Proficiency Test 2/10 TW O5 Special organic analytes in drinking water

Transcription

1 Analytische Qualitätssicherung Baden-Württemberg Proficiency Test 2/ TW O5 Special organic analytes in drinking water Metabolites of pesticides and Glyphosate provided by AQS Baden-Württemberg at Institute for Sanitary Engineering, Water Quality and Solid Waste Management, University of Stuttgart Bandtäle 2, 7569 Stuttgart-Büsnau, Germany on behalf of the Ministry of Rural Affairs, Food and Consumer Protection Baden-Württemberg and in cooperation with IWW Water Centre, Mülheim an der Ruhr Stuttgart, in May 2

2 Responsibilities: Scientific director AQS: Dr.-Ing. Dipl.-Chem. Michael Koch PT manager: Dr.-Ing. Frank Baumeister AQS Baden-Württemberg at Institute of Sanitary Engineering, Water Quality and Solid Waste Management at University of Stuttgart Bandtäle Stuttgart-Büsnau Germany Tel.: +49 ()7 / Fax: +49 ()7 / info@aqsbw.de

3 PT 2/ TW O5 page LIST OF CONTENTS. GENERAL PT DESIGN SAMPLE PREPARATION SAMPLE DISTRIBUTION ANALYTICAL METHODS SUBMISSION OF RESULTS EVALUATION PROCEDURE ASSESSMENT EVALUATION...6. EXPLANATION OF APPENDIX A...6. EXPLANATION OF APPENDIX B EXPLANATION OF APPENDIX C TRACEABLE REFERENCE VALUES INTERNET... Appendix A METABOLITE B... A- METABOLITE B... A-7 R478, NA SALT... A-3 R A-9 CGA A- CGA354742, NA-SALT... A-3 CGA369873, NA-SALT... A-37 M27, NA-SALT... A-43 BH A-49 BH A-55 CGA A-6 CGA A-67 CGA A-73 CGA354743, NA-SALT... A-79 N,N-DIMETHYLSULFAMIDE (DMS)... A-85 BH / 635M... A-9 GLYPHOSATE... A-97 AMPA... A-3

4 PT 2/ TW O5 page 2 Appendix B Appendix C METABOLITE B...C- METABOLITE B...C- R478, NA SALT...C-9 R C-28 CGA C-37 CGA354742, NA SALT...C-46 CGA369873, NA SALT...C-55 M27, NA SALT...C-64 BH C-73 BH C-82 CGA C-9 CGA896...C- CGA522...C-9 CGA354743, NA SALT...C-8 N,N-DIMETHYLSULFAMIDE (DMS)...C-27 BH / 635M...C-36 GLYPHOSATE...C-45 AMPA...C-54

5 PT 2/ TW O5 page 3. General This PT was provided in the context of the AQS Baden-Württemberg drinking water PT scheme. In collaboration with IWW Water Centre (Mülheim an der Ruhr) this interlaboratory comparison study on metabolites of pesticides and Glyphosate was also carried out under the umbrella of the NORMAN Network of Reference Laboratories for Monitoring of Emerging Environmental Pollutants ( Following metabolites were provided: metabolite metabolite of Desphenylchloridazon metabolite B Chloridazon Methyl-Desphenylchloridazon metabolite B R478, Na-salt Chlorothalonil R6965 CGA 5266 Dimethachloric acid Dimethachlor CGA Dimethachlor sulfonic acid, Na-salt CGA , Na-salt M27, Na-salt Dimethenamid-P BH Metazachloric acid Metazachlor BH Metazachlor sulfonic acid CGA (racemic form) Metalaxyl-M CGA 896 CGA 522 S-Metolachloric acid (racemic form) S-Metolachlor CGA S-Metolachlor sulfonic acid (racemic form), Na-salt DMS (N,N-Dimethylsulfamid) Tolylfluanid BH / 635M Tritosulfuron AMPA Glyphosate The PT was executed according to the recommendations of the German Federal Environment Agency from December 23. These recommendations for the execution of PTs for the measurement of chemical parameter and indicator parameter for the external quality control of drinking water laboratories (Bundesgesundheitsblatt 46 2, 94-95) require, that drinking water laboratories must demonstrate their competence for all parameters they are accredited for or they want to be accredited for by a successful participation in a PT round within a cycle of 2-3 years. The PT was executed and evaluated according to the requirements of the DIN 3842-A45 and ISO/TS 262.

6 PT 2/ TW O5 page 4 2. PT design Each participant received the following samples: 3 samples for the determination of the metabolites in -ml-glass bottles with screw cap; sample preservation by cooling. 3 samples for the determination of Glyphosate and AMPA in -ml-glass bottles with screw cap; sample preservation by cooling. 3 different concentration levels/batches were produced. All participants received each concentration level. 3. Sample preparation The samples for the determination of the metabolites and Glyphosate were based on a real ground water matrix. The ground water was filtered by using 5 µm and µm filter cartridges to eliminate particles. To reduce germs, the ground water was irradiated with ultraviolet light and was pasteurised at 8 C in a stainless steel vessel overnight. During pasteurisation the matrix was aerated with a mixture of carbon dioxide and nitrogen to prevent degassing of carbon dioxide followed by precipitation of calcium carbonate. For the preparation of the samples, the matrices were spiked with stock solutions and the concentrations covered drinking and ground water relevant ranges. The samples were cooled directly after preparation. The samples were dispatched with freezer packs added to the packages. 4. Sample distribution The samples were dispatched on 8 th March 2 by express service.

7 PT 2/ TW O5 page 5 5. Analytical methods The participants were free to choose a suitable method, but a limit of quantification of,5 µg/l for all analytes were required. The samples had to be analysed in duplicate over the complete method (sample preparation and measurement). The participants were asked to submit the results in µg/l with four significant digits. 6. Submission of results The deadline for the submission of results was on 26 th March Evaluation procedure The statistical evaluation was executed according to DIN 3842-A45 and ISO TS 262 Interlaboratory comparisons for proficiency testing of analytical chemistry laboratories. From the participants results a relative standard deviation was calculated for each concentration level and parameter using the Q-method. The Hampel estimator was used for the calculation of the consensus mean which was used as assigned value x a. The standard deviation for proficiency assessment σ ) was taken from the Q-method for the calculation of the z U -score according to DIN 3842-A45 or ISO TS 262 respectively in those cases, when the relative standard deviation was within the limits. σ ) was limited as follows: lower limit: 5 % upper limit: % The limits were used as σ ), when the relative standard deviation derived from the Q- method, was above the upper limit. A z-score was calculated for each measurement result derived from the assigned value x a and the standard deviation for proficiency assessment: result z score = σˆ x a The z-score was modified to a z U -score with a correction factor for proficiency assessment (as described in the standards mentioned above). The tolerance limit was defined as Iz U I= Assessment There was no overall assessment of the proficiency test round, but the single parameters were assessed. A parameter was assessed as successful, if more than half of the values were correctly determined (2 out of 3 values were within the tolerance limits). Not successful were:.) Values which were not determined (if the other samples of this parameter were analysed), 2.) Values, which were indicated with lower than limit of quantification, 3.) Values, which were subcontracted, 4.) Values, which were submitted after the deadline of submission of results.

8 PT 2/ TW O5 page 6 9. Evaluation Number of participants: 82 Number of reported values: 784 Number of accepted values: 528 (85,65 %) In the following figure the successful and not successful laboratories for each parameter are illustrated. succesful not successful number of labs metabolite_b metabolite_b R478_Na R6965 CGA_ CGA _Na CGA_369873_Na M27_Na BH_479_4 BH_479_8 CGA_62826 CGA_896 CGA_522 CGA_ DMS BH_635_4 635M glyphosate AMPA. Explanation of Appendix A Appendix A contains for each parameter parameter tables a figure of participants means versus the spiked amount for the determination of the recovery rate and the matrix content a figure of the relative standard deviations versus the concentrations a figure of the tolerance limits in the PT versus the concentrations the frequency of application of analytical methods the method specific evaluation a comparison of mean and reference values for each concentration level a comparison of the relative standard deviations of the different methods the statistical characteristics of the method specific evaluation a tabular comparison of the means with the reference values and their uncertainties

9 PT 2/ TW O5 page 7 Parameter tables In these tables the following values for each concentration level are listed: assigned value expanded uncertainty of the assigned value in %, calculated according to ISO 3528 using the formula rel. standard deviation U = 2. number of values standard deviation, calculated using robust statistical method standard deviation for proficiency assessment for the calculation of z U - scores rel. standard deviation for proficiency assessment tolerance limits above and below permitted deviations above and below in % number of values in this level number of not satisfactory values below and above the assigned value and the percentage of these values in total. Determination of recovery rate In the diagrams of the assigned values versus the spiked amount of analyte a linear regression line was calculated using a generalized least square regression which takes into account the uncertainties of the values in both directions. From these values the recovery rate for each parameter was determined (see diagrams). The slope of the line indicates the average recovery rate. The diagrams also contain the expanded uncertainty (k=2) of the mass values and the assigned values. Relative standard deviations and tolerance limits The diagrams for the relative standard deviation vs. the assigned value show the concentration dependency of the standard deviation and the tolerance limits in percent. The relative standard deviations calculated from participants data are the stars connected by an interrupted line, the rel. standard deviation taken from the variance function (and sometimes limited by the upper or lower limit) are given by squares, connected by a continuous line. Method specific evaluation For each parameter the methods used by the participants are shown in a diagram. In a second diagram for each method with a frequency of more than 5 %, values are sorted in 5 categories: too low results with z U -score < -2 low results with 2 z U -score < correct results with z U -score + high results with + < z U -score +2 too high results with z U -score > +2

10 PT 2/ TW O5 page 8 Comparison of means and reference values for each concentration level Finally the mean value calculated from all results (used as assigned value) is compared with mean values calculated for all methods separately (in this case using the Hampel estimator described in ISO/TS 262). Mean values were calculated only, if more than 7 results were within a z-score-range of ± 2. The means are reported with their expanded uncertainty calculated according to ISO Explanation of Appendix B Participants were asked to report expanded uncertainties of their results on a voluntary basis. In this diagram for each parameter the reported uncertainties for all concentration levels with the reproducibility standard deviation (horizontal line) are displayed. Values which deviate from the reproducibility standard deviation with a factor more than 2 are usually not realistic. 2. Explanation of Appendix C In the last part of the report, for all concentration levels the results of all participants are illustrated. Confidentiality of participants is ensured by using s. The lab codes were sent to participants with the certificates. In detail Appendix C contains: - a table with all data - figures with o all reported results o all z U -scores o all reported expanded uncertainties o all ζ scores Table with all data The assigned value with the expanded uncertainty and the tolerance limits for the concentration level is illustrated in the table. For each participant the following data are given: reported result measurement uncertainty of the value (if reported) ζ-score for this value, calculated with the following formula ζ = x x 2 2 u lab + u ref, with x x = difference from the measured value and the assigned value u lab = standard uncertainty of the value, reported by the participant u ref = standard uncertainty of the assigned value z U -score for proficiency assessment assessment of the value according to its z-score Meaning of ζ-scores: The assessment of ζ-scores is similar to that of z-scores. If the data are normally distributed and the uncertainties are well estimated, ζ-scores will lie between -2 and +2 with a probability of around 95 %. ζ-scores are mainly influenced by the measurement uncertainties reported by the laboratory. Therefore ζ-scores are usually not appropriate for the assessment of the reported results, unless the reported measurement uncertainty is checked for fitnessfor-purpose.

11 PT 2/ TW O5 page 9 Therefore we do not use the ζ-scores for the assessment of the laboratories. Nevertheless ζ-scores are appropriate to check the plausibility of the reported measurement uncertainty: If the z-score of a result is within the tolerance limit and the ζ-score is outside, then the measurement uncertainty is underestimated. If the z-score is outside the tolerance limits and the absolute value of the ζ-score is lower than two, then the requirements of the proficiency test were stronger compared with the reported measurement uncertainty. Diagrams of uncertainty data In the first figure for all s the measurement uncertainty (together with the reproducibility standard deviation) is illustrated. The second figure shows the associated ζ-scores. 3. Measurement uncertainty 22 (3,99 %) out of 7 laboratories with valid values reported measurement uncertainties. In total 3 (2, %) out of 784 valid values were given with the measurement uncertainty. The following table displays the number of values with measurement uncertainty against the accreditation status. Accreditation status of Number of Number of values with measurement the values values uncertainty accredited (37,5%) not accredited (5,67%) not specified (3,68%) We would like to put emphasis on the fact that reporting of measurement uncertainties in our PT scheme is absolutely voluntary. The only objective is to help all participants to reasonably handle measurement uncertainties and their estimation. The diagrams show that the spread of reported uncertainties is vast, from unrealistic low values up to very high. A plausibility check using reproducibility standard deviations of the PT round could be helpful here. If measurement uncertainties are underestimated values assessed as satisfactory in the PT ( z 2), will have a large ζ-score. ζ > 2 means that the own requirements (defined in terms of estimated uncertainty) are not fulfilled. 8 (5 %) of the 3 values reported with uncertainties and having a z-score z 2. had a ζ-score > 2.. This means that the requirements of the PT scheme have been fulfilled, but not the own requirements, the uncertainty is underestimated. 4. Traceable reference values Traceability of analytical results to national and international references is of increasing importance in all laboratories. This is not easy to realise for chemical analyses and often can only be done by analysing certified reference materials. But availability of these reference materials in the water sector is very limited. Therefore we try to provide reference values for the proficiency test samples, traceable to national and international references. Since our PT samples without exception are spiked, real water samples, reference values can be calculated from the sum of matrix content and spiked amount of analyte. For both summands traceable values and their uncertainty have to be determined. Thereby we assume that no unrecognised bias resulting from sample preparation and transport is present and that we recognise all uncertainty components.

12 PT 2/ TW O5 page Determination of the spiked amount and its uncertainty All spiking of samples was controlled gravimetrically. Conversion to concentration was done by measuring the density of the resulting samples using a pycnometer. This procedure allows the preparation of a complete uncertainty budget. The first step is the specification of the measurand with a formula. This shows the links between the result and all influence quantities. c lot = c standard ρ m standard standard m lot ρ lot with: c lot c standard m standard m lot ρ lot concentration of the analyte in the lot resulting from the spike in g/l concentration of the standard solution in g/l mass of the standard solution added into the lot in g total mass of the lot in g density of the lot in g/l Based on this formula the uncertainty budget can be prepared and all components be quantified. The following figure shows a typical distribution of the contributions, here for metabolite B as an example. The main contribution results from the purity of the standard solution. 2E- 4E- 6E- 8E- E-9,2E-9,4E-9,6E-9,8E-9 2E-9 conc. standard in g/l density standard in g/l w eighing into lot in g density lot in g/l total mass of the lot in g All weighings were done as difference weighings. The precision of these weighings was determined in experiments by multiple (2fold) measurements of mass pieces with similar masses as a type A uncertainty. The trueness of the weighing, that has to be considered twice for each weighing, was taken from the calibration certificate of the balance. Maintaining of these tolerances is assured by regular maintenance of the balances by a calibration laboratory and by supervision with our mass pieces (calibrated by an accredited calibration laboratory). The determination of the density was also made using weighings (of the pycnometer). The above said also applies here. Temperature measurement was made with a calibrated thermometer. The purity of the used metabolite (99,5 %) was taken from the certificate of the manufacturer. The manufacturer reported an expanded uncertainty of 2 %.

13 PT 2/ TW O5 page With all these uncertainty components the combined uncertainty, as described in the EURACHEM/CITAC-Guide Quantifying Uncertainty in Analytical Measurement, was calculated using the sensitivity coefficients determined by partial derivation of the formula to the respective influence quantities. So traceability was assured by using calibrated balances and thermometers. Determination of the matrix content Because the same matrix was used for preparation of all samples, the matrix content could be calculated from the mean values of the participants and the spiked amounts in a standard-addition-like way,2. The uncertainties of the spiked amounts were known from the uncertainty budgets. The expanded uncertainties of the mean values of participants result were calculated according to ISO 3528 (Statistical Methods for Use in Proficiency Testing by Interlaboratory Comparisons) as u mean with: = 2, sr n s R reproducibility standard deviation n number of data for this level 2 coverage factor for the expanded uncertainty, correction factor (according to ISO 3528 to be used for robust methods) The content of the matrix can be derived from a linear regression of means vs. spiked amounts. Since uncertainties of all data points were available for x- as well as y-direction a generalised least square regression was used as described in DIN EN 643. The computer program B_LEAST (from BAM) was used for this purpose. With this method a value for matrix and its uncertainty are obtained. Because of statistical variation of the input values the calculated matrix content might result in a negative value. From a scientific point of view this of course is nonsense. In those cases the matrix content is set to zero. The lower end of the uncertainty range of the matrix content also might be negative. Therefore the expanded uncertainty of the matrix content was set to the matrix content itself in this case. The matrix content is not directly traceable to national or international references, but it does not considerably compromise the traceability of the final content due to its comparably low contribution. 5. Internet The report is available on the following webpage: Rienitz, O., Schiel, D., Güttler, B., Koch, M., Borchers, U.: A convenient and economic approach to achieve SI-traceable reference values to be used in drinking-water interlaboratory comparisons. Accred Qual Assur (27) 2: Koch, M., Baumeister, F.: Traceable reference values for routine drinking water proficiency testing: first experiences. Accred Qual Assur (28) 3:

14 PT 2/ - TW O5 metabolite B level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],599 3,,57,4,,26,876,98-45, ,7 2,376 2,67,96,769,,4,6,98-45, , 3,56 3,,93,422,,89,38,98-45, , sum ,9 upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content metabolite B,7,6 assigned value in μg/l,5,4,3,2, -,,,2,3,4,5,6,7,8 added amount in μg/l Slope of the regression line:,853, recovery: 8,5 % neg. x-axis-intercept = matrix content:,6 μg/l expanded uncertainty of the matrix content:,6 μg/l = % page A- of A-8

15 PT 2/ - TW O5 Relative standard deviation and tolerance limits metabolite B rel. standard deviation in % ,,2,3,4,5,6 concentration in μg/l The standard deviation, calculated from the Q-method, reached the upper limit for all concentration levels. metabolite B 8 6 tolerance limits in % ,,2,3,4,5,6 concentration in μg/l page A-2 of A-8

16 PT 2/ - TW O5 Method specific evaluation used methods for metabolite B ,9 33,33 3 percentage ,68 5 5,26 2,63 LC-MS - ext. calibr. LC-MS - ext. calibr. + int. standard LC-MS - standard addition Others HPLC-MS - isotope dilution method method comparison for metabolite B frequency in % Others LC-MS - standard addition LC-MS - ext. calibr. + int. standard LC-MS - ext. calibr. too low low correct high too high The values determined with standard addition showed the least variation. page A-3 of A-8

17 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],599,2 3,,9,6 3, 2,376,39 2,7,387,69,8 3,56,772 3,6,78,9,3 comparison of means and reference values overall mean reference values HPLC-MS - external calibration HPLC-MS - external calibration + internal standard HPLC-MS - standard addition mean in μg/l,,2,5,,5 level page A-4 of A-8

18 PT 2/ - TW O5 comparison of means and reference values overall mean reference values mean in μg/l,5,4,3,2, HPLC-MS - external calibration HPLC-MS - external calibration + internal standard HPLC-MS - standard addition 2 level comparison of means and reference values overall mean reference values mean in μg/l,8,6,4,2 HPLC-MS - external calibration HPLC-MS - external calibration + internal standard HPLC-MS - standard addition 3 level page A-5 of A-8

19 PT 2/ - TW O5 rel. standard dev. method specific rel. standard dev. in % level overall mean HPLC-MS - external calibration HPLC-MS - external calibration + internal standard HPLC-MS - standard addition page A-6 of A-8

20 PT 2/ - TW O5 metabolite B level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],27 7,39,454,454 7,97,3532,6 39,79-33, ,5 2,5527 7,47,4,4 8,7,7753,367 4,27-33, ,5 3,7697 8,56,63,63 2,83,3, ,98-38, ,2 sum 3 3 4,4 upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content metabolite B,9,8 assigned value in μg/l,7,6,5,4,3,2, -,,,2,3,4,5,6,7,8,9 added amount in μg/l Slope of the regression line:,6, recovery:,6 % neg. x-axis-intercept = matrix content:,367 μg/l expanded uncertainty of the matrix content:,367 μg/l = % page A-7 of A-8

21 PT 2/ - TW O5 Relative standard deviation and tolerance limits metabolite B 3 rel. standard deviation in % 2 5 5,,2,3,4,5,6,7,8,9 concentration in μg/l The standard deviation, calculated from the Q-method, didn't reach the limits. metabolite B tolerance limits in % ,,2,3,4,5,6,7,8,9 concentration in μg/l page A-8 of A-8

22 PT 2/ - TW O5 Method specific evaluation used methods for metabolite B ,54 35,4 percentage ,22 8, 5 LC-MS - ext. calibr. LC-MS - ext. calibr. + int. standard LC-MS - standard addition Others method comparison for metabolite B frequency in % Others LC-MS - standard addition LC-MS - ext. calibr. + int. standard LC-MS - ext. calibr. too low low correct high too high The values determined with external calibration and internal standard showed the least variation. The external calibration delivered some results that are too low. page A-9 of A-8

23 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],27,87 7,4,7,22,9 2,5527,43 7,5,5439,33,6 3,7697,659 8,6,7732,44,6 comparison of means and reference values overall mean reference values HPLC-MS - external calibration HPLC-MS - external calibration + internal standard mean in μg/l,3,,2,5,,5 level page A- of A-8

24 PT 2/ - TW O5 comparison of means and reference values overall mean reference values mean in μg/l,7,6,5,4,3,2, HPLC-MS - external calibration HPLC-MS - external calibration + internal standard 2 level comparison of means and reference values overall mean reference values mean in μg/l,8,6,4,2 HPLC-MS - external calibration HPLC-MS - external calibration + internal standard 3 level page A- of A-8

25 PT 2/ - TW O5 rel. standard dev. method specific 3 rel. standard dev. in % level overall mean HPLC-MS - external calibration HPLC-MS - external calibration + internal standard page A-2 of A-8

26 PT 2/ - TW O5 R478, Na salt level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],2769 9,64,5,5 2,76,465,75 46,79-38,5 2,3 2,6439,52,434,434 22,26,973,38 5,69-4,6 28 3,6 3,8538,22,227,227 23,74,32,4 54,58-43,8 28, sum ,7 upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content R478, Na salt,9 assigned value in μg/l,8,7,6,5,4,3,2, -,,,2,3,4,5,6,7,8,9 added amount in μg/l Slope of the regression line:,9, recovery:,9 % neg. x-axis-intercept = matrix content:,395 μg/l expanded uncertainty of the matrix content:,395 μg/l = % page A-3 of A-8

27 PT 2/ - TW O5 Relative standard deviation and tolerance limits R478, Na salt 3 rel. standard deviation in % 2 5 5,,2,3,4,5,6,7,8,9 concentration in μg/l The standard deviation, calculated from the Q-method, didn't reach the limits. R478, Na salt 6 4 tolerance limits in % ,,2,3,4,5,6,7,8,9 concentration in μg/l page A-4 of A-8

28 PT 2/ - TW O5 Method specific evaluation used methods for R478, Na salt ,96 percentage ,92,3 5 4,82 LC-MS - ext. calibr. LC-MS - ext. calibr. + int. standard LC-MS - standard addition Others method comparison for R478, Na salt frequency in % too low low correct high too high LC-MS - standard addition LC-MS - ext. calibr. + int. standard LC-MS - ext. calibr. The values determined with standard addition showed the least variation. page A-5 of A-8

29 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],2769,267 9,6,274,4,5 2,6439,677,5,645,85,3 3,8538,958,2,8369,9,3 comparison of means and reference values overall mean reference values HPLC-MS - external calibration HPLC-MS - external calibration + internal standard mean in μg/l,35,3,,2,5,,5 level page A-6 of A-8

30 PT 2/ - TW O5 comparison of means and reference values overall mean reference values HPLC-MS - external calibration HPLC-MS - external calibration + internal standard,8 mean in μg/l,6,4,2 2 level comparison of means and reference values overall mean reference values mean in μg/l,2,8,6,4,2 HPLC-MS - external calibration HPLC-MS - external calibration + internal standard 3 level page A-7 of A-8

31 PT 2/ - TW O5 rel. standard dev. method specific rel. standard dev. in % level overall mean HPLC-MS - external calibration HPLC-MS - external calibration + internal standard page A-8 of A-8

32 PT 2/ - TW O5 R6965 level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],2,3,49,49 2,42,342,394 48,48-39,6 27 2, 2,427,35,7,7 23,59,6585, ,9-42, , 3,6323 3,4,763,58,,9989,3466,98-45,9 27 3, sum 8 7 2, upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content R6965,8,7 assigned value in μg/l,6,5,4,3,2, -,,,2,3,4,5,6,7,8 added amount in μg/l Slope of the regression line:,853, recovery: 85 % neg. x-axis-intercept = matrix content:,3772 μg/l expanded uncertainty of the matrix content:,3772 μg/l = % page A-9 of A-8

33 PT 2/ - TW O5 Relative standard deviation and tolerance limits R rel. standard deviation in % 2 5 5,,2,3,4,5,6,7 concentration in μg/l The standard deviation, calculated from the Q-method, reached the upper limit for one concentration. R tolerance limits in % ,,2,3,4,5,6,7 concentration in μg/l page A-2 of A-8

34 PT 2/ - TW O5 Method specific evaluation used methods for R ,74 percentage ,63 22,22 7,4 5 LC-MS - ext. calibr. LC-MS - standard addition LC-MS - ext. calibr. + int. standard Others method comparison for R6965 frequency in % Others LC-MS - ext. calibr. + int. standard LC-MS - standard addition LC-MS - ext. calibr. too low low correct high too high The differences between the methods, with exception of "Others", were not significant. page A-2 of A-8

35 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],2,236,3,27,9 7, 2,427,485,3,4897,96 4, 3,6323,848 3,4,767,28 2,7 comparison of means and reference values overall mean HPLC-MS - standard addition reference values HPLC-MS - external calibration mean in μg/l,35,3,,2,5,,5 level page A-22 of A-8

36 PT 2/ - TW O5 comparison of means and reference values overall mean HPLC-MS - standard addition reference values HPLC-MS - external calibration,6,5 mean in μg/l,4,3,2, 2 level comparison of means and reference values overall mean HPLC-MS - standard addition reference values HPLC-MS - external calibration mean in μg/l,9,8,7,6,5,4,3,2, 3 level page A-23 of A-8

37 PT 2/ - TW O5 rel. standard dev. method specific 35 3 rel. standard dev. in % level overall mean HPLC-MS - external calibration HPLC-MS - standard addition page A-24 of A-8

38 PT 2/ - TW O5 CGA5266 level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],2 7,93,38,38 7,38,326,482 38,33-32,24 3 2, 2,422 8,98,83,83 9,67,677, ,2-36, ,3 3,7667 9,8,542,542 2,2,3, ,6-36, ,3 sum ,2 upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content CGA5266,9,8 assigned value in μg/l,7,6,5,4,3,2, -,,,2,3,4,5,6,7,8,9 added amount in μg/l Slope of the regression line:,985, recovery: 98,5 % neg. x-axis-intercept = matrix content:,7 μg/l expanded uncertainty of the matrix content:,7 μg/l = % page A- of A-8

39 PT 2/ - TW O5 Relative standard deviation and tolerance limits CGA rel. standard deviation in % 2 5 5,,2,3,4,5,6,7,8,9 concentration in μg/l The standard deviation, calculated from the Q-method, didn't reach the limits. CGA tolerance limits in % ,,2,3,4,5,6,7,8,9 concentration in μg/l page A-26 of A-8

40 too low low correct high too high PT 2/ - TW O5 Method specific evaluation used methods for CGA , percentage ,67 23,33 5 3,33 LC-MS - ext. calibr. LC-MS - ext. calibr. + int. standard LC-MS - standard addition Others method comparison for CGA5266 frequency in % LC-MS - standard addition LC-MS - ext. calibr. + int. standard LC-MS - ext. calibr. The differences between the methods were not significant. page A-27 of A-8

41 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],2,74 7,9,227,44 2, 2,422,379 9,,434,63,4 3,7667,74 9,2,7744,,3 comparison of means and reference values overall mean reference values HPLC-MS - external calibration HPLC-MS - external calibration + internal standard mean in μg/l,3,,2,5,,5 level page A-28 of A-8

42 PT 2/ - TW O5 comparison of means and reference values overall mean reference values mean in μg/l,6,5,4,3,2, HPLC-MS - external calibration HPLC-MS - external calibration + internal standard 2 level comparison of means and reference values overall mean reference values mean in μg/l,8,6,4,2 HPLC-MS - external calibration HPLC-MS - external calibration + internal standard 3 level page A- of A-8

43 PT 2/ - TW O5 rel. standard dev. method specific rel. standard dev. in % level overall mean HPLC-MS - external calibration HPLC-MS - external calibration + internal standard page A-3 of A-8

44 PT 2/ - TW O5 CGA354742, Na salt level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],2345 7,95,45,45 7,7,3263,5 39,6-32, ,9 2,3 8,62,974,974 9,2,7247, ,87-35, , 3,77,43,79,79 23,23,8, ,24-42, , sum , upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content CGA354742, Na salt,9,8 assigned value in μg/l,7,6,5,4,3,2, -,,,2,3,4,5,6,7,8,9 added amount in μg/l Slope of the regression line:,9585, recovery: 95,9 % neg. x-axis-intercept = matrix content:,5 μg/l expanded uncertainty of the matrix content:,5 μg/l = % page A-3 of A-8

45 PT 2/ - TW O5 Relative standard deviation and tolerance limits CGA354742, Na salt 3 rel. standard deviation in % 2 5 5,,2,3,4,5,6,7,8,9 concentration in μg/l The standard deviation, calculated from the Q-method, didn't reach the limits. CGA354742, Na salt 6 4 tolerance limits in % ,,2,3,4,5,6,7,8,9 concentration in μg/l page A-32 of A-8

46 PT 2/ - TW O5 Method specific evaluation used methods for CGA354742, Na salt 45 4, percentage 3 2 5,8,8 5 6,45 LC-MS - ext. calibr. LC-MS - standard addition LC-MS - ext. calibr. + int. standard Others method comparison for CGA354742, Na salt frequency in % Others LC-MS - ext. calibr. + int. standard LC-MS - standard addition LC-MS - ext. calibr. too low low correct high too high The differences between the methods, with exception of "Others", were not significant. page A-33 of A-8

47 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],2345, 8,,24,53 2,2 2,3,437 8,6,577,78,5 3,77,84,4,82,,3 comparison of means and reference values overall mean reference values HPLC-MS - external calibration HPLC-MS - standard addition HPLC-MS - external calibration + internal standard mean in μg/l,3,,2,5,,5 level page A-34 of A-8

48 PT 2/ - TW O5 comparison of means and reference values overall mean reference values mean in μg/l,6,5,4,3,2, HPLC-MS - external calibration HPLC-MS - standard addition HPLC-MS - external calibration + internal standard 2 level comparison of means and reference values overall mean reference values mean in μg/l,8,6,4,2 HPLC-MS - external calibration HPLC-MS - standard addition HPLC-MS - external calibration + internal standard 3 level page A-35 of A-8

49 PT 2/ - TW O5 rel. standard dev. method specific rel. standard dev. in % level overall mean HPLC-MS - external calibration HPLC-MS - standard addition HPLC-MS - external calibration + internal standard page A-36 of A-8

50 PT 2/ - TW O5 CGA , Na salt level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],2394,8,492,492 2,,353,49 46,3-37, ,8 2,476 9,93,964,964 2,26,6926,89 45,52-37,2 26, 3,68 9,73,35,35 9,85,9838,43 44,47-36, ,8 sum ,6 upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content CGA , Na salt,8,7 assigned value in μg/l,6,5,4,3,2, -,,,2,3,4,5,6,7,8,9 added amount in μg/l Slope of the regression line:,978, recovery: 9,8 % neg. x-axis-intercept = matrix content:,954 μg/l expanded uncertainty of the matrix content:,954 μg/l = % page A-37 of A-8

51 PT 2/ - TW O5 Relative standard deviation and tolerance limits CGA , Na salt 3 rel. standard deviation in % 2 5 5,,2,3,4,5,6,7,8 concentration in μg/l The standard deviation, calculated from the Q-method, didn't reach the limits. CGA , Na salt tolerance limits in % ,,2,3,4,5,6,7,8 concentration in μg/l page A-38 of A-8

52 PT 2/ - TW O5 Method specific evaluation used methods for CGA , Na salt ,46 percentage ,77 23,8 7,69 5 LC-MS - ext. calibr. LC-MS - ext. calibr. + int. standard LC-MS - standard addition Others method comparison for CGA , Na salt frequency in % too low low correct high Others LC-MS - standard addition LC-MS - ext. calibr. + int. standard LC-MS - ext. calibr. too high The differences between the methods, with exception of "Others", were not significant. page A-39 of A-8

53 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],2394,24,,265,56 2, 2,476,473 9,9,546,76,5 3,68,663 9,7,7599,,3 comparison of means and reference values overall mean reference values HPLC-MS - external calibration HPLC-MS - external calibration + internal standard mean in μg/l,3,,2,5,,5 level page A-4 of A-8

54 PT 2/ - TW O5 comparison of means and reference values overall mean reference values mean in μg/l,6,5,4,3,2, HPLC-MS - external calibration HPLC-MS - external calibration + internal standard 2 level comparison of means and reference values overall mean reference values mean in μg/l,8,6,4,2 HPLC-MS - external calibration HPLC-MS - external calibration + internal standard 3 level page A-4 of A-8

55 PT 2/ - TW O5 rel. standard dev. method specific rel. standard dev. in % level overall mean HPLC-MS - external calibration HPLC-MS - external calibration + internal standard page A-42 of A-8

56 PT 2/ - TW O5 M27, Na salt level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],287 5,63,26,26 2,53,2647,592 26,83-23, ,7 2,47 5,4,54,54,44,5,373 24,34-2, ,7 3,6752 7,26,92,92 6,8,943,477 35,4-3, ,7 sum ,7 upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content M27, Na salt,8,7 assigned value in μg/l,6,5,4,3,2,,,2,3,4,5,6,7,8 added amount in μg/l Slope of the regression line:,9533, recovery: 95,3 % neg. x-axis-intercept = matrix content: μg/l expanded uncertainty of the matrix content:,3 μg/l = % page A-43 of A-8

57 PT 2/ - TW O5 Relative standard deviation and tolerance limits M27, Na salt 3 rel. standard deviation in % 2 5 5,,2,3,4,5,6,7,8 concentration in μg/l The standard deviation, calculated from the Q-method, didn't reach the limits. M27, Na salt 4 3 tolerance limits in % ,,2,3,4,5,6,7,8 concentration in μg/l page A-44 of A-8

58 PT 2/ - TW O5 Method specific evaluation used methods for M27, Na salt , percentage 3 2 5,8 9,35 9,68 5 LC-MS - ext. calibr. LC-MS - ext. calibr. + int. standard LC-MS - standard addition Others method comparison for M27, Na salt frequency in % Others LC-MS - standard addition LC-MS - ext. calibr. + int. standard LC-MS - ext. calibr. too low low correct high too high The differences between the methods, with exception of the others, were not significant. page A-45 of A-8

59 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],287,7 5,6,223,9,8 2,47,243 5,,4997,33,7 3,6752,49 7,3,784,45,6, comparison of means and reference values overall mean reference values HPLC-MS - external calibration,2 mean in μg/l,5,,5 level page A-46 of A-8

60 PT 2/ - TW O5 comparison of means and reference values overall mean reference values HPLC-MS - external calibration,6,5 mean in μg/l,4,3,2, 2 level comparison of means and reference values overall mean reference values HPLC-MS - external calibration mean in μg/l,8,7,6,5,4,3,2, 3 level page A-47 of A-8

61 PT 2/ - TW O5 rel. standard dev. method specific rel. standard dev. in % level overall mean HPLC-MS - external calibration page A-48 of A-8

62 PT 2/ - TW O5 BH479-4 level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],277 7,95,42,42 9,33,74,338 43,8-35, ,9 2,465,37,5,4,,69,2283,98-45, ,9 3,7236 6,6,84,84 4,99,9593,527 32, -28, ,9 sum 5 6 8,9 upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content BH479-4,9,8 assigned value in μg/l,7,6,5,4,3,2, -,,,2,3,4,5,6,7,8 added amount in μg/l Slope of the regression line:,973, recovery: 97,3 % neg. x-axis-intercept = matrix content:,98 μg/l expanded uncertainty of the matrix content:,98 μg/l = % page A-49 of A-8

63 PT 2/ - TW O5 Relative standard deviation and tolerance limits BH rel. standard deviation in % 2 5 5,,2,3,4,5,6,7,8 concentration in μg/l The standard deviation, calculated from the Q-method, reached the upper limit for one concentration. BH tolerance limits in % ,,2,3,4,5,6,7,8 concentration in μg/l page A-5 of A-8

64 PT 2/ - TW O5 Method specific evaluation used methods for BH ,35 percentage ,62 8,92 8, LC-MS - ext. calibr. LC-MS - ext. calibr. + int. standard LC-MS - standard addition Others method comparison for BH479-4 frequency in % Others LC-MS - standard addition LC-MS - ext. calibr. + int. standard LC-MS - ext. calibr. too low low correct high too high The differences between the methods, with exception of "Others", were not significant. page A-5 of A-8

65 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],277,65 7,9,22,3,6 2,465,432,4,44,28,6 3,7236,446 6,2,735,45,6 comparison of means and reference values overall mean reference values, HPLC-MS - external calibration HPLC-MS - external calibration + internal standard mean in μg/l,2,5,,5 level page A-52 of A-8

66 PT 2/ - TW O5 comparison of means and reference values overall mean reference values HPLC-MS - external calibration HPLC-MS - external calibration + internal standard,5 mean in μg/l,4,3,2, 2 level comparison of means and reference values overall mean reference values HPLC-MS - external calibration HPLC-MS - external calibration + internal standard mean in μg/l,8,6,4,2 3 level page A-53 of A-8

67 PT 2/ - TW O5 rel. standard dev. method specific rel. standard dev. in % level overall mean HPLC-MS - external calibration HPLC-MS - external calibration + internal standard page A-54 of A-8

68 PT 2/ - TW O5 BH479-8 level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],2 8,64,44,44 2,73,38,37 46,72-38, 36 3, 2,5349 6,7,, 6,7,72, ,6 -, ,4 3,7877 6,2,37,37 4,44,34,44 3,27-27, ,9 sum ,8 upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content BH479-8,9,8 assigned value in μg/l,7,6,5,4,3,2,,,2,3,4,5,6,7,8,9 added amount in μg/l Slope of the regression line:,998, recovery: 99,8 % neg. x-axis-intercept = matrix content: μg/l expanded uncertainty of the matrix content:,856 μg/l = % page A-55 of A-8

69 PT 2/ - TW O5 Relative standard deviation and tolerance limits BH rel. standard deviation in % 2 5 5,,2,3,4,5,6,7,8,9 concentration in μg/l The standard deviation, calculated from the Q-method, didn't reach the limits. BH479-8 tolerance limits in % ,,2,3,4,5,6,7,8,9 concentration in μg/l page A-56 of A-8

70 PT 2/ - TW O5 Method specific evaluation used methods for BH , percentage ,22 22,22 8,33 5 LC-MS - ext. calibr. LC-MS - standard addition LC-MS - ext. calibr. + int. standard Others method comparison for BH479-8 frequency in % too low low correct high too high Others LC-MS - ext. calibr. + int. standard LC-MS - standard addition LC-MS - ext. calibr. The values determined with standard addition showed the least variation. page A- of A-8

71 PT 2/ - TW O5 Comparison of means and reference values level mean [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%] reference value [μg/l] exp. uncertainty [μg/l] exp. uncertainty [%],2,84 8,6,227,45 2, 2,5349,358 6,7,556,54, 3,7877,474 6,,796,64,8 comparison of means and reference values overall mean HPLC-MS - standard addition reference values HPLC-MS - external calibration,,2 mean in μg/l,5,,5 level page A-58 of A-8

72 PT 2/ - TW O5 comparison of means and reference values overall mean HPLC-MS - standard addition reference values HPLC-MS - external calibration,6,5 mean in μg/l,4,3,2, 2 level comparison of means and reference values overall mean HPLC-MS - standard addition reference values HPLC-MS - external calibration mean in μg/l,9,8,7,6,5,4,3,2, 3 level page A-59 of A-8

73 PT 2/ - TW O5 rel. standard dev. method specific 3 rel. standard dev. in % level overall mean HPLC-MS - external calibration HPLC-MS - standard addition page A-6 of A-8

74 PT 2/ - TW O5 CGA level assigned value [μg/l] expanded uncertainty of the assigned value [%] standard deviation, calculated using robust statistics [μg/l] standard deviation for proficiency assessment [μg/l] standard deviation for proficiency assessment [%],2376,2,522,522 2,96,3562,424 49,9-4, ,8 2,4,82,224,22,,7596,2635,98-45,9 2,3 3,7362 9,67,533,533 2,83,82, ,97-38, ,8 sum ,6 upper tolerance limit [μg/l] lower tolerance limit [μg/l] upper tolerance limit [%] lower tolerance limit [%] number of results out below out above out [%] Recovery and matrix content CGA 62826,9,8 assigned value in μg/l,7,6,5,4,3,2, -,,,2,3,4,5,6,7,8,9 added amount in μg/l Slope of the regression line:,2, recovery:, % neg. x-axis-intercept = matrix content:,4599 μg/l expanded uncertainty of the matrix content:,4599 μg/l = % page A-6 of A-8

75 PT 2/ - TW O5 Relative standard deviation and tolerance limits CGA rel. standard deviation in % 2 5 5,,2,3,4,5,6,7,8 concentration in μg/l The standard deviation, calculated from the Q-method, reached the upper limit for one concentration. CGA tolerance limits in % ,,2,3,4,5,6,7,8 concentration in μg/l page A-62 of A-8

76 PT 2/ - TW O5 Method specific evaluation used methods for CGA , percentage ,59 2,69 6,9 5 LC-MS - ext. calibr. LC-MS - ext. calibr. + int. standard LC-MS - standard addition Others method comparison for CGA frequency in % Others LC-MS - standard addition LC-MS - ext. calibr. + int. standard LC-MS - ext. calibr. too low low correct high too high The values determined with external calibration and internal standard showed the least variation. page A-63 of A-8