An Effective Stability Indicating RP-HPLC Method for Simultaneous Estimation of Lamivudine and Raltegravir in Bulk and Their Tablet Dosage Form

Background: In the current study, asimple and specific stability indicating RP-HPLC method was developed and validated for the determination of Lamivudine and Raltegravir in bulk drug and it tablet dosage form using an UV-detector. Good separation was achieved by isocratic ally on a Zorbax SB-Phenyl (150 × 4.6 mm, 3.5 μ, 80 A°) column, using a mobile phase composition of buffer (0.1% v/v Phosporic acid in water): Acetonitrile (40:60 v/v) at a flow rate of 1.0 mL/min. The eluted analytes detected at 260 nm wavelength. Results: Lamivudine and Raltegravir were eluted at 3.1 and 5.4 min respectively with run time 7 min. Linearity in the method was measured in the concentration range of 30 – 70 μg/mL and 60 – 140 μg/mL for Lamivudine and Raltegravirrespectively. The percentage recoveries of Lamivudine and Raltegravirwere determined to be 100.30% and 100.53%, respectively. The validation of the developed method is carried as per USFDA and ICH guidelines, and the degradants were well resolved from Raltegravir and Lamivudine peaks. The developed RP-HPLC method was highly precise, specific, sensitive, and stability indicating. Original Research Article Kumar et al.; JPRI, 33(45B): 263-277, 2021; Article no.JPRI.75005 264 Conclusion: The results of the analysis prove that thedeveloped RP-HPLC method is simple, economical and widely acceptable, which can be used in routine quality control tests in the industry.


INTRODUCTION
The antiviral therapy with combination of multi drugs is a great advancement in the treatment of human immune virus (HIV) and hepatitis B diseases. The use of multiple drug therapy, i.e., at least three or more drugs alone or in combination daily is in practice to treat the HIV effectively [1][2]. However, extensive research on multiple drug therapy revealed that a t-drug regimen consisting of Lamivudine and Raltegravir controls the HIV disease effectively.
A sensitive, shot run and effective analytical methodsare required for a drug to analyze individually or simultaneously in combination with other drugs in pharmaceutical industry.On the converse based on the review of literature very few analytical methods such as RP-HPLC methods and UV methods were published for the estimation of Lamivudine and Raltegravir individually [12][13][14]. Further, there are some RP-HPLC methods available for simultaneous estimation of Lamivudine, Raltegravir, and tinofivirdisproxil fumarate or butcaver sulfate or abacavir in triple combination [15][16][17][18][19]. As per FDA official news, Lamivudine and Raltegravir fixed dose film-coated tablet got an approval in April 2019 for the treatment of HIV-1. To our knowledge, there is no short run time RP -HPLC method reported for simultaneous estimation of Lamivudine and Raltegravir in bulk drug and pharmaceutical formulations. Thus, efforts were made to develop selective, sensitive and fast analytical method for the estimation of Lamivudine and Raltegravir in their combined dosage form using reverse phase high performance liquid chromatographic method.

Materials
HPLC grade acetonitrile, Milli-Q water, Phosphoric acid and remaining analytical grade chemicals were obtained from Merck India Limited, Mumbai, India. API of Lamivudine and Raltegravir was provided by Fortune Pharma, Hyderabad, as a gift sample.

Chromatographic Conditions
RP-HPLC experiments were performed on WATERS 2695 with 2487 PDA detector with auto sampler, data-processing, and acquisition has done by using the Empower 2 software. Effective separation attained by injecting 10 μL of the standard solution into Zorbax SB-Phenyl (150 × 4.6 mm, 3.5 μ, 80 A°) column, using a mobile phase composed of buffer (0.1% v/v Phosporic acid in water): Acetonitrile (40:60) at a flow rate of 1.0 mL/min, and the eluted analytes were detected at 260 nm wavelength. Ambient temperature 30°C was maintained in auto sampler and in the analytical column. Standard, sample and mobile phase solutions were filtered through a 0.45-μm nylon filter prior to injecting into the HPLC system (Fig. 2).

Preparation of Standard Solution
Weigh accurately working standard equivalent to 50 mg of Lamivudine and 100 mg of Raltegravir transferred into 100 mL volumetric flask, add ~30 ml of diluent (acetonitrile and water (50:50)) and dissolve, further make up the volume with diluent. 1.0 ml of above solution was transferred into 10 mL volumetric flask, again volume made with diluents to obtain concentration of 50 μg/mL, and 100μg/mL for Lamivudine and Raltegravir, respectively, said as 100% level concentrations. Prior to injecting, standard solution was filtered through 0.4 μm Nylon filter (Fig. 3).

Preparation of Sample Solution
An amount of tablet (Dutrebis) powder (150 mg) equivalent to 50 mg of Lamivudine and 100 mg of Raltegravir was weighed and transferred into 100 mL volumetric flask, volume made with diluents to 100 mL. One milliliter of above solution was transferred into 10 mL volumetric flask, volume made with diluents to obtain concentration of 50 μg/mL, and 100μg/mL for Lamivudine and Raltegravir respectively. Prior to injecting, sample solution was filtered through 0.4 μm Nylon filter (Fig. 4).

System Suitability Test
The system suitability test of the current method was carried out by injecting 100% level of working standard concentration in 6 replicates, and parameters like percentage relative standard deviation (% RSD), USP tailing factors (T), USP plate count (N), and resolution (R) were evaluated for the obtained chromatograms.

Linearity
The Linearity is the ability of the method to elicit test results that are proportional to concentration of the analyte in the sample. The linearity of the present method has performed by injecting the series of working standard concentrations ranges from 30 μg/ml to 70 μg/ml of Lamivudine and 60 μg/mL to 140 μg/mL of Raltegravir into the HPLC system under optimized chromatographic conditions. Eventually, linearity graph was plotted for concentration vs peak area and determined regression coefficient (r2) value.

Precision
It is the closeness of test results obtained by the method to the true value. Usually, it was determined in the intraday and inter-day. Intraday and inter-day precision of the method were performed by injecting 100% level of working standard concentration for 6 times in a day and 3 times per day for three continuous days. Percentage RSD calculated for peak areas obtained.

Accuracy
The accuracy of the method was established by recovery of known amount sample solution spiked at three different standard concentration levels about 50, 100, and 150%, each level of solution injected in triplicate. The percentage mean recovery at three different levels of the drug solution was calculated.

Specificity
Specificity represents the ability of the method to determine or assess the intended drug in the presence of other substances without interferences. Ten microliter volume of prepared blank solution, 100% level pure working standard solution injected individually. The retention time (RT) of individual injection of standard sample solution alone and along with placebo was observed to assess any interference that has been happened with peaks of Lamivudine and Raltegravir in obtained chromatograms.

Sensitivity
The LOD and LOQ were calculated by implementation of standard deviation method, in which the following formulae were used.

LOD ¼ 3 σ=S LOQ ¼ 10 σ=S
Where σ is the standard deviation of the intercept, and S is the slope of the linear curve.

Robustness
The robustness of the method was checked by slightly and deliberately changing the flow rate, mobile phase composition, and maximum absorption wavelength. It can be performed by evaluating the system suitability parameters after changing the HPLC flow rate (± 0.1 mL/ min) and mobile phase ratio (± 1 mL).

FORCED DEGRADATION STUDIES
In forced degradation studies, intentionally drug substance is exposed to conditions more intense than accelerated conditions. Chemical stability of the drug molecule can be depicted with forced degradation studies, which helps in successful development of stable formulation with appropriate storage conditions. ICH guidelines emphasized certain degradation conditions like acid hydrolysis, base hydrolysis, oxidation, thermal degradation, and photo stability in ICH Q1A, QIB, and Q2B guidelines.

Acidic Degradation Solution
0.2 mL of 1 N HCl is added to 1.0 mL of stock solution and was reflux for 3 hours at 70 °C in water bath, cool the solution for 24 h at room temperature. Later the solution was neutralizing with 1 N NaOH and done the further dilution to get a solution having 50 μg/mL of Lamivudine and 100 μg/mL of Raltegravir (Fig. 5).

Alkali Degradation Solution
0.2 mL of 1 N NaOHis added to 1.0 mL of stock solution and was reflux for 3 hours at 70°C in water bath, cool the solution for 24 h at room temperature. Later the solution was neutralizing with 1 N HCl and done the further dilution to get a solution having 50 μg/mL of Lamivudine and 100 μg/mL of Raltegravir (Fig. 6).

Oxidative Degradation Solution
1.0 mL of 3% Hydrogen peroxide and 1.0 mL of stock solution taken into 100 ml of Volumetric flask and was reflux for 3 hours at 70 °C in water bath, cool the solution for 24 h at room temperature. Later, make up to10 mL with diluent to obtain concentration of 50 μg/mL and 100 μg/mL for Lamivudine and Raltegravir respectively (Fig. 7) [20].

Thermal Degradation Solution
100 mL of standard stock solution taken into 500 mL of Volumetric Flask and kept it in heating chamber for 24 h at 80°C/75% RH. Further above solution is diluted to 10 mL to obtain concentration of 50 μg/mL and 100 μg/mL for Lamivudine and Raltegravir respectively (Fig. 8).

Photolytic Degradation
1 mL of sample stock solution filtrate was transferred to 100 mL of Volumetric Flask; it was kept for 12 hours in UV-light. Then volume was made up to mark with Diluent and mixed well and injected. The representative chromatogram is shown in Fig. 9.

Assay
Assay of the Lamivudine and Raltegravircan be determined by injecting standard and sample solutions at the concentration about 50 μg/mL and 100 μg/ mL respectively. The preparation of sample and standard solutions was mentioned prior the in methods section.

Results
As per solubility studies, we found that Lamivudine was freely soluble in water, acetonitrile and partially soluble in methanol. Raltegravir was freely soluble in methanol and water. Based on the solubility data, water and acetonitrile in (50: 50) ratio selected as diluent to prepare sample and standard solutions.

METHOD OPTIMIZATION
Method optimization was successfully completed by applying trial and error method in such a way to obtain a chromatogram with good accepted number of USP plates,efficiency,resolution (R), and tailing factor. In this way, numerous trials have been done by altering flow rate, mobile phase composition and columns.Eventually, the methodwith Zorbax SB-Phenyl (150 × 4.6 mm, 3.5 μ, 80 A°) column, mobile phase composition of buffer (0.1% v/v Phosporic acid in water) :Acetonitrile (40:60) and a flow rate of 1.0 mL/min was selected as optimized method. The results obtained in the trial and error method were mentioned in Table 1; trial 7 selected as optimized conditions and optimized chromatogram shown in Fig. 10 [20].

System Suitability
100% level concentration of both drugs was prepared and injected into the HPLC system. The USP tailing factor (≤ 2), % RSD (≤ 2) and USP plate count (> 2000) values shown in Table  2 were satisfying the acceptance criteria as per Q2 specifications of ICH guidelines.

Linearity
The linearity was performed for the prepared standard solutions of lamivudine and raltegravirat various concentration range of 30 to 70 μg/mL and 60 to 140 μg/mL respectively (Fig. 11). It was determined by constructing calibration curve between peak area and concentration (

Accuracy
Percentage mean recovery of the Lamivudine and Raltegravir at three different concentration levels that were observed as 100% ± 2 illustrates the acceptance of the method as per Q2 specifications of ICH guidelines. Results were shown in Table 4.    At each percentage level mean percentage recovery in the acceptable limit of 98 to 102%

Precision
We found to be 0.45 and 0.77 percentage RSD values by injecting 100% level working standard solution of Lamivudine and Raltegravir respectively (Table 5), and depicts the precision of the method.

Sensitivity
The LOQ and LODfound as 11μg/mL and3.65μg/mL for Lamivudine and 5μg/mL and 1.6μg/mL for Raltegravir, respectively, which indicates that method has good sensitivity.

Robustness
The robustness was unaffected when small, deliberate changes like in mobile phase ratio, flow rate, and absorption maximum and all values obtained in acceptable limits as per ICH guidelines and results are summarized in Table 6.

FORCED DEGRADATION RESULTS AND DISCUSSION
Forced degradation conditions such as acidic, basic, oxidative, reduction, thermal, hydrolysis, and photolytic stresses were attempted as per the International Conference of Harmonization (ICH) guidelines Q1A (R2). There is an effect of assay results. The results are shown in Tables 7  & 8.

Percentage Assay
Lamivudine and Raltegravir assay percentage in tablets were found as 100% ± 15. Hence it indicates that the analyzed tablets have purity within the acceptance specification as per ICH guidelines. Results were shown in Table 9.

Discussion
Few methods were reported for the estimation of Lamivudine and Raltegravir in different combination [12][13][14]. In the present method, raltigravir was eluted at 5.46 min and Lamivudine was eluted at 3.13 min with a run time of 7 min. The developed procedure using reverse phase HPLC with UV-detector and the bestmobile phase composition was found to bebuffer (0.1% v/v Phosporic acid in water): Acetonitrile (40:60 v/v). The flow rate of the solvent system was kept at 1.0 mL/ min. TheLinearity was obtained in over the concentration range of30 -70 μg/mL and 60 -140 μg/mL for Lamivudine and Raltegravir respectively (R2=0.999) with a LOD and LOQ of 0.05 μg/mL and 0.1 μg/mL, respectively.

CONCLUSION
A specific, precise, accurate, stability-indicating, isocratic RP-HPLC method was developed for the estimation of Lamivudine and Raltegravir in bulk and tablet dosage form. The compound was evaluated by forced degradation pertaining to several stress conditions. The developed methodseparates both of the drugs and its degradation products successfully and quantifies the active contents at minute concentration levels. The developed methodfully validated according to ICH guidelines for all the parameters which were found within acceptance criteria. Hence, the proposed method can be adapted to regular analysis in pharmaceutical industry.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.