Determination of pyridoxal isonicotinoyl hydrazone, cefoperozone, and ampicillin in biological fluids by high-performance liquid chromatography

Analytical methods for the quantitation measurement of the concentrations of pyridoxal isonicotinoyl hydrazone (PIH), cefoperazone (CPZ), and ampicillin (AMP) were developed with the goal of implementation in pharmacokinetic studies. The assays were accomplished by reversed phase high performance liquid chromatography (HPLC), after suitable sample preparation. All the methods achieved separation using isocratic conditions, with absorbance detection as the means of quantitation. The quality of the chromatography was monitored through the determination of chromatographic figures of merit.;The antibiotic assays, AMP and CPZ, included matched internal standards to simplify sample preparation and quantitation. The recovery of drugs and internal standards from serum, and linearity of the methods were determined. The chromatographic retentions of other antibiotics were ascertained, and did not coelute with either analyte or internal standard.;PIH is an orphan drug proposed for the chelation treatment of iron overload conditions arising from transfusion therapy for an underlying anemia. Chromatography of standard solutions of PIH required an end-capped reversed phase column and eluent modifiers to produce well resolved peaks with precision. Recovery studies from biological fluids indicated alternative modes of drug disposition from those usually associated with drug metabolism. Evidence was obtained that indicated reaction of PIH with protein and non-protein fractions of serum. It was felt that a suitable internal standard could not be established at this time, quantitation was achieved through comparison to an external standard.;PIH was demonstrated to be present in the urine of subjects taking PIH orally, a pathway for elimination that was previously undescribed.