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B. Raj Kumar et al. / JGTPS/ 5(3)-(2014) 1827- 1832
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A NEW VALIDATED STABILITY-INDICATING RP-HPLC METHOD FOR THE
DETERMINATION OF TELBIVUDINE
B. Raj Kumar1*
Dr. K. V. Subrahmanyam2
Telbivudine 5'–triphosphate inhibits HBV DNA polymerase (reverse transcriptase) by competing with the natural substrate, thymidine 5'–triphosphate. This leads to the chain termination of DNA synthesis, thereby inhibiting viral replication. Department of Pharmaceutical Incorporation of telbivudine 5'–triphosphate into viral DNA also causes DNA chain Analysis, Dr. K.V.Subba Reddy termination, resulting in inhibition of HBV replication. Telbivudine inhibits anti Institute of Pharmacy, compliment or second-strand DNA. A novel, stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for the quantitative determination of Telbivudine in active pharmaceutical ingredients and in its 2Principal & Professor Pharmaceutical dosage form by using develosil C18, 5µm, 150 x 4.6 mm i.d. column with Department of Pharmaceutical a mobile phase containing a mixture of acetonitrile: phosphate buffer (pH 3.0) (40:60v/v) and conditions optimized were flow rate (1.0 ml/minute), wavelength (273 nm), Run time Analysis, Samskruthi College of was 10 min and a peak eluted at 3.52 min and column oven temperature was maintained Pharmacy, Ghatkeskar, Hyderabad ambient. Calibration curve was plotted with a range from 0-40µg/ml. Stress degradation conditions were established for Telbivudine by subjecting it to acid, base, oxidation and thermal stress. The stress samples were assayed against a qualified reference standard and the mass balance was close to 99.35%.The developed RP-HPLC method was validated according to the current International Conference on Harmonization (ICH) guidelines for specificity, LOD, LOQ, linearity, accuracy, precision, intermediate precision and robustness. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of in bulk drug and in its pharmaceutical dosage form.
Keywords: Tebivudine, RP-HPLC, ODS, ICH, LOD, LOQ
(hydroxymethyl) oxolan-2-yl]-5-methyl-1, 2, 3, 4- Telbivudine (Sebivo) is a synthetic thymidine nucleoside analog with specific activity against the Molecular formula: C10H14N2O5
hepatitis B virus. Telbivudine is orally administered, with Molecular weight: 242.2286
good tolerance, lack of toxicity and no dose-limiting side Category: .Antiviral drug
Solubility: Sparingly soluble in water (>20 mg/ml)
DMSO 48 mg/ml (198 mM);
PKa: 9.96
Mechanism of Action:
Telbivudine 5'–triphosphate inhibits HBV DNA
polymerase (reverse transcriptase) by competing with the
natural substrate, thymidine 5'–triphosphate. This leads to
the chain termination of DNA synthesis, thereby
inhibiting viral replication. Incorporation of telbivudine
5'–triphosphate into viral DNA also causes DNA chain
termination, resulting in inhibition of HBV replication.
Telbivudine inhibits anticompliment or second-strand
DNA.
Address for correspondence
EXPERIMENTAL METHODS 7-19
B. Raj Kumar*
CHARACTERIZATION OF TELBIVUDINE
Dr. K.V.Subba Reddy Institute of Pharmacy, Physiochemical characteristics
Description of Telbivudine: slightly yellowish
B. Raj Kumar et al, JGTPS, 2014, Vol. 5(3): 1827 - 1832
Solubility of Telbivudine: The solubility of drug sample
was determined according to I.P. 1996.
Fig 1: Showing the UV spectrum of Telbivudine
Two 10 ml and one 250 ml volumetric flasks were taken.
Preparation of standard solution of Telbivudine
Flask 1: 10 mg of Telbivudine was accurately weighed
25 mg of Telbivudine was weighed accurately and transferred to 10 ml volumetric flask. 0.1 ml of water and transferred into 25 ml volumetric flask. About 10 ml was added into it. The contents were mixed for one of mobile phase was added and sonicated to dissolve. The minute. The drug was slightly dissolved. Again 0.1 ml of volume was made up to the mark with same solvent. The water was added into the volumetric flask. The contents final solution contained about 1000 μg/ml of Telbivudine. were mixed for one minute. The solubility state was From the stock solution again 0.2 ml was taken in a 10 ml volumetric flask & volume was make up to the mark by Flask 2: 10mg of Telbivudine was accurately weighed
mobile phase. This solution contains 20 μg/ml of and transferred to 10 ml volumetric flask. 0.1 ml of Telbivudine which has been injected to HPLC.
methanol was added into it. The contents were mixed for one minute. The drug was slightly dissolved. Again 0.1 Initialization of the instrument
ml of methanol was added into the volumetric flask. The The HPLC instrument was switched on. The contents were mixed for one minute. The solubility state column was washed with HPLC water for 45 minutes. The column was then saturated with mobile phase for 45 Flask 3: Accurately weighed Telbivudine (10 mg) was
minute. The mobile phase was run to find the peaks. After transferred to 250 ml volumetric flask. Added 10 ml of 20 minutes the standard drug solution was injected in acetonitrile to it. Mixed the solution for one minute. The drug could not dissolve. Added more 90 ml of acetonitrile to the volumetric flask. Mixed the solution for two MOBILE PHASE PREPARATION
minutes. The drug could not dissolve. Added more 100 The mobile phase used in this analysis consists ml of water to the volumetric flask. Mixed the solution of a mixture of Buffer (0.05 M potassium dihydrogen for two minutes. The solubility state was noted.
phosphate & pH adjusted to 3.4 with orthophosphoric acid) and Acetronitrile in a ratio of 60:40. To the 600 ml DEVELOPMENT
of this buffer solution was added and properly mixed with VALIDATION FOR TELBIVUDINE BY RP-HPLC-
400 ml of acetronitrile and a homogenous solution is Selection of wavelength
achieved. This mobile phase was filled and sonicated for HPLC Instrumentation & Conditions:
15 minutes before using in the experiment The HPLC system employed was HITACHI L2130 with
D Elite 2000 Software with Isocratic with UV-Visible
Sample & Standard Preparation for the Analysis
Detector (L-2400).
10 mg of Tebivudine standard was transferred The standard & sample stock solutions were prepared into 10 ml volumetric flask, dissolved & make up to separately by dissolving standard & sample in a solvent volume with mobile phase. Further dilution was done by in mobile phase diluting with the same solvent.(After transferring 0.5 ml of the above solution into a 10ml optimization of all conditions) for UV analysis. It volumetric flask and make up to volume with mobile scanned in the UV spectrum in the range of 200 to phase. The sample was analysed by HPLC by using the 400nm. This has been performed to know the maxima of above method and a very nicely resolved peak has been Telbivudine, so that the same wave number can be obtained at a Retention Time of about 3.52 min. The utilized in HPLC UV detector for estimating the respective chromatogram is attached in the following Telbivudine. While scanning the Telbivudine solution we observed the absorption maxima was 273 nm. The UV spectrum has been recorded on ELICO, corp. make UV – Optimization of Chromatographic Conditions:
VIS spectrophotometer model UV-2450. The scanned The chromatographic conditions were optimized UV spectrum is attached in by different trails. (Using different column, different mobile phase, different flow rate, different detection wavelength & different diluents for sample preparation etc. The Optimum conditions obtained from experiments can be summarized as Develosil ODS HG-5 RP C18, 5m, 15cmx4.6mm I.D was used for analysis at column temperature 45°C. The mobile phase was pumped through the column at a flow rate of 1.0 mL/ min. The sample injection volume was 20 μL and the sample temperature was maintained at Ambient. The wavelength of UV-273 nm was set for Tebivudine and Chromatographic Gradient programme runtime was 10 minutes.
B. Raj Kumar et al, JGTPS, 2014, Vol. 5(3): 1827 - 1832
In nine different 10 ml volumetric flasks, 1 ml of the pre-analyzed capsule solution (100 g/ml) was taken and added 1, 2, 3 ml of standard solution of bulk (API) mixture (100g/ml) and the volume was made up to 10 ml with mobile phase. The solutions were then injected into the HPLC system and the peak areas were recorded. The data are shown in Table 2: Data of recovery studies
%Recovery
Statistical
Retention Time (min)
Pure drug
Fig 2: The chromatogram obtained after condition 7th
trail, Typical chromatogram of Telbivudine (Rt 3.52) Here the peaks were separated and shown better resolution, theoretical plate count and symmetry. The proposed chromatographic conditions were found appropriate for the quantitative determination of the Linearity and Range
Linearity range was found to be 0-40 µg/ml for Telbivudine. The correlation coefficient was found to be 0.995, the slope was found to be 14357 and intercept was found to be 24166 for Telbivudine.
The mean recovery was found to be 99.882% for Telbivudine. The limit for mean % recovery is 98-102% and as both the values are within the limit, hence it can be said that the proposed method was accurate.
The precision of each method was ascertained separately from the peak areas obtained by actual determination of six replicates of a fixed amount of drug Telbivudine. The percent relative standard deviations were calculated for Telbivudine are presented in the table-3.
Fig 3: Standard curve for Telbivudine
MEAN AUC (n=6)
Replicates of Telbivudine
Standard Deviation Table 3: Data showing repeatability analysis
Table 1: Standard curve for Telbivudine
B. Raj Kumar et al, JGTPS, 2014, Vol. 5(3): 1827 - 1832
INTERMEDIATE PRECISION
Ingredient (API). The API (Telbivudine) was subjected to For intra-day studies the drug having stress conditions in various ways to observe the rate and concentration value 80%, 100 % & 120% of the target extent of degradation that is likely to occur in the course concentration (n  3), were injected in triplicate into the of storage and/or after administration to body. This is one HPLC system and for inter-day studies the drug at above type of accelerated stability studies that helps us three concentrations were injected in triplicate into the determining the fate of the drug that is likely to happen HPLC system for three days. Data were subjected to after a long time storage, within a very short time as statistical treatment for the calculation of SD and RSD. compare to the real time or long term stability testing.
The data are shown in Table 4
The various degradation pathways studied are acid hydrolysis, basic hydrolysis, thermal degradation and Observed Conc. Of Telbivudine
oxidative degradation (µg/ml) by the proposed method
An accurately weighed 25 mg. of pure drug was transferred to a clean & dry 25 ml volumetric flask. To which 0.1 N Hydrochloric acid was added & make up to the mark & kept for 24 hrs. from that 0.2 ml was taken in to a 10 ml volumetric flask & make up to the mark with mobile phase, then injected into the HPLC system against Table 4: Data for Telbivudine analysis
a blank of HCL (after all optimized conditions) Intraday and interday studies show that the mean RSD (%) was found to be within acceptance limit (≤2%), so it was concluded that there was no significant difference for the assay, which was tested within day and between days. Hence, method at selected wavelength wasfound to be precise.
LIMIT OF DETECTION AND LIMIT OF
The detection limit (LOD) and quantitation limit (LOQ) may be expressed as: L.O.D. = 3.3(SD/S). L.O.Q. = 10(SD/S) Where, SD = Standard deviation of the response S = Slope of the calibration curveThe LOD was found to be 0.452 g/ml and LOQ was found to be 1.356 g/ml for Telbivudine which represents Retention Time (min)
that sensitivity of the method is high.
Fig 4: Chromatogram showing degradation for
SYSTEM SUITABILITY PARAMETER
Telbivudine in 0.1 N HCL System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical Peak results
operations and samples to be analyzed constitute an integral system that can be evaluated as such. Following system suitability test parameters were established. The data are shown in Table 5.
An accurately weighed 10 mg. of pure drug was transferred to a clean & dry 10 ml volumetric flask. To which 0.1 N Sodium hydroxide was added & make up to Theoretical plate N  2000 Telbivudine=6246 the mark & kept for 24 hrs. from that 0.2 ml was taken in to a 10 ml volumetric flask & make up to the mark with Table 5: Data of System Suitability Parameter
mobile phase, then injected into the HPLC system against a blank of . NaOH (after all optimized conditions) FORCED DEGRADATION STUDIES:
Following protocol was strictly adhered to for forced degradation of Telbivudine Active Pharmaceutical B. Raj Kumar et al, JGTPS, 2014, Vol. 5(3): 1827 - 1832
Peak results
Telbivudine 3.49 3129815 Oxidation with (3%) H2O2:
Accurately weighed 1 mg. of pure drug was taken in a clean & dry 100 ml. volumetric flask. 30 ml. of 3% H2O2 and a little methanol was added to it to make it soluble & then kept as such in dark for 24 hours. Final volume was made up to 100 ml. using water to prepare 100 ppm solution. The above sample was injected into the HPLC Retention Time (min)
Fig 5: Chromatogram showing degradation related
impurity in 0.1 N NaOH Peak results
1 Telbivudine 3.49 2917874 Retention Time (min)
Fig 7: Chromatogram showing oxidative degradation.
An accurately weighed 1 mg. of pure drug was transferred to a clean & dry 100 ml volumetric flask, Peak results
make up to the mark with mobile phase & was maintained at 50 0C for 24 hrs. Then injected into the HPLC system against a blank of mobile phase (after all Telbivudine 3.51 3158422 optimized conditions) Results of degradation studies:
The results of the stress studies indicated the specificity of the method that has been developed. Telbivudine was stable in photolytic & temperature stress conditions. The result of forced degradation studies are given in the following table.
degraded
Hydrolysis 24Hrs.
Hydrolysis 24Hrs.
Degradation 24Hrs.
Retention Time (min)
Fig 6: Chromatogram showing thermal degradation
Table 7: Results of force degradation studies of
Telbivudine API.
B. Raj Kumar et al, JGTPS, 2014, Vol. 5(3): 1827 - 1832
RESULT and DISCUSSION
To develop a precise, linear, specific & suitable stability indicating RP-HPLC method for analysis of Telbivudine, different chromatographic conditions were applied & the results observed are presented in previous Scholars Research Library Der Pharmacia Lettre, Isocratic elution is simple, requires only one 2012, 4 (1):76-86 ISSN 0974-248X USA pump & flat baseline separation for easy and reproducible results. So, it was preferred for the current study over Journal of Pharmaceutical and Biomedical gradient elution. Analysis Volume 21, Issue 2, 1 November 1999, In case of RP-HPLC various columns are available, but here Develosil ODS HG-5 RP C Tropical Journal of Pharmaceutical Research, 15cmx4.6mm i.d. column was preferred because using October 2009; 8 (5): 449-454 Pharmacotherapy this column peak shape, resolution and absorbance were 10. Instrumental Method of Analysis by Rabi Sankar, Mobile phase & diluent for preparation of various samples were finalized after studying the 11. Practical HPLC Method Development by Lloyd R. solubility of API in different solvents of our disposal Snyder et al; 2nd edition, P-503 (methanol, Acetronitrile, dichloromethane, water, 0.1N 12. Guidance for industry, Analytical Procedure and Method Validation, U.S. Department of Health The drug was found to be soluble in and Human Services FDA, August 2000 Acetronitrile & dichloromethane and partially soluble in methanol. Drug was insoluble in water. Using these 13. The United State Pharmacopeia 25/National solvents with appropriate composition newer methods can Formulary 20, ch. 1225, pg. 2256-2259 (The be developed and validated. United State Pharmacopeia Convention, Inc., Detection wavelength was selected after Rockville, Maryland, 2002) scanning the standard solution of drug over 200 to 14. ICH Q2B: Validation of Analytical Procedure; 400nm. From the U.V spectrum of Telbivudine it is Methodology (International Conferences on evident that most of the HPLC work can be accomplished Harmonization of Technical requirements for the in the wavelength range of 240-300 nm conveniently. registration of Drugs for Human use, Geneva, Further, a flow rate of 1 ml/min & an injection volume of Switzerland, May 1997) 20 ul were found to be the best analysis. 15. ICH Q2B: Validation of Analytical Procedure; The result shows the developed method is yet Methodology (International Conferences on another suitable method for assay which can help in the Harmonization of Technical requirements for the analysis of Telbivudine in different formulations registration of Drugs for Human use, Geneva, Switzerland, Nov 2003) 16. Journal of Chromatography .B, Analytical A sensitive & selective RP-HPLC method has Technologies in the Biomedical and life Sciences. been developed & validated for the analysis of 2008 March 1; 863(2): 258-265. published on Telbivudine API. Further the proposed RP-HPLC method has excellent sensitivity, precision and reproducibility. 17. Kassen Mussen nicht fur "Acomplia" zahlen. The result shows the developed method is yet another Tagesschau.de (2006-10-17). Retrieved on 2007- suitable method for assay, purity which can help in the analysis of Telbivudine in API.
18. Matheson A.J., Noble S., Drugs, Volume 59, Number 4, April 2000, pp. 829-835(7).
19. Dawei Zhou, Xuntian Jiang, and Xinping Fang, A Rapid LC-MS/MS Method for the Determination of Telbivudine in Human Plasma, XenoBiotic Laboratories, Inc., 107 Morgan Lane, Plainsboro, How to cite this article:
B. Raj Kumar1*Dr. K. V. Subrahmanyam2: a validated stability-indicating RP-HPLC method for the determination of Telbivudine: 5(3): 1827-32. (2014) All 20104 are reserved by Journal of Global Trends in Pharmaceutical Sciences
B. Raj Kumar et al, JGTPS, 2014, Vol. 5(3): 1827 - 1832

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