Concurrent estimation of lamivudine, tenofovir disoproxil fumarate, and efavirenz in blended mixture and triple combination tablet formulation by a new stability indicating RP-HPLC method

An easy, defined, rapid, and accurate reverse phase high-performance liquid chromatography method was developed and subsequently validated for the concurrent estimation of lamivudine, efavirenz, and tenofovir disoproxil fumarate in their pure blend and combined tablet formulation. An efficient and appropriate separation of the three analytes was attained with Zorbax eclipse XDB-Phenyl column, with a mobile phase of methanol: buffer (0.1% v/v formic acid in water) (73:27 v/v) at a flow rate of 1mL/min and isocratic elution by using 260nm as detection wavelength. Equal ratio of acetonitrile and water was used as diluent. The retention times of lamivudine, tenofovir disoproxil fumarate, and efavirenz were found at 2.6, 4.4, and 5.9 min respectively. The linear response for lamivudine, tenofovir disoproxil fumarate, and efavirenz was in the range of 15.0–45.0μg/mL, 15.0–45.0μg/mL, and 20.0–60.0 μg/mL respectively. The method validation was done in accordance to ICH guidelines and all validation parameters in compliance with ICH standards. The degradants produced by stress testing were well resolved from the peaks of active analytes, which stipulates the stability-indicating property of the method. The method has the ability to separate lamivudine, efavirenz, and tenofovir disoproxil fumarate concurrently in blended powder and their combined tablet. All degradants produced by application of stress conditions were separated with high resolution and determined with good sensitivity that ensures the stability-indicating property of the method. Thus, the projected method has high probability to adopt in the pharmaceutical industrial sector.

A competent analytical method is important for an analyte to determine alone or in blend with other analytes concurrently. The broad literature search disclosed that a small number of analytical methods like UV and RP-HPLC methods were at hand for determination of LAM. TDF and EVZ individually and in combined dosage form [3,5,[12][13][14][15][16]. In addition to those methods, few RP-HPLC methods were described for determination of LAM, TDF with dolutegravir, and other antiviral agents [17][18][19][20][21].
As of yet, a competent stabilityindicating RP-HPLC method was not available for the concurrent evaluation of LAM, EVZ, and TDF in pure blend and combined tablet formulation. Hence, research attempts have been done to develop an efficient, highly responsive and cost-effective RP-HPLC method to estimate the percentage purity and to evaluate the stability of LAM, EVZ, and TDF in blended bulk powder and tablet formulation concurrently. To assure the developed method, validation of the method was done as indicated by Q2R1 guidelines of ICH.

Formulation
Film-coated tablet dosage forms (Symfi Lo is a fixeddose combination product containing 400 mg of EVZ, 300 mg of LAM, and 300 mg of TDF) were purchased from local pharmacy in Hyderabad.

Chemicals and reagents
All HPLC grade and analytical grade solvents were purchased by local distributor of Merck India.

Instrument specifications
The method was done by using WATERS HPLC (2695model, PDA detector, and the Empower 2 solution software). In addition, an analytical balance (Denver instrument, TB-215D), a digital pH meter (MEZARIT), and water (Milli-Q) were used. Forty-five micrometers povidone filters were used to filter all the solutions and the solvents before introduced into the instrument.

Chromatographic method conditions
An efficient and appropriate separation of the three analytes was done with Zorbax eclipse XDB-Phenyl (250 × 4.6mm, 5μ) column, using a mobile phase consist of methanol: buffer (0.1% v/v formic in water) (73:27 v/v) at a flow rate of 1mL/min and a detection wavelength of  260.0 nm. Equal ratio of acetonitrile and water was used as diluents.

Preparation of standard solution
One hundred milligrams of LAM, 100.0mg of TDF, and 133.3mg of EVZ pure powders were accurately weighed and dissolved with diluent in a 100-mL volumetric flask. One milliliter of the above solution was diluted to 10mL. Three milliliters of the resultant solution was further diluted to 10mL to get a concentration of 30.0μg/mL, 40.0μg/mL, and 30.0μg/mL for LAM, EVZ, and TDF respectively.

Preparation of sample solution
The crushed tablet powder equivalent to 100mg of LAM, 100mg of TDF, and 133.3mg of EVZ pure powders was accurately weighed and dissolved with diluent in 100-mL volumetric flask. One milliliter of the above solution was diluted to 10mL. Three milliliters of the resultant solution was further diluted to 10mL to get a concentration of 30.0μg/mL, 40.0μg/mL, and 30.0μg/mL for LAM, EVZ, and TDF respectively.

Method validation
Validation is written evidence that provides assurance of the method or process with high degree level. To validate analytical method, Q2R1 guidelines of the ICH were taken into consideration.

System suitability test
The system suitability test of the present method was established by injecting standard solution in 6 consecutive injections, and the parameters such as percentage relative standard deviation (%RSD), tailing factor (T), resolution (R), and number of theoretical plates (N) were assessed for the chromatograms thus attained.

Linearity
The linearity of an analytical method signifies the direct proportional relationship between experimental results and the given concentrations. It was established for the solutions having concentrations ranging from 15.0 to 45.0 μg/mL of both LAM and TDF and 20.0 to 60.0μg/ mL of EVZ. A calibration curve was plotted between concentration and peak area to determine regression coefficient (R 2 ).

Precision
The precision of the method expresses the close agreement between the observed responses of homogenous samples on multiple sampling at identical conditions.

Specificity
Specificity is the ability of the analytical method to assess the analyte of interest in the presence of other substances including degradation products and excipients without interferences. It was performed by introducing consecutive injections of placebo, blank, standard, and placebo mixed with standard. The obtained chromatograms were examined for interference from the peaks of other substances with peaks of analytes to be determined.

Sensitivity
The LOQ and LOD were reckoned by using the following formulae: where σ is the SD of intercept and S is the slope of the linear curve.

Robustness
Method's robustness was assessed by changing the optimized conditions of the method to a little extent intentionally. In this case, optimized conditions like ratio of the mobile phase (± 1mL), flow rate of mobile phase (± 0.1 mL/min), and detection wavelength (± 2nm) were intentionally altered to a little extent.

Forced degradation studies
In forced degradation (FD) studies, chemical stability of the analyte can be assessed in the presence of more intensive conditions like acid and alkali hydrolysis, oxidative degradation, and thermal and photo stability as provided by ICH quality guidelines.

Oxidative degradation
Ten milliliters of standard stock solution was mixed with 2mL of 3% hydrogen peroxide and reflux the prepared solutions for 2h at 70°C, and kept it aside  for 24 h and further diluted in such a way to get a concentration of 30μg/mL, 40μg/mL, and 30 μg/mL for LAM, EVZ, and TDF respectively.

Thermal degradation
The standard stock solution was placed in hot air oven at 80°C/75% relative humidity for 24 h. One milliliter of the above exposed solution was further diluted in such a way to get a concentration of 30μg/mL, 40μg/mL, and 30μg/mL for LAM, EVZ, and TDF respectively.

Photo degradation
The standard stock solution was in ultraviolet chamber at 254.0 nm for 24 h. One milliliter of above exposed solution was further diluted in such a way to get a concentration of 30μg/mL, 40μg/mL, and 30μg/mL for LAM, EVZ, and TDF respectively. The above mentioned FD solutions were injected and computed for the percentage degradation of LAM, EVZ, and TDF. As per most of the researcher's suggestions, the considerable degradation of analyte is around 20% for the validation of stability-indicating HPLC method.

Assay
The assays of the LAM, EVZ, and TDF in commercial tablets were determined by injecting sample solution.

Results
The foremost step in the method development is to determine the solubility of the analyte. It was observed that LAM and TDF were freely soluble in water and acetonitrile and slightly soluble in methanol, and EVZ was soluble in methanol and acetonitrile and insoluble in water. On the basis of solubility of LAM, EVZ, and TDF, water and acetonitrile in 1:1 ratio was chosen as diluent.

Method optimization
Method optimization was completed after several trials. In this research, several trials have been done with different columns, mobile phases, and flow rates to attain a method with good resolution (R), N, T, and % RSD. At last, a method with Zorbax eclipse XDB-Phenyl column, with a mobile phase of methanol: buffer (0.1% v/v formic in water) (73:27 v/v) at a flow rate of 1mL/min and detection wavelength of 260.0nm was elected as optimized  Table 1. The optimized chromatogram is shown in Fig. 2.

Method validation System suitability
All parameters of the system suitability test were in compliance with standards of ICH (Table 2).

Linearity
The linear response of the LAM, TDF, and EVZ were in the series of 15.0 to 45.0 μg/mL, 15.0 to 45.0 μg/mL, and 20.0 to 60.0μg/mL. Linearity was confirmed by calculating R 2 value from linear plot constructed between concentration and peak area ( Table 3, Fig. 3). The R 2 values of the three analytes were in compliance with ICH limits.

Accuracy
The mean percentage recovery of the all the three analytes in spiked solutions of three specified levels were found to be 100%±2 (Table 4), which illustrates that method was highly accurate as it is in compliance with standards laid down by ICH.
Precision %RSD value of the peak areas of the LAM, EVZ, and TDF in replicate injections of the standard solution was assessed as ≤ 2 (Table 5), which describes that the current method was highly precise.

Sensitivity
The detection limit (LOD) and quantification limit (LOQ) responses were assessed to be 1.9μg/ml and 5.8μg/ml for LAM, 1.7μg/mL and 5.2μg/mL for TDF, and 2.3 μg/mL and 6.8μg/mL for EVZ respectively.

Robustness
Intentional changes in optimized procedure conditions to little extent could not influence the system suitability parameter results which were in compliance with the standards of ICH (Table 6), stipulates that the method has considerable robustness.

Forced degradation
The percentage degradations of the three analytes in stressed solutions are given in Table 7. Acid hydrolysis, alkali hydrolysis, oxidative degradation, photo degradation, and thermal chromatograms were mentioned in Figs. 4, 5, 6, 7, and 8 respectively. Those results were stipulating the stability-indicating property of the method.

Assay
The percentage assay of the LAM, EVZ, and TDF in the combined tablet form were within 100%±15 limit (Table 8), which are in compliance with standards of ICH.

Discussion
Many RP-HPLC procedures were available for simultaneous determination of LAM, EVZ, and TDF in combined tablets [12][13][14][15][16][17][18][19]. But no RP-HPLC method was subsisted with a simple solvent system, and less retention time existed. Along with those, the reported methods have drawbacks like less sensitivity and without FD studies. Therefore, endeavored to ascertain a stability-indicating method with simple solvent system [methanol 0.1% formic acid (73:27 v/ v)] and less RT (less than 6min). The RT of the analytes was less than the previously subsisted method which is regarded as cost-effective due to decrease in elution time and volume of solvent to be consumed. Therefore, analysis of sample time is reduced, and more number of samples can be analyzed.
The developed method was validated as per Q2R1 guidelines of ICH. The developed method has good LOD and LOQ of 1.9μg/ml and 5.8μg/ml for LAM, 1.7μg/mL and 5.2μg/mL for TDF, and 2.3 μg/mL and 6.8μg/mL for EVZ respectively. Those values revealed that the current method has good sensitivity than the previously reported methods. The FD studies helped in determination of the amount of the drug degraded by application of different stressed conditions, which represents the stability-indicating property of the method. The developed method validation parameters were in compliance with Q2R1 specifications of ICH.

Conclusion
An easy, accurate, unambiguous, insightful, and precised RP HPLC-PDA method with isocratic mobile phase was created to determine LAM, EVZ, and TDF concurrently in blended powder and their combined tablet. The current method has the ability to separate LAM, EVZ, and TDF, with effective resolution with less retention times. All degradants produced by application of FD degradation were separated with high resolution and determined with good sensitivity that ensures the stability-indicating property of the method. Thus, the projected method has high probability to adopt in the pharmaceutical industrial sector.