LC-LTQ-Orbitrap sweetly detects antimicrobial residues in honey

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  • Published: Jul 14, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: LC-LTQ-Orbitrap sweetly detects antimicrobial residues in honey

Antimicrobial residues in honey need accurate measurement

Various antimicrobial compounds can be used in beekeeping, but careful measurement of any residues of these agents in honey is necessary.

Some bee diseases can be combatted by antimicrobial compounds, but these may leave residues in the honey produced from treated hives. No official maximum limits have been set in the EU for antimicrobials, so no detectable residues are permitted. Other jurisdictions, such as Canada, have set maximum levels for various antimicrobial agents. The accurate determination of the levels of these residues in honey is therefore necessary. The researchers from Beirut and Fougères wanted to upgrade a validated method for residue determination of microbial compounds using basic LC-MS/MS to a new one using LC-Linear Ion Trap/Orbitrap (LC-LTQ-Orbitrap). The latter is a high resolution mass spectrometry method, in which ions are separated by a linear ion trap and then by an Orbitrap (where ions are separated between an outer electrode and an inner, spindle-shaped electrode). The LC-LTQ-Orbitrap method enables the simultaneous evaluation of a wide range of targets, while also being able to give accurate mass values for novel ‘unknowns’, which in many cases will enable their identification. The aim of the work was to transfer the method from the old to the new system, without having to undergo a completely new validation process.

New LC-LTQ-Orbitrap method compared with old LC-MS/MS method

Honey samples were spiked with an internal standard solution consisting of seven antimicrobial compounds, two of which were deuterated and two labelled with 13C. The honey was taken up in water and acidified methanol was added. The pH was adjusted to pH 2 using Na4EDTA (tetrasodium ethylenediaminetetraacetic acid). The mixture was centrifuged and solids removed. Primary secondary amine (PSA) sorbent was used to remove interfering species, the solid being removed by centrifugation after use.

HPLC for the initial LC-MS/MS method was carried out on an Agilent 1200 instrument fitted with an Agilent Zorbax SB-C18 column. A three-solvent gradient system was used, with the following eluting liquids: 100 mMol aqueous HFBA (heptafluorobutyric acid), kept at 10% by volume; acetonitrile, run from 5 to 80% in a series of gradients; and pure water, run from 85 to 10%. The MS/MS method employed an Agilent 6410 with positive electrospray ionisation (ESI) and multiple reaction monitoring (MRM).

HPLC for the new LC-LTQ-Orbitrap method was carried out with a Thermo Fisher Accela system using the same conditions as previously. A Thermo Fisher LTQ-Orbitrap instrument was employed, using positive electrospray ionisation.

The transfer protocol involved spiking honey with 19 microbial compounds at three different levels, with six replicates for each level, and then comparing the two analytical methods. Decision limits (CCα) and detection capabilities (CCβ) were tabulated for the 19 compounds using the two methods. The methods gave similar results, but the LC-LTQ-Orbitrap CCα values were typically about 1.3 times higher than those for LC-MS/MS. The recoveries from spiked samples were largely in line with EU regulations, the LC-LTQ-Orbitrap method giving adequate results for 14 out of the 19 compounds examined. The authors noted that improved LC-LTQ-Orbitrap instruments have become available since the purchase of their instrument, which was a 2008 model.

The new method was tested on a variety of real honey samples from various regions of Lebanon. All the residues detected by the LC-MS/MS method were also detected by the LC-LTQ-Orbitrap technique.

New Orbitrap method suitable for honey analysis

The authors concluded that the transfer of the method to the new LC-LTQ-Orbitrap protocol could be accepted. However, it should be noted that the overall performance of the new method tended to be slightly inferior to the old LC-MS/MS method. The LC-LTQ-Orbitrap technique can be more readily extended to other residues, so offers greater flexibility. It will be interesting to see how the latest LC-LTQ-Orbitrap model performs in these tests.

Related Links

Rapid Communications in Mass Spectrometry 2017, 31, 1103-1110. El Hawari et al. Design for the transfer of a validated liquid chromatography/tandem mass spectrometry analytical method for the determination of antimicrobial residues in honey from low-resolution to high-resolution mass spectrometry.

Food Additives & Contaminants: Part A 2017, 34, 582-597. El Hawari et al. Development and validation of a multiclass method for the determination of antibiotic residues in honey using liquid chromatography-tandem mass spectrometry.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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