NIST Micronutrients Measurement Quality …NISTIR 7880 NIST Micronutrients Measurement Quality...

NISTIR 7880

NIST Micronutrients Measurement Quality Assurance Program

Winter 2012 Comparability Studies

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

and Round Robin 36 Ascorbic Acid in Human Serum

David L. Duewer

Jeanice B. Thomas

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

NISTIR 7880 NIST Micronutrients Measurement

Quality Assurance Program Winter 2012 Comparability Studies

Results for Round Robin LXXI

Fat-Soluble Vitamins and Carotenoids in Human Serum and Round Robin 36 Ascorbic Acid in Human Serum

David L. Duewer

Jeanice B. Thomas Materials Measurement Laboratory

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

December, 2012

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

ii

(This page intentionally blank)

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 Winter 2012 MMQAP measurement comparability improvement studies: 1) Round Robin LXXI Fat-Soluble Vitamins and Carotenoids in Human Serum and 2) Round Robin 36 Total Ascorbic Acid in Human Serum. The materials for both studies were shipped to participants in mid-January 2012; participants were requested to provide their measurement results by April 16, 2012.

Keywords

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

Total Ascorbic Acid

iii

iv

Table of Contents Abstract............................................................................................................................................... iii Keywords ............................................................................................................................................ iii Table of Contents ............................................................................................................................... iv Introduction......................................................................................................................................... 1 Round Robin LXXI: Fat-Soluble Vitamins and Carotenoids in Human Serum ......................... 1 Round Robin 36: Vitamin C in Human Serum............................................................................... 2 References ............................................................................................................................................ 3 Appendix A. Shipping Package Inserts for RR71........................................................................ A1 Appendix B. Final Report for RR71 ..............................................................................................B1 Appendix C. All-Lab Report for RR71..........................................................................................C1 Appendix D. Representative Individualized Report for RR71 ................................................... D1 Appendix E. Shipping Package Inserts for RR36 .........................................................................E1 Appendix F. Final Report for RR36............................................................................................... F1 Appendix G. “All-Lab Report” for RR36 .................................................................................... G1 Appendix H. Representative “Individualized Report” for RR36............................................... H1

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 LXXI: Fat-Soluble Vitamins and Carotenoids in Human Serum Participants in the MMQAP Fat-Soluble Vitamins and Carotenoids in Human Serum Round Robin LXXI comparability study (hereafter referred to as RR71) 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 mid-January 2012. 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 RR71 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 All-Lab 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 the Individualized Report are described in detail elsewhere [3]. An example Individualized Report is reproduced as Appendix D.

Round Robin 36: Vitamin C in Human Serum Participants in the MMQAP Vitamin C in Human Serum Round Robin 36 comparability study (hereafter referred to as RR36) 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 mid-January 2012. 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 RR36 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 All-Lab 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 the Individualized Report are described in detail elsewhere [3]. An example Individualized Report is reproduced as Appendix H.

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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.

Appendix A. Shipping Package Inserts for RR71

The following three items were included in each package shipped to an RR71 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

January 2, 2012

Dear Colleague:

UNITED STATES DEPARTMENT OF COMMERCE National Institute of Standards and Technology Gaithersburg, Maryland 20899-

Enclosed are samples for the first fat-soluble vitamins and carotenoids in serum study (Round Robin LXXI) for the 2012 NIST Micronutrients Measurement Quality Assurance Program. The set of samples (Sera 382- 386) consists of one vial 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 Apri116, 2012. 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 May 2012.

Samples should be allowed to stand at 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 con.tact 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 • em): retinol, 1843 at 325 nm (ethanol); retinyl palmitate, 975 at 325 nm (ethanol); a -tocopherol, 75.8 at 292 nm (ethanol); y

tocopherol, 91.4 at 298 nm (ethanol); a-carotene, 2800 at 444 nm (hexane); 13-carotene, 2560 at 450 nm (ethanol), 2592 at 452 nm (hexane); and lycopene, 3450 at 472 nm (hexane).

Please report your results for Round Robin LXXI 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 atjbthomas@nist.

NISI A2

Participant #: ________ Date: _____________

Analyte 382 383 384 385 386 Units*total retinol

trans-retinolretinyl palmitate

-tocopherol-tocopherol-tocopherol

total -carotenetrans--carotene

total cis--carotenetotal -carotene

total lycopenetrans-lycopene

total -cryptoxanthintotal -cryptoxanthin

total luteintotal zeaxanthin

total lutein&zeaxanthin total coenzyme Q10

ubiquinol (QH2)

ubiquinone (Qox)phylloquinone (K1)

25-hydroxyvitamin DPhytoene

Phytofluene

* we prefer g/mL

Were the samples frozen when received? Yes | No

Comments:

Round Robin LXXI: Human SeraNIST Micronutrients Measurement Quality Assurance Program

Mail: M2QAP NIST, Stop 8392 Gaithersburg, MD 20899-8392

Fax: 301-977-0685Email: [email protected]

Please return results by16-Apr-2012

A3


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

Serum Form Reconstitute? Vial/Cap#382 Liquid frozen No 2 mL clear, forest green cap#383 Liquid frozen No 2 mL amber, gold cap#384 Liquid frozen No 2 mL amber, purple cap#385 Liquid frozen No 2 mL clear, black cap#386 Liquid frozen No 2 mL amber, blue cap

Please 1) Open the pack immediately2) Check that it contains all of the above samples3) Check if the vials are intact4) Store the sera at -20 ˚C or below until analysis5) Complete the following information6) 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 | No4) Did the samples arrive frozen? Yes | No5) At what temperature are you storing the serum samples? _______ ˚C6) When do you anticipate analyzing these samples? ________________________

Your prompt return of this information is appreciated.

The M2QAP Gang

Packing List and Shipment Receipt Confirmation Form

NIST Micronutrients Measurement Quality Assurance ProgramFat-Soluble Vitamins Round Robin LXXI



A4

Appendix B. Final Report for RR71

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

May 10, 2012

Dear Colleague:


Enclosed is the summary report of the results for round robin LXXI (RR71) of the 2012 NIST Micronutrients Measurement Quality Assurance Program (MMQAP) for the fat-soluble vitamins and carotenoids in human serum. Included in this report are: 1) 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 AllLab Report. Combined results rated 1 to 3 are within 1 to 3 standard deviations of the assigned value, respectively; those rated 4 are >3 standard deviations from the assigned 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.

Samples for the second fat-soluble vitamins and carotenoids in serum interlaboratory exercise (RR72) of the 2012 MMQAP will be shipped starting June 25,2012. Please contact us immediately if this schedule is problematic for your laboratory.

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

Analytical Chemistry Division Material Measurement Laboratory

Enclosures

cc: L.C. Sander

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

NISI B2

The NIST MMQAP Round Robin LXXI (RR71) report consists of:

Page All-Lab Report

1-4 A listing of all results and statistics for analytes reported by more than one participant. 5 A listing of the analytes reported by only one participant and a legend for the list of results

and statistics. 6 The text Comparability Summary (“Score Card”) of measurement performance.

Page Individualized Report

1 Your values, the number of labs reporting values, and our assigned values. 2 to

n “Four Plot” summaries of your current and past measurement performance, one page for each analyte you report that is also reported by at least eight other participants.

n+1 The graphical Comparability Summary (target plot) of measurement performance. Samples. Five samples were distributed in RR71.

Serum Description Prior Distributions

382 Fresh-frozen, native, multi-donor serum prepared in 2011. This material was designed to have relatively high contents of α- and β-carotene.

New material

383 Fresh-frozen, native, single-donor serum prepared in 2011. This material was designed to have low contents of α- and β-carotene and lycopene but a high content of β-cryptoxanthin.

New material

384 Fresh-frozen, native, single-donor serum prepared in 2011. This material was designed to have rather similar analyte contents as the #383 materials but with moderate contents of α-carotene and lycopene and a low content of β-cryptoxanthin.

New material

385 Fresh-frozen, native, multi-donor serum prepared in 2011. This serum was prepared as a 31:26 mixture of the #383:#384 materials.

New material

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

#358:RR66-9/09, #364:RR67-3/10, #374:RR69-3/11

Results

1) SRM 968e Stability. There was no significant change in the median level or measurement variability of any measurand in the SRM 968e Level II material.

2) Analyte additivity. The reported content of serum #385 is well predicted for most analytes by the reported contents for its two constituent materials, sera #383 and #384 and the 31:26 mixing ratio. However, the -carotene and -cryptoxanthin contents in #384 are slightly higher than expected. Since #383 was relatively higher in α-carotene than #384 but lower in β-cryptoxanthin, we’re still puzzling over this result.

B3

Appendix C. All-Lab Report for RR71

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.

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16

Tota

l Ret

inol

, μg/

mL

trans

-Ret

inol

, μg/

mL

Ret

inyl

Pal

mita

te, μ

g/m

Lα-

Toco

pher

ol, μ

g/m

Lγ/β-

Toco

pher

ol, μ

g/m

L

All

Lab

Rep

ort

Pag

e 1

/ 6

C2

Rou

nd R

obin

LX

XI L

abor

ator

y R

esul

ts

Lab

FSV

-BG

FSV

-BA

FSV

-BB

FSV

-BC

FSV

-BD

FSV

-BE

FSV

-BF

FSV

-BJ

FSV

-BK

FSV

-BL

FSV

-BM

FSV

-BN

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

-CG

FSV

-CI

FSV

-CZ

FSV

-DD

FSV

-DV

FSV

-EE

FSV

-EZ

FSV

-FK

FSV

-FZ N

Min

Med

ian

Max SD

CV

Npa

stM

edia

npas

tS

Dpa

st

NIS

T

NA

VN

AU

382

383

384

385

386

382

383

384

385

386

382

383

384

385

386

382

383

384

385

386

382

383

384

385

386

0.65

0.04

50.

058

0.05

20.

247

0.34

0.01

40.

036

0.02

40.

033

0.09

80.

068

0.08

00.

074

0.05

50.

760.

049

0.06

00.

058

0.28

40.

720.

046

0.05

60.

054

0.26

50.

040

0.00

30.

003

0.00

40.

019

0.42

0.01

10.

037

0.02

30.

036

0.06

40.

057

0.05

80.

059

0.04

30.

890.

041

0.08

70.

056

0.19

20.

550.

032

0.05

20.

038

0.16

60.

036

0.00

20.

004

0.00

20.

008

0.31

0.00

70.

032

0.01

60.

018

0.77

0.05

20.

064

0.06

80.

241

0.59

0.02

70.

039

0.02

70.

256

0.53

0.03

70.

027

0.03

60.

183

0.46

nq0.

026

0.02

8nq

0.57

0.04

40.

061

0.05

10.

250

0.38

0.01

20.

045

0.02

70.

047

0.53

0.04

70.

065

0.05

90.

239

0.28

0.00

90.

038

0.02

50.

031

0.52

0.08

90.

121

0.16

00.

247

0.32

0.02

30.

017

0.02

40.

057

≥0.4

86≥0

.07

≥0.0

74≥0

.084

≥0.2

010.

490.

070

0.07

40.

084

0.20

10.

240.

022

0.03

60.

032

0.03

60.

011

0.01

00.

010

0.01

00.

011

0.72

0.05

00.

078

0.06

40.

263

0.63

0.04

40.

068

0.05

60.

234

0.03

30.

003

0.00

40.

003

0.01

10.

220.

010

0.03

20.

020

0.02

80.

670.

054

0.06

50.

057

0.21

70.

430.

015

0.04

60.

030

0.02

70.

720.

049

0.06

40.

055

0.26

50.

630.

012

0.05

00.

029

0.03

60.

570.

032

0.04

60.

046

0.22

60.

440.

011

0.03

50.

023

0.02

2

0.44

0.14

40.

153

0.15

10.

253

0.28

0.00

40.

029

0.01

90.

019

0.70

0.04

00.

020

0.03

00.

220

0.59

30.

503

0.30

20.

435

0.34

30.

730.

047

0.06

20.

055

0.24

20.

670.

043

0.05

60.

052

0.22

00.

053

nqnq

nq0.

019

0.51

0.01

60.

045

0.03

20.

040

0.76

0.05

80.

066

0.05

50.

297

0.46

0.01

30.

041

0.02

70.

039

0.56

0.04

00.

050

0.05

00.

190

0.60

≤0.0

59≤0

.059

≤0.0

590.

206

44

44

419

1818

1819

55

55

54

33

34

1514

1515

140.

011

0.01

00.

010

0.01

00.

011

0.44

0.02

70.

020

0.02

70.

183

0.49

0.03

20.

052

0.03

80.

166

0.03

30.

002

0.00

30.

002

0.00

80.

220.

004

0.01

70.

016

0.01

80.

081

0.06

30.

069

0.06

70.

049

0.65

0.04

70.

063

0.05

50.

242

0.63

0.04

40.

056

0.05

40.

220

0.03

80.

003

0.00

40.

003

0.01

50.

380.

012

0.03

60.

025

0.03

40.

593

0.50

30.

302

0.43

50.

343

0.89

0.14

40.

153

0.16

00.

297

0.72

0.07

00.

074

0.08

40.

265

0.05

30.

003

0.00

40.

004

0.01

90.

630.

023

0.05

00.

032

0.05

70.

065

0.04

30.

052

0.04

80.

032

0.12

0.00

90.

013

0.00

70.

031

0.13

0.00

40.

007

0.00

40.

029

0.00

50.

006

0.12

0.00

40.

007

0.00

40.

009

8068

7572

6619

2021

1213

208

127

1314

3830

3420

1826

522

86

180.

081

0.23

90.

215

0.01

30.

031

0.02

00.

032

0.02

50.

003

0.01

2

0.68

0.05

30.

057

0.06

10.

251

0.37

0.01

00.

033

0.02

20.

028

0.08

10.

063

0.06

90.

067

0.04

90.

670.

050

0.06

00.

058

0.24

60.

630.

044

0.05

60.

054

0.22

00.

038

0.00

30.

004

0.00

30.

015

0.38

0.01

10.

035

0.02

40.

031

0.12

0.01

10.

014

0.01

20.

037

0.13

0.00

90.

009

0.00

90.

029

0.12

0.00

50.

012

0.00

90.

012

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

All

Lab

Rep

ort

Pag

e 2

/ 6

C3

Rou

nd R

obin

LX

XI L

abor

ator

y R

esul

ts

Lab

FSV

-BG

FSV

-BA

FSV

-BB

FSV

-BC

FSV

-BD

FSV

-BE

FSV

-BF

FSV

-BJ

FSV

-BK

FSV

-BL

FSV

-BM

FSV

-BN

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

-CG

FSV

-CI

FSV

-CZ

FSV

-DD

FSV

-DV

FSV

-EE

FSV

-EZ

FSV

-FK

FSV

-FZ N

Min

Med

ian

Max SD

CV

Npa

stM

edia

npas

tS

Dpa

st

NIS

T

NA

VN

AU

382

383

384

385

386

382

383

384

385

386

382

383

384

385

386

382

383

384

385

386

0.28

70.

180

0.32

90.

245

0.61

0.14

60.

092

0.17

30.

128

0.32

80.

067

0.20

30.

027

0.11

60.

059

0.25

10.

151

0.24

60.

220

0.52

0.13

10.

074

0.12

80.

110

0.28

20.

083

0.20

20.

028

0.13

10.

084

0.02

90.

032

0.01

60.

027

0.04

60.

247

0.13

20.

264

0.19

20.

430.

118

0.07

00.

134

0.09

80.

253

0.06

10.

146

0.02

20.

090

0.05

00.

018

0.01

80.

011

0.01

50.

019

0.27

80.

135

0.25

00.

178

0.55

0.06

40.

107

nq0.

061

nq

0.06

20.

178

0.02

90.

112

0.05

90.

180

0.15

30.

267

0.23

70.

460.

044

0.14

80.

012

0.08

50.

034

0.38

90.

257

0.47

20.

432

1.22

0.06

50.

138

0.03

40.

010

0.07

3

0.10

10.

065

0.11

40.

108

0.27

0.02

80.

065

0.10

80.

076

0.18

00.

065

0.16

10.

053

0.12

90.

075

0.27

30.

182

0.33

80.

260

0.60

0.13

20.

099

0.18

90.

146

0.35

60.

043

0.08

10.

018

0.05

40.

038

0.01

30.

010

0.01

10.

010

0.01

70.

283

0.17

10.

327

0.23

20.

560.

062

0.16

40.

016

0.08

30.

043

0.34

70.

195

0.36

80.

281

0.68

0.06

80.

217

0.01

90.

120

0.05

20.

242

0.17

10.

325

0.22

40.

650.

057

0.14

70.

017

0.09

90.

050

0.33

70.

248

0.34

40.

306

0.63

0.10

40.

237

0.03

90.

156

0.09

50.

029

0.03

50.

021

0.02

80.

041

0.36

90.

209

0.34

00.

275

0.59

0.16

50.

096

0.16

20.

128

0.29

80.

111

0.20

80.

038

0.13

00.

079

1313

1313

136

66

66

1414

1314

134

44

44

0.10

10.

065

0.11

40.

108

0.27

0.02

80.

065

0.10

80.

076

0.18

00.

043

0.08

10.

012

0.01

00.

034

0.01

30.

010

0.01

10.

010

0.01

70.

278

0.17

10.

327

0.23

70.

590.

132

0.08

30.

148

0.11

90.

290

0.06

50.

163

0.02

70.

106

0.05

90.

023

0.02

50.

013

0.02

10.

030

0.38

90.

257

0.47

20.

432

1.22

0.16

50.

099

0.18

90.

146

0.35

60.

111

0.23

70.

053

0.15

60.

095

0.02

90.

035

0.02

10.

028

0.04

60.

053

0.03

60.

060

0.05

60.

080.

021

0.01

90.

033

0.02

20.

055

0.00

50.

048

0.01

40.

034

0.02

30.

008

0.01

30.

004

0.01

00.

018

1921

1824

1416

2322

1919

829

5232

4036

5027

4759

178

174

0.62

0.29

40.

052

0.02

50.

090.

046

0.01

40.

007

0.28

20.

170

0.33

10.

253

0.51

0.06

90.

205

0.02

50.

095

0.04

5

0.28

00.

171

0.32

90.

245

0.55

0.13

20.

083

0.14

80.

119

0.29

00.

067

0.18

40.

026

0.10

00.

052

0.02

30.

025

0.01

30.

021

0.03

00.

064

0.04

30.

073

0.05

70.

130.

023

0.01

90.

033

0.02

20.

055

0.01

60.

056

0.01

40.

035

0.02

5

Tota

l Lyc

open

e, μ

g/m

Ltra

ns-L

ycop

ene,

μg/

mL

Tota

l β-C

rypt

oxan

thin

, μg/

mL

Tota

l α-C

rypt

oxan

thin

, μg/

mL

All

Lab

Rep

ort

Pag

e 3

/ 6

C4

Rou

nd R

obin

LX

XI L

abor

ator

y R

esul

ts

Lab

FSV

-BG

FSV

-BA

FSV

-BB

FSV

-BC

FSV

-BD

FSV

-BE

FSV

-BF

FSV

-BJ

FSV

-BK

FSV

-BL

FSV

-BM

FSV

-BN

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

-CG

FSV

-CI

FSV

-CZ

FSV

-DD

FSV

-DV

FSV

-EE

FSV

-EZ

FSV

-FK

FSV

-FZ N

Min

Med

ian

Max SD

CV

Npa

stM

edia

npas

tS

Dpa

st

NIS

T

NA

VN

AU

382

383

384

385

386

382

383

384

385

386

382

383

384

385

386

382

383

384

385

386

382

383

384

385

386

0.16

20.

115

0.11

20.

110

0.13

20.

207

0.16

40.

134

0.15

40.

186

0.17

10.

127

0.13

40.

128

0.13

60.

053

0.08

60.

048

0.06

80.

088

0.22

40.

213

0.18

20.

196

0.22

4

0.67

01.

060.

920

0.97

00.

940

3.95

50.

267

0.40

90.

330

0.87

9

0.13

80.

082

0.06

80.

069

0.08

90.

752

1.00

0.88

40.

936

0.95

9

0.15

40.

187

0.11

70.

139

0.13

90.

135

0.11

10.

084

0.10

20.

110

0.03

90.

014

0.02

60.

020

0.02

10.

175

0.12

60.

110

0.12

20.

131

0.73

21.

110.

961

1.01

41.

064

0.28

90.

136

0.18

40.

196

0.18

6

0.07

40.

075

0.05

70.

084

0.06

30.

117

0.08

80.

075

0.08

60.

089

0.65

51.

080.

965

1.05

11.

012

0.15

80.

110

0.09

20.

096

0.11

60.

810

1.12

0.96

51.

010

0.97

50.

152

0.14

50.

097

0.11

70.

134

0.10

90.

078

0.06

50.

084

0.07

80.

769

0.91

0.72

20.

993

0.89

2

0.17

30.

140

0.10

30.

138

0.14

4

0.22

50.

176

0.13

90.

163

0.17

10.

108

0.07

30.

063

0.06

90.

078

0.01

10.

035

0.00

80.

024

0.02

60.

119

0.10

80.

071

0.09

30.

104

0.69

01.

060.

920

0.99

00.

930

1.86

20.

099

0.19

00.

129

0.35

10.

780

1.20

1.16

01.

230

1.17

0

0.67

71.

130.

955

0.96

80.

941

44

44

43

33

33

1414

1414

149

99

99

22

22

20.

108

0.07

30.

063

0.06

90.

078

0.01

10.

014

0.00

80.

020

0.02

10.

074

0.07

50.

057

0.08

40.

063

0.65

50.

910.

722

0.93

60.

892

1.86

20.

099

0.19

00.

129

0.35

10.

137

0.09

70.

076

0.08

60.

100

0.03

90.

035

0.02

60.

024

0.02

60.

160

0.13

10.

107

0.12

00.

133

0.73

21.

080.

955

0.99

30.

959

2.90

90.

183

0.30

00.

230

0.61

50.

171

0.12

70.

134

0.12

80.

136

0.05

30.

086

0.04

80.

068

0.08

80.

289

0.21

30.

184

0.19

60.

224

0.81

01.

201.

160

1.23

01.

170

3.95

50.

267

0.40

90.

330

0.87

90.

022

0.02

80.

016

0.02

40.

024

0.06

20.

041

0.04

40.

044

0.05

00.

071

0.06

0.05

20.

034

0.04

316

2920

2924

3932

4137

3710

55

34

98

188

0.08

70.

030

0.12

50.

960

0.01

80.

013

0.02

90.

072

nq0.

110

0.07

50.

084

0.10

20.

042

0.03

00.

045

0.02

90.

032

nq0.

140

0.12

00.

113

0.13

4

0.13

70.

103

0.07

50.

085

0.10

10.

040

0.03

20.

036

0.02

70.

029

0.16

00.

136

0.11

30.

116

0.13

40.

732

1.08

0.95

50.

993

0.95

90.

030

0.01

60.

024

0.02

40.

011

0.01

00.

016

0.00

80.

009

0.06

20.

042

0.04

50.

045

0.05

00.

073

0.11

0.09

60.

099

0.09

6

Phy

lloqu

inon

e (K

1), n

g/m

LTo

tal L

utei

n&Ze

axan

thin

, μg/

mL

Coe

nzym

e Q

10, μ

g/m

LTo

tal L

utei

n, μ

g/m

LTo

tal Z

eaxa

nthi

n, μ

g/m

L

All

Lab

Rep

ort

Pag

e 4

/ 6

C5

Round Robin LXXI Laboratory Results

Analytes Reported By One Laboratory

Analyte Lab 382 383 384 385 38625-hydroxyvitamin D, μg/mL FSV-BN 0.0153 0.0046 0.0037 0.0027 0.0104

Phytofluene, μg/mL FSV-BS 0.093 0.101 0.104 0.094 0.132Phytoene, μg/mL FSV-BS 0.108 nd nd nd 0.122

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

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

Max Maximum (non-NIST) quantitative value reportedSD Adjusted median absolute deviation from the median of the non-NIST resultsCV 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)

NIST Mean of NIST results

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

NAU NIST Assigned Uncertainty: √(S2 + Sbtw2)

S is the maximum of (0.05*NAV, SD, SDpast, eSD) and Sbtw is the standard deviation between Median and NIST. 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 at or below the limit of quantification, x≥x Concentration greater than or equal to x

italics Not explicitly reported but calculated by NIST from reported values

All Lab Report Page 5 / 6C6

Round Robin LXXI Laboratory Results

Lab TR aT g/bT bC aC TLy TbX L&Z Label DefinitionFSV-BG 1 1 1 1 1 1 1 1 Lab Participant codeFSV-BA 1 1 1 1 1 1 1 1 TR Total RetinolFSV-BB 1 1 1 2 1 1 1 2 aT -TocopherolFSV-BC 1 g/bT -TocopherolFSV-BD 1 1 bC Total -CaroteneFSV-BE 2 3 1 aC Total -CaroteneFSV-BF 1 1 2 TLy Total LycopeneFSV-BJ 1 1 1 2 1 1 2 TbX Total -CryptoxanthinFSV-BK 1 1 L&Z Total Lutein & ZeaxanthinFSV-BL 1 2FSV-BM 2 2 n number of participants providing quantitative dataFSV-BN 1 1 1 1 1 1 % 1 Percent of CS = 1 (within 1 SD of medians)FSV-BO 2 1 2 1 1 1 1 1 % 2 Percent of CS = 2 (within 2 SD of medians)FSV-BP 1 2 4 2 4 2 2 % 3 Percent of CS = 3 (within 3 SD of medians)FSV-BQ 1 1 % 4 Percent of CS = 4 (3 or more SD from medians)FSV-BR 1 3FSV-BS 2 2 2 3 1 2FSV-BT 1 1 1 1 1 1 2 1FSV-BU 2 1 1 1 1 1 1 1FSV-BV 3 2 1 1 2 1 1 1FSV-BW 1 1 1 1 1 1 1 2FSV-CC 1 1FSV-CD 1 2 2 4 2 2 2 1FSV-CE 2 3 2FSV-CG 2 1 1 1 1 1 2 1FSV-CI 1 1 1 1 1 1

FSV-CZ 3 1 2 1FSV-DD 1FSV-DV 2 1FSV-EZ 2 1 1 1FSV-FK 1 1FSV-FZ 1 1 1

NIST 1 1 1 1 1 1 1 1

n 33 30 16 21 16 14 15 15

TR aT g/bT bC aC TLy TbX L&Z% 1 67 73 81 67 75 79 67 73% 2 27 17 19 24 25 7 33 27% 3 6 10 0 0 0 7 0 0% 4 0 0 0 10 0 7 0 0

Comparability Summary

“Comparability Score”

The Comparability Score (CS) summarizes your measurement performance for a given analyte relative to the consensus medians in this study. CS is the average distance (in units of standard deviation) of your measurement performance characteristics from the consensus performance. CS is calculated when the number of quantitative values you reported, Nyou, is at least two and at least six participants reported quantitative values for the analyte. We define CS as follows:

CS =

22 APC1INTEGER4,MINIMUM

C = Concordance = you

N

1i i

ii

NNAU

MedianYouyou

AP = Apparent Precision = 1N

NAUMedianYou

you

N

1i

2

i

iiyou

NAU = NIST Assigned Uncertainty 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.

All Lab Report Page 6 / 6C7

Appendix D. Representative Individualized Report for RR71

Each participant in RR71 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 RR71:

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

The following ten pages are the “Individualized Report” for the analytes evaluated at NIST.

D1

Set

34

of 3

4

Indi

vidu

aliz

ed R

ound

Rob

in L

XX

I Rep

ort:

NIS

T1S

umm

ary

Ser

um 3

82S

erum

383

Ser

um 3

84S

erum

385

Ser

um 3

86A

naly

teY

ouN

AV

nY

ouN

AV

nY

ouN

AV

nY

ouN

AV

nY

ouN

AV

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tal R

etin

ol0.

476

0.46

931

0.47

80.

469

310.

474

0.47

131

0.47

90.

474

310.

499

0.50

031

α-To

coph

erol

11.3

111

.45

297.

377.

2629

7.60

7.77

297.

317.

4129

10.3

610

.43

29γ/β-

Toco

pher

ol1.

413

1.38

915

1.78

31.

767

151.

803

1.80

715

1.83

91.

804

151.

416

1.41

915

Tota

l β-C

arot

ene

0.68

30.

675

190.

053

0.05

018

0.05

70.

060

180.

061

0.05

818

0.25

10.

246

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tal α

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oten

e0.

373

0.39

515

0.01

00.

011

140.

033

0.03

515

0.02

20.

024

150.

028

0.03

114

Tota

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open

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282

0.27

813

0.17

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171

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331

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244

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511

0.54

313

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130.

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54

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113

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D2

Set 34 of 34

Individualized RR LXXI Report: NIST1

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 Comments History#382 Fresh-frozen, native, multi-donor New#383 Fresh-frozen, native, single-donor New#384 Fresh-frozen, native, single-donor New#385 Mixture of (310 mL #383) and (260 mL #384) New#386 Fresh-frozen, native, multi-donor: SRM 968e II 66#358, 67#364, 69#374

Total Retinol, μg/mL38

2

383

385

384

358

364

374

386

[Ana

lyte

], µg

/mL

App

aren

t Pre

cisi

on, S

D

Concordance, SD

Median [Analyte], µg/mL

You

r [A

naly

te],

µg/m

L

Long-term Precision, SD

App

aren

t Pre

cisi

on, S

D

You, this RR

You, past RRs Others, this RR

Expectation

3rd Quartile (75%)

Median (50%)

1st Quartile (25%)

You, ≥x, this RR

You, ≥x, past RRs

Individualized Report ILR_RR71_NIST1.XLS, Page 2 / 10D3

Set 34 of 34




α-Tocopherol, μg/mL38

2

383

385

384

358

364

374

386

[Ana

lyte

], µg

/mL

App

aren

t Pre

cisi

on, S

D

Concordance, SD


You

r [A

naly

te],

µg/m

L


App

aren

t Pre

cisi

on, S

D

You, this RR


Expectation

3rd Quartile (75%)

Median (50%)

1st Quartile (25%)

You, ≥x, this RR

You, ≥x, past RRs


Set 34 of 34




γ/β-Tocopherol, μg/mL38

2

383

385

384

358

364

374

386

[Ana

lyte

], µg

/mL

App

aren

t Pre

cisi

on, S

D

Concordance, SD


You

r [A

naly

te],

µg/m

L


App

aren

t Pre

cisi

on, S

D

You, this RR


Expectation

3rd Quartile (75%)

Median (50%)

1st Quartile (25%)

You, ≥x, this RR

You, ≥x, past RRs


Set 34 of 34




Total β-Carotene, μg/mL38

2

383

385

384

358

364

374

386

[Ana

lyte

], µg

/mL

App

aren

t Pre

cisi

on, S

D

Concordance, SD


You

r [A

naly

te],

µg/m

L


App

aren

t Pre

cisi

on, S

D

You, this RR


Expectation

3rd Quartile (75%)

Median (50%)

1st Quartile (25%)

You, ≥x, this RR

You, ≥x, past RRs


Set 34 of 34




Total α-Carotene, μg/mL38

2

383

385

384

358

364

374

386

[Ana

lyte

], µg

/mL

App

aren

t Pre

cisi

on, S

D

Concordance, SD


You

r [A

naly

te],

µg/m

L


App

aren

t Pre

cisi

on, S

D

You, this RR


Expectation

3rd Quartile (75%)

Median (50%)

1st Quartile (25%)

You, ≥x, this RR

You, ≥x, past RRs


Set 34 of 34




Total Lycopene, μg/mL38

2

383

385

384

358

364

374

386

[Ana

lyte

], µg

/mL

App

aren

t Pre

cisi

on, S

D

Concordance, SD


You

r [A

naly

te],

µg/m

L


App

aren

t Pre

cisi

on, S

D

You, this RR


Expectation

3rd Quartile (75%)

Median (50%)

1st Quartile (25%)

You, ≥x, this RR

You, ≥x, past RRs


Set 34 of 34




Total β-Cryptoxanthin, μg/mL38

2

383

385

384

358

364

374

386

[Ana

lyte

], µg

/mL

App

aren

t Pre

cisi

on, S

D

Concordance, SD


You

r [A

naly

te],

µg/m

L


App

aren

t Pre

cisi

on, S

D

You, this RR


Expectation

3rd Quartile (75%)

Median (50%)

1st Quartile (25%)

You, ≥x, this RR

You, ≥x, past RRs

Individualized Report ILR_RR71_NIST1.XLS, Page 8/ 10D9

Set 34 of 34




Total Lutein&Zeaxanthin, μg/mL38

2

383

385

384

358

364

374

386

[Ana

lyte

], µg

/mL

App

aren

t Pre

cisi

on, S

D

Concordance, SD


You

r [A

naly

te],

µg/m

L


App

aren

t Pre

cisi

on, S

D

You, this RR


Expectation

3rd Quartile (75%)

Median (50%)

1st Quartile (25%)

You, ≥x, this RR

You, ≥x, past RRs


Set

34

of 3

4

Tota

l Ret

inol

α-To

coph

erol

Tota

l β-C

arot

ene

Tota

l Lyc

open

e

Ret

inyl

pal

mita

teγ/β-

Toco

pher

oltra

ns-β

-Car

oten

etra

ns-L

ycop

ene

Tota

l α-C

arot

ene

Tota

l β-C

rypt

oxan

thin

Tota

l Lut

ein&

Zeax

anth

inC

oenz

yme

Q10

Indi

vidu

aliz

ed R

ound

Rob

in L

XXI R

epor

t: N

IST1

Gra

phic

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ompa

rabi

lity

Sum

mar

y

Indi

vidu

aliz

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epor

tIL

R_R

R71

_NIS

T1.X

LS,

Page

10

/ 10

D11

Appendix E. Shipping Package Inserts for RR36

The following five items were included in each package shipped to an RR36 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

January 2, 2012

Dear Colleague:


The samples within this package constitute Vitamin C Round Robin 36 (RR36) ofthe 2012 Micronutrients Measurement Quality Assurance Program. RR36 consists offour vials offrozen serum test samples (#361, #362 #363, and #364), one vial of frozen conh·ol serum (CS #2), and one vial of ascorbic acid solid conh·ol material (Control). Please follow the attached protocols when you prepare and analyze these samples. If you cannot prepare the solid control solutions gravimetrically, please prepare equivalent solutions volumetrically and report the exact volumes used. (Routine 0.5 g gravimetric measurements are generally 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 for CS #2 is 28.05 J.lmoi/L of sample; the ::::95% confidence interval for the target value is 27.56 to f8.54 J.lmoi/L of sample.

Please be aware that sample contact with any 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 Chemishy 2001, 47(8), 1463-1464). You should suspect such degradation if you · observe unusually large variation in replicate analyses.

Please report your results (using the attached form) for RR36 by e-mail to [email protected] or fax to . 301-977-0685 by April16, 2012. If you have questions or comments regarding this study, please call me at (301) 975-3120 or e-mail me [email protected].

~ncerely, i \~ ]U U i 'OJYilGj anice Brown Thomas

ogram Coordinator/Research Chemist alytical Chemistry Division

Chemical Science and Technology Laboratory

Enclosures: Protocols, Preparation and Analysis of Control Materials and Analysis of Test Samples RR36 Report Form for Ascorbic Acid Solid Control Material Preparation RR36 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:

Solution Stock in Diluent gSolution Stock in AA g

Lmol/g 56785Solution Stock in AA gSolution Dilute in Solution Stock g [TAA]DS

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, & 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

Solution Stock in AA g1 Solution Dilute in Solution Stock g

Solution Stock in Diluent g Solution Stock in AA gA )

cmgdL(E max1%

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 #2 (see protocol below). The target values for this materials is 28.1 ±1.0 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 301-975-3120 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:

Please return by April 16, 2012 MMQAP Fax: 301-977-0685 100 Bureau Drive, Stop 8392 Email: [email protected] Gaithersburg, MD 20899-8392

Vitamin C Round Robin 36 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

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


Calculated [TAA]DS2......................................................................................__________ μmol/L DILUTE SOLUTION 3

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


Calculated [TAA]DS3......................................................................................__________ μmol/L

E5

Participant #: Date:

Please return by April 16, 2012 MMQAP Fax: 301-977-0685 100 Bureau Drive, Stop 8392 Email: [email protected] Gaithersburg, MD 20899-8392

Vitamin C Round Robin 36 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

CS #2 μmol/L of Sample Target: 28.05 μmol/L (27.56 μmol/L to 28.54 μmol/L;95 % confidence interval)

Serum Test Sample #361 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:

E6


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

Label FormVitC #361 Liquid frozen (1:1 serum:10% MPA) SRM970 Lv1VitC #362 Liquid frozen (1:1 serum:10% MPA) New1 (5)VitC #363 Liquid frozen (1:1 serum:10% MPA) New4 (31)VitC #364 Liquid frozen (1:1 serum:10% MPA) New5 (46)

CS #2 Liquid frozen (1:1 serum:10% MPA) SRM970 Lv2Control Solid AA

Please 1) Open the pack immediately2) Check that it contains one vial each of the above samples3) Check if the samples arrived frozen4) Store the samples at -20 ˚C or below until analysis5) Complete the following information6) 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 | No4) Did the samples arrive frozen? Yes | No5) At what temperature are you storing the samples? _______ ˚C6) When do you anticipate analyzing these samples? ________________________

Your prompt return of this information is appreciated.

The M2QAP Gang

Packing List and Shipment Receipt Confirmation Form

NIST Micronutrients Measurement Quality Assurance ProgramVitamin C Round Robin 36



E7

Appendix F. Final Report for RR36

The following five 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

May 10,2012

Dear Colleague:


Enclosed is the summary report of the results for Round Robin 36 (RR36) for the measurement of total ascorbic acid (T AA, 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 "adjusted median absolute deviation from the median" (MADe) is used to estimate the expected standard deviation, and we estimate the coefficient of variation (CV) as 100xMADe/median.

RR36 consisted of four test samples (S36:1, S36:2, S36:3, and S36:4), one serum control material (CS#2), and one solid control material for preparation ofTAA 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 www.nist.gov/srm; 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 second vitamin C round robin (RR37) of the 2012 MMQAP will be shipped starting June 25,2012. Please contact us immediately if this schedule is problematic for your laboratory.

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

ea ice Brown Tho Re arch Chemist

alytical Chemistry Division Material Measurement Laboratory

Enclosures

cc: L. C. Sander

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

F2

The NIST MMQAP Vitamin C Round Robin 36 (RR36) report consists of:

Page “Individualized” Report

1 Summary of 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 RR36 sample measurements.

Page “All-Lab” Report

1 A tabulation of results and summary statistics for total ascorbic acid [TAA] in the RR36 samples and control/calibration solutions.

Serum-Based Samples. One serum control and four test samples were distributed in RR36.

CS#2 SRM 970 level 2, ampouled in mid-1998 S36:1 Ampouled in late 2009, previously distributed as sample S34:4 (RR34, Spring 2010). This is

the same material as S36:4. S36:2 Ampouled in late 2009, previously distributed as sample S32:3 (RR32, Fall 2009) and S35:3

(RR35, Fall 2010) S36:3 SRM 970 level 2, ampouled in mid-1998, previously distributed as an “Unknown” in RRs 11

to 15, 18, 20 22, 25, and 29 S36:4 Ampouled in late 2009, previously distributed as sample S34:4 (RR34, Spring 2010). This is

the same material as S36:1. Results. 1) All participants who prepared the four 5% metaphosphoric acid (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 solution (≈1.03 g/mL), the observed wavelength maximum of “Dilute Solution #1” (≈244 nm), the observed absorbance at that maximum (≈0.58), and the calculated E1%#1” (≈560 dL/g·cm).

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 the root-mean-square (RMS) residual close to 0.0) and well calibrated (intercepts close to 0 and slopes close to 1).

3) The Measured = p+q*Median regression parameters for samples S36:1 to S36:4 (columns 23 to 26 of the All-Lab Report) confirm the linearity of all measurement systems (R2 close to 1 and RMS close to 0.0). However, the intercepts and slopes indicate that there are systematic differences in the response of the various measurement procedures to the TAA in the 5% aqueous metaphosphoric acid (MPA) of the test samples.

The consequences of these differences are directly visualized by comparing results for Samples S36:1 and S36:4. Figure 1 is a Youden Plot, displaying the reported values for S36:1 against those for S36:4. These two samples are the same material – the only difference being that the S36:1 vials represent the beginning of the production run and the S36:4 vials represent the end of the run.

Each solid blue dot in Figure 1 represents the average of the two replicate results for S36:1 and for S36:4 reported by one participant, with the thin blue “error bars” spanning the replicate range. The longer these lines, the less stable the participant's measurement process and/or the materials over the time course of the measurements. The difference between replicates is not a significant source of variation for the large majority of participants.

F3

49

51

53

55

57

59

61

63

65

49 51 53 55 57 59 61 63 65

[Total AA] in S36:1, μmol/L

[Tot

al A

A] i

n S

36:4

, μm

ol/L

MedianReported95 % CIY = X

Figure 1: Youden Plot of the Reported Results for S36:1 and S36:4

The black 45° line represents equality between measurements of the two samples. The red circle is an approximate 95 % confidence interval on the joint distribution of the within-sample averages, estimated using robust methods and assuming that all measurements are independent. The center of the circle is the median of all of the available TAA results for this material. The radius is defined from the adjusted median absolute differences from the median (MADe) of the same complete data set. The solid red diamond at the center of the 95 % CI circle marks the median of the average results for S36:1 and for S36:4. The medians are nearly equal, indicating that there is very little difference in TAA content between the vials taken from the beginning of the production run and those from the end.

If there was significant between-vial variation in the amount of TAA across the production run and/or if the participants’ measurement processes were not stable over the course of the serum test sample measurements, then the blue dots would be expected to be distributed in a rather wide band around the equality line. Since nearly all of the blue dots lie quite close to the equality line, we conclude that 1) the material is adequately homogenous and 2) all of the participants’ measurement processes are relatively stable.

If all of the participants’ measurement processes provided closely concordant results for the S36:1/S36:4 material, then no more than one of the blue dots would be expected to be located outside of the red circle. Three of the ten dots are along the line but well outside the circle, two quite high and one quite low. The measurement processes that produced these results are thus internally consistent but are significantly biased relative to those used by the other participants.

One reason for asking participants to prepare control/calibration solutions from the solid control material is to test whether use of a common calibration material would make results from the different measurement procedures more concordant. The information provided on the preparation and measurement of the control/calibration solutions provide that common reference –assuming that there are no significant differences in measurement system response to TAA in 5 % aqueous MPA and TAA in 1:1 mixtures of plasma and 10 % aqueous MPA.

F4

Figure 2 displays the results of re-calibrating the reported results to each participant's individually prepared control solutions. This calibration was accomplished by projecting the as-reported results onto the least-squares fit of the control solution results as a linear function of their preparative values. The values for these intercept, a, and slope, b, parameters are listed in the All-Lab Report. The calibrated results are defined as: Calibrated = (Measured - a)/b. The open blue dots represent the control-solution calibrated results, with the blue lines connecting calibrated and the reported values.

49

51

53

55

57

59

61

63

65

49 51 53 55 57 59 61 63 65

[Total AA] in S36:1 μmol/L

[Tot

al A

A] in

S36

:4, μ

mol

/LReportedCalibrated to Contols95 % CIY = X

A

Figure 2: Youden Plot of Control-Solution Calibrated Results for S36:1 and S36:4.

While calibration to the control solutions does reduce the differences among the results nearest the center of the circle, it does little to reduce the larger differences between this central group and all of the others. Thus the differences in results among the measurement procedures cannot be explained just by differences in measurement system response to TAA in 5 % aqueous MPA.

If the among-procedure differences arise from differences in response to 1:1 mixtures of plasma and 10 % aqueous MPA, then linear calibration to the two components of SRM 970 would be expected to improve concordance. Only one of the two components (Level 2, aka CS#2) was distributed in RR36 so this hypothesis cannot be directly tested using the S36:1 and S36:4 data. However, it can be indirectly tested using results from RR20 to RR25 where SRM 970 Level 1 (aka CS#1) and Level 2 were both distributed.

Figure 3 displays summary statistics for the 20 test samples distributed in these 6 RRs as a function of the mean values, with the bottom panel displaying SD as a function of analyte mean and the top panel displaying the same data as % CV (100·SD/Mean). The solid green diamonds represent the summary statistics estimated from the as-reported results, and the open pink diamonds represent the summaries after projecting the reported results onto the line connecting the results for the CS#1 and CS#2 serum controls. The green lines represent a best-fit model for the SD of the as-reported data; the pink lines represent a model for the serum control-transformed data. Between-participant variability is about halved by calibrating to SRM 970.

F5

0

5

10

15

20

25

30

%C

V [T

AA

], 10

0·S

D/M

ean

0

2

4

6

8

0 10 20 30 40 50 6

Mean [TAA], μmol/L

SD

[TA

A], μm

ol/L

0

As reported

Calibrated to serum controls

22 Mean)0.131.6SD

22 Mean)0.0630SD 95.

Figure 2: Precision Functions for the Results from RR20 to RR25.

Top) % CV as a function of Mean; Bottom) SD as a function of Mean

The reduction in between-participant variability following calibration to the reported results for the SRM 970 materials used as serum control solutions strongly suggests that some measurement procedures react rather differently to TAA in 5 % aqueous MPA than to 1:1 solutions of plasma and 10 % MPA. While such a difference does not necessarily indicate anything about the performance of a given measurement procedure for other sample matrices, it does suggest that there may be unexplained influences at work.

If you are concerned about the accuracy of your measurement procedure for TAA in the 1:1 plasma and 10 % MPA matrix, we recommend the following diagnostic procedure: 1) calibrate your measurement procedure in your routine manner, 2) measure both levels of SRM 970 enough times so that the standard deviation of the mean, SD/√n, of the measurements is less than about 0.5 mmol/L, 3) compare your measurements with the certified 95 % level of confidence intervals: (7.75 to 9.07) mmol/L and (27.56 to 28.54) mmol/L. If your mean values are not both within the appropriate certified range, then it is unlikely that your measurement process provides accurate results for TAA in 1:1 mixtures of plasma and 10 % aqueous MPA.

F6

Appendix G. “All-Lab Report” for RR36

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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G2

Appendix H. Representative “Individualized Report” for RR36

Each participant in RR36 received an “Individualized Report” reflecting their reported results. The following two pages are the “Individualized Report” for participant “VC-MA”.

H1

MPADensity

Date RR Method g/mL max Amax E1% Inter Slope R2 SEE09/10/09 31 HPLC-EC 1.036 244.0 0.566 546.1 -0.1 1.02 1.000 0.2002/24/10 32 HPLC-EC 1.035 242.0 0.566 545.1 0.3 1.03 1.000 0.4609/27/10 33 HPLC-EC 1.037 244.0 0.560 540.5 0.4 1.08 1.000 0.4302/28/11 34 HPLC-EC 1.039 244.0 0.575 555.2 0.6 1.14 1.000 0.7808/24/11 35 HPLC-EC 1.039 242.0 0.568 547.7 0.0 1.03 1.000 0.2402/02/12 36 HPLC-EC 1.035 244.0 0.561 550.5 -0.4 1.05 1.000 0.55

Mean 1.037 243.3 0.57 547.5 0.48SD 0.002 1.0 0.01 5.0CV 0.20 0.42 1.0 0.9

Date RR Sample Rep1 Rep2 Fadj Mean SDdup N Mean SDrepeat SDreprod

02/23/04 20 CS#2 25.8 26.2 1.0 26.0 0.3 12 28.4 0.4 1.609/13/04 21 CS#2 26.2 27.2 1.0 26.7 0.703/08/05 22 CS#2 29.0 29.0 1.0 29.0 0.010/17/05 23 CS#2 29.4 30.5 1.0 30.0 0.803/09/06 24 CS#2 29.2 29.1 1.0 29.2 0.108/28/06 25 CS#2 27.2 28.1 1.0 27.6 0.610/05/07 27 CS#2 28.1 27.4 1.0 27.7 0.508/11/08 29 CS#2 27.2 27.2 1.0 27.2 0.009/10/09 31 CS#2 31.8 32.2 1.0 32.0 0.302/24/10 32 CS#2 28.6 29.0 1.0 28.8 0.302/28/11 34 CS#2 28.3 27.6 1.0 27.9 0.502/02/12 36 CS#2 28.5 28.3 1.0 28.4 0.1

09/23/98 11 S11:2:A 50.7 47.7 0.5 24.6 1.1 11 26.6 0.5 1.904/02/99 12 S12:2:A 49.5 45.9 0.5 23.9 1.309/17/01 13 S13:2 27.6 27.7 1.0 27.7 0.109/27/01 14 S14:4 25.7 26.4 1.0 26.0 0.509/18/01 15 S15:2 25.4 25.6 1.0 25.5 0.203/20/03 18 S18:3 28.8 29.2 1.0 29.0 0.302/23/04 20 S20:4 25.9 25.2 1.0 25.5 0.503/08/05 22 S22:4 29.4 29.4 1.0 29.4 0.008/28/06 25 S25:2 27.6 27.4 1.0 27.5 0.108/11/08 29 S29:3 25.6 25.7 1.0 25.6 0.102/02/12 36 S36:3 28.3 28.5 1.0 28.4 0.1

02/24/10 32 S32:3 33.4 32.8 1.0 33.1 0.5 3 33.1 0.3 0.308/24/11 35 S35:3 33.4 33.4 1.0 33.4 0.102/02/12 36 S36:2 32.8 32.8 1.0 32.8 0.0

02/28/11 34 S34:4 58.1 58.4 1.0 58.3 0.2 3 59.3 0.2 0.902/02/12 36 S36:1 59.5 59.7 1.0 59.6 0.102/02/12 36 S36:4 59.7 60.2 1.0 60.0 0.3

Vitamin C "Round Robin" 36 Report: Participant VC-MA

[TAA] mmol/Lsample

Dilute Solution 1Ymeas = Inter + Slope* Xgrav

Control/Calibration SolutionsSpectrophotometry

Pooled SEE

Email: [email protected]

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

Micronutrients Measurement Quality Assurance ProgramNational Institute of Standards and Technology100 Bureau Drive Stop 6392Gaithersburg, MD 20899-6392 USA

Fax: (301) 977-0685

5/8/2012 [ILR_RR36_Coded.XLS]Summary Page 1/2

H2

Vitamin C "Round Robin" 36 Report: Participant VC-MA

Total Ascorbic Acid, µmol/mL


SampleS36:1 VitC #361, previously distributed in RR 34. Same material as S36:4S36:2 VitC #362, previously distributed in RRs 32, 35S36:3 VitC #363, SRM 970 Lv II. Previously distributed in RRs 11 to 15, 18, 20, 22, 25, 29S36:4 VitC #364, previously distributed in RR 34. Same material as S36:1

Comments

S36

:4S

36:1

S34

:4

S36

:2S

35:3

S13

:2

S32

:3

S36

:3

S11

:2S

12:2

S14

:4S

15:2

S18

:3S

20:4

S22

:4S

25:2

S29

:3

[Tot

al A

scor

bic

Acid

], µm

ol/L

App

aren

t Pre

cisi

on, S

D

Concordance, SD

Median [Total Ascorbic Acid], µmol/L

You

r [To

tal A

scor

bic

Aci

d], µ

mol

/L

You, this RR You, past RRs

NIST, RR 22 Others, this RR

3rd Quartile (75%)Median (50%)1st Quartile (25%)

5/8/2012 [ILR_RR36_Coded.XLS]Pics Page 1/2

H3

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