Development and validation of HPLC-DAD method for the simultaneous determination of amoxicillin, metronidazole and rabeprazole sodium. Application to spiked simulated intestinal fluid samples.

This work deals with the development, validation and application of an HPLC-DAD method for the determination of a ternary mixture containing amoxicillin (AX), metronidazole (MZ) and the proton pump inhibitor rabeprazole sodium (RB). This triple therapy is used for treatment of Helicobacter pylori infection. Effective chromatographic separation between the three drugs was achieved using Thermo Hypersil BDS-C8 (4.6×250mm, 5µm particle size) column and a mobile phase composed of phosphate buffer pH 7 and acetonitrile (70: 30, by volume). The mobile phase was pumped isocratically at a flow rate of 1 mL/min. Quantification of the analytes was based on measuring their peak areas at 230nm for both AX and RB, and at 319nm for MZ. AX, MZ and RB eluted at retention times 2.36, 3.55 and 8.72min respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness, detection and quantification limits. The linear dynamic ranges were 25-250, 25-250 and 5-50µg/mL for AX, MZ and RB respectively with correlation coefficients>0.9998. The validated method was successfully applied to the analysis of several laboratory-prepared mixtures as well as simulated intestinal fluid samples spiked with the three drugs.

Sensitive spectrofluorimetric and spectrophotometric methods for the determination of thonzylamine hydrochloride in pharmaceutical preparations based on coupling with dimethylbarbituric acid in presence of dicyclohexylcarbodiimide.

Two sensitive and selective spectrophotometric and spectrofluoimetric procedures were developed for the determination of thonzylamine hydrochloride (THAH) in tablets and nasal drops. The methods are based on König reaction which resulted in an orange-yellow fluorescent product. The orange-yellow product of the interaction between the dicyclohexylcarbodiimide (DCC), THAH and dimethylbarbituric acid (DMBA) showed an absorption maximum at 492 nm, a first-derivative signal at 494 nm and a fluorescence emission peak at 518 nm (lambda(ex)=492 nm). The orange-yellow color was found to be stable for at least 2 h. The reaction conditions were studied and optimized. The reaction obeys Beer's law over the ranges 8-20 and 0.2-2.0 microg ml(-1) for the derivative spectrophotometric and fluorimetric measurements, respectively. The detection limits were found to be 0.29 and 0.018 microg ml(-1) for the spectrophotometric and fluorimetric measurements, respectively. The proposed methods were applied to the analysis of pharmaceutical formulations containing THAH, either alone or in combination with naphazoline nitrate.

Application of H-point standard additions method to spectrophotometric and spectrofluorimetric determinations of glafenine and glafenic acid in mixtures.

H-point standard additions method (HPSAM), based on spectrophotometric and spectrofluorimetric measurements, was proposed for simultaneous determination of glafenine (G) and glafenic acid (GA). A study of the absorption spectra of G and GA in various pH media has been carried out. Reasonably resolved UV-absorption spectra were obtained with a solution adjusted at pH 4.5 with citric acid-phosphate buffer. Additionally, the fluorescence properties in aqueous micellar systems of anionic, cationic and non-ionic surfactants were investigated. Well resolved fluorescence spectra were established in aqueous Triton X-100 solution at pH 7.8 (citric acid-phosphate buffer). As a comparative method, UV-derivative spectrophotometry (based on zero-crossing technique) was suggested. First-derivative value at 352 nm ((1)D(352)) and second-derivative value at 366 nm ((2)D(366)) were selected for the quantification of G and GA, respectively. The relative standard deviations of the proposed methods approximate 2%. The proposed methods were evaluated through the analysis of commercial tablets. The results were accurate and precise.

Voltammetric, spectrofluorimetric and spectrophotometric methods to determine flufenamic acid.

Simple, rapid and sensitive voltammetric, spectrofluorimetric and spectrophotometric methods for determination of flufenamic acid (FF) in bulk powder and capsule dosage form are presented. The methods are based on the cyclisation reaction of FF with concentrated sulphuric acid to produce the corresponding acridone derivative. The voltammetric method is based on the adsorptive stripping differential pulse (DP) technique. The acridone derivative is determined over the concentration range of 8-60 ng ml(-1) using adsorptive preconcentration at the hanging mercury drop electrode (HMDE). The lower detection limit was found to be 1.02 ng ml(-1). The fluorimetric and spectrophotometric methods are based on the measurement of the fluorescence intensity at 450 nm (lambda(ex) = 400 nm)and peak-to-peak measurements of the first- (D1) and second-derivative (D2) curves, respectively. Beer's law is obeyed over the concentration ranges of 2-20 ng ml(-1) and 0.2-8.0 microg ml(-1) for the fluorimetric and spectrophotometric measurements, respectively. The three methods were proved to be accurate and reproducible as indicated by a relative standard deviation of <2%.

Adsorptive stripping voltammetric assay of phenazopyridine hydrochloride in biological fluids and pharmaceutical preparations.

A sensitive method for the measurement of phenazopyridine hydrochloride (PAP) by differential pulse polarography (DPP) based on adsorptive stripping technique, using a hanging mercury drop electrode (HMDE) is described. The voltammetric peak is obtained at -0.760 V, which corresponds to the reduction of the azo group in Britton-Robinson buffer. The redox behaviour is reversible. Optimum conditions were found to be: accumulation potential -50 mV (vs. Ag/AgCl), accumulation time 60 s, scan rate 5 mV s(-1), pulse amplitude -100 mV and supporting electrolyte Britton-Robinson buffer (0.04 M, pH=11). The relative standard deviation (at 20 ng ml(-1) level) was +/-0.6% for six measurements. The calculated detection limit was 0.0299 ng ml(-1) with a 60-s accumulation time. The applicability of such a method was evaluated through the assay of PAP in human plasma and urine samples after a simple extraction procedure and in pharmaceutical preparation. The mean recovery was 97+/-2 (100 ng ml(-1) plasma).

Spectrophotometric determination of benzene in ethanol.

A rapid, simple spectrophotometric method for the detection and determination of benzene in ethanol is described. Thus, using 1 cm cuvettes and applying the first and/or the second derivative methods, benzene can be determined in ethanol over the concentration range of 5-25 p.p.m. with a CV% less than 1% and with a detection limit of 2 p.p.m. This is considered to be an improvement over the method currently given in the British Pharmacopoeia.