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1.
Pure samples
The pure form of TDF (99.17%), DOR (99.3%) and LMV (99.4%) were procured from Spectrum pharma research solutions, Hyderabad.
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2.
Formulation
The DELSTRIGO™ (100 mg of Doravirine, 300 mg of Lamivudine, and 300 mg of Tenofovir disoproxil fumarate) tablets were purchased from local marketing agency upon request.
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3.
Chemicals and reagents
All the solvents of HPLC grade were procured from local distributor of Merck India Limited, Mumbai. All the chemicals of analytical grade and HPLC grade water were acquired from the Finar chemical distributor.
Instrumentation
The current method was carried out using WATERS Acquity UPLC system which is equipped with Tunable UV detector and HSS (C18 100 × 2.1 mm, 1.8 μ) column used as a stationary phase. Empower 2 software was used to process and integrate the data.
Preparation of standard solution
25 mg of DOR, 75 mg of LAM and 75 mg of TDF were accurately weighed and transferred into 50 mL volumetric flask and dissolve the above analytes with diluent consisting of equal volumes of water and 0.01 N Potassium dihydrogen ortho phosphate and make up to the volume with the diluent to get the standard stock solution concentration consisting of 500 µg/mL, 1500 µg/mL and 1500 µg/mL for DRV, LMV and TDF respectively. Dilute 1 mL of above solution to 10 mL with diluent to get working standard solution having concentration of 50 µg/mL, 150 µg/mL and 150 µg/mL for DOR, LAM and TDF respectively.
Preparation of sample solution
The tablet (Delstrigo™ consisting of 300 mg of Tenofovir disoproxil fumarate, 300 mg of Lamivudine, and 100 mg of Doravirine) powder is equivalent to 75 mg of LAM 75 mg of TDF and 25 mg of DRV were accurately weighed and placed in 50 mL volumetric flask. Above contents of analytes were dissolved with diluents consisting of equal volumes of water and 0.01 N Potassium dihydrogen ortho phosphate in equal proportions. 1 mL of the above solution is further diluted with 10 mL diluent to get a solution having concentration 150 µg/mL, 150 µg/mL and 50 µg/mL for LAM, TDF and DOR respectively. The possible particulate matter in sample was eliminated by using 0.45 µm Nylon filters.
Method development
Chromatographic method conditions:
The current method was carried out by using WATERS UPLC system with Tunable UV detector. DOR, LAM and TDF analytes were successfully separated by using HSS (C18 100 × 2.1 mm, 1.8 µ) column and with mobile phase consisting of 0.01 N Potassium dihyrogen ortho phosphate (pH-4.8) and Acetonitrile in 60:40 v/v pumping at 0.3 mL/min flow rate. Analytes were detected at a wavelength 260 nm. Both analytical column and injection device were maintained at the same temperature 30 °C. Before application of sample and mobile phase into the instrument, filtered through 0.45µ Nylon filters to dispose of particulate matter in it.
Method validation
The following method has been validated using below mentioned validation parameters according to ICH Q2 (R1) guidelines specifications [29].
System suitability
The system suitability of the proposed method has been validated by injecting 6 replicates of standard solution (150 µg/mL of LAM, 150 µg/mL of TDF and 50 µg/mL of DOR) into UPLC system. System suitability parameters like resolution (R), number of theoretical plates (N), tailing factor (T) were assessed by determining the percentage relative standard deviation (%RSD) of parameters for the recorded chromatograms.
Linearity
Linearity of the method assures a direct proportional relationship between input concentrations and the obtained output peak area responses. Linearity was estimated by assessing the correlation coefficient (r2) value for the triplicates of the series of working standard solution concentrations about 37.5, 75.0, 112.5, 150.0, 187.5, 225.0 µg/mL for both LAM and TDF and 12.5, 25.0, 37.5, 50.0, 62.5, 75.0 µg/mL for DOR, by plotting a linear response curve in between series of concentrations and obtained peak areas mentioned for each analyte.
Sensitivity
The limit of detection (LOD) and limit of quantification (LOQ) were assessed by following formulae
$$\begin{aligned} & {\text{LOD}} = 3.3\;\sigma /S \\ & {\text{LOQ }} = \, 10\;\sigma /S \\ \end{aligned}$$
where σ standard deviation (SD) obtained from the intercept of linear plot (n = 3). S average slope of the linear plot (n = 3).
Solution stability
Solution stability was performed by using standard solution stored at room temperature and evaluate the solution at specific intervals of time for 72 h.
Specificity
Specificity of the method was evaluated by analysis of different analytes which are not interfered by the presence of other impurities or degradation products. Specificity was determined by giving subsequent injections of blank, standard solution and placebo spiked in standard solution and make sure that no interferences from blank and placebo at the retention time of DOR, LAM, TDF.
Precision
Precision parameter represents the closeness relationship established among the obtained responses of same sample under similar conditions. It was done by giving six replicate injections of standard solutions in the same day (intra-day precision) and same standard solution injected two times in a day for three continuous days under similar conditions (inter-day precision). %RSD was calculated for peak areas of recorded chromatograms.
Accuracy
Percentage recovery method was adopted to ensure the accuracy of the proposed method, in which at three percentage levels sample solution was spiked into standard solution. Analysis was done by triplicate injections of each level spiked solution. At the three different levels, mean percentage recovery of DOR, LAM, TDF was calculated.
Robustness
Robustness parameter assures that to maintain originality of the method to produce response by modifying certain method conditions intentionally. In the proposed method, to validate the robustness parameter slight variations employed in method conditions are mobile phase ratio (± 10%), flow rate (± 10% mL/min) and temperature (± 5 °C). %RSD was assessed for the peak areas of recorded chromatograms.
Stability indicating studies
Forced degradation studies were conducted as per Q1A, QIB and Q2B guidelines of ICH [29]. Stability indicating studies were conducted with standard drug solution in order to assess the stability indicating power of the method and to predict the essential requirements of storage conditions for pure drug and its dosage form.
Acid degradations studies
In this study, equal volumes of standard stock solution (1.5 mg/mL of LAM, 1.5 mg/mL of TDF and 0.5 mg/mL of DOR) and 2 N HCl solution were mixed and the resultant solution was refluxed for 60 min at 60 °C. Neutralize the obtained solution with 2 N NaOH and dilution was made to accomplish concentration in the order of 150 µg/mL, 150 µg/mL and 50 µg/mL for TDF, LAM and DOR. Inject 1 µl of above solution into UPLC system to assess the percentage of drugs degraded under acid degradation conditions from the obtained chromatograms.
Alkali degradation studies
In this alkaline study, equal volumes of standard stock solution and 2 N NaOH were mixed properly and reflux the resultant solution for 60 min at 60 °C. The obtained solution was neutralized with 2 N HCl and diluted further to obtain concentration of 150 µg/mL, 150 µg/mL and 50 µg/mL for TDF, LAM and DOR respectively. From the above solution 1 µl was injected into UPLC system and the percentage of drugs degraded was assessed from the attained chromatograms under alkaline conditions.
Oxidative degradation studies
In this study, standard stock solution and 20% hydrogen peroxide solution were mixed in equal proportion and reflux for 60 min at 60 °C. The obtained solution was further diluted to attain 150 µg/mL, 150 µg/mL and 50 µg/mL for TDF, LAM and DOR respectively. A volume of 1 µl was introduced into UPLC system and evaluate the percentage of drugs degraded from the resultant chromatograms.
Photo degradation studies
In this study, expose the standard stock solution to a wavelength of 254 nm in UV chamber for 72 h with dark control. The above solution was to accomplish concentration of 150 µg/mL, 150 µg/mL and 50 µg/mL for TDF, LAM and DOR in that order. 1 µl of the diluted solution was introduced into the UPLC system and assessed the percentage of the drug degraded from the attained chromatograms.
Thermal degradation studies
In this study, held 5 mL of the standard solution in the oven at 1050C/75% RH for 6 h. A volume of 1 µl resultant solution was injected into the UPLC system and the percentage of the drug degraded was assessed from the attained chromatograms under thermal degradation conditions.
Neutral degradation studies
In this degradation study, standard stock solution and Milli-Q water were mixed properly in equal volumes to get the homogenous solution. Dilute the above solution was further to attain concentration of 150 µg/mL, 150 µg/mL and 50 µg/mL for TDF, LMV and DRV respectively. 1 µl of above solution was introduced and recorded the chromatograms to evaluate the percentage of the drug was degraded under neutral conditions.
Application of the method to Marketed formulation
The current method has been applied to find out the percentage purity of commercial tablets (DELSTRIGO™) by injecting subsequent injections of same concentration of both standard and sample solutions consecutively. The percentage purity of every analyte was evaluated from the peak areas of three analytes in both standard and sample solution.