Food & Beverage Analysis An Improved Evaporative Sample Preparation Methodology
for Determining Nitrofuran Antibiotic Residues in Foodstuffs A. Kaufmann; K. Maden and S. Walker
Nitrofuran antibiotics were banned from use in the European Union [EU] in 1995 due to concerns that their residues were carcinogenic. In 2002/3 the EU introduced a stringent testing regimen which calls for the use of highly sensitive methods to test food stuffs, principally meat, fi sh & shellfi sh, for the presence of this class of antibiotics. The Minimum Required Performance Limit [MRPL] laid down by the EU directive is 1mg per kg for edible tissues, and is enforced on all products whether produced locally or imported into the EU. Many papers detail methods and identify metabolites and derivatives of the drugs concerned and are listed by Vass, Hruska & Franek (2008). The analytical method calls for good upstream sample preparation to eliminate the effects of the matrix, and can be manual and time consuming, particularly where evaporation is concerned. This article describes operational benefi ts including workfl ow improvements gained by the offi cial food control authority of the canton of Zurich (Kantonales Labor Zurich) or KLZH during improvement of their upstream sample preparation methodology.
Sample Preparation Methodology Of the methods cited in the literature for upstream sample preparation, many
laboratories favour physico-chemical assays with chromatographic separation and mass spectrometry [MS] detection of metabolites. The general scheme for preparation and analysis of nitrofurans in foodstuffs at KLZH is as follows, and summarised in Figure 1:
1. Homogenisation of tissue sample 2. Acid hydrolysis to release tissue bound metabolites 3. Derivatisation of metabolites with orthonitrobenzaldehyde to increase molar mass and increase sensitivity of detection
4. Liquid:liquid extaction with ethyl acetate 5. Evaporation to dryness 6. Resuspension with water
7. Clean-up with Solid Phase Extraction to eliminate interfering matrix elements, such as lipids
8. Elution with ammoniacal methanol 9. Evaporation to dryness 10. Resuspension in a known small volume of solvent suitable for chromatography
11. Separation via ultra high performance liquid chromatography [UHPLC] and analysis with MS/MS
1 Sample
Preparation Methodology
Homogenise tissue 5 4 3 Acid hydrolysis 2 Traditional Evaporation Technique
At KLZH the evaporation part of the sample preparation method has traditionally relied upon use of a rotary evaporator. The rotary evaporator method for Nitrofuran testing was proven to give good recoveries. However it has a number of operational drawbacks. The biggest disadvantage was that the rotary evaporator is a single sample system which requires continuous monitoring to control the process and to ensure that no foaming or bumping occurs. Samples for Nitrofuran typically presented to KLZH in batches of 5 to 30. Where a small batch is managed in an assessable amount of time with a rotary evaporator, bigger batches would soon become impossible to process. Therefore, three batch process evaporators were evaluated in order to improve the productivity of this step.
New Evaporative Technologies Evaluated
Blow-Down Evaporation In these evaporator systems, an inert gas such as nitrogen is blown down through needles onto the samples in tubes to create a fl ow over the liquid surface. This alters the equilibrium between the vapour and liquid phases to favour the vapour phase. Heat is normally applied to the samples to hasten evaporation. Therefore the samples are hot during the process, being at the temperature of the heating block or bath, and consequently the technique offers poor recovery of volatile analytes. Although blow down evaporation is relatively fast for volatile solvents, it can be slow for solvents with high boiling points or those that are diffi cult to
evaporate such as water. Blow down evaporation requires continuous monitoring by the user to detect the end point of the drying process and to optimise the position of the gas jets, keeping them close to the liquid surface.
Evaporate to dryness 6 7
Liquid:liquid extraction with ethyl acetate
Derivatise with ortho-nitrobenzaldehyde 8 Vortex Evaporation Resuspend with water 11 10 Load on to SPE for clean-up 9 Elute with ammoniacal methanol Separation & analysis by UHPLC-MS-MS Resuspend in a small volume Figure 1. Sample preparation & analysis methodology
The most time consuming and labour intensive parts of this methodology are the extraction and the two evaporation-to-dryness steps.
Evaporate to dryness
These systems boil batches of samples under vacuum and therefore the samples are cold throughout evaporation, while swirling the sample tubes to create a vortex. The vortex created generates a large sample surface area for evaporation, making the process relatively fast. However, the resultant dried product is spread across the vessel walls, which can make sample recovery more diffi cult. Moreover, in contrast to centrifugal concentrators, the swirling movement generates insuffi cient g force to prevent solvent bumping and tends to aid foam formation. Hence vortex evaporators are prone to sample loss and cross contamination. Controls on the system can permit hands free operation once the vigour of the vortexing action has been correctly set.
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