Sample preparation for residue determination of gentamicin and neomycin by liquid chromatography

Abstract

The effect of sample preparation on the determination of gentamicin and neomycin residues in animal tissues was investigated. The extract was mixed with an ion-pair reagent and applied to an octadecyl cartridge. The cartridges were washed with buffer followed by water, and analytes were eluted with ion-pair buffer–acetonitrile mixture. The aminoglycosides were derivatized with 9-fluorenylmethyl chloroformate prior to liquid chromatography using a reversed-phase column and fluorescence detection. Under the conditions applied neomycin was fully separated from all the gentamicin compounds. The highest recoveries of gentamicin and neomycin from spiked tissues were obtained using trichloroacetic acid after initial extraction with phosphate-buffered saline. No interfering peaks from endogenous compounds of matrix were noted at the elution position of the analytes. An intra-laboratory validation of the whole procedure was performed. The calibration graphs were linear from 0.1 to 1.0 mg/kg for gentamicin, and from 0.2 to 1.0 mg/kg for neomycin. Limits of detection were 0.05 mg/kg and 0.10 mg/kg for gentamicin and neomycin, respectively. Limits of quantitation for gentamicin and neomycin were 0.1 and 0.20 mg/kg muscle, liver or kidney tissue, respectively. Recoveries of gentamicin spiked at levels of 0.1 mg/kg porcine tissues ranged from 76 to 86%. Recoveries of neomycin spiked at levels of 0.2 mg/kg porcine tissues ranged from 77 to 83%. The validated procedure was used to determine gentamicin concentrations in porcine tissue after dosing with gentamicin at a level of 5 mg/kg body mass.

Introduction

Gentamicin and neomycin are aminoglycoside antibiotics used to treat bacterial infections in animals because of their good spectrum of activity against gram-negative bacteria. Aminoglycosides are rapidly absorbed from an injection site but poorly absorbed after oral administration. They are not inactivated in the intestine and are eliminated quantitatively in feces. Gentamicin and neomycin, systemic compounds, are excreted almost entirely as parent compound. Aminoglycosides bind to tissue proteins and macromolecules via ionic bonds, but binding to plasma proteins is low (<25%). Gentamicin and neomycin in tissues are usually found in low concentrations, except in the renal cortex where they tend to concentrate. There is a human health risk associated with the use of gentamicin and neomycin, since they have both been found to cause damage to the cranial nerves, resulting in hearing loss [1], [2]. To keep harmful aminoglycoside residues out of the human food chain, international authorities require long withdrawal periods before dosed animals may be slaughtered. The maximum residue levels (MRLs) recommended by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) are 0.50–10.0 mg neomycin/kg, and 0.10–5.0 mg gentamicin/kg, depending on the different tissues [2]. The European Union [3] has fixed the MRLs in edible animal products for gentamicin from 0.1 to 1.0 mg/kg. In the case of neomycin the MRLs were set from 0.5 to 5.0 mg/kg. Regulatory methods for gentamicin and neomycin that could perform at these concentrations were needed, but development of such a method required overcoming challenges in sample preparation and analysis of drug content.

Chromatographic methods are capable of simultaneous determination of gentamicin and neomycin in animal tissues. However, separations are difficult to achieve because of the structural similarity of aminoglycoside moieties and some endogenous compounds. Additionally, gentamicin and neomycin have no UV or visible absorption and most procedures have to use pre- or post-column derivatization. The derivative reaction with o-phthaldialdehyde (OPA) [1], [4], [5], dansyl chloride [6], fluorescamine [7] or 9-fluorenylmethyl chloroformate (FMOC-Cl) [8], [9], [10] is the most desirable because fluorescence detection generally provides better sensitivity and selectivity than UV absorption.

The purpose of this study was to develop a simple and rapid method for the extraction of gentamicin and neomycin from tissue samples and a solid-phase extraction (SPE) as a clean-up procedure, which would yield a very high rate of analyte recovery and clean sample extracts for analysis by high-performance liquid chromatography (HPLC).