Open Access Peer-reviewed

Development and Validation of Newer High Performance Thin Layer Chromatographic Method for Quantification of Eflornithine Hydrochloride in Pharmaceutical Formulations

Amit Kumar1, Vijender Singh2, Praveen Kumar3,

1Department of Pharmaceutical analysis, NKBR College of Pharmacy & Research Centre, Meerut, India

2Department of Pharmaceutical analysis, BBS Institute of Pharmaceutical & Allied Sciences, Greater Noida, India

3Department of Pharmaceutical Chemistry, S. D. College of Pharmacy and Vocational Studies, Muzaffarnagar, India

American Journal of Pharmacological Sciences. 2013, 1(4), 47-52. DOI: 10.12691/ajps-1-4-1
Published online: August 25, 2017


A new, simple, sensitive, precise and robust high performance thin layer chromatography (HPTLC) method was developed for the estimation of eflornithine hydrochloride (DFMO) in pharmaceutical dosage forms. Estimation was performed on TLC aluminum plates precoated with silica gel 60F254 as stationary phase. Linear ascending development was carried out in twin trough glass chamber saturated with mobile phase consisting of methanol: chloroform: acetic acid: 1% triethylamine (4:6:0.1:0.5 v/v/v) at room temperature (25±2°C). Analysis of the plate in absorbance mode at 220nm was carried out. The system was found to give compact spots for DFMO with Rf (Retardation factor) value of 0.55 respectively. The data for calibration plots showed good linear relationship with correlation coefficient of 0.999 in the concentration range of 300–800ng mL-1 for DFMO respectively. The values of limit of detection (LOD) were 0.6238ng mL-1 and limit of quantification (LOQ) were 1.8903ng mL-1 for DFMO respectively. The accuracy of the method was 100.44%. The precision demonstrated a relative standard deviation of less than 2.5%. The results were satisfactory when compared with the literature. This new method was validated according to the International Conference on Harmonization (ICH) guidelines which include linearity, precision, accuracy, specificity, robustness, detection and quantitation limits. The developed methods found to be sufficiently precise and reproducible for established conditions and after validation may be used for routine analysis of eflornithine hydrochloride in pharmaceutical formulations.


eflornithine hydrochloride (DFMO), quantification, validation, ICH guidelines, HPTLC
[1]  Bacchi, C. J., Goldberg, B., Carofalo-Hannan, J., Rattendi, D., Lyte, P. and Yarlett, N, “In vivo effects of α-DL-Difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei,” J. Biochem., 309. 737-40. 1995.
[2]  Bitonti, A. J., Bacchi, C. J., McCann, P. P. and Sipertsma, A, “Intestinal changes caused by DL-α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase,” Biochem. Pharmacol., 34. 1773-78. 1985.View Article
[3]  McCann, P. P., Bbacchi, C. J. and Clarkson, A. B, “In vivo effects of α-DL-Difluoromethyl ornithine on the metabolism and morphology of Trypanosoma brucei,” Med. Biol., 59. 434-37. 1981. PubMed
[4]  Sjoerdsma, A, “Treatment of gambiense sleeping sickness in the Sudan with oral DFMO (DL-α-difluoromethylornithine), an inhibitor of ornithine decarboxylase,” Clin. Pharmacol. Ther., 30. 3-8. 1981.View Article  PubMed
[5]  Sjoerdsma, A. and Schechter, P, “Difiuoromethylornithine in the treatment of African trypanosomiasis,” Clin. Pharmacol. Ther., 35. 287-90. 1984.View Article  PubMed
[6]  Available: Term=eflornithine&Search_Button=Submit. [April 18, 1986].
[7]  Pepin, J., Guern, C., Milord, F. and Schechter, P. J, “Difluoromethylornithine for arseno-resistant trypanosoma brucei gambiense sleeping sickness,” The Lancet, 330. 1431-1433. 1987.View Article
[8]  Merali, S. and Clarkson, A. B. Jr, “Polyamine content of Pneumocystis carinii and response to the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine,” Antimicrob. Agents Chemother., 40. 973-978. 1996. PubMed
[9]  Balfour, J. A. and McClellan, K, “Topical eflornithine,” Am. J. Clin. Dermatol, 2(3). 197-201.View Article  PubMed
[10]  Cohen, J. L., Ko, R. J., Lo, A. T., Shields, M. D. and Gilman, T. M, “High-pressure liquid chromatographic analysis of eflornithine in serum,” J. Pharm. Sci., 78(2). 114-6. Feb. 1989.View Article  PubMed
[11]  Huebert, N. D., Schwartz, J. J. and Haegele, K. D, “ Analysis of 2-difluoromethyl-DL-ornithine in human plasma, cerebrospinal fluid and urine by cation-exchange high-performance liquid chromatography,” J. Chromatogr. A., 762(1-2). 293-8. Feb. 1997.View Article
[12]  Saravanan, C., Kumudhavalli, M. V., Kumar, M. and Jayakar, B, “ A new validated RP-HPLC method for estimation of eflornithine hydrochloride in tablet dosage form,” J. Phar. Res., 2. 1730-1731. 2009.
[13]  Kilkenny, M. L., Slavik, M., Christopher, M. R. and Stobaugh, J. F, “Plasma analysis of alpha-difluoromethylornithine using pre-column derivatization with naphthalene-2,3-dicarboxaldehyde/CN and multidimensional chromatography,” J. Pharm. Biomed. Anal., 17. 1205-1213. 1998.View Article
[14]  Jansson-Löfmark, R., Römsing, S., Albers, E. and Ashton, M, “Determination of eflornithine enantiomers in plasma by precolumn derivatization with o-phthalaldehyde-N-acetyl-l-cysteine and liquid chromatography with UV detection,” Biomed. Chromatogr., 24(7). 768-773. July 2010.View Article  PubMed
[15]  Malm, M. and Bergqvist, Y, “Determination of eflornithine enantiomers in plasma, by solid-phase extraction and liquid chromatography with evaporative light-scattering detection,” J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 846. 98-104. 2007.View Article  PubMed
[16]  Kumar, A., Venkatesh, Prasad, S. P., Mohan, S. and Kumar, P, “Spectrophotometric determination of eflornithine hydrochloride as active pharmaceutical ingredient using sodium 1,2-naphthoquinone-4-sulfonate as the derivative chromogenic reagent,” Trade Sci. Inc., 7, 2008.
[17]  Kumar, A., Venkatesh, Prasad, S. P., Mohan, S. and Singh, A. K, “Estimation of eflornithine hydrochloride by UV spectroscopy,” Trade Sci. Inc., 8. 2009.
[18]  Validation of Analytical Procedures, Methodology ICH Harmonised Tripartite Guideline, Having Reached Step 4 of the ICH Process at the ICH Steering Committee meeting on November 6, 1996.