How to deal with sweet matrix? – a method for LC-MS/MS analysis of antibiotics in honey, regardless of its kind

Julia Mironenka¹, Rafał Szewczyk^1,2, Katarzyna Krupczyńska-Stopa^1,2, Maciej Stopa ^1,2 

LabExperts sp. z o. o., Gdańsk, Poland; 2. Bioanalytic sp. z o. o., Gdańsk, Poland;

Introduction

Antibiotics are commonly used in therapy of infectious diseases. Their overuse to increase animal productivity, including honey production, has become a serious problem in food safety regulations. High doses of antibiotics used by farms, find their way into the end products that enter human consumption. Honey is a valuable product, often used in medicine and as a dietary ingredient. All the sweetness of honey disappears when it reaches the laboratory as a matrix for LC-MS/MS analysis, due to the great diversity of components content between each, or even the same kind of honey. In this work we have focused on the development of highly reproducible and precise method of 57 antibiotics determination by LC-MS/MS in honey, regardless of its origin.

Methods

Sample preparation is based on modified QuEChERS method. The honey samples after enrichment with known concentrations of analytes were hydrated using acidified water, heated and sonicated until sugar dissolved. Next, after mixing with acidified acetonitrile solution, samples were desalted using ready to use QuEChERS sachets and filtered with PVDF syringe filter.  Before the LC-MS/MS analysis samples were diluted with water. Data were acquired on QTRAP 5500+ (SCIEX) mass spectrometer coupled with ExionAC LC (SCIEX) and processed with SciexOS 2.2 software. The LC-MS/MS analysis was performed in positive ionization sMRM mode during reversed phase 12-min separation on Fortis H₂O C18 chromatographic column. Mobile phases consisted of water, acetonitrile and formic acid additive.

Bioanalytic: Fig 1. Sample preparation procedure.

Preliminary data

Honey is a complex matrix that consists of over 180 components. Depending on the origin, type and bees, the ingredients are present in different amounts, which undoubtedly affects its properties. Developing a repeatable extraction method for one compound becomes a challenge. In the present work a multimethod for 57 analytes from 10 different classes of antibiotics was developed. The optimization of the sample preparation method consisted of: selection of solvents and pH of the solutions, concentration of additives, time of sonication and extraction, dissolution ratio and method of cleaning the sample before LC-MS/MS analysis. The intensity and shape of signals of samples subjected to evaporation under nitrogen concentration and dissolved in the initial phase of the chromatographic method were compared to samples analyzed immediately after extraction with different water dilutions. The present method allows sample preparation without time-consuming stage of evaporation followed by residues dissolving.

The efforts devoted to method development resulted in obtaining averaged recovery of tetracyclkines, cefalosporins, sulfonamides and fluorochinones was found to be 106.4%. The linearity (R<0.995) covered wide concentration range 0.5-500 µg/kg. LLOQ for most macrolides (tylosin, spiramycin, tilmicosin) and tetracyclines (doxycycline, chlorotetracycline, 4-epi-chlorotetracycline) was 0.5 µg/kg, for some of beta-lactams (nafcillin, cloxacillin, oxacyllin) was 0.1 µg/kg and for some sulfonamides (sulfadimethoxine, sulfachinoxaline, sulfchloropyrazine) reached 0.05 µg/kg. Thanks to a laborious optimization process excellent repeatability for 10 different honeys obtained from local beekeepers was achieved. The accuracy and precision for the whole set of honeys was lower than 20% in the working range of the assay.

Bioanalytic: Table 1. The results of method validation.

Bioanalytic: Table 2. Ion source parameters; Table 3. sMRM scan parameters; Table 4. General LC-MS parameters.

Novel aspect

The effective analytical procedure for the determination of 57 antibiotics residues from different honey samples in one LC-MS/MS run.