NIST Micronutrients Measurement Quality Assurance …• A cover letter for the current study, a...

NISTIR 7880-6

NIST Micronutrients Measurement Quality Assurance Program

Summer 2009 Comparability Studies

Results for Round Robin LXVI Fat-Soluble Vitamins and Carotenoids in Human Serum

and Round Robin 31 Ascorbic Acid in Human Serum

David L. Duewer Jeanice B. Thomas

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http://dx.doi.org/10.6028/NIST.IR.7880-6

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NISTIR 7880-6

NIST Micronutrients Measurement Quality Assurance Program

Summer 2009 Comparability Studies

Results for Round Robin LXVI Fat-Soluble Vitamins and Carotenoids in Human Serum

and Round Robin 31 Ascorbic Acid in Human Serum

David L. Duewer Jeanice B. Thomas

Chemical Sciences Division Materials Measurement Laboratory

April, 2013

U.S. Department of Commerce Rebecca Blank, Acting Secretary

National Institute of Standards and Technology Patrick D. Gallagher, Under Secretary of Commerce for Standards and Technology and Director

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http://dx.doi.org/10.6028/NIST.IR.7880-6

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ii

Abstract

The National Institute of Standards and Technology coordinates the Micronutrients Measurement Quality Assurance Program (MMQAP) for laboratories that measure fat- and water-soluble vitamins and carotenoids in human serum and plasma. This report describes the design of and results for the Summer 2009 MMQAP measurement comparability improvement studies: 1) Round Robin LXVI Fat-Soluble Vitamins and Carotenoids in Human Serum and 2) Round Robin 31 Total Ascorbic Acid in Human Serum. The materials for both studies were shipped to participants in June 2009; participants were requested to provide their measurement results by September 28, 2009.

Keywords

Human Serum Retinol, α-Tocopherol, γ-Tocopherol, Total and Trans-β-Carotene

Total Ascorbic Acid

iii

Table of Contents

Abstract............................................................................................................................................... iii Keywords ............................................................................................................................................ iii Table of Contents ............................................................................................................................... iv Introduction......................................................................................................................................... 1 Round Robin LXVI: Fat-Soluble Vitamins and Carotenoids in Human Serum ......................... 1 Round Robin 31: Vitamin C in Human Serum............................................................................... 2 References ............................................................................................................................................ 3

Appendix A. Shipping Package Inserts for RR66........................................................................ A1 Appendix B. Final Report for RR66 ..............................................................................................B1 Appendix C. “All-Lab Report” for RR66 ......................................................................................C1 Appendix D. Representative “Individualized Report” for RR66 ............................................... D1 Appendix E. Shipping Package Inserts for RR31 ......................................................................... E1 Appendix F. Final Report for RR31............................................................................................... F1 Appendix G. “All-Lab Report” for RR31..................................................................................... G1 Appendix H. Representative “Individualized Report” for RR31............................................... H1

iv

Introduction

Beginning in 1988, the National Institute of Standards and Technology (NIST) has coordinated the Micronutrients Measurement Quality Assurance Program (MMQAP) for laboratories that measure fat- and water-soluble vitamins and carotenoids in human serum and plasma. The MMQAP provides participants with measurement comparability assessment through use of interlaboratory studies, Standard Reference Materials (SRMs) and control materials, and methods development and validation. Serum-based samples with assigned values for the target analytes (retinol, alpha-tocopherol, gamma/beta-tocopherol, trans- and total beta-carotene, and total ascorbic acid) and performance-evaluation standards are distributed by NIST to laboratories for analysis.

Participants use the methodology of their choice to determine analyte content in the control and study materials. Participants provide their data to NIST, where it is compiled and evaluated for trueness relative to the NIST value, within-laboratory precision, and concordance within the participant community. NIST provides the participants with a technical summary report concerning their performance for each exercise and suggestions for methods development and refinement. Participants who have concerns regarding their laboratory’s performance are encouraged to consult with the MMQAP coordinators.

All MMQAP interlaboratory studies consist of individual units of batch-prepared samples that are distributed to each participant. For historical reasons these studies are referred to as “Round Robins”. The MMQAP program and the nature of its studies are described elsewhere. [1,2]

Round Robin LXVI: Fat-Soluble Vitamins and Carotenoids in Human Serum

Participants in the MMQAP Fat-Soluble Vitamins and Carotenoids in Human Serum Round Robin LXVI comparability study (hereafter referred to as RR66) received five liquid-frozen human serum test samples for analysis. Unless multiple vials were previously requested, participants received one vial of each serum. These sera were shipped on dry ice to participants in June 2009. The communication materials included in the sample shipment are provided in Appendix A.

Participants are requested to report values for all fat-soluble vitamin-related analytes that are of interest to their organizations. Not all participants report values for the target analytes, and many participants report values for non-target analytes.

The final report delivered to every participant in RR66 consists of three documents:

• A cover letter for the current study, a brief description of the other two documents, and a discussion of our analysis of the overall results that may be of broad interest. This cover letter is reproduced as Appendix B.

• The “All-Lab Report” that lists all of the reported measurement results, a number of consensus statistics for analytes reported by more than one participant, and the mean median and pooled SD from any prior distributions of the serum. This report also provides a numerical “score card” for each participant’s measurement comparability for the more commonly reported analytes. This report is reproduced as Appendix C.

1

• An “Individualized Report” that graphically analyzes each participant’s results for all analytes reported by at least five participants. This report also provides a graphical summary of their measurement comparability. The graphical tools used in this report are described in detail elsewhere [3]. An example “Individualized Report” is reproduced as Appendix D.

Round Robin 31: Vitamin C in Human Serum

Participants in the MMQAP Vitamin C in Human Serum Round Robin 31 comparability study (hereafter referred to as RR31) received four frozen serum test samples, one frozen control serum, and a solid ascorbic acid control material for analysis. Unless multiple vials were previously requested, participants received one vial of each material. These sample materials were shipped on dry ice to participants in June 2009. The communication materials included in the sample shipment are provided in Appendix E.

The test and control serum materials were prepared by adding equal volumes of 10 % metaphosphoric acid (MPA) to human serum that had been spiked with ascorbic acid. While these samples contain some dehydroascorbic acid, its content is variable. Therefore, the participants report only total ascorbic acid (TAA, ascorbic acid plus dehydroascorbic acid). Participants are also encouraged to prepare calibration solutions from the supplied solid control to enable calibrating their serum measurements to the same reference standard.

The final report delivered to every participant in RR31 consists of three documents:

• A cover letter for the current study, a brief description of the other two documents, and a discussion of our analysis of overall results that may be of broad interest. This cover letter is reproduced as Appendix F.

• The “All-Lab Report” that summarizes all of the reported measurement results and provides several consensus statistics. This report is reproduced as Appendix G.

• An “Individualized Report” that graphically analyzes each participant’s results for TAA, including a graphical summary of their measurement comparability. The graphical tools used in this report are described in detail elsewhere [3]. An example “Individualized Report” is reproduced as Appendix H.

2

References

1 Duewer DL, Brown Thomas J, Kline MC, MacCrehan WA, Schaffer R, Sharpless KE, May WE, Crowell JA. NIST/NCI Micronutrients Measurement Quality Assurance Program: Measurement Repeatabilities and Reproducibilities for Fat-Soluble Vitamin-Related Compounds in Human Sera. Anal Chem 1997;69(7):1406-1413.

2 Margolis SA, Duewer DL. Measurement Of Ascorbic Acid in Human Plasma and Serum: Stability, Intralaboratory Repeatability, and Interlaboratory Reproducibility. Clin Chem 1996;42(8):1257-1262.

3 Duewer DL, Kline MC, Sharpless KE, Brown Thomas J, Gary KT, Sowell AL. Micronutrients Measurement Quality Assurance Program: Helping Participants Use Interlaboratory Comparison Exercise Results to Improve Their Long-Term Measurement Performance. Anal Chem 1999;71(9):1870-1878.

3

Appendix A. Shipping Package Inserts for RR66

The following three items were included in each package shipped to an RR66 participant: • Cover letter • Datasheet • Packing List and Shipment Receipt Confirmation Form

The cover letter and datasheet were enclosed in a sealed waterproof bag along with the samples themselves. The packing list was placed at the top of the shipping box, between the cardboard covering and the foam insulation.

A1

•• I

UNmD STATES DEPARTMENT OF COMMERl;E. National Institute of Standards and Technology Gaithersburg, Maryland 20899- '·

June 1, 2009

Dear Colleague:

Enclosed are samples for the second fat-soluble ·vitamins and carotenoids in serum study (Round Robin LXVI) for the 2009 NIST Micronutrients Measurement Quality Assurance Program. The set of samples (Sera 357 - 361) consists of one vial of each of five liquid-frozen serum samples for analysis along with a form for reporting your results. These samples should be stored in the dark at or below -20 °C upon receipt. When reporting your results, please submit one value for each analyte for a given serum sample. If a value obtained is below your limit of quantification, please indicate this result on the form by using NQ (Not Quantified). Results are due to NIST by September 28, 2009. Results received more than two weeks after the due date may not be included in the summary report for this round robin study. The feedback report concerning the study will be distributed in October 2009.

Samples should be allowed to stand a.t room temperature under subdued light until thawed. We recommend that sample mixing be facilitated with 3 to 5 min agitation in an ultrasonic bath or at least 15 min at room temperature with intermittent swirling. (CAUTION: Vigorous shaking will cause foaming and possibly interfere with accurate measurement. The rubber stopper contains phthalate esters that may leach into the sample upon intermittent contact of the liquid sample with the stopper. These esters absorb strongly in the UV region and elute near retinol in most LC systems creating analytical problems.) Water should not be added to the liquid-frozen samples.

For consistency, we request that laboratories use the following absorptivities (dL/g ·cm): retinol, 1843 at 325 nm (ethanol); retinyl palmitate, 975 at 325 nm (ethanol); a.-tocopherol, 75.8 at 292 nm (ethanol); ytocopherol, 91.4 at 298 nm (ethanol); a-carotene, 2800 at 444 nm (hexane); J3-carotene, 2560 at 450 nm (ethanol), 2592 at 452 run (hexane); and lycopene, 3450 at 472 nm (hexane).

' Please report your results for Round Robin LXVI by e-mail to [email protected] or fax to 301-977-0685. If you have questions or comments regarding this study, please call me at (301) 975-3120 or e-mail me [email protected].

ical Chemistry Division ical Science and Technology L.aboratory

Enclosures

NISI A2

Participant #: ________ Date: _____________

Round Robin LXVI: Human Sera NIST Micronutrients Measurement Quality Assurance Program

Analyte 357 358 359 360 361 Units* total retinol

trans-retinol didehydroretinol retinyl palmitate

α-tocopherol γ/β-tocopherol δ-tocopherol

total β-carotene trans-β-carotene

total cis-β-carotene total α-carotene

total lycopene trans-lycopene

total β-cryptoxanthin total α-cryptoxanthin

total lutein total zeaxanthin

total lutein&zeaxanthin total coenzyme Q10

ubiquinol (QH2) ubiquinone (Qox)

phylloquinone (K1) 25-hydroxyvitamin D

Other measurands?

* we prefer µg/mL Were the liquid-frozen samples (357 to 361) frozen when received? Yes | No

Comments:

Mail: M2QAP Please return results by NIST, Stop 8392 28-Sep-2009 Fax: 301-977-0685 Gaithersburg, MD 20899-8392 Email: [email protected]

A3


Fat-Soluble Vitamins Round Robin LXVI NIST Micronutrients Measurement Quality Assurance Program

Packing List and Shipment Receipt Confirmation Form

This box contains: one vial each of the following five FSV M2QAP sera

Serum Form Reconstitute? Vial/Cap #357 Liquid frozen No 2 mL amber, red cap #358 Liquid frozen No 2 mL amber, blue cap #359 Liquid frozen No 2 mL amber, green cap #360 Liquid frozen No 2 mL amber, green cap #361 Liquid frozen No 10 mL amber, silver cap

Please 1) Open the pack immediately 2) Check that it contains all of the above samples 3) Check if the vials are intact 4) Store the sera at -20 ˚C or below until analysis 5) Complete the following information 6) Fax the completed form to us at 301-977-0685

(or email requested information to [email protected])

1) Date this shipment arrived: ___________________________ 2) Are all five sera vials intact? Yes | No

If "No", which one(s) were damaged?

3) Was there any dry-ice left in cooler? Yes | No 4) Did the liquid frozen samples arrive frozen? Yes | No 5) At what temperature are you storing the serum samples? _______ ˚C 6) When do you anticipate analyzing these samples? ________________________

Your prompt return of this information is appreciated.

The M2QAP Gang

Mail: M2QAP NIST, Stop 8392 Fax: 301-977-0685 Gaithersburg, MD 20899-8392 Email: [email protected]

A4

mailto:[email protected]


Appendix B. Final Report for RR66

The following two pages are the final report as provided to all participants: • Cover letter. • An information sheet that:

o describes the contents of the “All-Lab” report, o describes the content of the “Individualized” report, o describes the nature of the test samples and details their previous distributions,

if any, and o summarizes aspects of the study that we believe may be of interest to the

participants.

B1

October 30, 2009

Dear Colleague:

UNnaJ STATES DEPARTMENT OF COMMERCE Nnianal lnsdtute of Standards and Technology Gaithersburg, Maryland 20899-

Enclosed is the summary report of the results for round robin LXVI (RR66) of the 2009 NIST Micronutrients Measurement Quality Assurance Program (M2QAP) for the fat-soluble vitamins and carotenoids in human serum. Included in this report are: I) a summary of data and measurement comparability scores for all laboratories, 2) a detailed graphical analysis of your results; and 3) a graphical summary of your measurement comparability.

Your overall measurement comparability is summarized in the "Score Card" summary, page 6 of the All Lab Report. Combined results rated 1 to 3 are within I to 3 standard deviations of the assigned value, respectively; those rated 4 are >3 standard deviations from the ~signed value. Similar information is presented graphically in the "target plots" that are the last page of your Individualized Report. If you have concerns regarding your laboratory's performance, please contact us for consultation.

We are in the process of finalizing the value-assignment for SRM 968e, Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum. This SRM will consist of three different levels for the core analytes (retinal, ci-tocopherol, and P-carotene). We will notify you when the SRM becomes available for purchase (estimated Spring 2010).

Samples for the first 2010 QA interlaboratory exercise will be shipped during the week of December 7, 2009. If you have any questions regarding this report, please contact Dave Duewer at [email protected] or me [email protected], tel: 301/975-3120, or fax: 301/977-0685.

Sincerely, /

Jeanice B. Thomas, M.B.A Research Chemist Analytical Chemistry Division Chemical Science and Technology Laboratory

Cc: L.C. Sander

David L. Duewer, Ph.D Research Chemometrician Analytical Chemistry Division Chemical Science and Technology Laboratory

NISI B2

The NIST M2QAP Round Robin LXVI (RR66) report consists of:

Page

1-4 A listing of all results and statistics for all analytes. 5 A legend for the list of results and statistics. 6 The text Comparability Summary (“Score Card”) of measurement performance.

Page “Individualized” Report

1 2 to

n n+1

Samples.

Serum

357

358

359

360

361

Results

“All Lab” Report

Your values, the number of labs reporting values, and our assigned values. “Four Plot” summaries of your current and past measurement performance, one page for each analyte you report that is also reported by at least 8 other participants. The graphical Comparability Summary (target plot) of measurement performance.

Five samples were distributed in RR66.

Description

Fresh-frozen, native, multi-donor, prepared in 2009. This is Level I of candidate SRM 968e.

Fresh-frozen, native, multi-donor, prepared in 2009. This is Level II of candidate SRM 968e.

Fresh-frozen, native, multi-donor, prepared in 2009. This is Level III of candidate SRM 968e.

Fresh-frozen, native, multi-donor, prepared in 2008

Fresh-frozen, native, multi-donor serum prepared in Fall, 2007 (SRM 968d)

Prior Distributions

#356:RR65-3/09

#341 & #344:RR63-3/08, #351:RR64-9/08

1) Sera Stability. There was no significant change in the median level or measurement variability of any measurand in either of the two previously distributed materials. However, measurement variability for some analytes appears to have increased in the SRM 968d material. We will closely monitor the stability of this material; we hope to replace it with in the very near future.

2) Candidate SRM 968e. Sera #357 to #359 are the components of candidate SRM 968e. All three of these materials were prepared by blending commercially available materials without spiking. The materials were designed to represent relatively low, middling, and relatively high levels of retinol, αTocopherol and β-carotene. We anticipate this material being available for purchase early-2010.

3) Environmental Stability. The initial set of samples for Lab 110 arrived thawed. After a replacement set was successfully delivered, both sets (listed as 110 and 110.1) were analyzed under reproducibility conditions. The is appreciable difference in the reported values for any analyte in any of the materials.

B3

Appendix C. “All-Lab Report” for RR66

The following six pages are the “All-Lab Report” as provided to all participants, with two exceptions:

• the participant identifiers (Lab) have been altered. • the order in which the participant results are listed has been altered.

The data summary in the “All-Lab Report” has been altered to ensure confidentiality of identification codes assigned to laboratories. The only attributed results are those reported by NIST. The NIST results are not used in the assessment of the consensus summary results of the study.

C1

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1.05

1.

53

1.66

0.

82

1.83

1.

48

0.24

2.

39

1.43

2.

04

1.58

2.

54

2.69

1.

67

1.82

1.

43

2.27

2.

41

1.47

1.77

1.

44

2.49

2.

41

1.30

1.84

1.

44

2.34

2.

50

1.46

2.

29

1.75

2.

70

2.91

1.

66

1.79

1.

43

2.28

2.

48

1.50

1.37

1.

01

1.62

1.

83

1.08

1.

36

1.08

1.

64

1.78

1.

06

1.60

1.

30

2.00

2.

10

1.30

1.70

1.

37

2.30

2.

23

1.23

1.

67

1.17

2.

00

2.26

1.

26

1.89

1.

39

1.95

2.

37

1.39

1.

69

1.27

2.

08

2.31

1.

25

1.61

1.

20

1.97

2.

11

1.30

1.50

1.

24

1.83

2.

13

1.29

1.

72

1.34

2.

18

2.26

1.

31

2.01

1.

55

2.50

2.

62

1.50

1.

75

1.30

2.

13

2.22

1.

29

N

33

33

33

33

33

5 5

5 5

5 11

9

12

11

9 34

34

34

34

33

21

21

21

21

21

M

in

0.29

3 0.

428

0.49

7 0.

600

0.27

5 0.

274

0.38

0 0.

455

0.42

1 0.

243

0.00

2 0.

014

0.05

7 0.

032

0.00

0 5.

47

8.76

13

.35

8.70

2.

41

1.13

0.

98

0.24

1.

59

0.82

M

edia

n 0.

351

0.49

7 0.

654

0.71

9 0.

327

0.33

0 0.

471

0.54

9 0.

675

0.28

9 0.

008

0.02

1 0.

090

0.04

3 0.

007

6.75

10

.30

18.4

8 10

.44

5.67

1.

72

1.34

2.

08

2.26

1.

30

Max

0.

493

0.66

0 0.

840

0.99

3 0.

700

0.65

5 0.

554

0.63

8 0.

704

1.07

5 0.

019

0.06

3 0.

219

0.05

3 0.

029

8.52

12

.96

23.9

6 13

.50

7.70

2.

29

1.75

2.

70

2.91

1.

67

SD

0.03

3 0.

049

0.05

2 0.

061

0.04

4 0.

032

0.06

4 0.

102

0.04

3 0.

069

0.00

5 0.

005

0.02

0 0.

004

0.00

9 0.

52

0.86

1.

39

0.85

1.

04

0.18

0 0.

158

0.37

5 0.

225

0.18

8 C

V 9

10

8 8

14

10

14

19

6 24

58

26

23

10

12

5 8

8 8

8 18

10

12

18

10

14

Npa

st

0 0

0 28

30

0

0 0

5 9

0 0

0 9

7 0

0 0

31

32

0 0

0 18

22

M

edia

npas

t 0.

760

0.34

4 0.

708

0.33

2 0.

045

0.01

0 10

.552

5.7

59

2.45

5 1.

437

SDpa

st

0.06

6 0.

025

0.13

0 0.

043

0.01

2 0.

006

0.81

2 0.

764

0.28

5 0.

144

NIS

T 0.

346

0.49

0 0.

672

0.70

2 0.

313

7.01

10

.25

19.5

6 10

.98

5.94

2.

01

1.50

2.

33

2.35

1.

40

NAV

0.

349

0.49

5 0.

662

0.71

0 0.

321

0.33

0 0.

471

0.54

9 0.

675

0.28

9 0.

008

0.02

1 0.

090

0.04

3 0.

007

6.90

4 10

.326

19.

057

10.7

16 5

.811

1.

865

1.41

9 2.

204

2.30

7 1.

351

NAU

0.

034

0.04

5 0.

056

0.06

7 0.

046

0.03

2 0.

064

0.10

2 0.

053

0.06

9 0.

010

0.01

1 0.

024

0.01

5 0.

010

0.60

8 0.

815

1.61

2 0.

895

1.11

5 0.

275

0.19

7 0.

414

0.23

9 0.

202

All L

ab R

epor

t Pa

ge 1

/ 6

Rou

nd R

obin

LXV

I Lab

orat

ory

Res

ults

δ-To

coph

erol

, μg/

mL

Tota

l β-C

arot

ene,

μg/

mL

trans

-β-C

arot

ene,

μg/

mL

Tota

l cis

-β-C

arot

ene,

μg/

mL

Tota

l α-C

arot

ene,

μg/

mL

C3

Lab

357

358

359

360

361

357

358

359

360

361

357

358

359

360

361

357

358

359

360

361

357

358

359

360

361

110.

2???

0.

094

0.26

5 0.

369

0.29

9 0.

071

0.00

5 0.

007

0.00

6 0.

007

0.00

4 11

0.3?

??

0.09

2 0.

277

0.37

3 0.

301

0.06

5 0.

006

0.00

8 0.

006

0.00

7 0.

004

119.

1???

nd

0.

001

0.00

3 0.

002

0.00

1 0.

090

0.25

5 0.

389

0.30

3 0.

083

0.09

0 0.

254

0.38

6 0.

301

0.08

2 0.

008

0.02

8 0.

020

0.03

8 0.

010

FSV-

BA 0

.097

0.0

67 0

.202

0.2

67 0

.073

0.

090

0.24

4 0.

415

0.29

5 0.

092

0.08

5 0.

231

0.39

9 0.

280

0.08

5 0.

005

0.01

3 0.

016

0.01

5 0.

007

0.00

9 0.

033

0.01

4 0.

054

0.01

1 FS

V-BB

0.0

84 0

.076

0.2

05 0

.255

0.0

85

0.08

6 0.

225

0.34

6 0.

265

0.07

9 0.

082

0.21

3 0.

330

0.24

9 0.

074

0.00

4 0.

012

0.01

6 0.

016

0.00

4 0.

005

0.02

8 0.

012

0.04

3 0.

009

FSV-

BC

FSV-

BD

FSV-

BE

0.07

8 0.

234

0.44

1 0.

273

0.06

5

FSV-

BF

0.06

9 0.

208

0.35

0 0.

252

0.05

3

FSV-

BG

0.09

4 0.

264

0.46

2 0.

311

0.08

2 0.

009

0.03

2 0.

020

0.04

8 0.

009

FSV-

BH

nq

nq

nq

0.03

4 nq

FS

V-BJ

0.

079

0.23

6 0.

387

0.24

1 0.

059

0.07

9 0.

224

0.37

1 0.

241

0.05

9 nq

0.

012

0.01

6 nq

nq

0.

088

0.26

9 0.

502

0.31

0 0.

092

nq

0.03

0 nq

0.

049

nq

FSV-

BK

FSV-

BL

FSV-

BM

FSV-

BN

0.00

5 0.

010

0.02

0 0.

015

0.00

8 0.

088

0.21

3 0.

364

0.25

8 0.

074

0.08

3 0.

203

0.34

4 0.

244

0.06

5 0.

012

0.03

1 0.

016

0.05

3 0.

011

FSV-

BNa

0.00

7 0.

016

0.01

8 0.

015

0.00

7 0.

092

0.23

5 0.

366

0.25

7 0.

070

0.08

5 0.

219

0.34

8 0.

242

0.06

3 0.

014

0.03

7 0.

017

0.05

3 0.

014

FSV-

BO

0.08

9 0.

219

0.30

7 0.

271

0.08

4 0.

003

0.02

2 0.

005

0.04

5 0.

008

FSV-

BP

0.06

3 0.

261

0.40

7 0.

287

0.05

6 nd

0.

028

0.01

8 0.

043

0.00

6 FS

V-BQ

FS

V-BR

FS

V-BS

≥0

.029

≥0

.145

≥0

.330

≥0

.234

≥0

.012

0.

029

0.14

5 0.

330

0.23

4 0.

012

0.00

6 0.

031

0.02

4 0.

060

0.01

2 FS

V-BT

0.0

66 0

.046

0.1

02 0

.106

0.0

33

0.09

1 0.

214

0.29

9 0.

257

0.07

9 0.

089

0.20

6 0.

291

0.25

2 0.

077

0.00

4 0.

013

0.01

5 0.

012

0.00

4 0.

008

0.02

8 0.

017

0.03

9 0.

010

FSV-

BU

0.10

2 0.

289

0.47

2 0.

310

0.10

0 0.

006

0.03

1 0.

006

0.04

4 0.

017

FSV-

BV

0.11

1 0.

292

0.44

4 0.

352

0.09

0 0.

010

0.04

3 0.

018

0.07

0 0.

010

FSV-

BW

0.09

9 0.

266

0.49

3 0.

314

0.06

4 0.

003

0.01

7 0.

004

0.04

0 nd

FS

V-C

C

FSV-

CD

0.

182

0.24

5 0.

321

0.25

0 0.

167

0.02

9 0.

037

0.02

7 0.

048

0.02

5 FS

V-C

E 0.

168

0.34

4 0.

585

0.48

6 0.

153

FSV-

CF

FSV-

CG

nq

nq

0.

179

0.23

8 0.

127

0.07

1 0.

205

0.28

3 0.

226

0.06

6 0.

067

0.19

2 0.

269

0.21

2 0.

061

nq

0.01

3 0.

014

0.01

4 nq

0.

009

0.03

5 0.

013

0.05

2 0.

011

FSV-

CI

0.07

5 0.

206

0.40

2 0.

229

0.05

5 0.

013

0.04

4 0.

043

0.05

2 0.

013

FSV-

CW

0.1

72 0

.100

0.2

62 0

.312

0.0

96

0.10

0 0.

239

0.38

6 0.

303

0.05

1 0.

005

0.02

7 0.

011

0.04

9 0.

002

FSV-

CZ

0.08

9 0.

199

0.27

7 0.

229

0.07

7

FSV-

DD

FS

V-D

V

FSV-

EE

N

4 4

5 5

5 24

24

24

24

24

9

9 9

9 9

5 8

8 7

6 18

20

19

21

18

M

in 0

.066

0.0

46 0

.102

0.1

06 0

.033

0.

063

0.19

9 0.

277

0.22

6 0.

051

0.02

9 0.

145

0.26

9 0.

212

0.01

2 0.

004

0.00

1 0.

003

0.00

2 0.

001

0.00

3 0.

007

0.00

4 0.

007

0.00

2 M

edia

n 0.

091

0.07

2 0.

202

0.25

5 0.

085

0.09

0 0.

241

0.38

6 0.

280

0.07

5 0.

083

0.21

3 0.

344

0.24

4 0.

065

0.00

5 0.

013

0.01

6 0.

015

0.00

6 0.

008

0.03

0 0.

016

0.04

8 0.

010

Max

0.1

72 0

.100

0.2

62 0

.312

0.1

27

0.18

2 0.

344

0.58

5 0.

486

0.16

7 0.

090

0.25

4 0.

399

0.30

1 0.

085

0.00

7 0.

016

0.02

0 0.

016

0.00

8 0.

029

0.04

4 0.

043

0.07

0 0.

025

SD 0

.023

0.0

22 0

.034

0.0

25 0

.018

0.

010

0.03

6 0.

070

0.03

8 0.

016

0.00

6 0.

016

0.03

9 0.

012

0.01

3 0.

001

0.00

1 0.

002

0.00

1 0.

003

0.00

4 0.

004

0.00

6 0.

007

0.00

3 C

V 25

31

17

10

21

11

15

18

14

21

7

8 11

5

19

26

6 12

6

46

51

14

39

15

33

Npa

st

0 0

0 4

5 0

0 0

20

22

0 0

0 7

9 0

0 0

5 5

0 0

0 17

15

M

edia

npas

t 0.

267

0.08

4 0.

268

0.07

6 0.

248

0.07

5 0.

013

0.00

5 0.

044

0.00

9 SD

past

0.

027

0.02

3 0.

020

0.01

3 0.

010

0.00

8 0.

003

0.00

2 0.

007

0.00

3

NIS

T

NAV

N

AU nq

nq

nq

0.

271

nq

0.11

5 0.

248

0.44

0 0.

266

0.07

4 0.

094

0.19

6 0.

380

nq

nq

0.02

1 0.

052

0.06

0 nd

nd

nq

0.

034

nq

0.04

1 0.

007

0.09

1 0.

072

0.20

2 0.

263

0.08

5 0.

036

0.04

4 0.

022

0.10

3 0.

243

0.41

3 0.

269

0.07

5 0.

024

0.03

9 0.

090

0.04

5 0.

018

0.08

8 0.

204

0.36

2 0.

244

0.06

5 0.

014

0.02

7 0.

047

0.02

7 0.

013

0.01

3 0.

032

0.03

8 0.

015

0.00

6 0.

012

0.03

3 0.

034

0.00

5 0.

003

0.00

8 0.

032

0.01

5 0.

044

0.00

8 0.

004

0.01

0 0.

008

0.01

6 0.

005

All L

ab R

epor

t Pa

ge 2

/ 6

Rou

nd R

obin

LXV

I Lab

orat

ory

Res

ults

Tota

l Lyc

open

e, μ

g/m

L tra

ns-L

ycop

ene,

μg/

mL

Tota

l β-C

rypt

oxan

thin

, μg/

mL

Tota

l α-C

rypt

oxan

thin

, μg/

mL

Tota

l Lut

ein,

μg/

mL

Lab

357

358

359

360

361

C4

110.

2???

0.2

19 0

.573

0.8

15 0

.312

0.1

92

110.

3???

0.2

04 0

.594

0.8

33 0

.338

0.1

63

119.

1???

0.2

43 0

.691

1.0

04 0

.437

0.3

04

FSV-

BA 0

.233

0.6

31 0

.966

0.4

05 0

.329

FS

V-BB

0.2

48 0

.640

0.9

36 0

.398

0.3

08

FSV-

BC

FSV-

BD

FSV-

BE

FSV-

BF

FSV-

BG 0

.242

0.6

65 1

.038

0.4

18 0

.285

FS

V-BH

0.2

36 0

.642

0.9

65 0

.375

0.2

54

FSV-

BJ 0

.234

0.5

94 1

.215

0.3

87 0

.270

FS

V-BK

FS

V-BL

FS

V-BM

FS

V-BN

0.2

55 0

.536

0.8

08 0

.355

0.2

05

FSV-

BNa

0.21

0 0.

558

0.81

8 0.

328

0.16

6 FS

V-BO

0.1

95 0

.471

0.6

32 0

.314

0.2

53

FSV-

BP 0

.268

0.9

33 1

.522

0.5

46 0

.182

FS

V-BQ

FS

V-BR

FS

V-BS

0.1

69 0

.490

1.0

55 0

.366

0.1

27

FSV-

BT 0

.241

0.5

75 0

.775

0.3

79 0

.305

FS

V-BU

0.2

50 0

.598

1.0

73 0

.380

0.3

08

FSV-

BV 0

.308

0.8

03 1

.113

0.5

15 0

.342

FS

V-BW

0.2

41 0

.682

1.1

40 0

.432

0.2

08

FSV-

CC

FS

V-C

D 0

.148

0.2

19 0

.303

0.1

78 0

.148

FS

V-C

E FS

V-C

F FS

V-C

G 0

.192

0.5

41 0

.696

0.3

26 0

.245

FS

V-C

I FS

V-C

W

FSV-

CZ

FSV-

DD

FS

V-D

V FS

V-EE

357

358

359

360

361

0.10

1 0.

283

0.42

4 0.

177

0.12

0 0.

127

0.33

9 0.

457

0.22

5 0.

156

0.10

1 0.

255

0.33

7 0.

162

0.11

0

0.14

1 0.

383

0.53

3 0.

248

0.15

1

0.11

3 0.

281

0.37

4 0.

175

0.10

1 0.

115

0.29

4 0.

383

0.17

3 0.

085

0.12

1 0.

279

0.30

4 0.

189

0.12

0

0.10

4 0.

296

0.33

7 0.

180

0.11

6

0.12

7 0.

287

0.36

1 0.

190

0.08

9

357

358

359

360

361

0.05

8 0.

080

0.02

6 0.

058

0.03

3 0.

048

0.05

3 0.

027

0.05

8 0.

031

0.03

2 0.

033

0.02

3 0.

038

0.02

8 0.

043

0.05

1 0.

031

0.05

8 0.

041

0.04

7 0.

050

0.03

4 0.

056

0.04

0

0.05

0 0.

051

0.03

2 0.

060

0.04

1 0.

059

0.07

1 0.

047

0.07

4 0.

045

0.03

6 0.

045

0.03

4 0.

056

0.03

4

0.04

7 0.

046

0.02

4 0.

055

0.03

3 0.

047

0.04

8 0.

025

0.05

4 0.

030

0.09

1 0.

050

0.02

8 0.

062

0.04

2 0.

044

0.06

4 0.

043

0.06

8 0.

036

0.01

5 0.

018

0.01

2 0.

032

0.02

9 0.

042

0.04

1 0.

028

0.05

1 0.

032

0.04

7 0.

050

0.02

9 0.

049

0.03

5 0.

049

0.04

8 0.

023

0.06

1 0.

037

0.04

1 0.

036

0.01

7 0.

075

0.02

9

0.04

1 0.

047

0.03

0 0.

045

0.03

5

0.05

6 0.

068

0.04

8 0.

072

0.05

2

0.05

2 0.

049

0.03

1 0.

060

0.04

8

357

358

359

360

361

0.01

5 0.

017

0.01

3 0.

017

0.01

6 0.

017

0.02

0 0.

014

0.01

7 0.

015

0.02

0 0.

027

0.03

1 0.

023

0.01

6

0.01

2 0.

022

0.01

6 <0

.01

<0.0

1

357

358

359

360

361

0.06

0 0.

059

0.06

7 0.

053

0.04

6

0.07

3 0.

102

0.14

0 0.

069

0.06

0

0.07

1 0.

088

0.11

4 0.

055

0.04

4 0.

064

0.08

7 0.

126

0.07

1 0.

048

0.07

3 0.

075

0.09

8 0.

058

0.04

4 0.

062

0.07

8 0.

096

0.05

9 0.

039

0.12

2 0.

127

0.14

3 0.

106

0.09

4

0.06

4 0.

059

0.07

1 0.

059

0.05

6

0.07

8 0.

092

0.11

7 0.

065

0.04

9 0.

108

0.11

4 0.

138

0.08

6 0.

096

N

19

19

19

19

19

9 9

9 9

9 20

20

20

20

20

4

4 4

3 3

10

10

10

10

10

Min

0.1

48 0

.219

0.3

03 0

.178

0.1

27

0.10

1 0.

255

0.30

4 0.

162

0.08

5 0.

015

0.01

8 0.

012

0.03

2 0.

028

0.01

2 0.

017

0.01

3 0.

017

0.01

5 0.

060

0.05

9 0.

067

0.05

3 0.

039

Med

ian

0.23

6 0.

594

0.96

5 0.

379

0.25

3 0.

115

0.28

7 0.

374

0.18

0 0.

116

0.04

7 0.

049

0.02

9 0.

058

0.03

5 0.

016

0.02

1 0.

015

0.01

7 0.

016

0.07

2 0.

088

0.11

5 0.

062

0.04

8 M

ax 0

.308

0.9

33 1

.522

0.5

46 0

.342

0.

141

0.38

3 0.

533

0.24

8 0.

156

0.09

1 0.

080

0.04

8 0.

075

0.05

2 0.

020

0.02

7 0.

031

0.02

3 0.

016

0.12

2 0.

127

0.14

3 0.

106

0.09

6 SD

0.0

25 0

.079

0.2

19 0

.061

0.0

81

0.01

7 0.

012

0.05

5 0.

013

0.02

2 0.

007

0.00

5 0.

006

0.00

6 0.

008

0.00

4 0.

004

0.00

2 0.

012

0.02

0 0.

031

0.01

0 0.

009

CV

11

13

23

16

32

15

4 15

7

19

16

10

21

11

22

23

18

15

16

23

27

17

19

Npa

st

0 0

0 15

17

0

0 0

7 8

0 0

0 16

18

0

0 0

5 7

0 0

0 8

12

Med

ianp

ast

0.37

2 0.

272

0.17

6 0.

116

0.05

9 0.

039

0.02

0 0.

015

0.05

5 0.

058

SDpa

st

0.03

3 0.

052

0.02

7 0.

018

0.01

0 0.

006

0.00

9 0.

002

0.01

0 0.

014

NIS

T 0.

175

0.38

9 0.

733

nq

0.26

2 0.

157

0.31

9 0.

603

0.18

7 nd

0.

042

0.03

8 0.

017

0.05

6 0.

026

0.06

7 0.

102

0.13

2 0.

056

0.04

7

NAV

0.2

05 0

.492

0.8

42 0

.377

0.2

58

0.13

6 0.

303

0.48

8 0.

184

0.11

6 0.

044

0.04

3 0.

023

0.05

7 0.

030

0.01

6 0.

021

0.01

5 0.

017

0.01

6 0.

070

0.09

5 0.

123

0.05

9 0.

048

NAU

0.0

70 0

.187

0.2

51 0

.082

0.0

79

0.03

6 0.

057

0.17

6 0.

032

0.02

2 0.

012

0.01

4 0.

011

0.01

4 0.

011

0.01

5 0.

023

0.03

3 0.

013

0.01

0

All L

ab R

epor

t Pa

ge 3

/ 6

Rou

nd R

obin

LXV

I Lab

orat

ory

Res

ults

Tota

l Zea

xant

hin,

μg/

mL

Tota

l Lut

ein&

Zeax

anth

in, μ

g/m

L C

oenz

yme

Q10

, μg/

mL

Phyl

loqu

inon

e (K

1), n

g/m

L 25

-hyd

roxy

vita

min

D, μ

g/m

L

C5

Lab

357

358

359

360

361

357

358

359

360

361

357

358

359

360

361

357

358

359

360

361

357

358

359

360

361

110.

2???

0.

154

0.14

8 0.

159

0.11

3 0.

083

110.

3???

0.

127

0.15

6 0.

164

0.11

4 0.

076

119.

1???

0.

032

0.03

4 0.

031

0.02

3 0.

035

0.11

9 0.

112

0.12

4 0.

104

0.08

5

FSV-

BA

0.10

5 0.

129

0.15

7 0.

102

0.08

6 0.

013

0.02

0 0.

015

0.01

3 0.

007

FSV-

BB

0.04

6 0.

059

0.03

9 0.

031

0.03

7 0.

110

0.14

8 0.

199

0.10

8 0.

091

FS

V-BC

FS

V-BD

FS

V-BE

0.

945

1.44

0 0.

806

0.65

6 0.

862

0.53

8 2.

971

1.17

4 0.

267

0.

454

FSV-

BF

FSV-

BG

0.10

5 0.

131

0.17

4 0.

097

0.07

9

FSV-

BH

0.02

8 0.

026

0.02

5 0.

020

0.03

9 0.

110

0.11

7 0.

141

0.08

0 0.

044

nq

0.01

7 0.

022

0.01

3 0.

012

FSV-

BJ

FSV-

BK

FSV-

BL

FSV-

BM

FSV-

BN

0.04

5 0.

044

0.03

9 0.

031

0.05

2 0.

112

0.10

9 0.

132

0.08

6 0.

065

FSV-

BNa

0.04

5 0.

042

0.04

0 0.

029

0.04

1 0.

093

0.11

1 0.

128

0.08

7 0.

058

FS

V-BO

0.

009

0.01

3 0.

007

0.00

4 0.

006

0.12

8 0.

136

0.15

6 0.

113

0.09

8

FSV-

BP

0.06

8 0.

139

0.10

5 0.

081

0.06

0

FSV-

BQ

FSV-

BR

FSV-

BS

0.13

3 0.

162

0.21

6 0.

164

0.14

7

FSV-

BT

0.02

7 0.

030

0.02

5 0.

022

0.02

9 0.

104

0.09

7 0.

133

0.09

5 0.

089

1.02

3 1.

199

1.12

4 0.

654

0.

979

FSV-

BU

0.11

6 0.

134

0.12

8 0.

095

0.07

4

FSV-

BV

0.10

4 0.

107

0.11

8 0.

088

0.06

9 FS

V-BW

0.

105

0.12

2 0.

142

0.09

4 0.

076

FS

V-C

C

FSV-

CD

0.

185

0.21

1 0.

236

0.15

7 0.

122

FS

V-C

E

FSV-

CF

FS

V-C

G

0.10

8 0.

122

0.12

0 0.

098

0.09

5

FSV-

CI

0.10

3 0.

114

0.13

9 0.

085

0.06

7 0.

022

0.02

2 0.

020

0.01

9 0.

025

0.44

0 2.

669

1.11

7 0.

163

0.36

0 FS

V-C

W

0.05

3 0.

048

0.05

2 0.

058

0.07

5 0.

183

0.16

7 0.

187

0.13

8 0.

153

1.14

1 1.

605

0.95

7 0.

663

0.

953

FSV-

CZ

0.

990

1.55

4 0.

943

0.70

1

0.87

1 FS

V-D

D

FSV-

DV

FS

V-EE

0.

663

1.33

6 0.

872

0.68

2 0.

887

N

9 9

9 9

9 20

20

20

20

20

5

5 5

5 5

2 2

2 2

2 1

2 2

2 2

Min

0.0

06 0

.009

0.0

13 0

.007

0.0

04

0.06

8 0.

097

0.10

5 0.

080

0.04

4 0.

862

0.66

3 1.

199

0.80

6 0.

654

0.36

0 0.

440

2.66

9 1.

117

0.16

3 0.

013

0.02

0 0.

013

0.01

2 M

edia

n 0.

037

0.03

2 0.

034

0.03

1 0.

023

0.11

0 0.

130

0.14

2 0.

098

0.08

1 0.

887

0.99

0 1.

440

0.94

3 0.

663

0.40

7 0.

489

2.82

0 1.

146

0.21

5 0.

007

0.01

5 0.

021

0.01

4 0.

013

Max

0.0

75 0

.053

0.0

59 0

.052

0.0

58

0.18

5 0.

211

0.23

6 0.

164

0.15

3 0.

979

1.14

1 1.

605

1.12

4 0.

701

0.45

4 0.

538

2.97

1 1.

174

0.26

7 0.

017

0.02

2 0.

015

0.01

3 SD

0.0

12 0

.019

0.0

15 0

.012

0.0

09

0.01

0 0.

027

0.02

6 0.

016

0.01

9 0.

037

0.06

7 0.

169

0.10

6 0.

013

CV

32

59

45

38

38

9 21

18

16

24

4

7 12

11

2

Npa

st

0 0

0 7

10

0 0

0 16

19

0

0 0

7 9

0 0

0 0

0 0

0 0

0 0

Med

ianp

ast

0.03

0 0.

024

0.09

7 0.

086

0.90

0 0.

618

SDpa

st

0.01

5 0.

009

0.02

4 0.

018

0.18

4 0.

147

NIS

T

NAV

N

AU 0.

027

0.03

0 0.

025

0.02

8 0.

023

0.09

5 0.

132

0.15

7 0.

084

0.07

0

0.03

2 0.

031

0.03

0 0.

030

0.02

3 0.

102

0.13

0 0.

149

0.09

1 0.

077

0.88

7 0.

990

1.44

0 0.

943

0.66

3 0.

014

0.01

9 0.

017

0.01

2 0.

009

0.02

5 0.

027

0.03

1 0.

023

0.02

0 0.

089

0.09

9 0.

169

0.10

6 0.

066

All L

ab R

epor

t Pa

ge 4

/ 6

Round Robin LXVI Laboratory Results

Term Legend N Number of (non-NIST) quantitative values reported for this analyte

Min Minimum (non-NIST) quantitative value reported Median Median (non-NIST) quantitative value reported

Max Maximum (non-NIST) quantitative value reported SD Standard deviation for (non-NIST) results: 0.741*(3rd Quartile - 1st Quartile) CV Coefficient of Variation for (non-NIST) results: 100*SD/Median

Npast Mean of N(s) from past RR(s) Medianpast Mean of Median(s) from past RR(s)

SDpast Pooled SD from past RR(s)

MeanNIST Mean of NIST results

Srep NIST's within-vial pooled standard deviation Shet NIST's among-vial pooled standard deviation

SNIST Combined standard deviation for NIST analyses: √(Srep 2 + Shet

2)

NAV NIST Assigned Value = (Median + MeanNIST)/2 for analytes reported by NIST analyst(s) = Median for analytes reported by ≥ 5 labs but not NIST

NAU NIST Assigned Uncertainty: √(S2 + Sbtw 2)

S is the maximum of (0.05*NAV, SD, SNIST, eSD) and Sbtw is the standard deviation between Median and MeanNIST. The expected long-term SD, eSD, is defined in: Duewer et al., Anal Chem 1997;69(7):1406-1413.

nd Not detected (i.e., no detectable peak for analyte) nq Detected but not quantitatively determined ≥x Concentration greater than or equal to x

italics Not explicitly reported but calculated by NIST from reported values

C6All Lab Report Page 5 / 6

Lab 110.2??? 110.3??? 119.1???

FSV-BA FSV-BB FSV-BC FSV-BD FSV-BE FSV-BF FSV-BG FSV-BH FSV-BJ FSV-BK FSV-BL FSV-BM FSV-BN

FSV-BNa FSV-BO FSV-BP FSV-BQ FSV-BR FSV-BS FSV-BT FSV-BU FSV-BV

FSV-BW FSV-CC FSV-CD FSV-CE FSV-CF FSV-CG FSV-CI

FSV-CW FSV-CZ FSV-DD FSV-DV FSV-EE

NIST n

% 1 % 2 % 3 % 4

Round Robin LXVI Laboratory Results

Comparability Summary

TR aT g/bT bC tbC aC TLy TbX TLu TZ L&Z 2 1 3 1 2 1 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 1 1 2 2 1 1 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 2 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 1 1 2 2 2 1 1 1 1 1 1 1 1 2 2 2 1 1 1 1 1 1 1 1 2 1 1 1 1 1 2 4 2 1 1 2 1 1 2 1 2 4 3 1 1 4 3 3 1 1 2 3 2 1 1 1 1 1 1 1 1 1 1 1 2 1 2 2 1 1 1 2 2 1 2 1 2 1 1 1 1 1 1 2 1 1 1 1 2 1 2 1 4 3 3 1 3 2 3 4 1 1 2 3 1 1 2 1 1 2 1 1 1 1 1 2 1 1 1 4 2 2 1 1 1 3 3 3 2 1 1 1 1 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 38 35 22 26 10 21 20 21 11 10 21

TR aT g/bT bC tbC aC TLy TbX TLu TZ L&Z 53 57 64 81 60 71 85 71 73 80 81 37 34 18 8 30 24 10 29 9 10 5 3 9 18 4 10 5 5 0 9 10 14 8 0 0 8 0 0 0 0 9 0 0

Label Lab TR aT

g/bTbC

tbC aC

TLy TbX TLu TZ

L&Z

n % 1 % 2 % 3 % 4

Definition Participant code Total Retinol α-Tocopherol γ/β-Tocopherol Total β-Carotene trans-β-Carotene Total α-Carotene Total Lycopene

Total β-Cryptoxanthin Total Lutein Total Zeaxanthin Total Lutein & Zeaxanthin

number of participants providing quantitative data Percent of CS = 1 (within 1 SD of medians) Percent of CS = 2 (within 2 SD of medians) Percent of CS = 3 (within 3 SD of medians) Percent of CS = 4 (3 or more SD from medians)

“Comparability Score”

The Comparability Score (CS) of summarizes your measurement performance for a given measurand, relative to the consensus medians. CS is the average distance, in standard deviation units, that your measurement performance characteristics are from the consensus performance. CS is calculated when the number of quantitative values you reported for a measurand, Nyou, is at least two and the measurand has been reported by 10 or more participants.

CS = MIN(4, INT(1 + C2 + AP 2 ))

yo uN You − Median C = Concordanc e = i i Ny ou ∑ NAU i i

yo u N You i − Median i 2

AP = Apparent Precision = ∑ (Ny ou − 1) NAU i i

NAU = NIST Assigned Uncertainty, our estimate of the overall measurement standard deviation for each sample. The estimate includes serum heterogeneity, analytical repeatability, and among-participant reproducibility variance components.

For further details, please see: Duewer DL, Kline MC, Sharpless KE, Brown Thomas J, Gary KT. Micronutrients Measurement Quality Assurance Program: Helping participants use interlaboratory comparison exercise results to improve their long-term measurement performance. Anal Chem 1999;71(9):1870-8.

C7All Lab Report Page 6 / 6

Appendix D. Representative “Individualized Report” for RR66

Each participant in RR66 received an “Individualized Report” reflecting their reported results. Each report included a detailed analysis for analytes that were assayed by at least five participants. The following analytes met this criterion in RR66:

• Total Retinol • trans-Retinol • Retinyl Palmitate • α-Tocopherol • γ/β-Tocopherol • Total β-Carotene • trans-β-Carotene • Total cis-β-Carotene • Total α-Carotene • Total Lycopene • trans-Lycopene • Total β-Cryptoxanthin • Total Lutein • Total Zeaxanthin • Total Lutein & Zeaxanthin • Coenzyme Q10

The following fourteen pages are the “Individualized Report” for the analytes evaluated by participant FSV-BA.

D1

Set

1 o

f 41

Indi

vidu

aliz

ed R

ound

Rob

in L

XVI R

epor

t: FS

V-BA

Su

mm

ary

Seru

m 3

57

Seru

m 3

58

Seru

m 3

59

Seru

m 3

60

Seru

m 3

61

D2

Anal

yte

You

NAV

n

You

NAV

n

You

NAV

n

You

NAV

n

You

NAV

n

Tota

l Ret

inol

0.3

66 0

.349

33

0.52

3 0.

495

33

0.67

5 0.

662

33

0.77

0 0.

710

33

0.35

7 0.

321

33

Ret

inyl

Pal

mita

te 0

.02

0.01

11

0.

1 0.

0 9

0.2

0.1

12

0.04

0.

04

11

0.03

0.

01

9 α-To

copherol 6

.62

6.90

34

9.

99

10.3

3 34

18

.18

19.0

6 34

10

.16

10.7

2 34

5.

88

5.81

33

γ/β-To

copherol 2

.041

1.8

65 2

1 1.

581

1.41

9 21

2.

540

2.20

4 21

2.

687

2.30

7 21

1.

666

1.35

1 21

δ-To

copherol 0

.097

0.0

91

4 0.

067

0.07

2 4

0.20

2 0.

202

5 0.

267

0.26

3 5

0.07

3 0.

085

5 To

tal β-Carotene

0.09

0 0.

103

24

0.24

4 0.

243

24

0.41

5 0.

413

24

0.29

5 0.

269

24

0.09

2 0.

075

24

trans-β-Carotene

0.08

5 0.

088

9 0.

231

0.20

4 9

0.39

9 0.

362

9 0.

280

0.24

4 9

0.08

5 0.

065

9 To

tal cis-β-Carotene

0.00

5 0.

013

5 0.

013

0.03

2 8

0.01

6 0.

038

8 0.

015

0.01

5 7

0.00

7 0.

006

6 To

tal α-Carotene

0.00

9 0.

008

18

0.03

3 0.

032

20

0.01

4 0.

015

19

0.05

4 0.

044

21

0.01

1 0.

008

18

Tota

l Lyc

open

e 0.

233

0.20

5 19

0.

631

0.49

2 19

0.

966

0.84

2 19

0.

405

0.37

7 19

0.

329

0.25

8 19

tra

ns-L

ycop

ene

0.12

7 0.

136

9 0.

339

0.30

3 9

0.45

7 0.

488

9 0.

225

0.18

4 9

0.15

6 0.

116

9 To

tal β-Cryptoxanthin 0

.043

0.0

44 2

0 0.

051

0.04

3 20

0.

031

0.02

3 20

0.

058

0.05

7 20

0.

041

0.03

0 20

To

tal α-Cryptoxanthin 0

.015

0.0

16

4 0.

017

0.02

1 4

0.01

3 0.

015

4 0.

017

0.01

7 3

0.01

6 0.

016

3 To

tal L

utei

n&Ze

axan

thin

0.1

05 0

.102

20

0.12

9 0.

130

20

0.15

7 0.

149

20

0.10

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Set 1 of 41

Individualized RR LXVI Report: FSV-BA

Total Retinol, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD

Median [Analyte], µg/mL

Long-term Precision, SD

3rd Quartile (75%) You, this RR You, ≥x, this RR NIST, this RR

Median (50%) You, past RRs You, ≥x, past RRs Others, this RR

1st Quartile (25%) Expectation

For details of the construction and interpretation of these plots, see: Duewer, Kline, Sharpless, Brown Thomas, Gary, Sowell. Anal Chem 1999;71(9):1870-8.

Serum History Comments #357 New Fresh-frozen, native, multi-donor #358 New Fresh-frozen, native, multi-donor #359 New Fresh-frozen, native, multi-donor #360 65:#356 Fresh-frozen, native, multi-donor #351 63:#341, 63:#344, 64:#351 Fresh-frozen, native, multi-donor

Individualized Report Page 2 / 14

D4

Set 1 of 41


Retinyl Palmitate, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D5

Set 1 of 41


α-Tocopherol, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D6

Set 1 of 41


γ/β-Tocopherol, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D7

Set 1 of 41


Total β-Carotene, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D8

Set 1 of 41


trans-β-Carotene, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D9

Set 1 of 41


Total cis-β-Carotene, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D10

Set 1 of 41


Total α-Carotene, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D11

Set 1 of 41


Total Lycopene, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D12

Set 1 of 41


trans-Lycopene, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D13

Set 1 of 41


Total β-Cryptoxanthin, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









D14

Set 1 of 41


Total Lutein&Zeaxanthin, μg/mL Ap

pare

nt P

recis

ion,

SD

[Ana

lyte]

, µg/

mL

357

358

359

356

360

341

344

351

361

Your

[Ana

lyte]

, µg/

mL

Appa

rent

Pre

cisio

n, S

D

Concordance, SD









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4

Appendix E. Shipping Package Inserts for RR31

The following five items were included in each package shipped to an RR31 participant: • Cover letter • Protocol for Preparation and Analysis of the Ascorbic Acid Solid Control

Material • Preparation and Validation of Ascorbic Acid Solid Control Material Datasheet • Analysis of Control Materials and Test Samples Datasheet • Packing List and Shipment Receipt Confirmation Form

The cover letter, preparation protocol, and the two datasheets were enclosed in a sealed waterproof bag along with the samples themselves. The packing list was placed at the top of the shipping box, between the cardboard covering and the foam insulation.

E1

June 1, 2009

Dear Colleague:

UNITED STATES DEPARTMENT OF ·COMMERCE Nation.-1 Institute of Standards and Technology Gaithersburg, Maryland 20899-

The samples withi,n this package constitute Vitamin C Round Robin 31 (RR31) of the 2009 Micronutrients Measurement Quality Assurance Program. RR.31 consists of four vials c:iffrozen serum test samples (#37, #47, #74,.and #114), one vial of frozen control serµm (CS #2), and 01;1e vial of ascorbic acid solid control material (Control). Please follow the attach~d protocols when you prepare and analyze these samples. lfyc;m cannot prepare the solid control solutions gravimetrically, please prepare equivalent ~olutions volumetrically an4 report the exact volumes used. (Routine 0.5 g gravimetric measurements are ge~erally 10-fold more accurate than routine 0.5 mL volumetric measurements.)

Please ·use the control serum to validate the performance of your measurement system before you analyze the test samples. The target value and ::::95% confidence interval for target value and ::::95% confidence interval for CS #2 is 28.1 ±1.0 µmol/L of sample.

The report for RR30 was e-mailed May 15, 2009. If you find your results for RR30 unsatisfactory, we recommend that you obtain Standard Reference Mat~rial (SRM) 970 Ascorbic Acid in Serum to validate yo.ur methodology and value assign in-house control materials. This SRM may be purchased from the Standard Materials Reference Program at NIST (Tel: 301-975-6776, Fax: 301-948-3 730, or e-mail: [email protected]).

Please be. aware that sample contact with ~y oxidant-contaminated surface (vials, glassware, etc.) may degrade your measurement system's performance (SA Margolis and E Park, "Stability of Ascorbic Acid in Solutions in Autosampler Vials", Clinical Chemistry 2001, 47(8), 1463-1464). You should suspect such degradation if you observe unusually large variation in replicate analyses.

If you have any questions or concerns about the Vitamin C Micronutrients Measurement Quality Assurance Program please contact Jeanice Brown Thomas at phone: 301-975-3120, fax: 301-977-0685, or e-mail: [email protected].

We ask that you return your results for these RR31 samples by September 28, 2009. We would appreciate receiving your results as soon as they become available. Please use the attached form. Your results will be kept confidential. ·

es rch Chemist Analytical Chemistry Division Chemical Science and Technology Laboratory

Enclosures: Protocols, Preparation and Analysis of Control Materials !l.Ild Analysis of Test Samples RR31 Report Form for Ascorbic Acid Solid Control Material Preparation RR31 Report Form for Control Material and Test Sample Analyses

NISI· E2

Micronutrient Measurement Quality Assurance Program for Vitamin C

Please Read Through Completely BEFORE Analyzing Samples

Protocol for Preparation and Analysis of the Ascorbic Acid Solid Control Material

The ascorbic acid solid control material (in the amber vial) should be prepared and used in the following manner:

1) Prepare at least 500 mL of 5% mass fraction metaphosphoric acid (MPA) in distilled water. This solution will be referred to as the “Diluent” below.

2) Weigh 0.20 to 0.22 g of the ascorbic acid solid control material to 0.0001 g (if possible), dissolve it in the Diluent in a 100 mL volumetric flask, and dilute with the Diluent to the 100 mL mark. Weigh the amount of Diluent added to 0.1 g. Record the weights. The resulting material will be referred to as the “Stock Solution” below.

3) Prepare three dilute solutions of the Stock Solution as follows:

Dilute Solution 1: Weigh 0.500 mL of the Stock Solution to 0.0001 g into a 100 mL volumetric flask; dilute with Diluent to the 100 mL mark. Record the weight.



4) Calculate and record the total ascorbic acid concentrations, [TAA], in these Dilute Solutions. If you follow the above gravimetric preparation directions, the [TAA] in μmol/L is calculated:

( ) ( ⋅ 56785 mol/g ⋅ L)g Stock Solution in Dilute Solution ⋅ g AA in Stock Solution ) ( µ[TAA] DS = (g AA in Stock Solution ) + (g Diluent in Stock Solution)

For example, if you prepared the Stock Solution with 0.2000 g of solid ascorbic acid and 103.0 g of Diluent, then 0.5 mL of the Stock Solution should weigh (0.2+103)/200 = 0.52 g and [TAA]DS1 = (0.52 g)(0.2 g)∙(56785 μmol/g·L)/(0.2 + 103 g) = 57.2 μmol/L. Likewise, 0.25 mL of the Stock Solution should weigh 0.26 g and [TAA]DS2 = 29.4 μmol/L and 0.125 mL should weigh 0.13 g and [TAA]DS3 = 14.2 μmol/L.

5) Measure the ultraviolet absorbance spectrum of Dilute Solution 1 against the Diluent as the blank using paired 1 cm path length cuvettes. Record the absorbance at 242, 243, 244, and 245 nm. Record the maximum absorbance (Amax) within this region. Record the wavelength (λmax) at which this maximum occurs.

The extinction coefficient (E1%) of ascorbic acid at λmax (using a cell with a 1 cm path length) of Dilute Solution #1 can be calculated:

E3

1% dL (A max ) ( ( g AA in Stock Solution ) + (g Diluent in Stock Solution )⋅ )E ( ) =

g ⋅ cm (g Stock Solution in Dilute Solution 1) (⋅ g AA in Stock Solution) If your spectrophotometer is properly calibrated, λmax should be between 243 and 244 nm and E1% should be 550 ± 30 dL/g·cm. If they are not, you should recalibrate the wavelength and/or absorbance axes of your spectrophotometer and repeat the measurements.

6) Measure and record the concentration of total ascorbic acid in all three dilute solutions and in the 5% MPA Diluent in duplicate using exactly the same method that you will use for the serum control materials and test samples, including any enzymatic treatment. We recommend that you analyze these solutions in the following order: Diluent, Dilute Solution 1, Dilute Solution 2, Dilute Solution 3, Dilute Solution 3, Dilute Solution 2, Dilute Solution 1, Diluent. a) Compare the values of the duplicate measurements. Are you satisfied that your measurement

precision is adequate? b) Compare the measured with the calculated [TAA] values. This is most conveniently done by

plotting the measured values on the y-axis of a scatterplot against the calculated values on the x-axis. The line through the four {calculated, measured} data pairs should go through the origin with a slope of 1.0. Are you satisfied with the agreement between the measured and calculated values?

Do not analyze the serum control materials or test samples until you are satisfied that your system is performing properly!

7) Once you have confirmed that your system is properly calibrated, analyze the serum control CS #1 (see protocol below). The target values for this materials is 8.4 ±0.7 µmol/L of sample.

If your measured values are not close to this value, please review your sample preparation procedure and whether you followed exactly the same measurement protocol the solutions prepared from the solid control material as you used for these serum controls. If the protocols differ, please repeat from Step 6 using the proper protocol. If the proper protocol was used, your measurement system may not be suitable for MPA-preserved samples; please contact us at 301975-3120 or [email protected]. Do not analyze the test samples until you are satisfied that your system is performing properly and is suitable for the analysis of MPA-preserved serum!

Protocol for Analysis of the Serum Control Materials and Test Samples

The serum control material and test samples are in sealed ampoules. They were prepared by adding equal volumes of 10% MPA to spiked human serum. We have checked the samples for stability and homogeneity. Only the total ascorbic acid is stable. While these samples contain some dehydroascorbic acid, its content is variable. Therefore, only total ascorbic acid should be reported. The serum control material and test samples should be defrosted by warming at 20 °C for not more than 10 min otherwise some irreversible degradation may occur.

Each serum test sample contains between 0.0 and 80.0 μmol of total ascorbic acid/L of solution. The total ascorbic acid in each ampoule should be measured in duplicate. Please report your results in μmol/(L of the sample solution) rather than μmol/(L of serum NIST used to prepare the sample).

E4


Participant #: Date:

Vitamin C Round Robin 31 NIST Micronutrient Measurement Quality Assurance Program

Preparation and Validation of Ascorbic Acid Solid Control Material

STOCK SOLUTION

Mass of ascorbic acid in the Stock Solution ..................................................__________ g

Mass of 5% MPA Diluent added to the 100 mL volumetric flask .................__________ g

DILUTE SOLUTION 1

Mass of added stock solution (0.5 mL) ..........................................................__________ g


Absorbance of Dilute Solution 1 at 242 nm...................................................__________ AU




Absorbance of Dilute Solution absorbance maximum ..................................__________ AU

Wavelength of maximum absorbance............................................................__________ nm

Calculated E1% ...............................................................................................__________ dL/g·cm

Calculated [TAA]DS1......................................................................................__________ μmol/L

DILUTE SOLUTION 2

Mass of added stock solution (0.25 mL) ........................................................__________ g



DILUTE SOLUTION 3

Mass of added stock solution (0.125 mL) ......................................................__________ g



Please return by September 28, 2009 MMQAP Fax: 301-977-0685 100 Bureau Drive, Stop 8392 Email: [email protected] Gaithersburg, MD 20899-8392

E5


Participant #: Date:

Vitamin C Round Robin 31 NIST Micronutrient Measurement Quality Assurance Program

Analysis of Control Materials and Test Samples

Sample Replicate 1 Replicate 2 Units

Dilute Solution 1 μmol/L of Dilute Solution



5% MPA Diluent μmol/L of Diluent

μmol/L of Sample CS #2 Target: 28.1 ±1.0 μmol/L

Serum Test Sample #37 μmol/L of Sample




Were samples frozen upon receipt? Yes | No

Analysis method: HPLC-EC | HPLC-Fluor DAB | HPLC-OPD | HPLC-UV | AO-OPD | Other If “Other”, please describe:

COMMENTS:

Please return by September 28, 2009 MMQAP Fax: 301-977-0685 100 Bureau Drive, Stop 8392 Email: [email protected] Gaithersburg, MD 20899-8392

E6



Vitamin C Round Robin 31 NIST Micronutrients Measurement Quality Assurance Program

Packing List and Shipment Receipt Confirmation Form

This box contains one vial each of the following six VitC M2QAP samples:

Label Form

VitC #37 Liquid frozen (1:1 serum:10% MPA) VitC #47 Liquid frozen (1:1 serum:10% MPA) VitC #74 Liquid frozen (1:1 serum:10% MPA)

VitC #114 Liquid frozen (1:1 serum:10% MPA) CS #2 Liquid frozen (1:1 serum:10% MPA) Control Solid AA

Please 1) Open the pack immediately 2) Check that it contains one vial each of the above samples 3) Check if the samples arrived frozen 4) Store the samples at -20 ˚C or below until analysis 5) Complete the following information 6) Fax the completed form to us at 301-977-0685

(or email requested information to [email protected])

1) Date this shipment arrived: ___________________________ 2) Are all of the vials intact? Yes | No

If "No", which one(s) were damaged?

3) Was there any dry-ice left in cooler? Yes | No 4) Did the samples arrive frozen? Yes | No 5) At what temperature are you storing the samples? _______ ˚C 6) When do you anticipate analyzing these samples? ________________________

Your prompt return of this information is appreciated.

The M2QAP Gang

Mail: M2QAP NIST, Stop 8392 Fax: 301-977-0685 Gaithersburg, MD 20899-8392 Email: [email protected]

E7



Appendix F. Final Report for RR31

The following three pages are the final report as provided to all participants: • Cover letter. • An information sheet that:

o describes the contents of the “All-Lab” report, o describes the content of the “Individualized” report, o describes the nature of the test samples and details their previous distributions,

if any, and o summarizes aspects of the study that we believe may be of interest to the

participants.

F1

October 30, 2009

Dear Colleague:

UNITED STATES DEPARTMENT OF COMMERCE Natiional lnadtut:a of kandarde and Technology Gaithersburg, Maryland 2089~

Enclosed is the summary report of the results for Round Robin 31 (RR3 l) for the measurement of total ascorbic acid (TAA, ascorbic acid plus dehydroascorbic acid) in human serum. Included in this report are a summary of data for all laboratories and an individualized summary of your laboratory's measurement performance. The robust median is used to estimate the consensus value for all samples, the "median absolute deviation from the median" (MADe) is used to estimate the expected standard deviation, and the coefficient of variation (CV) is defined as 1 OOxMADe/median.

RR31 consisted of four !est samples (#37, #47, #74, and #114), one serum control material (CS#2), and one solid control material for preparation of TAA control solutions. Details regarding the samples can be found in the enclosed report.

If you have concerns regarding your laboratory's performance, we suggest that you obtain and analyze a unit of Standard Reference Material (SRM) 970, Vitamin C in Frozen Human Serum. SRM 970 can be purchased from the NIST SRM Program at phone: 301-975-6776; fax: 301-948-3730. If your measured values do not agree with the certified values, we suggest that you contact us for consultation. ·

Samples for the first vitamin Cround robin (RR32) of the 2010 NIST_Micronutrients Measurement Quality Assurance Program will be shipped during the week of December 7, 2009.

If you have questions or concerns regarding this report, please contact David Duewer at 301-975-3935; e-mail: [email protected] or me at 301-975-3120; e-mail: [email protected]; or fax: 301-977-0685.

I

e i~e B. Thomas, M.B.A. Research Chemist Analytical Chemistry Division

David L. Duewer, Ph.D Research Chemometrician Analytical Chemistry Division

Chemical Science and Technology Laboratory Chemical Science and Technology Laboratory

Enclosures

Cc: L. C. Sander

NISI F2

The NIST M2QAP Vitamin C Round Robin 31 (RR31) report consists of

Page “Individualized” Report 1 Summarizes your reported values for the nominal 55 mmol/L solution you prepared from the

ascorbic acid solid control sample, the serum control sample, and the four serum test samples.

2 Graphical summary of your RR31 sample measurements.

Page “All Lab” Report 1 A tabulation of results and summary statistics for Total Ascorbic Acid [TAA] in the RR31

samples and control/calibration solutions.

Serum-based Samples. One serum control and four unknowns were distributed in RR31. CS#2 SRM 970 level 2, ampouled in mid-1998. S31:1 Serum 37, ampouled in late 2001, previously distributed as sample S17:2 (RR17, Fall 02),

S18:1 (RR18, Spring 03), S20:1 (RR20, Spring 04), S22:2 (RR22, Spring 05), S23:2 (RR23, Fall 05), S27:1 (RR27, Fall 07).

S31:2 Serum 47, ampouled in late 2001, previously distributed as sample S18:2 (RR18, Spring 03), S19:3 (RR19, Fall 03), S21:3 (RR21, Fall 04), S22:3 (RR22, Spring 05) and S24:2 (RR24, Spring 06)., S26:2 (RR26, Spring 07)

S31:3 SRM 970 level 1, ampouled in mid-1998 S31:4 Serum 114, ampouled in 1995, previously distributed as sample 188a in (RR9, Summer 96)

and 27:3 (RR27, Fall 07)

Results.

1) Most participants who prepared the four 5% MPA control/calibration solutions (the three “Dilute Solutions” and the “Diluent”) did so correctly. The criteria used to evaluate this success are: the density of the 5% MPA (≈1.03 gm/mL), the observed wavelength maximum of “Dilute Solution #1”(≈244 nm), the observed absorbance at that maximum (≈0.58 OD), the calculated E1% #1”(≈560 dL/g·cm). On the evidence of MPA density, one participant prepared the solutions in 2.5% MPA; this may have contributed to systematically low results for the unknowns. On the evidence of the maximum absorbance and calculated E1% #1, one participant had spectrophotometer issues; this had no impact on the [TAA] measurements.

2) The Measured = a+b*Gravimetric calibration parameters for the control/calibration solutions (columns 10 to 13 of the All Lab Report) indicate that the measurement systems for all participants are linear (R2 close to 1 and RMS close to 0.0) and reasonably well calibrated (intercepts range from -1.0 to 0.9 and slopes range from 0.96 to 1.14).

3) The Measured = p+q*Median regression parameters for samples S31:1 to S31:4 (columns 23 to 26 of the All Lab Report) confirm the linearity of most measurement systems (R2 close to 1 and RMS close to 0.0).

4) There is no evidence of sample degradation in any of the materials.

5) The Figure below displays the interlaboratory MADe (a robust estimate of the standard deviation) as a function of the interlaboratory median for both the “Unkowns” and the “Controls”. The interlab

F3

reproducibility precision, SDunk, for the unknowns is reasonably well described by the function

SDunk = 1.32 + (0.080 × [TAA ])2 .

That is, SDunk is 1) at least 1.3 μmol/L regardless of the [TAA] and 2) at high [TAA] levels the relative SDunk, RSDunk, is about 8%. The SDunk is displayed as a thick black line, RSDunk is displayed as a blue line.

Inte

rlabo

rato

ry S

D, μ

mol

/L

6

5

4

3

2

1

0

Unknowns Controls

15

10

5

0 R

elat

ive

Inte

rlabo

rato

ry S

D, %

0 10 20 30 40 50 60 70

Interlaboratory Median, μmol/L

While there are too few data for confident assessment, the reproducibility precision for the two materials that have been provided as Controls, SDCS, is consistently smaller than for the Unknowns. It appears, therefore, that there is “room for improvement” in the interlaboratory comparability of [TAA] measurements.

F4

Appendix G. “All-Lab Report” for RR31

The following single page is the “All-Lab Report” as provided to all participants, with two exceptions:

• the participant identifiers (Lab) have been altered. • the order in which the participant results are listed has been altered.

The data summary in the “All-Lab Report” has been altered to ensure confidentiality of identification codes assigned to laboratories.

G1

Mic

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In

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g/m

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S31:

3 S3

1:4

Inte

r Sl

ope

R2

RM

S λ m

ax

A max

VC-M

A 10

/09/

09

58.8

29

.6

14.8

60

.2

30.0

14

.9

0.0

-0.1

4 1.

02

1.00

0 0.

2 1.

036

244.

0.

5660

54

6.1

32.0

22

.1

33.8

7.

7 29

.4 -

1.09

1.

05

0.99

9 0.

5 VC

-MB

05/0

8/09

58

.2

28.5

14

.6

60.7

30

.2

15.5

0.

0 0.

23

1.04

1.

000

0.3

1.03

0 24

3.

0.58

00

565.

7 28

.4

23.2

35

.4

8.5

29.9

-0.

46

1.06

1.

000

0.2

VC-M

C

02/1

0/09

59

.1

29.6

15

.0

56.9

28

.6

14.2

0.

0 -0

.06

0.96

1.

000

0.1

1.01

6 24

3.

0.56

63

544.

0 27

.5

16.7

28

.5

7.6

23.2

-0.

14

0.82

0.

987

1.3

VC-M

E 23

/07/

09

56.7

28

.4

14.2

57

.8

28.6

14

.7

0.0

0.03

1.

02

1.00

0 0.

3 1.

031

243.

0.

5714

57

2.0

31.8

25

.7

38.8

9.

7 34

.9 -

0.17

1.

18

0.99

5 1.

1 VC

-MG

28

/08/

09

62.8

31

.5

16.4

64

.1

31.8

16

.1

0.0

-0.3

2 1.

02

1.00

0 0.

4 1.

028

243.

6 0.

6100

55

1.3

28.0

22

.1

33.7

8.

2 28

.6 -

0.31

1.

01

1.00

0 0.

1 VC

-MH

13

/10/

09

61.2

30

.5

14.9

61

.0

30.5

15

.1

0.0

0.08

1.

00

1.00

0 0.

1 1.

027

244.

1 0.

6038

56

0.3

28.2

21

.4

32.7

8.

3 28

.1

0.04

0.

97

1.00

0 0.

2 VC

-MI

23/0

9/09

55

.4

28.3

13

.9

56.7

27

.5

14.8

0.

0 -0

.06

1.02

0.

999

1.0

1.03

0 28

.4

21.5

35

.3

10.5

28

.7

1.55

0.

97

0.98

8 1.

4 VC

-MJ

25/0

9/09

59

.2

30.5

15

.2

67.2

33

.4

15.3

0.

0 -1

.01

1.14

0.

999

1.1

1.02

3 25

4a

0.36

8a

352.

7a

30.1

26

.0

37.8

11

.1

b 2.

23

1.06

1.

000

0.4

VC-M

K 16

/09/

09

57.9

29

.3

14.5

57

.7

29.3

15

.0

1.0

0.85

0.

98

1.00

0 0.

2 1.

029

245.

0.

1761

17

2.6

30.0

24

.5

36.0

10

.5

28.0

2.

29

0.97

0.

983

1.7

VC-M

N

24/0

9/09

63

.0

31.6

15

.8

62.5

30

.8

14.3

0.

0 -0

.64

1.00

0.

999

0.7

1.02

9 24

3.7

0.61

78

556.

7 28

.7

22.1

32

.7

11.3

27

.0

4.00

0.

83

0.99

5 0.

8 VC

-MP

04/0

8/09

21

.2

32.5

8.

5 31

.5

0.00

1.

01

0.97

2 2.

3 VC

-MU

11

/09/

09

25.9

18

.2

31.3

7.

4 24

.7 -

1.17

0.

93

0.98

9 1.

3 VC

-NE

02/1

1/09

60

.3

29.6

14

.2

60.3

30

.1

14.3

0.

0 0.

14

1.00

1.

000

0.3

1.03

2 24

3.5

0.59

50

560.

1 27

.9

21.5

32

.1

7.8

25.7

-0.

11

0.94

0.

995

0.9

G2

N

10

10

10

10

10

10

10

59.2

29

.8

14.9

60

.5

30.1

15

.0

0.1

2.5

1.2

0.7

3.4

1.7

0.6

0.3

N

10

8 8

8 11

12

12

12

11

Av

erag

e Av

erag

e 1.

028

243.

7 0.

5364

50

8.6

29.0

22

.1

34.0

9.

1 28

.5

SD

SD

0.00

5 0.

7 0.

1470

13

6.1

1.8

2.7

2.8

1.4

3.1

Min

55

.4

28.2

9 13

.9

58.0

28

.69

14.5

59

.0

29.6

4 14

.9

60.7

30

.53

15.1

63

.0

31.5

8 16

.4

2.4

1.5

0.5

4 5

3

56.7

27

.50

14.2

0.

0 57

.7

28.7

7 14

.7

0.0

60.4

30

.09

15.0

0.

0 62

.1

30.6

9 15

.3

0.0

67.2

33

.36

16.1

1.

0 4.

0 1.

7 0.

5 0.

0 7

6 3

Min

1.

016

243.

0 0.

1761

17

2.6

25.9

16

.7

28.5

7.

4 23

.2

%25

%

25

1.02

7 24

3.0

0.56

62

545.

6 28

.1

21.4

32

.6

8.1

27.5

M

edia

n M

edia

n 1.

029

243.

7 0.

5757

55

4.0

28.4

22

.1

33.7

8.

5 28

.6

%75

%

75

1.03

0 24

4.0

0.60

54

561.

7 30

.0

23.6

35

.5

10.5

29

.7

Max

M

ax

1.03

6 24

5.0

0.61

78

572.

0 32

.0

26.0

38

.8

11.3

34

.9

MAD

e eS

D

0.0

0.8

0.02

80

13.2

1.

4 1.

5 2.

3 1.

6 2.

0 C

V C

V 0.

22

0.33

4.

9 2.

4 5

7 7

18

7

a )

5% T

richl

oroa

cetic

aci

d so

lutio

n b

) M

isla

bele

d sa

mpl

e

All L

ab R

epor

t 1

/ 1

Appendix H. Representative “Individualized Report” for RR31

Each participant in RR31 received an “Individualized Report” reflecting their reported results. The following two pages are the “Individualized Report” for participant “VC-MA”.

H1

Set 1 of 16

Vitamin C "Round Robin" 31 Report: Participant VC-MA

MPA Dilute Solution 1 Control/Calibration Solutions Density Spectrophotometry Ymeas = Inter + Slope* Xgrav

Date RR Method 03/20/07 26 HPLC-EC 10/05/07 27 HPLC-EC 03/04/08 28 HPLC-EC 08/11/08 29 HPLC-EC 03/03/09 30 HPLC-EC 09/10/09 31 HPLC-EC

g/mL 1.033 1.032 1.035 1.037 1.037 1.036

λmax Amax E1%

244.0 0.573 554.3 242.0 0.561 557.2 243.0 0.572 562.2 243.0 0.567 553.2 242.0 0.569 555.6 244.0 0.566 546.1

Inter Slope R2 SEE 0.3 1.00 1.000 0.31

-0.1 0.99 1.000 0.14 0.7 1.03 0.999 0.99 0.3 1.03 1.000 0.64 0.2 1.03 1.000 0.40

-0.1 1.02 1.000 0.20 Mean 1.035 243.0 0.57 554.8 Pooled SEE 0.53

SD 0.002 0.9 0.00 5.3 CV 0.18 0.37 0.8 0.9

[TAA] mmol/Lsample

7 34.9 0.3 1.1

12 8.2 0.3 0.5

Date RR Sample 10/17/05 23 CS#2 03/09/06 24 CS#2 08/28/06 25 CS#2 10/05/07 27 CS#2 08/11/08 29 CS#2 09/10/09 31 CS#2

03/20/03 18 S18:2 11/13/03 19 S19:3 09/13/04 21 S21:3 03/08/05 22 S22:3 03/09/06 24 S24:2 03/20/07 26 S26:2 09/10/09 31 S31:2

09/23/98 11 S11:1:A 04/02/99 12 S12:1:A 09/17/01 13 S13:1 09/27/01 14 S14:3 09/18/01 15 S15:1 11/18/02 16 S16:1 11/13/03 19 S19:4 02/23/04 20 S20:3 10/17/05 23 S23:4 08/28/06 25 S25:1 08/11/08 29 S29:2 09/10/09 31 S31:3

06/19/96 09 S09:2

Rep1 Rep2 Fadj Mean SDdup

29.4 30.5 1.0 30.0 0.8 29.2 29.1 1.0 29.2 0.1 27.2 28.1 1.0 27.6 0.6 28.1 27.4 1.0 27.7 0.5 27.2 27.2 1.0 27.2 0.0 31.8 32.2 1.0 32.0 0.3

N Mean SDrepeat SDreprod

6 28.9 0.5 1.8

35.1 36.0 1.0 35.6 0.6 35.9 35.8 1.0 35.9 0.1 33.2 32.9 1.0 33.0 0.2 35.7 35.6 1.0 35.6 0.1 35.8 35.5 1.0 35.6 0.2 35.0 35.4 1.0 35.2 0.3 33.9 33.7 1.0 33.8 0.2

15.5 13.9 0.5 7.4 0.6 14.5 15.8 0.5 7.6 0.5

8.4 8.5 1.0 8.5 0.1 8.0 7.7 1.0 7.8 0.2 8.9 8.7 1.0 8.8 0.1 8.8 8.8 1.0 8.8 0.0 7.8 8.6 1.0 8.2 0.5 8.3 8.1 1.0 8.2 0.1 8.6 8.8 1.0 8.7 0.1 8.7 8.5 1.0 8.6 0.2 8.3 8.4 1.0 8.3 0.1 7.3 8.1 1.0 7.7 0.5

51.7 51.1 0.5 25.7 0.2 28.5 28.6 1.0 28.5 0.1 29.0 29.9 1.0 29.4 0.7

3 27.9 0.4 2.0 10/05/07 27 S27:3 09/10/09 31 S31:4

Please check our records against your records. Send corrections and/or updates to...

Micronutrients Measurement Quality Assurance Program National Institute of Standards and Technology 100 Bureau Drive Stop 8392 Fax: (301) 977-0685 Gaithersburg, MD 20899-8392 USA Email: [email protected] Individualized Report Page 1 / 2

H2


NIST, RR 22

H3

Set 1 of 16

Vitamin C "Round Robin" 31 Report: Participant VC-MA

Total Ascorbic Acid, µmol/mL [T

otal

Asc

orbi

c Ac

id],

µmol

/L

S17:

2S1

8:1

S20:

1S2

2:2

S23:

2 S2

7:1

S31:

1

S18:

2 S1

9:3

S21:

3 S2

2:3

Appa

rent

Pre

cisio

n, S

D S2

4:2

S26:

2S3

1:2

S11:

1S1

2:1

S13:

1S1

4:3

S15:

1S1

6:1

S19:

4 S2

0:3

S23:

4 S2

5:1

S29:

2 S3

1:3

S09:

2 S2

7:3

S31:

4

Your

[Tot

al A

scor

bic

Acid

], µm

ol/L

Median [Total Ascorbic Acid], µmol/L

Concordance, SD

3rd Quartile (75%) You, this RR Median (50%)

You, past RRs Others, this RR 1st Quartile (25%)


Sample Comments S31:1 VitC #37 previously distributed in RRs 17, 18, 20, 22, 23, 27 S31:2 VitC #47, previously distributed in RRs 18, 19, 21, 22, 24, 26 S31:3 VitC #74, previously distributed in RRs 11, 12, 13, 14, 15, 16, 19, 20, 23, 25, 29 S31:4 VitC #114, previously distributed in RRs 9, 27

Individualized Report 2 / 2

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