Quantitative trace analysis of a broad range of antiviral drugs in poultry muscle using column-switch liquid chromatography coupled to tandem mass spectrometry

Abstract

A liquid chromatography-tandem mass spectrometry method for the analysis of seven antiviral drugs, zanamivir, ribavirin, oseltamivir, oseltamivir carboxylate, amantadine, rimantadine and arbidol, in poultry muscle is reported. The antiviral drugs were extracted from the homogenized poultry muscle sample using methanol. The extract was purified using tandem solid-phase extraction combining a cation exchange cartridge and a phenylboronic acid cartridge. To prevent excessive matrix effects, the analytes were separated from the matrix constituents using a column-switch liquid chromatography system combining a reversed-phase and a Hypercarb analytical column. Detection was carried out using tandem mass spectrometry. The method was fully validated according to 2002/657/EC [1] and proved to be adequate for quantification and confirmation of zanamivir and ribavirin at 10 μg kg(-1), oseltamivir, oseltamivir carboxylate, amantadine and rimantadine at levels below 1.0 μg kg(-1) and for qualitative confirmatory analysis of arbidol at levels below 1 μg kg(-1).

Fig. 1

Chemical structures of a zanamivir, b ribavirin, c oseltamivir, d oseltamivir carboxylate, e rimantadine, f amantadine and g arbidol

Fig. 2

Schematic representation of (a) the dual SPE procedure and (b) the LC procedure. Z zanamivir, RB ribavirin, O oseltamivir, OC oseltamivir carboxylate, AM amantadine, RM rimantadine, AB arbidol

Fig. 3

Matrix effects (signal MMRS/signal standard) of the antiviral drugs when using zic-HILIC (light grey), C₁₈ (medium grey), Hypercarb (dark grey) and the column-switch set-up (black). 100% corresponds to no matrix effects

Fig. 4

Relative amount of antiviral drugs in the breakthrough during application of a 50 μg kg⁻¹ spiked poultry muscle sample (light grey), the breakthrough during washing (dark grey) and the elution (black) of (a) the Strata-X C and (b) the PBA SPE cartridge

Fig. 5

(a) Matrix effect (signal MMRS/signal standard) obtained for the antiviral drugs after the clean-up procedure when using RP C₁₈ (light grey) and the column-switch set-up (dark grey) as the chromatographic system (n = 5) and (b) the recovery of the sample clean-up procedure (n = 5)

Fig. 6

SRM chromatograms of (a) blank poultry muscle sample spiked with zanamivir and ribavirin at 10 μg kg⁻¹ including the internal standards and (b) blank poultry muscle sample spiked with the internal standards only

Fig. 7

SRM chromatograms of (a) blank poultry muscle sample spiked with oseltamivir, oseltamivir carboxylate, rimantadine, amantadine and arbidol at 2.5 μg kg⁻¹ including the internal standards and (b) blank poultry muscle sample spiked with the internal standards only