Pure samples
Lamivudine (purity—99.53%) and tenofovir disoproxil fumarate (purity—99.40%) were acquired as gift sample from Cipla Pvt. Ltd. Pune, India.
Formulation
The tablet matrix of Tenvir-L consisting of 300 mg of LVD and 300 mg of TDF was procured from local Indian market.
Chemical and reagents
Acetonitrile (ACN), methanol, orthophosphoric acid, and other reagent and chemicals were procured from Merck, Mumbai, India. Water for HPLC also procured from Merck, Mumbai, India.
Instrumentation
Chromatographic resolution was executed with the help of LC 20AD system (Shimadzu Corporation, Japan) which composed of LC-20 AD (binary solvent delivery pump) connected to 20 μL injection loop (a Rheodyne injector). SPD-M20A photodiode array detector and CTO 10 AS vp (thermostated column oven compartment). The data were collected and analyzed with LC-solution (Shimadzu Corporation, Japan). Ultrasonication of samples was conducted by means of an Ultrasonicator; ENERTECH Electronics Pvt. Ltd., India.
Chromatographic conditions
The chromatographic resolution of present analysis was studied on Hypersil BDS C18(250 mm × 4.6 mm) particle size 5 μm. Hypersil BDS column was equilibrated with solvent system comprised of 10 mM potassium dihydrogen phosphate: acetonitrile (60:40% v/v). Lastly, pH 4.0 was adjusted by mean of orthophosphoric acid. A 20-μL solution of standard was injected. The analysis was established at 30 oC at 1 mL/min rate for flow of mobile system through Hypersil BDS column. The analytes of column were monitored at 265 nm using PDA.
Preparation of buffer solution for mobile phase
Potassium dihydrogen phosphate was used for solvent system. The 10 mM potassium dihydrogen phosphate solution of buffer was prepared by accurately solubilising pre-determined amount of potassium dihydrogen phosphate in double distilled water (1000 mL) and solution of buffer was adjusted to pH 4.0 using orthophosphoric acid. Further, prepared buffer solution was filtered using 0.45 μm filter.
Preparation of standard stock solution
One milligram per milliliter stock solution of LVD and TDF were workout separately in 10 mL of calibrated flask in ACN. From prepared stock solution aliquots of 1 mL each were moved to neat and dry10 mL of calibrated flask and diluted up to the mark with same to achieved 100 μg/mL concentration for LVD and TDF, respectively.
Preparation of sample of LVD and TDF in marketed formulation
The present validated investigational RP-LC method was employed for quantification of LVD and TDF in tablet matrix. The tablet matrix of Tenvir-L consisting of 300 mg of LVD and 300 mg of TDF was procured from local Indian market. To estimating analyte in the marketed matrix; 20 tablets were weighed precisely ground into fine powder. Accurately measured tablet powder equivalent to (300 mg LVD and 300 mg TDF) was moved into a 100 mL of calibrated flask, 30 mL ACN was added and the calibrated flask was sonicated for 20 min. Correspondingly, volume of calibrated flask was made using ACN up to the mark to obtained 3000 μg/mL concentration of stock solution of tablet matrix. Then, after resulting solution of tablet matrix filtered using 0.45 μm filter, suitable volume was moved from resulting solution of tablet matrix into calibrated flask of 10 mL and the volume of it was diluted up to the mark using the solvent system to achieved 30 μg/mL sample solution concentration.
Validation of investigated reversed phase-liquid chromatography method
The present RP-LC method has been validated for the confirmation of LVD and TDF in marketed product. The investigated method has been effectively validated for the different parameters namely system suitability, accuracy, precision, sensitive (limit of detection and quantification), robustness, ruggedness, and for selectivity and specificity as stated in the Q2R1 procedure International Council for Harmonization (ICH) of Technical Requirements for Pharmaceuticals for Human Use [20].
System suitability
System suitability assessment is typically aimed to preventing the perceived instability of chromatographic elements like detector, type of column, and pump from negatively affecting official methods [21]. The 10 μg/mL concentration of LVD and TDF solution was introduced and examined as six replicates. Theoretical plate number, tailing factor, resolution, and relative standard deviation (% RSD) of retention time (Rt) and peak area values for LVD and TDF were estimated.
Calibration curve
Calibration curve for LVD and TDF for present investigation have been constructed in optimized chromatographic conditions. For calibration curve, appropriate volumes were moved from the previously prepared stock solution of 100 μg/mL in the range of 1–5 mL was moved to series of calibrated flask of 10 mL and volume was marked up to the mark using solvent system to give concentration in the range of 10–50 μg/mL for LVD and TDF, respectively. Through Hamilton Syringe’s help a fixed volume of 20 μL is injected into the LC. All measurements for every single concentration were replicated six times. Regression analysis for obtained results was conducted through the least-square method.
Accuracy
To evaluate the closeness of the measured value to the true value, the accuracy of an analytical method is established. A method’s accuracy is generally assessed through the drug candidate’s percentage recovery, which is spiked into a placebo matrix. Percent recovery of the commenced investigation has been performed at 80, 100, and 120% levels. It was done by the addition to the pre-studied sample a known amount of standard drug and further it was re-examined through the same investigation.
Precision
Precision of the commenced investigation was accomplished through the intra-day and inter-day precision and repeatability (system precision) and was assessed as RSD percentage. According to ICH Q2R1 procedure, RSD percentage value must be less than 2%. Three distinct concentrations 20, 30, and 40 μg/mL were selected for intra-day and inter-day precision and repeatability of present investigation was performed using 20 μg/mL.
Sensitivity
Sensitivity the commenced investigation was evaluated for LOD and LOQ. LOD and LOQ for LVD and TDF have being estimated by injecting the 10–30 μg/mL low concentrations solution of the LVD and TDF through the investigated method. The formulae’s were used to approximate the LOD = 3.3×ASD/S and LOQ = 10×ASD/S. Where, average standard deviation and slope of the linearity curve are ASD and S.
Robustness
According to ICH procedure, the concept of robustness of the analytical protocol is an appraisal potential to remain unaffected due to minor yet deliberate differences in the analytical protocol parameters. To evaluate the robustness of the investigation, flow rate of solvent system, column oven temperature, and pH of solvent system were selected as independent variables. The impact of each independent variable was observed over the responses, i.e., tailing factor, Rt, theoretical plates. Robustness evaluation has been investigated using concentration of 30 μg/mL.
Ruggedness
The ruggedness of the investigated protocol is the level of repeatability, under the same analytical and environmental conditions, of the test outcomes produced by the estimate of the sample of interest by two independent researchers. Ruggedness evaluation has been investigated using concentration of 30 μg/mL.
Selectivity and specificity
Specificity is a step for detecting of analytical sample of interest quantitatively throughout the context of ingredients that could be required to escape in the sample solid form; thus, the selectivity is the step for qualitatively distinguishing the analyte in the existence of ingredients officially available in the solid form. Selectivity and specificity of the present investigation evaluated using checking the resolution factor of LVD and TDF chromatographic peaks were assessed though the UV spectra generated by a UV detector.