Stability indicating high performance liquid chromatography method development and validation for simultaneous estimation of Glecaprevir and Pibrentasvir in bulk and pharmaceutical dosage form
Mohan Goud V1*, Tharun Goud G2
1Department of Pharmaceutical Analysis, Joginpally B. R Pharmacy College,
Yenkapally, Moinabad, R.R. Dist. Telangana.
2Department of Pharmacognosy, University College of Pharmaceutical Sciences, Palamuru University, Mahabubnagar, Telangana.
*Corresponding Author E-mail: mohanvanga@yahoo.com
ABSTRACT:
A simple, accurate and precise method was developed for the simultaneous estimation of the Glecaprevir [GPV] and Pibrentasvir [PNV] in Tablet dosage form by using HPLC. The chromatogram was run through Std. Zodiac column (150 x 4.6 mm, 5m) by using the mobile phase containing Buffer 0.1% OPA: Acetonitrile taken in the ratio 55:45. The flow rate maintained at 1 ml/min. and temperature set to 30°C. Optimized wavelength selected was 260 nm. The retention time of GPV and PNV were found to be 2.207 min and 3.263 min. %RSD of the GPV and PNV were and found to be 0.2 and 0.1 respectively. Average % Assay of marketed formulation of Glecaprevir and Pibrentasvir obtained was 99.56 and 99.25% respectively. LOD, LOQ values obtained from regression equations of GPV and PNV were 0.32, 0.97 and 0.04, 0.11 respectively. Regression equation of GPV was y = 23779x + 9673 and of PNV was y = 13508x + 2326. From the experimental results, the retention times were decreased and run time was decreased, so that the method developed was simple and economical that can be adopted in regular Quality control test in Industries.
KEYWORDS: Glecaprevir, Pibrentasvir, RP-HPLC, Method development and validation.
INTRODUCTION:
GPV is a direct acting antiviral agent and Hepatitis C virus NS3/4A protease inhibitor that targets the viral RNA replication. In combination with PNV, GPV is a useful therapy for patients who experienced therapeutic failure from other NS3/4A protease inhibitors. The IUPAC name of GPV is (1R,14E,18R,22R,26S,29S)-26-tert-butyl-N-[(1R,2R)-2-(difluoromethyl)-1-{[(1-methylcyclopropyl) sulfonyl] carbamoyl} cyclopropyl]-13,13-difluoro -24,27-dioxo-2,17,23-trioxa-4,11,25,28-tetraazapentacyclo [26.2.1.0 {3,12}.0 {5,10}.0 {18,22}] hentriaconta-3,5(10),6,8,11,14-hexaene-29-carboxamide. PNV is a direct acting antiviral agent and Hepatitis C virus NS5A inhibitor that targets the viral RNA replication and viron assembly. In combination with GPV, PNV is a useful therapy for patients who experienced therapeutic failure from other NS5A inhibitors. The IUPAC name of PNV, is (2S,3R)-1-[(2S)-2-{5-[(2R,5R)-1-{3,5-difluoro-4-[4-(4-fluorophenyl) piperidin-1-yl] phenyl} -5-{6-fluoro-2-[(2S)-1-[(2S,3R)-2-{[hydroxyl (methoxy) methylidene] amino} -3-methoxy butanoyl] pyrrolidin-2-yl]-1H-1,3-benzodiazol-5-yl} pyrrolidin-2-yl]-6-fluoro-1H-1,3-benzodiazol-2-yl}pyrrolidin-1-yl]-2-{[hydroxy(methoxy) methylidene] amino}-3-methoxybutan-1-one.[1-5]
Figure 1: Structures for GPV and PNV
Literature review [6- 11] reveals that different methods for its analysis in formulations. Our present plan is to develop a new, sensitive, robustand accurate method for its analysis in formulation, after a detailed study, a new RP-HPLC method was decided to be developed and validated as per ICH norms [12, 13].
MATERIALS
AND METHODS:
Materials:
GPV and PNV (Spectrum lab Private Ltd.), Combination of GPV
and PNV (Mavyret) tablet dosage forms, distilled water (milli-Q), Acetonitrile,
phosphate buffer and potassium dihydrogen phosphate buffer. All chemicals, HPLC
grade, Merck, are purchased from local distributor.
Instruments:
HPLC instrument used was of Waters HPLC 2965 SYSTEM with Auto Injector and PDA Detector. Software used is Empower 2. UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz was be used for measuring absorbance for GPV and PNV solutions. Sonicator (Ultrasonic sonicator), PH meter (Thermo scientific), Micro balance (Sartorius), Vacuum filter pump (Welch) are the other instruments used for this study.
Analytical
methodology:
Preparation
of buffer (0.1% OPA):
Accurately 1ml of OPA in a 1000ml of Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water.
Standard
preparation:
Accurately Weighed and transferred 25mg of GPV and 10mg of PNV working Standards into a 25ml clean dry volumetric flask, add 3/4th volume of diluent, sonicated for 5 minutes and make up to the final volume with diluents. 1ml from the above stock solution was taken into a 10ml volumetric flask and made up to 10ml.
Sample
preparation:
1tablet was weighed, powdered and then was transferred into a 100mL volumetric flask, 50mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered. From the filtered solution 1 ml was pipetted out into a 10 ml volumetric flask and made up to 10ml with diluent.
Linearity:
Linearity solutions are prepared such that 0.25, 0.5, 0.75, 1, 1.25, 1.5ml from the Stock solutions of GPV and PNV are taken in to 6 different volumetric flasks and diluted to 10ml with diluents to get 25ppm, 50ppm, 75ppm, 100ppm, 125ppm, 150ppm of GPV, and 10ppm, 20ppm, 30ppm, 40ppm, 50ppm, 60ppm of PNV.
Precision:
Accurately weighed 25mg of GPV 10mg of PNV and transferred to 25ml volumetric flasks and 3/4 th of diluents was added to these flasks and sonicated for 10 minutes. Flask were made up with diluents and labeled as Standard stock solution (1000µg/ml of GPV and 400µg/ml PNV). From this solution 1ml of each was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (100µg/ml of GPV and 40µg/ml of PNV).
Accuracy:
Accurately weighed 25mg of GPV 10mg of PNV and transferred to 25ml volumetric flasks and 3/4 th of diluents was added to these flasks and sonicated for 10 minutes. Flask were made up with diluents and labeled as Standard stock solution. (1000µg/ml of GPV and 400µg/ml PNV). From this solution 0.5, 1.0 and 1.5ml was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent to produce 50, 100, 150% of spiked solution respectively.
METHOD
DEVELOPMENT:
UV
spectra of GPV and PNV
Overlay spectra gave the optimized wavelength for these two drugs. Optimized wavelength selected was 260nm.
Figure 2: Overlay UV spectra of GPV and PNV
Optimized
method:
Trials were performed for the method development and the best peak with least fronting factor was found to be with RT=2.207 min for GPV and 3.263 min for PNV.
Table 1: Chromatographic conditions
|
Mobile phase |
55%OPA (0.1%): 45% Acetonitrile |
|
Flow rate |
1ml/min |
|
Column |
Zodiac C18 (4.6 x 150mm, 5µm) |
|
Detector wavelength |
260nm |
|
Column temperature |
30°C |
|
Injection volume |
10mL |
|
Run time |
6 min |
|
Diluent |
Water and Acetonitrile in the ratio 50:50 |
Figure
3: Optimized chromatogram
System suitability:
According to ICH guidelines plate count should be more than 2000, tailing factor should be less than 2 and resolution must be more than 2. All the system suitable parameters were passed and were within the limits. (Table-2).
Table 2: System suitability
parameters for GPV and PNV
|
S. No. |
GPV |
PNV |
|||||
|
Injection |
RT(min) |
USP Plate Count |
Tailing |
RT (min) |
USP Plate Count |
Tailing |
USP Resolution |
|
1 |
2.201 |
2773 |
1.40 |
3.250 |
6245 |
1.21 |
6.2 |
|
2 |
2.206 |
3119 |
1.38 |
3.259 |
6165 |
1.19 |
6.3 |
|
3 |
2.207 |
3810 |
1.35 |
3.261 |
5955 |
1.12 |
6.3 |
|
4 |
2.208 |
3896 |
1.38 |
3.263 |
5948 |
1.10 |
6.5 |
|
5 |
2.214 |
3752 |
1.31 |
3.263 |
6281 |
1.13 |
6.7 |
|
6 |
2.221 |
4170 |
1.47 |
3.268 |
6523 |
1.19 |
6.8 |
METHODS
FOR VALIDATION:
Linearity:
Six linear concentrations of GPV (5-30µg/ml) and PNV (30-180µg/ml) were
injected in a duplicate manner. Average areas were mentioned above and
linearity equations obtained for GPV was y = 23779x + 9673 and of PNV was y =
13508x + 2326 Correlation coefficient obtained was 0.999 for the two drugs.
Table 3: Linearity table for GPV and PNV
|
GPV |
PNV |
||
|
Conc
(μg/mL) |
Peak area |
Conc
(μg/mL) |
Peak area |
|
0 |
0 |
0 |
0 |
|
25 |
612051 |
10 |
145461 |
|
50 |
1212540 |
20 |
267958 |
|
75 |
1779048 |
30 |
412192 |
|
100 |
2397881 |
40 |
532867 |
|
125 |
2966606 |
50 |
675059 |
|
150 |
3583660 |
60 |
819337 |
Figure 4: Calibration curve of GPV
Figure 5: Calibration curve of PNV
Precision:
From a single volumetric flask of working standard solution
six injections were given and the obtained areas were mentioned above. Average
area, standard deviation and % RSD were calculated for two drugs. % RSD
obtained as 0.2% and 0.1% respectively for GPV and PNV. As the limit of
Precision was less than “2” the system precision was passed in this method.
(Table-4).
Accuracy:
Three levels of Accuracy samples were prepared by standard
addition method. Triplicate injections were given for each level of accuracy
and mean %Recovery was obtained as 98.90% and 99.61% for GPV and PNV
respectively. (Table-5).
Robustness:
Robustness conditions like Flow minus (0.9ml/min), Flow
plus (1.1ml/min), mobile phase minus (40B:60A), mobile phase plus (50B:50A),
temperature minus (25°C) and temperature plus(35°C) was maintained and samples
were injected in duplicate manner. System suitability parameters were not much
affected and all the parameters were passed. %RSD was within the limit.
(Table-6).
Table 4: System precision table of GPV and PNV
|
S. No |
GPV |
PNV |
||
|
Peak area |
Day-Day precision Peak area |
Peak area |
Day-Day precision Peak area |
|
|
1. |
2349978 |
2322409 |
532792 |
521485 |
|
2. |
2347236 |
2326327 |
532786 |
527090 |
|
3. |
2349229 |
2306047 |
532712 |
526593 |
|
4. |
2340195 |
2312188 |
532147 |
524145 |
|
5. |
2349034 |
2307286 |
532783 |
526290 |
|
6. |
2340524 |
2316050 |
531952 |
523429 |
|
Mean |
2346033 |
2315051 |
532529 |
524839 |
|
S.D |
4486.9 |
8146.8 |
377.4 |
2189.4 |
|
%RSD |
0.2 |
0.4 |
0.1 |
0.4 |
Table 5: Recovery studies for GPV
and PNV
|
% Concentration |
GPV |
PNV |
||||
|
50% |
100% |
150% |
50% |
100% |
150% |
|
|
Trail-I |
98.92 |
99.13 |
99.15 |
99.65 |
99.08 |
99.68 |
|
Trail-II |
98.44 |
98.87 |
99.24 |
99.95 |
99.51 |
99.69 |
|
Trail-III |
98.45 |
98.45 |
99.43 |
99.68 |
99.46 |
99.82 |
|
AVG (%Recovery) |
98.90 |
99.61 |
||||
|
SD |
0.38 |
0.25 |
||||
|
%RSD |
0.38 |
0.25 |
||||
Table 6: Robustness data for GPV and PNV
|
S. No. |
Condition |
%RSD of Glecaprevir |
%RSD of Pibrentasvir |
|
1 |
Flow rate (-) 0.9ml/min |
0.4 |
1.4 |
|
2 |
Flow rate (+) 1.1 ml/min |
1.5 |
0.7 |
|
3 |
Mobile phase (-) 35B:65A |
1.2 |
0.2 |
|
4 |
Mobile phase (+) 45B:55A |
0.9 |
0.6 |
|
5 |
Temperature (-) 25°C |
0.5 |
1.2 |
|
6 |
Temperature (+) 35°C |
1.1 |
1.2 |
LOD and
LOQ:
LOD and LOQ were estimated from the signal-to-noise ratio.
The LOD for GPV and PNV were found to be 0.32 and 0.04μg/ml and the LOQ
were 0.97 and 0.11μg/ml respectively. (Table-7).
Table 7: LOD AND LOQ of GPV and PNV
|
Molecule |
LOD |
LOQ |
|
GPV |
0.32 |
0.97 |
|
PNV |
0.04 |
0.11 |
Degradation
Data:
Degradation studies were performed with the formulation and
the degraded samples were injected. Assay of the injected samples was
calculated and all the samples passed the limits of degradation.
(Table-8).
Assay of
marked formulation:
Abbvie pharmaceuticals, Mavyret tablet bearing the label claim
Glecaprevir 100mg, Pibrentasvir 40mg. assay was performed with the above
formulation. Average % Assay for Glecaprevir and Pibrentasvir obtained was
99.56and 99.25% respectively. (Table-9).
Table 8: Degradation data of GPV and PNV
|
Type of degradation |
GPV |
PNV |
||||
|
% Drug Degraded |
Purity Angle |
Purity Threshold |
% Drug Degraded |
Purity Angle |
Purity Threshold |
|
|
Acids |
4.72 |
0.209 |
0.391 |
4.98 |
2.400 |
2.831 |
|
Base |
2.97 |
0.259 |
0.381 |
2.91 |
3.108 |
3.387 |
|
Peroxide |
1.71 |
0.176 |
0.360 |
1.85 |
2.173 |
2.490 |
|
Thermal |
0.85 |
0.183 |
0.336 |
0.64 |
2.154 |
2.423 |
|
UV |
0.58 |
0.168 |
0.369 |
0.68 |
2.975 |
3.346 |
|
Water |
0.48 |
0.339 |
0.383 |
0.92 |
1.730 |
2.137 |
Table 9: Assay Data of Marked Formulation of GPV and PNV
|
S. No |
GPV |
PNV |
||||
|
Standard area |
Sample area |
% Assay |
Standard area |
Sample area |
% Assay |
|
|
1 |
2352409 |
2349978 |
99.73 |
535485 |
532792 |
99.29 |
|
2 |
2336327 |
2347236 |
99.61 |
537090 |
532786 |
99.29 |
|
3 |
2366047 |
2349229 |
99.69 |
536593 |
532712 |
99.28 |
|
4 |
2352188 |
2340195 |
99.31 |
534145 |
532147 |
99.17 |
|
5 |
2357286 |
2349034 |
99.69 |
536290 |
532783 |
99.29 |
|
6 |
2346050 |
2340524 |
99.32 |
533429 |
531952 |
99.14 |
|
AVG (%Recovery) |
2351718 |
2346033 |
99.56 |
535505 |
532529 |
99.25 |
|
SD |
10060.1 |
4486.9 |
0.19 |
1447.2 |
377.4 |
0.07 |
|
%RSD |
0.4 |
0.2 |
0.19 |
0.3 |
0.1 |
0.07 |
RESULTS
AND DISCUSSION:
A simple, rapid and precise method has been developed and validated for the drug GPV and PNV. The estimation was carried out with a mixture of Phosphate buffer and Acetonitrile in the ratio of 55:45%v/v. Precision of the methods were studied by making repeated injections of the samples and system precision values were determined. The retention time was 2.207 min for GPV and 3.263 min for PNV. The calibration curve was linear over the concentration range of GPV (5-30µg/ml) and PNV (30-180µg/ml). The LOD for GPV and PNV were found to be 0.32 and 0.04μg/ml and the LOQ were 0.97 and 0.11μg/ml respectively. The high percentage of recovery and low percentage coefficient of variance confirm the suitability of the method. Hence it was concluded that the RP-HPLC method developed was very much suit for routine analysis.
Table 10: Result and Discussion data of GPV and PNV
|
S.No. |
Parameter |
Acceptance criteria |
Observed value |
|
|
GPV |
PNV |
|||
|
1 |
Linearity |
R2 not less than 0.99 |
R2=0.999 |
R2=0.999 |
|
2 |
Precision |
RSD within 2% |
0.2 |
0.1 |
|
3 |
Accuracy |
98-102% |
98.90% |
99.61% |
|
4 |
LOD |
S/N=3 |
0.32 |
0.97 |
|
5 |
LOQ |
S/N=10 |
0.04 |
0.11 |
CONCLUSION:
In the present investigation, from the above experimental results it was concluded that, the newly developed RP-HPLC method was simple, specific, accurate and precise. The method was effectively validated in terms of system suitability, linearity, precision, accuracy, range, LOD, LOQ and robustness and stability indicating studies according to ICH guidelines. Hence the developed method can use for estimation of Glecaprevir and Pibrentasvir in control department in pharmaceutical industries and testing laboratories.
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Received on 17.05.2020 Modified on 19.06.2020
Accepted on 10.07.2020 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2020; 10(3): 141-146.
DOI: 10.5958/2231-5675.2020.00025.3