ANNEX IIU.K.SAMPLE PREPARATION AND CRITERIA FOR METHODS OF ANALYSIS USED IN OFFICIAL CHECKING OF THE LEVELS OF FUSARIUM TOXINS IN CERTAIN FOODSTUFFS
1.PrecautionsU.K.
As the distribution of Fusarium toxins is non-homogeneous, samples shall be prepared, and especially homogenised, with extreme care.
All the material received by the laboratory must be used for the preparation of test material.
2.Treatment of the sample as received in the laboratoryU.K.
Each laboratory sample must be finely grinded and mixed thoroughly using a process that has been demonstrated to achieve complete homogenisation.
In case the maximum level applies to the dry matter, the dry matter content of the product shall be determined on a part of the homogenised sample, using a procedure that has been demonstrated to determine accurately the dry matter content.
3.Subdivision of samples for enforcement and defence purposesU.K.
The replicate samples for enforcement, trade (defence) and referee purposes shall be taken from the homogenised material unless such procedure conflicts with Member States’ rules on sampling.
4.Method of analysis to be used by the laboratory and laboratory control requirementsU.K.
4.1.DefinitionsU.K.
A number of the most commonly used definitions that the laboratory shall be required to use are the following:
The most commonly quoted precision parameters are repeatability and reproducibility.
=
Repeatability, the value below which the absolute difference between two single test results obtained under repeatability conditions, namely same sample, same operator, same apparatus, same laboratory, and short interval of time may be expected to lie within a specific probability (typically 95 %) and hence r = 2,8 × sr
=
Standard deviation, calculated from results generated under repeatability conditions
=
Reproducibility, the value below which the absolute difference between single test results obtained under reproducibility conditions, namely on identical material obtained by operators in different laboratories, using the standardised test method may be expected to lie within a certain probability (typically 95 %); R = 2,8 × sR
=
Standard deviation, calculated from results under reproducibility conditions
=
Relative standard deviation calculated from results generated under reproducibility conditions 
4.2.General requirementsU.K.
Methods of analysis used for food control purposes must comply with the provisions of items 1 and 2 of the Annex to Directive 85/591/EEC.
4.3.Specific requirementsU.K.
4.3.1.Performance CriteriaU.K.
Where no specific methods for the determination of Fusarium toxins levels in foodstuffs are required by Community legislation, laboratories may select any method provided the selected method meets the following criteria:
Performance characteristics for deoxynivalenol
| Level μg/kg | Deoxynivalenol | ||
|---|---|---|---|
| RSDr % | RSDR % | Recovery % | |
| > 100-≤ 500 | ≤ 20 | ≤ 40 | 60 to 110 |
| > 500 | ≤ 20 | ≤ 40 | 70 to 120 |
Performance characteristics for zearalenone
| Level μg/kg | Zearalenone | ||
|---|---|---|---|
| RSDr % | RSDR % | Recovery % | |
| ≤ 50 | ≤ 40 | ≤ 50 | 60 to 120 |
| > 50 | ≤ 25 | ≤ 40 | 70 to 120 |
Performance characteristics for Fumonisin B1 and B2
| Level μg/kg | Fumonisin B1 or B2 | ||
|---|---|---|---|
| RSDr % | RSDR % | Recovery % | |
| ≤ 500 | ≤ 30 | ≤ 60 | 60 to 120 |
| > 500 | ≤ 20 | ≤ 30 | 70 to 110 |
Performance characteristics for T-2 and HT-2 toxin
| Level μg/kg | T-2 toxin | ||
|---|---|---|---|
| RSDr % | RSDR % | Recovery % | |
| 50-250 | ≤ 40 | ≤ 60 | 60 to 130 |
| > 250 | ≤ 30 | ≤ 50 | 60 to 130 |
| Level μg/kg | HT-2 toxin | ||
|---|---|---|---|
| RSDr % | RSDR % | Recovery % | |
| 100-200 | ≤ 40 | ≤ 60 | 60 to 130 |
| > 200 | ≤ 30 | ≤ 50 | 60 to 130 |
The detection limits of the methods used are not stated as the precision values are given at the concentrations of interest.
The precision values are calculated from the Horwitz equation:
RSDR = 2(1-0,5logC)
where:
RSDR is the relative standard deviation calculated from results generated under reproducibility conditions
C is the concentration ratio (i.e. 1 = 100 g/100 g, 0,001 = 1 000 mg/kg
That is a generalised precision equation, which has been found to be independent of analyte and matrix but solely dependent on concentration for most routine methods of analysis.
4.3.2.‘Fitness-for-purpose’ approachU.K.
In the case where there are a limited number of fully validated methods of analysis, alternatively, a ‘fitness-for-purpose’ approach, defining a single parameter, a fitness function, to evaluate the acceptability of methods of analysis may be used. A fitness function is an uncertainty function that specifies maximum levels of uncertainty regarded as fit for purpose.
Given the limited number of methods of analysis, fully validated by a collaborative trial, especially for the determination of T-2 and HT-2 toxin, the uncertainty function approach, specifying the maximum acceptable uncertainty, may also be used to assess the suitability (the ‘fitness-for-purpose’) of the method of analysis to be used by the laboratory. The laboratory may use a method which produces results within the maximum standard uncertainty. The maximum standard uncertainty may be calculated using the following formula:
where:
Uf is the maximum standard uncertainty (μg/kg)
LOD is the limit of detection of the method (μg/kg)
α is a constant, numeric factor to be used depending on the value of C. The values to be used are set out in Table 3
C is the concentration of interest (μg/kg).
If the analytical method provides results with uncertainty measurements less than the maximum standard uncertainty the method shall be considered being equally suitable to one which meets the performance characteristics given in point 4.3.1.
Table 3
Numeric values to be used for α as constant in formula set out in this point, depending on the concentration of interest
| C (μg/kg) | α |
|---|---|
| ≤ 50 | 0,2 |
| 51-500 | 0,18 |
| 501-1 000 | 0,15 |
| 1 001-10 000 | 0,12 |
| > 10 000 | 0,1 |
4.4.Recovery calculation and reporting of resultsU.K.
The analytical result must be reported corrected or uncorrected for recovery. The manner of reporting and the level of recovery must be reported. The analytical result corrected for recovery shall be used for checking compliance (see Annex I, point 5).
The analytical result must be reported as x +/– U whereby x is the analytical result and U is the expanded measurement uncertainty.
U is the expanded uncertainty, using a coverage factor of 2 which gives a level of confidence of approximately 95 %.
4.5.Laboratory quality standardsU.K.
Laboratories must comply with Council Directive 93/99/EEC.