PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES RP-HPLC METHOD FOR DEVELOPMENT AND VALIDATION OF LISINOPRIL TABLETS Divyakant Patel*, Sharad Kumar, Yogesh Chand Yadav, A.K. Seth, Gajanan J. Deshmukh, Tejas K Ghelani Department of Pharmacy, Sumandeep Vidyapeeth, Piparia, Ta. Waghodia, Dist.Vadodara-391760, (Gujarat) India. ABSTRACT The study is focused to develop a validate High Performance Liquid Chromatographic (HPLC) method for the Lisinopril Tablets from their dosage forms. The wavelength of this drug is found to be 215. The accuracy and precision were determined and validated statistically. A simple Reverse phase liquid chromatographic method has been also developed and subsequently validated for the lisinopril Tablets. Here we use a mobile phase consisting of Potassium dihydrogen phosphate and acetonitrile. The column used was Inertsil C8 (250x4.6mm) 5µ with flow rate 1.0 ml/min using UV detection at 215nm. The retention time of Lisinopril is found to be 10.479 respectively. Result of analysis was validated statistically. The method shows good reproducibility & recovery with less than 1%, all the tests of above mentioned studies were found to be in acceptance criteria.the method was found to be rapid, specific, precise & accurate and can be successfully applied for routine analysis of Lisinopril dosage form. Keywords: Lisinopril, HPLC, UV. INTRODUCTION Lisinopril is an angiotensin converting enzyme inhibitor. ACE is a peptidyl dipeptidase that catalyzes the conversion of angiotensin-і to the vasoconstrictor substance angiotensin-іі. Angiotensin-ІІ also stimulates cortex inhibition of ACE which results in decreased plasma angiotensin-і and thus leads to vasopressor activity and to decreased aldosterone secretion.the latter decrease may result in small increase of serum potassium. The objective of the present work is to develop and validate new analytical methods for Lisinopril tablet dosage form. This communication forms the first report of simple, sensitive and reproducible method for the Lisinopril dosage form. Dosage are administrated once daily without regard to meal. In children greater than 6 year, the dose www.pharmasm.com IC VALUE 4.01 S - 16
is initially 0.007mg/kg up to 5 mg once daily. In adult, the dose is initially 10 mg daily. Chest pain, stomach pain, fatigue, in rare case heart palpitation, weight loss, skin rash, difficulty in breathing and swallowing are some side effects caused due to it. Plasma half life is 12 hours. It is excreted as unchanged form. Patient should undergo some precaution when treated with diuretic as symptomatic hypotension may occur occasionally. So diuretic should be stopped for 2 to 3 days during therapy with lisinopril. MATERIALS AND METHODS Chemicals and Reagents Lisinopril was obtained as a gift sample from Unimark Remedies limited respectively. Water, Acetonitrile, Methanol, Potassium dihydrogen phosphate, Orthophosphoric acid and Hexane sulphonic acid sodium were of HPLC grade. Instrumentation HPLC of Agilent 1100/1200 series with UV/DAD detector and isocratic/gradient pump, injector with a 20 μl loop was used for the injection of sample, Inertsil C8 (250x4.6mm)5µ column was used. EXPERIMENTAL Chromatographic Condition The mobile phase containing Acetonitrile: Phosphate solution, ph adjusted to 2 with Ortho-phosphoric acid. Ortho-phosphoric acid was used for ph adjustment of buffer. Ratio was 180:820. The mobile phase was filtered on a 0.45 Nylon filter and then ultrasonicated for 30 min. The flow rate was set to 1.0 ml/min. Drugs showed good absorbance at 215 nm, which was used as wavelength for further analysis. Phosphate solution: Dissolve a quantity about 4.1 g of monobasic potassium phosphate in 900 ml of water. Adjust the ph to 2.0 with ortho-phosphoric acid (88%) and make up the volume up to 1000 ml with water. Filter through 0.45 µ Nylon filter. Mobile phase: Dissolve 1.0 g of hexane sulphonic acid sodium (Make Spectrochem) in 820 ml of Phosphate solution. Add 180 ml of Acetonitrile and mix well. Degas before use. Diluent: Prepare a mixture of 800 volumes of water and 200 volumes of Methanol. www.pharmasm.com IC VALUE 4.01 S - 17
Standard preparation: Transfer an accurately weighed quantity about 22.0 mg Lisinopril working standard in to 100 ml volumetric flask. Add about 50 ml of diluent; dissolve and dilute to volume with diluent; mix well. Assay Preparation: Weigh accurately 10 tablets and transfer into 200ml volumetric flask. Add exact 125 ml of diluent, sonicate for 30min. Taking care to maintain temperature of ultrasonic bath at room temperature cool and centrifuge for 5 min, filter the clear supernatant liquid through 0.45µ Nylon filter. Discard first 3-4ml of the filtrate. Calculation: Au W 1 125 W 3 P X X X X As 100 L.C. 100 W 2 X 100 Where Au = Mean peak area due to Lisinopril obtained with assay preparation. As = Mean peak area due to Lisinopril obtained with standard preparation. W 1 W 2 W 3 = Weight of Lisinopril working standard taken in mg. = Weight of sample taken in mg. = An average weight of tablet in mg. L.C. = Label claim of Lisinopril in mg per tablet. P = Potency of Lisinopril working standard in percentage on as is basis. [7]. Figure 1 Final method www.pharmasm.com IC VALUE 4.01 S - 18
Validation of the method The developed method was validated in terms of linearity, accuracy, specificity, limit of detection, limit of quantification, intra-day and inter-day precision and repeatability of measurement. Figure 2 Assay preparation Figure 3 Standard preparation Method Precision: Prepare six set of Assay preparation as mentioned in the test method described in 4.0. Separately inject duplicate injection of Assay preparation in to liquid chromatography and record the chromatograms. Six Assay preparations were analyzed by proposed method. The results are recorded in following table. www.pharmasm.com IC VALUE 4.01 S - 19
TABLE 1: METHOD PRECISION Set No. Mean area Assay (% L.C.) Set-1 5374.65809 98.8 Set-2 5407.53381 99.5 Set-3 5399.46927 99.3 Set-4 5400.56744 99.3 Set-5 5401.48178 99.4 Set-6 5393.26540 99.3 Mean 99.3 Relative standard deviation 0.24% Accuracy (Recovery studies) To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 80%, 100% and 120%. Known amounts of standard Lisinopril were added to pre-analyze samples and were subjected to the proposed HPLC method. TABLE 2: ACCURACY (RECOVERY STUDIES) Theoretical Practical Set. No. Accuracy Mean area of amount of amount of % Level Lisinopril Lisinopril Lisinopril Recovery mg/tablet mg/tablet Set-1 4356.05325 31.98 99.9 Set-2 4355.63609 32.00 31.91 99.7 Set-3 80% 4363.12482 31.97 99.9 Mean 99.8 % RSD 0.12 Set-1 5476.95438 40.14 100.4 Set-2 5436.13750 40.00 39.84 99.6 Set-3 100% 5435.22341 39.84 99.6 Mean 99.9 % RSD 0.46 Set-1 6482.90506 47.52 99.0 Set-2 6495.91594 48.00 47.61 99.2 Set-3 120% 6543.30736 47.95 99.9 Mean 99.4 % RSD 0.48 Overall Mean 99.7 Overall % RSD 0.41 www.pharmasm.com IC VALUE 4.01 S - 20
System Suitability. Standard preparation was injected six time and results were recorded with respect to Lisinopril peaks. Observations are recorded in the following table. TABLE 3: SYSTEM SUITABILITY System Suitability Parameters Results Acceptance Criteria Relative standard deviation of six replicate injections for Lisinopril peak area 0.04 NMT 2.00% Average Tailing factor for Lisinopril peak 1.51 NMT 2.00 Average Theoretical plates (by tangent method) for Lisinopril peak Average Capacity factor K for Lisinopril peak 4.45 Average area of Lisinopril peak in replicate injections 3075 NLT 700 5419.86216 NA Should be more than 1.5 All the analytical performance parameters were checked as per the approved validation protocol and found well within specified acceptance results. Hence it is concluded that this method is capable to produce accurate and precise results during routine analysis of initial and stability samples. RESULTS AND DISCUSSION To develop a precise, accurate and suitable RP- HPLC method for the Development and validation of the Lisinopril tablets, different temperature and the column were tried and the proposed chromatographic conditions were found to be appropriate for the quantitative determination of the lisinopril. So by performing different trials of Temperature and column for Lisinopril we concluded final method with desired tailing and the temperature. Also concluded the separation of different impurity from main peak of Lisinopril. www.pharmasm.com IC VALUE 4.01 S - 21
TABLE 4: SUMMARY OF VALIDATION PARAMETERS Parameters Conclusions Specificity No peaks are detected at the retention time of Lisinopril peak in the chromatograms of diluent and placebo preparations. Lisinopril peak in Standard preparation and Assay preparation are spectrally pure. The retention time of the peak obtained due to Lisinopril in the Assay preparations corresponds with that obtained from Standard preparation. Stability of analyte in solution It is concluded that Standard preparation and Assay preparation are stable up to 9 hour at 23 C to 27 C when stored without light protection. Filter Saturation Minimum 3-4 ml of Assay preparation is suitable to discard from 0.45 µ Nylon filter prior to sample filtration during analysis. Precision System precision Based on Relative standard deviation data of Lisinopril peak area (RSD 0.04%), it is concluded that the system is precise. Method precision All the results meet the acceptance criteria for Assay limit. % Relative standard deviation of results is less than 0.50, indicates good degree of precision. Hence method is capable to give precise results. Accuracy The recovery at each level of Lisinopril is between 99.0% to 100.4%. Relative standard deviation for recovery at each level is less than 1.00%. Overall relative standard deviation for all the levels is less than 1.00% Range Based on precision and accuracy results, working range for Lisinopril is between concentration ranges of 160.00 µg/ml to 240.00 µg/ml. CONCLUSION The proposed method is simple, sensitive and reproducible and hence can be used in routine for Lisinopril determination in pharmaceutical preparations. Statistical analysis of the results has been carried out revealing high accuracy and good precision. The RSD for all parameters was found to be less than one, which indicates the validity of method www.pharmasm.com IC VALUE 4.01 S - 22
and assay results obtained by this method are in fair agreement. The developed method can be used for routine quantitative estimation of lisinopril pharmaceutical preparation. ACKNOWLEDGEMENT It gives me an immense pleasure to express my gratitude towards my Guide Mr. Sharad Kumar, Co-Guide Mr. Yogesh Chand Yadav & Dr A.K Seth, Director, Department of Pharmacy, Sumandeep Vidyapeeth, and their encouragement and positive attitude towards work has instilled more confidence in me. REFERENCES 1. Lawrence and Sujatha Ramkrishna: United States Pharmacopoeia, Volume 2007; 2483. 2. Hoboke, John Wiley & Sons: J. Pharm.Biomed, New Jersey, 2007; 4-11. 3. P.D.Sethi: Quantitative Analysis of Drugs in Pharmaceutical Formulation, 3 rd Ed.: CBS Publishers and Distributors, New Delhi; 1997. 4. Douglas S., James H. and Timothy N: Principles of Instrumental Analysis, 5 th Ed. Thomson Learning Inc., Singapore, 1998; 110, 300. 5. Gurdeep R Chatwal: Textbook of Pharmaceutical Analysis, 3 rd Ed., Himalaya Publishing House, Delhi; 2008. 6. Pawlak, Z. and Clark, B.J: J. Pharm. Biomed. Anal., 1992; 10, 329. 7. Sujatha Ramkrishna: United States Pharmacopoeia, Volume 2007; 2476. 8. Kasture AV, Wadodkar SG, Mahadik KR, More HM: Introduction to Instrumental Techniques, Vol. II, 12 th ed. Pune: Nirali Prakasan; 2005; 1-10. 9. Ewing GW: Instrumental Methods of Chemical Analysis, 5 th ed. New York: McGraw-Hill Book Company; 1985. p. 1-7. 10. Singh SS, Bakshi M: Development of Validated Stability Indicating Assay Methods: Critical Review. J Pharm Bio Anal, 2002; 28: 1011 1040. For Correspondence: Divyakant Patel Department of Pharmacy Sumandeep Vidyapeeth, Vadodara (Gujarat)-391760 E-mail: devamamy@gmail.com www.pharmasm.com IC VALUE 4.01 S - 23