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Year : 2015  |  Volume : 6  |  Issue : 1  |  Page : 27-32

Validated RP-HPLC-PDA method for simultaneous determination of Zidovudine, Lamivudine, and Nevirapine in pharmaceutical formulation

1 Research and Development, Jawaharlal Nehru Technological University, Kakinada and Department of Pharmaceutical Analysis, Balaji College of Pharmacy, Anantapur, Andhra Pradesh, India
2 Department of Pharmaceutical Analysis, SSJ College of Pharmacy, Hyderabad, Telangana, India
3 Centre for Pharmaceutical Sciences, IST, JNTU-H, Hyderabad, Telangana, India

Date of Web Publication8-Jan-2015

Correspondence Address:
K Vanitha Prakash
Department of Pharmaceutical Analysis, SSJ College of Pharmacy, Hyderabad - 500 075, AP
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2394-2002.148890

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Aim and Objectives: A simple, rapid, and sensitive high performance liquid chromatographic method with ultraviolet detection has been developed and validated according to the International Conference on Harmonization (ICH) guidelines for the quantitation and qualification of zidovudine (ZID), lamivudine (LAM), and nevirapine (NEV) in pharmaceutical dosage forms. Materials and Methods: The proposed method was based on the separation of the drugs in reversed phase mode using Water's C18 250 cm × 4.6 mm, 5 μ particle size column maintained at an ambient temperature. The optimum mobile phase consisted of Water: Methanol (70:30 v/v), pH adjusted to four with orthophosphoric acid (OPA). The flow rate of mobile phase was set 1.0 mL min -1 and photodiode array detection was performed at 275 nm with a total run time of 8 min which is very short for accurate analysis of simultaneous estimation of three analytes. The method was validated according to ICH guidelines. Results: The method was linear over the concentration range of 25-75 μg mL -1 with limit of quantifications (LOQ) of 13, 0.49, and 0.40 ng mL -1 for ZID, LAM and NEV respectively and limit of detection (LOD) of 4, 0.14, and 0.12 ng mL -1 for ZID, LAM and NEV respectively. Accuracy (% recovery studies) and precision values of both inter and intraday obtained from six different replicates for all the analytes ranged from 99.00% to 100.00% and % relative standard deviation of precision (assay) was between 0.64 and 1.28, respectively. All the three analytes and their combination drug product were exposed to thermal, photolytic, hydrolytic, reductive, oxidative and peroxide stress conditions and the stressed samples were analyzed by the proposed method. There were no interfering peaks from excipients, impurities or degradation products due to variable stress conditions and the proposed method is specific for the simultaneous estimation of ZID, LAM and NEV in the presence of their degradation products. Conclusion: The proposed method can be successfully applied in the quality control and stability samples of pharmaceutical dosage forms.

Keywords: lamivudine, Zidovudine, Nevirapine, methanol, orthophosphoric acid

How to cite this article:
Ishaq B M, Prakash K V, Mohan G K. Validated RP-HPLC-PDA method for simultaneous determination of Zidovudine, Lamivudine, and Nevirapine in pharmaceutical formulation. Drug Dev Ther 2015;6:27-32

How to cite this URL:
Ishaq B M, Prakash K V, Mohan G K. Validated RP-HPLC-PDA method for simultaneous determination of Zidovudine, Lamivudine, and Nevirapine in pharmaceutical formulation. Drug Dev Ther [serial online] 2015 [cited 2019 Aug 18];6:27-32. Available from: http://www.ddtjournal.org/text.asp?2015/6/1/27/148890

  Introduction Top

Human Immunodeficiency Virus (HIV) has been one of the most overwhelming blood-borne virus affecting a large population across the globe. Due to the development thereafter of various antiretrovirals (ARVs), it led to the significant decrease in the morbidity and mortality rates of the HIV infected population. The standard ARV therapy in treatment of these HIV-infections entails the use of combination drug therapy, usually a triple combination such as two-nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) as a backbone and a nonnucleoside reverse transcriptase inhibitor (NNRTI). [1]

Out of existing various ARV drugs currently suggested by World Health Organization for HIV infection in pediatric population in resource limited countries, the combination of zidovudine (ZID)/lamivudine(LAM) (NRTI backbone) and nevirapine (NEV) (NNRTI) is considered as one of the ideal ARV treatment. [2],[3] ZID is 3-azido-3'-deoxythymidine, are synthetic nucleoside analogs. LAM is (2R, cis)-4-amino-1- (2 - hydroxymethyl-1,3-oxathiolan-5-yl)-(1H) pyrimidin-2-one and NEV is 11-cyclopropyl-5, 11-dihydro-4-methyl-6H dipyrido [3,2 - b:2'3'-e] [1],[4] diazepin-6-one, is a NNRTI of HIV type 1 (HIV-1), [4] chemical structures of ZID, LAM and NEV were shown in [Figure 1] [Figure 2] [Figure 3].
Figure 1: Chemical structure of zidovudine

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Figure 2: Chemical structure of lamivudine

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Figure 3: Chemical structure of nevrapine

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Several analytical methods such as high performance liquid chromatographic (HPLC), [5],[6] LCMS [7] and radioimmunoassay [8],[9] have been reported for simultaneous estimation of ZID/LAM. Some authors have described the analysis of ZID/LAM/NEV in plasma based on HPLC, [10],[11],[12] as well as stability indicating. [13] Some authors have reported the analysis of ZID/LAM/NEV in plasma and fixed dose combinations based on HPLC, HPTLC. [14],[15],[16],[17],[18],[19],[20] Despite the fact that, there were some methods reported regarding the quantitation of ZID, LAM and NEV in pure form and pharmaceutical formulations, but suffers from few drawback like the run time of those methods is more than 20 min, which raises the cost of the mobile phase or very hectic sample preparation procedures etc.

Therefore, the aim of this study was to develop and validate an HPLC-ultraviolet (UV) method, to quantify ZID, LAM, and NEV in its tablet dosage form. Molar absorptivity of ZID, LAM and NEV in the UV region was found to be at 275 nm. The validated method was applied to the analysis of tablets (Brand: Duovir-N, Company: Cipla) containing ZID, LAM and NEV (300 + 150 + 200 mg).

  Material and Methods Top


The HPLC system consisted of a LC Waters (Waters, Milford, MA, USA) using a Water's C 18 250 mm × 4.6 mm, 5 μ column, a quaternary gradient system (600 controller), in line degasser (Waters, model AF). The system was equipped with a photodiode array (PDA) detector (Water, 2998 model) and auto sampler (Waters, model 717 plus). Data was processed using Empower Pro software (Waters, Milford, MA, USA). The mobile phase was pumped at a flow rate of 1.0 mL min -1 . The detection wavelength for analytes was 275 nm.

Chemicals and reagents

Lamivudine, ZID and NEV were supplied by Cipla Ltd., Goa, India with 100% purity. Tablets (Brand: Duovir-N, Company: Cipla) for analysis were purchased from local pharmacy. HPLC grade methanol was procured from Merck, Mumbai. Water used in this study was prepared by Millipore milli Q (Bedford, USA) water purification system. The mobile phase consisted of a mixture of water: Methanol pH 4 adjusted with orthophosphoric acid (OPA) (70:30, v/v) was delivered at a flow rate of 1.0 mL min -1 . The mobile phase was prepared daily, filtered through a 0.45 mm membrane filter (Millipore) and sonicated before use. The injection volume of sample was 10 μL. The mobile phase was used as diluent.

Preparation of stock and standard solutions

The standard stock solutions containing 1 mg mL -1 each of ZID, LAM and NEV were prepared separately by dissolving reference standards in mobile phase and diluting with the same. Diluted solution of 50 μg mL -1 was prepared from the stock solution. Calibration curve solutions containing 25-75 μg mL -1 each of ZID, LAM and NEV were prepared by diluting the standard stock solution to the appropriate volume with the mobile phase.

Preparation of test solution (analysis of zidovudine, lamivudine and nevirapine in tablets)

Ten tablets of Duovir-N were weighed and finely powdered. A quantity of the powder equivalent to one tablet content was accurately weighed, transferred into a 100 ml volumetric flask containing mobile phase. The above mixture was sonicated for about 10 min. For complete mixing. This solution was filtered through Whatman No.1 filter paper. From the filtrate different aliquots were taken in separate 10 ml volumetric flasks and diluted with mobile phase up to the mark so as to get a concentration ranging from 25 to 75 μg mL -1 each of ZID, LAM and NEV. Each of these solutions (10 μL) was then injected 3 times into the column. The mean peak areas of each drug were calculated and the drug contents in the tablets were quantified using the respective regression equations.

Forced degradation study of drug substance and drug product

In order to establish whether the analytical method and the assay were stability indicating, tablets and pure active pharmaceutical ingredient of ZID, LAM and NEV were stressed under thermolytic, photolytic, hydrolytic and oxidative stress conditions as shown by Snyder et al. 1997. [21]

  Results Top

Method development

The chromatographic conditions were optimized for the simultaneous determination of ZID, LAM and NEV in a tablet dosage form within a short analysis time (<8 min). To accomplish these objectives, Water's C18 250 cm × 4.6 mm, 5 μ particle size column was chosen based on peak shapes and resolution. Column was maintained at ambient temperature. Many different combinations of the mobile phase were tested until the suitable phase namely, Water: Methanol in the ratio of 70: 30 v/v, pH 4 (adjusted with OPA) was reached. The mobile phase was pumped at a flow rate of 1 mL min -1 . PDA detection was performed at 275 nm with a total run time of 8 min. After a steady baseline the standard solution were injected and chromatograms were recorded until the reproducibility of the peak areas were found. ZID, LAM and NEV were separated at the retention times of 2.2, 4.0 and 6.0 min respectively. The typical chromatograms of the standard solutions were recorded for the repeatability and the respective chromatogram was given in [Figure 4].
Figure 4: Typical chromatogram of zidovudine, lamivudine and nevrapine with detection at 275 nm

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Method validation after development of method, validation of the method for simultaneous estimation of LAM, ZID and NEV was performed in accordance with International Conference on Harmonization (ICH) guidelines (ICH 2000) which include system suitability, linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ), specificity and robustness.


Calibration graphs were constructed by plotting peak area versus concentration of ZID, LAM and NEV and the regression equations were calculated. The calibration graphs were plotted over five different linear concentrations in the range of 25-75 μg mL -1 for all the drugs. Aliquots (20 ml) of each solution were injected under the operating chromatographic condition described above [Number of replicates (n = 6)]. The method was found linear over the concentration range of 25-75 μg mL -1 for ZID, LAM and NEV. The parameters for the regression analysis are given in [Table 1]. Linearity curves of the three drugs were shown in [Figure 5] (a-c).
Figure 5: (a) Linearity curve of zidovudine. (b) Linearity curve of lamivudine. (c) Linearity curve of nevrapine

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Table 1: Data indicating linearity of the proposed method

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The accuracy of the method was established by recovery studies that is external standard addition method. The known amount of standard was added at three different levels to preanalyzed sample. Each determination was performed in triplicate. The mean recoveries obtained were 98.00% and 100.00% for ZID, LAM and NEV. The results of accuracy were tabulated in [Table 2].
Table 2: Summary of validation parameters

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The intraday and interday precision of the proposed method was determined by analyzing mixed standard solution of ZID, LAM and NEV at concentration 50 μg mL -1 3 times on the same day and on 3 different days. The results are reported in terms of relative standard deviation (RSD). The % RSD values for ZID, LAM and NEV were found to be 1.06% and 0.64% and 1.28% respectively.

Limit of detection and limit of quantitation

The limit of detection and LOQ of ZID, LAM and NEV were determined by calculating the signal-to noise ratio of 3:1 and 10:1, respectively according to ICH guidelines. LOD values for ZID, LAM and NEV were found to be 4 ng mL -1 , 0.14 ng mL -1 , and 0.12 ng mL -1 respectively. LOQ values for ZID, LAM and NEV were found to be 13 ng mL -1 , 0.49 ng mL -1 , 0.40 ng mL -1 respectively.


The robustness of the method was evaluated by assaying the test solutions after slight, but deliberate changes in the analytical conditions like flow rate (0.1 mL min -1 ), and pH of the mobile phase (±0.2). Stability of standard and test solution (prepared from the dosage form) was established by storage at 25°C and 15°C for 48 h. During the storage period, the test solutions were reanalyzed at intervals of 6, 12, 24, 36, and 48 h and assay was determined against appropriate fresh standard preparations.

Forced degradation study

As per ICH guidelines, the target degradation between 10% and 30% should be there for the stability indicating ability of the assay method and the same was tried in the present study. No interfering peaks were found due to degradation products at the drugs RTs [Figure 4]. [Figure 6] (a) and (b) shows the thermolytic and photolytic stress chromatograms.
Figure 6: (a) Chromatogram of thermolytic degradation studies. (b) Chromatogram of photolytic degradation studies

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Assay of the tablet dosage form

The proposed validated method was successfully applied to determine ZID, LAM and NEV in tablet dosage form. The result obtained was comparable with corresponding labeled amounts [Table 3].
Table 3: Assay of tablets

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  Discussion Top

The present research work was designed at developing a rapid, sensitive, precise and accurate HPLC method for the simultaneous estimation of ZID, LAM and NEV in pharmaceutical dosage forms. In order to affect analysis of the component peaks under isocratic conditions, mixtures of water and methanol in different combinations with different pH were tested as mobile phase on a Waters C18 stationary phase. A binary mixture of water and methanol (pH adjusted to 4.0 with OPA) in a ratio of 70:30 v/v was proved to be the most suitable of all the combinations since the chromatographic peaks obtained were better defined and resolved and free from tailing. A flow rate of 1.0 mL min -1 of the mobile phase was found to be suitable. The retention times for ZID, LAM and NEV were found to be 2.2, 4.0, and 6.0 min respectively. A Typical chromatogram showing the separation is given in [Figure 4].{Figure 4}

The method was validated by determining the specificity, linearity, precision and accuracy, LOD, LOQ, robustness, and system suitability as per ICH guideline. The chromatograms recorded for the drug matrix (mixture of drug and excipients) showed almost no excipient peaks within the retention time range of 8 min. All the three drugs were suitably resolved from excipients as shown in [Figure 3]. This indicates that, the method is specific and selective for their simultaneous determination. Six point calibration curves constructed with the working standard dilutions (n = 3) were found to be linear (r2 > 0.999) for each of the analytes in the concentration range of 25-75 μg mL -1 . The results are presented in [Table 1]. Both precision and accuracy were determined with standard quality control samples prepared in triplicates at different concentration levels covering the linearity range.

The accuracy of the proposed method was assessed by recovery studies. All solutions were prepared and analyzed in triplicate. The above procedure is adopted for all the three drugs and a high recovery values obtained [Table 2] indicate that the proposed method is highly accurate. All the three drugs were suitably recovered from the tablet dosage forms by the proposed method and the results are shown in [Tables 2] and [Table 3]. The method specificity was assessed by studying the chromatograms obtained for a mixture of the drugs, the common excipients and the degradation products. As none of the excipients or the degradation products interfered with the analytes of interest, the method was found to be suitable for analyzing the commercial formulation of these ARV drugs. The representative stress chromatograms were shown in [Figure 6] (a) and (b).

  Conclusion Top

A sensitive, specific and validated Isocratic HPLC assay method was developed for the simultaneous analysis of ZID, LAM and NEV in tablet dosage form using simple and economic chromatographic conditions, achieving very high resolution. This method can be easily applied for the routine quality control analysis of tablets containing ZID, LAM and NEV.


The authors are thankful to M/s Cipla Ltd., Goa, India for providing gift samples of lamivudine, zidovudine and nevirapine.

  References Top

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

  [Table 1], [Table 2], [Table 3]

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Analytical Chemistry Letters. 2018; 8(1): 131
[Pubmed] | [DOI]


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