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LABORATORY NAME: LABORATORY NUMBER:

Amino Acid Racemization Proficiency Study Report II: AMINO ACID D/L STANDARDS SOLUTION June 2012

AAR PT Report; Standards Solution THAA

Acknowledgements.

Firstly, thanks go to all the laboratories who agreed to take part in this study. Also to Matthew Collins, Kirsty Penkman, James Cussens at the University of York, UK and to Norman MacLeod at the Natural History Museum, London, UK for their support, to Richard Allen and Bea Demarchi for analytical technical assistance and to Ken Mathieson, FAPAS, Ferra, Sand Hutton, York for initial spreadsheet design ideas. This work was carried out at the NERC recognised North East Amino Acid Racemization Laboratory at the University of York and was funded by the Arts and Humanities Research Council (AHRC), UK with assistance from NHM in London.

Page 1 of 157

AAR PT Report; Standards Solution THAA

Contents

ACKNOWLEDGEMENTS. 1 1.1 1.2 INTRODUCTION Amino Acid Racemisation Proficiency Testing Organisation 1 9 9 10 11 12 12 12 12 13 14 14 14 15 25 25 27 27 28 29 77 77 77 78 78 79 80 . 80 80

1.2.1 2 2.1 2.2 2.3 2.4 3 3.1

TEST MATERIALS Preparation Homogeneity Distribution Result Submission HOMOGENEITY General Procedure Statistical analysis.

3.1.1 3.2 4 4.1 4.2

Evaluation of Standards Solution Test Material Homogeneity Data STATISTICAL EVALUATION; SUMMARY STATISTICS Precision Analysis Summary Statistics Experimental Standard Uncertainty of the Mean Setting the correct coverage factor for Expanded Uncertainty determination.

4.2.1 4.2.2 4.3 5 5.1

t-Distribution vs Normal Distribution STATISTICAL EVALUATION; ACCURACY & PERFORMANCE ANALYSIS Background to understanding Performance Evaluation z-Scores The Target Standard Deviation; σp Relative percentage bias

5.1.1 5.1.2 5.2

In the absence of Fitness-for-Purpose Criteria

5.2.1 5.3

The Assigned Value, The uncertainty of the Assigned value

5.3.1 5.4

Derivation of

for Amino Acids in Standards Solution Test Material

Page 2 of 157

1 6.AAR PT Report. Symbols.5 For a result from a single proficiency test.3: Summary Statistics for L and D Aspartic Acid / Asparagine Concentration Data (pM) Table 4. MEASUREMENT UNCERTAINTY Estimation of Measurement Uncertainty from Inter-laboratory comparisons.4 6.2 Appendix 1: Reverse Phase HPLC/ HPLC-Ion Exchange Gas Chromatography Internal Quality Control Appendix 2: Glossary of Abbreviations. Analytical Methods Used by Participants 6.2.1 6.5. Combined uncertainty ( Expanded Uncertainty (U).1 6.2: Summary Statistics for L and D Aspartic Acid / Asparagine Peak Area Data Table 4. Calculating Measurement Uncertainty for Amino Acids in Standards Solution Test Material Measurement Uncertainty Evaluation for a series of results using RMSbias.2 Interpreting Results . 81 105 105 106 106 107 107 108 108 108 109 135 135 138 140 142 142 142 143 152 152 152 153 154 157 6. For results from multiple proficiency tests ). Tables Table 3.3 6.1: Precision Estimates derived from Participants’ submitted results Table 4.a word of caution.5 6 6.4: Summary Statistics for L and D Aspartic Acid / Asparagine D/L Ratio Value Table 4. Standards Solution THAA 5. Standard uncertainty due to Bias ( ).2. Terms & Definitions Abbreviations Symbols Terms and Definitions Appendix 3: Tables of Critical Values Student t-distribution Factors F1 and F2 (95% significance level) Cochran’s Critical values (95% significance level) Appendix 4: References Contact Details.5: Summary Statistics for L and D Glutamic Acid / Glutamine Peak Area Data Table 4.1: Homogeneity D/L Values for Standards Solution Test Material Table 4.5.6: Summary Statistics for L and D Glutamic Acid / Glutamine Concentration Data (pM) 17 26 30 31 32 35 36 Page 3 of 157 .2 6. Measurement Uncertainty Evaluation for a single result.

3: Satisfactory Performance(Percentage within 95% Confidence Interval) 37 40 41 42 45 46 47 50 51 52 55 56 57 60 61 62 65 66 67 70 71 72 75 76 82 86 87 Table 6.13: Summary Statistics for L and D Arginine D/L Ratio Value Table 4.21: Summary Statistics for L and D Phenylalanine Concentration Data (pM) Table 4.24: Summary Statistics for D-Alloisoleucine/L-Isoleucine Concentration Data (pM) Table 4.27: Summary Statistics for L and D Leucine Concentration Data (pM) Table 4. Combined and Expanded Uncertainty for Amino Acids (using RMSbias% to access bias contributions) across ALL Laboratories.15: Summary Statistics for L and D Alanine Concentration Data (pM) Table 4.11: Summary Statistics for L and D Arginine Peak Area Data Table 4.1: Estimation of Relative Standard Uncertainty.22: Summary Statistics for L and D Phenylalanine D/L Ratio Value Table 4.AAR PT Report.20: Summary Statistics for L and D Phenylalanine Peak Area Data Table 4.8: Summary Statistics for L and D Serine Peak Area Data Table 4.18: Summary Statistics for L and D Valine Concentration Data (pM) Table 4.16: Summary Statistics for L and D Alanine D/L Ratio Value Table 4. Combined and Expanded Uncertainty Estimations for Individual Laboratories 111 Page 4 of 157 .29: Summary Statistics for L and D Methionine Peak Area Data Table 4.7: Summary Statistics for L and D Glutamic Acid / Glutamine D/L Ratio Value Table 4.2: Assigned Values.25: Summary Statistics for D-Alloisoleucine/L-Isoleucine D/L Ratio Value Table 4.12: Summary Statistics for L and D Arginine Concentration Data (pM) Table 4. 110 Table 6.14: Summary Statistics for L and D Alanine Peak Area Data Table 4. Standard Deviations and Standard Uncertainties Table 5.1: Results and Relative Percentage Bias for Total Hydrolysed Amino Acids in Standards Solution Test Material Table 5.30: Summary Statistics for HPLC Internal Standards.26: Summary Statistics for L and D Leucine Peak Area Data Table 4.2: Estimation of Relative Standard Uncertainty.23: Summary Statistics for D-Alloisoleucine/L-Isoleucine Peak Area Data Table 4.10: Summary Statistics for L and D Serine D/L Ratio Value Table 4.17: Summary Statistics for L and D Valine Peak Area / Height Data Table 4.9: Summary Statistics for L and D Serine Concentration Data (pM) Table 4.19: Summary Statistics for L and D Valine D/L Ratio Value Table 4. Standards Solution THAA Table 4. Peak Area/Height Data Table 5.28: Summary Statistics for L and D Leucine D/L Ratio Value Table 4.

1: Relationship between the t-distribution and the Normal distribution at a 95% Confidence Level. for low values of n (degrees of freedom (n-1) between 1-35). 54 Figure 4.df)) of the Mean D/L value for Phenylalanine (value of n displayed). Figure 3.12: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Arginine (value of n displayed).2: Homogeneity Amino Acid D/L Values.df)) of the Mean D/L value for Glutamic Acid / Glutamine (value of n displayed).14: Distribution of D/L Values submitted for Alanine 49 53 Figure 4.AAR PT Report.df)) of the Mean D/L value for Alanine (value of n displayed). 63 64 Figure 4.df)) of the Mean D/L value for Aspartic Acid / Asparagine (value of n displayed).05.3: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Aspartic Acid / Asparagine (value of n displayed).6: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Glutamic Acid / Glutamine (value of n displayed).1: Homogeneity Amino Acid D/L Values in Analytical Sequence Order.11: Distribution of D/L Values submitted for Arginine 48 Figure 4.22: Experimental Expanded Uncertainty (k=t(0. 64 Page 5 of 157 . 38 39 Figure 4.16: Experimental Expanded Uncertainty (k=t(0.05. 44 Figure 4. 49 Figure 4.10: Experimental Expanded Uncertainty (k=t(0.05. 39 Figure 4.19: Experimental Expanded Uncertainty (k=t(0.8: Distribution of D/L Values submitted for Serine 43 Figure 4.5: Distribution of D/L Values submitted for Glutamic Acid / Glutamine Figure 4.05.15: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Alanine (value of n displayed).df)) of the Mean D/L value for Valine (value of n displayed). 19 22 Figure 4. 34 Figure 4.df)) of the Mean D/L value for Arginine (value of n displayed).7: Experimental Expanded Uncertainty (k=t(0.05. 59 Figure 4.df)) of the Mean D/L value for Serine (value of n displayed). 29 Figure 4. 59 Figure 4. Standards Solution THAA Figures Figure 3.05.17: Distribution of D/L Values submitted for Valine 58 Figure 4.18: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Valine (value of n displayed).21: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Phenylalanine (value of n displayed).20: Distribution of D/L Values submitted for Phenylalanine Figure 4. 33 34 Figure 4. Paired Sub-samples showing Outliers.13: Experimental Expanded Uncertainty (k=t(0. 44 Figure 4.2: Distribution of D/L Values submitted for Aspartic Acid / Asparagine Figure 4. Figure 4. 54 Figure 4.05.9: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Serine (value of n displayed).4: Experimental Expanded Uncertainty (k=t(0.

7: Relative Percentage Bias for Arginine D/L Results (rpHPLC data only) in Standards Solution Test Material Figure 5.5: Relative Percentage Bias for Glutamic Acid / Glutamate D/L Results (rpHPLC data only) in Standards Solution Test Material 92 Figure 5.df)) of the Mean D/L value for DAlloisoleucine/L-Isoleucine (value of n displayed).4: Relative Percentage Bias for Glutamic Acid / Glutamate D/L Results (all data) in Standards Solution Test Material 91 Figure 5.05.12: Relative Percentage Bias for Phenylalanine D/L Results (all data) in Standards Solution Test Material 99 Figure 5.16: Relative Percentage Bias for Leucine D/L Results (all data) in Standards Solution Test Material 103 Page 6 of 157 .9: Relative Percentage Bias for Alanine D/L Results (rpHPLC data only) in Standards Solution Test Material 96 Figure 5.AAR PT Report. 74 Figure 5.6: Relative Percentage Bias for Serine D/L Results (all / rpHPLC data) in Standards Solution Test Material 93 Figure 5.10: Relative Percentage Bias for Valine D/L Results (all data) in Standards Solution Test Material 97 Figure 5.26: Distribution of D/L Values submitted for Leucine 68 69 69 73 Figure 4.23: Distribution of D/L Values submitted for D-Alloisoleucine/L-Isoleucine Figure 4.25: Experimental Expanded Uncertainty (k=t(0.24: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for DAlloisoleucine/L-Isoleucine (value of n displayed).1: Distribution of Participants’ Average Measurement Values Figure 5. Figure 4.df)) of the Mean D/L value for Leucine (value of n displayed).8: Relative Percentage Bias for Alanine D/L Results (all data) in Standards Solution Test Material 94 95 Figure 5.14: Relative Percentage Bias for D-Alloisoleucine/L-Isoleucine Results (all data) in Standards Solution Test Material 101 Figure 5.15: Relative Percentage Bias for D-Alloisoleucine/L-Isoleucine Results (rpHPLC data only) in Standards Solution Test Material 102 Figure 5.13: Relative Percentage Bias for Phenylalanine D/L Results (rpHPLC data only) in Standards Solution Test Material 100 Figure 5.11: Relative Percentage Bias for Valine D/L Results (rpHPLC data only) in Standards Solution Test Material 98 Figure 5.05.28: Experimental Expanded Uncertainty (k=t(0.27: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Leucine (value of n displayed).2: Relative Percentage Bias for Aspartic Acid / Asparagine D/L Results (all data) in Standards Solution Test Material 88 89 Figure 5. Figure 4.3: Relative Percentage Bias for Aspartic Acid / Asparagine D/L Results (rpHPLC data only) in Standards Solution Test Material 90 Figure 5. Standards Solution THAA Figure 4. 74 Figure 4.

3: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Aspartic acid / Asparagine D/L Values in Standards Solution Test Material 119 Figure 6.5: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Aspartic acid / Asparagine rpHPLC D/L Values in Standards Solution Test Material 120 Figure 6.AAR PT Report.13: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Arginine D/L Values in Standards Solution Test Material 124 Figure 6.10: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Serine D/L Values in Standards Solution Test Material 123 Figure 6.11: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Serine D/L Values in Standards Solution Test Material 123 Figure 6.16: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Alanine (rpHPLC) D/L Values in Standards Solution Test Material 126 Figure 6.12: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Arginine D/L Values in Standards Solution Test Material 124 Figure 6.14: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Alanine D/L Values in Standards Solution Test Material 125 Figure 6.15: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Alanine D/L Values in Standards Solution Test Material 125 Figure 6. 104 106 Figure 6. Standards Solution THAA Figure 5.7: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Glutamic acid / Glutamine D/L Values in Standards Solution Test Material 121 Figure 6.17: Relative Percentage Bias for Leucine D/L Results (rpHPLC data only) in Standards Solution Test Material Figure 6.6: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Glutamic acid / Glutamine D/L Values in Standards Solution Test Material 121 Figure 6.8: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Glutamic acid /Glutamine rpHPLC D/L Values in Standards Solution Test Material 122 Figure 6.4: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Aspartic acid / Asparagine rpHPLC D/L Values in Standards Solution Test Material 120 Figure 6.2: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Aspartic acid / Asparagine D/L Values in Standards Solution Test Material 119 Figure 6.1: Bias and Precision Components of Measurement Uncertainty Estimation.17: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Alanine (rpHPLC) D/L Values in Standards Solution Test Material 126 Page 7 of 157 .9: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Glutamic acid / Glutamine rpHPLC D/L Values in Standards Solution Test Material 122 Figure 6.

33: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Leucine rpHPLC D/L Values in Standards Solution Test Material 134 Page 8 of 157 .25: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Phenylalanine (rpHPLC) D/L Values in Standards Solution Test Material 130 Figure 6.18: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Valine D/L Values in Standards Solution Test Material 127 Figure 6.26: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for D-Alloisoleucine/L-Isoleucine Values in Standards Solution Test Material 131 Figure 6.AAR PT Report.29: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on DAlloisoleucine/L-Isoleucine rpHPLC Values in Standards Solution Test Material 132 Figure 6.21: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Valine (rpHPLC) D/L Values in Standards Solution Test Material 128 Figure 6.27: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on DAlloisoleucine/L-Isoleucine Values in Standards Solution Test Material 131 Figure 6.24: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Phenylalanine (rpHPLC) D/L Values in Standards Solution Test Material 130 Figure 6.28: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for D-Alloisoleucine/L-Isoleucine rpHPLC Values in Standards Solution Test Material 132 Figure 6.22: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Phenylalanine D/L Values in Standards Solution Test Material 129 Figure 6. Standards Solution THAA Figure 6.32: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Leucine rpHPLC D/L Values in Standards Solution Test Material 134 Figure 6.23: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Phenylalanine D/L Values in Standards Solution Test Material 129 Figure 6.31: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Leucine D/L Values in Standards Solution Test Material 133 Figure 6.19: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Valine D/L Values in Standards Solution Test Material 127 Figure 6.20: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Valine (rpHPLC) D/L Values in Standards Solution Test Material 128 Figure 6.30: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Leucine D/L Values in Standards Solution Test Material 133 Figure 6.

2008).AAR PT Report. most research tends to apply the technique as a relative stratigraphic tool within a defined locality using independently calibrated material. it becomes difficult to determine precise age estimates. with minimal loss and free from contamination. matrix and species specific. Similarly. the assumption being that if all sites share the same temperature history. Nonetheless. is a useful screening method with the potential to provide age estimates that go far beyond current radiocarbon timescales.1 Amino Acid Racemisation Amino Acid racemization (or epimerizationi for molecules with two carbon centres) is a diagenetic process that occurs naturally following protein synthesis. from six or seven using gas chromatography (GC) to ten or more routinely determined today using reverse-phase HPLC (rp-HPLC). any observed D/L differences can be interpreted as relative age differences. i Page 9 of 157 . This technique has been particularly successful in dating quaternary sediments using protein decomposition in fossil biominerals such as shell. it became possible to detect and measure increasing numbers of amino acids. the building blocks of proteins. Because the thermal history of a site is rarely known. These advances have continued to improve the precision in routine analysis and its acceptability as a valid dating method within the geochronology community. it becomes possible to use D/L values as indicators of relative temperature differences between same age sites. This process can take many thousands of years. from the Laevo (L-form) in life to the Dextro (D-form). if independently dated using other appropriate techniques. More recently a value of 30% representing 53-142 years in Holocene shells has been reported following the removal of outliers (Kosnik et al. The process involves the slow inter-conversion between the two chiral forms of amino acids. The unique mineral crystalline structure of shells trap original proteins. For this reason. thus the D/L ratio value can be used as an indicator of time. Note. 1987). Conversion of the L to D form continues until equilibrium is reached. Early research based on ion-exchange liquid chromatography (IE-LC) focused on the ratio between the D and L form of isoleucine but as methods developed. INTRODUCTION 1 INTRODUCTION 1. The last 30 years has seen significant changes in the analysis of amino acid racemization. The more general term ‘racemization’ will be used throughout this report to refer to both racemization and epimerization. The rates of racemization for the 20 or so different amino acids vary. 1992) with wide precision estimates for numerical ages up to 40-50% where the age equation was not calibrated locally. covering the entire quaternary period. Standards Solution THAA 1. AAR data is still often viewed dismissively. Important unaccounted differences between AAR age estimates and other dating methods have been previously reported (Wehmiller. are highly temperature dependent. AAR now requires mg sample sizes. for most amino acids this is usually equal to 1. is relatively fast and with inexpensive preparation and analytical costs.. improving to 15% when it is (McCoy.

However. performance. and it is timely to coordinate a new inter-laboratory study in support of current methodologies.htm Page 10 of 157 . Many laboratories continue to report uncertainty estimates as the CV of replicate instrumental measurements. or both. forensic science. 1984). participation in a proficiency test can provide a valuable alternative for laboratories. 1. whilst for multiple samples from the same sample location. between 3-5%. there remains inconsistency in the use and expression of precision estimates. Whilst most laboratories report CV% values between 2-5%. ambiguity in the reporting of uncertainty. are outside the scope of this report. The regular analysis of an independent quality control material forms a valuable part of external quality control (EQC) enabling comparability on a much wider scale with other laboratories. random error influencing poor repeatability. instrumental repeatability) is an important component of the overall uncertainty budget. http://www. chemical and geochemical analytical services.bam. A laboratory may be inaccurate due to systematic bias effects. and an absence of any assessment of method or laboratory bias.AAR PT Report..300 PT schemes worldwide are listed on the EPTISii website. In spite of these efforts. the accuracy of numerical age estimates relies heavily on the accuracy of analytical data. calibration and engineering. Proficiency testing is commonly encountered in sectors that rely heavily on regulation and compliance such as medicine and public health. through regular participation in proficiency tests. determination of method/laboratory precision through method validation or inter-laboratory collaborative trail. Wehmiller and Miller (2000) in their review of aminostratigraphic dating methods. It is with regard to these issues that the current study has been undertaken and attempts to address. food and feed industries. Proficiency testing (PT) is a specific type of inter-laboratory evaluation providing an objective and formalized evaluation of accuracy against a consensus value enabling an objective comparison with other laboratories’ data and is an important indicator of bias. not least due to the absence of a suitable reference material. there are often significant differences between laboratories that would result in substantial numerical age differences of 25% or greater. it is usually amongst one of the smallest contributions and is often negligible compared to method and laboratory precision estimates.de/en/about/what_is_eptis/index. between 5-10%.eptis. Although analytical precision (i. INTRODUCTION Clearly. Today more than 1. manufacturing industries. Experience within other industry sectors has demonstrated. and call for the need for a common working standard with D/L reference values. Previous inter-laboratory studies have focused on comparing individual laboratory precision estimates derived from replicate instrumental measurements (Wehmiller.e. Participation in such a scheme is also a requirement of analytical laboratories seeking accreditation to ISO 17025 (2005). report intralaboratory precision estimates for repeated instrumental determinations of the same hydrolysate of 2%. Accuracy and by inference. Standards Solution THAA 1. analysts ii European Proficiency Testing Information Service. is characterized by elements of both precision and trueness.2 Proficiency Testing It has long been widely appreciated that participation in inter-laboratory studies is a valuable tool enabling method comparisons and development. These studies have demonstrated the variability in precision between different amino acids and methods. In the absence of Certified Reference Materials (CRMs) for bias determination. 1984). that analytical performance improves over time. It is now nearly thirty years since the last inter-laboratory study was carried out using powdered fossil material (Wehmiller. for multiple analyses of different fragments of the same material.

The spread of results from a laboratory over a period of time should be compatible with that laboratory’s own evaluation of uncertainty. it has minimal bias associated with it and a known uncertainty.2. Section 6 then turns to the subject of measurement uncertainty and discusses the requirement for bias estimation in addition to precision estimates for uncertainty determination.2. 1.AAR PT Report. see Section 6. Page 11 of 157 . Whilst performance in individual rounds can identify unexpected error influences needing investigation. The next section. The standard deviation of the differences between the laboratory values and the assigned values providing a means of evaluating the standard uncertainty (Eurachem 2000). instrumental repeatability) and the percentage relative standard uncertainty (RSU%).1 Organisation This report is organized in to a number of sections. As such. percentage relative standard deviations (RSD%) otherwise referred to as the coefficient of variation (CV%). Test materials left over after the end of a proficiency test can also act as suitable matrix specific reference materials in the absence of CRMs. Standards Solution THAA 1.2.e. The section demonstrates how proficiency test data can be used to derive indicative standard uncertainty contributions and values for combined and expanded uncertainty estimates. details how test materials were prepared and distributed. it is an essential element of any laboratory’s Quality Assurance (QA) programme. instrumental replicate standard uncertainty estimates (u) representing precision from repeated measurements. long term trends are probably of greater value and can be observed using control charts (Thompson et al. relevant statistical tables and references. 2006). Finally method details as provided by the participants have been collated and together with the glossary of terms and symbols used in this report. Section 2. and Section 3 presents the homogeneity data and discusses some of the issues encountered with the assessment of homogeneity for this test material. make up the Appendices at the end of the report. Values for peak area and peak height together with concentrations and D/L values are tabulated with individual laboratory standard deviations. A summary evaluation of submitted results is presented in Section 4.. Section 5 assesses the accuracy of the results compared to the assigned value and calculates the relative percentage bias as an indication of performance. Because the value of the analyte has been determined by a consensus.. (i. INTRODUCTION and methods. together with the use of validated methods and internal quality control (IQC) procedures. The last section.

TEST MATERIALS 2 TEST MATERIALS Standards Solution 2.Participants were subsequently notified of the error. Phe. Participants receiving multiple test materials were asked to pool the contents to get the required quantity rather than simply having a larger sample sent Page 12 of 157 . Standards Solution THAA 2. AA-S-18. Arg. those using ion-exchange HPLC (HPLC-IE) were sent three individual test materials (60mg total) and those using gas chromatography (GC) were sent ten individual test materials (200mg total).2 Homogeneity Ten randomly selected test materials were sub-sampled to give 10 duplicate samples (10 x a and b).5 μmol/ml in 0. were measured into sterile plastic 3 ml eppendorf tubes and labeled. The original standards solution was made up by the addition of thirteen D-amino acid powders (D-Ala.AAR PT Report. Each test material was then dried over-night using a centrifugal evaporator and stored at room temperature to avoid condensation. (Penkman. 1998).1 N HCl). containing L-amino acids at a concentration of 2.5 (approximately). dissolved in HPLC grade water. Ser.F. prior to distribution. this frozen bulk solution was similarly diluted using HPLC grade water and thoroughly mixed prior to dispensing the individual 20 μl aliquots for the test materials. For the preparation of the test material. The results. 2005)). diluted to 0. 2. Leu. The test material was originally INCORRECTLY labeled as a racemic mix and should have been referred to as a D/L standards mix. D-amino acid powders were added to give final D/L ratios of 0. The organizers would like to apologise for any confusion (and concern!) caused to laboratories.. This bulk solution was subsequently stored at -4oC and is periodically re-sampled and diluted by a further factor of 10 prior to routine analysis and use as an in-house standard. Met. are given in Section 3. Asp. to a diluted liquid L-amino acid standard (Sigma. which were then analysed for total hydrolysable amino acids (THAA) using reverse phase HPLC (rpHPLC) according to the standard method (Kaufman and Manley W. His. Glu. Those using rpHPLC were sent a single individually numbered 20mg test material.3 Distribution Participants were previously asked to notify the organizer with details of their proposed analytical method and were sent the appropriate number of individual test materials necessary to give sufficient bulk material required by the different methods. together with their statistical evaluation.500-d. Thr and Val). 2. Pro. Aile.0001 M with HPLC grade water.1 Preparation Individual 20 μl sub-samples of an existing in-house standard solution (ref 0.

which would not be lost when pooled. instrumental repeatability standard uncertainty estimates have been determined and plotted to demonstrate the effect of the expanded uncertainty at a 95% confidence level (2 std deviations approximately) on the mean value. These are tabulated in Section 5. In this report only data given for the total hydrolysable amino acid fraction (THAA). Each set of results was given a unique laboratory number.4 Result Submission Participants were asked to submit results and method information on electronic documents sent following dispatch and no later than October 2010. Where results were submitted as the mean and standard deviation. together with an evaluation of performance. Instrumental replicate measurements provided by individual laboratories have been averaged as necessary to give a single value for each amino acid in the test material supplied. a results proforma was prepared enabling the submission of peak area and height data. 2. As a result this increased the possible number of sets of results up to twenty three. Due to the delay in results being submitted and the time required in assessing the data.e. the additional information has been summarized and tabulated in Section 4 but not evaluated. Participants were asked to indicate their primary means of determination. This way. a number of laboratories also asked to submit raw chromatogram data. The final set of results was submitted mid-December but three participants were unable to return any results on this occasion due to instrumental difficulties or other commitments. Where more than one replicate value was submitted. Due to the small number of participants in the study. assessed as the relative percentage bias. those using more than one method and those who had more than one member of staff available to carry out the analysis. The analytical methods used by each participant are summarised in Appendix I. have been evaluated. Consequently. using peak areas. Page 13 of 157 . Standards Solution THAA 2. heights or concentrations. together with concentrations and D/L values. which are also presented as histograms at the end of the section. Whilst the original intention of this study was to determine performance for only D/L amino acid values. Test materials were dispatched to eight laboratories located around the world on 15 July 2010.AAR PT Report. these values have been used for the calculation of the standard uncertainty directly. TEST MATERIALS because of the risk of heterogeneity in larger sub-samples. a defined minimum measure of homogeneity could be assured between individual sub-samples of a specified weight.. additional sets of test materials were provided to those laboratories who had more than one instrument. One laboratory provided free amino acid data (FAA) but these have not been assessed or tabulated on this occasion. i. A total of fifteen sets of results were submitted.

it is reasonable to start with the assumption that test materials are homogeneous and by carrying out homogeneity testing we are looking for evidence of heterogeneity. Standards Solution THAA 3.1 Statistical analysis. Page 14 of 157 . HOMOGENEITY 3 HOMOGENEITY Standards Solution Test Material 3. It is helpful to plot data in run order to identify trends. The Cochran’s test statistic is determined by the ratio of the maximum squared difference to the sum of squared differences. C is the Cochran’s statistic. Each individual sub-sample is then prepared according to the appropriate method and analysed in a random order under repeatability conditions. Due to the time and expense of preparing homogeneous test materials and carrying out the analysis. Resulting data should be scrutinized first for obviously anomalous values eg values greater or less than 10 times the average. (i.…. at the same time or in as short a time as possible..AAR PT Report. stability issues or measurement problems. as a single batch on the same day by the same analyst on the same instrument etc).e. The following procedure for the assessment of homogeneity follows that given in the standard ISO 13528:2005. characterized by the sampling standard deviation is negligible compared to the variation in measurement determinations carried out by participants of the proficiency test. a) Data are initially subjected to a Cochran’s outlier test. and the 2006 IUPAC International Harmonized Protocol (Thompson et al). rather than vice versa. and is the difference between each pair of duplicates.1 General Procedure The purpose of carrying out homogeneity testing. However. is the largest difference between duplicates.10a & 10b etc. assuming no problems are identified the data should be sorted and sub-samples re-paired to undergo the following statistical evaluation.1. It is recommended that ten (and no fewer than seven) randomly selected prepared and packaged test materials are selected at random using a random number generator. 2a & 2b. is to prove that any variation in composition between individual test materials. Where. Each sample is then individually homogenized and two separate portions are removed and labeled 1a and 1b. 3.

1 is analogous to the repeatability standard deviation. 2001) is analogous to the between-sample standard deviation. Note. If . the pair is identified as a Cochran’s outlier and removed from the data set. b) Evaluation of Analytical Variance Occasionally. It is therefore recommended to evaluate the analytical precision first to ensure that the method is sufficiently precise to detect inhomogeneity. between the two sub-sample values (eg. The analytical variance Note how where = within groups mean square. 1a & 1b).1. 2006. Satisfactory analytical precision is assumed if the analytical deviation is less than half the target value for standard deviation (σp) for the proficiency test (Fearn and Thompson. in Section 4. due to the absence of an external target value for standard deviation (σp). a target value for homogeneity (σh) has been determined such that c) Evaluation of Sampling Variance.. the sampling variance has not exceeded the allowable fraction of the target standard deviation. For aspartic acid / asparagine. Standards Solution THAA 3. 2001). Each test material was divided into two sub-samples and the twenty individual sub-samples where then randomized and analysed as a single batch under repeatability conditions using reverse-phase HPLC.AAR PT Report. Fearn and Thompson. If . The D/L results for the 20 sub-samples for each amino acid were plotted in run order to identify trends or problems with the data and are shown in Figure 3.2 Evaluation of Standards Solution Test Material Homogeneity Data Ten test materials were selected at random from the bulk of previously prepared individual test materials. m-1. where m is the number of duplicate pairs.e. i. . There is no evidence of inhomogeneity and the test has been passed. Thompson et al. Page 15 of 157 .10). in Section 4. HOMOGENEITY The C-value is then compared against tabulated critical values based on the required confidence level and the degrees of freedom. 1 . The sampling variance Or as Note how where = between groups mean square.1. This can mask significant between-sample differences (eg. Data are assessed using a one-way ANOVA to estimate the analytical variance. 3.. 2001). results for samples 6 and 7 and for alanine sample 5 were identified as Cochran’s oultiers and so removed from the statistical evaluation. Calculate the permissible sampling variance Calculate the critical value (c) for the test using tabulated values for F1 and F2 (ISO 13528:2005.e. i. if the above estimate is negative (Fearn & Thompson. genuine inhomogeneity between samples is missed due to large within-sample analytical variances.

Outliers that were removed from the statistical evaluation are shown as empty symbols on the charts. Figure 3..e. HOMOGENEITY The D/L results and statistical evaluation are given in Table 3. the target standard deviation (for sufficient homogeneity). In all cases. Standards Solution THAA 3.1. was set as the minimum value necessary to ensure fitness-for-purpose. Removed values and those identified as outliers have been coloured red in the tables. i. .2 shows the paired D/L values for each amino acid. that was at least twice the analytical precision (repeatability) and that the allowable sampling variance was sufficient to accommodate the observed between-sample differences. Page 16 of 157 .AAR PT Report.

3. HOMOGENEITY-

**Table 3.1: Homogeneity D/L Values for Standards Solution Test Material
**

sample id Asx D/L

replicate 1 replicate 2

analyte Glx D/L

replicate 1 replicate 2

Ser D/L

replicate 1 replicate 2

Arg D/L

replicate 1 replicate 2

Ala D/L

replicate 1 replicate 2

**1 2 3 4 5 6 7 8 9 10 mean, N origin of target sd (σh) abs. target sd (σh) & as RSD% san san / σh san / σh <0.5?
**

2 ssam

0.500 0.500 0.500 0.502 0.501 0.492 0.499 0.501 0.500 0.501 0.501 perception 0.0012 0.0006 0.4843 yes 6.93E-09 1.19E-07 6.29E-07 ACCEPT

0.500 0.500 0.500 0.500 0.501 0.501 0.502 0.501 0.502 0.501 16 C C

0.554 0.557 0.557 0.556 0.557 0.555 0.557 0.554 0.560 0.555 0.556 perception

0.557 0.558 0.555 0.555 0.558 0.555 0.556 0.555 0.554 0.556 20

0.404 0.405 0.405 0.404 0.406 0.404 0.406 0.406 0.402 0.405 0.405 perception

0.407 0.405 0.405 0.404 0.407 0.405 0.405 0.406 0.405 0.405 20

0.353 0.351 0.358 0.358 0.357 0.384 0.350 0.346 0.350 0.386 0.365 perception

0.384 0.365 0.382 0.351 0.386 0.357 0.360 0.386 0.387 0.341 20

0.471 0.471 0.469 0.471 0.471 0.471 0.471 0.470 0.469 0.470 0.470 perception

0.470 0.470 0.469 0.470 0.465 0.471 0.469 0.470 0.470 0.472 18 C

AAR PT Report; Standards Solution THAA

0.23

0.0035 0.0017 0.4928 yes 0.00E+00 1.10E-06 5.09E-06 ACCEPT

0.63

0.0019 0.0010 0.4917 yes 3.59E-07 3.40E-07 1.56E-06 ACCEPT

0.48

0.0412 0.0206 0.4991 yes 0.00E+00 1.53E-04 7.14E-04 ACCEPT

11.3

0.0014 0.0007 0.4878 yes 2.19E-07 1.67E-07 8.16E-07 ACCEPT

0.29

σall

2

critical

2 ssam <critical?

Page 17 of157

3. HOMOGENEITY

**Table 3.1: Homogeneity D/L Values for Standards Solution Test Material (continued).
**

sample id Val D/L

replicate 1 replicate 2

analyte PheD/L

replicate 1 replicate 2

D-Aile/L-Ile

replicate 1 replicate 2

Leu D/L

replicate 1 replicate 2

**1 2 3 4 5 6 7 8 9 10 mean, N origin of target sd (σh) abs. target sd (σh) & as RSD% san san / σh san / σh <0.5?
**

2 ssam

0.591 0.595 0.595 0.592 0.594 0.593 0.593 0.594 0.590 0.590 0.591 perception 0.0054 0.0027 0.4987 yes 0.00E+00 2.66E-06 1.24E-05 ACCEPT

0.590 0.595 0.587 0.590 0.591 0.593 0.593 0.586 0.589 0.588 20

0.485 0.484 0.483 0.484 0.485 0.485 0.484 0.486 0.485 0.485 0.485 perception

0.484 0.485 0.485 0.486 0.485 0.485 0.485 0.486 0.485 0.484 20

0.563 0.556 0.560 0.563 0.561 0.563 0.559 0.562 0.558 0.559 0.562 perception

0.561 0.562 0.557 0.568 0.563 0.560 0.559 0.568 0.563 0.571 20

0.585 0.585 0.585 0.586 0.584 0.588 0.585 0.585 0.583 0.586 0.586 perception

0.585 0.585 0.586 0.589 0.585 0.584 0.588 0.584 0.588 20 0.585

AAR PT Report; Standards Solution THAA

0.92

0.0013 0.0006 0.4891 yes 6.96E-08 1.43E-07 6.53E-07 ACCEPT

0.26

0.0077 0.0038 0.4975 yes 1.60E-07 5.33E-06 2.48E-05 ACCEPT

1.37

0.0028 0.0014 0.4964 yes 4.59E-07 6.82E-07 3.17E-06 ACCEPT

0.47

σall

2

critical

2 ssam <critical?

Page 18 of 157

3. HOMOGENEITY

Figure 3.1: Homogeneity Amino Acid D/L Values in Analytical Sequence Order.

Asx D/L

0.45

0.55 0.54 0.53 0.52

D/L Value

Ser D/L

0.44 0.43 0.42

D/L Value

0.51 0.5 0.49 0.48 0.47 0.46 0.45 9b 8b 6b 1a 10a 5b 5a 3a 2b 1b 10b 7a 4b 8a 6a 3b 4a 9a 7b 2a Sub-sample run order Asx D/L Asx mean

0.41 0.4 0.39 0.38 0.37 0.36 0.35 9b 8b 6b 1a 10a 5b 5a 3a 2b 1b 10b 7a 4b 8a 6a 3b 4a 9a 7b 2a Sib-sample run order Ser D/L Ser mean

Glx D/L

AAR PT Report; Standards Solution THAA

0.6 0.59 0.58 0.57

D/L Value

0.42 0.41 0.4 0.39

Arg D/L

Arg D/L

D/L Value

0.56 0.55 0.54 0.53 0.52 0.51 0.5 9b 8b 6b 1a 10a 5b 5a 3a 2b 1b 10b 7a 4b 8a 6a 3b 4a 9a 7b 2a Sub-sample run order Glx D/L Glx mean

0.38 0.37 0.36 0.35 0.34 0.33 0.32 9b 8b 6b 1a 10a 5b 5a 3a 2b 1b 10b 7a 4b Sub-sample run order 8a 6a 3b 4a

Arg mean

9a 7b

2a

Page 19 of 157

43 0.59 0.48 0.47 D/L Value D/L Value Phe D/L 0.3.47 0.61 0.51 0.43 0.5 9b 8b 6b 1a 10a 5b 5a 3a 2b 1b 10b 7a 4b 8a 6a 3b 4a 9a 7b 2a Sub-sample run order D-Aile/L-Ile D-Aile/L-Ile Page 20 of 157 .45 0. HOMOGENEITY Figure 3.4 9b 8b 6b 1a 10a 5b 5a 3a 2b 1b 10b 7a 4b 8a 6a 3b 4a 9a 7b 2a Sub-sample run order Ala D/L Ala mean 9b 8b 6b 1a 10a 5b 5a 3a 2b 1b 10b 7a 4b 8a 6a 3b 4a 9a 7b 2a Sub-sample run order AAR PT Report.5 0.6 Val D/L Val mean 0.58 0.1: Homogeneity Amino Acid D/L Values in Analytical Sequence Order (continued).48 0.46 0.56 0.42 0. Standards Solution THAA Val D/L 0.59 0.62 D/L Value D-Aile/L-Ile 0.57 0.42 0. Ala D/L 0.44 0.56 0.49 0.65 0.64 0.54 0.41 0.5 0.45 0.4 Phe D/L Phe mean 0.53 0.46 0.58 0.55 0.57 D/L Value 0.63 0.44 0.41 0.52 0.55 9b 8b 6b 1a 10a 5b 5a 3a 2b 1b 10b 7a 4b 8a 6a 3b 4a 9a 7b 2a Sub-sample run order 0.6 0.49 0.

(continued) Leu D/L 0. Standards Solution THAA 3.62 D/L Value Leu D/L Leu mean 0.61 0. HOMOGENEITY Figure 3.59 0.6 0.AAR PT Report.1: Homogeneity Amino Acid D/L Values in Analytical Sequence Order.56 0.63 0.55 9b 8b 6b 1a 10a 5b 5a 3a 2b 1b 10b 7a 4b 8a 6a 3b 4a 9a 7b 2a Sub-sample run order Page 21 of 157 .65 0.57 0.58 0.64 0.

39 outlier 2 rep 1 outlier 1 0.35 0 1 2 3 4 5 6 7 Sample Number 8 9 10 11 12 Page 22 of 157 . Paired Sub-samples showing Outliers.41 0.56 0. Standards Solution THAA 3.37 0.43 D/L Value rep 2 0. Asx D/L 0.54 0.55 0.53 D/L Value 0. HOMOGENEITY Figure 3.47 0.58 D/L Value 0.45 0 1 2 3 4 5 6 7 Sample Number 8 9 10 11 12 outlier 1 Glx D/L 0.45 0.5 0 1 2 3 4 5 6 7 Sample Number 8 9 10 11 12 Ser D/L 0.51 0.52 rep 2 outlier 2 rep 1 outlier 1 0.6 0.49 rep 2 outlier 2 rep 1 0.AAR PT Report.2: Homogeneity Amino Acid D/L Values.

3 0 1 2 3 4 5 6 7 Sample Number 8 9 10 11 12 outlier 1 Ala D/L 0.38 D/L Value 0. Paired Sub-samples showing Outliers.4 0.44 0.55 0 1 2 3 4 5 6 7 Sample Number 8 9 10 11 12 outlier 1 Page 23 of 157 . HOMOGENEITY Figure 3.48 D/L Value 0.AAR PT Report. Standards Solution THAA 3. Arg D/L 0.32 0.34 rep 2 outlier 2 rep 1 0.63 D/L Value 0.61 rep 2 outlier 2 rep 1 0.5 0.57 0.42 0.46 0.36 0.59 0.65 0.4 0 1 2 3 4 5 6 7 Sample Number 8 9 10 11 12 rep 2 outlier 2 rep 1 outlier 1 Val D/L 0.2: Homogeneity Amino Acid D/L Values.

Phe D/L 0.5 0 1 2 3 4 5 6 7 Sample Number 8 9 10 11 12 outlier 1 Leu D/L 0.58 D/L Value 0.61 0. Paired Sub-samples showing Outliers.48 D/L Value 0.54 rep 2 outlier 2 rep 1 0.42 0.59 0.63 D/L Value 0.44 0. HOMOGENEITY Figure 3.52 0.55 0 1 2 3 4 5 6 7 Sample Number 8 9 10 11 12 Page 24 of 157 .AAR PT Report.56 0.46 0.5 0.57 rep 2 outlier 2 rep 1 outlier 1 0. Standards Solution THAA 3.6 0.4 0 1 2 3 4 5 6 7 Sample Number 8 9 10 11 12 rep 2 outlier 2 rep 1 outlier 1 D-Aile/L-Ile 0.65 0.2: Homogeneity Amino Acid D/L Values.

incorporates both the within and between laboratory variability and is a single measure of the variability or uncertainty of the measurement procedure associated with precision. have also been evaluated separately. AOAC. It should be noted that where all data have been used in the evaluation of precision estimates in Table 4. 2010). Estimates were calculated using a one way analysis of variance (ANOVA). As GC data were submitted as average D/L values. Results from the analysis of relative bias presented in Section 5. the (or ) given in Tables 4. all rpHPLC data and HPLC-IE data for D-alloisoleucine/L-isoleucine. because all HPLC-IE data came from the same laboratory. ( .30) but smaller than method repeatability which includes additional variability arising from the analysis of different samples of the same material by a single laboratory. This reflects the variability that a single laboratory might be expected to achieve for replicate measurements of the same sample. is the overall inter-laboratory between sample standard deviation. 2000) known as the “top-down” approach to uncertainty estimation (RSC Page 25 of 157 . it was not possible to determine comparable GC specific precision estimates. 1995. participants were invited to submit peak information and concentration data in addition to the D/L value data requested for the proficiency study. suggest possible empirical differences between methods. Typically. this includes GC D/L values derived from both peak area and height data where given. Therefore.1 summarises indicative values of repeatability and reproducibility precision estimates for each amino acid derived from all participants’ individual D/L values. Standards Solution THAA 4. is a measure of the overall within laboratory precision derived from all participating laboratories. Wehmiller. is the inter-laboratory reproducibility standard deviation and a measure of the overall precision for any given amino acid in the specified test material. reproducibility ( ) values should more correctly be interpreted as an intra-laboratory reproducibility or intermediate precision estimate. this may be slightly larger than instrumental precision estimates derived from a single laboratory (i. due to random error effects. Consequently a substantial quantity of information was captured. Due to time constraints it was not possible to evaluate all of this additional data. Often the is more conveniently given as the relative repeatability standard deviation expressed as a percentage. allowing for unequal replicate numbers. Table 4. Such determinations are more commonly used to assess data from method specific collaborative trials (Horwitz. On this occasion.2 – 4.AAR PT Report. although the laboratory subsequently confirmed that in practice only peak area data would be used for chronology building.1 Precision Analysis In keeping with the style of previously conducted inter-laboratory comparisons (Wehmiller.1. However. this represents an interlaboratory approximation of the instrumental precision only.e. under repeatability conditions. Summary Statistics 4. STATISTICAL EVALUATION – Summary Statistics 4 STATISTICAL EVALUATION. 1984. and indicates the level of agreement between participants. The repeatability standard deviation (Table 4.1). although a comparison of L and D amino acid concentrations would be enlightening.

43 0.0019 0.28 1.01 0.41 11. The relative standard deviation of reproducibility ( ) obtained from a collaborative trial may then be used for the assessment of proficiency test data as it provides an external value for the target standard deviation.561 0.69 0.0034 0.21 Glx D/L-rpHPLC Ser D/L-rpHPLC Arg D/L-rpHPLC Ala D/L-All Ala D/L-rpHPLC Val D/L-All Val D/L-rpHPLC Phe D/L-All Phe D/L-rpHPLC D-Aile/L-Ile -allb D-Aile/L-Ile -rpHPLC D-Aile/L-Ile -HPLC-IE D-Aile/L-Ile -GC Leu D/L-all a a Not determined 0.56 1.83 2.00 0.03 0.02 0.01 0.02 9.0122 0.67 0.0039 0.03 11.0037 0. Table 4.60 5.43 0.0037 0. and can be found in (ISO 5725. i.03 0.01 0. It should be noted that these values have not been used in the later performance evaluation but are given for information and indicative purposes only.566 0.05 0.02 9.70 1.03 0.29 6.e.69 0.01 0.03 0.0037 0.491 0.53 1.438 0. Precision estimates are calculated using ANOVA. STATISTICAL EVALUATION – Summary Statistics Analytical Methods Committee.0042 0.0007 0.402 0.78 1.03 0.0105 1.32 1.00 0. thus.75 0.66 0.05 0.0105 1.42 6.01 0.97 3.1: Precision Estimates derived from Participants’ submitted results amino acid no of sets of results total no of mean replicates Sr RSDr% SL RSDL% SR RSDR% (m) Asx D/L-all Glx D/L-all a (N) 27 23 27 23 23 16 27 23 26 23 27 23 28 23 4 21 18 0.01 0.37 5.01 0.43 6.01 0.55 0.06 0.517 0.11 0.0019 0.06 0.04 0.29 5.493 0. ISO 21748. 1995).49 11.79 0. All submitted results have been included in this evaluation without removal of outliers as would otherwise be the case with collaborative trail data.36 0.74 1.06 0.15 3.564 0.00 0.81 0.75 0. in the case of empirical methods.499 0.0034 0.68 0.74 12.AAR PT Report.576 0..17 2. GC and HPLC-IE data Page 26 of 157 . albeit being slightly exaggerated due to additional method variation between participants. Standards Solution THAA 4.01 0.597 b Asx D/L-rpHPLC a 14 10 14 10 10 8 14 10 13 10 14 10 15 10 2 11 8 0.72 12.75 0.01 0.24 0. precision evaluation of the submitted PT results will help give an indication of the agreement between laboratories.84 0.587 0.376 0.59 1. (Note.0042 0.52 1. However.435 0.31 6.01 0.76 Leu D/L-rpHPLC = rpHPLC and GC data = rpHPLC. 1994.03 0.0042 0. Further details on the calculations of .27 4.90 2.64 1. 2010).71 11. On this occasion it is the intention to observe the behaviour of all submitted results rather than to define best practice.492 0.489 0.74 1.553 0. PT data should be assessed against method specific precision estimates).04 0.74 0.01 0.0037 0. in the absence of a collaborative trial.0042 0.06 0. it describes how the data is expected to behave under conditions of best practice.40 0.03 0.03 0.

Experimental standard uncertainty of the mean is obtained from. In this report.2. has been evaluated for each amino acid and data are presented in figures to illustrate the effect of uncertainty on the mean value of submitted replicate data.2-4. and these have been displayed as the mean value with associated error bars on the charts. It is important to appreciate that is the uncertainty associated with the mean of replicate instrumental results only. the greater the confidence in the mean as an estimate of the true value μ. also known as the coefficient of variance. As a standard uncertainty. including internal standard data provided by participants. for comparison. This gives little confidence as in nearly one out of every three occasions.. Standard uncertainty is always expressed as a standard deviation. i. Note. Individual laboratory replicate D/L values as submitted.30 for rpHPLC peak areas and concentrations. However. in practice it is often more helpful to consider a confidence interval equivalent to 2 standard deviations or a Page 27 of 157 . represents a confidence level equivalent to 68% or 1 standard deviation. Measurement uncertainty determination is discussed this in more detail in Section 6 later in the report. and the smaller the uncertainty. Calculations have been carried out on each laboratory’s results to give the instrumental precision estimate as the standard deviation ( ) and relative standard deviation. where. there are various names used to describe ( ) as mentioned above. Standards Solution THAA 4. It contributes to the bias component of the overall combined uncertainty associated with the measurement system (see Figure 6. Thus. will be referred to as the experimental standard uncertainty of the mean and reflects the confidence in the mean of replicate values. 4. (Strictly speaking this should be determined using independent repeated measurements and not replicate measurements of the same sample). expressed as a percentage relative to the mean. . for each amino acid.e. This means that 68 percent of the means of repeated replicate results will fall within these limits either side of the mean determined by . Only one laboratory reported data for the free amino acids and this has not been included in this report.1) but is only one component of the uncertainty that should be reported with the mean of analytical results. the experimental standard deviation (or standard error or standard uncertainty) of the mean ( ) and the relative standard uncertainty of the mean ( ). the larger the value of n.2 Summary Statistics Summary statistics are presented in Tables 4. the mean is likely to fall outside of this range. Each laboratory’s expanded uncertainty to 2 std deviations or an approximate 95% confidence level. Which. peak-height values for HPLC-IE and D/L values for all participants.AAR PT Report. are also shown graphically against the assigned values determined in Section 5. The observed standard deviation of replicate instrumental measurements describes the distribution of data and is not the same as the uncertainty estimate for the mean. thus either experimental standard deviation or standard uncertainty of the mean would be acceptable.1 Experimental Standard Uncertainty of the Mean Depending on information sources. It should be noted that GC data was submitted as the mean of n replicates with a stated standard deviation. s. Additionally. Data are presented as submitted on the result proforma for each of the total hydrolysed amino acids. STATISTICAL EVALUATION – Summary Statistics 4. have been determined.

However. s. no uncertainty estimation can be made. Standards Solution THAA 4. A comparison of Page 28 of 157 .05). it would be possible to calculate the range of values within which repeated experimental means of n measurements were likely to fall with a certain level of confidence. To compensate for the use of the sample standard deviation. it may also be helpful to observe the effect of uncertainty on individual elements to aid method development or quality improvements. n=2. 2005) then the distribution of mean values conforms with the expectation of normality. However. As discussed above..7 and the expanded uncertainty becomes over six times larger than otherwise predicted if k=2! Thus the range in which the true value lies with 95% confidence broadens and becomes. laboratories can often be found to overlook this t-value correction by quoting expanded uncertainties derived from the more favorable k=2. However for decreasing values of n. and its role in determining the Expanded uncertainty is now considered in more detail below. i. Where the number of replicate measurements is large. 4. t=3. The value of t depends on the value of n and the required level of confidence and can be read from any two-tailed t-table in statistical texts.4% probability level in experimental design (usually rounded to 95% for simplicity). Thus for n=5 (degrees of freedom=4) at 95% confidence level (α=0. a 2 standard deviation or approximately 95% confidence level is usually acceptable. Note that where a single replicate value is reported. . the true value of μ and σ cannot be known and the aim of experimental investigations is to get the best estimate of μ from the sample mean. for most general applications. in real terms. rather than the population standard deviation σ. or for a pair of replicates.e.AAR PT Report. if analytical results represented an entire population and the true value μ and standard deviation σ were known.df) and the more frequently used k=2 have been calculated and values expressed as a percentage.96σ) and the relevant confidence interval where (approx) 95% of values would lie would be in the range. data are given in tables and presented as two comparative figures. The differences observed in expanded uncertainties between different amino acids for a single laboratory highlights the ease or difficulty of analysis and instrument repeatability.2 Setting the correct coverage factor for Expanded Uncertainty determination. t=12. k=2 is replaced by the critical t-value as a correction term. is the coverage factor set according to the required confidence Expanded uncertainty is more usually determined following the combination of all individual standard uncertainty components as demonstrated in Section 6. Relative Expanded uncertainties of the submitted results using both k=t(0. df=1.2. In practice and often for simplicity rather than intent. The coverage factor. To determine these extended limits of confidence an Expanded Uncertainty (U) is calculate thus. Theoretically. STATISTICAL EVALUATION – Summary Statistics 95.05.7% confidence or 1 in 300. the characteristic bell shaped curve of the normal distribution flattens and widens reflecting the reduced confidence in the value as the best estimate of μ and our uncertainty estimate increases. n=30 or more (Currell and Dowman. Thus in this instance (actually its 1. .18 compared to the original value of k=2. where level. This equates to a 1 in 20 chance of falling outside the range. For each amino acid. 3 standard deviations would be equivalent to 99.

Figure 4. Demands for quality and lower uncertainty estimates must be balanced against the extra cost and time incurred by increasing replicate numbers not to mention material availability and often it is financial and resource constraints that become deciding factors.AAR PT Report. 4. Thus the effect of increasing the number of replicate values from 2 to 3 will make a substantial reduction in the expanded uncertainty estimate. It can be clearly seen that for a pair if replicate values.35 where n is the sample size). The contribution of a particular standard uncertainty estimate to the overall uncertainty budget.2. the effect of the number of replicates is an important practical consideration. (degrees of freedom (n-1) between 1 . t-values are given in Appendix 3. for low values of n (degrees of freedom (n-1) between 1-35). It therefore makes more sense to put time into increasing the number or individual samples tested than spending the same time increasing the number of instrumental replicates.1. the t-value correction becomes 3. It illustrates the t-distribution deviation (red line) away from normal (black line) for low sample numbers. the contribution of instrumental analytical precision is likely to me much smaller than the contribution from method precision between different samples. as there is more to gain in reducing the expanded uncertainty. The t-value given on the y-axis is used as the correction term in the calculation of expanded uncertainty. Increasing the number of replicate values to n =3 (df = 2).3. should also be borne in mind. 13 12 11 10 9 8 t-value 7 6 5 4 95% confidence 2 std deviation 3 2 1 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Degrees of freedom (n-1) Page 29 of 157 . introducing a correction factor more than 10x larger (t-value = 12. For example.3 t-Distribution vs Normal Distribution The relationship between the t-distribution and the Normal or Gaussian distribution at 2 standard deviations (95% confidence) is shown below in Figure 4. The level of benefit gained by increasing the numbers of replicates gradually diminishes until normality is achieved at about n = 25. Whilst these effects are interesting to observe analytically. but the difference will not be quite as significant. whilst increasing the number of replicates from 3 to 4 will still make an improvement. and for n = 4 (df = 3). Standards Solution THAA 4. there is a significant deviation from normal. STATISTICAL EVALUATION – Summary Statistics expanded uncertainties across all laboratories for any individual amino acid also demonstrates the effect of different methods or even using different numbers of replicates for the same method. reduces the t-value correction to 4. (df = 1).7) on the standard uncertainty estimate.1: Relationship between the t-distribution and the Normal distribution at a 95% Confidence Level.

60 2.87 8.84 44.6 2.04 3.7 97.7 314.93 12.710 12.7 3.1 2.61 34.92 69.2 38.2 191.7 66.0 161.21 L-Asx peak area 1 2 3 4 5 6.17 33.76 282.40 26.90 264.46 5.74 2.89 7.7 62.32 12.54 5.71 6.53 549.4 47.81 Uncertainty of Mean & Expanded U at 95% CL std u 3000.8 68.36 40. Standards Solution THAA 1 2 3 4 5 6.48 63.2 7.710 t critical (0.35 8.41 12.710 31.9 75.1 6.7 88.48 2.01 6.8 245.92 3.710 12.3 135.47 4.2: Summary Statistics for L and D Aspartic Acid / Asparagine Peak Area Data Lab No method a 8976 2483 2733 b 9738 2608 2554 c 2233 Submitted Replicate data d 12335 e 20794 f g h i j Standard Deviation mean 10815 2546 2643 n 5 2 2 std dev 6709.42 Exp U% (k=2) 55.50 10.75 3.73 7.20 2.df) 2.1 7.1 157.05.17 4.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 9354 5886 5548 a 4473 1270 1407 9883 6568 5857 b 4777 1325 1313 c 1108 d 5929 e 10133 f g h i j 9619 6227 5702 mean 5284 1297 1360 2 2 2 n 5 2 2 373.4. STATISTICAL EVALUATION – Summary Statistics Table 4.22 6.21 4.710 12.710 12.4 std dev 3251.83 CV% 61.0 97.74 .75 5.710 12.11 3.4 2.8 482.0 138.90 6.20 5.99 4.91 6.9 106.4 127.65 11.1 6.00 3.1 68.42 Exp U% (k=tcrit) 76.710 12.46 3.777 12.64 35.1 CV% 62.710 30.59 40.83 5.6 3.710 12.85 53.0 3.1 154.710 12.0 27.01 8.44 5.95 5.66 5.93 71.710 12.40 5.0 223.43 4.05.52 4.710 12.71 RSU% 27.df) 2.710 12.710 12.9 222.51 4.53 2.1 7.6 173.9 RSU% 27.81 Page 30 of 157 22049 5705 2994 2946 23149 5260 3310 3142 22599 5482 3152 3044 2 2 2 2 777.9 113.03 31.00 7.52 2.2 7.05 5.3 341.80 t critical (0.7 48.93 51.03 4.4 std u 1454.40 Exp U% (k=2) 55.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 11005 2841 1508 1461 4663 2932 2759 11571 2614 1645 1557 4934 3279 2910 11288 2728 1577 1509 4798 3105 2835 2 2 2 2 2 2 2 400.08 4.5 89.36 3.49 4.3 218.710 Exp U% (k=tcrit) 77.777 12.710 34.22 43.10 10.710 12.03 D-Asx peak area AAR PT Report.04 55.16 4.

4 0.710 12.710 12.06 Exp U% (k=2) 2.63 1.25 10.6 2.53 AAR PT Report.710 12.55 3.1 0.2 8 9 10 11 12 13 14 15 107 141 124 a 25 132 143 128 b 26 c 26 d 24 e 26 f g h i j 120 142 126 mean 26 2 2 2 n 5 18.777 135.777 Exp U% (k=tcrit) 2.40 RSU% 1.3 2.1 9.7 15.1 1.710 12.21 0.1 6.29 0.9 1.37 Uncertainty of Mean & Expanded U at 95% CL std u 0.5 std dev 0.84 12.52 12.61 17. STATISTICAL EVALUATION – Summary Statistics Table 4.710 12.26 1.4 1.710 8.1 6.71 2.6 RSU% 1.76 1. Standards Solution THAA D-Asx Conc 1 2 3 4 5 6.2 7.14 Page 31 of 157 62 59 61 61 63 62 62 61 61 2 2 2 0.3: Summary Statistics for L and D Aspartic Acid / Asparagine Concentration Data (pM) Lab No method a RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 51 b 53 c 53 Submitted Replicate data d 51 e 54 f g h i j Standard Deviation mean 52 n 5 std dev 1.36 15.22 136.73 9.65 0.8 0.10 0.95 L-Asx Conc 1 2 3 4 5 6.710 10.05.8 0.66 4.52 1.98 37.0 0.91 0.710 12.5 0.3 10.2 0.12 t critical (0.21 1.05.df) 2.79 Exp U% (k=2) 2.33 0.1 6.27 21.73 8.51 21.90 1.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 31 30 30 53 70 62 31 31 30 66 71 64 31 30 30 60 71 63 2 2 2 2 2 2 0.97 CV% 2.51 2.1 7.97 0.710 12.3 0.76 0.7 0.95 0.6 0.710 12.68 0.54 29.8 1.41 5.2 7.1 7.8 0.74 Exp U% (k=tcrit) 3.2 1.4 1.83 2.2 1.27 2.00 4.35 1.07 1.17 3.8 std u 0.4.54 12.2 CV% 2.32 .80 3.04 17.37 0.710 t critical (0.df) 2.710 12.36 1.70 12.

0046 0.0005 0.4: Summary Statistics for L and D Aspartic Acid / Asparagine D/L Ratio Value Lab No D/L Asx 1 2 3 4 5 1 6.777 2.29 0. Standards Solution THAA = submitted as the mean and standard deviation of n results.09 Uncertainty of Mean & Expanded U at 95% CL std u 0.02 2.34 0.0006 0.511 0.13 t critical (0.82 0.504 0.498 0.56 8.508 0.65 0.647 0.498 0.01 1.38 0.4.96 6.497 0.0099 0.0003 RSU% 0.0201 0.496 5 1 5 2 2 2 2 2 0.481 e 0.599 0.496 0.0009 0.647 0.0007 0.497 0.01 3.710 1.0001 8.510 0.710 12.14 0.496 0.1 1 7.500 0.2 1 7.05 0.0009 0.31 0.69 0. STATISTICAL EVALUATION – Summary Statistics Table 4.34 0.01 8.0006 0.10 0.df) 2.0140 0.2 8 9 10 11 12 13 14 15 1 method a RP RP RP IE IE GCA GCHt GCA GCHt RP RP RP RP RP RP RP RP 0.499 0.10 .01 0.0002 0.710 12.497 0.49 0.514 c 0.491 0.79 4.11 0.86 2.487 f g h i j Standard Deviation mean 0.500 0.17 0.591 0.497 0.0032 0.08 0.496 Submitted Replicate data d 0. GCA= derived using peak area GCHt = derived using peak height Page 32 of 157 0.20 0.16 0.0032 0.710 12.92 0.499 0.656 0.0071 0.25 1.710 12.491 0.25 0.06 Exp U% (k=2) 1.12 0.0003 0.710 Exp U% (k=tcrit) 1.499 0.27 1.515 n 5 2 2 std dev 0.500 0.65 0.30 0.14 3.0450 0.17 0.0005 CV% 1.05.710 12.0006 0.07 0.499 0.710 11.591 0.0025 0.0004 0.656 0.07 1.498 0.56 0.0580 0.497 2 2 2 0.25 0.777 12.64 0.13 8.498 0.0000 4.0018 0.02 6.09 12.710 12.44 0.497 0.01 1.52 21.515 b 0.1 1 6.60 AAR PT Report.599 0.777 12.710 12.12 0.499 0.0259 0.

48 0.71 0.49 0.45 0.69 0.2 8 9 10 11 13 14 15 Submitted Value Submitted mean & std dev Assigned value (rpHPLC only) Assigned value (all data) Laboratory Number Page 33 of 157 D/L Value .54 0. Standards Solution THAA 0.65 0.68 0.58 0.2: Distribution of D/L Values submitted for Aspartic Acid / Asparagine RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP AAR PT Report.53 0.47 0.52 0.50 0.64 0.55 0.2 7.67 0.66 0.62 0.60 0.44 0.4.43 1 2 3 4 5 6.1 6.59 0.70 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.46 0.73 0.72 0.56 0.61 0.63 0.1 7.51 0.57 0.

45 0.69 0. RP 0.53 2 2 0.71 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.67 5 0.59 0.43 1 2 3 4 5 6.61 2 1 D/L Value 0.49 0.2 7.1 7.49 0.51 2 5 2 2 2 2 2 2 0.3: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Aspartic Acid / Asparagine (value of n displayed).43 1 2 3 4 5 6.67 5 0.51 2 5 2 2 2 2 2 2 0.69 0.2 8 9 10 11 13 14 15 Laboratory Number Page 34 of 157 . STATISTICAL EVALUATION – Summary Statistics Figure 4.57 0.55 0.57 0.73 Replicate means 0. Standards Solution THAA 4.2 7.53 2 2 0.73 Replicate means 0.05.47 0. RP 0.71 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.63 0.65 5 0.4: Experimental Expanded Uncertainty (k=t(0.59 0.df)) of the Mean D/L value for Aspartic Acid / Asparagine (value of n displayed).1 6.AAR PT Report.47 0.65 5 0.45 0.63 0.1 6.1 7.55 0.61 2 1 D/L Value 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 4.

3 91.87 5.710 t critical (0.04 5.91 RSU% 27.99 2.31 53.777 12.5: Summary Statistics for L and D Glutamic Acid / Glutamine Peak Area Data Lab No method a 8670 2317 2511 b 9476 2447 2378 c 2139 Submitted Replicate data d 11999 e 19829 f g h i j Standard Deviation mean 10423 2382 2445 n 5 2 2 std dev 6394.88 9.9 2.36 8.710 32.53 6.23 3.1 6.36 5.87 3.65 5.710 12.3 148.57 61.54 4.1 113.8 38.710 34.710 12.08 8.4 315.95 5.4 201.43 9.2 std u 1599.05.6 30.86 Uncertainty of Mean & Expanded U at 95% CL std u 2859.90 12.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 11712 2945 1534 1527 4774 3036 2891 12357 2695 1694 1647 5073 3380 3066 12034 2820 1614 1587 4924 3208 2978 2 2 2 2 2 2 2 456.84 3.90 10.2 3.21 4.30 47.7 84.41 38.0 304.1 7.28 7.44 63.08 10.72 2.710 12.1 6.777 12.4 215.44 2.2 7.54 4.1 155.710 Exp U% (k=tcrit) 76.44 2.59 5.6 87.18 34.70 5.97 Exp U% (k=tcrit) 76.7 149.48 69.66 L-Glx peak area 1 2 3 4 5 6.30 7. Standards Solution THAA D-Glx peak area 1 2 3 4 5 6.7 242.4 445.44 4.72 12.34 265.6 219.710 12.76 3.49 Page 35 of 157 20602 5322 2779 2771 21677 4892 3064 2981 21140 5107 2922 2876 2 2 2 2 760.6 94.75 7.710 12.82 Exp U% (k=2) 55.96 7.5 211.7 4.710 12.69 4.32 4.96 6.2 54.04 29.4 std dev 3577.2 142.04 5.5 43.2 7.79 6.710 12.6 124.35 2.68 4.73 Exp U% (k=2) 54.35 3.30 12.4 3.6 64.17 322.710 12.7 RSU% 27.9 2.7 171.55 34.98 37.2 80.710 12.710 12.65 67.1 7.44 5.93 46.2 177.710 12.52 2.98 3.99 AAR PT Report.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 8736 5481 5179 a 4870 1250 1342 9267 6111 5489 b 5185 1327 1280 c 1204 d 6715 e 11090 f g h i j 9002 5796 5334 mean 5813 1289 1311 2 2 2 n 5 2 2 375.4 59.09 36.3 CV% 61.83 38.89 5.56 4.11 CV% 61.07 56. STATISTICAL EVALUATION – Summary Statistics Table 4.04 5.710 12.4.45 t critical (0.8 66.df) 2.4 104.37 .6 125.95 5.df) 2.710 37.9 2.99 4.16 537.17 7.710 12.87 5.05.

32 0.2 8 9 10 11 12 13 14 15 104 137 121 a 28 130 139 125 b 28 c 28 d 28 e 29 f g h i j 117 138 123 mean 28 2 2 2 n 5 18.95 0.82 10.4 0.42 6.52 12.4 15.02 1.19 Exp U% (k=2) 1.0 0.71 2.0 0.710 11.0 1.6 1.2 2.8 2.710 12.1 7.05.16 15.4 0.50 1.777 138.01 3.710 12.07 36.64 21.7 0.1 1.10 2.3 7.1 6.710 13.23 4.14 8.75 t critical (0. Standards Solution THAA D-Glx Conc 1 2 3 4 5 6.41 12.4 1.6 1.4.97 0.20 5.52 20.95 Uncertainty of Mean & Expanded U at 95% CL std u 0.26 7.5 1.4 1.710 12.710 12.00 0.4 9.9 0.60 RSU% 0.4 RSU% 0.0 std u 0.30 Exp U% (k=tcrit) 1.710 12.5 1.710 12.68 21.87 Exp U% (k=2) 1.05.69 1.40 20.710 12.710 12.2 10.95 .82 Page 36 of 157 61 57 60 60 61 61 60 59 61 2 2 2 0.76 1.51 0.64 2.4 0.710 12.59 1.51 10.2 7.94 1.95 37.90 1.2 7. STATISTICAL EVALUATION – Summary Statistics Table 4.70 1.7 0.84 2.df) 2.5 1.38 0.17 1.36 12.74 5.df) 2.42 139.7 2.777 Exp U% (k=tcrit) 2.1 7.42 L-Glx Conc 1 2 3 4 5 6.0 CV% 1.8 std dev 0.6: Summary Statistics for L and D Glutamic Acid / Glutamine Concentration Data (pM) Lab No method a RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 49 b 51 c 50 Submitted Replicate data d 49 e 51 f g h i j Standard Deviation mean 50 n 5 std dev 1.18 3.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 34 32 33 57 76 68 33 34 34 71 77 70 33 33 33 64 76 69 2 2 2 2 2 2 0.1 6.87 2.55 4.710 t critical (0.84 0.88 0.28 12.26 CV% 1.89 AAR PT Report.

552 0.558 0.553 0.1 1 7.690 0.551 0.88 1.710 8.710 1.0005 0.11 0.19 10.0014 0.0021 0.60 14.710 12.08 12.10 2.710 12.12 0.541 0.0007 0.0028 0.68 0.599 0.14 0.777 12.52 Uncertainty of Mean & Expanded U at 95% CL std u 0.7: Summary Statistics for L and D Glutamic Acid / Glutamine D/L Ratio Value Lab No D/L Glx 1 2 3 4 5 1 6.47 .542 0.0010 0.558 0.0002 0.08 0.710 12.777 2.599 0.09 0.551 0.23 2.41 3. STATISTICAL EVALUATION – Summary Statistics Table 4.563 Submitted Replicate data d 0.2 8 9 10 11 12 13 14 15 1 method a RP RP RP IE IE GCA GCHt GCA GCHt RP RP RP RP RP RP RP RP 0.710 Exp U% (k=tcrit) 1.05.547 0.09 0.38 1.0005 0.534 b 0.32 0.623 0.23 0.0018 0.0003 0.552 0.568 0.559 0.15 0.559 f g h i j Standard Deviation mean 0.592 0.0015 0.562 0.553 0.13 0.2 1 7.0063 0.18 1.25 0.0304 0.710 12.0183 0.35 0.554 0.27 0.0013 0.06 0.39 0.74 t critical (0.15 0.560 e 0.93 11.538 c 0.592 0.04 0.554 0.0006 3.0007 0.0680 0.45 0.690 0.19 0.623 0.12 6.0410 0.540 0.1 1 6.35 0.02 0.552 0.710 12.18 0.547 0.777 12.4.536 n 5 2 2 std dev 0.22 0.0006 0.558 2 2 2 0.570 0.0008 0.12 0.51 0.18 0.552 0.0020 RSU% 0.569 0.16 0.55 0. Standards Solution THAA = submitted as the mean and standard deviation of n results.23 2.552 5 1 5 1 2 2 2 2 0. GCA= derived using peak area GCHt = derived using peak height Page 37 of 157 0.52 AAR PT Report.df) 2.546 0.49 4.547 0.0009 6.0028 CV% 1.553 0.710 12.37 Exp U% (k=2) 1.10 5.

50 1 2 3 4 5 6.56 0.4.54 0.68 0.52 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.57 0.58 Assigned value (all data) AAR PT Report.1 7.66 0.69 0.55 0.2 8 9 10 11 13 14 15 Laboratory Number Page 38 of 157 0.63 0.70 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.60 0.59 Submitted Value Submitted mean & std dev Assigned value (rpHPLC only) 0.1 6.61 0.5: Distribution of D/L Values submitted for Glutamic Acid / Glutamine RP 0.53 0.2 7.64 0.67 0.65 0.62 D/L Value .51 0. Standards Solution THAA 0.

df)) of the Mean D/L value for Glutamic Acid / Glutamine (value of n displayed).1 6.64 1 0.52 0.54 0.66 0.66 0.2 7.62 D/L Value 5 0.54 2 2 2 2 2 2 2 0.2 8 9 10 11 13 14 15 Laboratory Number Page 39 of 157 .70 1 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.60 5 0.1 7.68 0.AAR PT Report. STATISTICAL EVALUATION – Summary Statistics Figure 4.62 D/L Value 5 0.6: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Glutamic Acid / Glutamine (value of n displayed).7: Experimental Expanded Uncertainty (k=t(0.58 2 5 0.68 0.05.64 1 0.70 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 1 Replicate means 0.1 7.2 7. Standards Solution THAA 4.2 8 9 10 11 13 14 15 Laboratory Number Figure 4. RP 0.56 2 0.1 6.58 5 0.60 5 0.50 1 2 3 4 5 6. RP 0.50 1 2 3 4 5 6.56 2 2 2 2 2 2 2 2 2 0.52 0.

6 156.97 5.52 8778 2219 1164 1162 3673 2283 2167 9245 2044 1286 1218 3879 2593 2293 9012 2131 1225 1190 3776 2438 2230 2 2 2 2 2 2 2 330.71 5.2 7.17 9.46 12.710 12.1 85.98 RSU% 27.4 std dev 2536.73 80.0 63.5 RSU% 27.7 138.60 3.50 5.35 2.df) 2.2 CV% 60.67 12.13 4.710 12.91 62.710 12.8 303.04 Page 40 of 157 .710 Exp U% (k=tcrit) 75.21 73.710 12.710 12.81 8.05.38 11.76 2.35 2.45 L-Ser peak area 1 2 3 4 5 6.06 9.33 3.99 4.4.9 219.71 5.69 5.1 6.1 2.7 28.0 103.86 8.34 5.6 195.777 12.81 4.12 2.52 63.777 12.710 12.73 3.3 350.2 214.67 2.710 12.7 495.710 75.11 37. Standards Solution THAA D-Ser peak area 1 2 3 4 5 6.2 2.01 233.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 21316 5613 2960 2824 9194 5736 5478 a 3523 946 1026 22458 5183 3274 3101 9703 6437 5815 b 3746 1002 977 c 877 d 4885 e 7883 f g h i j 21887 5398 3117 2963 9448 6086 5647 mean 4183 974 1002 2 2 2 2 2 2 2 n 5 2 2 807.72 Uncertainty of Mean & Expanded U at 95% CL std u 2805.6 359.1 238.33 34.66 5.710 33.52 5.95 2.710 12.00 3.3 254.90 2.78 7.1 6.8 39.1 168.710 12.94 29.05 33.40 571.91 34.1 60.94 51.0 91.11 3.16 2.64 4.18 8.8 64.41 35.4 28.2 123.710 12.90 3.80 2.1 7.73 6.710 12.8: Summary Statistics for L and D Serine Peak Area Data Lab No method a 8678 2319 2514 b 9271 2451 2385 c 2175 Submitted Replicate data d 12014 e 19508 f g h i j Standard Deviation mean 10329 2385 2449 n 5 2 2 std dev 6273.2 155.97 12.710 12.710 12.4 221.22 CV% 60.62 7.22 7.6 145.61 3.84 5.2 7.69 5.1 89.94 AAR PT Report.24 5.4 93.1 7.63 Exp U% (k=2) 54.0 34.5 87.710 12.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 54.32 5.77 36.0 3.17 50.4 3.30 36.90 4.40 5.95 10.5 40.59 4.6 65.710 32. STATISTICAL EVALUATION – Summary Statistics Table 4.11 6.90 59.03 4.75 2.92 30.26 t critical (0.5 std u 1134.08 4.3 24.

67 RSU% 0.3 1.26 6.710 12.18 21.29 2.2 1.710 12.84 15.05.15 0.45 21.89 4.2 7.710 12.37 CV% 1.92 0.710 12.3 1.1 5.66 1.df) 2.10 12.55 21.df) 2.23 0.51 1.2 7.52 1.710 t critical (0.0 0.777 8.1 std dev 0.59 10.13 0.00 1.2 0.05 4.0 1. STATISTICAL EVALUATION – Summary Statistics Table 4.91 1.1 7.1 6.39 t critical (0.35 Exp U% (k=2) 1.74 2.5 18.05.3 RSU% 0.710 12.1 13.8 0.4.2 0.2 7.94 L-Ser Conc 1 2 3 4 5 6.8 3.08 2.93 135.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 25 24 25 44 57 50 25 25 25 54 58 52 25 25 25 49 58 51 2 2 2 2 2 2 0.52 135.7 1.56 Uncertainty of Mean & Expanded U at 95% CL std u 0.3 1.710 9.62 0.2 std u 0.7 0.9: Summary Statistics for L and D Serine Concentration Data (pM) Lab No method a RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 49 b 50 c 51 Submitted Replicate data d 49 e 50 f g h i j mean 50 Standard Deviation n 5 std dev 0.2 0.54 19.55 1.710 12.710 12.1 7.24 2.710 12.1 6.25 1.02 1.73 10.26 Exp U% (k=tcrit) 1.01 38.82 3.24 19.710 12.85 1.15 7.44 15.9 0.2 8 9 10 11 12 13 14 15 64 61 61 109 142 127 a 20 63 64 63 135 145 132 b 20 c 21 d 20 e 20 f g h i j 63 63 62 122 144 130 mean 20 2 2 2 2 2 2 n 5 0.9 1.70 Exp U% (k=2) 1.8 CV% 1.05 12.03 3.3 2.4 1.33 3.14 2.777 Exp U% (k=tcrit) 1.36 Page 41 of 157 .03 11.1 0.1 1. Standards Solution THAA D-Ser Conc 1 2 3 4 5 6.41 37.00 3.710 12.63 3.53 AAR PT Report.11 21.6 2.48 5.

412 0.0024 0.32 0.df) 2.4.395 0.405 0.710 12.408 b 0.0006 0.710 0.44 1.710 12.395 2 2 2 0.393 0.22 0.409 n 5 2 2 std dev 0.0006 0.21 0.00 0.46 .00 0.2 8 9 10 11 12 13 14 15 0.15 0.16 0.0002 0.710 12.412 0.1 6.1 7.00 0.0132 0.0006 0.403 0.2 7.93 D/L Serine 1 2 3 4 5 6.46 t critical (0.05.69 1.0014 0.28 0.404 0.35 0.0002 0.40 0.710 Exp U% (k=tcrit) 0.52 7.08 2.400 0.30 12.15 0.15 4.398 0.06 0.10: Summary Statistics for L and D Serine D/L Ratio Value Lab No method a RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 0.408 0.04 0.0008 0.409 0.33 Uncertainty of Mean & Expanded U at 95% CL std u 0.400 0.00 1.90 AAR PT Report.402 2 2 2 2 0.08 1.60 0.0000 0.404 f g h i j Standard Deviation mean 0.0013 CV% 0.403 Submitted Replicate data d 0.86 0.16 0.395 0.0004 0.0008 0.21 0.412 0.394 0.0003 0. STATISTICAL EVALUATION – Summary Statistics Table 4.393 0.21 0.710 12.23 Exp U% (k=2) 0.777 12.11 3.11 0.408 0.394 0.393 0.396 0.87 2.393 0.32 0.96 29.710 12. Standards Solution THAA Page 42 of 157 0.412 0.0006 0.710 0.0009 RSU% 0.0004 0.0093 0.0000 0.0034 0.410 c 0.29 0.63 12.407 e 0.406 0.710 12.400 0.400 0.

31 0.40 Submitted Value Assigned value (all data) 0.41 0.46 0.42 0.44 0.32 0.48 0.50 0.49 0.43 0.34 0.2 8 9 10 11 13 14 15 Laboratory Number Page 43 of 157 D/L Value . Standards Solution THAA 0.4.39 0.2 7.45 0.35 0.1 6.47 0.36 AAR PT Report.37 0.38 0.8: Distribution of D/L Values submitted for Serine RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.30 1 2 3 4 5 6.1 7.33 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.

AAR PT Report; Standards Solution THAA

4. STATISTICAL EVALUATION – Summary Statistics

**Figure 4.9: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Serine (value of n displayed).
**

RP 0.50 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP

Replicate means 0.48

0.46

0.44

0.42

D/L Value

5

2

2

2

2 2 2

2

2

2

0.40

0.38

0.36

0.34

0.32

0.30 1 2 3 4 5 6.1 6.2 7.1 7.2 8 9 10 11 13 14 15

Laboratory Number

**Figure 4.10: Experimental Expanded Uncertainty (k=t(0.05,df)) of the Mean D/L value for Serine (value of n displayed).
**

RP 0.55 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP

0.53 0.51

0.49

0.47

0.45 0.43

D/L Value

0.41

0.39 0.37

5

2

2

2 2 2

2 2 2 2

0.35

0.33 0.31

0.29

0.27

Replicate means 0.25 1 2 3 4 5 6.1 6.2 7.1 7.2 8 9 10 11 13 14 15

Laboratory Number

Page 44 of 157

4. STATISTICAL EVALUATION – Summary Statistics

**Table 4.11: Summary Statistics for L and D Arginine Peak Area Data
**

Lab No method

a b c

**Submitted Replicate data
**

d e f g h i j

Standard Deviation

mean n std dev CV%

**Uncertainty of Mean & Expanded U at 95% CL
**

std u RSU% Exp U% (k=2) 6.99 4.91 t critical (0.05,df) 12.710 12.710 Exp U% (k=tcrit) 44.40 31.19

**L-Arg peak area
**

1 2 3 4 5 6.1 6.2 7.1 7.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP

2873 2358

3081 2245

2977 2301

2 2

147.0 79.9

4.94 3.47

104.0 56.5

3.49 2.45

11847 7197 6157 a

12321 7096 7066 b c d e f g h i j

12084 7146 6611 mean

2 2 2 n

334.9 70.9 643.1 std dev

2.77 0.99 9.73 CV%

236.8 50.2 454.8 std u

1.96 0.70 6.88 RSU%

3.92 1.40 13.76 Exp U% (k=2) 6.04 5.11

12.710 12.710 12.710 t critical (0.05,df) 12.710 12.710

24.91 8.92 87.42 Exp U% (k=tcrit) 38.41 32.50

AAR PT Report; Standards Solution THAA

**D-Arg peak area
**

1 2 3 4 5 6.1 6.2 7.1 7.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP

1027 1124

1092 1068

1059 1096

2 2

45.3 39.6

4.27 3.62

32.0 28.0

3.02 2.56

2424 1278 1230 4084 2491 2341

2263 1410 1325 4242 2738 2517

2344 1344 1278 4163 2615 2429

2 2 2 2 2 2

114.1 93.4 67.0 111.8 174.3 124.3

4.87 6.95 5.24 2.68 6.67 5.12

80.7 66.0 47.4 79.0 123.3 87.9

3.44 4.91 3.71 1.90 4.71 3.62

6.88 9.83 7.42 3.80 9.43 7.24

12.710 12.710 12.710 12.710 12.710 12.710

43.74 62.44 47.12 24.13 59.92 46.00

Page 45 of 157

6969 3402 3187

6639 3740 3551

6804 3571 3369

2 2 2

233.7 239.0 257.8

3.44 6.69 7.65

165.3 169.0 182.3

2.43 4.73 5.41

4.86 9.46 10.82

12.710 12.710 12.710

30.87 60.14 68.76

4. STATISTICAL EVALUATION – Summary Statistics

**Table 4.12: Summary Statistics for L and D Arginine Concentration Data (pM)
**

Lab No method

a RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP b c

**Submitted Replicate data
**

d e f g h i j

Standard Deviation

mean n std dev CV%

**Uncertainty of Mean & Expanded U at 95% CL
**

std u RSU% Exp U% (k=2) t critical (0.05,df) Exp U% (k=tcrit)

L-Arg Conc

1 2 3 4 5 6.1 6.2 7.1 7.2 8 9 10 11 12 13 14 15

138 176 141 a

169 157 158 b c d e f g h i j

153 167 149 mean

2 2 2 n

21.5 13.1 11.8 std dev

14.00 7.84 7.88 CV%

15.2 9.2 8.3 std u

9.90 5.55 5.57 RSU%

19.80 11.09 11.14 Exp U% (k=2)

12.710 12.710 12.710 t critical (0.05,df)

125.82 70.48 70.81 Exp U% (k=tcrit)

AAR PT Report; Standards Solution THAA

D-Arg Conc

1 2 3 4 5 6.1 6.2 7.1 7.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP

27 26 26 48 61 54

27 27 27 58 61 56

27 27 26 53 61 55

2 2 2 2 2 2

0.0 1.1 0.3 7.4 0.1 1.8

0.14 4.08 1.08 13.91 0.19 3.26

0.0 0.8 0.2 5.2 0.1 1.3

0.10 2.88 0.76 9.84 0.13 2.31

0.19 5.77 1.53 19.68 0.26 4.62

12.710 12.710 12.710 12.710 12.710 12.710

1.23 36.65 9.71 125.05 1.68 29.33

Page 46 of 157

78 69 68

79 72 71

79 71 69

2 2 2

1.0 2.7 2.4

1.30 3.82 3.49

0.7 1.9 1.7

0.92 2.70 2.47

1.83 5.40 4.94

12.710 12.710 12.710

11.65 34.35 31.38

03 0.09 7.341 0.df) 12.0170 1.710 12.06 5.386 0.1 6.41 3.26 2.0198 0.710 12.710 12.377 0.348 0.354 0.21 t critical (0.373 0.27 2.10 0.477 0.356 0.0017 0.344 0.18 1.0007 0.710 12.83 6.68 0.0010 0.0035 0.2 7.0065 0.99 1.47 0.0049 0.78 68.88 2.380 0.710 12.15 0.710 12.386 0.36 3.43 0.344 0.476 2 2 0.356 0.0003 0.1 7.12 10.0005 0.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP method a b c Submitted Replicate data d e f g h i j Standard Deviation mean n std dev CV% Uncertainty of Mean & Expanded U at 95% CL std u RSU% Exp U% (k=2) 0.53 12.710 12.0092 0.710 Exp U% (k=tcrit) 5.41 0.0002 0.346 0.0120 1.366 0.94 0.41 0.476 0.71 0.4.0007 0.62 0. STATISTICAL EVALUATION – Summary Statistics Table 4.376 0.13: Summary Statistics for L and D Arginine D/L Ratio Value Lab No D/L Arg 1 2 3 4 5 6.53 AAR PT Report.344 0.81 41.67 0.0280 0.380 0.01 0.345 0.0024 0.05.368 2 2 2 2 2 2 0.376 0.31 21.32 0.358 0. Standards Solution THAA Page 47 of 157 .66 4.

42 0.35 0.38 Submitted Value Assigned value (all data) AAR PT Report.2 8 9 10 11 13 14 15 Laboratory Number Page 48 of 157 D/L Value .45 0.48 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.50 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.49 0.31 0.36 0.37 0.2 7.33 0.46 0.39 0.32 0.41 0.44 0.40 0.1 6.43 0.4.34 0.11: Distribution of D/L Values submitted for Arginine RP 0. Standards Solution THAA 0.30 1 2 3 4 5 6.47 0.1 7.

48 0.12 0.1 7.20 0.34 0.df)) of the Mean D/L value for Arginine (value of n displayed).58 0.42 0.1 6.22 0.1 7.24 0.10 1 2 3 4 5 6.54 0.30 1 2 3 4 5 6.44 0.44 0.12: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Arginine (value of n displayed).56 0.38 2 0.18 0.36 2 2 2 0.60 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.32 0.28 0.32 0.46 0.40 2 2 2 0. RP 0.46 0.16 0.13: Experimental Expanded Uncertainty (k=t(0.1 6.AAR PT Report. STATISTICAL EVALUATION – Summary Statistics Figure 4. RP 0.2 8 9 10 11 13 14 15 Laboratory Number Page 49 of 157 .30 0.50 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.48 2 0.38 2 2 2 2 0.50 0.2 7.05.62 0.52 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 4.2 7. Standards Solution THAA 4.42 D/L Value 0.26 0.36 0.34 0.40 2 2 2 2 D/L Value 0.14 0.

df) 2.34 7.710 Exp U% (k=tcrit) 75.37 6.0 std u 1715.7 69.51 RSU% 27.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 10005 6207 5882 a 5322 1458 1565 10510 6953 6310 b 5643 1557 1516 c 1309 d 7364 e 11903 f g h i j 10258 6580 6096 mean 6308 1507 1541 2 2 2 n 5 2 2 356.6 84.78 1.46 5.16 12.df) 2.6 112.10 9.64 20.73 6.84 51.14: Summary Statistics for L and D Alanine Peak Area Data Lab No method a 9753 2635 2819 b 10489 2794 2720 c 2413 Submitted Replicate data d 13397 e 21926 f g h i j Standard Deviation mean 11596 2715 2769 n 5 2 2 std dev 7051.710 31.710 33.4 3.7 49.710 12.6 79.710 12.56 2.87 3.01 12.00 6.8 69.710 12.9 228.08 AAR PT Report.01 18.1 66.79 4.19 3.6 115.02 4.01 4.63 2.13 9.1 6.2 373.777 12.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 12948 3334 1755 1696 5595 3407 3218 13565 3074 1924 1829 5764 3803 3417 13256 3204 1839 1763 5679 3605 3318 2 2 2 2 2 2 2 436.3 2.3 280.78 Exp U% (k=2) 54. Standards Solution THAA D-Ala peak area 1 2 3 4 5 6.710 12.50 3.710 12. STATISTICAL EVALUATION – Summary Statistics Table 4.78 4.6 334.7 236.88 38.57 3.2 119.710 t critical (0.52 Uncertainty of Mean & Expanded U at 95% CL std u 3153.29 5.8 34.93 1.4.8 2.47 5.68 7.28 1.81 4.55 3.08 44.2 7.48 41.2 99.33 4.710 12.25 72.1 6.49 5.77 5.710 12.05 4.710 12.2 183.67 3.8 84.4 198.20 2.10 7.05.12 48.02 Exp U% (k=2) 54.59 5.6 163.85 5.1 7.08 984.15 7.61 Exp U% (k=tcrit) 75.8 CV% 60.710 12.88 69.2 3.39 5.18 12.4 2.3 141.51 37.66 L-Ala peak area 1 2 3 4 5 6.7 302.7 527.75 6.96 CV% 60.710 12.58 51.777 12.1 3.65 .22 Page 50 of 157 35995 6063 3177 3097 37964 5589 3500 3328 36979 5826 3339 3212 2 2 2 2 1392.58 4.710 12.0 49.30 22.05.06 4.97 11.1 7.4 24.48 8.66 4.34 2.5 129.23 252.84 3.1 214.0 94.65 8.66 61.25 308.2 7.710 12.92 11.6 119.4 RSU% 27.6 std dev 3834.54 45.49 58.57 t critical (0.33 8.15 2.710 12.8 161.710 29.

03 AAR PT Report.43 0.4 1.1 6.44 5.7 0.3 1.48 Uncertainty of Mean & Expanded U at 95% CL std u 0.7 4.1 7.710 12.5 1.52 27.70 1.72 2.40 0.777 Exp U% (k=tcrit) 1.4 12.777 9.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP method a 55 b 57 c 57 Submitted Replicate data d 55 e 57 f g h i j Standard Deviation mean 56 n 5 std dev 0.4.6 2.60 119.09 1.79 1.16 35.20 RSU% 0.54 0.4 0.05.710 12.8 0.60 1.0 0.33 0.1 6.32 10.92 14.6 1.5 0. Standards Solution THAA D-Ala Conc 1 2 3 4 5 6.69 1.4 1.7 0.2 0.66 4.1 7.30 20.15: Summary Statistics for L and D Alanine Concentration Data (pM) Lab No L-Ala Conc 1 2 3 4 5 6.02 3.84 64 60 62 110 143 127 a 63 64 63 136 146 133 b 31 c 31 d 30 e 31 f g h i j 63 62 62 123 144 130 mean 31 2 2 2 2 2 2 n 5 0. STATISTICAL EVALUATION – Summary Statistics Table 4.65 1.0 std dev 0.62 1.0 0.710 12.40 Exp U% (k=2) 0.39 12.2 2.83 9.66 Exp U% (k=2) 1.74 12.df) 2.1 1.18 13.3 1.10 10.11 CV% 0.4 RSU% 0.93 2.42 5.67 18.8 1.710 8.710 12.84 8.1 0.83 0.710 12.32 21.2 7.710 12.df) 2.710 t critical (0.17 3.54 Page 51 of 157 .8 CV% 1.86 1.3 7.74 8.710 12.2 7.710 12.05.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 30 30 28 29 52 67 59 29 30 29 63 68 61 30 29 29 58 67 60 2 2 2 2 2 2 0.31 3.99 32.2 5.710 12.71 2.81 0.98 0.94 Exp U% (k=tcrit) 1.65 10.9 std u 0.23 132.37 1.39 0.00 3.83 2.32 t critical (0.6 18.710 12.

1 1 7.0074 0.468 0.0013 0.06 0.20 Exp U% (k=2) 0.26 0.500 0.357 0.99 4.32 0.97 0.35 1.550 e 0.777 12.500 0.459 0.16: Summary Statistics for L and D Alanine D/L Ratio Value Lab No D/L Ala 1 2 3 4 5 1 6.0043 0.710 Exp U% (k=tcrit) 0.18 3.03 0.0021 0.19 2.710 12.556 n 5 2 2 std dev 0.1 1 6.22 3.466 0.0046 0.0012 4.20 6.466 0.43 2.0015 0.29 Uncertainty of Mean & Expanded U at 95% CL std u 0.468 0.547 0.71 0.27 0.01 0.41 t critical (0.02 0.547 0.360 0.0033 1.710 12.0072 0.467 0.38 0.563 0.710 12.461 2 2 2 0.72 0.474 0.466 0.25 0.0011 RSU% 0.543 f g h i j Standard Deviation mean 0.710 12.0019 0.37 .2 1 7.97 7.36 0.38 2.555 0.0016 CV% 0.710 3. GCA= derived using peak area GCHt = derived using peak height Page 52 of 157 0.42 0.538 0.0210 0.710 12.80 0.553 0.710 12.465 0.38 0.48 3.df) 2.777 12.42 0.4.0340 0.0001 0.464 0.555 b 0.59 0.59 1.469 0.557 c 0.17 3.0065 0.466 0.0001 0.468 0.48 0.50 1. Standards Solution THAA = submitted as the mean and standard deviation of n results.54 0.2 8 9 10 11 12 13 14 15 1 method a RP RP RP IE IE GCA GCHt GCA GCHt RP RP RP RP RP RP RP RP 0.0019 0.34 0.465 0.0160 0.44 4.58 0.68 0.25 5.563 0.95 0.0023 0.51 15.53 0.0018 0.17 0.01 12.546 0.359 0.465 8 3 5 1 2 2 2 2 0.466 0.0196 0.20 6.0027 0.710 12.0008 0. STATISTICAL EVALUATION – Summary Statistics Table 4.557 0.34 2.37 2.303 2.464 0.05.0009 1.365 4.40 AAR PT Report.84 0.464 0.464 0.0011 0.542 Submitted Replicate data d 0.544 0.

39 0.53 0.60 0.1 6.31 0.51 0.52 0.2 8 9 10 11 13 14 15 D/L Value Submitted Value Submitted mean & std dev Assigned value (all data) Assigned value (rpHPLC only) Laboratory Number Page 53 of 157 .47 0.38 0.59 0.54 0.56 0.45 0.43 0.40 0.36 0. Standards Solution THAA 0.32 0.37 0.34 0.44 0.58 0.4. STATISTICAL EVALUATION – Summary Statistics Figure 4.55 0.41 0.2 7.48 0.42 0.50 0.49 0.1 7.57 0.35 0.33 0.30 1 2 3 4 5 6.46 0.14: Distribution of D/L Values submitted for Alanine RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP AAR PT Report.

60 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.52 0.38 2 0.1 7.54 2 2 1 3 5 D/L Value 0.46 0.40 0.56 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 4.58 0.16: Experimental Expanded Uncertainty (k=t(0.1 7.64 0. Standards Solution THAA 4.70 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.48 0.54 2 5 2 3 1 0.30 1 2 3 4 5 6.56 0.34 0.32 0.05.2 7.1 6.38 2 0.30 1 2 3 4 5 6.60 0.40 0.1 6.48 5 2 2 2 2 2 2 D/L Value 0.66 0.50 0.46 8 5 2 2 2 2 2 2 0.36 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.15: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Alanine (value of n displayed).52 8 0.34 0.42 0.42 0.58 0.df)) of the Mean D/L value for Alanine (value of n displayed).2 8 9 10 11 13 14 15 Laboratory Number Page 54 of 157 . RP 0.68 0.36 0.44 0.50 0.AAR PT Report. RP 0.44 0.32 0.62 Replicate means 0.2 7.

2 49.710 12.710 Exp U% (k=tcrit) 75.07 5.710 12.65 4.18 7.10 Exp U% (k=2) 54.91 5.7 2.710 12.277 14.32 3.78 7.05.98 L-Val peak area 1 2 3 4 5 6.70 13.4 312.710 12.76 5.17 7.52 2.05.1 132.80 3.2 80.67 47.64 1.2 150.31 2.75 5.29 2.2 54.00 3.7 187.1 7.56 11735 3084 1592 1554 5033 3086 2934 a 12374 2842 1759 1661 5335 3463 3104 b c d e f g h i j 12054 2963 1676 1608 5184 3274 3019 mean 2 2 2 2 2 2 2 n 452.8 RSU% 27.12 8.90 7.05.09 70.9 std u 2.655 16.df) 33.51 652.710 12.03 5.2 7.59 3.710 12.68 4.710 t critical (0.3 255.76 2.4 563.48 5.8 120.62 Exp U% (k=2) 12.710 t critical (0.53 5.12 34.00 73.777 12.277 1 1 Page 55 of 157 D-Val peak area Exp U% (k=tcrit) .86 50.17: Summary Statistics for L and D Valine Peak Area / Height Data Lab No method a 10480 3059 3194 b 11168 3221 3125 c 2584 Submitted Replicate data d 14296 e 23556 f g h i j Standard Deviation mean 12417 3140 3160 n 5 2 2 std dev 7576.49 Exp U% (k=2) 54.7 84.1 6.18 9.710 12.3 213.df) 2.4 118.98 CV% 319.4 185.0 261.24 2.74 RSU% 27.3 180.81 RSU% 5. STATISTICAL EVALUATION – Summary Statistics Table 4.1 7.82 11.20 5. Standards Solution THAA 1 2 3 4 5 6.2 171.85 35.74 Exp U% (k=tcrit) D+L Val peak height 004 005 IE IE 14.5 38.df) 2.710 12.2 CV% 61.9 188.98 10.710 12.99 5.710 12.4 75.1 std dev 3487.2 441.1 398.99 6.710 12.15 3.87 AAR PT Report.52 5.3 15.50 42.1 6.777 12.710 12.7 121.56 Uncertainty of Mean & Expanded U at 95% CL std u 3388.2 83.20 t critical (0.3 364.66 5.62 5.09 3.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 75.98 63.15 2.09 5.88 CV% 60.02 3.7 53.31 8.2 114.13 12.72 64.64 7.710 12.4 std u 1559.2 22.1 std dev 3.57 5.23 35.63 33.71 51.710 32.8 34.4 266.24 2.71 1.32 13.76 32.62 1.52 11.710 12.91 3.57 1.76 2.2 3.4.2 7.07 4.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 24573 6708 3452 3383 10995 6821 6393 a 4853 1424 1488 25878 6193 3822 3648 11619 7618 6754 b 5120 1501 1457 c 1183 d 6640 e 10829 f g h i j 25225 6450 3637 3516 11307 7220 6574 mean 5725 1463 1473 2 2 2 2 2 2 2 n 5 2 2 922.07 37.1 257.655 16.

df) 2.29 6.710 12.05.84 3.95 2.48 37.2 0.12 1.68 19.4 1.710 12.1 1.06 0.93 0.1 7.2 7.6 0.6 std dev 0.9 CV% 1.710 12.1 0.7 0.20 Exp U% (k=tcrit) 1.64 12.710 9.1 7.33 1.710 12.9 0.86 Exp U% (k=2) 0.1 6.92 1.05.30 2.37 10.38 1.02 CV% 0.2 15.00 12.75 2.2 7.90 8.0 1.48 Uncertainty of Mean & Expanded U at 95% CL std u 0.05 4.68 1.20 0.18: Summary Statistics for L and D Valine Concentration Data (pM) Lab No method a RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 60 b 61 c 61 Submitted Replicate data d 59 e 61 f g h i j Standard Deviation mean 60 n 5 std dev 0. Standards Solution THAA 1 2 3 4 5 6.8 0.79 11.43 RSU% 0.84 0.8 0.35 2.df) 2.65 3.84 L-Val Conc 1 2 3 4 5 6. STATISTICAL EVALUATION – Summary Statistics Table 4.710 12.6 2.5 0.33 t critical (0.69 0.1 7.10 D-Val Conc AAR PT Report.710 12.777 Exp U% (k=tcrit) 1.710 12.9 1.80 12.33 1.4 1.52 15.710 8.1 10.7 0.16 0.8 1.4 RSU% 0.97 0.50 1.4.71 4.91 4.65 137.66 Exp U% (k=2) 1.49 5.9 0.73 21.12 0.710 12.1 5.67 1.89 13.4 2.07 Page 56 of 157 65 61 63 64 64 64 65 62 63 2 2 2 0.44 .710 t critical (0.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 27 25 26 46 59 53 27 27 26 57 60 54 27 26 26 52 60 53 2 2 2 2 2 2 0.710 12.777 135.2 8 9 10 11 12 13 14 15 112 145 127 a 28 138 147 131 b 28 c 28 d 27 e 28 f g h i j 125 146 129 mean 28 2 2 2 n 5 18.5 0.3 1.12 0.1 6.22 38.9 std u 0.88 10.56 18.40 21.82 1.

710 12.427 0.10 0.49 0.0003 0.0013 0.777 12.05 0.403 0.00 1. Standards Solution THAA = submitted as the mean and standard deviation of n results.07 1.0110 0.466 b 0.409 0.0004 0.412 0.0014 0.10 0.571 2.2 8 9 10 11 12 13 14 15 1 0.464 e 0.0005 0.31 0.03 Exp U% (k=2) 0.91 0.07 0.13 0.0009 0.413 0.57 0.14 2.458 0.20 0.413 0.df) 2.415 0.96 1.0000 0.78 1.777 12.1 1 7.478 0.408 0.466 0.0029 0.29 0.414 0.0002 RSU% 0.0006 0.91 3.05.478 0.458 Submitted Replicate data d 0.412 0.47 0.19: Summary Statistics for L and D Valine D/L Ratio Value Lab No method a RP RP RP IE IE GCA GCHt GCA GCHt RP RP RP RP RP RP RP RP 0.0004 2.00 0.710 12.414 0.0027 0.403 0.0019 0.00 0.414 0.466 n 5 2 2 std dev 0.2 1 7.35 0.09 0.463 0.413 0.0000 0.710 Exp U% (k=tcrit) 0.26 1.478 0.414 0.710 12.461 0.365 2.4.42 D/L Valine 1 2 3 4 5 1 6.413 0.14 0.460 f g h i j Standard Deviation mean 0.0039 0.15 0.0003 0.0041 0.18 0.710 12.88 1.94 0.410 0. STATISTICAL EVALUATION – Summary Statistics Table 4.414 0.10 0.55 0.427 0.0006 0.466 c 0.0140 0.415 0.710 12.0002 0.12 0.45 0.64 0.21 0.05 Uncertainty of Mean & Expanded U at 95% CL std u 0.79 0.07 t critical (0.87 AAR PT Report. GCA= derived using peak area GCHt = derived using peak height Page 57 of 157 .413 0.25 0.11 0.414 8 6 5 2 2 2 2 2 2 2 0.415 0.710 12.710 2.0002 CV% 0.34 3.466 0.23 2.00 0.62 0.46 4.64 0.0004 0.710 12.17 5.0010 0.30 0.0063 0.1 1 6.66 2.410 0.0100 0.91 3.466 0.

50 0.1 7.52 0.51 0.37 0.2 8 9 10 11 13 14 15 Laboratory Number Page 58 of 157 0.49 0.1 6.36 0.44 0.47 D/L Value .53 0. Standards Solution THAA 0.55 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.54 0.42 0.35 1 2 3 4 5 6.40 0.38 0.17: Distribution of D/L Values submitted for Valine RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.2 7.48 0.39 0.41 0.43 Submitted mean & std dev Assigned value (all data & rpHPLC only) AAR PT Report.45 Submitted Value 0.4.46 0.

STATISTICAL EVALUATION – Summary Statistics Figure 4.47 2 D/L Value 0.41 0.37 0.2 7.2 8 9 10 11 13 14 15 Figure 4.49 2 2 5 2 0.43 2 2 2 2 0.05.45 0.1 7.45 6 8 2 5 2 0.55 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.47 D/L Value 0.53 0.1 6.49 2 5 2 0. RP 0.1 6.2 7.39 0.2 8 9 10 11 13 14 15 Laboratory Number Page 59 of 157 .df)) of the Mean D/L value for Valine (value of n displayed).43 8 0.35 1 2 3 4 5 6.53 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.19: Experimental Expanded Uncertainty (k=t(0. RP 0.1 Lab No 7.37 0.41 6 2 5 2 2 2 2 2 0.51 0.18: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Valine (value of n displayed).39 0.55 Replicate means 0.AAR PT Report. Standards Solution THAA 4.35 1 2 3 4 5 6.51 0.

3 485.4 328.710 12.5 202.53 3.0 114.20 1.95 5.3 135.710 32.2 84.6 118.6 48.9 12.710 12.1 6.02 3.84 3.0 51.67 4.710 32.3 142.41 Exp U% (k=tcrit) 74.80 566.11 2.710 12.63 Exp U% (k=2) 53.5 191.0 109.24 3.6 237.65 5.64 10.45 28.7 97.41 5.32 t critical (0.16 7.74 12.82 5.710 12.40 7.9 128.42 6.5 3.df) 2.73 61.1 7.94 1.17 .14 38.7 72.79 12.1 std dev 3539.12 2.84 4.24 4.9 CV% 60.57 63.0 232.1 2.2 161.710 t critical (0.22 312.48 7.20 2.52 4.98 65.47 48.9 2.710 28.4.96 4.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 11789 3104 1587 1550 5132 3128 2964 12413 2875 1755 1652 5404 3445 3103 12101 2990 1671 1601 5268 3287 3033 2 2 2 2 2 2 2 441.32 4.6 158.6 91.08 29.8 224.710 12.60 5.64 6.83 2.24 3.17 5.1 6.23 278.13 6.77 48.05.710 12.777 12.9 62.4 168.32 1.27 CV% 60.67 9.6 RSU% 27.58 3.92 3.0 34.98 2.06 1.64 Uncertainty of Mean & Expanded U at 95% CL std u 3226.05.710 12. STATISTICAL EVALUATION – Summary Statistics Table 4.77 11.71 32.87 5.15 9.85 40.1 43.3 2.2 7.710 12.6 154.75 62.53 43.20: Summary Statistics for L and D Phenylalanine Peak Area Data Lab No method a 10138 2884 3018 b 10748 3066 2949 c 2478 Submitted Replicate data d 13788 e 22457 f g h i j Standard Deviation mean 11922 2975 2983 n 5 2 2 std dev 7214.56 12.777 12.1 7.84 6.10 4.28 5.2 7.7 3.1 343.97 0.16 Exp U% (k=2) 54.06 3.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 10438 6361 6035 a 5031 1434 1496 10994 7047 6320 b 5284 1522 1470 c 1248 d 6712 e 11067 f g h i j 10716 6704 6178 mean 5868 1478 1483 2 2 2 n 5 2 2 393.2 3.90 37.710 12.95 Page 60 of 157 24701 6291 3247 3108 25833 5826 3583 3327 25267 6058 3415 3218 2 2 2 2 800.710 12.9 std u 1583.57 4.40 4.9 69.1 18.73 L-Phe peak area 1 2 3 4 5 6.67 7.86 14.710 Exp U% (k=tcrit) 75.df) 2.05 6.710 12.31 RSU% 26.710 12.710 12. Standards Solution THAA D-Phe peak area 1 2 3 4 5 6.04 AAR PT Report.19 10.

29 133.01 0.02 t critical (0.4. STATISTICAL EVALUATION – Summary Statistics Table 4.99 0.710 12.710 12.52 21.df) 2.90 21.24 36.89 0. Standards Solution THAA D-Phe Conc 1 2 3 4 5 6.76 AAR PT Report.3 RSU% 0.67 4.02 0.56 0.55 2.07 38.6 1.710 t critical (0.49 2.73 Exp U% (k=tcrit) 2.81 3.710 12.51 Exp U% (k=2) 1.5 2.53 0.9 0.00 Exp U% (k=2) 1.2 0.710 12.48 2.3 std u 0.7 0.710 12.710 12.98 5.6 0.06 3.710 12.2 8 9 10 11 12 13 14 15 67 62 63 116 148 131 a 29 66 66 63 143 149 134 b 29 c 29 d 28 e 29 f g h i j 66 64 63 129 148 133 mean 29 2 2 2 2 2 2 n 5 0.1 6.93 12.51 0.05.9 0.df) 2.69 4.2 1.710 12.777 Exp U% (k=tcrit) 1.45 10.95 5.09 0.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 33 30 31 57 73 64 33 32 31 70 73 66 33 31 31 64 73 65 2 2 2 2 2 2 0.1 9.27 Page 61 of 157 .23 0.0 0.3 0.6 0.4 1.78 0.14 Uncertainty of Mean & Expanded U at 95% CL std u 0.99 0.97 0.9 0.777 6.99 0.37 0.3 13.75 133.3 0.06 0.2 7.41 L-Phe Conc 1 2 3 4 5 6.6 0.74 5.7 CV% 1.9 std dev 0.89 0.39 1.00 RSU% 0.05.21: Summary Statistics for L and D Phenylalanine Concentration Data (pM) Lab No method a RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 58 b 58 c 58 Submitted Replicate data d 57 e 58 f g h i j Standard Deviation mean 58 n 5 std dev 0.42 CV% 2.3 0.99 12.710 12.1 6.50 12.1 7.02 1.6 0.64 14.1 7.3 1.14 12.26 10.710 12.4 19.2 7.28 1.1 6.22 0.86 0.02 1.37 14.0 0.710 6.4 0.

475 0.74 1.29 0.32 2.493 f g h i j Standard Deviation mean 0.22: Summary Statistics for L and D Phenylalanine D/L Ratio Value Lab No D/L Phe 1 2 3 4 5 1 6.10 0.0052 0.0003 0.491 2 2 2 0.489 0.1 1 7.64 0.09 0.475 0.777 12.0001 0.0090 0.0006 0.0010 2.0148 0.499 0.0063 0.492 0.496 0.0020 0.303 2.496 0.492 0.17 1.28 0.02 0.478 0.0005 0.98 0.57 0.95 0.491 0.15 0.04 0.0370 0.09 3.491 0.478 0.497 0.710 12.0014 7.15 0.492 0.0330 0.df) 2.29 Exp U% (k=2) 1.69 0.26 3.02 0.57 0. GCA= derived using peak area GCHt = derived using peak height Page 62 of 157 0.39 2.47 4.710 12.2 8 9 10 11 12 13 14 15 1 method a RP RP RP IE IE GCA GCHt GCA GCHt RP RP RP RP RP RP RP RP 0.0015 0.710 12.710 12.46 .31 0.496 0.07 12.0131 0.19 5.497 0.492 0.20 6.777 12.493 0.497 0.498 8 3 5 1 2 2 2 2 0.0001 0.0007 0.710 0.90 6.2 1 7.490 0.494 0.0001 0.493 0.46 AAR PT Report.63 0.74 1.494 0.497 n 5 2 2 std dev 0.0002 0.480 0.12 0.11 0.63 3.27 0.4.47 2.487 e 0.41 Uncertainty of Mean & Expanded U at 95% CL std u 0.710 12.21 0.710 Exp U% (k=tcrit) 1.44 0.492 0.17 0.58 t critical (0.473 0.0021 0.03 0.73 8.59 1.05 0.04 0.1 1 6.03 0.0001 0.481 0.489 0.40 0.0004 0.0028 0.0014 0.496 0.0014 RSU% 0.504 Submitted Replicate data d 0.05.473 0.496 b 0.490 0.710 6.489 0.10 3. Standards Solution THAA = submitted as the mean and standard deviation of n results. STATISTICAL EVALUATION – Summary Statistics Table 4.01 0.0020 CV% 1.365 4.499 c 0.710 12.21 0.478 0.

53 0.51 0.47 0.59 0.41 0.44 0.42 0.45 0.60 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.54 0.40 1 2 3 4 5 6.4.56 0.1 6.20: Distribution of D/L Values submitted for Phenylalanine RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.58 0. Standards Solution THAA 0.2 7.1 7.43 0.46 0.48 Assigned value (rpHPLC only) AAR PT Report.52 D/L Value 0.50 Submitted Value submitted mean & std dev Assigned value (all data) 0.49 0.55 0.57 0.2 8 9 10 11 13 14 15 Laboratory Number Page 63 of 157 .

2 7.44 0.46 0. Standards Solution THAA 4.42 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 4.60 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.58 0.AAR PT Report.1 6. RP 0.1 7.05.42 0.2 8 9 10 11 13 14 15 Laboratory Number Page 64 of 157 .52 D/L Value 0.50 5 2 2 1 2 2 2 2 2 2 2 8 0.22: Experimental Expanded Uncertainty (k=t(0.2 7.46 0.48 3 5 0.56 0.40 1 2 3 4 5 6.56 0.52 D/L Value 0.1 7.54 0.58 0.50 5 2 2 1 2 2 2 2 2 2 2 8 0.1 6.60 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.21: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Phenylalanine (value of n displayed).40 1 2 3 4 5 6.df)) of the Mean D/L value for Phenylalanine (value of n displayed). RP 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.44 0.48 3 5 0.54 0.

4 3.63 CV% 60.1 28.68 66.8 162.07 5.85 12.3 RSU% 26.21 5.21 Page 65 of 157 26139 6941 3527 3436 27515 6399 3915 3710 26827 6670 3721 3573 2 2 2 2 973.13 8.83 5. Standards Solution THAA D-Aile peak area 1 2 3 4 5 6.37 5.47 5.53 8.14 403.4 2.710 33.14 Exp U% (k=2) 54.710 12.21 3.6 274.710 12.76 4.02 9.2 7.60 51.3 3.05.83 8.40 4.6 115.48 2.2 71.0 271.80 2.2 7.777 12.5 244.710 12.77 2.1 7.71 5.710 12.71 .710 12.74 5.710 12.df) 2.22 6.710 12.1 2.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 11363 6937 6553 a 6065 1779 1849 11995 7792 6899 b 6347 1879 1821 c 1478 d 8244 e 13482 f g h i j 11679 7365 6726 mean 7123 1829 1835 2 2 2 n 5 2 2 447.23: Summary Statistics for D-Alloisoleucine/L-Isoleucine Peak Area Data Lab No method a 10749 3082 3223 b 11465 3261 3166 c 2649 Submitted Replicate data d 14698 e 23575 f g h i j Standard Deviation mean 12627 3172 3195 n 5 2 2 std dev 7559.41 11.0 383.87 3.8 std u 1940.1 7.61 5. STATISTICAL EVALUATION – Summary Statistics Table 4.6 126.710 t critical (0.4 172.68 37.81 0.88 60.79 9.4.77 t critical (0.57 RSU% 27.43 7.05.4 223.93 5.90 3.11 316.3 193.3 std dev 4338.1 50.62 1.8 89.55 5.24 2.63 5.57 12.3 20.81 3.90 1.710 12.98 AAR PT Report.1 229.56 4.710 34.96 5.0 CV% 59.76 0.0 139.25 L-Ile peak area 1 2 3 4 5 6.0 98.25 Uncertainty of Mean & Expanded U at 95% CL std u 3380.9 109.40 73.11 2.66 12.51 1.35 10.777 12.3 316.0 136.42 688.99 69.84 55.1 6.1 6.710 12.65 Exp U% (k=tcrit) 75.7 81.77 6.0 604.710 12.13 10.66 4.62 35.75 7.97 1.71 11.df) 2.79 5.76 32.9 173.710 12.710 32.710 12.18 33.710 12.56 52.9 2.78 7.4 14.67 5.8 245.2 194.6 154.0 40.1 427.34 35.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 14762 3846 1972 1913 6311 3860 3618 15570 3522 2170 2077 6658 4308 3836 15166 3684 2071 1995 6484 4084 3727 2 2 2 2 2 2 2 571.710 Exp U% (k=tcrit) 74.33 8.7 3.21 3.75 4.89 Exp U% (k=2) 53.25 48.

df) 2.710 12.64 15.5 1.2 1.05 1.6 0.7 std u 0.72 3.18 0.77 134.1 7.777 Exp U% (k=tcrit) 1.93 12.78 12.2 10.2 7.710 12.46 21.28 3.71 L-Ile Conc 1 2 3 4 5 6. STATISTICAL EVALUATION – Summary Statistics Table 4.710 t critical (0.22 12.11 5.4.1 6.1 7.44 6.2 8 9 10 11 12 13 14 15 120 153 136 a 34 148 156 139 b 34 c 35 d 34 e 35 f g h i j 134 155 137 mean 34 2 2 2 n 5 20.23 12.0 0.5 2.51 Exp U% (k=2) 0.26 RSU% 0.00 0.df) 2.710 12.8 1.61 0.6 1.78 CV% 1.64 1.4 std dev 0.1 6.21 1.05 2.8 1.2 2.89 1.54 Page 66 of 157 70 64 66 69 68 67 69 66 66 2 2 2 0.2 7.710 12.8 CV% 1.4 1.32 21.62 Exp U% (k=2) 1.05.6 11.81 8.36 1.1 0.710 12.7 3.64 0.21 15.39 34.1 2.29 D-Aile Conc AAR PT Report.710 12.5 1.49 3.3 1.4 RSU% 0.710 12.11 20.4 0.74 1.2 0.777 135.4 15.96 1.47 2.02 4.04 14.16 10.47 11.0 0.97 Exp U% (k=tcrit) 1.23 t critical (0.92 2.7 1.62 2.38 Uncertainty of Mean & Expanded U at 95% CL std u 0.710 12.50 1.9 0.28 1.28 0.710 12.710 9.4 7.26 0.24: Summary Statistics for D-Alloisoleucine/L-Isoleucine Concentration Data (pM) Lab No method a RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 61 b 62 c 62 Submitted Replicate data d 60 e 61 f g h i j Standard Deviation mean 61 n 5 std dev 0.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 39 36 37 67 85 75 38 38 38 82 86 77 38 37 37 74 86 76 2 2 2 2 2 2 0.05.18 40.29 .37 1.77 14.710 13.87 1. Standards Solution THAA 1 2 3 4 5 6.90 2.

08 0.1 1 7.0140 0.564 0.33 0.556 0.72 12.10 4.0002 0.2 8 9 10 11 12 13 14 15 1 0.555 0.0028 0.05 0.36 1.20 2.571 0.577 0.554 n 5 2 2 1 1 8 6 5 2 2 2 2 2 2 2 std dev 0.df) 2.561 e 0.06 1.27 D/L Aile/Ile 1 2 3 4 5 1 6.0022 1.574 0.92 0.11 0.0025 0.85 1.552 b 0.63 0.55 2.566 0.0031 0.83 0.51 0.0005 0.66 0.3.05.0007 0.587 0.557 0.571 0. STATISTICAL EVALUATION – Summary Statistics Table 4.555 0.0050 0.554 0.03 0.556 0.32 0.18 0.27 0.0003 0.71 3.555 0.558 Submitted Replicate data d 0.710 12.566 0.567 0.565 0.623 0.587 0.575 c 0.39 0.710 12.365 2. Standards Solution THAA = submitted as the mean and standard deviation of n results.00 2.777 12.14 Uncertainty of Mean & Expanded U at 95% CL std u 0.20 t critical (0.76 0.555 0.710 12.06 0.557 0.13 0.0022 0.47 0.710 12.09 0.85 0.0026 0.0025 0.47 0.1 1 6.777 12.710 Exp U% (k=tcrit) 1.562 0.10 Exp U% (k=2) 1.70 1.0035 0.05 0.567 0.558 0.0004 0.553 0.710 1.710 0.56 AAR PT Report.0057 0.0025 0.38 0.41 4.0003 0.554 0.574 0.571 2.0015 0.0070 0.53 0.623 0.10 0.36 0.55 0.577 0.0018 0.710 12.584 0.42 0.25: Summary Statistics for D-Alloisoleucine/L-Isoleucine D/L Ratio Value Lab No method a 0.559 0.25 0.710 12.584 0.12 4.0069 0.2 1 7.0008 CV% 1.21 5.45 0. GCA= derived using peak area GCHt = derived using peak height Page 67 of 157 RP RP RP IE IE GCA GCHt GCA GCHt RP RP RP RP RP RP RP RP 0.554 0.576 0.23 0.552 0.65 0.90 0.572 f g h i j Standard Deviation mean 0.560 .46 0.0020 0.550 0.0018 0.0006 RSU% 0.

60 Submitted Value Submitted mean & std dev Assigned value (rpHPLC only) 0.23: Distribution of D/L Values submitted for D-Alloisoleucine/L-Isoleucine RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.69 0.52 0.67 0.63 0.2 8 9 10 11 13 14 15 Laboratory Number Page 68 of 157 D/L Value .65 0. Standards Solution THAA 0.56 0.53 0.55 0.57 0.58 Assigned value (all data) AAR PT Report.68 0.4.66 0.50 1 2 3 4 5 6. STATISTICAL EVALUATION – Summary Statistics Figure 4.62 0.70 0.1 6.59 0.54 0.64 0.61 0.1 7.2 7.51 0.

64 6 0.25: Experimental Expanded Uncertainty (k=t(0.60 8 0.56 2 2 2 0.56 2 2 2 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.50 1 2 3 4 5 6.52 0.2 7.62 D/L Value 0.68 0. RP 0.58 5 2 5 2 1 1 2 2 2 2 0.50 1 2 3 4 5 6.2 8 9 10 11 13 14 15 Laboratory Number Page 69 of 157 .24: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for D-Alloisoleucine/L-Isoleucine (value of n displayed).1 7. Standards Solution THAA 4.1 6.AAR PT Report.66 0.58 5 2 5 2 1 1 2 2 2 2 0.1 7.54 0.df)) of the Mean D/L value for D-Alloisoleucine/L-Isoleucine (value of n displayed).68 0.70 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.1 6.2 7.64 6 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 4.05.66 0.70 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.54 0. RP 0.60 8 0.62 D/L Value 0.52 0.

430 2 2 2 2 2 2 2 n 1 1 549.75 5.710 12.40 176.12 3.3 1494.1 std u 3.26 7.7 127.04 561.710 12.df) 2.52 2.32 L-Leu peak area 1 2 3 4 5 6.66 4.13 Exp U% (k=2) 54.1 3.70 7.49 12.3 RSU% 27.98 CV% 62.05.31 5.20 4.05 CV% 388.2 7.99 4.09 5.1 6.57 7.62 5.21 8.20 88.df) 2.0 102.710 12.1 127.2 90.26: Summary Statistics for L and D Leucine Peak Area Data 4.4 std dev 4.710 12.8 195.2 2113.28 70.4 149.430 12522 202* 1818 1751 5652 3650 3238 b c d e f g h i j 12134 1696 1728 1678 5524 3455 3148 mean 6.72 Exp U% (k=2) 12.80 3.df) 40.00 10.710 t critical (0.35 6.710 t critical (0.3 89.00 5.2 7.3 127.710 12.1 210.6 CV% 60.616 6.777 33.03 55.55 2.710 12.26 t critical (0.7 339.62 Exp U% (k=2) 55.1 6.66 7.0 2.63 11. STATISTICAL EVALUATION – Summary Statistics Lab No method a 8693 b 9314 c 2126 Submitted Replicate data d 11851 e 19389 f g h i j Standard Deviation mean 10275 n 5 std dev 6232.7 208.710 12.777 Exp U% (k=tcrit) 75.85 65.53 124.17 10.8 276.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP AAR PT Report. removed for subsequent statistical analysis Page 70 of 157 .39 8. Standards Solution THAA 11745 3191 1639 1606 5397 3259 3058 a 6.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 20985 5419 2794 2739 8915 5485 5151 a 5205 22108 5002 3096 2941 9394 6126 5449 b 5615 c 1312 d 7547 e 12162 f g h i j 21547 5211 2945 2840 9154 5805 5300 mean 6368 2 2 2 2 2 2 2 n 5 794.710 12.710 12.13 50.13 3.Table 4.04 29.14 35.81 RSU% 27.1 7.9 239.05.86 RSU% 6.7 320.11 5.710 12.25 5.16 33.41 71.33 2.68 1119.1 7.8 std dev 3951.27 7.03 3.0 std u 1767.710 12.0 100.0 453.18 45.82 36.616 6.8 72.05.5 151.24 11.4 294.6 142.65 2.66 Uncertainty of Mean & Expanded U at 95% CL std u 2787.8 180.59 66.710 12.61 4.30 5.04 5.74 Exp U% (k=tcrit) 77.04 D-Leu peak area 1 2 3 4 5 6.69 5.37 Exp U% (k=tcrit) D+L Leu peak height 004 005 IE IE *= aberrant value.8 213.

26 19.50 RSU% 1.77 40.50 1.710 12.8 1.6 21.1 2.83 D-Leu Conc AAR PT Report.0 1.710 t critical (0.28 10.25 0. Standards Solution THAA 1 2 3 4 5 6.10 1.70 L-Leu Conc 1 2 3 4 5 6.92 0.38 0.70 *= aberrant value.710 12.95 2.61 3.5 2.61 Exp U% (k=2) 1.05.1 6.1 0.55 174.48 4.81 1.df) 2.24 17.10 0.9 1.86 14.8 12.1 6.93 4.31 6.710 12. removed for subsequent statistical analysis Page 71 of 157 .34 39.8 0.710 12.710 12.63 6.777 8.68 1.2 7.3 RSU% 0.11 8.07 Exp U% (k=tcrit) 2.7 3.55 0.96 14.6 3.1 std dev 0.7 0.48 1.37 Uncertainty of Mean & Expanded U at 95% CL std u 0.20 1.710 12.02 1.4.28 0.17 1.12 CV% 2.710 12.39 10.2 8 9 10 11 12 13 14 15 73 68 70 126 162 143 a 30 72 73 71 156 165 147 b 30 c 31 d 31 e 31 f g h i j 73 70 71 141 163 145 mean 31 2 2 2 2 2 2 n 5 0.19 20.05.3 0.1 1.710 12.3 1.1 7.777 Exp U% (k=tcrit) 1.7 0.22 21.3 87.50 1.710 1109.39 7.4 1.66 3.1 1.710 12.78 20.00 Exp U% (k=2) 2.4 14.33 2.22 t critical (0.3 0. STATISTICAL EVALUATION – Summary Statistics Table 4.1 7.6 8.35 3.2 std u 0.14 2.2 0.58 19.32 3.61 10.710 12.04 12.df) 2.27: Summary Statistics for L and D Leucine Concentration Data (pM) Lab No method a RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 49 b 51 c 50 Submitted Replicate data d 49 e 50 f g h i j Standard Deviation mean 50 n 5 std dev 0.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 43 40 41 76 96 85 3* 43 42 94 98 87 23 41 42 85 97 86 2 2 2 2 2 2 28.2 7.7 CV% 1.10 12.64 130.9 123.75 134.

16 0.589 0.710 12.0006 0.33 0.560 0.30 0.637 e 0. GCA= derived using peak area GCHt = derived using peak height *= aberrant value.0060 0.63 AAR PT Report.595 0.040 0.67 0.56 0.07 174.594 0.43 0.0004 0.79 1.26 0.710 12.77 0.14 6.315 0.566 *0.523 0.0161 CV% 2.602 0.32 0. STATISTICAL EVALUATION – Summary Statistics Table 4.365 2.87 1.589 0.95 1.3878 0.594 0.75 123.23 0.594 8 6 5 2 2 1 2 2 2 2 2 0.0003 0.627 f g h i j Standard Deviation mean 0.05.78 0.07 0. Standards Solution THAA = submitted as the mean and standard deviation of n results.11 1.1 1 6.596 0.594 0.529 0.68 0.604 0.19 0.540 0.4.10 0.df) 2.523 0.0011 0.0046 0.599 b 0.603 c 0.595 0.07 0.05 0.710 12.0024 0.91 1.617 Submitted Replicate data d 0.617 n 5 std dev 0.0008 0.45 0.86 9.0050 0.2 8 9 9 10 11 12 13 14 15 1 method a RP RP RP IE IE GCA GCHt GCA GCHt RP RP RP RP RP RP RP RP RP 0.26 0.571 2.2742 0.72 0.710 12.605 0.89 3.529 0.710 12.28: Summary Statistics for L and D Leucine D/L Ratio Value Lab No D/L Leu 1 2 3 4 5 1 6.61 0.1 1 7.77 1107.0018 0.2 1 7. removed for subsequent statistical analysis Page 72 of 157 .0040 0.540 0.33 t critical (0.0065 0.54 1.777 12.586 0.0018 0.777 Exp U% (k=tcrit) 3.0004 0.0026 0.587 0.10 2.710 0.0072 RSU% 1.53 87.710 12.13 1.16 Exp U% (k=2) 2.0030 0.60 Uncertainty of Mean & Expanded U at 95% CL std u 0.589 0.17 2.586 0.587 0.13 0.563 0.10 1.78 0.0090 0.0042 0.591 0.

Standards Solution THAA 0.2 8 9 10 11 13 14 15 Laboratory Number Page 73 of 157 .4.2 7.25 Assigned value (all data) AAR PT Report.60 0.05 0. removed from subsequent statistical analysis 0.55 0.1 6.20 Aberrant value.40 0.30 0. STATISTICAL EVALUATION – Summary Statistics Figure 4.15 0.10 0.26: Distribution of D/L Values submitted for Leucine RP 0.45 D/L Value 0.70 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.00 1 2 3 4 5 6.1 7.65 0.50 0.35 Submitted Value Submitted mean & std dev Assigned value (rpHPLC only) 0.

40 1. RP 4.80 1.df)) of the Mean D/L value for Leucine (value of n displayed).2 8 9 10 11 13 14 15 Laboratory Number Figure 4.80 3.20 2. RP 1.40 3. STATISTICAL EVALUATION – Summary Statistics Figure 4.00 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP Replicate means 0.20 8 6 5 2 2 2 2 2 2 2 0.20 Replicate means 3.30 0.40 D/L Value 2.2 8 9 10 11 13 14 15 Laboratory Number Page 74 of 157 .20 0.80 5 0.80 2.40 0.1 7.1 7.00 1 2 3 4 5 6.AAR PT Report.27: Experimental Expanded Uncertainty (k=2) of the Mean D/L value for Leucine (value of n displayed).50 0.90 0.60 2 2 2 2 2 8 D/L Value 6 2 5 0.20 1.60 1.60 2.60 3. Standards Solution THAA 4.70 5 0.05.80 0.1 6.60 0.00 1.40 2 0.2 7.00 1 2 3 4 5 6.00 0.00 2.10 0.00 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 3.1 6.2 7.28: Experimental Expanded Uncertainty (k=t(0.

8 55.28 Exp U% (k=2) 12.710 12.710 12.16 3.710 12.90 7.49 12.49 3.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 3292 1771 1714 5524 3384 3184 3061 1903 1792 5802 3713 3363 3176 1837 1753 5663 3549 3274 2 2 2 2 2 2 163.82 58.34 45.18 5.38 31.88 115.05.10 193.47 6.6 148.710 12.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 9491 5854 5491 a 9963 6417 5789 b c d e f g h i j 9727 6136 5640 mean 2 2 2 n 333.23 2.710 12.47 4.33 33.3 196.75 7. Standards Solution THAA D-Met peak area 1 2 3 4 5 6. STATISTICAL EVALUATION – Summary Statistics Table 4.1 139.4 127.55 36.1 6.0 87.4.710 45.43 6.21 58.79 12.65 3.46 4.05.3 89.16 5.710 12.710 12.9 281.1 164.1 7.09 10.83 .64 RSU% 4.710 t critical (0.710 12.07 65.29: Summary Statistics for L and D Methionine Peak Area Data Lab No method a b c Submitted Replicate data d e f g h i j Standard Deviation mean n std dev CV% Uncertainty of Mean & Expanded U at 95% CL std u RSU% Exp U% (k=2) t critical (0.4 std dev 3.3 3.2 210.8 93.02 7.4 5.73 CV% 235.2 7.8 66.df) Exp U% (k=tcrit) L-Met peak area 1 2 3 4 5 6.3 39.59 2.0 227.710 46.29 7.42 4.22 4.6 232.90 Page 75 of 157 5658 2961 2925 5270 3283 3099 5464 3122 3012 2 2 2 274.1 7.91 9.55 3.85 9.8 std u 2.31 5.6 398.1 6.85 34.8 161.10 3.29 4.55 5.df) 30.88 28.53 Exp U% (k=tcrit) AAR PT Report.9 3.61 2.16 2.710 12.1 5.2 7.26 5.7 123.63 2.

09 54.42 .06 29.710 12.6 n 11.4.5 11.2 9.4 12.83 37.94 6.33 2.0 19.85 1.710 12.34 2.1 6.96 1676.67 4.710 12.91 9.9 15.06 5.1 7.417 1 1 Page 76 of 157 1701 941 894 1590 980 948 1646 961 921 2 2 2 77.25 6.2 7.1 7.45 3.6 38.04 2.df) 2.05. Peak Area/Height Data Lab No method a b c Submitted Replicate data d e f g h i j Standard Deviation mean n std dev CV% Uncertainty of Mean & Expanded U at 95% CL std u RSU% Exp U% (k=2) t critical (0.2 27.51 25.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP a 5279 397 446 5532 416 428 1274 7289 11626 6200 406 437 5 2 2 3748.df) AAR PT Report.411 0.73 2.0 CV% 7.61 L-homoArginine peak area 1 2 3 4 5 6.66 26. STATISTICAL EVALUATION – Summary Statistics Table 4.87 4.68 t critical (0.710 12. Standards Solution THAA Norleucine peak height 1 2 3 4 5 6.95 4.411 0.19 1627 776 829 1387 856 851 a b c d e f g h i j 1507 816 840 2 2 2 m ea n 169.417 0.5 9.89 12.31 std u 15.16 55.710 Exp U% (k=2) 101.05.85 1.5 27.1 3.710 12.1 6.03 2.62 RSU% 12.9 39.69 4.69 2.710 Exp U% (k=tcrit) 75.4 4.710 42.07 4.2 13.8 27.30 2.777 12.86 std dev 119.11 61.2 7.5 55.59 16.30: Summary Statistics for HPLC Internal Standards.9 60.2 8 9 10 11 12 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP RP 0.

|z|≤2.. matrix matched CRM with a known value and uncertainty. ISO 13528. The results of a typical chemical analysis will be normally distributed about the mean with a known standard deviation. such that. STATISTICAL EVALUATION-Accuracy & Performance Analysis 5 STATISTICAL EVALUATION. 2005). It follows that if a participant’s z-score lies outside |z| >2 there is about a 1 in 20 chance that their result is in fact an acceptable result from the extreme of the distribution. σp = the target standard deviation. Satisfactory performance is indicated by achieving a z-score no greater than 2.1 z-Scores Participation in a proficiency test provides the opportunity to evaluate analytical bias by comparing an individual laboratory’s result against the assigned value for the test material. Approximately 95% of data will be expected to lie within 2 standard deviations either side of the mean and 99. 2006). However.. where = the mean of participant’s reported replicate results (or simply reported result) = the assigned value.1. Page 77 of 157 . but may give rise to unrealistically small uncertainties.. In routine analysis a laboratory’s evaluation of analytical competence is often restricted to intra-laboratory precision evaluation of repeated analyses or the evaluation of bias using certified reference materials (CRM’s). evaluation of method and/or laboratory bias can be impossible without the cooperation of additional laboratories. Accuracy & Performance Analysis 5. 2006. in the absence of a suitable.7% within ± 3 standard deviations. calculated using the submitted result. Performance is traditionally determined by the calculation of a z-score.AAR PT Report. Estimations of precision may be excellent when taken in isolation.1 Background to understanding Performance Evaluation The purpose of this evaluation is to provide a clear and independent statistical evaluation and comparison of participants’ results. using a procedure recommended in the IUPAC/ISO/AOAC International Harmonised Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories (Thompson et al. i. If a participant’s z-score lies outside |z| >3 the chance that their result is actually acceptable is only about 1 in 300 (Thompson et al. a reference or assigned value and the target value for standard deviation. is the calculation for bias. it is considered ‘satisfactory’ if a participant’s z-score lies within this range. for a single and Note that. Thus.e. 5. Standards Solution THAA 5.

Horwitz. 2006).2 The Target Standard Deviation. such that . mg/Kg = 10-6. Horwitz. there has not been an inter-laboratory collaborative trial carried out according to international guidelines (AOAC. it can be assumed that participants will be hoping to achieve a satisfactory performance and achieve fitness-for-purpose. the standard deviation of the assigned value.1. expressed as % ˆ . the assigned value. in the absence of an external value for target standard deviation. The Horwitz equation requires the measurement units to be expressed as a mass fraction. expressed in relevant concentration.1) should be small by comparison to the target standard deviation. . σp The target standard deviation ( ) describes how the data is expected to perform for a given analyte and / or test material and determines the limits of satisfactory performance. It is therefore not an unreasonable expectation that the distribution of submitted results (i. should be close to the limits of satisfactory performance.. Page 78 of 157 . STATISTICAL EVALUATION-Accuracy & Performance Analysis 5. 5.. (Note. 2000. p where and RSDR c 100 RSDR c = = Relative Standard Deviation of Reproducibility from collaborative trial data. 1982. and that the target value is not overly restrictive. the use of perception or fitness-for-purpose criteria may need to be employed. RSC Analytical Methods Committee.e. which is not appropriate in the current study as D/L results are expressed as a ratio and are thus dimensionless. 1980. i. A further comment is made in the International Harmonised Protocol concerning the uncertainty of the assigned value to ensure it is sufficiently small so as not to overly influence the calculation of z-scores.AAR PT Report. 2004) is widely accepted as a suitable predictive measure for the target standard deviation in chemical analysis. .e.. These values are often obtained from collaborative trials as the reproducibility standard deviation ( ). it was necessary to use perception using fitness-for-purpose criteria. Standards Solution THAA 5. the Horwitz function is not necessarily suited to every type of chemical analysis and in the absence of a suitable alternative. It is recommended that which approximates to as also recommended in ISO 13528 (2005). This ensures that the data are sufficiently tight to give a measure of confidence in the assigned value. Therefore.3. which describes best practice for a specified method for a given matrix/analyte/ concentration (Thompson et al. X units. ( ). As a general rule. However. The International Harmonized Protocol (2006) states that if then “laboratories are having difficulty achieving the required reproducibility precision in results from a single population. 1995) to determine single method precision parameters for amino acid racemization analysis on fossil material. ). or that two or more discrepant populations may be represented in the result”.. In the absence of collaborative trial data. The distribution of submitted results and uncertainty of the assigned value ( (see section 5.2 In the absence of Fitness-for-Purpose Criteria To date. The exact value chosen represents the appropriate order of magnitude although the exact value is to some extent discretionary). i.e. the Horwitz equation (Horwitz et al. taking into consideration any uncertainty in homogeneity of test materials.

The relative value of expressed as a percentage. The distributions of the mean values are presented as dot plots in Figure 5. the homogeneity target value was too wide compared to the submitted data for the test. Laboratory results were calculated from the mean of submitted replicate data so as not to dominate and unfairly influence the distribution by a single method. but requires the target value to be scaled appropriately to the individual analyte or matrix.e. i. analyst or single test material.e. STATISTICAL EVALUATION-Accuracy & Performance Analysis The target value chosen during homogeneity evaluation.1. Page 79 of 157 . ( ) is an excellent indication of the observed variation within test materials and reflects the uncertainty due to matrix plus the analytical method used for their determination.1. During the statistical evaluation of data. When calculating z-scores. the RSD%. in the absence of such data. such that. Whilst an inter-laboratory collaborative trial reproducibility standard deviation (RSDR%) would also reflect an additional laboratory component of variation. performance has not been determined by the calculation of z-scores but rather an evaluation of bias has been carried out. it was observed that for some amino acids in some test materials provided in this series of studies.1 Relative percentage bias Whilst these observations were surprising. as the denominator acts to normalize results. still permits a comparison between analytes and test materials but on a common percentage scale.. However. Standards Solution THAA 5. representing 95% confidence limits. it posed some difficulties in using objective fitness for purpose criteria for the determination of the target values for standard deviation. This enables performance between different analytes or between different test materials to be compared on a common scale. . the use of a standard deviation. suggesting that the precision between different laboratories in some instances was better than that observed between samples analysed by a single laboratory under repeatability conditions for homogeneity! 5. On this occasion.AAR PT Report. is a more useful value and can be used to set the minimum permissible value for . using the assigned value as the denominator.2 – 5.17. with the 2 std deviation satisfactory range being given as percentage values rather than ±2.2.. ± 2 In this way it was possible to represent participant’s results graphically as histograms in a similar way to z-score charts. assigned value. and shown as histograms in Figures 5. and calculating the relative percentage bias. In order to overcome this problem and in order to ensure consistency between test materials. thus providing perhaps a slightly more intuitive presentation of observed bias for individual results. Laboratory mean values and relative percentage bias for each amino acid are given in Table 5. in the absence of independently determined performance criteria it was decided to present the data as an assessment of relative bias (%). it none the less makes a good starting point for evaluating submitted results and provides a minimum fitness-for-purpose target value. i. Satisfactory performance was assessed as plus or minus twice the standard deviation of the ˆ.

1 The uncertainty of the Assigned value . (SEM). In such instances. RSC Analytical Methods Committee. 1989. 2002a.3. 2001) was strongly influenced by extreme values resulting in a skewed distribution with a high or low end tail. When determining the appropriate measure of central tendency. equates to the consensus from expert laboratories! Whilst assessing the data. Standards Solution THAA 5. a consensus of expert laboratories. the robust mean is often used as the best estimate in a large data set as it minimises the effect of outliers and gives a fairer estimate of central tendency. then this can have an adverse impact by exaggerating observed bias. For the mode. If there is too much uncertainty associated with the assigned value. which due to the low numbers of participants involved. 2006. STATISTICAL EVALUATION-Accuracy & Performance Analysis 5. or the consensus of submitted results..AAR PT Report. RSC Analytical Methods Committee. Page 80 of 157 . This appeared largely influenced by method and on occasions by an individual laboratory where more than one result was submitted using the same method. RSC Analytical Methods Committee. in many cases it became clear that the robust mean (Ellison. Depending on the nature of a test material. this can be done in a number of different ways. the use of the median may be more suitable or even the mode. but carried out using a different instrument or analyst. 5. when determining the mode (Ellison. ˆ . for example the use of a reference value from a Certified Reference Material. For the robust mean and median: Where and m ˆ = = the number of laboratory results used to calculate the robust mean or median the standard deviation of the robust mean or median absolute deviation (sMAD). additional modes were identified due to only a couple of values and in some cases only a single data point. In addition. i. 2002b. either m is too small or the distribution of results is too large. whilst the robust mean may still be preferable to the standard mean.4 Derivation of for Amino Acids in Standards Solution Test Material In this study all assigned values have been determined as the consensus of submitted data.e. X analyte. (Note this is not the same as the target standard deviation used for calculating z-scores (σp)). ) is taken to be directly equivalent to the standard error of the mode. In determining the assigned value for a specific analyte. However. for small data sets such as here. Lowthian and Thompson.3 The Assigned Value. it became clear that due to the low numbers of results. 5. the effect of the uncertainty of the assigned value ( on performance assessment also needs to be given consideration. is the best estimate of the true concentration of each The reference or assigned value. 2002a) are shown against each histogram for amino acids with eight or more data points. Plots showing the modal distributions derived using the kernel density Excel add-in (Ellison. 2002). the influence of extreme values may still be significant.

Regrettably submitted results by GC were limited making it difficult to know whether the observed differences are genuine method differences or simply extreme values. There is no judgment being made as to which set of results is ‘correct’. Caution should be exercised when evaluating the results from this study. phenylalanine. together with the number of ˆ and the standard uncertainty laboratory results m. it would not be appropriate to penalise these laboratories by comparison against an assigned value determined from the primary or first mode. The medians used to set the assigned values for all amino acids. The extent of any such differences between GC and HPLC or even between rpHPLC and HPLC-IE for the analysis of amino acid racemization. glutamic acid/ glutamine. Proficiency tests in principle tend not to be method prescriptive unless methods are known to be empirical and produce different results. the standard deviation of the assigned value. define reference values for the use of any remaining material in place of CRMs enhancing quality control processes.3 then gives the percentage of laboratories with mean values falling within ± 2 standard deviations of the assigned value. 5. and permit the objective assessment of participants’ PT data in future studies. of the assigned value. GC data have been included with HPLC values and initially evaluated against the same assigned value. such that the method defines the output. rpHPLC results have also been evaluated separately for comparison. In situations such as this where the method may be empirical. STATISTICAL EVALUATION-Accuracy & Performance Analysis In cases where there were two evenly matched modes or where a smaller second mode was predominated by data using a specific method such as GC. However. A greater number of laboratories submitting GC data may have helped to answer this. The report indicates a number of issues such as the level of agreement between HPLC and GC or even between reverse phase HPLC and ion-exchange HPLC methods. alloisoleucine/isoleucine and leucine GC data can be seen to contribute to high or low end tails. and whether these approaches should be considered empirical. have not been fully established to date. the mode should not be used. it would not be appropriate to calculate performance for GC results using an assigned value determined from HPLC values if the GC data clustered differently. Therefore.5 Interpreting Results . Whilst in this test material GC results for alanine and valine appear to fall within the general distribution of the data. for consistency with other test materials in this series. Table 5. for aspartic acid/asparagine. where GC data has been provided. For these reasons. This is suggested from results of a number of amino acids. therefore. Determination of method specific assigned values would therefore provide truer estimates of bias and uncertainty and a more accurate performance evaluation. the median has been used as the most appropriate measure of central tendency for all amino acids. Standards Solution THAA 5.2. As an indicator of best practice this would provide guideline uncertainty estimates (so long as a laboratory’s repeatability complied with published values).AAR PT Report. Insufficient data prevented a separate evaluation for GC or HPLC-IE methods individually. Page 81 of 157 .a word of caution. This allows for any asymmetry arising from bimodality to be seen in the histograms but makes no judgment as to the correct mode. Obtaining an independent and externally derived precision estimate for the target standard deviation such as the reproducibility standard deviation obtained from a collaborative trial becomes paramount for the future. results from all statistical evaluations should be treated for information only due to the absence of external reference data and the uncertainty surrounding assessment parameters. The median ignores the effect of outliers and assumes a normal distribution placing data symmetrically placed either side of the mid-point. in this proficiency test. are given in Table 5. Whilst every effort has been made to provide a statistically sound and informative comparison and assessment of data. .

1 -2.2 8 9 10 11 13 14 15 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.9 Total Hydrolysed Amino Acid (THAA) Asx D/L (rpHPLC) assigned value result D/L 0.558 3.2 3.0 -0.9 1 2 3 4 5 6.497 29.552 0.5 Glx D/L (rpHPLC) assigned value result D/L 0.2 19. Page 82 of 157 .8 24. or as submitted by participants.499 0.623 0.3 0.1 2. STATISTICAL EVALUATION-Accuracy & Performance Analysis Table 5.2 1.1 -0.0 -0.547 0. where a mean value was provided.0 0.491 0.499 0.0 0.569 0.554 0.558 0.5.497 0.6 0.0 0.0 0.2 -0.5 0.1 -1.1 7.6 2.1 -0.500 0.552 0.2 7.5 0.592 0.499 0.541 0.3 -0.1 6.569 0.8 -0.552 0.496 0.9 2.547 0.2 31.3 -0.552 0.7 -1.3 0.496 0.599 0.500 relative bias % -1.6 -0.1 7.647 0.552 relative bias % 1.4 18.7 -0.500 0.656 0.6 -0.0 -2.541 0.500 0.4 -2.552 0.554 0.2 Glx D/L (all) assigned value result D/L 0.536 0.7 -3.510 0.558 0.4 -0.6 0.4 0.1: Results and Relative Percentage Bias for Total Hydrolysed Amino Acids in Standards Solution Test Material Lab No.536 0.552 0.515 AAR PT Report.690 0.599 0.9 1.5 12.8 -0.558 6.499 0.2 -0.591 0.1 Results shown are the average of replicate values where more than one value was given. method Asx D/L (all) assigned value result D/L 0.510 0.556 relative bias % 0.515 0.491 0.499 relative bias % -1.500 0.497 0. Standards Solution THAA 0.497 0.

4 -0. Standards Solution THAA 8 9 10 11 13 14 15 Results shown are the average of replicate values where more than one value was given. where a mean value was provided. or as submitted by participants. Page 83 of 157 .367 relative bias % Ala D/L assigned value result D/L 0.1 -0.1 6.402 0.6 -0.2 AAR PT Report.6 0.368 -6.0 19.7 16.500 0.555 0.344 0.1 0.405 0.461 -23.393 0.1 -0.3 0.1 -23.469 0.400 0.7 -1.1 20.3 0.380 0.2 -0.3 0.1: Results and Relative Percentage Bias for Total Hydrolysed Amino Acids in Standards Solution Test Material (continued) Lab No.5 18.2 7.466 0.9 0.8 1.464 0.467 0.1 7.461 6.401 relative bias % 0.468 0.555 0.5.556 0.2 -0.7 Ala D/L (rpHPLC) assigned value result D/L 0.408 0.6 -0.8 0.469 relative bias % 16.2 -0.563 0.1 0.4 -0.2 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0. method Ser D/L (rpHPLC) assigned value result D/L 1 2 3 4 5 6.1 0. STATISTICAL EVALUATION-Accuracy & Performance Analysis Table 5.0 18.464 0.2 2.544 0.359 0.469 0.544 0.5 3.344 0.467 0.6 0.4 -6.9 -1.466 0.395 2.7 -0.359 0.7 Total Hydrolysed Amino Acid (THAA) Arg D/L (rpHPLC) assigned value result D/L 0.5 -1.476 -3.409 0.547 0.400 0.356 0.9 1.556 0.5 19.5 -0.6 -2.376 0.412 0.0 29.465 0.467 relative bias % 16.2 -1.465 0.366 0.395 0.

1 0.414 relative bias % 11.6 Phe D/L assigned value result D/L 0.478 0.3 0.2 -0.493 0.496 0.5.3 0.490 0.489 0.491 relative bias % 0. where a mean value was provided.5 0.491 -2.478 0.6 12.497 0.6 0.494 0.480 0.2 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0. method Val D/L assigned value result D/L 1 2 3 4 5 6.413 0.2 3.415 0.7 -3.2 Phe D/L (rpHPLC) assigned value result D/L 0.6 1.413 0.412 0.412 0.498 0.1 1. or as submitted by participants.0 0.1 -0.6 Total Hydrolysed Amino Acid (THAA) Val D/L (rpHPLC) assigned value result D/L 0.414 0. STATISTICAL EVALUATION-Accuracy & Performance Analysis Table 5.2 -0.2 -0. Standards Solution THAA 8 9 10 11 13 14 15 Results shown are the average of replicate values where more than one value was given.3 0.409 0.493 relative bias % 0.9 -2.3 -0.478 0.6 1.466 0.4 1.497 0.473 0.415 0.461 0.2 7.4 -0.497 0.1 7.498 0.4 -0.2 -1.427 0.0 0.461 0.491 -2.475 0.494 0.466 0.2 -2.4 0.5 0.0 0.414 15.413 0.3 12.403 -0.3 0.409 0.2 -1.0 0. Page 84 of 157 .414 0.492 0.414 15.489 0.5 -0.0 -0.6 12.493 0.490 0.466 0.1 6.497 0.3 0.7 -3.3 0.9 AAR PT Report.6 0.480 0.492 0.3 -0.3 12.466 0.4 -0.1: Results and Relative Percentage Bias for Total Hydrolysed Amino Acids in Standards Solution Test Material (continued) Lab No.9 0.414 relative bias % 11.

0 -2.2 Total Hydrolysed Amino Acid (THAA) D-Aile/L-Ile (rpHPLC) assigned value result D/L 0.3 2.3 -11.523 -10. STATISTICAL EVALUATION-Accuracy & Performance Analysis Table 5.3 10.4 -1.567 0.2 RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.617 0.589 relative bias % 4.7 0.4 0.557 0.577 0.1 6.591 0.555 0.6 -1.3 -1.587 0.562 0.4 -1.558 0.595 0.540 0. Page 85 of 157 .623 0.5 -0.558 relative bias % 0.4 3.529 0.1 0.7 3.574 0.562 0. Standards Solution THAA 8 9 10 11 13 14 15 Results shown are the average of replicate values where more than one value was given.6 0.566 relative bias % -0.563 0.0 0.7 -1.566 0.5 1.584 0.571 0.587 0.595 0.594 -5.1 0. where a mean value was provided.577 0.1 -0.589 0.9 AAR PT Report.566 0.604 0. or as submitted by participants.7 Leu D/L (rpHPLC) assigned value result D/L 0.5. method D-Aile/L-Ile (all) assigned value result D/L 1 2 3 4 5 6.6 1.9 -2.7 2.3 3.2 -8.558 0.0 -0.563 0.1 0.555 0.1 -0.3 0.555 0.574 0.2 7.587 0.1 7.617 0.552 0.591 0.9 0.552 0.554 1.1 -0.557 0.3 0.604 0.2 3.594 -4.0 -0.0 1.8 -1.0 Leu D/L (all) assigned value result D/L 0.0 0.4 1.589 0.8 0.4 -0.554 0.593 relative bias % 3.1: Results and Relative Percentage Bias for Total Hydrolysed Amino Acids in Standards Solution Test Material (continued) Lab No.555 0.

48 2.02 0.367 0.76 6.0026 0.593 sMAD ( ) 0.491 0.0019 0. Standards Solution THAA 5.008 0.558 0.589 0.0019 0.006 0.0042 0.28 1.499 0.53 0.016 0.0044 0.006 0.0027 0.007 RSD % 2.40 0.0013 0.552 0.47 0.2: Assigned Values.0020 0.0039 0.38 4.01 3.469 0. GC and HPLC-IE data m = number of replicate mean values sMAD = median absolute deviation CV% = coefficient of variation expressed as a percentage RSU% = Relative standard uncertainty expressed as a percentage Page 86 of 157 .53 0.0062 0.493 0.010 0.47 0.556 0.41 0. Standard Deviations and Standard Uncertainties analyte m Asx D/L (all ) Asx D/L (rpHPLC) Glx D/L (all ) Glx D/L (rpHPLC) Ser D/L (rpHPLC) Arg D/L (rpHPLC) Ala D/L (all ) Ala D/L (rpHPLC) Val D/L (all ) Val D/L (rpHPLC) Phe D/L (all ) Phe D/L (rpHPLC) D-Aile/L-Ile (all ) D-Aile/L-Ile (rpHPLC) Leu D/L (all ) Leu D/L (rpHPLC) a a b a a a a a assigned value Median ( ) 0.70 0.017 0.414 0.64 0.0008 0.008 0.401 0.0024 RSU % 0.022 0.006 0.90 1.500 0. STATISTICAL EVALUATION-Accuracy & Performance Analysis Table 5.0066 0.0030 0.85 2.566 0.15 0.79 0.0022 0.40 14 10 14 10 10 8 14 10 13 10 14 10 15 10 11 8 b = rpHPLC and GC data = rpHPLC.49 1.414 0.008 0.0077 0.002 0.27 0.AAR PT Report.54 0.50 2.94 1.13 Std uncertainty of median ( ) 0.71 1.32 1.029 0.12 1.015 0.12 0.467 0.010 0.0018 0.68 1.69 1.75 1.

500 0.558 0. STATISTICAL EVALUATION-Accuracy & Performance Analysis Table 5.414 0. Standards Solution THAA 5.AAR PT Report.467 0.414 0.566 0. GC and HPLC-IE data Page 87 of 157 .401 0.593 b 10 7 10 9 10 7 8 6 9 6 12 9 14 8 8 6 14 10 14 10 10 8 14 10 13 10 14 10 15 10 11 8 m = number of participants’ results 71% 70% 71% 90% 100% 88% 57% 60% 69% 60% 86% 90% 93% 80% 73% 75% = rpHPLC and GC data = rpHPLC.552 0.3: Satisfactory Performance(Percentage within 95% Confidence Interval) analyte Median ( ) Satisfactory m assigned value Total number of m Percent satisfactory Asx D/L (all ) Asx D/L (rpHPLC) Glx D/L (all ) Glx D/L (rpHPLC) Ser D/L (rpHPLC) Arg D/L (rpHPLC) Ala D/L (all ) Ala D/L (rpHPLC) Val D/L (all ) Val D/L (rpHPLC) Phe D/L (all ) Phe D/L (rpHPLC) D-Aile/L-Ile (all ) D-Aile/L-Ile (rpHPLC) Leu D/L (all ) Leu D/L (rpHPLC) a a b a a a a a 0.493 0.499 0.469 0.556 0.589 0.491 0.367 0.

5) Ala D/L Arg D/L Ser D/L Glx D/L Asx D/L 0 0. Standards Solution THAA 5.6 0.3 0.2 0.8 0.1 0. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.9 1 .4 0.7 0.1: Distribution of Participants’ Average Measurement Values Tyr D/L Leu D/L D-Aile/L-Ile Phe D/L Val D/L Amino Acid Page 88 of 157 AAR PT Std sol (D/L≈ 0.AAR PT Report.5 D/L Value 0.

6 0.520 D/L 2.0 8 7 6 5 32.0 8. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.AAR PT Report.2 7.2 6.1 11 -6.0 30. Standards Solution THAA 5.480 D/L 9 8 2 3 6.500 D/L RP RP RP RP RP RP 0.0 26.0 20.0 RP 0.2: Relative Percentage Bias for Aspartic Acid / Asparagine D/L Results (all data) in Standards Solution Test Material 10 9 34.0 6.0 14.7 0.0 4.0 -4.0 1 Laboratory Number Page 89 of 157 15 14 13 10 .0 12.0 GC GC 4 3 2 24.0 1 0 0 0.3 0.0 0.0 28.4 0.0 10.5 0.0 -2.1 0.1 7.0 RP RP RP 0.8 GC GC Relative bias (%) 16.0 22.0 18.2 0.

0 0 0.49 0.0 1.5 0.3: Relative Percentage Bias for Aspartic Acid / Asparagine D/L Results (rpHPLC data only) in Standards Solution Test Material 120 100 80 5.51 0.499 D/L -1.0 60 4.504 D/L 0.48 0.0 40 20 RP 3.AAR PT Report.494 D/L -2.53 RP Relative bias (%) 2. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.0 RP 0.0 1 Laboratory Number Page 90 of 157 9 8 2 3 11 15 14 13 10 .0 RP RP 0.52 0.0 RP RP RP RP 0. Standards Solution THAA 5.0 RP -3.

556 D/L RP RP RP RP 0.0 22.4: Relative Percentage Bias for Glutamic Acid / Glutamate D/L Results (all data) in Standards Solution Test Material 16 14 12 26.0 4. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.2 0.1 7.0 8.4 0.0 14.3 0.0 3 2 9 RP 0.0 -4.0 16.7 0.0 10 GC 24.0 0.5 0.0 4 2 0 0 0.0 8 6 20.5.0 -6.589 D/L 2.6 0.523 D/L 1 8 6.2 7.0 12.0 GC GC AAR PT Report.0 -8.2 Laboratory Number Page 91 of 157 13 11 10 14 15 .8 GC Relative bias (%) 10.1 6.0 18.1 0.0 -2.0 RP RP RP RP RP 0. Standards Solution THAA 6.

58 0.0 RP 0.0 3.0 3 2 Laboratory Number Page 92 of 157 9 1 13 11 10 14 15 .59 4.0 5. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.5.0 -8.0 0.0 RP AAR PT Report. Standards Solution THAA -3.569 D/L RP RP RP RP RP Relative bias (%) 1.56 0.55 0.536 D/L -5.0 6.0 RP RP 0.0 -1.57 0.52 0.51 0.5: Relative Percentage Bias for Glutamic Acid / Glutamate D/L Results (rpHPLC data only) in Standards Solution Test Material 50 45 40 35 30 8.0 -6.087 D/L -2.0 25 20 15 7.0 0.53 0.0 2.0 -7.0 -4.0 10 5 0 0.54 0.

0 0 0.37 0.0 5 5.382 D/L -8.0 0. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.43 0.38 0.0 6.4 0. Standards Solution THAA -2.0 -1.401 D/L RP AAR PT Report.0 RP RP RP 0.0 RP -5.0 -4.39 0.420 D/L RP RP 4.0 2.6: Relative Percentage Bias for Serine D/L Results (all / rpHPLC data) in Standards Solution Test Material 50 45 40 35 30 25 8.0 RP RP RP Relative bias (%) 1.0 -7.0 0.42 0.0 -6.0 20 15 10 7.41 0.0 3.5.0 9 Laboratory Number Page 93 of 157 1 2 3 8 10 15 13 14 11 .0 -3.

4 0.0 26.0 6.0 30.0 10.0 -2.1 0.0 -6.0 8.332 D/L 9 Laboratory Number Page 94 of 157 2 3 13 14 15 10 11 .7: Relative Percentage Bias for Arginine D/L Results (rpHPLC data only) in Standards Solution Test Material 18 16 14 AAR PT Report.6 0. Standards Solution THAA 32.0 -4.0 24.0 14.0 -12. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.0 4.0 12.402 D/L RP RP RP 0.0 0.367 D/L RP RP RP RP 0.0 12 10 8 6 RP 4 2 0 Relative bias (%) 0 0.0 2.2 0.0 -10.3 0.0 -8.0 16.0 18.0 20.0 28.5 0.0 22.5.

4 0.2 0.5.0 -10.0 3 RP RP GC RP GC 2 1 0 0 0.8: Relative Percentage Bias for Alanine D/L Results (all data) in Standards Solution Test Material 8 7 6 5 25.2 15 14 11 10 Laboratory Number Page 95 of 157 13 .0 0.520 D/L GC Relative bias (%) RP 0.0 RP RP RP RP RP GC 0.0 10.0 5.418 D/L AAR PT Report.6 0.5 0.0 RP -30.3 0.2 2 3 7.1 1 6.7 0. Standards Solution THAA -15.1 0.0 15.469 D/L -5.0 -20.0 8 9 7.1 6.0 4 20.0 -25. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.

0 6 5 20.5 0.478 D/L 0.0 -15.0 -20.7 5.4 0.0 RP RP RP RP -5.2 0.3 0. Standards Solution THAA Laboratory Number Page 96 of 157 8 9 1 2 3 15 14 11 10 13 .0 15.6 0.0 RP 4 RP RP 3 2 10.455 D/L 0.1 0. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.0 -25.0 RP AAR PT Report.9: Relative Percentage Bias for Alanine D/L Results (rpHPLC data only) in Standards Solution Test Material 8 7 25.0 RP RP 0.467 D/L 0.0 -10.5.0 1 Relative bias (%) 0 0 0.

5.0 14.0 2.2 1 2 3 8 7.399 D/L Laboratory Number Page Page97 97of of157 157 6.0 15.0 -6.0 10.414 D/L GC RP GC 0.428 D/L RP RP RP RP RP 0.6 Relative bias (%) GC 0.0 1.0 12.0 -1.0 -5.0 0.0 4.4 0.3 0.5 0.0 14 RP 12 10 8 6 4 RP RP RP 2 0 0 0.0 6.1 9 14 11 13 15 10 .10: Relative Percentage Bias for Valine D/L Results (all data) in Standards Solution Test Material 20 18 16 AAR PT Report.0 13.0 5.0 -4.1 6.0 3.2 0.0 -3. Standards Solution THAA 17.1 0.0 16.0 11.0 8.0 7.0 9.0 -2. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.

4.0 4.0 16.402 D/L Laboratory Number Page Page98 98of of157 157 2 3 8 14 11 13 15 10 9 1 . Standards AAR PT Report.0 -6.0 0.0 -4.0 6.4 0.6 RP RP RP Relative bias (%) 10.0 RP 0. STATISTICAL 5.0 10 8 18.414 D/L -2.2 0.1 0.5 0.0 8.0 6 4 RP 2 0 0 0. STATISTICAL Accuracy Accuracy Analysis Figure 5.0 12.427 D/L 2.THAA Solution THAA Solution Standards EVALUATIONEVALUATION& Performance & Performance Analysis R PT Report.0 RP RP RP RP RP 0.11: Relative Percentage Bias for Valine D/L Results (rpHPLC data only) in Standards Solution Test Material 16 14 12 20.0 14.0 0.3 0.

5.0 -6.12: Relative Percentage Bias for Phenylalanine D/L Results (all data) in Standards Solution Test Material 60 50 40 4.52 RP RP 6.0 GC Laboratory Number Page 99 99 of of 157 157 Page 0.0 10 0. Standards Solution THAA -3.1 6.5 0.0 GC GC 0.491 D/L RP RP RP Relative bias (%) -1.48 0.478 D/L -4. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.0 RP RP GC RP RP 0.0 RP AAR PT Report.2 2 3 10 14 15 13 11 .0 0 1.51 0.2 7.1 8 9 1 7.505 D/L 2.47 0.46 0.45 0.0 30 20 3.0 -2.0 -5.0 0.49 0.

0 3.475 0.0 8 RP RP 0.51 9 1 2 3 10 14 15 13 11 .0 -5.465 0. Standards Solution THAA 0.505 0.0 -1.0 -3.0 50 40 4.0 -4.49 0.484 D/L Laboratory Number Page 100 of 157 0.493 D/L RP RP RP AAR PT Report.47 0.501 D/L 0.5.0 RP RP RP RP RP 0.5 0.0 1.0 30 20 10 0 Relative bias (%) 2.13: Relative Percentage Bias for Phenylalanine D/L Results (rpHPLC data only) in Standards Solution Test Material 90 80 70 60 5.485 0.48 0.495 0. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.0 -2.

1 6.0 10 GC 5 0 0 0.1 7.0 9.0 10.566 D/L RP -4.592 D/L GC RP IE RP GC 4.0 -6.0 IE RP AAR PT Report.0 8.0 0.6 0.0 -5.0 9 Laboratory Number Page 101 of 157 Page 101 of 157 1 8 5 4 3 2 6.0 0.0 2.4 0.1 0.2 0.0 -3. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.0 6.0 15 11.14: Relative Percentage Bias for D-Alloisoleucine/L-Isoleucine Results (all data) in Standards Solution Test Material 30 25 20 12.5.0 RP RP RP RP RP RP 0.0 Relative bias (%) 3.539 D/L -8.0 5.0 -1.0 1.3 0.2 15 14 13 10 11 . Standards Solution THAA 0.5 0.0 -7.7 7.0 -2.

0 -5.6 0.57 0.0 Laboratory Number Page 102 of 157 1.0 30 RP 20 RP 3.0 0.59 0.0 AAR PT Report.569 D/L RP RP Relative bias (%) 0.0 0 0.15: Relative Percentage Bias for D-Alloisoleucine/L-Isoleucine Results (rpHPLC data only) in Standards Solution Test Material 60 50 40 4.56 0.0 -4. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.5.53 0.0 RP RP RP 0. Standards Solution THAA -2.58 0.0 RP 9 1 8 3 2 15 14 13 10 11 .54 0.0 10 2.546 D/L -3.558 D/L RP RP -1.55 0.

8 2 5.0 6 4 0.0 Laboratory Number Page 103 of 157 7. Standards Solution THAA 0.1 0. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.6 0.0 RP 0.0 RP AAR PT Report.16: Relative Percentage Bias for Leucine D/L Results (all data) in Standards Solution Test Material 14 12 10 8 10.1 6.2 0.2 8 9 1 10 11 15 14 13 .0 GC GC -15.545 D/L GC -10.4 0.7 0.589 D/L -5.5 0.3 0.5.0 0 RP RP RP RP Relative bias (%) RP 0.633 D/L RP 0 0.1 6.

0 -9.4 0.0 -4.0 3. Standards Solution THAA -6.5 0.0 6 4.0 -3.609 D/L 1.0 -10.5.577 D/L AAR PT Report.0 0 0.1 0.593 D/L RP Relative bias (%) -2.6 0.0 -5.0 RP 0.2 0.17: Relative Percentage Bias for Leucine D/L Results (rpHPLC data only) in Standards Solution Test Material 20 18 16 14 12 10 8 5.0 -8.0 RP 0.0 -7. STATISTICAL EVALUATION-Accuracy & Performance Analysis Figure 5.0 RP 4 2 0 2.7 RP RP 0.0 8 9 Laboratory Number Page 104 of157 1 10 11 15 14 13 .3 0.0 -1.0 RP RP 0.

MEASUREMENT UNCERTAINTY 6 MEASUREMENT UNCERTAINTY Standards Solution Test Material 6. For those readers unfamiliar with measurement uncertainty estimation. Thus. and is referred to as the ‘top-down’ approach to measurement uncertainty determination (Barwick and Ellison.. It is widely recognised that evaluation of PT data can be a valuable addition to the determination of measurement uncertainty. Proficiency test data can provide a valuable indication of method and laboratory bias in routine analysis. EUROLAB.pdf http://www. The uncertainty estimate is likely to be larger than that resulting from the analysis of a CRM. 2004) In addition. Below helps to illustrate the sources and relevance of iii iv http://www. 2006. To simply use the standard deviation from replicate results submitted for the proficiency test is not a realistic representation of the overall method and laboratory precision.net/nordtestfiler/tec537.AAR PT Report. performance in a proficiency test can provide verification of a laboratory’s own uncertainty estimates. Alternatively. bias and the uncertainty due to bias associated with a PT. 2004) produced as a handbook for the Nordic environmental testing laboratories and Eurolab’s Technical reportsiv Nos 1/2006 and 1/2007 (EUROLAB. the Nordtest Report TR 537iii (Magnusson et al.1 Estimation of Measurement Uncertainty from Inter-laboratory comparisons. 2000) on exactly how this should be done. However in the absence of such data the result can be used as a direct indication of bias itself.eurolab. EURACHEM / CITAC.html Page 105 of 157 . This. distinguishing the various uncertainty components can be somewhat baffling.. 2008. Standards Solution THAA 6. can be used directly in place of the combined standard uncertainty. It should be recognised that due to the uncertainty of the assigned value. All documents are freely downloadable and recommended for further reading on the subject. however there is very little information provided by the main guidance documents (JCGM 100:. It is recommended that long term bias trends are observed to lessen the impact from a single proficiency test result and at least 6 rounds of testing are used to evaluate bias estimates (Magnusson et al. which should be compatible with the spread of their PT results over time. an estimation of the between laboratory reproducibility standard deviation (SR) determined using an analysis of variance (ANOVA) on results from a collaborative trial. which together with an estimate of precision such as the intra-laboratory reproducibility standard deviation (SRw).org/pub/i_pub. 2000). Where such validation data is available. it is recommended that intra-laboratory precision estimates (SRw) are determined from replicate analyses of samples under reproducibility conditions over an extended period of time to take account of between run and general day to day variability. together with the determination of internal precision estimates (intra-laboratory reproducibility standard deviation (SRw)) can define the overall combined uncertainty for a measurement system (uC). 2007). The following methodology is therefore derived from two main sources.nordicinnovation. can provide a value for the combined uncertainty. Bias (bias) and its associated uncertainty (u(bias))is often evaluated as part of a laboratory’s method validation process by analysis of a certified reference material (CRM) or from spiking experiments.

1 Standard uncertainty due to Bias ( For a result from a single proficiency test. and number of laboratories’ results contributing to the assigned value. bias should be accounted for and corrected for significant systematic effects. However in circumstances where this is not done by convention and the method is said to be empirical.2 6. z etc.X x1 x3 x4 Precision (μ ) true value Standard deviation (s) 6. Assigned Value (consensus of labs x. Where = = = = standard deviation of the laboratory’s submitted result. y. any significant uncorrected bias should contribute to the combined uncertainty budget.2. Note. In routine analysis. The simplest expression for the bias uncertainty (u(bias)) is the experimental uncertainty of the laboratory mean plus the uncertainty of the assigned value where . if a CRM was used as the test material. Page 106 of 157 (x x Uncertainty due to Bias Standard uncertainty of the Assigned Value (u(X) = σ/√m) x Mean (of lab x replicate values) Standard uncertainty of the mean (u( = s/√n ) x2 . Figure 6. number of laboratory replicates.1: Bias and Precision Components of Measurement Uncertainty Estimation. MEASUREMENT UNCERTAINTY the different contributions due to precision and particularly those elements due to bias. can be taken from the specifications directly. mean values ) Bias = x . together with the calculation of the combined standard uncertainty and expanded uncertainty estimates. Standards Solution THAA 6.AAR PT Report. standard deviation of the assigned value. These will now be expanded on in the remainder of this section. ).

In this case the uncertainty associated with a Page 107 of 157 . MEASUREMENT UNCERTAINTY To determine whether the observed bias is significant or not. 6. there will be no overall bias associated with the laboratory’s performance.2 For results from multiple proficiency tests When multiple results have been obtained from several proficiency tests then the contribution due to bias and the uncertainty due to bias (i. some may be positive and others negative. that is. However. t is calculated as.2. the experimental uncertainty of the replicate mean ).. |z|=2. the t statistic is calculated and compared to the 2-tailed critical value for n-1 degrees of freedom. 6. then the bias is significant and an additional term to account for uncorrected bias in the result needs to be included in the combined uncertainty estimate (EURACHEM / CITAC. where . a bias correction is the same as u(bias) given above. can be replaced by the bias root mean square ( ). where The average standard deviation for the assigned values and the average number of participants across all the tests can be determined and used to calculate an average uncertainty value for the tests. Where is the intra-laboratory reproducibility precision estimate. “The use of an RMS value is equivalent to an estimated standard deviation around an assumed value of bias equal to zero. 2007). i.e. thus.3 Combined uncertainty ( ). Standards Solution THAA Bias is determined as . This implies that the RMS value takes into account both the bias and the variation of bias”. Thus. (EUROLAB. Note concerning z-scores. or as a relative value Where and = laboratory result (or the mean of replicate values) = the assigned value. The combined uncertainty is therefore calculated as. 6. tcrit . If t is greater than or equal to the critical value. a CRM and and usually represents the recovery associated with the analysis of remains uncorrected. If . 2000). Rec is significantly different from 1 and the result term needs to be included in the combined uncertainty estimate. this scenario is to some extent academic as the uncertainty of the assigned value in a proficiency test is likely to be much larger than that of a CRM (if one were available) and it is recommended to include the bias contribution in the uncertainty evaluation at all times regardless of whether or not (Magnusson et al..e. for laboratories performing within the satisfactory range. the bias uncertainty now becomes. where there is a normal distribution of z-scores . 2004).AAR PT Report..

degrees of freedom of individual uncertainty components. “Where the combined standard uncertainty is dominated by a single contribution with fewer than six degrees of freedom. The use of this equation is covered in detail in Annex G of the Guide to Uncertainty Measurement or “GUM”.1 Measurement Uncertainty Evaluation for a series of results using RMSbias. result ( ) ± U (at 95% confidence. were negligible and where the uncertainty of replicate values. One approach is to calculate an effective degree of freedom using the Welch-Satterthwaite formula where the effective degree of freedom is less than or equal to the sum of the individual values. The final step in determining the measurement uncertainty is to calculate the Expanded uncertainty U by multiplying the combined uncertainty with a coverage factor k. where the uncertainty of the assigned values. the following evaluations have been carried.4 Expanded Uncertainty (U). i. This is due to a number of reasons. then the uncertainty associated with the laboratory’s result is simply equivalent to σp.. plus the precision contribution . combined standard uncertainty individual uncertainty components. Also because of the absence of true intralaboratory precision estimates and the fact that not all laboratories supplied analytical replicate values. for PT results with no overall bias ( ). Nonetheless.e. For a discussion of the appropriate value of k. A combined uncertainty brings together uncertainty contributions from different sources.e. This is to simplify the calculations whilst considering uncertainty components from various sources but also in order to enable direct comparability between laboratories and across analytes.5 Calculating Measurement Uncertainty for Amino Acids in Standards Solution Test Material To illustrate how precision and bias components can be used to provide an estimate of analytical uncertainty. where level.05. However. Eurachem make the following recommendation. 6. Where the uncertainty of the assigned value and /or the uncertainty of the result is considered negligible compared to the target standard deviation used for assessment (σp). For smaller data sets k=t(0.. which in this case is equivalent to the target standard deviation. Results should be expressed as. such as the relatively small data set. σp. at a 95% or 2 standard deviation confidence level. 6. MEASUREMENT UNCERTAINTY result will be based on the uncertainty of that result. 2000). ( ) . is the coverage factor set according to the required confidence Where = = = = the effective degrees of freedom. were small Page 108 of 157 . the data presented in the following tables demonstrates how it can be possible to determine measurement uncertainty using proficiency test data and provides some interesting indicative values.2. individual laboratory expanded uncertainties (U) have been determined using a coverage factor k=2. therefore determining k becomes a little more tricky as there is no single value for the degrees of freedom. The information thus presented should perhaps be considered more as an information exercise than a definitive measure of uncertainty. for a large. normally distributed data set. i. k=2. In all cases.df).3. As already mentioned in Section 6. 2008). However. using k=2) 6. . the “uncertainty” surrounding the empirical nature of the results and the effect on the confidence in the assigned value. or it’s RSD value expressed as a percentage. see Section 4. (JCGM 100:.AAR PT Report. plus the uncertainty of the assigned value . Standards Solution THAA 6. it is recommended that k be set equal to the two-tailed value of the Student’s t for the number of degrees of freedom associated with that contribution and for the level of confidence required…” (EURACHEM / CITAC.5. .2.

However..3). In the absence of this and to avoid the risk of under-valuing the precision contribution. They represent the distributions of the laboratories’ means and were used to set the satisfactory limits (i.1 demonstrates how this could be carried out using a series of results. ( ).e. by poor repeatability of the replicate results and extreme values. in reality it is probably a little unrealistic. the instrumental repeatability (i. given in Table 4. the RSD% or CV%) derived from the replicate values might be used. MEASUREMENT UNCERTAINTY compared to intra-laboratory precision estimates .but are not as influenced as the reproducibility standard deviations (SR and RSDR%) given in Table 4.1. From this the average combined and expanded uncertainties for each amino acid for this test material can be derived. Table 6. Page 109 of 157 . if this information where known. SRW.e. Standards Solution THAA 6.5.2. It would be far more appropriate to assess the bias components and include them in the uncertainty budget. it can be helpful to observe unexpected random error effects between rounds of proficiency testing. the reproducibility value derived from all participant’s results. i. the average uncertainty for each amino acid calculated across all the laboratories still provides some interesting results which can be compared to the individual values calculated next.2.2 then looks at individual laboratory uncertainty estimates for each amino acid. In addition. Individual laboratory standard uncertainty components have been presented as histograms. In practice it is perhaps more likely that a single laboratory would want to assess their own data from a series of proficiency tests carried out.e. Under these circumstances and assuming the absence of bias described above still holds.2 Measurement Uncertainty Evaluation for a single result. ideally with an additional term included to take into account the expected variability between samples. give more appropriate approximations. Whilst the above scenario may be ideal. Here the precision estimates used are the standard deviations for the assigned values. The data shown uses the RMSbias% (see 6. Table 6. no values for target standard deviation.. have been given.2) determined from all the submitted results by all the laboratories for any given amino acid.. Nonetheless. This would assume that all laboratories were performing at the stated level of precision and makes no allowance for those that were performing better or worse than this. ± 2 std dev). However it should be noted that precision based on instrument repeatability is likely to be small compared to any long term true intra-laboratory reproducibility (intermediate precision) estimate and may contribute to smaller expanded uncertainties than might be otherwise expected.AAR PT Report. In this example we are using results from a number of laboratories in a single round of testing to obtain an average uncertainty for the amino acid in the test material.1 at the beginning of the report. expanded uncertainty confidence intervals have been determined and plotted for each amino acid to illustrate the effect of uncertainty on the mean of submitted results. at least until the analyst is confident that analytical results are free from bias.. In the absence of this. Here the individual bias components have been assessed separately as discussed in Section 6. (Although in practice each laboratory should use their own intra-laboratory reproducibility (SRW) precision estimate for the analyte in question and the different laboratories would be replaced by results from different rounds of testing for any given laboratory). might be used as a compromise. in this report. even if their overall contribution is small. sMAD (see Section 5. then the standard uncertainty for laboratories within the satisfactory range would be equivalent to the target standard deviation. . Although this approach is not recommended and long term trends (as described above). 6. the uncertainty of laboratories’ mean values would be equivalent to each laboratory’s own intra-laboratory reproducibility . in place the laboratory’s own estimation of precision for that analyte. together with each laboratory’s combined uncertainty value and the average combined uncertainty for the test material described in the previous section and given in Table 6.1.1 and the CV% or RSD% determined from instrumental replicates have been used where available.

64 0.12 12. expressed as a percentage (given in Table 5.44 8.50 2.79 13.74 1.40 0..81 2.AAR PT Report.40 3 as RSD% Asx D/L (alla) Asx D/L (rpHPLC) Glx D/L (alla) Glx D/L (rpHPLC) Ser D/L (rpHPLC) Arg D/L (rpHPLC) Ala D/L (all ) Ala D/L (rpHPLC) Val D/L (all ) Val D/L (rpHPLC) Phe D/L (all ) Phe D/L (rpHPLC) D-Aile/L-Ile (allb) D-Aile/L-Ile (rpHPLC) Leu D/L (alla) Leu D/L (rpHPLC) Notes for Table 6.2).48 2.79 24.36 8.6 2.38 1.77 1.76 6. i.41 0.75 1.71 1.68 1.38 2.69 1.54 0.32 1.88 16.12 1.15 0.57 5.01 3.90 1.28 1.48 2.69 Expanded 27. Std uncertainty contributions Precision 1 1 2 analyte Combined & Expanded uncertainties 2.35 7.37 28.15 1.47 0.24 16.58 1.36 4.62 8.2).49 1.1: Estimation of Relative Standard Uncertainty.24 24. 3 = RMSbias is the observed uncertainty due to bias of the submitted results Page 110 of 157 .71 8.98 15.84 1.62 5.89 12.3 Bias components as RSU% 0.e.02 3.33 2.53 0.41 2. the median absolute deviation (sMAD).42 7.53 0.47 0.79 0.41 combined % 13.70 0.12 0.49 7.20 1.47 11.85 2.18 1. 2 = is the uncertainty of the assigned value expressed as a percentage. GC and HPLC-IE data 1 = is the standard deviation for the assigned value. (given in Table 5.62 12. Standards Solution THAA 6.19 14.54 1.20 2.40 5.82 4. MEASUREMENT UNCERTAINTY Table 6.65 4.94 1.40 3. Combined and Expanded Uncertainty for Amino Acids (using RMSbias% to access bias contributions) across ALL Laboratories.27 0.02 0.09 12.13 b = rpHPLC and GC data = rpHPLC.76 2.38 4.1: a a a a RMSbias% 13.89 6.

28 2.54 as 6 RSU% 0.591 0.54 0.10 0.497 0.06 Relative 7 bias % 1.499 0.1 7.78 64.11 2.53 0.11 20.54 0.15 0.2 7.54 0.35 0.496 0. (see Section 4).12 0.09 Combined & Expanded uncertainties combined % 2.15 0.2: Estimation of Relative Standard Uncertainty.17 combined % 2.49 0.1 6.49 0.19 3.17 0.76 1.01 0.18 0.496 0.73 = is the standard deviation of submitted results.01 62.37 1.97 as 5 RSU% 0.81 0.15 0. = is the uncertainty of the assigned value expressed as a relative % i.17 0.05 1.510 0.65 0.2 8 9 10 11 13 14 15 4 5 0.44 0.34 0.26 0.599 0.10 0.497 8.50 0.15 1.85 0.21 19.44 0.e.497 0.41 0.01 0.15 0.65 0.17 0.07 0.64 0.22 40.11 0.01 0.01 1.05 29..02 0.27 1.60 0.87 1.08 0.56 32.06 Relative 7 bias % 1.33 0.55 0.15 as 6 RSU% 0.54 4.56 4.51 2.e.86 2.64 0.491 0.656 0.07 0.28 3. expressed as a relative % i.54 0..26 0.2 7.21 0.6.63 0.500 0.01 n=1 3.54 0.54 0. Standards Solution THAA 6. Page 111 of 157 .12 0.64 0.1 6.54 0.26 0.00 0.497 0.54 0.54 0.500 0.12 0.14 0.96 n=1 6.647 0..500 0.15 0.499 0.57 0.34 0.95 2.AAR PT Report.2 8 9 10 11 13 14 15 0.92 0.499 0.15 0.05 0.54 0.499 0.15 0.63 2.09 as 5 RSU% 0.61 0.55 6.08 0.28 0.12 0.7 Precision Bias components std dev 4 as CV% 1.07 1.54 0.21 5.25 1.92 Expanded 5. Combined and Expanded Uncertainty Estimations for Individual Laboratories laboratory number Asx D/L 1 2 3 4 5 6.26 0.36 1.15 0.11 0. MEASUREMENT UNCERTAINTY Table 6.01 4.14 0.34 0.1 7.01 0.21 31.56 31.92 0.e.500 0.41 18.491 0.54 0.15 0.03 0.15 0.02 0. (see Section 5) (see Section 4).510 0.95 Asx D/L rpHPLC std dev 4 as CV% 1.515 mean result Std uncertainty contributions 4 5.09 0.18 Expanded 1 2 3 4 5 6.32 0.73 2.09 0.36 0.52 2. 6 = is the bias standard deviation for submitted results expressed as a relative % 7 = Relative bias expressed as a % i.65 0.19 1.50 2.22 0.17 0.515 4.02 0.

1 6.14 0.25 0.04 3.552 0.12 0.47 as 6 RSU% 0.47 0.93 n=1 11.82 Glx D/L rpHPLC std dev 4 as CV% 1.06 5.10 1.552 0.74 1. Combined and Expanded Uncertainty Estimations for Individual Laboratories (continued).12 0.16 0.09 0.552 0.79 0.52 Expanded 1 2 3 4 5 6.12 0.89 combined % 1.554 0.47 0.67 3.27 0.06 0.11 3.47 0.18 1.32 0.67 0.552 0.552 0.37 Relative 7 bias % 1.20 3.08 n=1 0.57 1.52 3.623 0.64 7..79 0.51 12. expressed as a relative % i.569 0.03 2.77 24.42 4.18 0.47 0.14 2.7 Precision Bias components std dev 4 as CV% as 5 RSU% 0.AAR PT Report.09 0.79 3.51 0.66 as 6 RSU% 0. (see Section 5) (see Section 4).79 0.46 10.21 6.47 0.13 0.05 2.03 14.12 0.558 0.02 1.558 0.79 0. = is the uncertainty of the assigned value expressed as a relative % i.42 = is the standard deviation of submitted results.23 0.37 Relative 7 bias % 0.541 0.62 0.08 0.79 0.11 0.90 0.39 0.547 0..04 2.23 0.10 n=1 5.79 0.47 0.15 0.1 7. laboratory number Glx D/L mean result Std uncertainty contributions 4 5.2 8 9 10 11 13 14 15 0.51 0.23 1.04 6.541 0..79 Combined & Expanded uncertainties combined % 1.03 0.592 0.42 2.71 2.18 0.29 6.6. (see Section 4).16 0.33 0.2 7.e.39 0.558 0.569 0.536 1.79 0.09 0.554 0.11 0. 6 = is the bias standard deviation for submitted results expressed as a relative % 7 = Relative bias expressed as a % i.96 0.10 0.09 0.79 0.00 0.62 0.37 5.14 3.1 6.47 0.2: Estimation of Relative Standard Uncertainty.690 0.1 7.37 0.42 0.13 0.52 1.79 0. Page 112 of 157 .51 0.2 7.47 0.10 2.e.52 1.599 0.03 0.552 0.11 1.09 0.e.68 2.04 0.82 3.27 0.23 1.06 0.14 0. MEASUREMENT UNCERTAINTY Table 6.47 0.91 20.02 1.21 2. Standards Solution THAA 6.79 0.25 0.79 0.09 7.547 0.32 0.2 8 9 10 11 13 14 15 4 5 0.79 0.52 as 5 RSU% 0.16 1.80 1.07 2.558 6.35 n=1 0.55 1.60 0.00 Expanded 1 2 3 4 5 6.53 2.05 29.79 0.04 2.536 3.15 0.

380 0.68 3.14 0.408 0.43 8.393 0.41 0.405 0.62 1.21 1.52 3.21 0.59 2.30 6.18 1.75 0.2 7.1 7.53 5.78 5.395 0.75 1.344 0.81 6.32 0.73 7.41 3.86 0.400 0.75 0.15 0. laboratory number mean result Std uncertainty contributions 4 5.65 3.75 0.00 0.1 6.75 0.21 0.79 2.68 1.16 0.2 8 9 10 11 13 14 15 2.11 3.15 2.05 4.56 29.67 0.31 1.55 Arg D/L rpHPLC std dev 4 as CV% as 5 RSU% as 6 RSU% Relative 7 bias % combined % Expanded 1 2 3 4 5 6.52 3.06 0.72 2.41 3.58 4.28 0.41 0.75 0.47 0.344 0.86 1. Page 113 of 157 .09 0.368 1. Standards Solution THAA 6.09 7. MEASUREMENT UNCERTAINTY Table 6.15 1.21 0.03 29.75 0..35 0.06 5.68 1.11 0.2 8 9 10 11 13 14 15 4 5 0.376 0.21 4.08 2.AAR PT Report.366 0.08 0.62 12.395 0.68 1.75 0.68 1.e.66 4.61 3.00 0.6.e.30 6.2: Estimation of Relative Standard Uncertainty.16 0.40 6.43 0.91 13.30 0.18 1.33 Combined & Expanded uncertainties combined % 1.62 2.76 1.356 0. (see Section 5) (see Section 4). = is the uncertainty of the assigned value expressed as a relative % i.06 11.75 0. 6 = is the bias standard deviation for submitted results expressed as a relative % 7 = Relative bias expressed as a % i.66 1.85 4.82 = is the standard deviation of submitted results.71 0.2 7.476 0.86 1.68 1.60 0.75 0.46 0.89 Expanded 1 2 3 4 5 6.07 Ser D/L as 5 RSU% 0.1 7.68 1.402 0.01 0.26 2.21 1. Combined and Expanded Uncertainty Estimations for Individual Laboratories (continued).75 as 6 RSU% 0.68 0.10 3. (see Section 4).23 Relative 7 bias % 0..68 1.04 0. expressed as a relative % i.412 0.16 0.409 0.1 6.77 19.10 9.400 0..39 9.19 2.e.7 Precision Bias components std dev 4 as CV% 0.27 6.12 59.93 2.

2 8 9 10 11 13 14 15 0.79 0.555 0.64 1.25 0.90 2.27 0.25 1.91 1.01 18.59 0.555 0.64 1.563 0.20 6.55 0.AAR PT Report.97 0.57 1.36 0.7 std dev 4 as CV% as 5 RSU% 1.26 4.71 3.461 0.48 3.36 0.80 0.25 combined % 16.40 0.15 0.51 0.17 0.e.40 0.20 18.84 0.02 19.46 3.09 0. 6= is the bias standard deviation for submitted results expressed as a relative % 7 = Relative bias expressed as a % i.59 0.2: Estimation of Relative Standard Uncertainty.13 23.1 6.11 37.78 as 6 RSU% 0.18 47.50 23.59 1.19 0.12 18.29 as 5 RSU% 0.12 23. Standards Solution THAA 6.51 0.64 1.1 7.71 1.42 0.80 0.29 32.465 0.38 0.35 1.2 7.467 0.09 0.14 20.48 3.71 0.33 0.64 1.50 6.48 0.64 1.27 0.22 3.20 Relative 7 bias % 16.465 0.68 1.86 Ala D/L (rpHPLC) std dev 4 as CV% 0.15 3.09 23.69 16. laboratory number Ala D/L mean result Std uncertainty contributions Precision4 Bias components5.469 0.359 0.55 0.38 0.61 1.26 1.42 0.2 7.40 0.64 1. MEASUREMENT UNCERTAINTY Table 6.15 38.73 1..40 36.53 n=1 0.466 0.461 4.02 0.58 19..67 1.2 8 9 10 11 13 14 15 4 5 0.64 1.48 46.14 0.42 n=1 0.20 Relative 7 bias % 16.19 0.46 1.83 1.06 38.40 0.69 4. (see Section 5) (see Section 4).48 0.1 7.468 0.464 0.544 0.26 1.34 0.556 0.40 0.53 8.40 0.95 = is the standard deviation of submitted results.6.23 3.40 0.64 1.464 0.97 0.34 0.42 0.500 0.547 0.02 0.64 1.55 18..40 as 6 RSU% 0.36 0. (see Section 4).62 0.47 18.25 0.70 Expanded 1 2 3 4 5 6.17 0.40 0.556 33. expressed as a relative % i.38 0.359 0.64 1.544 0.43 16.84 2.24 37.e.60 0.25 Expanded 1 2 3 4 5 6.e.34 0.01 0.74 1.38 0.64 1.79 4. Page 114 of 157 .40 0.467 0.03 19.469 0.57 8.01 0.71 0.64 1.55 19.36 2. = is the uncertainty of the assigned value expressed as a relative % i.17 0.64 Combined & Expanded uncertainties combined % 16.64 1.466 0. Combined and Expanded Uncertainty Estimations for Individual Laboratories (continued).1 6.

00 0.72 25.415 0.26 3.05 0.478 0.03 2.64 0.12 1.03 = is the standard deviation of submitted results.6.62 15.53 0. Combined and Expanded Uncertainty Estimations for Individual Laboratories (continued).409 0.15 0.AAR PT Report.53 0.21 0.54 5.34 3. laboratory number Val D/L mean result Std uncertainty contributions Precision4 Bias components5.07 0.00 0.414 0.403 0.412 0. (see Section 4). 6 = is the bias standard deviation for submitted results expressed as a relative % 7 = Relative bias expressed as a % i.e.7 std dev 4 as CV% as 5 RSU% 0.461 0.14 0.47 0.413 0..53 0.76 8.414 0.409 0.29 0.15 0.35 0.40 12.03 1.03 Relative 7 bias % 11.09 0.08 0.45 0.1 6.28 0.55 0. Page 115 of 157 .69 1. Standards Solution THAA 6.57 12.46 combined % 11.413 0.25 0.33 12.63 Expanded 1 2 3 4 5 6.18 2.08 1.2 7.53 0.05 0.53 0.47 Expanded 1 2 3 4 5 6. (see Section 5) (see Section 4).45 0.36 12.07 0.41 12.1 7.53 0.413 0.17 15.47 as 6 RSU% 0.94 0..64 0.53 0.478 0.2 8 9 10 11 13 14 15 0.20 9.1 7.66 2.51 30.21 0.58 12.90 1.10 4.16 25.64 as 6 RSU% 0.466 0.19 1.43 0.415 0.35 0.00 0.52 1.04 1.414 0.20 3.05 as 5 RSU% 0.53 0.47 0.e. expressed as a relative % i.28 0.e.47 0.88 4.05 22.461 0.00 0.53 0.10 0.30 31.2: Estimation of Relative Standard Uncertainty.02 2.51 0.10 0.52 0.65 15.47 0.47 0. = is the uncertainty of the assigned value expressed as a relative % i.29 0.17 1.11 1.29 0.56 0.04 0.25 0.53 0.47 0.47 0.62 1.08 Val D/L (rpHPLC) std dev 4 as CV% 0.47 0.62 1.03 Relative 7 bias % 11.58 0.15 0.466 0.1 6.10 0.2 8 9 10 11 13 14 15 4 5 0.15 0.00 0.16 12.09 0.47 0.53 0.10 0.45 0.35 0.64 0.33 1.95 0.47 0.13 0.20 12.23 2.2 7.55 0.07 15..414 2.14 0.83 24.43 0.13 0.53 Combined & Expanded uncertainties combined % 11.52 0.63 1.34 0.466 22.39 24.53 0. MEASUREMENT UNCERTAINTY Table 6.96 1.25 2.64 0.07 0.466 0.412 0.427 0.

12 0.28 0.2 7.6.480 0.12 1.42 0.39 0.41 0.74 1.41 0.33 0.31 0.65 0.45 0.05 0.18 0.68 0.24 0.41 as 6 RSU% 0.03 0.AAR PT Report.497 0.497 3.475 0.70 16.41 0.41 0.494 0.32 2.29 0.95 n=1 0.73 3.71 3.20 1.90 6. Page 116 of 157 .41 0.e.12 0.93 1. = is the uncertainty of the assigned value expressed as a relative % i.27 as 6 RSU% 0.1 6.27 0.16 2.10 0.02 0.01 1.50 0.2 8 9 10 11 13 14 15 0.27 0.40 0.15 0.496 0.27 0.05 0.41 0.01 0.04 2.498 0.88 0.57 0.06 8.41 5. expressed as a relative % i. Combined and Expanded Uncertainty Estimations for Individual Laboratories (continued).492 0.491 7.02 0.04 2.40 0.7 Precision Bias components std dev 4 as CV% 1.27 0.71 0.36 Phe D/L as 5 RSU% 0.1 7.83 1.33 0.41 0.01 0.27 0.1 7.57 0.490 0.58 1.41 0.19 1.84 Phe D/L (rpHPLC) std dev 4 as CV% 1.29 0.35 0.493 0. Standards Solution THAA 6.66 1.60 1.27 0.27 0.494 0.480 0.41 0.63 5.489 0.41 2.86 2.02 0.66 4.12 0.41 2.44 0.30 2...41 0.40 1.65 1.74 1.64 1.28 0.72 1.02 0.31 0.2 7.497 0.09 0.1 6.45 0.41 0.491 0. 6 = is the bias standard deviation for submitted results expressed as a relative % 7 = Relative bias expressed as a % i.478 0.29 Relative 7 bias % 0.95 2.27 0. MEASUREMENT UNCERTAINTY Table 6.490 0.15 0.09 0.e.19 0.27 0.498 0.94 combined % 1.10 Expanded 1 2 3 4 5 6. (see Section 5) (see Section 4).82 = is the standard deviation of submitted results.43 2.35 0.41 as 5 RSU% 0.28 0.59 0.41 Combined & Expanded uncertainties combined % 1.43 0.489 0.31 8.27 0.19 Expanded 1 2 3 4 5 6.95 1.47 0.04 0.492 0.44 0.71 0.11 n=1 0..10 0.19 0.21 0.06 0.27 0.10 3.42 17. (see Section 4).2: Estimation of Relative Standard Uncertainty.59 1.29 Relative 7 bias % 0.21 0.497 2.03 0.41 0.20 2.493 0.33 0.2 8 9 10 11 13 14 15 4 5 0.77 0.18 0.41 0.72 0.e.35 8. laboratory number mean result Std uncertainty contributions 4 5.473 0.43 0.

55 0.2 7.73 D-Aile/L-Ile as 5 RSU% 0. expressed as a relative % i.75 3.27 1.95 2.05 Expanded 1 2 3 4 5 6.05 0.87 1.27 0.e.14 n=1 n=1 1.80 0.25 4.558 0.03 combined % 1.70 0.1 6.71 1.70 0.552 0.13 2.41 0.71 2.32 0.577 0.56 1.83 0.57 3.03 0.98 0..46 0.05 0.51 0.555 0.15 0.2: Estimation of Relative Standard Uncertainty.10 1.e.574 3.70 as 6 RSU% 0.85 0.47 3. Combined and Expanded Uncertainty Estimations for Individual Laboratories (continued).567 0.70 0.32 0.11 0.00 2.554 3.587 0. = is the uncertainty of the assigned value expressed as a relative % i.70 0.27 3.05 1.1 7.27 0.16 0.32 0.10 Relative 7 bias % 0.51 Combined & Expanded uncertainties combined % 1. Standards Solution THAA 6.53 0.02 Expanded 1 2 3 4 5 6.25 0.08 0.52 1.32 4.17 3. Page 117 of 157 .555 0.32 as 6 RSU% 0. 6 = is the bias standard deviation for submitted results expressed as a relative % 7 = Relative bias expressed as a % i.623 0.45 0.15 0.85 0.39 0.70 0.2 8 9 10 11 13 14 15 4 5 0.82 1.48 3.70 0.566 0.1 6.93 2.32 0.45 0.562 0.20 2.558 0.48 1.32 0.95 0.98 1.66 2.32 0. (see Section 5) (see Section 4).58 10.70 0.95 1.36 1.46 0.70 0.92 0.32 0.70 0.04 3.63 0.7 Precision Bias components std dev 4 as CV% 1.23 0.05 0. (see Section 4).93 2.92 6.66 1.59 0..14 as 5 RSU% 0.32 0.554 0.2 8 9 10 11 13 14 15 0.70 0.75 3.42 0.52 0.70 0.32 0.23 0.70 0.55 0.577 0.571 0.05 0.33 0.13 0.95 7. laboratory number mean result Std uncertainty contributions 4 5.38 0.86 = is the standard deviation of submitted results.AAR PT Report.09 0.67 1.35 1.555 0.16 20.574 0.70 0.39 0.557 0.45 D-Aile/L-Ile rpHPLC std dev 4 as CV% 1.10 n=1 n=1 0.562 0.36 Relative 7 bias % 0.18 1.14 6.1 7.13 0. MEASUREMENT UNCERTAINTY Table 6..97 0.89 4.09 0.22 7.20 3.04 3.584 0.46 3.e.03 0.58 0.95 2.31 1.08 0.552 0.44 3.566 0.63 0.47 0.32 0.47 0.43 0.70 0.557 0.555 0.42 5.2 7.32 0.27 10.52 1.33 0.6.61 1.

604 0. (see Section 4).60 Combined & Expanded uncertainties combined % 5.42 20.07 0.e.56 0.12 1.2 7.75 0.587 0.50 1. = is the uncertainty of the assigned value expressed as a relative % i.591 0.17 4.587 0.08 0.75 n=1 0.595 0.17 1.563 0.40 0.12 as 6 RSU% 1.14 1..1 6.12 1.40 0.10 1.12 1.72 Expanded 1 2 3 4 5 6.77 9.60 as 5 RSU% 0.56 1.6.12 1.529 0.74 1.07 0.13 5.25 0. laboratory number mean result Std uncertainty contributions 4 5.1 7.05 5.12 1.40 0.e.57 5.34 2.00 0.07 1.61 1.595 0.10 0.1 6.e.38 4.26 8. Page 118 of 157 .523 0.77 0.45 11.18 1.53 16.28 11.40 as 6 RSU% 1.36 2.59 3.19 0.12 1.11 1.45 0.05 10..87 10.12 0.12 1.63 Leu D/L as 5 RSU% 1.2 8 9 10 11 13 14 15 4 5 0.19 0.78 0.589 0.12 2.72 0.33 0.1 7. expressed as a relative % i.40 0.12 1.13 0.75 n=1 0.06 0. 6 = is the bias standard deviation for submitted results expressed as a relative % 7 = Relative bias expressed as a % i. MEASUREMENT UNCERTAINTY Table 6.89 22.38 0.40 0.92 3..06 0.604 0.53 n=1 0.32 0.7 Precision Bias components std dev 4 as CV% 0.12 0.540 0.28 2.16 Relative 7 bias % 3.74 1.40 0.89 Expanded 1 2 3 4 5 6.594 0.2 7.07 0.95 1. (see Section 5) (see Section 4).16 Relative 7 bias % 4.2 8 9 10 11 13 14 15 11.10 0.40 1.43 0.22 2.40 0.46 1.13 0.30 0.43 10. Combined and Expanded Uncertainty Estimations for Individual Laboratories (continued).10 1.12 1.594 0.15 8.87 0.617 9.43 0.95 combined % 4.86 = is the standard deviation of submitted results.2: Estimation of Relative Standard Uncertainty.617 2.34 3.53 n=1 0.78 0.589 0.33 0. Standards Solution THAA 6.77 0.591 0.79 2.30 0.84 Leu D/L rpHPLC std dev 4 as CV% 2.79 1.AAR PT Report.563 0.

85 0.2 8 9 10 11 13 14 15 Laboratory Number Page 119 of 157 .3: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Aspartic acid / Asparagine D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 1.20 0.1 6.10 1.30 0.1 6.90 0.35 0.05 0.55 0.AAR PT Report.50 0.2 7.2 7.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.65 0.00 1 2 3 4 5 6. Standards Solution THAA 6.1 7.60 0.05 replicate mean assigned value (all data) 1.75 0.15 0.45 0.2: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Aspartic acid / Asparagine D/L Values in Standards Solution Test Material relative bias (x-X/X %) rel uncert of assigned value (u(X)/X%) 45 rel uncert of submitted results (u(x)/x%) rel std dev of submitted result (CV%) 40 combinded uncertainty Asx D/L (RMS%) u combined 35 standard uncertainty contribution as % 30 25 20 15 10 5 0 1 2 3 4 5 6.10 0.70 D/L Value 0.95 0.15 1.80 0.00 0.40 0. MEASUREMENT UNCERTAINTY Figure 6.25 0.1 7.

1 6.4: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Aspartic acid / Asparagine rpHPLC D/L Values in Standards Solution Test Material 10 relative bias (x-X/X %) rel uncert of assigned value (u(X)/X%) 9 rel uncert of submitted results (u(x)/x%) rel std dev of submitted result (CV%) 8 combinded uncertainty Asx D/L rpHPLC (RMS%) u combined standard uncertainty contribution as % 7 6 5 4 3 2 1 0 1 2 3 4 5 6.00 RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 0.30 0.00 1 2 3 4 5 6.2 7.70 0.2 8 9 10 11 13 14 15 Laboratory Number Page 120 of 157 .95 0.1 7. MEASUREMENT UNCERTAINTY Figure 6.35 0.AAR PT Report.75 0.60 D/L Value 0.85 replicate mean assigned value (HPLC-rp only) 0.65 0.25 0.20 0.1 6.1 7.05 0.80 0.90 0.10 0.5: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Aspartic acid / Asparagine rpHPLC D/L Values in Standards Solution Test Material RP 1.15 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.40 0.55 0.45 0.2 7. Standards Solution THAA 6.50 0.

2 8 9 10 11 13 14 15 Laboratory Number Page 121 of 157 .45 0.90 replicate mean assigned value (all data) 0.30 0.10 0.1 6.55 0.85 0.70 0.25 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.65 0.60 D/L Value 0.6: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Glutamic acid / Glutamine D/L Values in Standards Solution Test Material relative bias (x-X/X %) 30 rel uncert of assigned value (u(X)/X%) rel uncert of submitted results (u(x)/x%) rel std dev of submitted result (CV%) combinded uncertainty 25 Glx D/L (RMS%) u combined standard uncertainty contribution as % 20 15 10 5 0 1 2 3 4 5 6.05 0.15 0. MEASUREMENT UNCERTAINTY Figure 6.00 1 2 3 4 5 6.2 7.80 0.50 0.7: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Glutamic acid / Glutamine D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 1.35 0.1 7.1 7.20 0.00 0.1 6.95 0. Standards Solution THAA 6.75 0.2 7.40 0.AAR PT Report.

65 0.8: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Glutamic acid /Glutamine rpHPLC D/L Values in Standards Solution Test Material relative bias (x-X/X %) 10 rel uncert of assigned value (u(X)/X%) rel uncert of submitted results (u(x)/x%) 9 rel std dev of submitted result (CV%) combinded uncertainty 8 Glx D/L rpHPLC (RMS%) u combined standard uncertainty contribution as % 7 6 5 4 3 2 1 0 1 2 3 4 5 6.85 replicate mean assigned value (HPLC-rp only) 0.25 0.45 0.55 0.70 0.20 0.2 8 9 10 11 13 14 15 Laboratory Number Page 122 of 157 .50 0.1 7.80 0.1 7.1 6.00 1 2 3 4 5 6.60 D/L Value 0.2 7.40 0.9: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Glutamic acid / Glutamine rpHPLC D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 1. Standards Solution THAA 6.95 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.05 0.90 0.10 0.2 7.15 0.00 0.30 0.1 6.35 0. MEASUREMENT UNCERTAINTY Figure 6.AAR PT Report.75 0.

AAR PT Report; Standards Solution THAA

6. MEASUREMENT UNCERTAINTY

Figure 6.10: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Serine D/L Values in Standards Solution Test Material

relative bias (x-X/X %) 10

rel uncert of assigned value (u(X)/X%)

**rel uncert of submitted results (u(x)/x%)
**

9

rel std dev of submitted result (CV%) combinded uncertainty

8

Ser D/L (RMS%) u combined

standard uncertainty contribution as %

7

6

5

4

3

2

1

0

1

2

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4

5

6.1

6.2

7.1

7.2

8

9

10

11

13

14

15

Laboratory Number

Figure 6.11: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Serine D/L Values in Standards Solution Test Material

RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP

1.00 0.95

0.90

replicate mean

assigned value (all data)

0.85 0.80

0.75

0.70

0.65 0.60

D/L Value

0.55

0.50

0.45 0.40

0.35

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0.00 1 2 3 4 5 6.1 6.2 7.1 7.2 8 9 10 11 13 14 15

Laboratory Number

Page 123 of 157

AAR PT Report; Standards Solution THAA

6. MEASUREMENT UNCERTAINTY

Figure 6.12: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Arginine D/L Values in Standards Solution Test Material

40 relative bias (x-X/X %)

rel uncert of assigned value (u(X)/X%) 35

**rel uncert of submitted results (u(x)/x%)
**

rel std dev of submitted result (CV%)

standard uncertainty contribution as %

30

combinded uncertainty

Arg D/L (RMS%) u combined

25

20

15

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5

0

1

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9

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Laboratory Number

Figure 6.13: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Arginine D/L Values in Standards Solution Test Material

RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP

1.00 0.95

0.90 replicate mean

assigned value (all data)

0.85 0.80

0.75

0.70

0.65 0.60

D/L Value

0.55

0.50

0.45 0.40

0.35

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0.00 1 2 3 4 5 6.1 6.2 7.1 7.2 8 9 10 11 13 14 15

Laboratory Number

Page 124 of 157

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6. MEASUREMENT UNCERTAINTY

Figure 6.14: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Alanine D/L Values in Standards Solution Test Material

30 relative bias (x-X/X %)

rel uncert of assigned value (u(X)/X%)

**rel uncert of submitted results (u(x)/x%)
**

25 rel std dev of submitted result (CV%)

combinded uncertainty

standard uncertainty contribution as %

**Ala D/L (RMS%) u combined
**

20

15

10

5

0

1

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Laboratory Number

Figure 6.15: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Alanine D/L Values in Standards Solution Test Material

RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP

1.00 0.95

0.90 replicate mean

assigned value (all data)

0.85 0.80

0.75

0.70

0.65 0.60

D/L Value

0.55

0.50

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Laboratory Number

Page 125 of 157

30 0.1 6. MEASUREMENT UNCERTAINTY Figure 6.20 0.15 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.75 0.1 7.2 8 9 10 11 13 14 15 Laboratory Number Page 126 of 157 .60 D/L Value 0.70 0.10 0.AAR PT Report.16: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Alanine (rpHPLC) D/L Values in Standards Solution Test Material relative bias (x-X/X %) 40 rel uncert of assigned value (u(X)/X%) rel uncert of submitted results (u(x)/x%) rel std dev of submitted result (CV%) 35 combinded uncertainty Ala D/L rpHPLC (RMS%) u combined standard uncertainty contribution as % 30 25 20 15 10 5 0 1 2 3 4 5 6.00 0.1 7.50 0.05 0.55 0.2 7.80 0.95 0.1 6.00 1 2 3 4 5 6.85 replicate mean assigned value (HPLC-rp only) 0.35 0.45 0.65 0. Standards Solution THAA 6.2 7.90 0.17: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Alanine (rpHPLC) D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC RP RP RP RP RP RP RP RP 1.40 0.25 0.

19: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Valine D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 1.70 0.20 0.1 6.75 0.95 0.1 6. MEASUREMENT UNCERTAINTY Figure 6.2 7.2 7.18: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Valine D/L Values in Standards Solution Test Material 20 relative bias (x-X/X %) rel uncert of assigned value (u(X)/X%) 18 rel uncert of submitted results (u(x)/x%) rel std dev of submitted result (CV%) 16 combinded uncertainty standard uncertainty contribution as % Val D/L (RMS%) u combined 14 12 10 8 6 4 2 0 1 2 3 4 5 6.1 7.1 7.AAR PT Report.90 replicate mean assigned value (all data) 0.10 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.00 1 2 3 4 5 6.55 0.15 0.25 0. Standards Solution THAA 6.05 0.85 0.80 0.30 0.00 0.60 D/L Value 0.50 0.40 0.45 0.65 0.35 0.2 8 9 10 11 13 14 15 Laboratory Number Page 127 of 157 .

1 7.15 0.20 0.70 0.05 0.35 0.10 0.1 6.80 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.20: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Valine (rpHPLC) D/L Values in Standards Solution Test Material 20 relative bias (x-X/X %) 18 rel uncert of assigned value (u(X)/X%) rel uncert of submitted results (u(x)/x%) 16 rel std dev of submitted result (CV%) combinded uncertainty standard uncertainty contribution as % 14 Val D/L rpHPLC (RMS%) u combined 12 10 8 6 4 2 0 1 2 3 4 5 6.65 0.40 0.95 0.50 0.1 7.85 0.45 0.2 8 9 10 11 13 14 15 Laboratory Number Page 128 of 157 .60 D/L Value 0.21: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Valine (rpHPLC) D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC RP RP RP RP RP RP RP RP 1.2 7.00 0.30 0. Standards Solution THAA 6.2 7.00 1 2 3 4 5 6.1 6.90 replicate mean assigned value (all data) 0.75 0.25 0.55 0. MEASUREMENT UNCERTAINTY Figure 6.AAR PT Report.

1 6.2 7.15 0. Standards Solution THAA 6.35 0.2 7.05 0.65 0.20 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.70 0.25 0.45 0.60 D/L Value 0.2 8 9 10 11 13 14 15 Laboratory Number Page 129 of 157 .55 0.10 0.30 0.00 1 2 3 4 5 6.40 0.1 6.95 0.75 0.22: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Phenylalanine D/L Values in Standards Solution Test Material relative bias (x-X/X %) 20 rel uncert of assigned value (u(X)/X%) rel uncert of submitted results (u(x)/x%) 18 rel std dev of submitted result (CV%) combinded uncertainty Phe D/L (RMS%) u combined 16 standard uncertainty contribution as % 14 12 10 8 6 4 2 0 1 2 3 4 5 6.1 7.90 replicate mean assigned value (all data) 0.50 0.00 0.AAR PT Report.85 0. MEASUREMENT UNCERTAINTY Figure 6.80 0.1 7.23: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Phenylalanine D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 1.

55 0.24: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Phenylalanine (rpHPLC) D/L Values in Standards Solution Test Material 10 relative bias (x-X/X %) 9 rel uncert of assigned value (u(X)/X%) rel uncert of submitted results (u(x)/x%) 8 rel std dev of submitted result (CV%) combinded uncertainty standard uncertainty contribution as % 7 Phe D/L rpHPLC (RMS%) u combined 6 5 4 3 2 1 0 1 2 3 4 5 6.00 0.2 8 9 10 11 12 13 14 15 Laboratory Number Figure 6.25: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Phenylalanine (rpHPLC) D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC RP RP RP RP RP RP RP RP replicate mean 1.60 D/L Value 0.05 0.20 0.90 assigned value (all data) 0. MEASUREMENT UNCERTAINTY Figure 6.30 0.25 0.80 0.2 7.00 1 2 3 4 5 6.AAR PT Report.40 0.45 0.2 8 9 10 11 12 13 14 15 Laboratory Number Page 130 of 157 .15 0.1 7.1 6.35 0. Standards Solution THAA 6.50 0.10 0.1 6.1 7.75 0.95 0.2 7.85 0.70 0.65 0.

1 6.1 7.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.15 0.2 7.70 0.65 0.95 0.40 0.85 0.1 7.1 6.30 0.45 0.2 8 9 10 11 13 14 15 Laboratory Number Page 131 of 157 .10 0.00 0.35 0.20 0.2 7.80 0.25 0.00 1 2 3 4 5 6. MEASUREMENT UNCERTAINTY Figure 6.55 0.AAR PT Report.60 D/L Value 0.50 0.26: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for D-Alloisoleucine/LIsoleucine Values in Standards Solution Test Material 20 relative bias (x-X/X %) rel uncert of assigned value (u(X)/X%) 18 rel uncert of submitted results (u(x)/x%) rel std dev of submitted result (CV%) 16 combinded uncertainty D-Aile/L-Ile (RMS%) u combined standard uncertainty contribution as % 14 12 10 8 6 4 2 0 1 2 3 4 5 6.75 0.90 replicate mean assigned value (all data) 0.05 0. Standards Solution THAA 6.27: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on D-Alloisoleucine/L-Isoleucine Values in Standards Solution Test Material RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 1.

1 6.AAR PT Report.50 0.55 0. Standards Solution THAA 6.29: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on DAlloisoleucine/L-Isoleucine rpHPLC Values in Standards Solution Test Material RP RP RP IE IE GC GC GC GC RP RP RP RP RP replicate mean RP RP 1.85 0.1 7.40 0.10 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.30 0.80 0.25 0.15 0.2 7.28: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for D-Alloisoleucine/LIsoleucine rpHPLC Values in Standards Solution Test Material 10 relative bias (x-X/X %) 9 rel uncert of assigned value (u(X)/X%) rel uncert of submitted results (u(x)/x%) rel std dev of submitted result (CV%) 8 combinded uncertainty standard uncertainty contribution as % D-Aile/L-Ile rpHPLC (RMS%) u combined 7 6 5 4 3 2 1 0 1 2 3 4 5 6.75 0.1 6.65 0.70 0.2 8 9 10 11 13 14 15 Laboratory Number Page 132 of 157 .90 assigned value (HPLC-rp only) 0.20 0.95 0.35 0.00 1 2 3 4 5 6.00 0. MEASUREMENT UNCERTAINTY Figure 6.1 7.2 7.05 0.60 D/L Value 0.45 0.

2 8 9 10 11 13 14 15 Laboratory Number Figure 6.80 0.25 0.35 0.20 0.85 replicate mean assigned value (all data) 0.60 D/L Value 0.00 0.1 7.90 0.15 0.2 7.1 6. MEASUREMENT UNCERTAINTY Figure 6.10 0.65 0.2 8 9 10 11 13 14 15 Laboratory Number Page 133 of 157 .70 0.1 7.50 0.55 0.75 0.95 0.40 0.30: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Leucine D/L Values in Standards Solution Test Material 20 relative bias (x-X/X %) rel uncert of assigned value (u(X)/X%) 18 rel uncert of submitted results (u(x)/x%) rel std dev of submitted result (CV%) combinded uncertainty 16 Leu D/L (RMS%) u combined standard uncertainty contribution as % 14 12 10 8 6 4 2 0 1 2 3 4 5 6.00 1 2 3 4 5 6. Standards Solution THAA 6.05 0.31: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Leucine D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 1.30 0.1 6.45 0.AAR PT Report.2 7.

AAR PT Report.00 0.50 0.40 0.32: Standard Uncertainty Contributions and Combined Uncertainty for each Laboratory against an Estimated Average Combined Uncertainty for Leucine rpHPLC D/L Values in Standards Solution Test Material 10 relative bias (x-X/X %) rel uncert of assigned value (u(X)/X%) 9 rel uncert of submitted results (u(x)/x%) rel std dev of submitted result (CV%) 8 combinded uncertainty standard uncertainty contribution as % Leu D/L rpHPLC (RMS%) u combined 7 6 5 4 3 2 1 0 1 2 3 4 5 6. MEASUREMENT UNCERTAINTY Figure 6.35 0.1 7.90 0.25 0.30 0.1 6.85 replicate mean assigned value (HPLC-rp only) 0.45 0.2 7.10 0. Standards Solution THAA 6.00 1 2 3 4 5 6.95 0.20 0.75 0.33: Effect of Expanded Uncertainty for each Laboratory at 95% Confidence on Leucine rpHPLC D/L Values in Standards Solution Test Material RP RP RP IE IE GC GC GC GC RP RP RP RP RP RP RP 1.80 0.15 0.70 0.65 0.60 D/L Value 0.1 7.05 0.2 8 9 10 11 13 14 15 Laboratory Number Figure 6.2 7.2 8 9 10 11 13 14 15 Laboratory Number Page 134 of 157 .1 6.55 0.

005 009. 015 008 004. 015 Pre-column Derivatization Reagent constituents and their concentrations (M or mM): OPA 170 mM IBLC 260 mM Potassium borate buffer 1M pH adjusted to: 10. Standards Solution THAA APPENDIX I – Analytical Methods Appendix 1: Analytical Methods Used by Participants Reverse Phase HPLC/ HPLC-Ion Exchange REFERENCES Please give details of any method relevant references. 015 001. 011. 015 001. 015 001. 012. 005 006. 009. 009. 010. 009. Flame Ionization 001. 003. 003. 014. 014. 012. 003. 013 002. 002. 005 001. 013. 010. 014. 002.AAR PT Report. 010. 002. 010. 008. 008. 009. 015 Page 135 of 157 . 008. 014.4 Sample injection volume (μl) 2 μl 4 μl 20 μl 001. 008. 011. 008. 011. 014. 002. 002. 013. 015 004. 012. 013. 010. 013. Kaufman & Manley 1998 ANALYSIS Please state method used Reverse phase HPLC Ion Exchange HPLC Instrument used Agilent 1100 Series Agilent / Hewlet Packard 1100 Series Agilent 1200 Series Agilent 6890 GC. 014. 007 001. 012. 011. 003. 011. 008. 013. 014. 011. 013. 009. 011. 014. 012. 013. 009. 002. 010. 003. 003. 012. 012. 015 004. 012. 010. 011. 009. 003. 010.

010. Standards Solution THAA HPLC COLUMN Column Make/Type & Phase(i. 005 004. 014. 009. 010. 015 002. 015 002. 009. 005 001. 008 001. 013. 011. 011. 015 001.56ml/min 76. 015 002. 002. 013. 008. 003 008 009. 011. 002. 005. 005.00) Methanol Acetonitrile Sodium citrate buffer (pH 3. 002. 012.86) Sodium chloride buffer (pH 11. 011. 003. 010.00) Gradient: Starting % | Final %| time (mins) | flow rate (ml/min) Page 136 of 157 . 010. 009. 010. 013. 015 001. 010. 014. 003. 003. 013. 015 004. 009. 015 Mobile phase components (please state. 009. 009. 005 001a 001b 002. 003. 014.6%|31mins|0. 005 004. 003.. 008. 005. 004. 013. 014. 002. 012. 004. 014.6%|46. 012. 005 009.AAR PT Report. 003. 013.e. 012. 008 001. Hypersil BDS)/ Batch Number: Thermo/Hypersil BDS C18/0742018X Hypersil BDS Hypersil BDS /5/120/4772 Pickering Labs Sodium Cation Exchange Supelcosil LC-18-DB(rp)/6520/5-1452 Column Packing: Silica Sodium Functional group. 004. 015 001.12) Sodium citrate buffer (pH 3. 012. 011. 015 001.5) 95%|76. 003. 013. 013.56ml/min o o APPENDIX I – Analytical Methods 001 009. i. 011. 002. 002. 005 008 002. 008. 008. 011.500ml/min 95%|50%|88min|0. 012. 010.2%|95min|0. 013. sodium acetate buffer/ methanol/ acetonitrile): Sodium acetate Buffer (pH 6. 010. 002.560ml/min 95%| %|95min|0. 003. 004. 012. 009. 010.60ml/min 95%|5%|83min|0. 002. 003 004. 012. 005 001. 011. 014. 014. 012.e. C18 End capped (Yes) Column width (mm) 3mm 5mm Column length (mm) 250mm Guard Column not used No HPLC Column Temperature ( C): 25 C o 30 C MOBILE PHASE Mobile phase programme: Gradient Sodium acetate Buffer (pH 6. 010. 011. 008 004. 014. 012. 011. 014. 004. 003. 013. 014. 015 001. 003. 003..

013. 003. 004.005 004. 012.12) Gradient: Starting % | Final %| time (mins) | flow rate (ml/min) 100%|0%|99mins|0. 010.500ml/min 5%|45%|88min|0. 012.4%|5%|83min|0.5M OPA 0.140ml/min 004.8%|95min|0. 015 002. 009. 003 008 009. 011.500ml/min 0%|5%|88min|0. 015 004. 015 004. 010. 015 Acetonitrile Gradient: Starting % | Final %| time (mins) | flow rate (ml/min) 0%|0.140ml/min 004. 014. 010. 012.56ml/min 0. 005 008.86) Gradient: Starting % | Final %| time (mins) | flow rate (ml/min) 0%|0%|99mins|0. 011. 013. 013.4 DETECTION Detector Type: Fluorescence Excitation wavelength (nm): 230 250 335 340 Emission wavelength (nm): 410 445 455 002. 009.0075M Ethanol 1% 2-mercapthoethanol 0. 003 001 004. 011.560ml/min 0%|5%|95min|0.56ml/min 23%|48. 014. 002. 003 001. 003 008 009. Standards Solution THAA MOBILE PHASE continued APPENDIX I – Analytical Methods Methanol Gradient: Starting % | Final %| time (mins) | flow rate (ml/min) 5%|23%|31mins|0. 005 001. 005 Sodium chloride buffer (pH11.005 004.00075% pH adjusted to 10.56ml/min 001a 001b 002. 010.4%|31mins|0.56mi/min 001a 001b 002.005 Page 137 of 157 . 008.60ml/min 5%|95%|83min|0. 005. 008. 012. 005 Post-column Derivatization Reagent constituents and their concentrations (M or mM): Boric Acid 0. 014. 011. 013. 014.5) Gradient: Starting % | Final %| time (mins) | flow rate (ml/min) 0%|100%|99mins|0. 009. 005 Sodium citrate buffer (pH3.560ml/min 5%|50%|95min|0.005 004. 015 Sodium citrate buffer (pH3.AAR PT Report. 011. 012.005 004. 014. 013.4%|5%|95min|0. 010.140ml/min 004.60ml/min 0.

Standards Solution THAA APPENDIX I – Analytical Methods Gas Chromatography REFERENCES Please give details of any method relevant references. 007 006. in continuous use Column Packing: Chiral Phase: Chirasil-val Column width (mm) 0. Serial # 5653. 007 006. 007 006. 007 006. 007 006. purchased in 1998. 007 Page 138 of 157 . 007 o 006. 007 006.AAR PT Report. Goodfriend 1991 with modifications ANALYSIS Sample injection volume (μl) 1 -3 μl GC injection mode: Splitless GC COLUMN Column Type.25mm Column length (mm) 25m Column Temperature ( C): See below for program Mobile phase / Carrier gas Helium Mobile phase flow rate (ml/min): Flow variable with temperature. pressure 7. Capillary Column Make / Batch Number: Alltech.6psi 006. 007 006. Catalog #13633. 007 006. 007 006.

then cyclohexane is added to ready the derivative for GC injection. hold for 10 minutes. This complete derivative is then usually stored overnight prior toGC analysis. but there is no “formula” for this because the GC analysis is not quantitative. 7) The DCM/TFA solution is transferred to a small GC vial. Page 139 of 157 . Injection amounts are usually 1 ul.) prior to esterification with isopropanol. 20 deg/min up to 80 deg. then further dried in a vacuum oven (1 hour. All important peaks are eluted within 100 minutes. hydrolyse for 22 hoursat 105 deg. dried with N2. 007). 0. 5 deg/min to 160.AAR PT Report. 3) After hydrolysis. last phases of temperature program are to clean out the column. 4) Dried residue is transferred using ~0. 5) Isopropanol esterification – one hour at 105 deg. The amount of cyclohexane is variable depending on the sample size. 007 NDP not used for these samples. or DCM) and TFA are added. recycle. solution is transferred to another plastic tube for N2 drydown in a heating block (~60 deg). This solution is dried with N2. APPENDIX I – Analytical Methods 006. HCl solution is transferred to plasticcentrifuge tube and appropriate amount of HF is added to remove Ca. hold for one minute. seal hydrolysis tube. 2 or even 3 ul will be injected. 50 deg. Derivatives remain in the cyclohexane solution until GC injection – in most cases. 007). 1 minute hold. five or six chromatograms are obtained over a period of one to two weeks. Standards Solution THAA DETECTION Detector Type: Flame ionisation Comments received (006. 8) GC temperature program: inject at 60 deg. 10 minutes hold. if samples are small. Drydown requires about one hour. 6) Isopropanol is then dried down with N2 in 50 deg heating block (~10 minutes). then methylene chloride (Dichloromethane. but used in previous studies – both NPD and FID give same D/L values ANYTHING ELSE? Please use this space for any additional information you would like to record concerning method details not covered above: Comments received (006. HCl to bringfinal solution to 6N 2) Purge with N2. Summary of the preparation sequence: 1) Dissolution in stoichiometric amount of conc. After centrifuging.85 deg/min to 135 deg.2 ml 1N HCl to a screwcap vial.

007. 010. 006. 004. 011. 010. 011. 005 006. as component of rehydration fluid Internal Standard Used?: L-homo-Arginine Norleucine No Concentration of Internal std used (M): 0. 009. 013. 014. prior to analytical run Yes. 004. 012. 014. 005. 002. 003. 015 If Yes. 015 001. 009. 013. 009. 003. 008. 005 001. 014. 004. 009. 013. 015 004. type of calibration: Calibration curve/std addition-single level Calibrated by Agilent Technician 001 008 001 008 002. 012.AAR PT Report. 004. 013. 010. 005. 015 001 001 Page 140 of 157 . 011. 014.01mM 6. 009. 002. what reference materials / standards are used? In-house std solution(s) NB: Solution prepared from single powdered AA standards Source of reference materials/standards: Sigma RECOVERY OR INTERNAL STANDARD Was % recovery determined? No If No. 002. 011.03 mM 0. 008.25 mM Source / supplier of internal standard: Sigma Sigma Aldrich (Fluka) 001. 008. within the last year No If Yes. 012. 014. 010. 005 008 001 002. 007 001. 011. 012. 002. 003. 003. 015 004. Standards Solution THAA APPENDIX I – Analytical Methods Internal Quality Control INSTRUMENT CALIBRATION Was the instrument calibrated prior to analysis? Yes. was an internal standard used? Yes. 003. 003. 005. 013. 008. 010. 012.

012. how often do you determine the MU? Routinely per run Approx once a month When its needed As the SD of multiple chromatograms from each derivative. 010. 010. 008. 005. 015 001. 012. 011. 009. 007 001. 009. 002. 012. 014. 006. 012. 011. 010. Standards Solution THAA D/L RATIO CALCULATION Do you routinely calculate concentrations? Yes No APPENDIX I – Analytical Methods 001. 013. 011. 005.F. 015 004. 003. 012. 005. 011. 009. 013. 009. 004. 014. 001. 003. 002. 008 009. 002. 004. 014. 005. 007 001. 014. 011.are they: In-house std solution(s) (Matrix-matched) ILC stds (Wehmiller) Source of QC materials: Sigma J.Wehmiller How do you use QC materials? Control charts Visual inspection of chromatograms/data D/L comparison to lit Comparison in ILC’s with long term mean MEASUREMENT UNCERTAINTY How do you determine Measurement Uncertainty (MU) of your data As the standard deviation 001. 015 Page 141 of 157 . 010. 014. 006. 004. 003. 013. 008. 010. 011. 011. 011. 008. 007. 006. 012. 015 002. 012. 010. 015 008 006. 008 Comments received. 004. 004. 002. 014. 010. 011. 014. 003. 003. 014. 002. 003. 006. 005. 013. 003. 005 008. 007. 007. 007): Only peak areas are reported under most circumstances but both are measured to check for reliability and peak distortion/overload.AAR PT Report. 004. 010. 013. 004. 013. 011. D/L values are routinely calculated using: Peak heights Peak areas Concentrations based on peak areas QUALITY CONTROL Do you routinely use lab QC materials or standards. 005. 007. 013. 015 If you do. 007. 009. 015 001. 013. 014. 012. 012. 003. 010. 009. 007. 004. 012. 014. 006. 015 002. 009. 004. 015 001. (001) Concentration of a single enantiomer in solution (milimol/L)= (enenatiomer area x Internal Standard concentration )/ Internal Standard area Concentration of a single enantiomer in the sample (picomol/mg)= [Concentration of enantiomer in solution (milimol/L) x Volume of rehydration fluid added (L) x 10-9 picomol/milimol)]/sample weight (mg) (006. 014. 003. 003. 011. 013. 012. 005. 013. 013. 010. 007. Yes If Yes. 006. 008 002. 005. 009. 006. 015 002. 008. 015 006. 002. 010. 009. 005. 009.

Standards Solution THAA APPENDIX 2 . Terms & Definitions Abbreviations ANOVA CRM Analysis of Variance Certified Reference Material Coefficient of Variation EQC IQC MU PT QA QC External Quality Control Internal Quality Control Uncertainty of Measurement / Measurement Uncertainty Proficiency test Quality Assurance Quality Control Symbols Coverage Factor Bias Root Mean Square Relative Between Sample Standard Deviation (expressed as a percentage) Relative Standard Uncertainty (expressed as a percentage) Relative standard deviation (expressed as a percentage) Relative Repeatability standard deviation (expressed as a percentage) Relative Reproducibility standard deviation (expressed as a percentage) (Homogeneity) Analytical Precision (Homogeneity) Analytical Variance (Homogeneity) Sampling Precision (Homogeneity) Sampling Variance (Homogeneity) Total Permissible Sampling Variance Standard Deviation Between-sample standard deviation Repeatability Standard Deviation Reproducibility Standard Deviation (Inter-Laboratory) Reproducibility Standard Deviation (Intra-Laboratory) or Intermediate Precision Target Standard Deviation Homogeneity Target standard deviation Assigned Value standard deviation Standard Uncertainty Page 142 of 157 .AAR PT Report.Glossary Appendix 2: Glossary of Abbreviations. Symbols.

1994) on the ‘Accuracy (trueness and precision) of measurement methods and results’. Readers are recommended to consult these documents for additional notes and comments not included here. Accuracy describes random and systematic error effects and as such composed of both precision and bias components. Accuracy is a concept that cannot be directly quantified. 2005). eg. Page 143 of 157 .refers to (ISO 5725-1. Accuracy closeness of agreement between a measured result and the true value (if it could be known). NOTE 1. A one-way ANOVA uses the F-test to compare the effect of one factor plus the experimental precision. (between-sample variance) against the inherent experimental precision (within-sample variance). (VIM 2.Glossary Terms and Definitions Specific references for terms that can be found in International Standards or guidance documents have been given in brackets at the end of each definition. a reference value from a reference laboratory or the consensus value from participants’ results calculated as the robust mean. (Currell and Dowman. Standards Solution THAA Standard Uncertainty of the Assigned Value Standard Uncertainty due to Bias Standard Uncertainty of Participant’s Results Combined (standard) Uncertainty Expanded Uncertainty Submitted Result or Value Measurement Result / Mean submitted result Assigned Value APPENDIX 2 . This may be the certified reference value of a CRM. Here. is Analysis of Variance (ANOVA) A group of statistical techniques that enable the different contributions from various sources of the observed variance in experimental data to be separated and estimated. It does not possess a numerical value. 2008). Assigned Value The best estimate of the true value of the measurand. NOTE. Miller and Miller. or a reference value. median or mode. 2008) and ISO (1). GUM refers to the ‘Guide to the expression of uncertainty in Measurement’ (JCGM 100:. 2005. Terms shown in bold indicate further definitions that may be found in this section. the effect of the measurement process on different samples. NOTE 2. NOTE 2. Whilst it is possible to carry out the analysis by hand more commonly statsistical software packages are more convenient such as the Excel Data Analysis tools as this also carries out the F-test evaluation at the same time.13) NOTE 1.AAR PT Report. VIM refers to ‘International vocabulary of metrology’ (JCGM 200:.

. a reference material accompanied by certified traceable measurement and uncertainty values determined using validated procedures (VIM 5. and is a component of reproducibility standard deviation. This may be the robust standard deviation.14) Cochran’s Test A statistical test that detects extreme variances between observations by calculating the Cochran’s (C) value as the ratio between the largest squared difference ( ) to the sum of all the squared differences ( ) and comparing this against tabulated critical values. It is determined using ANOVA.18) Bias Root Mean Square ( ) A component of the bias standard uncertainty taking into account both the bias and bias variation. Eg. (ISO 5752-2: 1994) Coefficient of Variation ( ) (expressed as a percentage).3. etc. Standards Solution THAA Assigned Value standard deviation ( ) Standard deviation of the assigned value. The precision or dispersion between independent measurements carried out on different samples of the same material under reproducibility conditions. (GUM 2. Page 144 of 157 . a). There are two common rules for the combination of standard uncertainty values which depend on the model used for deriving the measurement value. such that. i. is given by.e. then the combined standard uncertainty. between-instruments. between-day.e. If the model involves the addition or subtraction of values. Eg..4) NOTE 1. is given by.AAR PT Report. If the model involves the product or quotient of values. APPENDIX 2 . Bias estimate of a systematic measurement error (VIM 2. or then the combined standard uncertainty. and between-laboratory variability’s. Certified Reference Material (CRM).Glossary NOTE. sMAD (median absolute deviation) or SEM (standard error of the mode) Between-sample standard deviation ). See Standard uncertainty due to bias ( ). i. NOTE: it includes the between-operator. See Relative standard deviation ( ) Combined (standard) Uncertainty ( ) The combined standard uncertainty of a measurement result taking into account various contributions from different standard uncertainty sources. b).

For smaller data sets where the distribution of measurement results is better described by a t-distribution. See Standard Uncertainty ( ) External Quality Control (EQC) See Quality Control (QC). NOTE. and Thompson. thus k=2. Values for F1 and F2 may be derived from statistical tables.df) . Expanded Uncertainty ( ) A quantity defined by a specified interval (i.Glossary NOTE 2.16) NOTE 1. 95% confidence) about the measurement result and describes the dispersion where a large number of repeated measurement results would be expected to lie.. 2 standard deviations) or confidence level (i.e. For proficiency testing the format given in the first example has been used. and = the combined uncertainty Experimental standard deviation of the mean. where k = the coverage factor. only a best estimation of it from measurement determinations.. thus. Most often a 95% or 2 standard deviation level of confidence is required for the reporting of measurement results.AAR PT Report. thus k=t(0.5. Where. 2001). F1 and F2 Are constants used to test the hypothesis that there is no significant evidence that the sampling standard deviation exceeds the allowable fraction of the target standard deviation and that the test for sufficient homogeneity has been passed (Fearn. = uncertainty due to precision.e. For large data sets where the distribution approximates to normality the value of k to use is taken from the level of confidence required in the measurement result. T. Standards Solution THAA APPENDIX 2 . M... and = i. the equivalent t-value is used as the multiplier. Error measured quantity value minus a reference value or true value (VIM 2. To some extent the concept of error is a theoretical one as it is not possible to be sure of a measurand’s true value. Coverage Factor ( ) Factor used to multiply the combined uncertainty by in order to derive the Expanded uncertainty value. If a reference value is to be used then it is more accurate to determine the precision and bias as estimates of random and systematic error contributions which can be quantified.e. where m = the number of samples measured in duplicate Page 145 of 157 . the uncertainty due to bias.

is derived from the homogeneity target standard deviation (either ). different operators. different locations over any given period of time. a target value sufficient homogeneity ( )can be determined using fitness-for-purpose criteria. Standards Solution THAA APPENDIX 2 . Calculated from the ANOVA between and within group mean square values.Glossary NOTE. F-statistic (Homogeneity) Analytical Precision ( ) The homogeneity within-sample standard deviation for the replicate values (i.N.AAR PT Report. The (Fisher) F-Test is a test for significant differences between the variances of two data sets and compares random error effects. 2005) Thus. Miller.. different operating conditions. & Miller.C. Intermediate conditions Independent measurement results obtained for identical test items using the same measurement procedure under a specified set of conditions within the same laboratory that include. Calculated from the ANOVA within group mean square. (Homogeneity) Sampling Precision ( ) The homogeneity between-sample standard deviation for the samples (i. J. A. (Homogeneity) Sampling Variance ( ) The square of the sampling precision. Page 146 of 157 . In the absence of an external value for target standard deviation ( ). (Homogeneity) Total Permissible Sampling Variance ( ) The total allowable between-sample variance that must not be exceeded by the sampling variance in order for the test materials to be considered homogeneous... (Currell. 1. 2…10) used in the test for sufficient homogeneity of the test materials. (Homogeneity) Analytical Variance ( ) The square of the analytical precision. Calculated from the ANOVA within group mean square. a and b) used in the test for sufficient homogeneity of the test materials. (VIM 2. G. Homogeneity Target standard deviation ( ). The F-test may also be used within other tests such as ANOVA... J. .e.e.22). & Dowman. See Reproducibility Standard Deviation (Intra-Laboratory) or Intermediate Precision ( ) Internal Quality Control (IQC) See Quality Control (QC) Measurement Result / Mean submitted result ( The average of an individual participant’s replicate measurement results for the same analyte in the proficiency test.2005. Calculated from the ANOVA between and within group mean square values.

Specific activities that are carried out in order to implement the procedures documented under the Quality Assurance programme.Glossary Precision closeness of agreement between repeated measurement results on the same material under specified conditions (VIM 2. Proficiency test (PT). This may be in the form of Internal Quality control (IQC) that are carried out internally by the organization such as method validation.15) NOTE 1. Random error component of measurement error that in replicate measurements varies unpredictably (VIM 2. Relative Bias % (expressed as a percentage) Bias divided by the assigned value (x 100) x 100 Relative Between Sample Standard Deviation ( ). This may be presented as z-scores or other assessment of bias. Documented procedures that describe a quality management system designed to control activities and maintain a quality output. proficiency tests or collaborative trails. or External Quality Control (EQC) coordinated by an external organization such as interlaboratory comparisons eg. A random error value is determined as the precision that would result from a number of replicate measurements of the same measurand. Quality Control (QC). Specific measurement conditions can be repeatability.19) NOTE 1. An external quality control (EQC) procedure through which the accuracy of a laboratory’s measurement result can be objectively evaluated. intermediate or reproducibility conditions. Standards Solution THAA APPENDIX 2 . (expressed as a percentage) The standard uncertainty divided by the (average) measurement result (x 100) Relative standard deviation ( ) or Coefficient of Variation ( ) (expressed as a percentage) The standard deviation divided by the (average) measurement result (x 100) Page 147 of 157 . NOTE. control charts. Quality Assurance (QA). NOTE 2. (expressed as a percentage) The between-sample standard deviation divided by the (average) measurement result (x 100) Relative Standard Uncertainty ( ). etc.AAR PT Report. variance. expressed as a distribution. relative std dev or CV and is a measure of random error. Performance is assessed by providing a comparison of trueness with other participating laboratories NOTE: Trueness is determined through the evaluation of laboratory bias against a reference value. calibration. Precision can be quantified and usually expressed as a measure of imprecision such as standard deviation.

a). under repeatability conditions.18). same operators. Independent measurement results are obtained for identical test items under a specified set of conditions that include the same measurement procedure.Within-sample (or instrumental/analytical) repeatability standard deviation is the dispersion of replicate instrumental measurements carried out on the same sample in the same analytical run. Eg. same measurement system or laboratory. (VIM 2. expressed as a percentage The Reproducibility standard deviation divided by the (average) measurement result (x 100) Repeatability conditions . an individual laboratory’s replicate PT results.15) NOTE. Inter-laboratory repeatability (laboratory+method+analytical) standard deviation is the dispersion of independent measurements carried out by more than one laboratory on different samples of the same material. a). The Inter-laboratory reproducibility standard deviation ( ) obtained from a collaborative trial represents the maximum dispersion for the measurement procedure carried out across laboratories and provides an estimate of best practice for the measurement procedure for a specified matrix / analyte/ concentration. Intra-laboratory (or method + analytical) repeatability standard deviation is the dispersion of independent measurements carried out by a single laboratory on different samples of the same material. Often calculated using ANOVA from the within group mean square (MS). eg. Providing a laboratory’s own repeatability is in agreement with the inter-laboratory repeatability precision estimate. different locations over any given period of time. (expressed as a percentage) The repeatability standard deviation divided by the (average) measurement result (x 100) Relative Reproducibility standard deviation ( ). c). eg.AAR PT Report.Glossary Relative Repeatability standard deviation ( ). See Repeatability Standard Deviation ( ) Repeatability Standard Deviation ( ) The dispersion or precision of replicate measurement values carried out under repeatability conditions ( ISO (1) 3. same operating conditions. different operators. Standards Solution THAA APPENDIX 2 . same location and in as short a time as period as possible. collaborative trial precision data. different operating conditions. ISO (1) 3. such that. eg.19) Thus. then the laboratory can claim the Reproducibility Page 148 of 157 . (VIM 2.14). different measurement systems and laboratories. From Intra-laboratory method validation data or homogeneity analytical precision data . under repeatability conditions. Eg. ISO (1) 3. Reproducibility Conditions. See Reproducibility Standard Deviation (Inter-Laboratory) ( ) Reproducibility Standard Deviation (Inter-Laboratory) ( ) The overall dispersion or precision of independent measurement values carried out on different samples of the same material by different laboratories. Independent measurement results obtained for identical test items using the same measurement procedure under a specified set of conditions that include.24. under reproducibility conditions and incorporates both within (repeatability) and between-sample precision estimates (ISO (1) 3. b).20.

AAR PT Report; Standards Solution THAA

APPENDIX 2 - Glossary

standard deviation from a collaborative trial as their own standard uncertainty estimate. Reproducibility Standard Deviation (Intra-Laboratory) or Intermediate Precision ( ) The overall dispersion or precision of independent measurement values carried out on different samples of the same material by the same laboratory, under reproducibility conditions and incorporates both within (repeatability) and between-sample precision estimates (VIM 2.23) Thus; Eg; Intra-laboratory reproducibility standard deviation ( ) represents the maximum dispersion for the measurement procedure carried out by an individual laboratory and is often used in method validation as the method precision for a particular matrix / analyte /concentration and used as the standard uncertainty. Standard Deviation ( ) A term used to describe the dispersion or spread of measurement values and has the same units as the measurement value. NOTE; by convention the symbol used for standard deviation depends on whether it is describing sample statistics or population parameters. Thus; Sample statistics; Population parameters; Where = individual measurement values = average measurement value for the sample = population mean = number of measurement values or population size )

Standard Error of the Mean. See Standard Uncertainty (

Standard Uncertainty ( ) The uncertainty of a measurement result expressed as a standard deviation, (GUM 2.3.1) NOTE; When determined from a series of repeated measurements this can also be found referred to in texts as the experimental standard deviation or standard error of the mean. Thus; Standard Uncertainty of the Assigned Value ( ) The uncertainty of the Assigned Value, expressed as a standard deviation, (GUM 2.3.1). where and NOTE; = the assigned value std dev m = the number of participants’ measurement results .

is also a component of the standard uncertainty due to bias

Standard Uncertainty due to Bias ( ). The uncertainty of the bias component of a participant’s measurement result, expressed as a standard deviation, (GUM 2.3.1). NOTE 1; An individual laboratory’s standard uncertainty due to bias for a single proficiency test, is given as;

Page 149 of 157

AAR PT Report; Standards Solution THAA

APPENDIX 2 - Glossary

NOTE 2; An individual laboratory’s standard uncertainty due to bias over multiple proficiency tests, is given as;

where;

= the bias root mean square and given as;

and

= the average standard uncertainty of the assigned value;

m = the number of proficiency tests or number of bias values, and n = the number of participants’ measurement results in each PT. NOTE 3; It often helps to carry out these calculations as the relative percentage values. Standard Uncertainty of Participant’s Results ( ) The uncertainty of a participant’s submitted replicate results, expressed as a standard deviation, (GUM 2.3.1). where and NOTE; = the std dev of replicate values n = the number of replicate values submitted .

is also a component of the standard uncertainty due to bias

Submitted Result or Value ( ) An individual participant’s submitted measurement result for the proficiency test. Systematic Error component of measurement error that in replicate measurements remains constant or varies predictably (VIM 2.17) NOTE 1; A systematic error value is determined as the bias, i.e.; the difference between a measured result and the true or reference value. Measurement results should always be corrected where significant bias is detected. Target Standard Deviation ( ) The target value for standard deviation for the proficiency test used to calculate z-scores and assess homogeneity data. NOTE; often determined independently from data external to the proficiency test, such as the reproducibility standard deviation (RSDR%) from a collaborative trail or using a predictive model such as the Horwitz function when appropriate of fitness-for purpose criteria. The target std dev is usually matrix / analyte specific. Eg; a) From a collaborative trial; where and

p

RSDR c 100

RSDR = Relative Standard Deviation of Reproducibility from collaborative trial data, expressed as %

ˆ , expressed in relevant units. c = concentration, i.e. the assigned value, X

Eg; b) Using the Horwitz equation; Or modified form; for concentrations less than 120ppb (1.2x10-7); and for concentrations greater than 13.8% (0.138); Page 150 of 157

AAR PT Report; Standards Solution THAA

APPENDIX 2 - Glossary

Where the concentration (c) is expressed as a mass fraction as shown in () above. Trueness closeness of agreement between the average of a large number of replicate measurement results and the true value (if it could be known) or a reference value (VIM 2.14) NOTE 1; Trueness is a concept that cannot be directly quantified. It does not possess a numerical value. NOTE 2; Trueness is usually expressed as bias and a measure of systematic error. t-value 2-tailed t-value is used as a correction factor in the determination of confidence intervals for small values of n. Derived from the t-distribution for sample data sets and described using , compared to the normal distribution for populations described as Values for t may be obtained from statistical tables. (Currell and Dowman, 2005, Miller and Miller, 2005). Such that, for a 95% confidence interval; NOTE; The (student’s) t-Test is a test for significant differences between the mean of two data sets and compares systematic error effects. Thus; t-statistic

Uncertainty of Measurement / Measurement Uncertainty (MU) A parameter associated with a measurement result (taken as the best estimate of the true value) and characterizes the dispersion of values that could be attributed to the measurement result, taking into account both random and systematic error contributions from all possible sources and represents the degree of doubt associated with the measurement result (GUM 2.2). Welch-Satterthwaite formula Formula used for deriving the effective degrees of freedom for the calculation of Expanded uncertainty, when various standard uncertainties are combined with differing degrees of freedom.

Where

= = = =

the effective degrees of freedom, degrees of freedom of individual uncertainty components, combined standard uncertainty individual uncertainty components.

z-Score A standardized measure of laboratory bias derived from the assigned value and target standard deviation, enabling a comparison of performance between laboratories. Satisfactory performance is considered if a |z|≤2.

z

ˆ) (x X

p

Page 151 of 157

3646 2.9768 2.8982 2.0595 99% 63.1009 2.64 0.1824 2.2281 2.59 0.0141 2.43 (Fearn and Thompson.0181 2.88 1.75 0.62 17 1.7333 2.7633 2.7765 2.0106 2.7787 2.0227 2.0321 3.8408 4.0639 2.8453 2.7564 2.7874 df 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 95% 2.8188 2.1058 3.9208 2.94 1.2622 2.0687 2.7970 2.7045 2.0086 99% 2.01 9 1.72 0.7116 2.0129 2.7440 2.57 19 1.0796 2.83 0.1315 2.0167 2.7012 2.60 0.4995 3.2010 2.69 0.0860 2.0301 2.0739 2.6923 2.0211 2.75 13 1.AAR PT Report.0322 2.7284 2.3554 3.0345 2.0195 2.6981 2.7195 2.7154 2.7074 3.6896 2.3060 2.79 0.8073 2.6951 2.1199 2.0262 2.0545 3.7707 2.11 8 2.0123 2.1448 2.25 7 2.10 1.9250 5.5706 2.0555 2.6846 2.1788 2.8314 2.6778 Factors F1 and F2 (95% significance level) m F1 F2 20 1.4469 2.62 0.6600 9.68 15 1.93 10 1.7238 2.0244 2.7079 2. Standards Solution THAA APPENDIX 3 – Critical Values Tables Appendix 3: Tables of Critical Values Student t-distribution df 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 95% 12.71 14 1.0369 2.6822 2.0117 2.0096 2.59 18 1.8609 2.80 12 1.0281 2.6041 4.1098 2.8784 2.9467 2.1693 3.7100 4.0395 2.0452 2.0484 2.7500 2. 2001) Page 152 of 157 .01 1.67 0.86 11 1.0930 2.3027 3.0518 2.6870 2.1604 2.2498 3.7385 2.0154 2.64 16 1.0423 2.6800 2.

2 37.2 33.AAR PT Report.8 68.2 43.7 84.1 78.7 39.9 96.1 51.5 41.1 51.3 38.1 45.2 47.1 35.1 76.8 44.1 72.6 47.5 49.9 30.3 28.9 97.9 60.1 27.5 29. Standards Solution THAA APPENDIX 3 – Critical Values Tables Cochran’s Critical values (95% significance level) No of Samples (m) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 No of sample replicates (n) 2 3 99.4 61.5 31.6 56.1 (ISO 5725-2. 1994) Page 153 of 157 .0 63.8 40.7 68.4 41.7 90.5 87.2 57 54.

HORWITZ. Kernal density estimation based on RSC AMC Technical Brief No 4. Available from. A.0e. ISO 5725-2 (1994) Accuracy (trueness and precision) of measurement methods and results. Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method. Appendix D: Guidelines for Collaborative Study Procedures to Validate Characteristics of a Method of Analysis. T. ISO 5725-1 (1994) Accuracy (trueness and precision) of measurement methods and results.AAR PT Report. John Wiley & Sons Ltd. 126.htm.asp. Available to download from: http://www. 1/2007.rsc. & THOMPSON. CURRELL. http://www. Available from. L. W.asp.org/docs/technical%20report/EL_11_01_06_387%20Technical% 20report%20-%20Guide_Measurement_uncertainty. . http://www. (1995) IUPAC Protocol for the design. Robust Statistics Tool Kit based on RSC AMC Technical Brief No 6.pdf. (2005) Essential Mathematics and Statistics for Science. 2 ed. Available from. International Standards Organisation. & BOUYER. conduct and interpretation of methodperformance studies.html. 63. & ELLISON. FEARN.. S. 67A-76A. S. ELLISON. EUROLAB (2006) Technical Report No. (2002a) Kernal. http://www. Analytical Chemistry. W. W. Standards Solution THAA APPENDIX 4 – References Appendix 4: References AOAC (2000) AOAC Official Methods Program Manual Part 12. R. BARWICK.org/Membership/Networking/InterestGroups/Analytical/AMC/Softw are/index. 47-53. M. EURACHEM / CITAC (2000) Guide CG 4: Quantifying Uncertainty in Analytical Measurements.org/vmeth/omamanual/omamanual. http://www. AOAC International. EUROLAB (2007) Technical Report No. Comparability and Reliability in Chemical Measurement. (2000) The evaluation of measurement uncertainty from method validation studies.. version1. G.AOAC.pdf. 5. R. L. 1/2006.xla version 1. Chichester.0... Accreditation and Quality Assurance: Journal for Quality. K.org/Membership/Networking/InterestGroups/Analytical/AMC/Softw are/index. International Standards Organisation. (1982) Evaluation of analytical methods used for regulation of foods and drugs. J. HORWITZ.xla. The Analyst. W. Available to download from..eurolab.org/pub/i_pub. S. 54. Measurement uncertainty revisited: Alternative approaches to uncertainty evaluation. ISO 5725 (1994) Accuracy (trueness and presicion) of measurement methods and results Part 2. HORWITZ.citac. & DOWMAN.rsc. ELLISON. 1344-1354. J. Guide to the evaluation of measurement uncertainty for Quantitative test results.cc/QUAM2000-1. (2001) A new test for 'sufficient homogeneity'. (1980) Quality assurance in the analysis of foods and trace constituents. V. 1414-1417. Available from. International Standards Organisation. KAMPS. http://www. Basic method for the determination of repeatability and reproducibility of a standard measurement method. Part 1: General principls and definitions. RSC. Page 154 of 157 .aoac.eurolab. (2002b) Robstat.

2303-2308. S. Handbook for calculation of measurement uncertainty in Environmental Laboratories. International Standards Organisation.pdf. http://www. & MILLER. 120.asp. 3.pdf. D. The Analyst. HOVIND. http://www. http://www. MAGNUSSON.org/Membership/Networking/InterestGroups/Analytical/AMC/Tech nicalBriefs. Available from. RSC ANALYTICAL METHODS COMMITTEE (2006) AMC Technical Briefs No 4. (2002) Bump-hunting for the proficiency tester searching for multimodality.net/nordtestfiler/tec537. JCGM 200: (2008) International Vocabulary of Metrology . Available from. J.rsc. http://www. (2005) Statistics and Chemometrics for Analytical Chemistry. MCCOY. M. & KRYSELL. LOWTHIAN.rsc.org/Membership/Networking/InterestGroups/Analytical/AMC/Tech nicalBriefs..Basic and general concepts and associated terms (VIM). RSC ANALYTICAL METHODS COMMITTEE (2004) AMC Technical Briefs No 17.asp. International Standards Organisation. The Analyst..Guide to the expression of uncertainty in measurement (GUM).bipm. Quaternary Geochronology. NAYKKI. MILLER. ISO 21748 (2010) Guidance for the use of repeatability. M. N. Standards Solution THAA APPENDIX 4 – References ISO 13528 (2005) Statistical Methods for use in Proficiency Testing by Inter-Laboratory Comparisons. 1359-1364. 114. Q. University of Newcastle. Quaternary Geochronology. Page 155 of 157 . A. Available from.. (1998) A New Procedure for determining DL amino acid ratios in fossils using reverse phase liquid chromatography. E. (1987) The Precision of Amino Acid Geochronology and Paleothermometry. Age calibration curves. 6. 127. England. W. T. KAUFMAN. reproducibility and trueness estimates in measurement uncertainty estimation. 2 ed. Representing data distributions with kernal density estimates. Quaternary Science Reviews. RSC ANALYTICAL METHODS COMMITTEE (1995) Uncertainty of measurement: implication of its use in analytical science. Available from. D. Unpublished thesis. H. Robust Statistics: a method of coping with outliers. P.F. (2008) Identifying outliers and assessing the accuracy of amino acid racemization measurements for geochronology: I.org/utils/common/documents/jcgm/JCGM_100_2008_E. Robust Statistics-how not to reject outliers: Part 1. (2005) Amino acid geochronology: a closed system approach to test and refine the UK model. Pearson Education Ltd. Available from. & HUA. RSC ANALYTICAL METHODS COMMITTEE (2001) AMC Technical Briefs No 6. Available from.bipm. (2004) NORDTEST Report TR 537. 1 ed. & MANLEY W. ISO / IEC 17025 (2005) General requirements for the competence of testing and calibration laboratories. K. H. RSC ANALYTICAL METHODS COMMITTEE (1989) AMC Technical Briefs.AAR PT Report. 1693-1697. J. 43-54. The Amazing Horwitz function. 308-327. The Analyst. http://www. D. International Standards Organisation. 987-1000.. Harlow. J. B. S. KOSNIK. 17.org/en/publications/guides/vim. PENKMAN. & THOMPSON. JCGM 100: (2008) Evaluation of measurement data .nordicinnovation. M. C.html KAUFMAN.. Basic concepts..

Quaternary Research. & WOOD. (2000) Aminostratigraphic Dating Methods in Quaternary Geology. J. J. Page 156 of 157 . M. G. I. F. Reference Shelf Series 4. THOMPSON. (Eds. & PIERCE. 22. 109-120. (2010) AAR Interlaboratory comparison of fossil hydrolysates (unpublished). F. 78. F. H.iupac. J. p 317-321. (Eds. WEHMILLER. M. R.http://www. S. Pure and Applied Chemistry. IN FLETCHER. F. J.. WEHMILLER. Washington DC.asp. R. Special Edition No 48. H. J. F. S. M. 145-196. & WEHMILLER. American Geophysical Union. Standards Solution THAA APPENDIX 4 – References http://www..rsc. S. J.) Quaternary Geochronology: methods and Applications. C. Available from. COLMAN.org/publications/pac/2006/pdf/7801x0145. L. K. (2006) The International Harmonized Protocol for the Proficiency Testing of Analytical Chemistry Laboratories. WEHMILLER.) Quaternary Coats of the United States: Marine and Lacustrine Systems. Tusla. J.. WEHMILLER. IN NOLLER.org/Membership/Networking/InterestGroups/Analytical/AMC/Tech nicalBriefs. L.AAR PT Report. (1992) Aminostratigraphy of Southern California Quaternary Marine Terraces. & MILLER. (1984) Interlaboratory Comparison of Amino Acid Enantiomeric Ratios in Fossil Pleistocene Mollusks. ELLISON. SEPM (Society for Sedimentary Geology). SOWERS.pdf.

University of York. Dept of Archaeology. Biology S Block. UK tel. Please direct communications regarding this Report to.uk Page 157 of 157 . YORK YO10 5DD.Contact Details. Wentworth Way. +44(0)1904 328806 email: jp588@york.ac. Jo Powell BioArCh.

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