Liquid Chromatographic Separation of Isoniazid, Pyrazinamide and Rifampicin on a Reversed- Phase Silica Column

G N Thoithi; I O Kibwage; O K King'ondu; J Hoogmartens

G N Thoithi Department of Pharmaceutical Chemistry, University of Nairobi
I O Kibwage Department of Pharmaceutical Chemistry, University of Nairobi
O K King'ondu Department of Pharmaceutical Chemistry, University of Nairobi
J Hoogmartens Laboratorium voor Farmaceutische Chemie en Analyse van Geneesmiddelen, Faculteit Farmaceutische Wetenschappen, Katholieke Universiteit Leuven. E. Van Evenstraat 4, 3000 Leuven. Belgium.

Keywords: Reversed-phase chromatography, gradient, isoniazid, pyrazinamide, rifampicin, gradient.

Abstract

A gradient liquid chromatographic method which can separate isoniazid,
pyrazinamide and rifampicin is described. A Hypersil Cis, 5 pm, 250 mm x 4.6
mm internal diameter column was maintained at 40°C. The method was
developed by systematic evaluation of the influence of the buffer concentration,
column temperature and the mobile phase pH. The method proposed uses
isocratic elution with potassium phosphate buffer (pH 6.0; 0.05 M) for 10 min,
followed by linear gradient to potassium phosphate buffer (pH 6.0; 0.05 M)-
methanol (40:60, v/v) in 5 min, isocratic elution at the same composition for a
further 15 min and then linear gradient back to potassium phosphate buffer (pH
6.0; 0.05 M) in 5 min. The flow-rate was 1 mllmin and UV detection was at 254
nm. The method was validated and it has been used for routine analysis of
tablets containing isoniazid, pyrazinamide and rifampicin. Analysis time is 35
minutes.