pibrentasvir was found to be less than 2%. The plate count was found to be more than 2000, tailing factor was found to be less than 2 for both drugs respectively. The resolution between the two peaks exceeded 2.0. demonstrating the method is robust.

7) LOD and LOQ: LOD value for Glecaprevir is 0.34µg/mL and for Pibrentasvir is 0.16 µg/mL. LOQ for Glecaprevir is 1.02µg/mL and for Pibrentasvir is 0.48 µg/mL

Table No. 8: LOQ and LOD of Glecaprevir and Pibrentasvir

Parameter	Glecaprevir	Pibrentasvir
LOD	0.34	0.16
LOQ	1.02	0.48

8) Forced Degradation studies: More degradation was observed in the acid stage and results were reported in Table No. 9. The method is capable of separating the main peak from degradants in stressed conditions reflecting that the method is stable.

Table No. 9: Forced degradation data of Glecaprevir and Pibrentasvir

Parameter	% Assay of Glecaprevir	% Assay of Pibrentasvir
Acid	94.26	94.47
Base	95.62	95.98
Peroxide	96.82	96.10
Thermal	97.92	97.51
UV	99.09	98.53
Water	99.42	99.43

CONCLUSION:

The current study developed and validated a stress-induced UPLC technique for estimation in API and tablets using ICH Q2R1 recommendations and results were within the acceptability limits. The developed stability indicating UPLC technique used a minimal volume of the mobile phase, less drug, and a shorter analysis time with sensitive detection limits. Therefore, the method helps to estimate Glecaprevir and Pibrentasvir in routine quality control analyses.

CONFLICT OF INTEREST STATEMENT:

The authors affirm that this manuscript does not involve any conflicts.

ACKNOWLEDGEMENTS:

The author wishes to express their gratitude to Spectrum Labs, Hyderabad, for giving free samples of Glecaprevir and Pibrentasvir.

LIST OF ABBREVIATIONS:

UPLC: Ultra Performance Liquid Chromatography

LOD: Limit of detection

LOQ: Limit of Quantification

SST: System suitability

Nm: Nanometer

mL: Milliliter

Min: minute

˚C: Celsius

V: Volume

Mg: Milligram

Rt: retention time

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Received on 20.01.2024 Modified on 28.03.2024

Research J. Pharm. and Tech 2024; 17(11):5356-5360.

DOI: 10.52711/0974-360X.2024.00818

Parameter	Glecaprevir			Pibrentasvir			Resolution
Parameter	Rt	Plate count	Tailing factor	Rt	Plate count	Tailing factor	Resolution
Flow (0.4 ml/min)	1.098	4621	1.14	1.467	5277	1.21	5.2
Flow (0.2 ml/min	1.132	4570	1.18	1.516	5371	1.24	5.4
M.P (25:75%v/v)	1.118	4328	1.14	1.607	5280	1.23	6.3
M.P (35:65 %v/v)	1.102	4636	1.15	1.399	5247	1.24	4.4
Temp (32˚C)	1.117	4289	1.15	1.563	5316	1.22	5.8
Temp (28˚C)	1.102	4523	1.16	1.420	5382	1.23	4.4