FORMULATION AND EVALUATION OF FIXED-DOSE COMBINATION OF BILAYER TABLETS OF ATAZANAVIR SULFATE AND RITONAVIR 300 MG/100 MG

Objective: The objective of this study is to formulation and development of fixed-dose combination as a single dosage regimen by using the design of experiments (DOE) approach against the single dose of reference listed drugs of brand reyataz capsule 300 mg (atazanavir sulfate) and norvir tablets 100 mg (ritonavir tablets) to treat human immunodeficiency virus (HIV) Infections. Methods: Formulation was developed with each blend of ritonavir by using hot-melt extrusion and atazanavir sulfate by wet granulation process and compressed by bilayer technology followed by film coating. Formulation and process optimization by design of experiments (DOE) to evaluate dissolution and related substances of the finished product. Fractional factorial (22+3) and full factorial design (33+3) by using a design expert (version 11.0) were used to evaluate the formulation and process variables to prepare a robust formulation. Results: Results indicate that the sorbitan monolaurate range has played a key role to achieve the dissolution for ritonavir formulation. The studied temperature range and interaction of temperature and feed rate, temperature and screw speed during the hot-melt extrusion process impact on the related substances of the bi-layer tablet. Analysis of variance (ANOVA) also finding the P-value less than 0.0500 and the studied range was significant. Design space was established for the significant factors to control the results within the acceptable limits. The studied formulation and wet granulation process for atazanavir sulfate have no significant impact on dissolution and related substances of the finished product. Further, the studied hardness range of 16-28kp for bi-layer tablets has no critical impact on the dissolution. Optimum formulation and process of bi-layer tablets in F37 yielded similar drug release and related substances against the reference drug product. Conclusion: The present invention of fixed-dose combination can be recommended as a single dosage regimen with the consistent drug release and control of the unknown impurities in the prototype formulation against the individual reference drug product.

A SELECTIVE AND SENSITIVE LC-MS/MS METHOD FOR QUANTIFICATION OF FOUR POTENTIAL GENOTOXIC IMPURITIES IN ATAZANAVIR SULFATE DRUG SUBSTANCE IN TRACE LEVEL

Objective: The main objective of current research work is to develop and validate a rapid, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the trace analysis of four potential genotoxic impurities in Atazanavir Sulfate drug substance. Methods: LC-MS/MS analysis of four potential genotoxic impurities was done on Acquity UPLC CSH C18 (100 mm × 2.1 mm, 1.7 μm) column. In this method, mobile phase A (10 mM ammonium acetate) mobile phase B (methanol: acetonitrile (90:10, v/v) with gradient run with the flow rate of 0.2 ml/min. The method was developed with the short run time of 13 min. Triple quadrupole mass detector coupled with positive electrospray ionization was used for the quantification of genotoxic impurities in multiple reaction monitoring (MRM) mode. Results: The method was linear in the range of 0.3 ppm to 4.5 ppm for BOC Hydrazine Acid impurity, BOC Epoxide and Keto impurity with a correlation coefficient not less than 0.9994. The accuracy of the method was in the range of 99.26% to 105.71% for all four potential genotoxic impurities (PGIs). No impurities were identified in the Atazanavir Sulfate active pharmaceutical ingredient sample. Conclusion: The proposed method is specific, linear, precise, accurate, robust and stable for the quantification of the four genotoxic impurities at very low levels.

Crystal structure of atazanavir, C38H52N6O7

The crystal structure of atazanavir has been solved and refined using synchrotron X-ray powder diffraction data and optimized using density functional techniques. Atazanavir crystallizes in space group P21 (#4) with a = 15.33545(7), b = 5.90396(3), c = 21.56949(13) Å, β = 96.2923(4)°, V = 1941.134(11) Å3, and Z = 2. Despite being labeled as “atazanavir sulfate”, the commercial reagent sample consisted of atazanavir free base. The structure consists of an array of extended-conformation molecules parallel to the ac-plane. Although the atazanavir molecule contains only four classical hydrogen bond donors, hydrogen bonding is, surprisingly, important to the crystal energy. Both intra- and intermolecular hydrogen bonds are significant. The hydroxyl group forms bifurcated intramolecular hydrogen bonds to a carbonyl oxygen atom and an amide nitrogen. Several amide nitrogens act as donors to the hydroxyl group and carbonyl oxygen atoms. An amide nitrogen acts as a donor to another amide nitrogen. Several methyl, methylene, methyne, and phenyl hydrogens participate in hydrogen bonds to carbonyl oxygens, an amide nitrogen, and the pyridine nitrogen. The powder pattern is included in the Powder Diffraction File™ as entry 00-065-1426.

Development and validation of a UPLC-MS method for determination of atazanavir sulfate by the “analytical quality by design” approach

A UPLC-MS method for the estimation of atazanavir sulfate was developed using the “analytical quality by design” approach. The critical chromatographic quality attributes identified were retention time, theoretical plates and peak tailing. The critical method parameters established were percent of organic modifier, flow rate and injection volume. Optimization performed using Box-Behnken Design (BBD) established 10 % organic modifier, 0.4 mL min−1 flow rate and 6-µL injection volume as the optimum method conditions. Atazanavir sulfate eluted at 5.19 min without any interference. Method validation followed international guidelines. The method has proven linearity in the range of 10–90 µg mL−1. Recovery was between 100.2–101.0 % and precision within the accepted limits (RSD 0.2–0.7 %). LOD and LOQ were 2.68 and 8.14 µg mL−1, resp. Stress testing stability studies showed atazanavir sulfate to degrade under acidic and basic conditions. The suggested technique is simple, rapid and sustainable. It is, therefore, suggested for routine analysis of atazanavir sulfate.

SIMULTANEOUS ESTIMATION OF ATAZANAVIR SULFATE AND RITONAVIR IN MARKETED FORMULATIONS – UV SPECTROSCOPIC THREE WAVELENGTH METHOD

Three-wavelength spectrophotometric method has been developed for the simultaneous estimation of atazanavir sulfate (ATZ) and ritonavir (RIT) in pharmaceutical preparations. The absorbance value at 279.7nm was used for the estimation of ATZ where RIT showed zero absorbance. The absorbance value for RIT was estimated by taking difference in absorbance at two wavelengths 248 nm and 252 nm. This method obeyed Beer’s law in the concentration range of 9-45 μg/mL for ATZ, and 3-15 µg/mL for RIT. The results of analysis have been validated statistically and recovery studies confirmed the accuracy of the proposed method. The percentage recoveries were found to be 101.3±0.4 for ATZ and 99.8± 0.2 for RIT. The assay values of atazanavir and ritonavir were found to be 100.7±0.7 and 103.0±1.2, respectively. The method was found to be simple, rapid, accurate, precise and sensitive. Hence, this method can be used for routine analysis of these drugs in formulations.