Development and validation of UPLC method for simultaneous estimation of Darunavir, Cobicistat, Emtricitabine and Tenofovir alafenamide in bulk drug and pharmaceutical dosage form
Vamsi Dadi*, G. Sowjanya
Department of Pharmaceutical Analysis, GITAM School of Pharmacy, GITAM (Deemed to be University), Rushikonda, Visakhapatnam, India.
*Corresponding Author E-mail: vamsidadi88@gmail.com
ABSTRACT:
The main objective of the present study was to develop and validate a multicomponent analysis of Darunavir, Cobicistat, Emtricitabine and Tenofovir alafenamide in bulk drug and pharmaceutical dosage form by ultra-performance liquid chromatography (UPLC). The drugs were separated using HSS C18 (100mm × 2.1mm, 1.8µ particle size) column with mobile phase consisting of potassium dihydrogen phosphate, pH adjusted to 5.4 and acetonitrile in the composition of 60:40 %v/v operated in isocratic mode at a flow rate of 0.3 ml/min. The column temperature maintained at 30˚C and detection wavelength used was 260 nm. The retention time for Darunavir was found to be 1.04min, for Cobicistat it was 1.82 min, for Emtricitabine it was 2.28 min and for Tenofovir it was found to be 1.39min. The developed method was validated in accordance to the ICH guidelines. The method obeyed Beer’s law in the concentration range of 50 µg/ml- 300µg/ml for Darunavir, 7.5 µg/ml – 45 µg/ml for Cobicistat, 25µg/ml - 150µg/ml for Emtricitabine and 0.625 µg/ml – 3.75µg/ml for Tenofovir alafenamide, with correlation coefficient of 0.999. The % relative standard deviation for all the drugs was found to be less than two indicating the method to be precise. The mean % recovery was found to be within the limits for all the drugs indicating that the method to be accurate. The method was also found to be specific, robust and stable. From the forced degradation studies, it was concluded that the drugs were found to be stable when exposed to different stress conditions as the net degradation was found within the acceptance criteria. The developed method for the multicomponent analysis of Darunavir, Cobicistat, Emtricitabine and Tenofovir alafenamide using UPLC can be used for quality control and routine analysis in bulk drug and pharmaceutical dosage form.
KEYWORDS: Darunavir, Cobicistat, Emtricitabine, Tenofovir alafenamide, UPLC, Validation.
INTRODUCTION:
Darunavir ethanolate1-3 (Figure 1a) with chemical name [(1S,2R)-3-[[(4-aminophenyl)sulfonyl](2-methylpropyl)amino]-2-hydroxy-1-(phenylmethyl)propyl]-carbamic acid (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl ester monoethanolate, is as anti-retroviral agent. It has a molecular formula of C27H37N3O7S.C2H5OH with molecular weight 593.73.
It is used in the treatment of HIV infections as it inhibits the human immunodeficiency virus (HIV-1) protease enzyme4-6. Cobicistat7-9 (Figure 1b) with chemical name 1,3-thiazol-5-ylmethyl [(2R,5R)-5-{[(2S)-2-[(methyl{[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl}carbamoyl)amino]-4-(morpholin-4-yl)butanoyl]amino}-1,6-diphenylhexan-2-yl]carbamate and it belongs to antiviral category. It is used for the treatment of HIV infection and AIDS10-12. Emtricitabine 13-15 (Figure 1c) with chemical name 4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one, is a white to off-white powder, soluble in water and methanol and practically insoluble in methylene chloride with pKa value of 2.65. It is used as antiretroviral drug in the treatment of HIV and AIDS as it inhibits the nucleoside reverse-transcriptase enzyme16-18. Tenofovir alafenamide19-21 (Figure 1d) with chemical name isopropyl (2S)-2-[[[(1R)-2-(6-amino-9H-purin-9-yl)-1-methyl-ethoxy]-methyl-phenoxy-phosphoryl]amino]propanoate belongs to anti-viral category. It has an empirical formula of C21H29O5N6P with molecular weight 476.474. It is used in the treatment of chronic hepatitis B virus infection as it acts by inhibiting hepatitis B virus nucleoside analog reverse transcriptase enzyme22-25. According to the literature study, it was found that there were methods developed for two drug combination like Darunavir and Cobicistat26 or Emtricitabine and Tenofovir alafenamide27 but only two analytical methods28-32 were developed till now for the simultaneous estimation of Darunavir, Cobicistat, Emtricitabine and Tenofovir alafenamide in pharmaceutical dosage form. The main objective of the proposed study was to develop and validate an UPLC method for the multicomponent analysis of Darunavir, Cobicistat, Emtricitabine and Tenofovir alafenamide in pharmaceutical dosage form.
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Figure 1a: Chemical structure of Darunavir |
Figure 1b: Chemical structure of Cobicistat |
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Figure 1c: Chemical structure of Emtricitabine |
Figure 1d: Chemical structure of Tenofovir alafenamide |
MATERIALS AND METHODS:
Chemicals and reagents: Darunavir, Cobicistat, Emtricitabine and Tenofovir alafenamide working standards were received from Hetero Labs Pvt. Ltd., Hyderabad, Telangana as gift samples. Symtuza tablets (marketed formulation) was purchased from the local pharmacy. All the chemicals used in the method were of analytical grade and procured from Merck, India and the solvents used were of HPLC grade and purchased from Merck, India.
Instruments and Chromatographic conditions:
The acquity UPLC system equipped with binary solvent manager, sample manager, ultra violet (UV) detector, with empower 2 software was used as the instrument for the separation of drugs. Optimized chromatographic conditions were HSS C18 (100mm × 2.1mm, 1.8µ particle size) column with potassium di-hydrogen phosphate, pH 5.4 and acetonitrile (60:40%v/v) as mobile phase run in isocratic mode at a flow rate of 0.3ml/min. The column temperature was maintained at 30˚C with detection wavelength set at 260nm. Other instruments used were pH meter (Lab India), weighing machine (Sartorius) and digital ultra sonicator (Labman). Glassware such as volumetric flasks, pipettes, burettes and beakers made of borosil was used.
Preparation of phosphate buffer:
Accurately weighed 1.36g of potassium dihydrogen phosphate was dissolved in 1000ml of distilled water and the pH was adjusted to 5.4 using dilute ortho phosphoric acid.
Preparation of mobile phase:
Mobile phase was prepared by mixing 60% of above prepared phosphate buffer and 40% of acetonitrile.
Preparation of standard and sample solution: Accurately weighed 100mg of Darunavir, 18.75mg of Cobicistat, 25mg of Emtricitabine and 1.25mg of Tenofovir alafenamide working standards were transferred into four 50ml volumetric flask separately and the volume was made up to the mark with diluent. Pipette out 1ml each from above four stock solutions and dilute to 10ml with diluent to prepare standard solution.
Average weight of 5 symtuza tablets were calculated and an amount equivalent to 100mg Darunavir was dissolved in 50ml of diluent. The solution was sonicated for 25min and later filtered using 0.45µ filters. From the above stock solution, 1ml was diluted to 10ml using diluent to the sample solution.
Method validation33,34:
System suitability: The prepared standard solution was injected into the UPLC five times and the system suitability parameters such as % relative standard deviation (RSD), tailing factor, plate count and resolution were estimated.
Specificity:
Specificity of the method was determined by injecting the placebo solution into the chromatographic system and the interference of placebo peaks with drug peaks was checked.
Linearity:
Linearity of the method was detected by preparing serial dilutions in the concentration range of the 50µg/ml - 300µg/ml for Darunavir, 7.5µg/ml - 45µg/ml for Cobicistat, 25µg/ml - 150µg/ml for Emtricitabine and 0.625µg/ml – 3.75µg/ml for Tenofovir alafenamide, and for these concentrations, peak area was measured by inject in to the chromatographic system. Later a graph was plotted between peak area and concentration values to detect the correlation coefficient.
Accuracy:
In order to determine the accuracy of the developed method, three concentration levels 50%, 100% and 150% was prepared by spiking method and the % recovery was evaluated.
Precision:
Repeatability: For the determination of repeatability, % RSD was calculated from the peak areas obtained by the injecting the six replicate sample solutions.
Intermediate precision: For the estimation of intermediate precision, %RSD was calculated from the peak areas obtained by injecting the six replicates of sample solution on two different days under same conditions.
Limit of detection (LOD) and Limit of quantitation (LOQ):
LOD and LOQ were calculated by using the slope of the calibration curve and standard deviation values.
Robustness:
Robustness of the method was determined by slightly altering the optimized chromatographic conditions such as ±5% organic phase variation in mobile phase composition, ±0.1ml/min flow rate and ±5°C column temperature and calculating the %RSD.
Forced degradation studies:
Under forced degradation studies35,36,37, the standard drug solution was exposed to different stress conditions such as acidic (2N hydrochloric acid for 30mins at 60˚C), basic (2N sodium hydroxide for 30mins at 60˚C), peroxide (20% hydrogen peroxide for 30 min at 60˚C), neutral (refluxing the drug in water for 6 hours at 60ºC), photolytic (105°C for 6h) and thermal (exposing the drug solution to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m2 in photo stability chamber) conditions.
RESULTS AND DISCUSSION:
Initially based on trial and error method, many mobile phase compositions were tried and finally phosphate buffer, pH 5.4 and acetonitrile in the ratio 60:40%v/v was selected as optimized mobile phase. Similarly many columns were tried and finally HSS C18 (100mm × 2.1mm, 1.8µ) column was selected as they gave good separation with acceptable values. The detection wavelength was selected as 260nm based on the UV overlay spectrum as shown in figure 2.
Figure 2: Overlay UV spectrum of Darunavir, Cobicistat, Emtricitabine and Tenofovir
After finalizing the chromatographic conditions, the prepared standard and sample solutions were injected in the UPLC and the values were noted. The retention time for Darunavir, Cobicistat, Emtricitabine and Tenofovir was found to be 1.04 min, 1.82 min, 2.28 min and 1.39 min respectively. The chromatograms of standard and sample solutions were presented in figure 3a and 3b respectively. After the development of the method, the method was validated as per the ICH guidelines. The system suitability parameters like % RSD, plate count, tailing factor and resolution were evaluated and the results were found to be within the acceptance criteria and summarized in Table 1. The method was found to be specific as there was no interference of excipient peaks with the drug peaks when placebo solution was injected into the UPLC.
Figure 3a: Standard chromatogram of Darunavir, Cobicistat, Emtricitabine and Tenofovir
Figure 3b: Sample chromatogram of Darunavir, Cobicistat, Emtricitabine and
Tenofovir
Table 1: Validation parameter results
Parameters |
Darunavir |
Cobicistat |
Emtricitabine |
Tenofovir |
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Specificity |
Specific, no interference |
Specific, no interference |
Specific, no interference |
Specific, no interference |
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Linearity |
Regression equation, y=mx+c |
y = 16538x + 12186 |
y = 33193x + 7100.1 |
y = 34913x + 5334.4 |
y = 87354x + 2258.4 |
|
Correlation coefficient (r) |
0.9998 |
0.9999 |
0.9999 |
0.9997 |
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Accuracy (recovery) n=3 |
Level I (50%) |
99.98% |
99.97% |
100.25% |
100.30% |
|
Level II (100%) |
99.39% |
100.68% |
100.32% |
99.42% |
||
Level III (150%) |
99.16% |
98.82% |
99.93% |
101.27% |
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Precision, Repeatability (%RSD) n=5 |
1.0 |
1.0 |
1.0 |
0.7 |
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Intermediate Precision (%RSD) n=5 |
Day 1 |
0.5 |
0.6 |
1.3 |
1.5 |
|
Day 2 |
0.9 |
1.5 |
0.7 |
1.7 |
||
Limit of Detection (LOD) |
0.79µg/mL |
0.16µg/mL |
0.27µg/mL |
0.02µg/mL |
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Limit of Quantitation (LOQ) |
2.40µg/mL |
0.48µg/mL |
0.81µg/mL |
0.06µg/mL |
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Robustness |
Robust |
Robust |
Robust |
Robust |
||
Solution stability |
Stable |
Stable |
Stable |
Stable |
||
System Suitability |
USP Plate Count |
3432 |
4763 |
5056 |
3525 |
|
USP Tailing |
1.36 |
1.19 |
1.15 |
1.29 |
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Resolution |
- |
4.3 |
3.9 |
4.2 |
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The placebo chromatogram was shown in Figure 4. The developed method was found to be linear in the concentration range of 50-300 µg/ml for Darunavir, 7.5-45 µg/ml for Cobicistat, 25-150 µg/ml for Emtricitabine and 0.625-3.75 µg/ml for Tenofovir alafenamide, as the correlation coefficient was found to be 0.999. The linearity plots of the drugs were presented in Figure 5a, 5b, 5c and 5d.
Figure 4: Chromatogram of placebo
Figure 5: Linearity
The method was found to be accurate, as the % recovery for Darunavir was found to be 99.16% - 99.98%, for Cobicistat % recovery was found to be 98.82% - 100.68%, for Emtricitabine % recovery was found to be 99.93% - 100.32% and for Tenofovir % recovery was found to be 99.42% - 101.27% with all the reading falling within the acceptance criteria. The % RSD values for all the drugs under repeatability and intermediate precision studies were found to be less than 2%, indicating the method to be precise. The method was found to be robust as the % RSD results were within the acceptable range i. e. less than two when the optimized conditions were slightly altered by varying the organic phase in mobile phase composition by ±5%, flow rate by ±0.1ml/min and column temperature by ±5°C.
When the standard drug solution was exposed to various stress conditions like acidic, basic, oxidative, neutral, photolytic and thermal conditions, and then analyzed the net degradation was to be within the limits, indicating that the drug solution was found to be stable. The results for forced degradation studies were summarized in Table 2 and the degradation chromatograms were presented in Figure 6.
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|
a: Acid degradation |
b: Base degradation |
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c: Peroxide degradation |
d: Thermal degradation |
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e: Photolytic degradation |
f: Neutral degradation |
Figure 6: Chromatograms during forced degradation study
Table 2: Forced degradation study results
Drug |
|
Stress condition |
|||||
Acidic |
Basic |
Peroxide |
Thermal |
Photolytic |
Neutral |
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Darunavir |
% Assay |
94.37 |
95.09 |
95.75 |
97.90 |
98.59 |
99.42 |
% degradation |
5.63 |
4.91 |
4.25 |
2.10 |
1.41 |
0.58 |
|
Cobicistat |
% Assay |
94.61 |
95.50 |
96.64 |
97.72 |
98.31 |
99.12 |
% degradation |
5.39 |
4.50 |
3.36 |
2.28 |
1.69 |
0.88 |
|
Emtricitabine |
% Assay |
94.11 |
95.59 |
95.75 |
97.68 |
98.97 |
99.55 |
% degradation |
5.89 |
4.41 |
4.25 |
2.32 |
1.03 |
0.45 |
|
Tenofovir |
% Assay |
94.51 |
95.24 |
95.62 |
96.37 |
98.06 |
99.38 |
% degradation |
5.49 |
4.76 |
4.38 |
3.63 |
1.94 |
0.62 |
CONCLUSION:
A new specific, precise and accurate UPLC method was developed for the multicomponent analysis of Darunavir, Cobicistat, Emtricitabine and Tenofovir alafenamide in the pharmaceutical dosage form. The developed method was validated as per the ICH guidelines, indicating the method to be linear, precise, accurate and robust. From the forced degradation studies, it was evident that the drugs were stable when exposed to forced degradation conditions, with less degradation. This developed method can be used for the routine analysis of Darunavir, Cobicistat, Emtricitabine and Tenofovir in quality control of various pharmaceutical dosage forms.
ACKNOWLEDGEMENT :
The authors are thankful to the Hetero Labs Pvt. Ltd., Hyderabad for providing the gift samples and to the Spectrum labs, Hyderabad, India for providing facilities to carry out this research work. The authors declare that there is no conflict of interest.
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Received on 08.03.2023 Modified on 12.04.2023
Accepted on 10.05.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(5):2336-2342.
DOI: 10.52711/0974-360X.2023.00384