Method development for the determination of antibiotics in milk samples using an on-line solid phase extraction UHPLC-MS/MS

Friday, October 9, 2015

Authors: Dr. Béla Kmellár, Judit Susán


1. Summary

Our objective was to develop a UHPLC-MS/MS method for the measurement of the most common antibiotics under regulation in milk, with which the time-consuming solid phase extraction sample preparation can be automated, and the sample can be enriched. This helps to achieve lower limits of detection, compared to off-line sample preparation. During method development, a routine method was developed that can be beneficial for any food testing laboratory with its excellent performance characteristics and low limits of detection. Its cost efficiency can also be noteworthy, because both time, equipment and chemical costs can be reduced by it.


2. Introduction

Analysis of trace contaminants in foods has been gaining ground in the last decades with the development of analytical techniques. The allowable amount of antibiotics in foods of animal origin is strictly regulated by the European Union [1]: MRL values are determined that can vary by matrix, as well as by compound. The list is updated from time to time: new antibiotics are covered by the regulation or the limit value of an already regulated one is altered, therefore, the relevant modification is always the latest one. It is the task of food testing laboratories to measure these trace quantities. The authority laboratory then gets involved in the food chain, depending on the results, since concentrations of antibiotics residues exceeding limit values are considered a food safety issue. Results are communicated to the customer by service laboratories. Because of the low limit values, low concentrations have to be measured reliably by the analytical laboratory, which requires sensitive and selective techniques.


From a cost efficiency point of view, it is an important objective to determine as many compounds as possible in one analysis, which is limited by the often very diverse physico-chemical properties of the components. There are several methods in the literature for the determination of individual groups of antibiotics in products of animal origin [2, 3], however, multi-methods capable of determining more than one group at a time are more uncommon.


One of the critical points of the analysis is sample preparation: target compounds have to be separated from the sample using some kind of extraction method and, at the same time, the presence of the matrix have to be reduced to a minimum as much as possible. A frequently used procedure for sample preparation is solid phase extraction (SPE), during which a given volume of the extract is passed through a sorbent layer. Components that are valuable for us are bound by the sorbent, while the interfering matrix is not, and so the latter will not be there on the column during solvent desorption and will not be present in the eluate. Laboratory time demand of the SPE technique is quite large, it can be performed manually (even on several samples in parallel). There was already an attempt to automate SPE, but it tried to automate the individual steps and not to use trapping, and a separate instrument, coupled to the HPLC, was needed [5].

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