24.P.Nagaraju et al.pdf

IAJPS 2017, 4 (02), 359-367 P.Nagaraju et al CODEN (USA): IAJPBB ISSN 2349-7750 ISSN: 2349-7750 I NDO A MER I CAN J OUR NA L OF P HA R MA CEUT I CA L SCI ENCES http://doi.org/10.5281/zenodo.375937 Available online at: http://www.iajps.com Research Ar ticl e STABILITY-INDICATING METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF LAMIVUDINE, ABACAVIR, DOLUTEGRAVIR IN PHARMACEUTICAL DOSAGE FORMS SK. Shaista Fatima, P. Nagaraju*, V. Mounika, G. Indira Priyadarshini and V. Vasu Naik Professor, Department of Pharmaceutical Analysis, Hindu College of Pharmacy, Amaravathi Road, Guntur – 522002, Andhra Pradesh, India. Received: 20 February 2016 Accepted: 26 February 2017 Published: 28 February 2017 Abstract: A simultaneous stability-indicating reversed-phase high performance liquid chromatography (HPLC) method for analysis of lamivudine (LAMI), abacavir (ABA), dolutegravir (DOLU) in the bulk drug and in the formulation was developed. Compounds were separated on Inersil ODS (4.6 × 250 mm x 5 mm). A gradient program of mobile phase at different proportions of Phosphate Buffer pH-7 & Acetonitrile was used. The retention times of LAMI, ABA & DOLU were 2.555, 4.282, 7.101 minutes (mins.) respectively. The drugs were subjected to the stress conditions of acid, base, oxidative & thermal degradation. The degradation products were well resolved from main peak and its impurities, proving the stability-indicating ability of the method. The method was linear in the concentration range of 0– 150μg/mL, 0-300 μg/mL and 0-25 μg/mL for LAMI, ABA & DOLU respectively. The method was accurate and precise with a limit of detection and limit of quantitation of 0.31 and 0.94 µg/ mL, 0.21 and 0.65 µg/ mL and 0.48 and 1.46 µg/ mL for LAMI, ABA & DOLU respectively. The method was applied for the analysis of LAMI, ABA & DOLU in the presence of its degradation products and commonly used excipients and was found to be specific. The developed method is stability indicating, precise and specific which can be applied for the routine analysis. Key words: Lamivudine, Abacavir, Dolutegravi and stability-indicating Corresponding author: P. Nagaraju, Professor, Department of Pharmaceutical Analysis, Hindu College of Pharmacy, Amaravathi Road, Guntur – 522002, Andhra Pradesh, India. Contact: 09985304304 E-mail: pappulanagaraju@gmail.com QR cod e Please cite this article in press as -.Nagaraju et al, Stability-Indicating Method Development and Validation of RP-HPLC Method for Simultaneous Estimation of Lamivudine, Abacavir, Dolutegravir in Pharmaceutical Dosage Forms, Indo Am. J. P. Sci, 2017; 4(02). www.ia jps .com Page 359 IAJPS 2017, 4 (02), 359-367 P.Nagaraju et al ISSN 2349-7750 INTRODUCTION [1-10]: Lamivudine (LAMI) which is chemically 4-amino1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5yl]-1,2-dihydropyrimidin-2-one is an analogue of cytidine. It can inhibit both types (1 and 2) of HIV reverse transcriptase and also the reverse transcriptase of hepatitis B virus. It is phosphorylated to active metabolites that compete for incorporation into viral DNA. They inhibit the HIV reverse transcriptase enzyme competitively and act as a chain terminator of DNA synthesis. The lack of a 3'-OH group in the incorporated nucleoside analogue prevents the formation of the 5' to 3' phosphodiester linkage essential for DNA chain elongation, and therefore, the viral DNA growth is terminated. combination [11-34]. Hence LC method that was developed in the present work is advantageous because it enables stability indicating, accurate, specific and reproducible analysis of LAMI, ABA & DOLU Fig 1a: Lamivudine Abacavir, (ABA) which is chemically [(1S,4R)-4[2-amino-6-(cyclopropylamino)-9H-pu rin-9yl]cyclopent-2-en-1-yl]methanol, is a carbocyclic synthetic nucleoside analogue. Intracellularly, it is converted by cellular enzymes to the active metabolite carbovir triphosphate. Carbovir triphosphate is an analogue of deoxyguanosine-5`triphosphate (dGTP). Carbovir triphosphate inhibits the activity of HIV-1 reverse transcriptase (RT) both by competing with the natural substrate d GTP and by its incorporation into viral DNA Standard stock solution: Accurately Weighed and transferred 30 mg of Lamivudine, 60 mg of Abacavir & 5 mg of Dolutegravir working Standards into individual 10 ml clean dry volumetric flask, diluent was added and sonicated for 10 minutes and make up to the final volume with diluent. From the above stock solution, 1 ml, 1ml, 1ml was pipetted out in to a 10 ml volumetric flask and then make up to the final volume with diluent. From above solution, 3ml, 3ml, 3ml was pipetted out into a 10 mL volumetric flask and then make up the final volume with diluent and thus we have (90 μg/ml Lamivudine, 180 μg/ml Abacavir & 15 μg/ml dolutegravir). Fig 1b: Abacavir Sample stock solution: 20 tablets were weighed and calculate the average weight of each tablet then the weight equivalent to 1 tablet was transferred into a three different 10 mL volumetric flask, 3/4th volume of diluent added and sonicated for 30 min, further the volume made up with diluent. From the above stock solution, 0.1 ml, 0.1ml, 0.1ml was pipetted out in to a 10 ml volumetric flask and then make up to the final volume with diluent. From above stock solution, 3ml, 3ml, 3ml was pipetted out into a 10 mL volumetric flask and then make up the final volume with diluent Dolutegravi r(DOLA) which is chemically ((4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2Hpyrido [1',2':4,5] pyrazino[2,1-b] [1,3]oxazine-9carboxamide, inhibits HIV integrase by binding to the integrase active site and blocking the strand transfer step of retroviral DNA integration which is essential for the HIV replication cycle. There are several reported spectrophotometric and chromatographic methods in the literature for analysis of LAMI, ABA & DOLU individually but there is no reported method for the simultaneous stability indicating assay of the drugs in www.ia jps .com MATERIALS AND METHODS: Instrumentation and Reagents: Liquid chromatography was performed with a Waters 2695 series High performance liquid chromatography instrument equipped with Auto Sampler and DAD or UV detector. Empower 2 software was applied for data collection and processing. The separation was achieved on a Inertsil ODS (4.6 x 250mm, 5mm) analytical column. LAMI, ABA & DOLU was obtained as a gift sample from Hetero Labs, Hyderabad, India. Preperation of solutions: Preparation of diluents: A mixture of Potassium Dihydrogen Orthophosphate of PH-7and Acetonitrile was prepared and used as mobile phase.(Gradient Mode) Optimized Chromatographic Conditions: Compounds were separated on a Inertsil ODS (4.6 x 250mm, 5mm) column with gradient program of Phosphate Buffer pH-7: Acetonitrile [Table 1] as mobile phase at a flow rate of 1.2 mL/min. Chromatography was performed at room temperature and the detection was carried out at 254 nm. Page 360 IAJPS 2017, 4 (02), 359-367 P.Nagaraju et al ISSN 2349-7750 Table 1: Column with gradient program of Phosphate Buffer pH-7: Acetonitrile TIME 0 5.0 8.5 FLOW ml/min 1.2 1.2 1.2 A(BUFFER pH-7.0) 75 30 75 Forced Degradation Studies: Intentional degradation (n = 3) was attempted by using, heat, acid, base, and oxidizing agent. For acid degradation, 3 mL of working standard solution was refluxed with 2N hydrochloric acid (HCl) at 60°C for 1hour and then neutralized by adjusting pH to 7.0 with 0.1N sodium hydroxide (NaOH). For alkali degradation, 3 mL of working standard solution was refluxed with 0.1N NaOH at 60°C for 1hour and then neutralized by adjusting pH to 7.0 with 0.1N HCl. For oxidative degradation, 3 mL of working standard solution was refluxed with 3%w/v hydrogen peroxide (H2O2) by heating on water bath at 60°C for 1hour. For thermal degradation, 2mL of working standard solution was exposed to temperatures at 110°C for 24 hours. All these solutions except for were prepared in amber volumetric flasks. After completion of the degradation treatments the samples were cooled to room temperature, diluted with the diluent, and injected for chromatographic analysis. Method Validation: The method was validated in accordance with recognized guidelines. System suitability: The suitability of the system was studied by the values obtained for Theoretical plate, Resolution and tailing factor of the chromatogram of standard drugs and presented in the Table 3. The selectivity of the method was revealed by the repeated injection of mobile phase and no interference was found. Linearity: Six solutions containing LAMI, ABA & DOLU were prepared in diluents. Peak area and concentration data were treated by least squares linear regression analysis (n = 3). Precision Method precision was evaluated by injecting sample preparation at test concentration level of LAMI, ABA & DOLU for 6 times (n=3) on different HPLC system. The system precision of test method was performed by injecting three portions from a standard solution on to the analytical column and the peak area data obtained then %RSD was calculated. www.ia jps .com B(ACETONITRILE) 25 70 25 Accuracy: Accuracy of the method confirmed by studying recovery at 3 different concentrations 50%, 100%, and 150% of these expected, in accordance with ICH guidelines, by replicate analysis (n=3). Standard drug solution was added to a pre analyzed sample solution and percentage drug content was measured. LOD and LOQ: LOD and LOQ were determined as the amounts for which the signal-to-noise ratios were 3:1 and 10:1, respectively. Robustness: Robustness of method was carried out with variation in pH, mobile phase composition and detection wavelength (± 2 nm). RESULTS AND DISCUSSION: The retention times of LAMI, ABA & DOLU under the chromatographic conditions described above were 2.555, 4.282, 7.101 mins respectively [Fig. 2.a]. Assay calculations are given in Table no. 2. System suitability data is given in table no. 3 where it is evaluated by theoretical plates and tailing factor. The peaks of the degradation products were well resolved from that of LAMI, ABA & DOLU [Fig. 2c–f]. There was no interference from the excipients commonly present in the formulation and from the mobile phase. It may therefore be inferred that no degradation of LAMI, ABA & DOLU in the pharmaceutical formulation was detected by using this method. In validation of the assay, blank, yielded clean chromatograms [Fig.2.b]; with no interference from the excipients and mobile phase; this is indicative of the specificity of the method. The LOD and LOQ was 0.31, 0.94 and 0.21, 0.65 µg/ mL and 0.48, 1.46 µg/ mL, for LAMI, ABA & DOLU respectively. A plot of drug peak area against concentration [Fig. 3a 3b 3c] of LAMI, ABA & DOLU was linear over the concentration range 0– 150µg/mL, 0 -300µg/ mL and 0- 25µg/ mL respectively. The regression equation was calculated by the least-square method for LAMI, Y = 22044X - 46592; correlation coefficient 0.999, for ABA, Y = 15639X+50671; correlation coefficient 0.999 and for DOLU Y = 32920X+50138; correlation coefficient 0.999. The method was found to be precise as the RSD <2 [Table 4]. Page 361 IAJPS 2017, 4 (02), 359-367 P.Nagaraju et al The recovery studies were carried out by comparing method of three individual standards with each of three samples with same procedure from the formulation and injecting. The percentage recovery and percentage relative standard deviation of the percentage recovery were calculated and presented in Table 5. From the data obtained, added recoveries of standard drugs were found to be accurate. This quantitative recovery of the drugs indicates that there was no interference from excipients present in the formulation and the ISSN 2349-7750 method is accurate whose results are shown in Table 5. LAMI, ABA & DOLU were found to be stable in the mobile phase for a period of 24hours, because no peaks corresponding to degradation products were observed and there was no significant change in the peak area of the drug (RSD <1%). The deliberate changes in the method have not much affected the peak tailing, theoretical plates and the percent assay. This indicates that the present method is robust (Table 6). Results of Degradation Studies are given in Table 7. Chromatograms obtained from drugs and its degradation products: Fig. 2a: Chromatogram of Standard Fig. 2:b Chromatogram of Blank Fig. 2c: Acid Degradation www.ia jps .com Page 362 IAJPS 2017, 4 (02), 359-367 P.Nagaraju et al ISSN 2349-7750 Fig. 2d: Alkali Degradation Fig. 2e: Thermal Degradation Fig.2f: Peroxide Degradation Fig.3a: Calibration Plot of Lami www.ia jps .com Page 363 IAJPS 2017, 4 (02), 359-367 P.Nagaraju et al ISSN 2349-7750 Fig.3b: Calibration Plot of Aba Fig.3c: Calibration Plot of Dolu Table 2: Assay Result of LAMI, ABA & DOLU S.No. Drug Lamivudine 1. Abacavir 2. Dolutegravir 3. * Mean of Three Determinations Label Claim Amount Found* %RSD 300 mg 600 mg 50 mg 299.65mg 591.84mg 49.1mg 99.88 98.64 98.0 Table 3: System Suitability Parameters Tailing factor No. of theoretical plates Retention time (min) Resolution Lamivudine 1.48 2638 2.555 -- Abacavir 1.31 8195 4.282 8.90 Dolutegravir 1.21 22798 7.101 14.98 Acceptance Criteria NMT 2.0 NLT 2000 NLT 2.0 Table 4: Precision Data of the Proposed Method Injection 1. 2. 3. 4. 5. Mean S.D %RSD Lamivudine Method Precision 1923988 1893524 1899965 1930246 1889827 1914466 2368.562 1.23 www.ia jps .com System Precision 1880157 1839839 1876570 1870271 1837622 1860891.8 2055.254 1.10 Abacavir Method Precision 2964962 2999786 2927107 2971382 2908039 2960767 3640.389 1.22 System Precision 2930353 2847284 2901585 2885735 2922475 2987486.4 3309.09827 1.14 Dolutegravir Method Precision 584343 582205 575084 584822 572613 580850.3 5617.704 0.96 System Precision 572535 559339 570178 569599 579735 570277.2 7325.668174 1.28 Page 364 IAJPS 2017, 4 (02), 359-367 P.Nagaraju et al ISSN 2349-7750 Table 5: Accuracy Data (Triplicate Values at 50, 100 and 150 Percent Levels) of LAMI, ABA & DOLU % Recovery of Lamivudine Recovery Level (%) Fixed concentration (µg/ml) 90 90 90 50 100 150 Spiked concentration (µg/ml) 60 90 120 Recovered concentration (µg/ml) 59.88 89.90 119.70 % Recovery 99.8 99.9 99.7 % Recovery of Abacavir Recovery Level (%) 50 100 150 Fixed concentration (µg/ml) 180 180 180 Spiked concentration (µg/ml) 150 180 210 Recovered concentration(µ g/ml) 149.8 178.9 209.03 Spiked concentration (µg/ml) 7.5 15 22.5 Recovered concentration (µg/ml) 7.51 15.23 22.48 % Recovery 99.6 99.3 99.5 % Recovery of Dolutegravir Recovery Level (%) 50 100 150 Fixed concentration (µg/ml) 15 15 15 % Recovery 100.1 101.5 99.9 Table 6: Robustness Data of LAMI, ABA & DOLU S.no Robustness Condition 1. Flow rate 1.0 ml/min 2. 3. 4. Flow rate 1.4 ml/min Wavelength 252nm Wavelength 256nm Stress condition Peak Area of Lamivudine 2136130 1784775 2766836 1855712 2791292 2001906 3111378 Table 7: Forced Degradation Data Lamivudine % Assay % Degradation Control Acid 100 88.81 Alkaline Peroxide Thermal 87.62 97.33 93.74 of Peak Area of Dolutegravir 692435 568517 526489 636050 Abacavir Dolutegravir 11.19 % Assay 100 86.84 13.16 %Assa y 100 90.43 12.38 2.67 6.26 85.69 92.99 93.74 14.31 7.01 6.36 92.51 88.52 98.27 CONCLUSION: This RP-HPLC method for assay of lamivudine abacavir and dolutegravir is precise, specific, rapid, and stability-indicating. The method may be used to assess the stability of lamivudine abacavir and dolutegravir as the bulk drug and in its pharmaceutical formulation. Chromatographic analysis time of less than 20 min was advantageous for use of the method in routine analysis. 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