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1.
The pharmaceutical-grade standards of TAF (99.7%), COBI (99.8%), FTC (99.9%), DRV (99.9%) are obtained from Spectrum Pharma Lab, Hyderabad.
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2.
Marketed formulation (symtuza, manufactured by Patheon Inc. for Janssen Pharmaceuticals, New Jersey, and Batch. no. 608) is obtained from Canada market.
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3.
Chemicals: Acetonitrile (HPLC grade, Merck India Ltd, Mumbai), monopotassium phosphate (AR grade), methanol (HPLC grade) were procured from Merck Chemical division (Mumbai, India). Purified HPLC-grade water was obtained from Milli-Q, USA.
Instrumentation
The separation was done on acquity UPLC system equipped with quaternary pumps, acquity TUV detector along with auto sampler. The data processor was Waters Empower 2 software. The column of SB C8 column of 50 × 2.1, 1.8μ dimensions was used for separation. UV Spectrophotometer (Shimadzu UV 1800, Japan), electronic balance (Afcoset ER-200A, Mumbai, India), ultrasonicator (Enertech) and pH meter (AD 1020, Adwa Hungary ft, Hungary) were used for the study.
UPLC optimised conditions
Analytes can be separated by using combination of 0.01 N potassium dihydrogen ortho phosphate (pH -4.8) buffer and acetonitrile (60:40 v/v) at a flow rate of 0.3 mL/min through column maintained at a temperature of 30 °C at a detection wavelength of 267 nm. The column was equilibrated with mobile phase before injecting the solution. The injection volume was 2 µL.
Composition of analytical solutions
Mobile phase
The buffer and acetonitrile in the ratio of 60:40 v/v were used as mobile phase.
Constitution of 0.01 N potassium dihydrogen ortho phosphate buffer (pH-4.8): 1.36 g of potassium dihydrogen ortho phosphate was weighed accurately and added to 1000 mL of Milli-Q water filtered through 0.45-µ membrane filter sonicated to degas, and made volume up to mark.
Diluent
Water and acetonitrile in the ratio of 50:50 were used as diluent.
Standard solution
1.25 mg, 18.75 mg, 25 mg and 100 mg of tenofovir, cobicistat, emtricitabine and darunavir working standards were accurately weighed and transferred into four different 25-mL clean dry volumetric flask, add 10 mL of diluent, sonicate for 30 min, and make up to the final volume with diluent. The solution contains 50 µg/mL tenofovir, 750 µg/mL cobicistat, 1000 µg/mL emtricitabine, 4000 µg/mL darunavir.
1 mL of above stock solution was pipetted out in to a 10-mL volumetric flask, and then, make up to the final volume with diluent. The solution contains 5 µg/mL tenofovir, 75 µg/mL cobicistat, 100 µg/mL emtricitabine, 400 µg/mL darunavir, and solution was dribbled through 0.45-µm PVDF syringe filter, supplied by Allpure Biotechnology.
Sample solution
Estimate the average weight of 10 symtuza tablets, powder equivalent to one tablet was taken in to 100-mL volumetric flask, and 50 mL of diluent was added, ultrasonicated for 25 min and made volume with water: acetonitrile 50:50v/v (diluent) and filtered. From this solution, pipette out 0.5 mL in to 10 mL of volumetric flask and contrive the volume with diluent. The solution was dribbled through the Allpure brand PVDF syringe filter (0.45 µ). The resultant solution contains 5 µg/mL TAF, 75 µg/mL COBI, 100 µg/mL FTC, 400 µg/mL of DRV.
Validation parameters of the UPLC method
The optimised approach was validated conferring to the ICH guidelines in Q2(R1) [19], and validation results show system suitability of the method developed, sensitivity, accuracy, precision, linearity, detection limit (DL), quantitation limit (QL), robustness and ruggedness. Stress conditions are induced, and forced degradation studies are carried on according to ICH guidelines Q2 (R1). According to ICH guidelines in Q1A (R2) [20], the stability testing of new drug products facilitates the identification of degradation products and the stability of analyte. Guidelines for the photostability studies are outlined in ICH guidelines Q1B [21].
System suitability
It is done to check the suitability of method to system as a part of estimating system performance. It was estimated on five replicate injections of freshly prepared standard solutions (5 μg/mL, 75 μg/mL, 100 μg/mL and 400 μg/mL of TAF, COBI, FTC and DRV, respectively), and the parameters like plate count, asymmetry and resolution were estimated. % RSD of area for five replicate injections should be NMT 2%.
Selectivity
Selectivity can be estimated to check for any interference noticed at the retention times of the drugs TAF, COBI, FTC and DRV. Selectivity can be known by comparing the results of standard solution, sample solution, blank sample and placebo sample.
Linearity
By constructing calibration curves of peak area versus concentration range, we can assess linearity of drugs in concentration range of: 1.25–7.5 μg/mL for TAF, 18.75–112.5 μg/mL for COBI, 25–150 μg/mL for FTC and 100–600 μg/mL for DRV. The linearity can be determined by calculating the regression equation from the calibration curve constructed by using six standard concentrations.
Accuracy
It was the closeness of observed value of the approach to the true value. It was estimated as percentage of analyte recovered for assay after adding a known amount of respective drugs. The known amounts of standard solutions of drugs were spiked to pre-analysed sample solutions injected into UPLC system. Accuracy was estimated at 50%, 100% and 150% concentrations in triplicate, and % recovery was estimated.
Precision
System precision or injection repeatability was estimated by injecting six replicate injections of standard solutions containing 5 μg/mL, 75 μg/mL, 100 μg/mL and 400 μg/mL of TAF, COBI, FTC and DRV. The % RSD of peak area response was calculated.
Method precision or repeatability was estimated by injecting sample solutions in six replicate injections of the sample solution containing 5 μg/mL, 75 μg/mL, 100 μg/mL and 400 μg/mL of TAF, COBI, FTC and DRV. Precision of the method was estimated by the RSD of the percentage assay of the analytes.
Intermediate precision was accessed by injecting six replicate samples which contain 5 μg/mL of TAF, 75 μg/mL of COBI, 100 μg/mL of FTC and 400 μg/mL of DRV on same day and consecutive day.
Detection limit and quantification limit
Detection limit also known as limit of detection was the lowest concentrations of TAF, COBI, FTC and DRV which can be reliably detected from background noise, and LOQ was the minimum concentration of the drugs quantified with accuracy and precision. LOD and LOQ can be estimated by the formulas and also from calibration curve.
$$\begin{aligned} & {\text{LOD}} = 3.3 \times \frac{{{\text{Std}}\;{\text{Dev}}\;{\text{of}}\;{\text{regression}}\;{\text{line}}}}{{{\text{Slope}}}} \\ & {\text{LOQ}} = 10 \times \frac{{{\text{Std}}.{\text{Dev}}\;{\text{of}}\;{\text{regression}}\;{\text{line}}}}{{{\text{Slope}}}} \\ \end{aligned}$$
Robustness
It provides evidence of reliability during usual handling and is determined by analysing different parameters like alternating flow rate of eluent (± 0.01 mL), constitution of mobile phase (± 2%) and changes in column temperature (± 2 °C). The effects of the changes in experimental conditions on retention times and % recoveries were recorded.
Accelerated degradation studies
ICH guidelines recommended stress testing to know the intrinsic stability of drug substance. In accelerated degradation studies, the solution of the standard was subjected to acid hydrolysis (2 N HCl, refluxed at 60o C for 30 min), alkali hydrolysis (2 N NaOH, refluxed at 60 °C for 30 min), peroxide degradation (20% hydrogen peroxide heated at 60 °C for 30 min), thermal degradation (samples arranged in hot air oven at 105 °C for 6 hrs), photolytic degradation (sample in UV chamber for 7 days), neutral degradation (water, refluxed at 60 °C for 6 h). The so stressed samples were observed for the peak areas and compared with peak areas of standard.
Standard solution stability
The results obtained after analysing the standard solution stored after 24 h must be compared with the results obtained for fresh solution (at 0 h). The average %RSD of the area of solution was considered stable if the results do not deviate more than 2% with respect to fresh solution.
Assay procedure
Separately inject 2 µL of standard solution (of pure drugs) and sample solution (extracted from Symtuza tablets) in to the UPLC. The amount of the drugs present was estimated against the peak area of recorded chromatograms.