International Journal of Innovative Pharmaceutical Sciences and Research

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1 International Journal of Innovative Pharmaceutical Sciences and Research ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPLC METHOD FOR ESTIMATION OF FIXED DOSAGE FORM OF ATROPINE SULPHATE, EPINEPHRINE BITARTARATE AND LIGNOCAINE HYDROCHLORIDE INJECTION 1 S.Abraham Jebaraj*, 2 R.Prasanna, 3 T.Sivakumar 1 Department of pharmaceutical Analysis, Karpagam university.coimbatore, INDIA 2 Adcock Ingram limited, Bangalore. INDIA 3 Prinicipal, Nandha college of pharmacy.erode. INDIA Abstract A Simple, Fast, precise, accurate stability indicating high performance liquid chromatography (HPLC) method has been developed for estimation of fixed dosage form of Atropine sulphate, Epinephrine bitartarate, Lignocaine hydrochloride injectable dosage form. The separation for lignocaine was obtained using a mobile phase composition at the ratio of 50:50 (v/v) of water and acetonitrile by adjusting ph 3.3 with ortho phosphoric acid on sunfire C18 Column (250 X4.6 mm, 5 µm ) with PDA detection at 254 nm at flow rate of 1 ml/min. The linear calibration was found to be for lignocaine. The separation for Epinephrine was obtained using a mobile phase composition at the ratio of 50:50 (v/v) of water and Methanol by adjusting to ph 3.2 with ortho phosphoric acid on sunfire C18 Column (250 X4.6 mm, 5 µm) with PDA detection at 280 nm at flow rate of 1 ml/min. The linear calibration was found to be for epinephrine. The separation for Atropine was obtained using a mobile phase composition at the ratio of 50:50 (v/v) of Methanol and water by adjusting ph 3.8 with ortho phosphoric acid on sunfire C18 Column (250 X4.6 mm, 5 µm ) with PDA detection at 229 nm at flow rate of 0.5 ml/min. The linear calibration was found to be for atropine. The method validation data showed excellent results for precision, linearity, specificity and robustness. The present method can be successfully used for routine quality control and stability studies. Keywords: Atropine, Epinephrine, Lignocaine, Injectable dosage, Precision, Linearity, Specificity. Corresponding Author: S.Abraham jebaraj Department of pharmaceutical Analysis Karpagam university.coimbatore,tamilnadu, INDIA abrahamjebaraj@rediffmail.com Phone: Available online: July Issue 1337

2 INTRODUCTION Atropine sulphate a potent parasympatholytic agent for use in producing cycloplegia and mydriasis. Useful for cycloplegic refraction or for pupil dilation in acute inflammatory conditions of the iris and uveal tract. Epinephrine (also known as adrenaline or adrenalin) is a hormone and a neurotransmitter. Epinephrine has many functions in the body, regulating heart rate, blood vessel and air passage diameters, and metabolic shifts; epinephrine release is a crucial component of the fight-or-flight response of the sympathetic nervous system. Lignocaine is a Local anesthetic which reversely blocks the nerve fibers. It binds to the intracellular portion of sodium channels and blocks sodium influx into nerve cells, which prevents depolarization. Chemically, Lignocaine Hydrochloride is 2-diethylaminoaceto-2,6 -xylidide hydrochloride monohydrate, Epinephrine bitartrate is (R)-1-(3,4-dihydroxyphenyl)-2- methylaminoethanol hydrogen tartrate and Atropine sulphate is (RS)-(1R,3r,5S)-3-tropoyloxytropanium sulphate monohydrate. Literature survey reveals that several HPLC methods are reported for the determination of Atropine sulphate, epinephrine bitartrate, and lignocaine hydrochloride individually. However no method reported for above drugs that stated below in HPLC. The proposed method validated stability indicating method can be used for routine quality control and stability studies of the subjected formulation. MATERIALS AND METHODS Instrumentation The high pressure liquid chromatography (HPLC) system was used of Waters e2695 alliance, auto-sampler with PDA detector supported with Empower Software. A column Sunfire C18 HPLC (250 X 4.6 mm, 5 µm ) was used as stationary phase. Materials Working standards of Atropine Sulphate, Epinephrine bitartrate and Lignocaine Hydrochloride, HPLC grade Methanol manufactured by Loba chemicals, HPLC grade acetonitrile manufactured merck, HPLC grade water manufactured by merck. Ortho phosphoric acid rankem analytical grade. Injection contains Lignocaine Hydrochloride mg/ml, Epinephrine bitartrate 6 µg/ml, Atropine sulphate 9 µg/ml. Chromatographic Conditions The analysis was carried out on Waters HPLC system, using the column Sunfire (250 X 4.6 mm, 5 µm) With PDA detector at 254 nm for lignocaine hydrochloride, 280 nm for epinephrine Available online: July Issue 1338

3 bitartrate and 229 nm for atropine sulphate at ambient temperature using 1 µl for Lignocaine Hydrochloride, 1 µl for epinephrine bitartrate, 10 µl for Atropine sulphate injection volume. A degassed mixture of (50:50 V/V) of water and acetonitrile adjusted the ph 3.3 with ortho phosphoric acid solution Epinephrine Bitartrate: A degassed mixture of (50:50 V/V) of water and methanol adjusted the ph 3.2 with ortho phosphoric acid solution. Atropine Sulphate: A degassed mixture of (50:50 V/V) of water and methanol adjusted the ph 3.8 with ortho phosphoric acid solution. Standard solution Lignocaine Hydrochloride: Standard stock solution of lignocaine hydrochloride was prepared by dissolving mg of Lignocaine hydrochloride in 100 ml volumetric flask dissolve it with water and make up the required volume. Epinephrine Bitartrate Standard stock solution of Epinephrine Bitartrate was prepared by dissolving mg of Epinephrine Bitartrate in 100 ml volumetric flask dissolve it with water and make up the required volume. From that above solution take 10 ml of Epinephrine Bitartrate solution transfer into 100 ml volumetric flask and make up the required volume. From that above solution take 10 ml of Epinephrine Bitartrate solution transfer into 100 ml volumetric flask and make up the required volume. Atropine Sulphate Standard stock solution of Atropine Sulphate was prepared by dissolving mg of Atropine Sulphate in 100 ml volumetric flask dissolve it with water and make up the required volume. From that above solution take 10 ml of Atropine Sulphate solution transfer into 100 ml volumetric flask and make up the required volume. From that above solution take 10 ml of Atropine Sulphate solution transfer into 100 ml volumetric flask and make up the required volume. Validation Parameter The Method validation was carried out as per ICH guidelines. Parameters as follows Specificity, Linearity, Precision, Ruggedness, Robustness, System suitability Specificity Specificity of the method was evaluated by injecting the blank, individual Lignocaine Hydrochloride, Epinephrine Bitartrate, Atropine Sulphate and sample solution prepared by Available online: July Issue 1339

4 spiking the know concentration and injected into HPLC system to check the co-elution,if any, at the retention time of Lignocaine Hydrochloride, Epinephrine Bitartrate and Atropine Sulphate peak. Linearity: Calibration cure obtained by the least square regression analysis between average peak area and concentration showed linear relationship with a correlation coefficient for Lignocaine Hydrochloride, Epinephrine Bitartrate and Atropine Sulphate. A. Lignocaine Hydrochloride linear calibration range was found to be 600 µg/ml to 1400µg/ml. B. Epinephrine Bitartrate calibration range was found to be 6 µg/ml to 14 µg/ml. C. Atropine Sulphate linear calibration range was found to be 6 µg/ml to14 µg/ml. Table 1 : Linear regression least square fit data obtained from the calibration curves Lignocaine Hydrochloride Epinephrine Bitartrate Atropine Sulphate S.No Concentratio Area Concentration Area Concentratio Area n n 1 60 % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % Correlation Coefficient : Correlation coefficient : Correlation Coefficient : Precision: Lignocaine Hydrochloride, Epinephrine Bitartrate and Atropine Sulphate Repeatability (method precision) and Intermediate Precision (Ruggedness) was performed. The Available online: July Issue 1340

5 RSD was found to be NMT 2 %. The compiled data are given in table 2. Robustness Lignocaine Hydrochloride: The slight change in the parameter done in the method and perform the analysis. The RSD was found to be NMT 2 %. The compiled data are given in table 3. Table 3 Table 2 Lignocaine Hydrochloride Epinephrine Bitartrate Atropine Sulphate S.No Area S.No Area S.No Area Mean Mean Mean SD SD SD RSD RSD RSD Lignocaine Hydrochloride ph Mobile phase composition water : Acetonitrile 55:45 S.No Area Area S.No Area Area Mobile phase composition water : Acetonitrile 45:55 Mean Mean SD SD RSD RSD Epinephrine Bitartrate: The slight change in the parameter done in the method and perform the analysis. The RSD was found to be NMT 2 %. The compiled data are given in table 4. Available online: July Issue 1341

6 Table 4 Epinephrine Bitartrate ph Mobile phase composition water : Methanol 55:45 S.No Area Area S.No Area Area Mean Mean Atropine Sulphate: The slight change in the parameter done in the method and perform the analysis. The RSD was found to be NMT 2 %. The compiled data are given in table 5. Accuracy Table 5 Lignocaine Hydrochloride: Added the know quantity of sample into the solution and perform the recovery. Recovery of the sample was between 98 % to 102 % v/v. The compiled data are given in table 6. Mobile phase composition water : Methanol 45:55 SD SD RSD RSD Atropine Sulphate ph Mobile phase composition water : Methanol 55:45 S.No Area Area S.No Area Area Mobile phase composition water : Methanol 45:55 Mean Mean SD SD RSD RSD Available online: July Issue 1342

7 Table 6 S.No Concentration Area Amount Added Recovery 1 80 % % % % % % % % % mg/ml 99.5% 41.4 mg/ml 98.5% mg/ml 98.2 % Epinephrine Bitartrate: Added the know quantity of sample into the solution and perform the recovery. Recovery of the sample was between 98 % to 102 % v/v. The compiled data are given in table 7. Table 7 S.No. Concentration Area Amount added Recovery 1 80 % % µg % 3 80 % % % µg % % % % µg % % Atropine Sulphate: Added the know quantity of sample into the solution and perform the recovery. Recovery of the sample was between 98 % to 10 2% v/v. The compiled data are given in table 8. Available online: July Issue 1343

8 Table 8 S.No. Concentration Area Amount added Recovery 1 80 % % % % % % % % % µg/ml 101.5% 45 µg/ml % 45 µg/ml % System Suitability Lignocaine Hydrochloride: System suitability was performed by injecting standard solution on various days of the validation. Tailing factor and number of theoretical plates was found to be 1.2 and 1524 respectively. Epinephrine Bitartrate: System suitability was performed by injecting standard solution on various days of the validation. Tailing factor and number of theoretical plates was found to be 1.01 and 1595 respectively. Atropine Sulphate: System suitability was performed by injecting standard solution on various days of the validation. Tailing factor and number of theoretical plates was found to be 1.56 and 1721 respectively. RESULT AND DISCUSSION In estimation of Atropine, Epinephrine, Lignocaine from injectable dosage form. The several combination of buffer tried like acetate buffer, phosphate buffer there is no elution. The separation for lignocaine was obtained using a mobile phase composition at the ratio of 50:50 (v/v) of water and acetonitrile by adjusting ph 3.3 with ortho phosphoric acid. The separation for Epinephrine was obtained using a mobile phase composition at the ratio of 50:50 (v/v) of water and Methanol by adjusting to ph 3.2 with ortho phosphoric acid. The separation for Atropine was obtained using a mobile phase composition at the ratio of 50:50 (v/v) of Methanol and water by adjusting ph 3.8 with ortho phosphoric acid. The linear calibration was found to be Available online: July Issue 1344

9 for lignocaine, for epinephrine, for atropine. All the three drugs have no interference with placebo. The precision was found to be for lignocaine hydrocloride, for epinephrine bitrate, for atropine sulphate.the robustness was found to be satisfactory. System suitability was found to be with in the limit for all three drugs. B.No 2 - Atropine Injection B.No 3- Atropine Standard B.No 3 - Atropine Injection Atropine Placebo B.No 1 - Epinephrine Standard B.No 1 - Epinephrine Injection B.No 2 - Epinephrine Standard B.No 2 - Epinephrine Injection B.No3 - Epinephrine Standard Available online: July Issue 1345

10 B.No 3 - Epinephrine Injection Epinephrine Placebo B.No 1- Lignocaine standard B.No 1- Lignocaine Injection B.No 2- Lignocaine standard B.No 2- Lignocaine Injection B.No 3- Lignocaine standard B.No 3- Lignocaine Injection Lignocaine Placebo Available online: July Issue 1346

11 CONCLUSION From above results and discussion conclude that this method is suitable for routine analysis of atropine sulphate, epinephrine bitatrate, lignocine hydrochloride injection and this method is used in combination determination and also individual determination of the above injection, when compare to pharmacopeia method the cost of this method is very less i.e. cost is 1/3 rd of the pharmacopeia method. REFERENCES: 1. Modi Foram P, Patel Priyal R. Formulation, Optimization & Evaluation of Fixed Dose Combination Moisture Barrier Film Coated Bilayer Tablet of Artesunate & Amodiaquine Hydrochloride, International Journal of PharmTech Research, coden (USA): IJPRIF, Vol.3, No.4, Oct-Dec 2011, p Nagaraja Y. S., Nagaraja T. S., Bharathi D. R and Manjunatha T. O., Formulation and Evaluation of Ofloxacin Aqueous Injection, International Journal of Pharmacy & Life Sciences, CODEN (USA),Oct 2012, IJPLCP,3(10). 3. Krunal Bhatt., Priyal R., Patel, Formulation and Evaluation of Diclofenac Sodium Injection Using 2-Hydroxy Propyl Beta Cyclodextrin, International Journal of Pharmacy and Pharmaceutical Sciences, Vol 3, Suppl 5, Prasanna S., Puranik SB., Preparation and Evaluation of Bendamustine Hydrochloride Aqueous Formulations, International Journal for Pharmaceutical Research Scholars (IJPRS),2013,Vol-2, I Margriet., Handriks Jan M.W.B., DE Boer H., Age K. Smilde(editors), Robustness of Analytical Chemical Technological Products, Data Handling in Science and Technology, 1996,Vol -19 p Ermer J., and McB J. H., Miller (Editors), Method Validation In Pharmaceutical Analysis,2005, p Donald A., Wellings., A Practical Handbook of Preparative HPLC, 2006, p Richard J. Smith, Michael L. Webb, Analysis of Drug Impurities, 2007, p Graham Currell, Analytical Instrumentation Performance Characteristics and Quality 2000, p Available online: July Issue 1347

12 10. Mueller-Harvey and Baker R. M., Chemical Analysis in the Laboratory, A Basic Guide 2002, p Satinder Ahuja, Michael W.Dong, Handbook of Pharmaceutical Analysis by HPLC, Vol- 6, p Paul C. Sadek., Illustrated Pocket Dictionary of Chromatography p Kealey D., Haines P. J., Instant Notes Analytical Chemistry , p Ashutosh Kar, Pharmaceutical Drug Analysis,New age international (P) limited,new Delhi,Bangalore,Chennai, , p Available online: July Issue 1348

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