A Validated RP-HPLC Method for the Determination of Atazanavir in Pharmaceutical Dosage Form

A validated RP HPLC method for the estimation of atazanavir in capsule dosage form on YMC ODS 150 x 4.6 mm, 5 μ column using mobile phase composition of ammonium dihydrogen phosphate buffer (pH 2.5) with acetonitrile (55:45 v/v). Flow rate was maintained at 1.5 mL/min with 288 nm UV detection. The retention time obtained for atazanavir was at 4.7 min. The detector response was linear in the concentration range of 30 – 600 μg/mL. This method has been validated and shown to be specific, sensitive, precise, linear, accurate, rugged, robust and fast. Hence, this method can be applied for routine quality control of atazanavir in capsule dosage forms as well as in bulk drug.


Experimental
Shimadzu HPLC with DAD detector was used with LC solution software.Milli-Q water, HPLC grade methanol and acetonitrile (Rankem Ltd) and GR grade ammonium dihydrogen phosphate and orthrophosphoric acid were used.The capsule formulation containing equivalent of 300 mg of atazanavir (Atavir®-300 mg, Cipla) was procured from local market.

Chromatographic conditions
Chromatographic separation was achieved on YMC ODS (150 x 4.6 mm i.d. 5 µ) analytical column at ambient temperature with mobile phase consisting of 2.0 g of ammonium dihydrogen phosphate in 1000 mL of Milli-Q water, adjusted pH to 2.5 with orthophosphoric acid and acetonitrile (55:45 v/v).The flow rate was maintained at 1.5 mL/min with injection volume 10 µL and the detection was made at 288 nm.Diluent was prepared by mixing 600 mL of methanol with 400 mL of buffer.

Preparation of standard solution
Accurately weighed 34 mg of atazanavir sulphate standard into a 100 mL volumetric flask added about 60 mL of diluent and sonicated to dissolve and then made up to the volume with diluent to get 300 µg/mL standard solution.

Preparation of sample solution
Contents of 20 capsules weighed and mixed and equivalent to 30 mg of atazanavir was taken into 100 mL volumetric flask, about 75 mL of diluent was added sonicated for 25 min at room temperature with intermediate shaking and then made up to volume with diluent.The sample solution was centrifuged at 7500 rpm for 5 min to get a clear solution to get a concentration of 300 µg/mL.

Assay
Sample solution of 10 µL was injected into HPLC (n=6) and recorded the chromatograph.The amount of drug present per capsule was calculated by comparing the peak area of the sample solution with that of the standard solution.Ruggedness of the method was checked on different days on different system following the proposed procedure.The data presented in Table 1.

Linearity
Several aliquots of standard stock solution (1 mL=2600 µg/mL) of atazanavir were taken in different 25 mL volumetric flasks and diluted up to the mark with diluent to obtained concentration of 30, 60, 150, 210, 300, 360, 455 and 600 µg/mL of atazanavir.Peak areas were recorded for all the peaks and a calibration curve was obtained by plotting peak areas versus concentrations of atazanavir (Figure 1).

Recovery studies
Accuracy was determined by spiking the known amount of atazanavir drug substance to the pre analyzed samples and subjected to the proposed HPLC Method.The study was done at 20%, 50% and 150% of test concentration levels (i.e. 6, 15 and 45 mg respectively) in triplicates.Results of recovery study are shown in Table 2.

Forced degradation
Degradation studies are performed on drug product under acidic, alkali, oxidative, thermal and photolytic stress conditions.Each stress condition samples are analyzed in the proposed method and peak purity data is recorded to check the homogeneous nature of the drug.

Robustness
Standard solution was prepared and injected into HPLC following the proposed method in different variable conditions such as Flow (±10%), pH (±0.2units), wavelength (±5 nm) and organic composition in mobile phase (± 2% absolute) and checked system suitability criteria in the each variable condition.

Results and Discussion
Atazanavir was freely soluble in methanol and has λ max at 288 nm, shown in Figure 2. System suitability parameters were carried out on freshly prepared standard stock solution of atazanavir as per the USP-XXVIII and reported in Table 3. Retention time achieved at 4.7 min.Regression equation y=6978.1x-18657with concentration range of 30 -600 µg/mL and the correlation coefficient (r=0.9999)obtained for atazanavir shows that the method is Linear.The assay results (n=6) of atazanavir in capsules were found to be 99.9% for method precision, 99.7% for ruggedness and 99.9% for recovery studies.System suitability parameters are within the acceptance criteria in robustness study indicates the method is robust.All the stress condition samples were analyzed for 30 min and checked for co elution -peak purity passes in each stress condition indicates there is no co-elution with the main analyte.Above parameters demonstrates that the developed HPLC method was specific, fast, simple, precise, accurate, sensitive, linear, accurate, robust and rugged.Thus the developed method can be easily used for the routine quality control of bulk and capsule dosage form of atazanavir within a short analysis time.

Figure 1 .
Figure 1.Calibration curve of atazanavir by HPLC

Figure 2 .
Figure 2. Spectrum of atazanavir standard by HPLC Table 3. Validation and system suitability parameters

Table 2 .
Results of recovery studies.

Table 3 .
Validation and system suitability parameters