Example method validation and MU approach for Cadmium in seafood test method

Transcription

1 Example method validation and MU approach for Cadmium in seafood test method Charun Yafa National Institute of Metrology (Thailand) (NIMT) 8th APMP TCQM-DEC MiC Workshop, Kobe, Japan December 011

2 QA Measures Analyst Equipment Laboratory Quality control Method Sample Sampling RM Report Envi. condition

3 ISO/IEC 1705 Technical requirements

4 Sample handling labeling storage condition

5 Environmental conditions Temperature Moisture Dust Air flow

6 Analyst Educated Trained Skilled staff

7 Choice of Method/Equipment/Standard AOAC Official methods AOAC Official Method Lead, Cadmium, Zinc, Copper, and Iron in Foods Atomic Absorption Spectrophotometry after Microwave Digestion AOAC Official Method Applicable for determination of Zn, Cu, and Fe in a variety of foods by microwave digestion and flame atomic absorption spectrophotometry (FAAS), and Cd and Pb by microwave digestion and graphite furnace atomic absorption spectroscopy (GFAAS). Method is capable of determining these elements at concentrations above approximately Pb(0.1), Cd(0.01), Zn(4), Cu(0.) and Fe(7) mg/kg. Method is not applicable to foods with a fat content > 40%. Not applicable to milk powder.

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9 A. Principle Products are digested with HNO 3 and H O under pressure in a closed vessel heated by microwaves. Solution is diluted with H O. Pb and Cd are determined by GFAAS. Zn, Cu, and Fe are determined by FAAS B. Apparatus a)atomic absorption spectrophotometer b)hollow cathode or electrodeless discharge lamps-for Pb, Cd, Zn, Cu, and Fe c)microwave oven

10 Reagent a) Water. Redistilled or demonized, >18 MΩ cm. b) Nitric acid. 65% (w/w) c) Hydrogen peroxide. 30% (w/w) d) Cadmium standard solution. 1 mg/ml. Dissolve mg Cd in 14 ml water + 7 ml HNO 3, in 1 L volumetric flask and dilute to volume with water. Commercially available standard solution for AAS may be used for all metal standard solutions e) Working standard solutions For graphite furnace analysis. Dilute Cadmium standard solution with 0.1 M HNO 3, to a range of standards that covers the linear range of the element to be determined.

11 Procedure a) Cleaning procedure (1) For glass and plastic ware.- Water. Acid solution: 500 ml concentrated HNO ml deionized water. Wash first with water and detergent. Rinse with tap water, followed by deionized water, then with acid solution. Finally rinse 4 5 times with deionized water. () For teflon digestion vessels. Rinse with acetone, wash with deionized water, keep vessels covered with 0.1 M HNO3 for at least 30 min, rinse with deionized water, and let vessels dry. b) Pre-treatment. c) Drying Moisture content d) Homogenization

12 e) Digestion. Weigh g dry material into digestion vessel. If water-containing materials are used, maximum weight is restricted to g, but dry matter content must never exceed 0.5 g. Add 5 ml HNO 3, and ml H O. Close vessels, place vessels in holder, place vessel holder in microwave oven, and close door. Set oven program according to given parameters Remove digestion vessels from microwave oven and let cool thoroughly before opening them. Open vessel and rinse down lid and walls into container. Transfer solution to 5 ml volumetric flask and dilute to mark with deionized water. Then transfer solution to plastic container. Treat blanks in the same way as tests. One blank should be included in every set. f) Dilution. If the test solution needs to be further diluted (due to high metal concentrations), dilute with 3 M HNO 3 in order to maintain same acid concentration prior to metal determination

13 Sample size used depends on water content of sample concentration of analyte vessel of microwave system

14 Equipment Instruments Manual Instruction Training Maintenance /Calibration program Record Intermediate check

15 Equipment Microwave digestion system Contract services Calibration Cleaning of vessel Cleaning run Appropriate temperature program Appropriate sample size

16 Equipment Muffle Furnace/ Hot air oven Calibration Temperature check

17 Reagent Grade Major content Minor and trace content (microwave digestion) Handling: strong acid/base, toxic chemical, etc. Labeling

18 Equipments Glassware Cleaning: soaking in 0% HNO 3 overnight rinse with 0% HNO 3 rinse with DI water 4-5 times Calibration: volumetric flask pipette/automatic pipette

19 Traceability/Reference material Reference material Certified reference material Calibrant (Pure material) Matrix CRM QC sample

20 Instrument Calibration/Source of traceability Analyte Supplier Stated Concentration Traceable to SI? Cd NIST SRM 10,000 mg/kg Yes Cd Sigma-Aldrich 1,000 mg/kg Yes

21 When is method validate/revalidate Necessary? New method developed for particular problem Established method revised to incorporate improvements or extended to a new problem When quality control indicates and established method is changing with time Established method used in a different laboratory, or with different analyst or different instrumentation To demonstrate the equivalence between two methods Major instruments replaced New batches of standards Used of validated method after out-of use for a long time

22 Method Validation Requirements Performance Characteristics Instrument calibration function and range Method linearity Confirmation of identity Method selectivity Method detection limit (LOD) Method quantification limit (LOQ) Repeatability Accuracy Ruggedness/Robustness Measurement uncertainty Reproducibility Metrological traceability

23 Validation Parameters for Specific Tasks Key performance characteristic Major compoundsquantitative Major compounds and tracesquantitative Tracesqualitative Tracesquantitative LOD N N Y N LOQ N Y N Y Linearity Y Y N Y Range Y Y N N Precision Accuracy Y Y Y Y N N Y Y Specificity Ruggedness Y Y Y Y Y N Y Y From: Validation and Qualification in Analytical Laboratories by Ludwig Huber

24 Method Validation Requirements Performance Characteristics Instrument calibration function and range Method linearity Confirmation of identity Method selectivity Method detection limit (LOD) Method quantification limit (LOQ) Repeatability Accuracy Ruggedness/Robustness Measurement uncertainty Reproducibility Metrological traceability

25 Confirmation of Identity and Selectivity/Specificity Procedure Analyze samples and reference materials by candidate and other independent methods Analyze samples containing various suspected interferences in the presence of the analyte of interest If detection or quantification is inhibited by the interferences, further method development will be required Eurachem Guide

26 Linearity and Working Range 1. Blank plus RMs or fortified sample blanks at various concentrations (at least 6 conc. Plus blank), single measurement at each concentration Plot the responds against concentration of standard Different concentrations should be prepared independently, and not from aliquots of the same master solution. At least 6 different concentrations within the linear range, 3 replicate measurements at each conc. - Plot the responds against concentration of standard - Calculate regression coefficient - Calculate and plot the residues at each concentration Random distribution of residual about the straight line confirms linearity. Systematic trends indicate non-linearity

27 Accuracy and Trueness/ Do we get the right answer? 1. Analyze reagent blank and CRM using candidate method 10 replicates Calculate the average concentration of CRM (with blank subtraction) determined by candidate method Difference between values obtained by candidate method and certify value can be used to estimate method bias Method s Bias. Analyze reagent blank and RM using candidate and independent method preferably primary method - 10 replicates - Calculate the average concentration of RM (with blank subtraction) determined by candidate method - Calculate the average concentration of RM (with blank subtraction) determined by independent/primary method - Difference between results obtained from these methods can be used to estimate method bias Method s Bias relative to independent/primary method

28 Precision and Reproducibility /Can we reproduce the result? Repeatability: Same lab, same analyst, same instrument, relative short time span At least 6 determinations of 3 different matrices at or 3 different concentrations the calculate the RSD (ICH) At least 9 replicates covering the complete specified range e.g. 3 concentrations with 3 injections at each concentration (ICH) At least 10 injections with RSD <1% (US EPA) 95% confidence interval on the difference between duplicate results measured under repeatability conditions Repeatability Limit r = s r =. 8s r

29 Limit of Detection (LOD)/Limit of Quantitation (LOQ) What is the smallest concentration that can be detected?/what is the smallest concentration that can be measured with a given measurement uncertainty? Sample blank produce signal Single measurement of each 10 independent sample blank, then calculate mean and standard deviation, s LOD = mean of sample blank + 3s LOQ = mean sample blank value + 5s (or 6s or 10s) No signal from sample blank Single measurement of each 10 independent sample blank fortified at lowest acceptable concentration, LOD = 0 + 3s, LOD = s (Eurachem Guide page 17-18) LOQ = 0 + 5s (or 6s or 10s) LOD determined from calibration curve 3 s y / x c dl = is the standard error of the regression b is the slope of the linear calibration

30 Method Validation Approach Approach used Confirmation of Identity and Selectivity/Specificity Linearity and Working Range Accuracy and Trueness Repeatability Method LOD and LOQ Analyze samples and reference materials by candidate and other independent methods At least 6 different concentrations within the linear range, 3 replicate measurements at each conc. - Calculate regression coefficient, Calculate and plot the residues at each concentration Analyze reagent blank and CRM or fortified samples using candidate and independent method preferably primary method At least 10 determinations of 3 different matrices at or 3 different concentrations the calculate the RSD Single measurement of each 10 independent sample blank, then calculate mean and standard deviation, s

31 Quality control Internal Quality control External Quality control PT

32 Measurement Uncertainty 4 Approaches - Bottom-up method: the GUM approach - Top-down method using results from an interlaboratory study by the harmonized IUPAC/AOAC protocol or ISO Estimation by applying the Horwitz formula relating the relative standard deviation to concentration - The supplement GUM approach using Monte Carlo calculation ISO/IEC 1705:005, Interpretation and suggestions for implementation by Prof. Dr. Ir Peter Bode 6/10/55 Slide 3

33 Horwitz Trumpet Coefficient of Variation (%) Pharmacueticals 10-1 Major 10 Nutrients 10% 1% 0.01% Drugs in Feeds Pesticide Residues Trace -3 Minor Nutrients 1 ppm Elements Aflatoxins 1 ppb Concentration 1 Horwitz, W., Evaluation of Analytical Methods Used for Regulation of Food and Drugs, Anal Chem, 51(1), 67A-76A 33

34 Estimation of measurement uncertainty according to the Top down approach The two main uncertainty sources are: Uncertainty u sys, [u(bias)] which is due to the systematic effects laboratory bias and method bias. This component is found from analysis of CRM-s or from participating in ILC-s. Uncertainty u rnd, [u(rw)] which is due to the random effects within-laboratory between-day reproducibility. This component is found from routine between-day reproducibility monitoring of the method (using e.g. a control chart). Nordtest Report TR 537. Handbook for Calculation of Measurement Uncertainty in Environmental Laboratories. B. Magnusson, T. Näykki, H. Hovind, M. Krysell. Available on the web at

35 Top down approach

36 Estimation of measurement uncertainty according to the Top down approach The quantification of the uncertainty components is carried out according to the following formula: u u + u c urel _ rec + urel _ meth = u c sys rnd rnd = 100% u = u + u sys u = ref s n d u dev = n d = c l c ref ref dev u rel_rec relative uncertainty of recovery [%] u rel_meth relative uncertainty of analysis method [%] c Cd concentration in a reference sample as determined by the measurement procedure [mg/kg] c ref reference concentration of Cd in the reference sample [mg/kg] d difference in concentration between our laboratory and reference value [mg/kg] s the standard deviation for reference value [mg/kg] n l the number of laboratories who took part in ILC n number of completed ILCs (n = 1 in our case)

37 The Uncertainty Estimation Process according to ISO GUM Step 1 Step Step 3a Step 3b Step 4 Specification Identify Uncertainty Sources Quantify Uncertainty Component Convert to Standard Deviation Combine the Uncertainties Write down a clear statement of what is being measured and the relationship between it and the parameters on which it depends. List sources of uncertainty for each part of the process/parameter. Estimate the size of each uncertainty. At this stage, approximate values suffice; significant values can be refined at subsequent stages. Express each component as a standard deviation. Combine the uncertainty components, either using a spread-sheet method or algebraically. Identify significant components. Re-evaluate the significant components Yes Do the significant components need re-evaluating? No REPORT Ref : EMU Course V.3 March 005 Commonwealth of Australia

38 c((())dfcd(mg/kg))measurand Specification Practical example: Determination of Cd content in seafood by AASm= mg/lv 0 mlgc 0 = Cd concentration in final solution (from calibration curve), mg/l V = volume of final solution = 5 ml. m = sample mass = 0.5 g. 6/10/55 Slide 38

39 Specification Practical example: Determination of Cd content in seafood by AAS Method Summary Weighing sample 0.5 g Digestion in the microwave digestion system Make up volume to 5 ml. 1 ml. to 10 ml AAS determination Stock solution, As 1000 mg/l 1 ml. to 100 ml Intermediate Std. Sol., As 10 mg/l 10, 5, 50, 75, 100 μl to 100 ml Calibration Std. Sol., As 1.0,.5, 5.0, 7.5, 10.0 μg/l AAS calibration curve C 0 39

40 Identification Practical example: Determination of As content in seafood by AAS Final cause and effect diagram Std M sample Dilution Type A, WS, 5 ppb Cal Type B, Stock C (mg/kg) Cal T Vsample vol R C 0 Method Precision 40

41 41 Uncertainty Source Value (Xi) Std uncert (u Xi) RSU (Xi / uxi) RSU^ [ (Xi / uxi)^ ] Repeatability Recovery Calibration std Type B Calibration std Type A Sample volume Sample mass C C 6.8 u C 0.4 Combining of uncertainty/practical example: Determination of Cd content in seafood by AAS = m V c c = repeat u R u c u c u v u m u c u c u repeat R calstd c calstd Ccalstd v m c c typeb calstdtypeb typea typea = repeat u R u c u c u v u m u c u c u repeat R calstd c calstd Ccalstd v m c c typeb calstd typeb typea typea ( ) ( ) ( ) ( ) ( ) ( ) ( ) = u c 0.4 = u c

42 Example Concentration of Cd in the sample is 6.8 ± 0.8 mg/kg Measurement uncertainty is the expanded uncertainty at coverage factor of which is approximately 95 % confidence interval

43 Contribution to total uncertainty C0 M Paramete V Std A Std B R Repeat 0% 5% 10% 15% 0% 5% 30% 35% 40% %contribution to total variance

44 Metrological Traceability Diagram MOLE METRE, KILOGRAM Method Validation Instrument calibration Repeatability Reproducibility -intra-laboratory -inter-laboratory Recovery/spiked samples Ruggedness Confirmation of Identity Selectivity Accuracy/CRMs Linearity LOD, LOQ Test result (state result and MU) Calibration Solution - (provide concentration and uncertainty) Pure standard (state manufacturer/certificate details) Volumetric apparatus (state calibration procedure) Gravimetric apparatus (state calibration procedure)