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uΔ um uCRM u + 2 2 m CRM u u = Equation 8 Δ combined standard uncertainty of the measurement result and certified value (= uncertainty of Δm: absolute difference between mean measured value and certified value, Equation 6) standard uncertainty of the measurement result standard uncertainty of the certified value Note: In case of asymmetric uncertainties of the certified value, the uncertainty concerned has to be taken. For example, if the measured value is above the certified value the 'plus' uncertainty of the certified value has to be taken. The expanded uncertainty UΔ, corresponding to a confidence level of approximately 95 %, is obtained by multiplication of uΔ by a coverage factor k = 2. UΔ uΔ U = 2 × u Equation 9 Δ Δ expanded uncertainty of difference between result and certified value combined standard uncertainty of the measurement result and certified value Note: For most purposes it is recommended that a coverage factor of k = 2 is used as the statistical observations have a degree of freedom of at least 6 [19]. If Δm (= the absolute difference between the mean measured value and the certified value) is smaller or less than UΔ (= the expanded uncertainty of the difference between results and certified value), then there is no significant difference between the measurement result and the certified value, meaning that the method does not have a bias. In case a bias is found, the cause has to be investigated and preferably eliminated. Approaches to calculate a bias can be found in the Guide to the expression of uncertainty in measurements (GUM) [19], but have to be considered carefully as a bias may be a constant or may be proportional to the measured GM concentration. In the case that no CRM with the required concentration level is available for bias control, CRMs certified for their GM purity can be used. The standard uncertainty of a sample produced by the laboratory has to be estimated using also the purity data of the CRMs used. Example: No CRM with the required GM concentration level is available and a sample with 10 g/kg (or 1 (m/m) %) GM content has to be produced by the laboratory using available CRMs certified for their GM purity. The laboratory has to estimate the standard uncertainty for the sample produced. The available CRMs certified for their purity are: • pure GM material certified to contain with 95 % probability > 985 g/kg (or 98.5 (m/m) %) of the GM event; • a non-GM material certified to contain with 95 % probability < 1 g/kg (or 0.1 (m/m) %) of the GM event. The required sample will be weighed at mass level of the powders using an analytical balance. The example given here refers to the weighing of 0.01 g of GM powder and 0.99 g of the non-GM powder. The nominal or target value of the mixture produced by the 22
laboratory is 1 (m/m) % GM (0.01 g / (0.99 + 0.01) g). The denominator is composed of 0.99 g non-GM material and 0.01 g GM material. In order to avoid the introduction of a bias due to different water contents of the GM and non-GM material, it is recommended to equilibrate the water content of both powders for 24 h (spread the powder on a dish and expose to the air in the laboratory). Four standard uncertainty sources have to be considered: • uncertainty arising from the purity of the non-GM material, • uncertainty arising from the purity of the GM material, • uncertainty arising from the weighing of the non-GM material and • uncertainty arising from the weighing of the GM material. The uncertainty of the GM and non-GM material purity is taken from the certificate. In this example the standard uncertainty arising from the purity of the GM material is 1.5 % (100 % - 98.5 % = 1.5 %) and 0.1 % for the non-GM material. The accredited calibration service reported for the used analytical balance a standard uncertainty of 1.3 %. This uncertainty contributes to the combined standard uncertainty during each weighing step. The combined uncertainty is calculated by taking the square root of the sum of the individual standard uncertainties: u = u + u + u + u Equation 10 2 2 2 2 p( 1) p( 2) w( 1) w( 2) u combined standard uncertainty up(1) standard uncertainty related to the purity of the non-GM material up(2) standard uncertainty related to the purity of the GM material uw(1) standard uncertainty related to the weighing of the non-GM material standard uncertainty related to the weighing of the GM material uw(2) u = (0.99 g × 0.015) 2 + (0.01g × 0.001) 2 + (0.99 g × 0.013) 2 2 + (0.01g × 0.013) The combined standard uncertainty is calculated to be 0.12955 g. The real GM value of the produced sample and its rounded standard uncertainty is estimated to be: 1.00 ± 0.13 g/kg Note: The above calculations concern standard uncertainties as required for bias control. If expanded uncertainties are required, the obtained uncertainties have to be multiplied with the appropriate coverage factor [18]. Likewise, attention has to be paid to the DNA extraction step. As further outlined in Section 7.2.3 a co-extraction procedure should be applied in order to avoid the need for a mixing step at powder level, which could easily lead to non-sufficiently homogeneous samples. In case only intact seed CRMs are available, it is advised to crush the GM and non-GM seeds separately in mortars and to allow them to equilibrate to the same water content for 24 h. 23
Page 1 and 2: European technical guidance documen
Page 3: European technical guidance documen
Page 6 and 7: 7.2.2 GM and non-GM CRMs available
Page 8 and 9: Abstract The aim of this guidance d
Page 10 and 11: 2 Introduction Legislation in the E
Page 12 and 13: • Range of measurements For some
Page 14 and 15: In each case, the laboratory would
Page 16 and 17: and the Compendium of reference met
Page 18 and 19: For the impact of the particle size
Page 20 and 21: 5.2.1.9 Limit of detection Procedur
Page 22 and 23: The absolute within-laboratory repr
Page 26 and 27: 5.2.2.4 Estimation of the uncertain
Page 28 and 29: 6.1 Calibration of qPCR measurement
Page 30 and 31: mass fractions for the calibration
Page 32 and 33: Example: Solution A is DNA extracte
Page 34 and 35: up the calibration curve and to use
Page 36 and 37: should also be checked in accordanc
Page 38 and 39: • detection and quantification of
Page 40 and 41: References [1] ISO/IEC 17025:2005 G
Page 43 and 44: ` European Commission EUR 25484 - J

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