| Popis: |
A hydrophilic liquid chromatography-tandem mass spectrometry method was developed and validated for determination of four macrolide antibiotics including erythromycin, spiramycin, tilmicosin and tylosin in honey samples in the mass fraction range from 0.50 to 50.0 µg kg-1. Parameters affecting extraction efficiency were examined during sample preparation, including the pH of extraction solvent as well as composition and volume of elution solvent. Furthermore, various chromatographic separation parameters (chromatographic column type, column temperature, mobile phase composition, flow rate) and mass spectrometric parameters (spray voltage, sheath gas pressure, capillary temperature), were optimized. Finally, macrolides were extracted from honey with PBS buffer 0.1 M, pH 8 and sample extracts were further cleaned up and analyte concentrated using solid-phase extraction on OASIS HLB columns. Separation was performed on a XBridge Amide column 3.5µm protected by a guard column using acetonitrile:water as mobile phase, (90:10, v/v) in the first segment and (70:30, v/v) in the second segment of run, at a flow rate of 0.3 mL min-1. Detection was achieved by triple quadrupole mass spectrometry using heated electrospray ionization interface. Depending on macrolide chemical structures, protonated molecules form single and/or double charged precursor ions in the positive ionization mode. The analysis under MS/MS was performed using multiple reactions monitoring of two characteristic ion transitions for each analyte and their ratios were key criteria of macrolides presence confirmation in incurred samples. Quantification was performed using matrix-matched standards with the use of roxithromycin as an internal standard. This approach provided efficiently correction of losses during sample preparation as well as the matrix effects. The developed and optimized method was validated by determination of following validation parameters: selectivity, linearity, precision, accuracy, recovery, limits of detection and quantification and stability of macrolide antibiotics in honey extract. The recoveries of macrolide antibiotics from honey spiked at 0.50, 5.0 and 50.0 µg kg-1 were within the range of 78.0 to 110.3%, intra-day precisions were ≤ 2.41%, inter-day precisions were ≤ 3.36% and accuracy was below 10%. The limit of detection and quantification was 0.20 µg kg-1 and 0.50 µg kg- 1 respectively for all studied macrolides. Through the analysis of different honey sample types regarding colour and floral origin the suitability of the developed method for purposes of determination food safety was confirmed. |
