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 Table of Contents  
Year : 2022  |  Volume : 14  |  Issue : 4  |  Page : 200-208

Response surface methodology-based quantification of lamivudine and zidovudine using reverse-phase high-performance liquid chromatography in pharmaceutical formulation

1 Department of Quality Assurance, L.J Institute of Pharmacy, L.J University, Ahmedabad, Gujarat, India
2 Department of Regulatory Affairs, L.J Institute of Pharmacy, L.J University, Ahmedabad, Gujarat, India

Date of Submission06-Sep-2022
Date of Decision27-Sep-2022
Date of Acceptance19-Oct-2022
Date of Web Publication16-Dec-2022

Correspondence Address:
Darshil B Shah
Department of Quality Assurance, L.J Institute of Pharmacy, L.J University, S.G Road, Ahmedabad - 382 210, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajprhc.ajprhc_74_22

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Objective: A novel reverse-phase high-performance liquid chromatography (RP-HPLC) method for estimation of lamivudine and zidovudine by using an experimental design approach applying the response surface technique was developed and validated using a C18 column and its application in marketed formulation. Multivariate optimization of experimental conditions was achieved using experimental design employing organic content in the mobile phase, pH, and flow rate of the mobile phase as three independent variables. The aim of this study was to apply response surface methodology and to study the effect of the independent variables on the separation and estimation of both drugs by the RP-HPLC method using a faced central composite experimental design. Materials and Methods: Derringer's desirability function was used to optimize the retention period of the last eluting peak and peak symmetry, and it was discovered that the optimal conditions were potassium dihydrogen phosphate buffer and acetonitrile in an isocratic ratio of 55:45 w/v, pH 3.5, and 0.4 ml/min flow rate. Baseline separation of both medications with good resolution and a run time of more than 7 min was accomplished using this ideal condition. Results: For lamivudine and zidovudine, a linear response was seen over the concentration range of 2–12 and 4–24 μg/mL, respectively. Lamivudine's limit of detection and limit of quantitation were determined to be 3.08 and 9.18 μg/mL, respectively, whereas zidovudine's values were 3.24 and 10.17 μg/mL. According to the ICH guidelines acceptance criteria for linearity, accuracy, precision, specificity, and robustness, the method was successfully validated.

Keywords: Design of experiments, design space, lamivudine, response surface methodology, reverse-phase high-performance liquid chromatography, validation, zidovudine

How to cite this article:
Chauhan AA, Yadav NJ, Shah AG, Shah DB, Maheshwari DG, Shah JS. Response surface methodology-based quantification of lamivudine and zidovudine using reverse-phase high-performance liquid chromatography in pharmaceutical formulation. Asian J Pharm Res Health Care 2022;14:200-8

How to cite this URL:
Chauhan AA, Yadav NJ, Shah AG, Shah DB, Maheshwari DG, Shah JS. Response surface methodology-based quantification of lamivudine and zidovudine using reverse-phase high-performance liquid chromatography in pharmaceutical formulation. Asian J Pharm Res Health Care [serial online] 2022 [cited 2023 Jun 8];14:200-8. Available from: http://www.ajprhc.com/text.asp?2022/14/4/200/363943

  Introduction Top

Acquired immunodeficiency syndrome is a chronic, potentially life-threatening condition caused by the human immunodeficiency virus (HIV).

HIV is a set of the genus Lentivirus under the family of Retroviridae or subfamily thoretrovirinae. Based on the genetic characteristics and various types of viral antigens, HIV is classified into types 1 and 2 (HIV-1, HIV-2).[1]

Zidovudine is recommended as initial therapy or HIV infection 1 and is generally tolerated well, but clinical studies show that its beneficial effects are limited in duration, it is because of the development of resistance by HIV.

Lamivudine or (-)-2-deoxy-3-thiacitidine (also known as 3TC), a reverse transcriptase inhibitor, exhibits in vitro activity against various HIV-1 isolates, including the zidovudine-resistant virus. Combination therapy with antiretroviral drugs may provide a more persistent antiviral effect, reduce the development of drug resistance, and affect a wider cell or tissue reservoir of HIV infection.[2],[3]

Decades from treatment with a single NRTI drug, followed by double NRTIs to a combination of three drugs of at least two drug classes known as ART or highly active antiretroviral therapy. ZDV and 3TC are the NRTI backbone of ART regimens for patients who have not received antiretroviral treatment for several years, based on favorable efficacy and safety information from multiple randomized controlled clinical trials. Patients did not receive antiretroviral treatment (either efavirenz, raltegravir, or ritonavir-boosted darunavir or atazanavir).

A unique consideration of ZDV and 3TC is their long-term safety history. It is one of the best-understood antiretroviral drugs in use today.[4]

On the bases of literature reports, reverse-phase high-performance liquid chromatography (RP-HPLC) methods have been developed and reported for the quantification of lamivudine and zidovudine individually and in the combination dosage form. However, all the reported methods were based on the one factor at a time (OFAT) approach. The experiment would be helpful to pharmaceutical organizations implementing the concept of DOE to improve the quality of products and processes in a systemic risk-based manner rather than determining the test quality into product retrospectively.[5]

The current research utilized an experimental design approach for evaluating the significance of the studied parameters and optimizing the chromatographic conditions and developing a sensitive, rapid, effective, and straight HPLC method employing the DOE approach of estimation of lamivudine and zidovudine in combined dosage formulation. The experimental design was used for the optimization of mobile phase, pH, and flow rate variables, and their effect was seen at a retention time of both drugs.[6],[7]

Design of experiment (DOE) base full factorial design was applied to quantify lamivudine and zidovudine in pharmaceutical formulation by RP-HPLC method. All chromatographic parameters like mobile phase, and flow rate can easily be optimized using the DOE which is called the Quality By Design approach. The main purpose of the study is to optimize various chromatographic parameters for better separation and quantification of lamivudine and zidovudine in a combined dosage formulation. The design of experiments is a multifactorial approach. It shows a distinct advantage over conventional OFAT methods. It aims to impart quality into the process.[7]

The DOE is used for statistically designed experiments for getting quality results. It provides knowledge about the design space to work within for achieving quality. It aims to impart quality into the process. It is a safe zone where the method variables have no significant influence on the quality of the product. It is developed to determine the design space.[8]

  Materials and Methods Top


Lamivudine and zidovudine were obtained as gift samples from Dr. Reddy's Laboratories, India. Potassium dihydrogen phosphate was used to prepare phosphate buffer and phosphoric acid was used to adjust pH of the mobile phase. Ultra-purified HPLC-grade water and ACN were used to prepare all solutions. The formulation was prepared in a Cipla Ltd. which has 150 mg of lamivudine and 300 mg of zidovudine.


A Shimadzu HPLC_2010-CHT instrument with a 20 μL loop volume was used. The system also includes a UV–VIS (Shimadzu, SPD-20A) detector operated at a wavelength of 271 nm. Data were acquired and processed by using LC-solution software. Chromatographic separation was performed using the C18 column (250 mm × 4.6 mm × 5.0 μm).


Experimental design (Faced central composite [FCC]), desirability function, and data analysis calculations were performed by using Design-Expert version 13. Microsoft Excel 13 was used for the calculation of the standard deviation and relative standard deviation (RSD) of validation data.

  Preparation of Solutions Top

Preparation of standard stock solution

A quantity of 10 mg of lamivudine and 10 mg of Zidovudine API (Active pharmaceutical ingredient) were weighed and transferred into a 100 ml volumetric flask and dissolved in sufficient quantity of mobile phase and made to volume to obtain 100 μg/ml of both lamivudine and zidovudine. Standard stock solutions were diluted using the mobile phase to obtain working standard solutions (2–12 μg/mL and 4–24 μg/mL), and both were protected from light during analysis.

Preparation of sample solution

Powder equivalent to 10 mg of tablet formulation was taken into 100 ml of a volumetric flask. Sonicate it for about 15 min with intermittent shaking. Allow it to come to room temperature, diluted up to the mark, and mixed. For assay determination, 6 μg for lamivudine and 12 μg for zidovudine was prepared in the mobile phase.

Chromatographic Procedure

The C18 column (250 mm × 4.6 mm × 5.0 μm) was used for chromatographic separations. The mobile phase employed in chromatographic separation was a mixture of ACN and potassium dihydrogen phosphate buffer (45:55) and its pH (3.5) was adjusted with phosphoric acid (H3PO4). The flow rate of 0.4 ml/min, injection volume of 10 μL, and maximum wavelength of 271 nm was used for analysis.

Experimental design and response surface methodology

The design space is the established range of process parameters that have been demonstrated to provide assurance of quality. Working within the design space is not generally considered a change of the approved ranges for process parameters and formulation attributes. Movement out of the design space is considered to be a change and would normally initiate a regulatory postapproval change process. DOEs is an efficient procedure for planning experiments so that the data obtained can be analyzed to yield valid and objective conclusions. A structured, organized method for determining the relationship between factors affecting a process and the output of that process is known as the “DOE.” DOEs is used to determine the causes of variation in the response, find conditions under which the optimal (maximum or minimum) response is achieved, compare responses at different levels of controlled variables and develop a model for predicting response.[9]

  Results and Discussion Top

Method development and optimization

RP-HPLC Method was used for the separation and analysis of lamivudine and zidovudine drug mixture because both drugs are polar. We considered the C18 column for the separation of both drugs due to better resolution and peak symmetry.

The overlay UV spectra of lamivudine and zidovudine, indicate that 271 nm is the optimum wavelength to detect lamivudine and zidovudine with good detector sensitivity and minimum baseline noise. pH of the mobile phase is an important factor for the selection of the analytical method. We tried pH from 3.0 to 3.8 for better separation of the lamivudine and zidovudine based on their pKa value and literature review, wherein the mobile phase with pH 3.5 was finally optimized. Maximum tailing (>2) was observed for lamivudine and zidovudine. Better separation, resolution, and less interference were observed with a mobile phase combination of ACN and phosphate buffer (45:55), at pH 3.5 and flow rate of 0.4 ml/min.

Design of experiment and design space

The central composite design concept has been came out in the process of optimization in which the best possible outcome can be obtained by employing various response variables. CCD model is well known as Box-Wilson Central Composite Design in which the development of center points is to be carried out using a group of star points which helps in the estimation of curvature. FCC Design matrix consisting of 15 optimized experiments has been developed. A two-level factorial design having center points within the experimental region is developed and used in the method development. Optimization of acetonitrile concentration and flow rate of the mobile phase was done based on the responses obtained and was finalized between 45% v/v and 55% v/v of organic component modifier and 0.2 to 0.4 ml/min of flow rate, respectively, to obtain better peak symmetry for both the drugs and accurate quantification of drugs with minimum run time. The pH range was optimized in the range of 3.5–3.8 due to improvement in peak symmetry, as higher peak tailing was occurring at pH more than 3.8, and the shelf life of the column was decreasing at pH <3.5 [Table 1] and [Table 2]. The adjusted R2 value was obtained well within the acceptable limits with probability P.[10]
Table 1: Dependent variables in high-performance liquid chromatography for central composite design

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Table 2: Experimental conditions and responses for central composite design

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[Table 3] illustrates the interaction term with the largest absolute coefficient among the fitted models is 0.05AC of the Rt model. By applying a regression model, the positive interaction between A and C was statistically significant (P = 0.001) for Rt. By performing various trials, it was revealed that by increasing ACN concentration, there was decrease in retention time of both at any level of buffer pH.
Table 3: ANOVA-based statistical parameter and regression model

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In addition, it was found that at lower concentrations of factor A, a slight increase in buffer pH causes a slight decrease in the retention time of lamivudine (Rt). It was determined that this interaction was synergistic because it resulted in a reduction in the analysis run time. Every factor has a moderate impact on zidovudine (T) tailing, which was clearly Model T second reaction serves as evidence.

Interpretation of perturbation plots

After all data had been processed using the Design Expert software, design space was produced.

The perturbation graphs in [Figure 1] make it easier to comprehend the findings. Design space was produced following the processing of all data using the modeling program Design Expert. [Figure 2] and [Figure 3] show two-dimensional color maps with warm “red” for high retention time and tailing and cold “blue” for poor retention and peak tailing.[11] The shortest retention duration of lamivudine and the tailing of the peak of zidovudine were used to visually select the working point from the created Design Space. The retention duration of lamivudine declines toward pH 3.5, the flow rate is 0.6 ml/min, and the percentage of ACN is 45%, as shown in [Figure 2], [Figure 3], [Figure 4], [Figure 5]. The tailing of the zidovudine peak was simultaneously shifted toward high ACN content and an acidic pH.
Figure 1: (a) Perturbation plot showing effect of each factor on retention time of lamivudine (b) Perturbation plot showing effect of each factor on tailing of zidovudine

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Figure 2: (a) 2D design space for tailing factor of lamivudine ACN (%w/v)_pH, (b) 2D design space for tailing factor of lamivudine ACN (%w/v)_Flowrate (ml/min), (c) 2D design space for tailing factor of lamivudine pH_Flowrate (ml/min)

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Figure 3: (a) 2D design space for tailing factor of zidovudine ACN (%w/v)_pH, (b) 2D design space for tailing factor of zidovudine ACN (%w/v)_Flow rate (ml/min), (c) 2D design space for tailing factor of zidovudine pH_Flow rate (ml/min)

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Figure 4: (a) 3D design space for tailing factor of lamivudine ACN (%w/v)_pH, (b) 3D design space for tailing factor of lamivudine ACN (%w/v)_Flowrate (ml/min), (c) 3D design space for tailing factor of Lamivudine pH_Flowrate (ml/min)

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Figure 5: (a) 3D design space for tailing factor of zidovudine ACN (%w/v)_pH, (b) 3D design space for tailing factor of zidovudine ACN (%w/v)_Flow rate (ml/min), (c) 3D design space for tailing factor of zidovudine pH_Flow rate (ml/min)

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Derringer's desirability function

The best method to use when several replies need to be optimized for various targets is the Derringer desirability (D) function, which is defined as the geometric mean, weighted or not, of the individual desirability functions. [Table 4] lists the suggested criteria for choosing the best experimental circumstances to optimize individual responses. The retention time was given high importance as an important factor in the method development and was optimized using Design Expert. Maximum desirability value (D = 1) was obtained with ACN 45% w/v, buffer pH 3.5, and flow rate of 0.4 ml/min as optimized coordinates for the proposed method.[12]
Table 4: The comparison of the experimental and predictive value of different objective functions under optimal condition

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Method validation

Method validation provides a high degree of assurance that a process, system, equipment or assay will meet the specified requirements for the intended analytical applications. Linearity, specificity, precision, accuracy, system suitability, the limit of detection (LOD) and limit of quantification (LOQ), robustness, and ruggedness were all taken into consideration during the validation of the developed Doe-based analytical method in accordance with the guidelines provided by ICH and FDA regulations.[13],[14],[15],[16]


The capacity of a method to assess the analyte response while its degradants and Excipients are present is known as specificity.[17] To determine whether there would be any interference with the retention period of lamivudine and zidovudine, a mixture of excipients was produced and injected. There was no evidence of excipient interference with lamivudine and zidovudine retention times.[14]


The suggested HPLC methods linearity was investigated at concentrations of 2–12 μg/mL for lamivudine and 4–24 μg/mL for zidovudine. The standard stock of 100 μg/mL lamivudine was diluted with mobile phase to acquire 2, 4, 6, 8, 10, and 12 μg/mL lamivudine and 100 μg/mL zidovudine was diluted with mobile phase to obtain 4, 8, 12, 16, 20, and 24 μg/mL zidovudine. The six replicates of each injection were carried out. The calibration curve of the mean area under curves versus concentration was plotted, and the regression coefficient was calculated. The results were R2 = 0.999 for lamivudine and R2 = 0.999 for zidovudine as shown in [Figure 6]. According to the regulations established by the International Conference on Harmonization and the Food and Drug Administration, compliance requires a regression coefficient, R2 >0.999.[15] The findings of the linearity of both medications are shown in [Table 5].
Table 5: System suitability parameters for lamivudine and zidovudine

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Figure 6: (a) Calibration curve of lamivudine, (b) calibration curve of zidovudine

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System suitability

The resolution between the lamivudine and zidovudine was at least 2. Lamivudine and zidovudine were suitable because their tailing factors were not 2, thus indicating suitability [Table 6].
Table 6: Linearity data of lamivudine and zidovudine

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Accuracy of the proposed method was evaluated by spiking standard stock solution containing lamivudine and zidovudine into placebo in amounts comparable to the quantity present in sample preparation to obtain 50%, 100%, and 150% of the desired concentration.[17] The placebo solution was mixed with the standard solution, and the % recovery was calculated. The mean recovery of both the drugs were found in the range of 98-100% with RSD as imposed by regulatory guideline, indicates that the proposed method was accurate for both drugs [Table 7].
Table 7: Accuracy data of lamivudine and zidovudine

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The proposed HPLC method precision was investigated by conducting a repeatability study and intraday precision for both drugs. The repeatability research was carried out by injecting lamivudine at 6 μ/ml and zidovudine at 12 μ/ml (n = 6). The intraday precision was investigated by injecting 8, 10, and 12 μ/mL of lamivudine and 16, 20, and 24 μ/mL of zidovudine (n = 3) and calculating the percent RSD. The results are shown in [Table 6]. The results were within the regulatory guidelines specified target threshold (2%) [Table 8].
Table 8: Precision data of lamivudine and zidovudine

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Limit of detection and limit of quantificationμ

According to ICH guidelines Q2 (R1), the LOD and LOQ for lamivudine and zidovudine were established. While LOQ is the smallest concentration of analyte that can be detected, LOD is the smallest concentration of analyte that generates observable reaction (the signal to noise ratio for LOD is 3.3) (10 is the signal to noise ratio). Lamivudine has a LOD value of 0.15 and a LOQ value of 0.46 and LOD value for zidovudine is 1.50 and LOQ value is 4.56, respectively [Table 9].
Table 9: LOD and LOQ data of lamivudine and zidovudine (n=3)

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Assay of marketed formulation

Three batches of the marketed formulation were subjected to the proposed HPLC method. By dissolving the equivalent amount of tablet powder in the mobile phase, a stock solution of 1000 μg/mL for lamivudine and 100 μg/mL for zidovudine was prepared. 0.4 mL of the solution was transferred to a volumetric flask with a volume of 10 mL filled with mobile phase. The sample was injected into the HPLC system, and the area under the curve was compared to that of standard solutions. The percentage of drug was calculated, as per the standard deviation. The results show that the formulation's label claim is well accepted [Table 10].
Table 10: Assay of marketed formulation

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  Conclusion Top

To optimize significant parameters for the estimation of lamivudine and zidovudine in combined dosage form using RP-HPLC, Central Composite design of DoE approach was used. Derringer's Desirability Function was used to optimize independent variables affecting retention time as method response. This method was evaluated for linearity, precision, accuracy, and selectivity, and it proved to be convenient and effective for Lamivudine and Zidovudine quality control in combination. The experimentally observed LOD and LOQ values of both drugs were lower, demonstrating a high degree of practical utility for estimating combination drugs in pharmaceutical dosage forms.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Campbell-Yesufu OT, Gandhi RT. Update on human immunodeficiency virus (HIV)-2 infection. Clin Infect Dis 2011;52:780-7.  Back to cited text no. 1
Levy JA. Pathogenesis of human immunodeficiency virus infection. Microbiol Rev 1993;57:183-289.  Back to cited text no. 2
Anderson PL, Rower JE. Zidovudine and lamivudine for HIV infection. Clin Med Rev Ther 2010;2:a2004.  Back to cited text no. 3
Eron JJ, Benoit SL, Jemsek J, MacArthur RD, Santana J, Quinn JB, et al. Treatment with lamivudine, zidovudine, or both in HIV-positive patients with 200 to 500 CD4+ cells per cubic millimeter. North American HIV Working Party. N Engl J Med 1995;333:1662-9.  Back to cited text no. 4
Kalariya PD, Namdev D, Srinivas R, Gananadhamu S. Application of experimental design and response surface technique for selecting the optimum RP-HPLC conditions for the determination of moxifloxacin HCl and ketorolac tromethamine in eye drops. JSCS 2017;21:S373-82.  Back to cited text no. 5
Bardan MY, Shakya AK. Stability indicating hplc-pda determination of febuxostat utilizing design of experiments and its application to formulation and dissolution studies. Indian J Heterocycl Chem 2016;26:23-35.  Back to cited text no. 6
Krishna MV, Dash RN, Reddy BJ, Venugopal P, Sandeep P, Madhavi G. Quality by Design (QbD) approach to develop HPLC method for eberconazole nitrate: Application oxidative and photolytic degradation kinetics. JSCS 2016;20:S313-22.  Back to cited text no. 7
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Habte G, Hymete A, Mohamed AM. Simultaneous separation and determination of lamivudine and zidovudine in pharmaceutical formulations using the HPTLC method. Anal lett 2009;42:1552-70.  Back to cited text no. 10
Darshil BS, Patel BH, Shah JS. Response surface methodology based development and quantification of celecoxib and amlodipine using RP-HPLC. Int J Life Sci Pharma Res 2022;12:75-85.  Back to cited text no. 11
Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 2008;76:965-77.  Back to cited text no. 12
ICH Q2 (R1), Validation of Analytical Procedure: Text and Methodology, International Conference on Harmonization, IFPMA, Geneva, Switzerland, 2005.  Back to cited text no. 13
ICH Expert Working Group. ICH harmonised Tripartite Guideline–Validation of Analytical Procedures text and Methodology: Q2 (R1). Geneva: International Conference on Harmonisation of Technical Requirements for Registrataion of Pharmaceuticals for Human Use; 2005.  Back to cited text no. 14
Food US. Drug Administration Analytical Procedures and Methods Validation for Drugs and Biologics: Guidance for Industry. Silver Spring, MD: US Food and Drug Administration; 2015.  Back to cited text no. 15
Elsonbaty A, Madkour AW, Abdel-Raoof AM, Abdel-Monem AH, El-Attar AMM. Computational design for eco-friendly visible spectrophotometric platform used for the assay of the antiviral agent in pharmaceutical dosage form. Spectrochim Acta A Mol Biomol Spectrosc 2022;271:120897.  Back to cited text no. 16
Modi PB, Shah NJ. DoE Approach: A stability indicating RP-HPLC method for simultaneous estimation of methylparaben, mometasone furoate and eberconazole nitrate in topical formulations. JAPS 2014;4:020-5.  Back to cited text no. 17


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]


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