Quantitative determination of ceftiofur in milk by liquid chromatography–electrospray mass spectrometry

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Abstract

A liquid chromatography–electrospray mass spectrometry (LC–ES-MS) was developed for the quantitation of ceftiofur in milk at the 50 ppb tolerance level set by the US Food and Drug Administration (FDA) for the drug. The method used ultrafiltration as a simple and rapid means to prepare the sample for analysis. A 100 μl volume of ultrafiltrate containing ceftiofur was concentrated on-column for LC–MS analysis. The LC separation was accomplished using an acetonitrile gradient with the ion-pair reagent heptafluorobutyric acid (HFBA). Propionic acid was added after the LC column to minimize electrospray signal suppression, enhancing the response for ceftiofur by a factor of 10. The transmission ions from the electrospray interface to the MS was enhanced by a factor of 7 by using a Rf ion guide. The development method could detect ceftiofur to 10 ppb and quantitate the antibiotic from 25–200 ppb (linear correlation coefficient of 0.993). The analysis indicated that bovine milk collected 32 h after dosing with ceftiofur was above the FDA tolerance of 50 ppb, while milk collected 48 h after dosing was found to contain 24–31 ppb of ceftiofur.

Introduction

A number of veterinary drugs which are frequently used to prevent infections, can contaminate meat and milk products destined for human consumption. Ceftiofur (Fig. 1) is a cephalosporin β-lactam antibiotic which is finding widely increased use in the treatment of bacterial infections. While residues of ceftiofur and its metabolites have been reported in bovine milk and serum 1, 2, 3, mass spectrometry (MS) methodology has not been developed for the confirmation of this antibiotic at the 50 ppb tolerance level set by US Food and Drug Administration (FDA) [4]. Based on the presence of a basic nitrogen on ceftiofur and its polar and thermally unstable properties, electrospray positive-ion detection (analyzing ceftiofur at low pH to protonate the molecule in solution) should be feasible 5, 6. Also, ceftiofur and other antibiotics from the cephalosporin class have been analyzed by positive-ion detection electrospray mass spectrometry (ES-MS) 7, 8, 9.

To achieve the ES-MS sensitivity necessary to detect residues of ceftiofur in milk, ES-MS required the use of an Rf ion guide to improve ion transport. The Rf ion guides offer advantages over electrostatic lens systems for transmissions of ions into the mass analyzer 10, 11, 12, 13, 14, 15. Rf ion guides involving quadrupole, hexapole and octapole configurations are now commonly used in the ion transport region of the atmospheric pressure ionization (API) interface for introduction of ions into a mass analyzer.

This paper presents the research into developing a quantitative confirmation methodology based on liquid chromatography–electrospray-mass spectrometry (LC–ES-MS) for ceftiofur in milk at the 25 ppb level.

Section snippets

Materials and reagents for LC–MS analysis

Water was purified with a Milli-Q water system (Millipore, Bedford, MA, USA) prior to use. Acetonitrile (ACN) was of LC–GC grade quality (Baxter Healthcare, Muskegon, MI, USA). The ion-pair reagent, heptafluorobutyric acid (HFBA) was obtained from Aldrich (Milwaukee, WI, USA). Ceftiofur hydrochloride standard was supplied by the Upjohn (Kalamazoo, MI, USA). The FDA milk samples collected 32 and 48 h after dosing with ceftiofur were supplied by the FDA Center of Veterinary Medicine, Beltsville,

Sample preparation

The development of the method took into account the need to handle large numbers of samples. For this reason the simple and rapid procedure of ultrafiltration using a 10 000 molecular-mass cutoff filter was chosen for sample clean-up. A 100-μl aliquot of the filtrate was concentrated on column. Ultrafiltration has been previously optimized in our laboratory for the extraction of β-lactams in milk [16]. This optimization demonstrated the need of the addition of an organic solvent to the milk to

Acknowledgements

This work was supported by the Food and Drug Administration under Cooperative Agreement Number FD-U-000589. Also, the authors thank both Hewlett-Packard and Analytica of Branford for technical assistance and instrumentation used in this work.

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