Journal Menu
Submit Manuscript via ScholarOne

Eurasian Journal of Analytical Chemistry
Volume 11, Issue 4 (July 2016), pp. 197-210

DOI: 10.12973/ejac.2016.132a

Downloaded 1337 times.

Research Article

Published online on Jul 28, 2016

How to reference this article?


A Validated Stability Indicating RP-HPLC Method for Determination of Phenobarbital and Acefylline Piperazine in Bulk Drug and Combined Dosage Form

Gamal Ragab, Hanaa Saleh, Magda el-Henawee & Omnia F. Elsayed


A simple, sensitive, precise and stability indicating RP-HPLC method was developed for determination of Phenorbarbital and Acefylline Piperazine in bulk drug and in combined syrup. Isocratic, HPLC method , using Thermo Hypersil C18 gold column with mobile phase of Acetonitrile - 0.1%  orthophosphoric acid (30:70,v/v)  was investigated to separate the drugs from their stressed degredation products. The flow rate was 1.5 ml/min. Detection was carried out at a wavelength of 240 nm. The method was validated for precision, accuracy, robustness and recovery. Phenobarbital and Acefylline Piperazine were exposed to acidic, basic, neutral, oxidative and photolytic stress conditions and the stressed samples were analyzed by the proposed method. Good linear relationship in the concentration range of (1-100) µg/ml and (4-250) µg/ml for Phenobarbital and Acefylline Piperazine respectively with correlation coefficient of 0.9999 for both drugs. Intra- and inter-day precision were less than 1% for both analytes. the method was tested for specifity for determination of target analytes in presence of excipients like Methylparaben and Propylparaben also The stressed sample chromatograms demonstrate the specificity of the proposed method for the determination of target analytes in presence of degradants.

Keywords: stability indicating , HPLC, Phenobarbital, Acefylline Piperazine

  1. Majesty’s, H. and Stationery Office, The British Pharmacopoeia. 2013: London, UK.
  2. Kwan, P. and M.J. Brodie (2004) Phenobarbital for the treatment of epilepsy in the 21st century: a critical review. Epilepsia, 45(9), 1141-1149.
  3. (2011) Kinetex®Core-Shell Technology Ultra-High Performance from HPLC to UHPLC.
  4. El-Gindy, A. (2005). HPLC and chemometric assisted spectrophotometric methods for simultaneous determination of diprophylline, phenobarbitone and papaverine hydrochloride. Il Farmaco, 60(9), 745-753.
  5. Xia, W., L. Daojie, and S. Jiling (2003) Simultaneous Determination of Seven Ingredients in Compound Theophylline Tablets by Reversed Phase High Performance Liquid Chromatography Gradient Elution. Chinese Journal of Analytical Chemistry, 31(5), 577-579.
  6. Fu, S., S. Duan, and Q. Ren (1997) Simultaneous analysis of theophylline, phenobarbital, amobarbital and carbamazepine in serum by high performance liquid chromatography. Se pu= Chinese journal of chromatography/Zhongguo hua xue hui, 15(2), 178-179.
  7. Hashem, H., A.A. Gouda, and H. Saleh (2013) Development And Validation Of Rapid Stability Indicating Hplc-Determinations Of Antiepileptic Drugs Phenobarbital In Suppositories And Phenytoin In Capsules As Well As In Urine Sample. Journal of Liquid Chromatography & Related Technologies, 36(16), 2292-2306.
  8. Pandey, S., A.K. Yadav, S. Singh, H. Gautam, and S. Sharma (2012). Bio-analytical method development and its validation for estimation of phenobarbital in human plasma using liquid chromatography coupled with tandem mass. Int. J. Pharm. Pharm. Sci, 4(5): p. 288-92.
  9. Dalmora, S.L., M.d.S. Sangoi, D.R. Nogueira, F.B. D'Avila, R.A. Moreno, C.E. Sverdloff, R.A.d. Oliveira, and N.C. Borges (2010). Determination of phenobarbital in human plasma by a specific liquid chromatography method: application to a bioequivalence study. Química Nova, 33(1): p. 124-129.
  10. Johnson, L.L. and U. Garg (2010). Quantitation of amobarbital, butalbital, pentobarbital, phenobarbital, and secobarbital in urine, serum, and plasma using gas chromatography-mass spectrometry (GC-MS), in Clinical Applications of Mass Spectrometry. Springer. p. 65-74.
  11. TANG, L. and J.-b. YING (2012). GC-MS/MS Determination of Barbiturate Drugs in Blood with Accelerated Solvent Extraction [J]. Physical Testing and Chemical Analysis (Part B: Chemical Analysis), 7,  030.
  12. El-Gindy, A., S. Emara, and G.M. Hadad (2003). Determination of two ternary mixtures containing phenobarbitone by second derivative of the ratio spectrum-zero-crossing and HPLC methods. Il Farmaco, 58(5), 377-390.
  13. Haque, A., X. Xu, and J.T. Stewart (1999). Determination of ephedrine, theophylline and phenobarbital in a tablet dosage form by capillary electrophoresis. Journal of pharmaceutical and biomedical analysis, 21(5), 1063-1067.
  14. Lima, J.L., M.C. BSM Montenegro, and A. Da Silva (1990) A phenobarbital ion-selective electrode without an inner reference solution, and its application to pharmaceutical analysis. Journal of pharmaceutical and biomedical analysis, 8(8), 701-704.
  15. Ni, Y., C. Liu, and S. Kokot (2000) Simultaneous kinetic spectrophotometric determination of acetaminophen and phenobarbital by artificial neural networks and partial least squares. Analytica chimica acta, 419(2), 185-196.
  16. Boeris, M., J. Luco, and R. Olsina (2000) Simultaneous spectrophotometric determination of phenobarbital, phenytoin and methylphenobarbital in pharmaceutical preparations by using partial least-squares and principal component regression multivariate calibration. Journal of pharmaceutical and biomedical analysis, 24(2), 259-271.
  17. El-Gindy, A., S. Emara, and H. Shaaban (2007) Development and validation of chemometrics-assisted spectrophotometric and liquid chromatographic methods for the simultaneous determination of two multicomponent mixtures containing bronchodilator drugs. Journal of pharmaceutical and biomedical analysis, 43(3),  973-982.
  18. Sved, S., I. McGilveray, and N. Beaudoin (1981) The assay and absorption kinetics of oral theophylline− 7‐acetic acid in the human. Biopharmaceutics & drug disposition, 2(2), 177-184.
  19. Rabouan-Guyon, S., P. Courtois, and D. Barthes (1996). Determination of acefylline heptaminol in pharmaceutical preprarations by high performance liquid chromatography. Il Farmaco, 51(11), 739-746.
  20. Owen, J. and K. Nakatsu (1978) Spectrophotometry of theophylline-7-acetic acid and theophylline. Clinical chemistry, 24(2), 367-368.
  21. Tsvetkova, B., J. Tencheva, and P. Peikov (2003) Alkaline, Enzymatic Hydrolysis and Physicochemical Properties of 7-Theophyllineacetic Acid Derivatives. Journal of Food and Drug Analysis, 11(3), 191-194.
  22. Levi, L. (1957) The Barbituric-Acids, Their Chemical-Structure, Synthesis And Nomenclature. Bulletin on Narcotics, 9(1), 30-40.
  23. ICH, Validation of analytical procedures , Text and Methodology. 2005, Geneva: International conference on harmonization.
  24. Miller, J.N. and J.C. Miller, (2005). Statistics and chemometrics for analytical chemistry. Pearson Education.
  25. Guidance, R., (1994). Validation of chromatographic methods. Center for Drug Evaluation and Research, Food and Drug Administration. 669, 671.