Development and validation of RP-UPLC method for simultaneous estimation of Cobicistat and Darunavir

 

S. Madhavi^*, A. Prameela Rani

University College  of  Pharmaceutical Sciences, Acharya  Nagarjuna  University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India. - 522 510.

 

ABSTRACT:

A new, simple, precise and stability-indicating UPLC (Ultra Performance Liquid Chromatography) method was developed and validated for the simultaneous determination of anti-viral drugs Cobicistat and Darunavir. The method was developed using Acquity UPLC HSS C18 x 1.8 m.with isocratic elution. 0.1% orthophosphoric acid buffer and acetonitrile (35:65 v/v) were used as mobile phase with 0.4 ml/min flow rate at 30°C temperature. The detection wavelength was fixed at 250 nm, the run time was within 2 min. The Retention time of Cobicistat and Darunavir were 1.06 and 0.79 min respectively. The method was validated in terms of linearity, precision, accuracy and robustness. Calibration plots were linear over the range of 7.5-45 μg/ml for Cobicistat and 40-240 μg/ml for Darunavir. Recovery was in the range of 98-102% with the relative standard deviation of less than 2% for both drugs. The limit of detection and the limit of quantification for the Cobicistat were found to be 0.161 and 0.488 μg/ml, respectively, and for Darunavir 0.55 and 1.67 μg/ml respectively. The specificity of the method was analyzed by the stress degradation study in acidic, alkaline, oxidative, thermal, UV and hydrolytic conditions. The suggested method was suitable for determination of Cobicistat in bulk and pharmaceutical dosage forms.

 

 

 

INTRODUCTION:

Darunavir (Figure.1), chemically [(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-henylbutan-2-yl]carbamate, is a protease enzyme inhibitor. It works by retarding the spread of HIV in the body. Some analytical methods [3-10] have been reported for the determination of Darunavir in pure drug, pharmaceutical dosage forms and in biological samples using spectrophotometry liquid chromatography, electro kinetic chromatography high performance thin layer chromatography either in single or in combined forms^1-11.

 

Cobicistat (Figure.2) is a chemical known as (1,3-thiazol-5-yl) methyl N-[(2R,5R) -5-[(2S) -2-{[methyl ({[2-(Propan-2-yl) -1,3-thiazol-4-yl] methyl}) carbamoyl] amino} -4-(morpholin-4-yl) butanamido] -1,6-diphenylhexan-2-yl]. Although it does not possess any anti-HIV activity, Cobicistat acts as a pharmacokinetic enhancer by inhibiting cytochrome P450 3A isoforms (CYP3A) and thus increases the systemic exposure of co administered agents that are metabolized by CYP3A enzymes. More specifically, Cobicistat is indicated to increase systemic exposure of atazanavir or Darunavir (once daily dosing regimen) in combination with other antiretroviral agents in the treatment of HIV-1 infection. Increasing systemic exposure of anti-retrovirals (ARVs) without increasing dosage allows for better treatment outcomes and a decreased side effect profile. The literature survey revealed that there are a very few HPLC and spectroscopic methods avail­able for the determination of Cobicistat in single and in combination with other antiviral drugs^12-20.

 

Figure.1: Darunavir structure

 

Figure.2: Cobicistat structure

 

From the various literature findings, it has been seen that Darunavir in combination with Cobicistat were analytically validated by few chromatographic techniques^21-25. Moreover, there is no UPLC method reported for Simultaneous estimation of Darunavir and Cobicistat. It is, thus, felt necessary to acquire a novel method for simultaneous determination of both the drugs with shorter run time. The major aims of this research is to develop a novel, convenient UPLC method for determination of Darunavir and Cobicistat in tablet dosage form, in accordance with international conference harmonized guidelines (ICH).

 

MATERIALS  AND METHODS:

Instruments and Apparatus:

The chromatography was performed on a WATERS UPLC SYSTEM equipped with quaternary pumps, TUV detector and Auto sampler integrated with Empower 2 Software. UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2 mm and 10mm and matched quartz cells integrated with UV win 6 Software was used for measuring absorbances of Darunavir and Cobicistat solutions.

 

Reagents and materials:

Working standards of Darunavir and Cobicistat were gifted by spectrum labs (Hyderabad, India). Dosage form PREZCOBIX tablets purchased from the market. HPLC-grade acetonitrile, methanol, ortho-phosphoric acid (OPA) purchased from Merck Ltd, Mumbai, India were used in the study.

Preparation of solutions:

Preparation of Standard stock solutions:

Accurately weighed 7.5mg of Cobicistat, 40mg of Darunavir transferred to 25ml of volumetric flasks separately. 20ml of diluent was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution 1and 2. (300 µg/ml of Cobicistat and 1600 µg/ml of Darunavir).

 

Preparation of Standard working solutions (100% solution):

1ml from each stock solution was piped out and taken into a 10ml volumetric flask and made up with diluents (30µg/ml of Cobicistat and 160µg/ml of Darunavir).

 

Preparation of marketed Sample stock solutions:

10 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 500ml volumetric flask, 250ml of diluent was added and sonicated for 25 min, further the volume was made up with diluent and filtered by UPLC filters (300µg/ml of Cobicistat and 1600µg/ml of Darunavir).

 

Preparation of Sample working solutions (100% solution):

1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (30 µg/ml of Cobicistat and 160µg/ml of Darunavir).

 

Buffer preparation (0.1%OPA):

1ml of Ortho phosphoric acid solution taken in a 1000ml of Volumetric flask, add about 100ml of milli-Q water and final volume make up to 1000 ml with milli-Q water

 

Diluent:

Based upon the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in the ratio of 50:50.

 

Method Development:

Chromatography:

Chromatographic analysis was performed on Acquity UPLC HSS C18 x 1.8 m. The mobile phase consists of 0.1%ortho phosphoric acid buffer: acetonitrile (35:65 v/v) was used throughout the analysis. The flow rate was 0.4 ml/min, the injection volume was 1 μl, column temperature was 30ºC and detection was performed at 250 nm using a TUV detector.

 

Determination of maximum wavelength for Cobicistat and Darunavir using a TUV detector:

The maximum wavelength for the Cobicistat and Darunavir were observed at 250 nm using a PDA detector in UPLC. At 250 nm both drugs were showing absorbance. So for the estimation of this combination we have selected 250 nm for Cobicistat and Darunavir respectively.

 

Calibration curve of Cobicistat:

Aliquots of working standard solution (300μg/ml) of Cobicistat (0.25, 0.5, 0.75, 1, 1.25 and 1.5ml) were transferred into a series of 10 ml volumetric flasks and volume was adjusted to the mark with diluent to get concentrations 7.5,15,22.5,30,37.5 and 45μg/ml. Solutions were injected into the system with stated chromatographic conditions. The graph of area of peak obtained versus respective concentration was plotted. The mean area and its standard deviation were calculated.

 

Calibration curve of Darunavir:

A liquots of working standard solution (1600μg/ml) of Darunavir (0.25, 0.5, 0.75, 1, 1.25 and 1.5ml) were transferred into a series of 10 ml volumetric flasks and volume was adjusted to the mark with diluent to get concentrations 40,80,120,160,200 and 240μg/ml. Solutions were injected into the system with stated chromatographic conditions. The graph of area of peak obtained versus respective concentration was plotted. The mean area and its standard deviation were calculated.

 

Method validation:

System suitability parameters:

The system suitability parameters were determined by preparing standard solutions of Cobicistat (30 μg/ml) and Darunavir (160 μg/ml) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined.

 

Linearity:

Linearity was demonstrated from 50% to 150% of standard concentration using minimum six calibration levels (50%, 60%, 80%, 100%, 120% and 150%) for both the title drugs. The method of linear regression was used for data evaluation.

 

Accuracy:

The accuracy of an analytical method expresses the nearness between the reference value and found value. The accuracy of the method was evaluated in triplicate at three concentration levels, i.e. 50%, 100%, and 150% of standard solutions of Cobicistat and Darunavir

 

LOD AND LOQ:

Increasingly dilute solution of each drug was injected into the chromatograph and signal to noise (S/N) ratio was calculated at each concentration. The limit of detection (LOD) and limit of quantitation (LOQ) was calculated on the basis of signal to noise ratio of 3:1 and 10:1 respectively.

Robustness:

Robustness of the method was determined to ensure its capacity to remain unaffected by small deliberate variation in the method parameters such as mobile phase ratio and flow rate of the mobile phase.

 

Forced degradation studies:

A thorough verification of method selectivity was carried out by forced degradation studies, also known as stress testing. They are performed to determine possible degradation products, and confirm the ability of the developed method to detect and separate impurities, which can possibly arise during the lifetime of an API or drug product. Stress tests are conducted in conditions exceeding those used in accelerated stability testing. Stress studies were performed under conditions of dry heat (thermal studies), hydrolysis (in the presence of acidic, alkaline and water), oxidation, and photolysis. A minimum of four samples were generated for every stress condition, the blank solution stored under normal conditions. 100% sample solution subjected to stress treatment. Hydrolytic decomposition of Cobicistat and Darunavir was conducted at 30ºC in 2N HCl, water, and 2N NaOH. For oxidative stress studies, sample was dissolved in 20% H₂O₂ and kept for one day at room temperature. Photolytic studies, drug solution was exposed to UV light for one day.

 

RESULTS AND DISCUSSION:

System suitability:

The column efficiency, resolution and peak symmetry were calculated for the standard solutions. (Table.1). The peaks obtained for Cobicistat and Darunavir were sharp and have clear baseline separation. It was observed from the results that the system suitability parameters meet the requirement of method validation.

 

Linearity:

The calibration curves plotted for Cobicistat and Darunavir were linear over the concentration range of 7.5-45µg/ml for Darunavir 40-240µg/ml (Table.2). Peak areas were plotted against concentrations, and linear regression analysis performed for the resultant curve. The correlation coefficient values of Cobicistat and Darunavir are 0.999 and 0.999.

 

Precision:

The precision of an analytical method gives information on the random error. It expresses of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions. The percentage RSD values for the precision study was 0.7%, 0.6% (inter-day precision) and 1.0%, 1.0% (intra-day precision) for Cobicistat and Darunavir respectively. This is confirming good precision of the method. The results are summarized in (Table 3).

Accuracy:

Percentage recovery of Cobicistat ranged from 98.90% to 101.57% and for Darunavir 98.50% to 101.03% showing better accuracy of the method. The results are summarized in Table.4 and 5.

 

LOD and LOQ:

The lower limit of detection for Cobicistat and Darunavir was found to be 0.161μg/ml and 0.488 μg/ml respectively. The lowest limit of quantitation for Cobicistat and Darunavir was found to be 0.488  μg/ml and 1.67 μg/ml respectively.

 

Robustness:

No significant effect was observed on system suitability parameters such as theoretical plates, purity angle, and purity threshold, when small but deliberate changes were made for chromatography conditions such as change in flow rate (± 5%)  and organic content (± 2%). The results are summarized in Table.6.

 

Forced degradation studies:

The specificity of the method was also evaluated by the forced degradation study. The peak purity angle is smaller than that of peak threshold angle means there was no interface with the analyte peak from degradation products. Major degradation up to 4.15 % occurred under acid hydrolysis condition. Under thermal condition the drug was degraded by approximately 0.8 %, 2.72 % degraded under alkali condition, 0.5 % degradation occur under UV degradation condition and 1.65 % degradation occurred under oxidative condition. The results are summarized in Table.7.

 

Figure.3: Chromatogram of blank.

 

Figure.4: Chromatogram of standard.

 

Figure.5: A typical UPLC chromatogram of Cobicistat and Darunavir from tablets.

 

Figure.6: Linearity curve for Cobicistat.

 

Figure.7: Linearity curve for Darunavir

 

 

Table. 1: System suitability parameters for Cobicistat and Darunavir

 

Table.2: Linearity table for Cobicistat and Darunavir.

 

Table.3: Method precision

 

Table.4:Accuracy data for Cobicistat

 

 

 

Table.5: Accuracy data for Darunavir

 

Table.6: Robustness data for Cobicistat and Darunavir

 

Table.7: Degradation data of Cobicistat and Darunavir

 

Table.7: Assay of tablet

 

CONCLUSION:

There are several methods were reported for the determination of Cobicistat and Darunavir, but there is no any RP-UPLC method revealed, so the recent work is useful for stability indicating, simultaneous determination of Cobicistat and Darunavir, in combined dosage form. It is a unique isocratic RP-UPLC method with TUV detector. Both the drugs shown the higher response and area of peak at the 250 nm of wavelength with shorter run time. This method gives higher and speedy throughput analysis. Stress degradation studies were carried out by conditions such as acidic, alkaline, oxidative, hydrolytic, thermal and photolytic for specificity. By the study of validation parameters, it is confirmed that the results obtained from this analytical method by UPLC- TUV are reliable, within the acceptance criteria and the proposed method is immensely robust as per the ICH guideline.

 

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Accepted on 14.10.2017      © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(12): 4343-4349.