|Year : 2018 | Volume
| Issue : 1 | Page : 92-97
Analytical Method Development and Validation for the Simultaneous Estimation of Abacavir and Lamivudine by Reversed-phase High-performance Liquid Chromatography in Bulk and Tablet Dosage Forms
Sufiyan Ahmad Raees Ahmad1, Lalit Patil1, Mohammed Rageeb Mohammed Usman2, Mohammad Imran3, Rashid Akhtar4
1 Department of Quality Assurance, Gangamai College of Pharmacy, Dhule, India
2 Department of Pharmacognosy, Smt. S. S. Patil College of Pharmacy, Chopda, India
3 Department of Pharmaceutical Chemistry, KBHSS Trusts Institute of Pharmacy, Nasik, Maharashtra, India
4 Department of Pharmacognosy, Royal College of Pharmaceutical Education and Research, Nasik, Maharashtra, India
|Date of Web Publication||19-Feb-2018|
Dr. Sufiyan Ahmad Raees Ahmad
781/1, Islampura, Malegaon, District Nasik, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: A simple rapid, accurate, precise, and reproducible validated reverse phase high performance liquid chromatography (HPLC) method was developed for the determination of Abacavir (ABAC) and Lamivudine (LAMI) in bulk and tablet dosage forms. Methods: The quantification was carried out using Symmetry Premsil C18 (250 mm × 4.6 mm, 5 μm) column run in isocratic way using mobile phase comprising methanol: water (0.05% orthophosphoric acid with pH 3) 83:17 v/v and a detection wavelength of 245 nm and injection volume of 20 μl, with a flow rate of 1 ml/min. Results: In the developed method, the retention times of ABAC and LAMI were found to be 3.5 min and 7.4 min, respectively. The method was validated in terms of linearity, precision, accuracy, limits of detection, limits of quantitation, and robustness in accordance with the International Conference on Harmonization guidelines. Conclusion: The assay of the proposed method was found to be 99% – 101%. The recovery studies were also carried out and mean % recovery was found to be 99% – 101%. The % relative standard deviation from reproducibility was found to be <2%. The proposed method was statistically evaluated and can be applied for routine quality control analysis of ABAC and LAMI in bulk and in tablet dosage form.
Keywords: Abacavir, dosage forms, lamivudine, method development, reverse phase high performance liquid chromatography, validation
|How to cite this article:|
Raees Ahmad SA, Patil L, Mohammed Usman MR, Imran M, Akhtar R. Analytical Method Development and Validation for the Simultaneous Estimation of Abacavir and Lamivudine by Reversed-phase High-performance Liquid Chromatography in Bulk and Tablet Dosage Forms. Phcog Res 2018;10:92-7
|How to cite this URL:|
Raees Ahmad SA, Patil L, Mohammed Usman MR, Imran M, Akhtar R. Analytical Method Development and Validation for the Simultaneous Estimation of Abacavir and Lamivudine by Reversed-phase High-performance Liquid Chromatography in Bulk and Tablet Dosage Forms. Phcog Res [serial online] 2018 [cited 2021 May 18];10:92-7. Available from: http://www.phcogres.com/text.asp?2018/10/1/92/225827
Abbreviations Used: HPLC: High-performance liquid chromatography, UV: Ultraviolet, ICH: International Conference on Harmonization, ABAC: Abacavir, LAMI: Lamivudine, HIV: Human immunodeficiency virus, AIDS: Acquired immunodeficiency syndrome, NRTI: Nucleoside reverse transcriptase inhibitors, ARV: Antiretroviral, RSD: Relative standard deviation, RT: Retention time, SD: Standard deviation.
- Attempts were made to develop RP-HPLC method for simultaneous estimation of Abacavir and Lamivudine for the RP-HPLC method. The developed method was validated according to the ICH guidelines. The linearity, precision, range, robustness were within the limits as specified by the ICH guidelines. Hence the method was found to be simple, accurate, precise, economic and reproducible. So the proposed methods can be used for the routine quality control analysis of Abacavir and Lamivudine in bulk drug as well as in formulations.
| Introduction|| |
Abacavir (ABAC) and lamivudine (LAMI) are synthetic nucleoside analogs that show a potent and synergistic effect on the inhibition of human immunodeficiency virus-1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). HIV encodes at least three enzymes: protease, reverse transcriptase, and endonuclease. ABAC and LAMI belong to the class of nucleoside reverse transcriptase inhibitors (NRTIs). New therapeutic strategy of AIDS treatment requires the combination of these antiretroviral (ARV) drugs. The introduction of highly effective combination regimens of ARV drugs has led to substantial improvements in morbidity and mortality. ABAC tablets in combination with other ARV agents in tablet form are indicated for the treatment of HIV-1 infection. ABAC should not be added as a single agent when ARV regimens are changed due to loss of virologic response. Intracellularly, ABAC is converted by cellular enzymes to the active metabolite, carbovir triphosphate, an analog of deoxyguanosine-5' triphosphate. Intracellularly, LAMI is phosphorylated to its active 5'-triphosphate metabolite, lamivudine triphosphate. Chemically, ABAC sulfate is (1S, cis)-4-[2-amino-6-(cyclopropyl amino)-9H-purin-9-yl]-2-cyclopentene-1-methanol sulfate, and LAMI is (2R, cis)-4-amino-1-(2-hydroxymethyl-1, 3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one. [Figure 1] and [Figure 2] show the structures of ABAC and LAMI, respectively. Numerous analytical methods have been employed for the quantitative determination of single- or multi-component NRTIs in pharmaceutical dosage forms. These methods include ultraviolet (UV)-visible spectrophotometric high-performance thin-layer chromatography and high-performance liquid chromatography (HPLC).,,,,,,,,,,,, HPLC was considered the best method of assay since this method is the most accurate of all chromatographic and other separation methods. The reported method differs with respect to extraction procedure, eluent used for reverse-phase HPLC (RP-HPLC), and UV detection wavelength. The development and validation of a simple, rapid, accurate, and precise method of assay for ABAC and LAMI in tablet formulations are now reported in this work using RP-HPLC with UV detection at 245 nm.
| Materials and Methods|| |
Materials and reagents
The analysis of the drug was carried out on Youngline (S.K.) Gradient System UV Detector. This study was equipped with reverse phase (Grace) C18 column (4.6 mm × 250 mm; 5 μm), a SP930D pump, a 20 μl injection loop, UV730D Absorbance detector, and running autochro-3000 software. ABAC and LAMI were procured from CIPLA. Orthophosphoric acid (OPA), methanol, acetonitrile (HPLC Grade Merck Specialties Pvt. Ltd. Shiv Sager Estate “A” Worli, Mumbai.), water, 0.45 μm filter (Millipore, Bangalore) were also used. A combination of ABAC (600 mg) and LAMI (300 mg) in tablet formulation was procured from local pharmacy (ABAMUNE-L, Cipla).
Column C18 (250 mm × 4.6 mm); particle size packing 5 μm; detection wavelength of 245 nm; flow rate 1.00 ml/min; temperature ambient; sample size 20 μl; mobile phase methanol: water (OPA 0.05%) (83:17); run time of 10 min.
Preparation of standard stock solution
20 mg of ABAC and 10 mg of LAMI were weighed accurately and transferred to a 10-ml volumetric flask dissolved in methanol and diluted to 10 ml with the mobile phase (methanol: water, 83:17 v/v) to give a stock solution of 2000 μg/ml ABAC and 1000 μg/ml LAMI [Table 1] shows the details of chromatogram of standard mixture ABAC and LAMI and [Figure 3] and [Figure 4] show the chromatogram of standard ABAC and LAMI. [Figure 5] shows the chromatogram of standard mixture of ABAC and LAMI.
|Table 1: Details of chromatogram of standard mixture abacavir and lamivudine|
Click here to view
|Figure 5: Representative chromatogram of abacavir and lamivudine using methanol + water (acetic acid 0.05% [orthophosphoric acid]) (83% +17%) v/vas mobile phase, showing retention time 3.5 min and 7.4 min|
Click here to view
Method development and validation
Serial dilutions were done to prepared various concentration stock working standard of various concentrations was prepared by taking aliquots of standard solution and diluted to get required concentration for calibration plot and which was injected.
Assay preparation for commercial formulation
For analysis of the tablet dosage form, 20 tablets were weighed individually and their average weight was determined. After that, they were crushed to fine powders and powder equivalent to 1 mg was taken and transferred to 10 ml volumetric flask and diluted with 10 ml methanol; from the above solution, 0.2 ml was taken and diluted to 10 ml methanol. The solutions were shaken vigorously for 10 min and filtered through 0.45 μg nylon membrane filters. Then, volume was made up to the mark with methanol: water (83:17); the amounts of ABAC and LAMI per tablet were calculated from the calibration curve. Analysis procedure was repeated five times with tablet formulation. Results are shown in [Table 2] and [Table 3] that show the analysis of marketed formulation and details of chromatogram of ABAC and LAMI in tablet formulation. [Figure 6] shows the chromatogram of ABAC and LAMI in tablet formulation.
|Table 3: Details of chromatogram of abacavir and lamivudine in tablet formulation|
Click here to view
| Results|| |
Linearity and range
From ABAC and LAMI standard stock solution, different working standard solutions (20–100 μg/ml) were prepared in the mobile phase. Likewise from ABAC and LAMI standard stock solution, different working standard solutions (10–50 μg/ml) were prepared in the mobile phase. 20 μl of sample solution was injected onto the column using fixed volume loop injector. Chromatograms were recorded. The area for each concentration was recorded in [Table 4], [Table 5], [Table 6] that show linearity study. [Figure 7] and [Figure 8] show the calibration curve of ABAC and LAMI, respectively.
Recovery studies were performed to validate the accuracy of developed method. To a preanalyzed tablet solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analyzed [Table 7]. Statistical validation of recovery studies is shown in [Table 8] and [Figure 9], [Figure 10], [Figure 11].
System suitability parameters
To ascertain the resolution and reproducibility of the proposed chromatographic system for estimation of ABAC (600 mg) and LAMI (300 mg), system suitability parameters were studied. The results are shown in [Figure 12] and [Table 9].
The method was established by analyzing various standards of ABAC and LAMI. All the solutions were analyzed thrice to record any intraday and interday variation in the result. The results obtained for interday and intraday variation are shown in [Table 10] and [Figure 13].
|Table 10: Intra- and inter-day precision studies on high-performance liquid chromatography method for abacavir and lamivudine|
Click here to view
The robustness is a measure of its capacity to remain unaffected by small and deliberate variations in method parameters and provides an indication of its reliability during normal usage; hence, the following are performed by slight variations in parameters. The assay content of the sample was measured by change in the flow rate of 0.90–1.10 ml/min. The results indicate that less variability in retention time and tailing factor were observed [Table 11] and [Table 12].
| Discussion|| |
The proposed methods for simultaneous estimation of ABAC and LAMI in tablet dosage forms were found to be simple, accurate, economical, and rapid. The method was validated as per the International Conference on Harmonization Q2 (R1) guidelines. Standard calibration yielded correlation coefficient (r2) 0.999 for both ABAC and LAMI at all the selected wavelengths. The values of % relative standard deviation are within the prescribed limit of 2%, showing high precision of methods, and recovery was close to 100% for both drugs. Results of the analysis of pharmaceutical formulations reveal that the proposed method is suitable for their simultaneous determination, with virtually no interference of any additive present in pharmaceutical formulations. Hence, the above methods can be applied successfully for simultaneous estimation of ABAC and LAMI in formulations.
| Conclusion|| |
The developed HPLC methods in that linearity, precision, range, and robustness were found to be more accurate, precise, and reproducible. The methods were found to be simple and time saving. All proposed methods could be applied for routine analysis in quality control laboratories.
We are thankful to the Principal, Gangamai College of Pharmacy, Nagaon, Dist. Dhule, for providing necessary facilities for research work. They are also grateful to Cipla Ltd., Goa, for giving gift samples of pure drugs.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
WHO Public Assessment Report. Abacavir Sulfate, Lamivudine and Zidovudine Tablets 300mg/150mg/300mg. Part 7; 2009. p. 1-2.
Intracellularly, abacavir is converted by cellular enzymes to the active metabolite, carbovir triphosphate. Indian Pharmacopoeia 1996;1:A-68-70.
Srinivas Rao M, Ravi Kumar D, Vardhan SV, Ramachandran D. Development of validated spectrophotometric method for the assay of Abacavir sulfate in bulk and pharmaceutical dosage formulations. Int J Chemtech Res 2011;3:1200-2.
Venkatamahesh R, Dhachinamoorthi D. Visible spectrophotometric determination of abacavir sulphate in bulk drug and tablet dosage form. Int J Pharm Tech Res 2011;3:356-9.
Srihari G, Rami Reddy N, Chakravarthi IE. Validate spectrophotometric method for the determination of Abacavir sulfate in pharmaceutical preparations. Global J C 2011;5:172-5.
Sudha T, Ravikumar VR, Hemalatha PV. Validated HPTLC method for simultaneous determination of lamivudine and Abacavir sulphate in tablets dosage form. Int J Pharm Sci Res 2010;1:101-11.
Sparidans RW, Hoetelmans RM, Beijnen JH. Liquid chromatographic assay for simultaneous determination of abacavir and mycophenolic acid in human plasma using dual spectrophotometric detection. J Chromatogr B Biomed Sci Appl 2001;750:155-61.
Ravitch JR, Moseley GC. High-performance liquid chromatographic assay for Acavir and its two major metabolites in human urine and cerebrospinal fluid. J Chromatogr 2001;762:165-73.
Aymard G, Legrand M, Trichereau N, Diquet B. Determination of twelve antiretroviral agents in human plasma sample using reversed-phase high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 2000;744:227-40.
Veldkamp IA, Sparidans WR, Hoetelmans WM, Beijnen HJ. Quantitative determination of Abacavir, a novel nucleoside reverse transcriptase inhibitor, in human plasma using isocratic RP-HPLC with ultraviolet detection. J Chromatogr 1999;734:123-8.
Anantha Kumar D, Srinivasa Rao G, Seshagiri Rao JV. Simultaneous determination of Lamivudine, Zidovudine and Abacavir in tablet dosage form by RP-HPLC method. E J Chem 2010;7:180-4.
Lewis SR, White CA, Bartlett MG. Simultaneous Determination of Abacavir and Zidovudine from rat tissue using HPLC with UV detection. J Chromatogr 2007;85:45-52.
Ozkan Y, Savaser A. Simple and reliable HPLC method of abacavir determination in pharmaceutical, human serum and drug dissolution studies from tablets. J Liquid Chrom Relat Technol 2005;28:23-37.
Raja T, Lakshmana Rao A. Development and validation of RP-HPLC method for the estimation of abacavir, lamivudine and zidovidine in pharmaceutical disage form. Int J Pharm Tech Res 2011;3:852-7.
Sudha T, Ravi Kumar VR, Hemalatha PV. RP-HPLC method for simultaneous estimation of lamivudine and abacavir sulfate in tablet form. Int J Pharm Biomed Res 2008;1:108-13.
ICH – Guidelines Q2A, Validation of Analytical Procedures: Definition and terminology (CPMP III/5626/94). Geneva, Switzerland; March, 1995. ICH –Guidelines Q2B, Validation of Analytical Procedures: Methodology, (CPMP/ICH/281/95). Geneva, Switzerland; November, 1996.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12]