Precision & Bias in Ochratoxin Measurement Uncertainty
Utilizing data from precision and bias experiments in wine analysis to understand and quantify measurement uncertainty, focusing on factors influencing test results, sources of uncertainty, and calculation of combined uncertainty. The process involves a general approach to specify the measurand, identify sources of uncertainty, and calculate overall uncertainty using statistical techniques. Additionally, exploring within-laboratory validation methods and the significance of reproducibility in determining uncertainty levels at different analyte levels.
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Presentation Transcript
Ochratoxin in Wine USING DATA FROM PRECISION & BIAS EXPERIMENTS Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Measurement Uncertainty General Approach Single Within-Laboratory Validation Approach Precision Data Uncertainty at Different Levels Bias Data CRMs, Recoveries, Proficiency Testing Example Ochratoxin in Wine Other Sources Common Sense Check Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
General Approach Specify measurand What is being measured, how is it being measured Identify uncertainty sources Survey of all factors that may influence the test result Quantify uncertainty components Usually collectively through validation, but large or dominant factors may need to be taken into account individually Calculate combined uncertainty Using appropriate statistical techniques Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Single Within-Laboratory Validation Eurolab Technical Report No 1/2007. Measurement Uncertainty Revisited: Alternative Approaches to Uncertainty. Measurement accuracy = precision + trueness Measurement uncertainty = within-laboratory reproducibility + uncertainty on bias 2+ ?2 ? = ?? Where u = standard uncertainty, sR is reproducibility of method, b is bias of method at level of test result. Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Precision Data The within-laboratory standard deviation of reproducibility, sR, determined from validation studies can be taken directly as a standard uncertainty Use of single run repeatability data does not give true estimates of uncertainty Reproducibility experiments should be designed to include as many within laboratory variables as possible Different runs, analysts, equipment, reagents, standards etc. Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Levels Precision may vary with the level of analyte Separate estimates of uncertainty may be necessary at different levels Especially for low level measurements Where relative precision is constant over the range of levels, a single relative uncertainty may be estimated Back-calculated for specific test results Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Bias The bias of a method, and its uncertainty, may be estimated from Analysis of CRMs Recovery experiments with fortified samples Analysis of proficiency testing materials Give priority to approach offering best traceability Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Bias The general formula for the uncertainty from bias is: 2+ ????2+?2 ? = ? Where = measured bias, uref = uncertainty in reference value (e.g. CRM), s = standard deviation of bias measurements, n = number of bias measurements Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Bias Components Where no bias is detected = 0, but there may remain some uncertainty in the bias estimation ???? may be derived from CRM certificates, or estimated in-house for spiking experiments ?2 ? is calculated from bias experiments. Where n is large the value becomes small. Where no bias is found in a robust experiment, b becomes small compared to sRw (<10%) it may be ignored. Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Correcting for Bias It is assumed that where bias is detected either: Method improvement is undertaken to reduce the bias, and/or A robust correction is applied to compensate for a small and well characterised residual bias The above approach is applicable where there is a small residual bias after correction, if needed. There is no well agreed approach to the uncertainty in a large uncorrected bias Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Example Ochratoxin in Wine by HPLC Solvent extraction, column clean-up, quantification by comparison of peak heights using fluorescence detection. Precision study Two samples over a period of months using different analysts and equipment Bias study Recovery experiments under same conditions Other sources Above studies include all sources except purity of standard Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Reproducibility Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Uncertainty from Reproducibility The relative standard deviations from the two materials are similar (F test) so can be pooled together to give a reproducibility figure that applies across the range sR = 0.0842 Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Bias Batch Date 15-Nov-99 01-Feb-00 03-Feb-00 08-Feb-00 17-Feb-00 24-Mar-00 19-May-00 25-May-00 30-May-00 31-May-00 07-Jul-00 11-Jul-00 08-Aug-00 14-Aug-00 28-Aug-00 04-Oct-00 10-Oct-00 08-Jan-01 Sample % Recovery Bias -0.26 0.11 0.03 -0.03 -0.11 -0.13 0.01 -0.07 -0.2 -0.12 -0.21 0.07 -0.11 -0.28 -0.1 -0.06 -0.02 -0.18 White wine (29894) White Grape Juice (29055) Red Grape Juice (29056) Red Grape Juice (29056) Red Grape Juice (29056) white wine (29891) Red Wine (29900) Red Wine (29900) White Wine (29891) Red Wine (29900) Red Wine (29900) White Grape Juice (29054) White wine (29891) Red Wine (29900) Red Wine (29900) Red Wine (29900) White wine (29891) Red Wine (593) 74 111 103 97 89 87 101 93 80 88 79 107 89 72 90 94 98 82 Mean Bias SD Bias -0.0922 0.1092 n 18 Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Contribution from Bias Bias (relative) contribution 0.09222+ ????2+0.10922 ? = 18 We could estimate ???? from uncertainties of volumes, weights etc. but it is likely to be small so we will disregard it, so 0.00850 + 0 + 0.00066 = 0.0957 ? = Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Purity of Standard The purity of standards may not be included in the precision experiments Stated purity 99-100% or 99.5% 0.5% Range is converted to a standard uncertainty by assuming a rectangular distribution: ??=0.005 3=0.0029 This is small compared to the contributions from reproducibility and bias so can be disregarded Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Combining Uncertainties The contributions from reproducibility and bias are now combined: 0.08422+ 0.0957 2= 0.1275 ????= THE EXPANDED RELATIVE UNCERTAINTY IS ????= 2 0.1275 = 0.2550 The uncertainty for a test result of 205 pg/ml is 205 x 0.255 Ochratoxin (205 52) pg/ml Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Common Sense Check Does the estimate we have made make sense? sR = 0.0842, ????= 0.1275 Uncertainty for Ochratoxins at ppb level 20% Excludes sampling We need to exercise our professional judgement about the estimates of measurement uncertainty we produce. Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
Measurement Uncertainty General Approach Single Within-Laboratory Validation Approach Precision Data Uncertainty at Different Levels Bias Data CRMs, Recoveries, Proficiency Testing Example Ochratoxin in Wine Other Sources Common Sense Check Les Coveney, INAB Calibration & Uncertainty Day, 18th June 2018
