Development and validation of novel stability indicating methods for estimation of amylocaine hydrochloride in bulk and dosage form Full Paper INTRODUCTION Amylocaine HCl (AM), (1-(Dimethylaminomethyl)- 2-methylbutan-2-yl) benzoate hydrochloride (Figure 1) is a benzoic acid ester local anaesthetic used mainly for relief of painful anorectal conditions and has been included in oral mixtures for the relief of coughs. It blocks Hanan Ahmed Merey* 1 , Fahima A.Morsy 2 , Mohammed A.Mohammed 3 , Maissa Y.Salem 1 1 Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El Aini St. Cairo, 11562, (EGYPT) 2 National Organization for Drug Control and Research (NODCAR), 6 Abou Hazem Street, Pyramids, P.O. Box 29, (EGYPT) 3 Medical Union Pharmaceuticals, Abu Sultan, Ismailia, 7010, (EGYPT) E-mail: bibatofa@yahoo.com Received: 14 th July, 2012 ; Accepted: 14 th October, 2012 KEYWORDS Derivative ratio spectrophotometry; Spectro-densitometry; High performance liquid chromatography; Amylocaine HCl; Benzoic acid. ABSTRACT Three sensitive and selective methods were developed and validated as stability indicating methods for the determination of amylocaine HCl in presence of its degradation product. The first method is based on the use of derivative ratio spectrophotometry ( 1 DD) for the determination of amylocaine HCl in presence of its degradation product and of methyl and propyl parabens by measurement of the peak amplitudes of the first de- rivative of ratio spectra at 234 and 247 nm using the spectrum 20 g of total parabens as a divisor. The second method is a spectro-densitometric method for the determination of amylocaine HCl after separation from its degrada- tion product and additives of pharmaceutical dosage form using toluene: methanol: chloroform: 10% NH 3 (5:3:6:0.1 v/v) followed by detection at 234 nm. The third method is an isocratic high performance liquid chromato- graphic method (HPLC) on a reversed phase C 18 column using mobile phase consisting of distilled water: acetonitrile: triethylamine (530: 470: 0.1v/v) and the pH was adjusted to 3 by o-phosphoric acid. The proposed meth- ods were successfully applied for the analysis of amylocaine HCl in labo- ratory prepared mixtures and in pharmaceutical dosage form and the re- sults obtained were assessed by applying the standard addition tech- nique. Statistical comparison between the results obtained by applying the proposed methods and manufacturer's method for amylocaine HCl in its pure powder form was done and no significant difference was found at p = 0.05.  2013 Trade Science Inc. - INDIA Trade Science Inc. ACAIJ, 12(3) 2013 [103-110] An Indian Journal Volume 12 Issue 3 Analytical CHEMISTRY Analytical CHEMISTRY ISSN : 0974-7419 O O H 3 C CH 3 N CH 3 CH 3 HCl Figure 1: Chemical structure of amylocaine HCl. the initiation and conduction of nerve impulse by inhibiting