Flow injection spectrophotometric determination of amoxicillin in pharmaceutical samples by coupling with diazotized p- nitroaniline

A spectrophotometric method using flow injection analysis (FIA) is described for the determination of amoxicillin (AMX). Its detection limit, linearity and reproducibility were examined.The automated method is based on a coupling reaction between AMX and diazotized paranitroaniline (DPNA) in alkaline medium to form an intense reddishorange, water-soluble dye that is stable and has a maximum absorption at 478 nm.The calibration graph was linear over the concentration range of 0.5-100 μg.ml with a detection limit of 0.104 μg.ml and sample throughput of 60 h. The proposed method was successfully applied to the determination of AMX in injections and capsules. : Introduction Amoxicillin (AMX), 6-(p-hydroxy-α-amino phenyl acetoamido) penicillanic acid, is the p-hydroxy analogue of ampicillin but possesses some significant advantage over ampicillin, include more complete gastrointestinal absorption, and littile or no effect on absorption of food (1).The British pharmacopaeia recommended a liquid chromatography (LC) and spectrophotometric (using imidazol-mercury reagent) methods for the determination of AMX in raw material and dosage forms respectively (2). The literature reported several analytical procedures for the determination of AMX in pure form or in pharmaceutical formulations as well as in biological fluids. It included spectrophotometric (3; 4), polarographic (5; 6), flourimetric (7; 8), flow injection analysis (9) and HPLC methods (10; 11). Recently a flow injection spectrophotometric method is developed for the determination of AMX in capsules and injections based on using N,Ndimethyl-p-phenylenediamine and potassium hexacyanoferrate (III) in alkaline medium to form a blue soluble product that can be measured at λ max of 660 nm (12). Various FIA-spectrophotometry methods based on diazotization and * Part of M.Sc. thesis for the second author Iraqi. J. Mark. Rec. Cons. Protection. Vol. 1. No. 1. Sep. 2009 ١٠٦ coupling reactions have been developed for the determination of drugs such as metronidazole (13), sulphonamide (14), acetaminophene and dopamine (15). The purpose of the present investigation is to develop a simple and sensitive method for the determination of AMX in pharmaceutical preparations using diazotization coupling reaction and FIA-spectrophotometer. The proposed method is based on a coupling reaction between AMX and DPNA in alkaline medium (16) to form an intense reddish-orange color product which shows an absorption maximum at 478 nm. Reaction mechanism of the method: Aِmoxicillin forms a reddish-orange colored product (λ max at 478 nm) with DPNA in alkaline medium. Due to the phenolic nature of AMX, it can readily be coupled with a chromogenic reagent (DPNA) according to scheme 1. The reddish-orange dye product was only formed in alkaline medium (sodium hydroxide) since AMX is converted into its salt (phenoxide ion). The later is more stable than phenol ( resonance) leading to a more stable intermediate with DPNA (17). Scheme 1: Proposed mechanism of the reaction between AMX and DPNA NO 2 NH 2 N aN O 2 / HC l NO 2