GRADIENT HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF RELATED SUBSTANCES IN [(3aS, 4R, 6aR)-2, 3, 3a, 4, 5, 6a-HEXAHYDROFURO [2, 3-b] FURAN-4-YL] N-[(2S, 3R)-4-[(4-AMINOPHENYL) SULFONYL-(2-METHYLPROPYL) AMINO]-3-HYDROXY-1-PHENYLBUTAN-2YL] CARBAMATE DOSAGE FORM

GRADIENT HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF RELATED SUBSTANCES IN [(3aS, 4R, 6aR)-2, 3, 3a, 4, 5, 6a-HEXAHYDROFURO [2, 3-b] FURAN-4-YL] N-[(2S, 3R)-4-[(4-AMINOPHENYL) SULFONYL-(2-METHYLPROPYL) AMINO]-3-HYDROXY-1-PHENYLBUTAN-2YL] CARBAMATE DOSAGE FORM

B. Karuna Kumar ^{* 1}, V. Guna Sekaran ² and K. B. Chandra Shekar ³

Department of Pharmaceutical Analysis ¹, Sri Venkateswra College of Pharamacy, Chittoor - 517127, Andhra Pradesh, India.

Department of Pharmaceutical Analysis ², Rao’s College, Nellore - 524001, Andhra Pradesh, India.

Department of Chemistry ³, Jawaharlal Nehru Technological University, Anantapur- 515002, Andhra Pradesh, India.

ABSTRACT: A simple, selective, linear, precise and accurate RP-HPLC method was developed and validated for quantification of Darunavir in bulk and its tablet dosage form. The separation was carried out on Zorbax Eclipse XDB C18 (150 × 4.6 mm; 5 µm) column at 35 °C temperature using ammonium acetate buffer as mobile phase-A, acetonitrile (100%) as mobile phase-B. The flow rate was 1.0 ml/min and effluent was detected at 265 nm. The retention time of Darunavir propylene glycolate was 31.34 min. The percentage recovery was within the range between 95.46% and 100.17% for Darunavir propylene glycolate. The linear ranges were found to be 0.086-3.084 µg/ml (r² = 0.9997) for Darunavir propylene glycolate. The percentage relative standard deviation for accuracy and precision was found to be less than 5.0%. Hence, the method could be successfully applied for routine analysis of Darunavir in pharmaceutical formulations.

RP-HPLC, Forced degradation, Darunavir

INTRODUCTION: Darunavir is a white to off-white hygroscopic powder. Its solubility in organic solvents varies significantly and it is very slightly soluble in aqueous solution (solubility increases with decreasing pH). Therefore, the particle size is likely to be important to the rate and possibly to the extent of absorption of Darunavir. It contains 5 chiral centers, however the manufacturing process leads, in a consistent way, to the single enantiomer 3R, 3aS, 6aR, 1S, 2R ^1-2.

The absolute configuration has been confirmed by X-ray diffraction analysis. Under commercial synthesis conditions, darunavir is isolated as a crystalline ethanolate (1:1 solvate). It can exist as a non-solvated amorphous form and as a hydrate form as well Darunavir is a protease inhibitor used to treat HIV. It acts on the HIV aspartyl protease which the virus needs to cleave the HIV polyprotein into its functional fragments. It is used under the brand name of Prezista and it is used with other combination drug like Darunavir ethanolate, cobicistat, ritonavir but a few combinations give good results in the treatment of HIV.

MATERIALS AND METHODS:

Instrumental and Analytical Conditions:

Reagents and Chemicals: The drug Darunavir substances and its process related impurities were gifted by Active Pharma Labs. (Hyderabad, India). Buffer salts were purchased from Merck and Sigma Aldrich, India. Highly purified water for HPLC was obtained from Milli Q plus water purifying system, Millipore. Methanol and acetonitrile of HPLC grade were obtained from RANKEM, India. Mobile phase was vacuum filtered through a 0.45µm Polyvinyl Dene Fluoride (PVDF) filter membrane and degassed using a sonicator to remove the dissolved gases.

FIG. 1: CHEMICAL STRUCTURE OF DARUNAVIR

Equipment: Waters e2695 gradient system with Empower-3 software and 2996 module Photo Diode Array detectors equipped with a quaternary solvent delivery pump, automatic sample injector and column thermostat was used for the analysis.

Chromatographic Conditions:

Column                : Zorbax Eclipse XDB C18 (150

mm × 4.6mm), 5µ or equivalent

Pump mode           : Gradient

Flow rate                  : 1.0 mL/min.

Detection                   : UV, 265 nm.

Injection volume         : 20 µL.

Column oven temp       : 35 °C

Sample cooler temp         : 6 °C

Date acquisition time        : 55 min

TABLE 1: GRADIENT PROGRAMME

Preparation of Solutions:

System Suitability Solution: Dissolve 1.6 mg of Darunavir enriched with cyclic carbamate reference sample in 1 ml of diluent.

Standard Solution: Weigh and transfer about 57 mg of Darunavir propylene glycol ate working standard (equivalent to about 50 mg of darunavir) in to a 50 mL clean, dry volumetric flask, add about 35 mL of diluent and sonicate to dissolve. Dilute to volume with diluent and mix. Diluent 5 mL of this solution to 50 mL with diluent and mix. Dilute 5 mL of this solution to 100 mL with diluent and mix. Filter the solution through Millipore PVDF 0.45 µm membrane filter or suitable ^5-6.

Sample Solution: Weigh and finely powder not less than 10 tablets using a suitable mortar and pestle. Weigh and transfer tablet powder equivalent to about 200 mg of Darunavir in to a 200 mL clean, dry volumetric flask. Add about 150 mL of diluent and sonicate for 30 min at room temperature with intermittent shaking at about each 5 min interval. Dilute to volume with diluent and mix. Filter the solution through Millipore PVDF 0.45 µm membrane filter or suitable ⁷.

Placebo Solution: Weigh and transfer placebo powder equivalent to about 50 mg of Darunavir into a 50 mL clean, dry volumetric flask. Add about 30 mL of diluent and sonicate for 30 min at room temperature with intermittent shaking at about each 5 min interval. Dilute to volume with diluent and mix. Filter the solution through Millipore PVDF 0.45 µm /mdi Nylon 0.45 µm membrane filter or suitable ⁸.

TABLE 2: ELUTION ORDER

Method Validation: The above method was validated according to ICH guidelines to establish the performance characteristics of a method (expressed in terms of analytical parameters) to meet the requirements for the intended application of the method ⁹.

Validation Parameters: ^10-12

1. System suitability
2. System & method precision
3. Intermediate precision
4. LOD and LOQ
5. Linearity
6. Robustness
7. Specificity

Spectrum Index for All impurity Mix: Injected all impurity mix solution in to HPLC and pick the spectrums of all impurities Fig. 2.

FIG. 2: SPECTRUM INDEX

Conclusion: As per spectrum index the ƛ(Lamda) max concluded 265nm.

System Suitability Solution: Dissolve 1.6 mg of Darunavir enriched with cyclic carbamate reference sample in 1 ml of diluent. All the system suitability parameters are within range and satisfactory as per ICH guidelines ^6-8.

System Precision:

Preparation of Solution: Weigh and transfer about 57 mg of Darunavir propylene glycolate working standard into a 50 mL of clean, dry volumetric flask, add about 35 mL of diluent and sonicate to dissolve. Dilute to volume with diluent and mix. Dilute 5 mL of this solution to 50 mL of with diluent and mix. Dilute 5 mL of this solution to 100 mL with diluent and mix (Concentration: about 5 ppm). The standard solution was injected for six times and the areas of chromatograms for all six injections were measured in HPLC. The % RSD for the area of six replicate injections was found to be within the specified limits ^9-10.

TABLE 3: SYSTEM SUITABILITY STUDIES OF DARUNAVIR METHOD

TABLE 4: SYSTEM PRECISION RESULTS FOR DARUNAVIR

% RSD peak areas of darunavir obtained from six replicate injections of standard solutions should not more than 5.0%.

Conclusion: % RSD of six standard injections was found 0.2.

Method Precision: Six sample solutions were prepared individually using single batch of Darunavir tablets spiked known related substances at specification level and injected into HPLC ^9-12.

TABLE 5: METHOD PRECISION RESULTS FOR DARUNAVIR

TABLE 6: RUGGEDNESS RESULTS FOR DARUNAVIR

% RSD should not be more than 10.0 for the results of related substances and Darunavir from the six determinations ^9-12.

Conclusion: % RSD of all the Impurities was found within the limit. The above results that the test method is precise for the determination of related substances in Darunavir tablets.

% RSD should not be more than 10.0 for the results of Related Substances and Darunavir from the six determinations.

Conclusion: % RSD of all the Impurities was found within the limit.

The above results that the test method is precise for the determination of related substances in Darunavir tablets.

Limit of Detection and Limit of Quantitation: The limit of detection (LOD) and limit of quantitation (LOQ) values of Darunavir and its known related substances were determined using the values of slope, standard deviation and responses of individual analytes that have been obtained from the linearity study carried out from 1% to 150% of specification level for known related substances and for 1% to 150% standard concentration level for Darunavir ^11-12.

TABLE 7A: LOD AND LOQ RESULTS FOR DARUNAVIR

TABLE 7B: LOD AND LOQ RESULTS FOR DARUNAVIR

Accptance Criteria:

LOD: RSD should not be more than 33.0.

LOQ: RSD should not be more than 10.0%.

Conclusion: The above results within acceptance criteria the above reported LOQ values for Darunavir and its known related substances are below 50% of specification level for known related substances and below 50% of standard concentration level for Darunavir. Hence, the method is precise for the Quantitation of the related substances in Darunavir tablets.

Robustness: Standard and sample solution spiked with known related substance at specification level were prepared as per test method and injected into HPLC at different deliberately varied conditions to evaluate method ability to remain unaffected ^5-8.

TABLE 8A: ROBUSTNESS RESULTS FOR DARUNAVIR

TABLE 8B: ROBUSTNESS RESULTS FOR DARUNAVIR

TABLE 8C: ROBUSTNESS RESULTS FOR DARUNAVIR

The varied conditions include change in flow rate by ± 10%, column oven temperature by ± 5% °C, gradient composition by ± 1% absolute with respect to mobile phase-B, wavelength by ± 5 nm, organic composition in mobile phase-A by ± 1 and pH of the buffer ± 0.1 units ^8-12.

Conclusion: The system suitability result at each of the varied conditions complied with the requirements as per the test procedure. Also, it was observed from the chromatograms of sample spiked with known related substances obtained from different robust conditions outlined above that, there is no significant variation in relative retention times (RRT) of related substances obtained at each varied conditions.

Hence, it can be concluded that the test method is robust for Related Substances in Darunavir tablets across the extent of changes studied for each of the above parameters.

Recovery:

1. Diamino Alcohol: Potency-96.40%

Stock-1: Weigh and transfer 0.86 mg of Di amino Alcohol impurity in 20 mL clean dry volumetric flask and dilute with Diluent and mix.

Stock-II: Pipette out 5 mL from Stock-I solution in 25 mL and dilute with diluent and mix.

Preparation of Linearity Levels:

5% Level 1- Pipette out 0.50 mL from Stock-II solution in 50 mL and dilute with diluent and mix. Concentration 0.086(µg/mL).

25% Level 2- Pipette out 1.20 mL from Stock-II solution in 25 mL and dilute with diluent and mix. Concentration 0.414(µg/mL)

50% Level 3- Pipette out 1.00 mL from Stock-II solution in 10 mL and dilute with diluent and mix. Concentration 0.863(µg/mL)

100% Level 4- Pipette out 2.00 mL from Stock-II solution in 10 mL and dilute with diluent and mix. Concentration 1.727(µg/mL)

125% Level 5- Pipette out 2.50 mL from Stock-II solution in 10 mL and dilute with diluent and mix. Concentration 2.158(µg/mL)

150% Level 6- Pipette out 3.00 mL from Stock-II solution in 10 mL and dilute with diluent and mix. Concentration 2.590(µg/mL) ^9-12

Concentration (µg/mL) =Weight of impurity/ Dilution × volume taken/ Dilution×Potency/100×1000

TABLE 9A: ACCURACY RESULTS OF DARUNAVIR AND ITS IMPURITIES

TABLE 9B: ACCURACY RESULTS OF DARUNAVIR AND ITS IMPURITIES

Forced Degradation: Forced degradation is a process whereby the natural degradation rate of a product is increased by the application of more stress. Forced degradation studies are used to identify reactions which may occur to degrade a processed product. Long term storage tests are usually used to measure similar properties when final formulations are involved because of the stringent FDA regulations. Degradation of Darunavir was found to occur under acidic condition (2M HCl, 120 min at 85 °C), alkaline condition (2M NaOH, 15 min at RT), oxidative condition (10% H₂O₂, 15 min at 85 °C), photolytic degradation (White Fluorescent light, 1.2 million Lux for 24 h), humidity degradation (90% RH/ 25°C /120 h) and thermal degradation (the oven at 60 °C for 120 h). The developed RP-HPLC method can be used to analyze from its degradation products and hence found to be specific for Darunavir ⁹.

TABLE 10: FORCED DEGRADATION

Acceptance Criteria: Darunavir peak should be homogeneous and there should be no co-eluting peaks. Peak purity of Darunavir peak should pass as per Acceptance criteria ¹⁴.

System Suit Chromatogram:

FIG. 3: SYSTEM SUIT CHROMATOGRAM

Linearity:

FIG. 4: LINERITY-5%                                                          

FIG. 5:  RF-LINERITY-25%

FIG. 6: LINERITY-50%                                                          

FIG. 7:  RF-LINERITY-100%

FIG. 8: LINERITY-125%                                                        

FIG. 9:  RF-LINERITY-150%

Calibration Curves:

FIG. 10: DIAMINO ALCOHOL                                                

FIG. 11: PROPYL ANALOG

FIG. 12: DARUNAVIR                                                      

FIG. 13: CYCLIC CARBAMATE

FIG. 14: STEREO ISOMER                                                 

FIG. 15: N-BIS THF DARUNAVIR

FIG. 16: O-BIS THF DARUNAVIR                                      

CONCLUSION: A simple, Accurate, precise method was developed for the determination of the Darunavir in Tablet dosage form. Retention time of Darunavir were found to be 31.34 min. % RSD of the Darunavir were and found to be 0.2.% Recovery was Obtained as 98.40% Darunavir. LOD, LOQ values were obtained from regression equations of Darunavir were % RSD-3.4, and 0.9 respectively. Regression equation Darunavir is y = 42504.x + 51.43. The method developed was simple and economical that can be adopted in regular Quality control test in Industries.

ACKNOWLEDGEMENT: The authors are thankful to the Active Pharma Labs. (Hyderabad, India) for providing the necessary facilities to carry out for the research work.

COMPETING INTEREST: The authors declare no conflict of interest.

REFERENCES:

1. Correa JC, Sera CH, Salgado HR: Stability study of darunavir ethanolate tablets applying a new stability-indicating HPLC method. Chromatog Res Int 2013; 1: 1-7.
2. Reddy BV, Jyothi G, Reddy BS, Subhash K, Rambabu C. J Chromatogr Sci 2012; 51: 471-76.
3. Das NR and Vidyasagar G: BioMedRx 2013; 1(5): 529-35.
4. Balamuralikrishna K: Development and validation of analytical procedure for the simultaneous estimation of efavirenz, lamivudine and zidovudine through new RP-HPLC method. J Pharm Res 2011; 4: 3766-8.
5. Reddy BVR and Jyothi G: Journal of Chromatographic Science 2013; 51(5): 471-76.
6. Reddy AVB and Yusop Z: Journal of Pharmaceutical and Biomedical Analysis 2016; 128(5): 141-48.
7. Ramesh B, Ramakrishna S, Reddy RKK, Babu KH, MaheswaraSarma VU and Devi PS: HPTLC method for determination of Darunavir in rat plasma and its application in pharmacokinetic studies. J Liq Chromatogr Relat Technol 2013; 36(2): 167-79.
8. Shinde VR, Gosavi SA, Pawar SS, Kasture VS, Musmade DS and Shinde VR: Development and validation of UV spectroscopic method for determination of Darunavir in bulk and tablet formulation. Inventi Rapid: Pharm Anal Qual Assur 2013; 12: 586.
9. Gupta A, Singhal P, Shrivastav PS and Mallika S: Application of a validated ultra performance liquid chromatography-tandem mass spectrometry method for the quantification of Darunavir in human plasma for a bioequivalence study in Indian subjects. J Chromatogr B 2011; 879(24): 2443-53.
10. British Pharmacopoeia: the Stationary Office, London, 2005.
11. “Martindale - The Extra Pharmacopoeia”, The Pharmaceutical Press, London, Edition 33^rd, 2002; 7.
12. D'Avolio A, Simiele M, Siccardi M, Baietto L, Sciandra M and Oddone V: A HPLC-MS method for the simultaneous quantification of fourteen antiretroviral agents in peripheral blood mononuclear cell of HIV infected patients optimized using medium corpuscular volume evaluation. J of Pharmaceutical & Biomedical Anal 2011; 54: 779-88.
13. Else L, Watson V, Tjia, Hughes A, Siccardi M and Khoo S: Validation of a rapid and sensitive High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) assay for the simultaneous determination of existing and new antiretroviral compounds. Journal of Chromatography B 2010; 878: 1455-65.
14. Raveendra BG, Ramprasad LA, Pamidi S, Jayachandra RP and Rao JVLNS: New RP-HPLC method for the determination of Darunavir in tablet dosage form. Asian Journal of Pharmaceutical Research 2011; 1: 10-14.
    

    ---

    How to cite this article:

    Kumar BK, Sekaran VG and Shekar KBC: Gradient High Performance Liquid Chromatography method for determination of related substances in [(3aS, 4R, 6aR)-2, 3, 3a, 4, 5, 6a-hexahydrofuro [2, 3-b] furan-4-yl] N-[(2S, 3R)-4-[(4-aminophenyl) sulfonyl-(2-methyl propyl) amino]-3-hydroxy-1-phenylbutan-2yl] carbamate dosage form. Int J Pharm Sci & Res 2019; 10(12): 5673-82. doi: 10.13040/IJPSR.0975-8232.10(12).5673-82.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---