INTRODUCTION:

Cobicistat is a potent pharmacokinetic enhancer, is a effective mechanism based inhibitor of cytochrome P450 3A4, an enzyme that metabolizes medicinal compounds in the body. Inhibition of CYP3A mediated metabolism by cobicistat enhances the systematic exposure of CYP3A4 substrates for mainly drugs where bioavailability is decreased and half life is reduced by CYP3A dependent metabolism¹. The chemical name of cobicistat is 1,3-thiazol-5-ylmethyl[(2R,5R)-5-{[(2S)2-[(methyl{[2-(propan-2-yl)-1,3-thiazol-yl]methyl}carbamoyl)amino]-4-(morpholin-4yl)butanoyl]amino}-1,6-diphenylhexan-2-yl]carbamate¹ and molecular formula is C40 H53 N7O5S2² Molecular mass is 776.023 g/mol.

The chemical structure is as follows:

Fig 1 Chemical structure for Cobicistat

Cobicistat is a licensed drug for use in the treatment of infection with the human immunodeficiency virus (HIV)³ It was granted by US FDA in Aug 2012 as a part of coformulation that includes integrase inhibitor like elvitegravin, tenofovir, emtricitabine. In Sept, 2014, cobicistat is approved for use as a free standing agent in combination with drugs like atazanavir or darunavir⁴. By combining cobicistat with elvitegravir, higher concentrations of the latter are achieved in the body with lower dosing, theoretically enhancing elvitegravir’s viral suppression while diminishing its adverse side effects³. Cobicistat is approved under the trade name Tybost⁵. There are methods available for estimation of Cobicistat in dosage forms in the literature but no method is available for analysis of Cobicistat and its related impurities in bulk and pharmaceutical dosage forms, hence present work aims for analysis of drug and also its related impurities.

MATERIALS AND METHODS:

Chemicals and materials: Cobicistat was obtained as a gift sample from Mylan Laboratories. HPLC grade double distilled water, analytical grade potassium dihydrogen phosphate, acetonitrile were obtained from Qualigens Fine chemicals Ltd, Mumbai.

Instrumentation:

The analytical separations were carried out on liquid chromatograph equipped with UV detector and the output of signal was monitored and integrated using LC solution software. The analytical column used was Inertsil ODS-3V (250 mmX4.6 mm, 5µ). Mobile phase consisted of 0.02 M potassium dihydrogen phosphate in 1000 ml of water(PH 2.5) and acetonitrile in the ratio of 30:70. The flow rate was1.0ml/min and runtime was 15 min. The column was monitored at ambient temperature. UV detection was measured at 240 nm and the volume of sample injected was 20µl.

Preparation of standard preparation:

Transfer 3000.2mg of cobicistat and 15.2 mg of hydroxy impurity of working standard into 100 ml volumetric flask and dilute with diluents (water:acetonitrile 30:70), take 5 ml and dilute to 50 ml and take 10 ml of above solution and dilute to 20 ml with diluent.

Preparation of test solution:

Transfer 6000.0 mg of Tybost formulation into 100 ml and dilute with diluents to make up the volume. Take 5 ml and dilute to 50 ml.Take 10 ml of above solution and dilute to 20 ml with the diluent.

Assay:

20µl of standard and sample solutions were injected into the chromatographic system and the areas of peaks for Cobicistat and impurity were measured and the % assay was calculated using the formula

AT X WS X DT X P X Avg wt X 100 = % assay

AS X DS X WT X 100 X Label claim

Where:

AT = average area count of sample preparation

AS = average area count of standard preparation

WS = weight of working standard taken in mg

WT = weight of working sample taken in mg

DS = dilution of standard

DT = dilution of test sample

P = percentage purity of working standard Label claim in mg/ml.

Label claim in mg/ml.

Method validation

Validation parameters like system suitability, linearity, accuracy, precision, specificity, limit of detection, limit of quantitation and robustness were performed as per ICH guidelines⁶.

RESULTS AND DISCUSSION:

Method development and optimization:

To optimize the chromatographic conditions, the effect of mobile phase is studied with various solvent system combinations for the determination of Cobicicstat and its impurity (hydroxy impurity) in bulk and pharmaceutical dosage forms. A mixture of 0.02 M potassium dihydrogen phosphate in 1000ml water (PH 2.5) and acetonitrile (30:70) was selected as it gave best resolution. The effect of flow rate was studied in the range of 0.9 to 1.2 ml/min and 1.0ml/min was preferred to be effective. Under these conditions, the analyte peak obtained was well defined and free from tailing. The retention time (RT) was found to be 7.076min for Cobicistat and 6.482 min for hydroxy impurity. The optimized parameters were listed in Table 1. Chromatogram for standard solutions of Cobicistat and hydroxy impurity were presented in Fig 2 and 3 respectively.

Fig2 Chromatogram for Cobicistat

Fig 3Chromatogram for hydroxy impurity

Table 1: Optimized chromatographic parameters

Elution Isocratic

Mobile phase 0.02M potassium dihydrogen phosphate in water(PH 2.5) and acetonitrile (30:70)

Column Inertsil ODS – 3V (250 mm ×4.6 mm, 5 µ)

Flow rate 1.0 ml/min

Detection 240 nm

Injection volume 20 µl

Temperature ambient

Retention time 7.076

Run time 15 min

System suitability studies:

These tests are an integral part of method development and are used to ensure adequate performance of chromatographic system. Retention time (RT), number of theoretical plates (N), tailing factor (T) and resolution were evaluated. The system suitability method acceptance criteria set in each validation run were capacity factor >2.0, tailing factor ≤ 2.0 and theoretical plates > 2000. In all cases, the relative standard deviation (RSD) for analyte peak area < 2.0%. System suitability parameters were shown in Table 2. Chromatogram for system suitability studies is presented in Fig 4.

Table 2 System suitability parameters

S.No Parameters Cobicistat hydroxy

impurity

1 Retention time 7.076 min 6.482 min

2 Theoretical plates 22911.473 33645.621

3 Tailing factor 1.429 1.292

4 Resolution 3.617 0.000

5 Peak area 10756679 55788

Fig 4 Chromatogram for system suitability studies

Linearity:

Aliquots of standard solutions of drug were taken and diluted to get the concentration range of 600µg - 1800µg/ml and standard solution of impurity was diluted to get concentration range of 3.04µg – 9.12µg/ml. Each of these drug solutions (20µl) was injected into the column and peak areas and retention times were recorded. A calibration graph was obtained by plotting graph between peak area versus concentration. Excellent correlation was obtained between peak area and concentration with R² = 0.999 for active ingredient and for hydroxy impurity, it is found to be 1.000. Results are shown in Table 3 and 4 and the calibration curves for linearity are shown in Fig 5 and 6.

Table 3 Linearity results for Cobicistat

Concentration (µg/ml) Peak area

600 6093670

900 7624436

1200 8999817

1500 10634089

1800 11766619

Table 4 Linearity results for Hydroxy impurity

Concentration (µg/ml) Peak area

3.04 28955

4.56 37203

6.08 46215

7.60 55655

9.12 64715

Fig 5 Calibration curve for Cobicistat

Fig 6 Calibration curve for Hydroxy impurity

Accuracy:

Accuracy studies were done by standard addition method. Accuracy is expressed as % recovery of the standard spiked to previously analysed test sample of tablet. It was measured in drug products by spiking known amounts (80%, 100%, 120%) of the analyte into the analyzed tablet powder and calculating the percent recovered. The closeness of obtained value to true value indicates that the proposed method is accurate. The recovery data for accuracy studies was shown in Table 5. The accuracy chromatograms for the respective concentrations were shown in Fig 7

Table 6 Precision results for standard and test sample of Cobicistat

S.No Retention time (min) Peak area (AU)

Standard Sample Standard Sample

1 7.079 7.085 10757266 10743233

2 7.079 7.085 10753768 10737474

3 7.083 7.088 10749069 10725396

4 7.083 7.087 10778582 10746878

5 7.082 7.090 10755682 10754814

6 7.091 7.088 10766060 10724114

AVERAGE 7.083 7.087 10760071 10738652

%RSD 0.061 0.030 0.099 0.113

S.D 0.004 0.002 10644 12152

Table 7 Precision results for standard and test sample of impurity

S.No Retention time (min) Peak area (AU)

Standard Sample Standard Sample

1 6.483 6.490 57660 56897

2 6.483 6.488 57593 57071

3 6.487 6.491 57331 56875

4 6.486 6.491 58185 56873

5 6.487 6.494 57358 57113

6 6.494 6.491 58184 57926

AVERAGE 6.486 6.491 57719 57126

%RSD 0.066 0.027 0.663 0.710

S.D 0.004 0.002 383 405

Fig 8 Chromatogram for precision of standard solutions of Cobicistat and impurity

Fig 9 Chromatogram for precision of test solutions of Cobicistat and impurity

Table 7 Robustness study for Cobicistat

Parameters studied Retention time Peak area % RSD

Standard Sample Standard Sample Standard Sample

Different column 7.082 7.075 1123395 10880111 0.086 0.043

Flow decrease

(0.9ml/min) 7.475 7.477 11884832 11872281 0.137 0.076

Flow increase

(1.1ml/min) 6.730 6.727 9908553 9923454 0.159 0.113

Table 8 Robustness study for hydroxy impurity

Parameters studied Retention time Peak area % RSD

Standard Sample Standard Sample Standard Sample

Different column 6.488 6.482 82556 82217 0.344 0.177

Flow decrease

(0.9ml/min) 6.886 6.887 62241 62659 0.239 0.896

Flow increase

(1.1ml/min) 6.136 6.133 73020 73074.3 0.155 0.069

Fig 10 Chromatogram for standard solutions with different column

Fig 11 Chromatogram for test solutions with different column

Fig 12 Chromatogram for standard solutions with flow decrease

Fig 13 Chromatogram for test solutions with flow decrease

Fig 14 Chromatogram for standard solutions with flow increase

Fig 15 Chromatogram for test solutions with flow increase