Dietary Supplement Laboratory 

Quality Assurance Program: 

Exercise H Final Report

Melissa M. Phillips

Catherine A. Rimmer

Laura J. Wood

Anthony F. Marlow

Michele M. Schantz

John R. Sieber

http://dx.doi.org/10.6028/NIST.IR.7903

NISTIR 7903
ii

Dietary Supplement Laboratory

Quality Assurance Program:

Exercise F Final Report

Melissa M. Phillips

Catherine A. Rimmer

Laura J. Wood

NISTIR 7903

Dietary Supplement Laboratory 

Quality Assurance Program: 

Exercise H Final Report

Melissa M. Phillips

Catherine A. Rimmer

Laura J. Wood

Anthony F. Marlow

Michele M. Schantz

John R. Sieber

Chemical Sciences Division

Material Measurement Laboratory

http://dx.doi.org/10.6028/NIST.IR.7903

December 2012

U.S. Department of Commerce

Rebecca M. Blank, Acting Secretary

National Institute of Standards and Technology

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

TABLE OF CONTENTS

ABSTRACT....................................................................................................................................1

INTRODUCTION..........................................................................................................................1

OVERVIEW OF DATA TREATMENT AND REPRESENTATION

Statistics...............................................................................................................................2

Individual Data Table ..........................................................................................................3

Summary Data Table ...........................................................................................................4

Graphs..................................................................................................................................4

Data Summary View................................................................................................4

Sample/Control Comparison View..........................................................................5

NUTRITIONAL ELEMENTS IN FOODS AND SUPPLEMENTS

Study Overview ...................................................................................................................6

Sample Information .............................................................................................................6

Oyster tissue.............................................................................................................6

Ca supplement..........................................................................................................6

Study Results .......................................................................................................................7

Technical Recommendations...............................................................................................7

Table 1. Individual data table (NIST) for nutritional elements in foods and dietary 

supplements..............................................................................................................9

Table 2. Data summary table for calcium in foods and dietary supplements. .................10

Table 3. Data summary table for copper in foods and dietary supplements. ...................11

Table 4. Data summary table for manganese in foods and dietary supplements. ............12

Figure 1. Calcium in SRM 1566b Oyster Tissue (data summary view). .........................13

Figure 2. Calcium in candidate SRM 3532 Calcium Dietary Supplement (data summary 

view). .....................................................................................................................14

Figure 3. Copper in SRM 1566b Oyster Tissue (data summary view)............................15

Figure 4. Copper in candidate SRM 3532 Calcium Dietary Supplement (data summary 

view). .....................................................................................................................16

Figure 5. Manganese in SRM 1566b Oyster Tissue (data summary view). ....................17

Figure 6. Manganese in candidate SRM 3532 Calcium Dietary Supplement (data 

summary view).......................................................................................................18

Figure 7. Calcium in SRM 1566b Oyster Tissue and candidate SRM 3532 Calcium 

Dietary Supplement (sample/sample comparison view)........................................19

Figure 8. Copper in SRM 1566b Oyster Tissue and candidate SRM 3532 Calcium 

Dietary Supplement (sample/sample comparison view)........................................20

Figure 9. Manganese in SRM 1566b Oyster Tissue and candidate SRM 3532 Calcium 

Dietary Supplement (sample/sample comparison view)........................................21

PAHs IN GREEN TEA

Study Overview .................................................................................................................22

Sample Information ...........................................................................................................22

Neat solution ..........................................................................................................22
iv

Green tea ................................................................................................................22

Study Results .....................................................................................................................23

Technical Recommendations.............................................................................................23

Table 5. Individual data table (NIST) for PAHs in green tea. .........................................24

Table 6. Data summary table for naphthalene in green tea..............................................25

Table 7. Data summary table for fluorene in green tea....................................................26

Table 8. Data summary table for phenanthrene in green tea............................................27

Table 9. Data summary table for anthracene in green tea................................................28

Table 10. Data summary table for fluoranthene in green tea. ..........................................29

Table 11. Data summary table for pyrene in green tea. ...................................................30

Table 12. Data summary table for benz[a]anthracene in green tea..................................31

Table 13. Data summary table for chrysene in green tea .................................................32

Table 14. Data summary table for triphenylene in green tea. ..........................................33

Table 15. Data summary table for chrysene + triphenylene in green tea.........................34

Table 16. Data summary table for benzo[a]pyrene in green tea. .....................................35

Figure 10. Naphthalene in SRM 1647e PAH Solution (data summary view). ................36

Figure 11. Naphthalene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data 

summary view).......................................................................................................37

Figure 12. Fluorene in SRM 1647e PAH Solution (data summary view). ......................38

Figure 13. Fluorene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary 

view). .....................................................................................................................39

Figure 14. Phenanthrene in SRM 1647e PAH Solution (data summary view). ..............40

Figure 15. Phenanthrene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data 

summary view).......................................................................................................41

Figure 16. Anthracene in SRM 1647e PAH Solution (data summary view)...................42

Figure 17. Anthracene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data 

summary view).......................................................................................................43

Figure 18. Fluoranthene in SRM 1647e PAH Solution (data summary view) ................44

Figure 19. Fluoranthene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data 

summary view).......................................................................................................45

Figure 20. Pyrene in SRM 1647e PAH Solution (data summary view) ..........................46

Figure 21. Pyrene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary 

view) ......................................................................................................................47

Figure 22. Benz[a]anthracene in SRM 1647e PAH Solution (data summary view).......48

Figure 23. Benz[a]anthracene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data 

summary view).......................................................................................................49

Figure 24. Chrysene in SRM 1647e PAH Solution (data summary view). .....................50

Figure 25. Chrysene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary 

view) ......................................................................................................................51

Figure 26. Sum of chrysene and triphenylene in SRM 1647e PAH Solution (data 

summary view).......................................................................................................52

Figure 27. Sum of chrysene and triphenylene in SRM 3254 Camellia sinensis (Green 

Tea) Leaves (data summary view).........................................................................53

Figure 28. Benzo[a]pyrene in SRM 1647e PAH Solution (data summary view)............54

Figure 29. Benzo[a]pyrene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data 

summary view).......................................................................................................55
v

Figure 30. Phenanthrene in SRM 1647e PAH Solution and SRM 3254 Camellia sinensis

(Green Tea) Leaves (sample/control comparison view)........................................56

Figure 31. Anthracene in SRM 1647e PAH Solution and SRM 3254 Camellia sinensis

(Green Tea) Leaves (sample/control comparison view)........................................57

Figure 32. Benz[a]anthracene in SRM 1647e PAH Solution and SRM 3254 Camellia 

sinensis (Green Tea) Leaves (sample/control comparison view) ..........................58

Figure 33. Chrysene in SRM 1647e PAH Solution and SRM 3254 Camellia sinensis

(Green Tea) Leaves (sample/control comparison view)........................................59

Figure 34. Sum of chrysene and triphenylene in SRM 1647e PAH Solution and SRM 

3254 Camellia sinensis (Green Tea) Leaves (sample/control comparison view)..60

CHOLINE IN FOODS

Study Overview .................................................................................................................61

Sample Information ...........................................................................................................61

Soy Flour................................................................................................................61

Whole egg powder.................................................................................................61

Study Results .....................................................................................................................61

Technical Recommendations.............................................................................................62

Table 17. Individual data table (NIST) for choline in foods............................................63

Table 18. Data summary table for choline in foods. ........................................................64

Figure 35. Choline in candidate SRM 3234 Soy Flour (data summary view).................65

Figure 36. Choline in candidate SRM 1845a Whole Egg Powder (data summary view)66

Figure 37. Choline in SRM 3234 Soy Flour and candidate SRM 1845a Whole Egg 

Powder (sample/sample comparison view) ...........................................................67

Figure 38. Expanded view of choline in SRM 3234 Soy Flour and candidate SRM 1845a 

Whole Egg Powder (sample/sample comparison view). .......................................68

TOCOPHEROLS IN FOODS

Study Overview .................................................................................................................69

Sample Information ...........................................................................................................69

Carrot extract .........................................................................................................69

Egg powder............................................................................................................69

Study Results .....................................................................................................................69

Technical Recommendations.............................................................................................70

Table 19. Individual data table (NIST) for tocopherols in foods.....................................71

Table 20. Data summary table for α–tocopherol in foods................................................72

Table 21. Data summary table for β–tocopherol in foods................................................73

Table 22. Data summary table for γ–tocopherol in foods................................................74

Table 23. Data summary table for δ–tocopherol in foods................................................75

Table 24. Data summary table for total tocopherol in foods............................................76

Figure 39. α-Tocopherol in SRM 3276 Carrot Extract in Oil (data summary view).......77

Figure 40. α-Tocopherol in candidate SRM 1845a Whole Egg Powder (data summary 

view) ......................................................................................................................78

Figure 41. β-Tocopherol in SRM 3276 Carrot Extract in Oil (data summary view).......79

Figure 42. γ-Tocopherol in SRM 3276 Carrot Extract in Oil (data summary view).......80

Figure 43. γ-Tocopherol in candidate SRM 1845a Whole Egg Powder (data summary 
vi

view) ......................................................................................................................81

Figure 44. δ-Tocopherol in SRM 3276 Carrot Extract in Oil (data summary view).......82

Figure 45. Total tocopherols in SRM 3276 Carrot Extract in Oil (data summary view).83

Figure 46. Total tocopherols in candidate SRM 1845a Whole Egg Powder (data 

summary view).......................................................................................................84

Figure 47. α-Tocopherol in candidate SRM 1845a Whole Egg Powder and SRM 3276 

Carrot Extract in Oil (sample/control comparison view).......................................85

Figure 48. γ-Tocopherol in candidate SRM 1845a Whole Egg Powder and SRM 3276 

Carrot Extract in Oil (sample/control comparison view).......................................86

Figure 49. Total tocopherols in candidate SRM 1845a Whole Egg Powder and SRM 

3276 Carrot Extract in Oil (sample/control comparison view) .............................87

FATTY ACIDS IN BOTANICAL OILS

Study Overview .................................................................................................................88

Sample Information ...........................................................................................................88

Flaxseed oil ............................................................................................................88

Perilla oil................................................................................................................88

Study Results .....................................................................................................................88

Technical Recommendations.............................................................................................89

Table 25. Individual data table (NIST) for fatty acids in botanical oils...........................90

Table 26. Data summary table for linoleic acid in botanical oils.....................................91

Table 27. Data summary table for α-linolenic acid in botanical oils...............................92

Table 28. Data summary table for γ-linolenic acid in botanical oils................................93

Table 29. Data summary table for arachidonic acid in botanical oils..............................94

Figure 50. Linoleic Acid [C18:2, n-6] in SRM 3274-3 Flaxseed Oil (data summary 

view) ......................................................................................................................95

Figure 51. Linoleic Acid [C18:2, n-6] in SRM 3274-4 Perilla Oil (data summary view)96

Figure 52. α-Linolenic Acid [C18:3, n-3] in SRM 3274-3 Flaxseed Oil (data summary 

view) ......................................................................................................................97

Figure 53. α-Linolenic Acid [C18:3, n-3] in SRM 3274-4 Perilla Oil (data summary 

view) ......................................................................................................................98

Figure 54. γ-Linolenic Acid [C18:3, n-6] in SRM 3274-3 Flaxseed Oil (data summary 

view) ......................................................................................................................99

Figure 55. γ-Linolenic Acid [C18:3, n-6] in SRM 3274-4 Perilla Oil (data summary 

view) ....................................................................................................................100

Figure 56. Arachidonic Acid [C20:4, n-6] in SRM 3274-3 Flaxseed Oil (data summary 

view) ....................................................................................................................101

Figure 57. Arachidonic Acid [C20:4, n-6] in SRM 3274-4 Perilla Oil (data summary 

view) ....................................................................................................................102

Figure 58. Linoleic acid in SRM 3274-3 Flaxseed Oil and SRM 3274-4 Perilla Oil

(sample/control comparison view).......................................................................103

Figure 59. α-Linolenic acid in SRM 3274-3 Flaxseed Oil and SRM 3274-4 Perilla Oil

(sample/control comparison view).......................................................................104

PHYTOSTEROLS IN SAW PALMETTO

Study Overview ...............................................................................................................105
vii

Sample Information .........................................................................................................105

Saw palmetto extract............................................................................................105

Saw palmetto berries............................................................................................105

Study Results ...................................................................................................................105

Technical Recommendations...........................................................................................106

Table 30. Individual data table (NIST) for phytosterols in saw palmetto......................107

Table 31. Data summary table for campesterol in saw palmetto ...................................108

Table 32. Data summary table for β-sitosterol in saw palmetto.....................................109

Table 33. Data summary table for stigmasterol in saw palmetto...................................110

Figure 60. Campesterol in SRM 3251 Serenoa repens Extract (data summary view)..111

Figure 61. Campesterol in SRM 3250 Serenoa repens (Fruit) (data summary view) ...112

Figure 62. β-Sitosterol in SRM 3251 Serenoa repens Extract (data summary view)....113

Figure 63. β-Sitosterol in SRM 3250 Serenoa repens (Fruit) (data summary view).....114

Figure 64. Stigmasterol in SRM 3251 Serenoa repens Extract (data summary view)..115

Figure 65. Stigmasterol in SRM 3250 Serenoa repens (Fruit) (data summary view) ...116

Figure 66. Campesterol in SRM 3250 Serenoa repens (Fruit) and SRM 3251 Serenoa 

repens Extract (sample/control comparison view) ..............................................117

Figure 67. β-sitosterol in SRM 3250 Serenoa repens (Fruit) and SRM 3251 Serenoa 

repens Extract (sample/control comparison view) ..............................................118

Figure 68. Stigmasterol in SRM 3250 Serenoa repens (Fruit) and SRM 3251 Serenoa 

repens Extract (sample/control comparison view) ..............................................119
1

ABSTRACT

The NIST Dietary Supplement Laboratory Quality Assurance Program (DSQAP) was 

established in collaboration with the National Institutes of Health (NIH) Office of Dietary 

Supplements (ODS) in 2007 to enable members of the dietary supplements community to 

improve the accuracy of measurements for demonstration of compliance with various 

regulations. Exercise H of this program offered the opportunity for laboratories to assess their 

in-house measurements of nutritional elements (Ca, Cu, and Mn), contaminants (polycyclic 

aromatic hydrocarbons [PAHs]) water-soluble vitamins (choline), fat-soluble vitamins 

(tocopherols), fatty acids, and phytosterols in foods and/or botanical dietary supplement 

ingredients and finished products.

INTRODUCTION

The dietary supplement industry in the US is booming, with two-thirds of adults considering 

themselves to be supplement users.1

 Consumption of dietary supplements, which includes 

vitamin and mineral supplements, represents an annual US expenditure of more than $25 billion. 

These figures represent an increasing American trend, and as a result, it is critically important 

that both the quality and safety of these products are verified and maintained.

The Dietary Supplement Health and Education Act of 1994 (DSHEA) amended the Food, Drug 

and Cosmetic Act to create the regulatory category called dietary supplements. The DSHEA 

also gave the FDA authority to write current Good Manufacturing Practices (cGMPs) that 

require manufacturers to evaluate the identity, purity, and composition of their ingredients and 

finished products. To enable members of the dietary supplements community to improve the 

accuracy of the measurements required for compliance with these and other regulations, NIST 

established the Dietary Supplement Laboratory Quality Assurance Program (DSQAP) in 

collaboration with the NIH ODS in 2007.

The program offers the opportunity for laboratories to assess their in-house measurements of 

active or marker compounds, nutritional elements, contaminants (toxic elements, pesticides, 

mycotoxins), and fat- and water-soluble vitamins in foods as well as botanical dietary 

supplement ingredients and finished products. Reports and certificates of participation are 

provided and can be used to demonstrate compliance with the cGMPs. In addition, NIST and the 

DSQAP assist the ODS Analytical Methods and Reference Materials program (AMRM) at the 

NIH in supporting the development and dissemination of analytical tools and reference materials. 

In the future, results from DSQAP exercises could be used by ODS to identify problematic 

matrices and analytes for which an Official Method of Analysis would benefit the dietary 

supplement community.

NIST has experience in the area of quality assurance programs, but the DSQAP takes a unique 

approach. In other NIST quality assurance programs, a set of analytes is measured repeatedly 

over time in the same or similar matrices to demonstrate laboratory performance. In contrast, the 

 1 Walsh, T. (2012) Supplement Usage, Consumer Confidence Remain Steady According to New Annual Survey from 

CRN. Council for Responsible Nutrition, Washington, DC.
2

wide range of matrices and analytes under the “dietary supplement” umbrella means that not 

every laboratory is interested in every sample or analyte. The constantly changing dietary 

supplement market, and the enormous diversity of finished products, makes repeated 

determination of a few target compounds in a single matrix of little use to participants. Instead, 

participating laboratories are interested in testing in-house methods on a wide variety of 

challenging, real-world matrices to demonstrate that their performance is comparable to that of 

the community and that their methods provide accurate results. In an area where there are few 

standard methods, the DSQAP offers a unique tool for assessment of the quality of 

measurements, provides feedback about performance, and can assist participants in improving 

laboratory operations.

This report summarizes the results from the eighth exercise of the DSQAP, Exercise H. 

Seventy-five laboratories responded to the call for participants distributed in January 2011. 

Samples were shipped to participants in March 2012, and results were returned to NIST by June 

2012. This report contains the final data and information to be disseminated to the participants 

in October 2012.

OVERVIEW OF DATA TREATMENT AND REPRESENTATION

Statistics

The individual data table and graphs contain information about the performance of each 

laboratory relative to that of the other participants in this study and relative to a target around the 

expected result (if available). The consensus mean and standard deviation are calculated 

according to the robust algorithm outlined in ISO 13528:2005(E), Annex C.2

 The algorithm is 

summarized here in simplified form.

Initial values of the consensus mean, x*, and consensus standard deviation, s*, are estimated as

x* = median of xi (i = 1, 2,...,n)

s* = 1.483 × median of |xi – x*| (i = 1, 2,...,n).

These initial values for x* and s* are updated by first calculating the expanded standard 

deviation, δ, as

δ = 1.5 × s*.

Then each xi is compared to the expanded range and adjusted to xi* as described below to reduce

the effect of outliers.

If xi < x* – δ, then xi* = x* – δ.

If xi > x* + δ, then xi* = x* + δ.

Otherwise, xi* = xi.

New values of x*, s*, and δ are calculated iteratively until the process converges. Convergence 

is taken as no change from one iteration to the next in the third significant figure of s* and in the 

equivalent digit in x*:
3

x* = ∑ xi n ∗ i=1

n

s* = 1.134 × �∑ �xi

∗−x∗� n

i=1

n−1 .

Individual Data Table

The data in this table is individualized to each participating laboratory and is provided to allow 

participants to directly compare their data to the summary statistics (consensus or community 

data as well as NIST certified, reference, or estimated values). The upper left of the data table 

includes the randomized laboratory code. Tables included in this report are generated using 

NIST data to protect the identity and performance of participants.

Section 1 of the data table contains the laboratory results as reported, including the mean and

standard deviation when multiple values were reported. A blank indicates that NIST does not 

have data on file for that laboratory for a particular analyte or matrix. An empty box for standard 

deviation indicates that only a single value was reported and therefore that value was not 

included in the calculation of the consensus data.2

Also in Section 1 are two Z-scores. The first Z-score, Zcomm, is calculated with respect to the 

community consensus value, using x* and s*:

Zcomm = xi−x∗

s∗ .

The second Z-score, ZNIST, is calculated with respect to the target value (NIST certified, 

reference, or estimated value), using xNIST and U95 (the expanded uncertainty) or sNIST, the 

standard deviation of NIST measurements:

ZNIST = xi−xNIST

U95

or

ZNIST = xi−xNIST

sNIST

.

The significance of the Z-score is as follows:

• |Z| < 2 indicates that the laboratory result is considered to be within the community 

consensus range (for Zcomm) or NIST target range (for ZNIST).

• 2 < |Z| < 3 indicates that the laboratory result is considered to be marginally different 

from the community consensus value (for Zcomm) or NIST target value (for ZNIST).

• |Z| > 3 indicates that the laboratory result is considered to be significantly different from 

the community consensus value (for Zcomm) or NIST target value (for ZNIST).

 2 ISO 13528:2005(E), Statistical methods for use in proficiency testing by interlaboratory comparisons, pp 14-15.
4

Section 2 of the data table contains the community results, including the number of laboratories 

reporting more than a single value for a given analyte1

, the mean value determined for each 

analyte, and a robust estimate of the standard deviation of the reported values.

3

 Consensus 

means and standard deviations are calculated using the laboratory means; if a laboratory reported 

a single value, the reported value is not included.1 Additional information on calculation of the 

consensus mean and standard deviation can be found in the previous section.

Section 3 of the data table contains the target values for each analyte. When possible, the target

is a certified or reference value determined at NIST. Certified values and the associated

expanded uncertainty (U95) have been determined with two independent analytical methods at 

NIST, by collaborating laboratories, or in some combination. Reference values are assigned 

using NIST values obtained from the average and standard deviation of measurements made 

using a single analytical method. For both certified and reference values, at least six samples 

have been tested and duplicate preparations from the sample package have been included, 

allowing the uncertainty to encompass variability due to inhomogeneity within and between 

packages. For commercial products, the analytes are measured at NIST using an appropriate 

method. The NIST value represents the mean of at least three replicates.

Summary Data Table

This data table includes a summary of all reported data for a particular analyte in a particular 

study. Participants can compare the raw data for a single laboratory to the other participating 

laboratories or to the consensus data. A blank indicates that the laboratory signed up and 

received samples for that particular analyte and matrix, but NIST does not have data on file for 

that laboratory.

Graphs

Data Summary View

In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). Data points that are unfilled represent laboratories that only reported a 

single value for that analyte and therefore were not included in the consensus mean. The black 

solid line represents the consensus mean, and the black dotted lines represent the consensus 

variability calculated as one standard deviation about the consensus mean. The gray shaded 

region represents the target zone for “acceptable” performance, which encompasses the NIST 

certified, reference, or estimated value bounded by twice its uncertainty (U95) or standard 

deviation. For the purpose of the DSQAP, a target range spanning twice the uncertainty in the 

NIST value is selected because participants are only asked to make a limited number of 

observations. The size of the y-axis on the data summary view graph represents the consensus 

mean bounded by 2δ. In this view, the relative locations of individual laboratory data and 

consensus zones with respect to the target zone can be compared easily. In most cases, the target 

zone and the consensus zone overlap, which is the expected result. One program goal is to 

reduce the size of the consensus zone and center the consensus zone about the target value. 

Analysis of an appropriate reference material as part of a quality control scheme can help to 

identify sources of bias for laboratories reporting results that are significantly different from the 

target zone.

 3 ISO 13528:2005(E), Statistical methods for use in proficiency testing by interlaboratory comparisons, Annex C.
5

Sample/Control Comparison View (Sample/Sample Comparison View)

In this view, the individual laboratory results for a control (NIST SRM with a certified value) are 

compared to the results for an unknown (another NIST SRM with a more challenging matrix, a 

commercial sample, etc.). The error bars represent the individual laboratory standard deviation. 

The solid red box represents the target zone for the control (x-axis) and unknown sample (yaxis). The dotted blue box represents the consensus zone for the control (x-axis) and the 

unknown sample (y-axis). This view emphasizes trends in the data that may indicate potential 

calibration issues or method biases. One program goal is to identify such calibration or method 

biases and assist participants in improving analytical measurement capabilities. In some cases, 

when two equally challenging materials are provided, the same view (sample/sample 

comparison) can be helpful in identifying commonalities or differences in the analysis of the two 

materials.
6

NUTRITIONAL ELEMENTS IN FOODS AND SUPPLEMENTS

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 1566b Oyster Tissue and

candidate SRM 3532 Calcium Dietary Supplement. Participants were asked to use in-house 

analytical methods to determine the mass fractions of three nutritional elements (calcium, 

copper, and manganese) in each of the matrices and report values on an as-received basis.

Sample Information

Oyster tissue. Participants were provided with six vials, each containing approximately 1 g of 

freeze-dried, powdered oyster tissue. The material was prepared from oysters collected in the 

Gulf of Mexico that had been shucked, rinsed, and blended both before and after freeze drying. 

Before use, participants were instructed to thoroughly mix the contents of the vial and use a 

sample size of at least 0.25 g. Participants were asked to report a single value from each pair of 

vials and store the material at controlled room temperature, 10 °C to 30 °C. Approximate 

analyte levels were not reported prior to the study. NIST certified values in SRM 1566b were 

determined using inductively coupled plasma mass spectrometry (ICP-MS), isotope dilution

ICP-MS, instrumental neutron activation analysis (INAA), and radiochemical neutron activation 

analysis (RNAA). The certified values and uncertainties for Ca, Cu, and Mn in SRM 1566b are 

outlined in the table below, both on a dry-mass basis and an as-received basis following 

adjustment for the moisture content of the material, 2.9 %.

Certified Mass Fraction (mg/kg) Adjusted Mass Fraction (mg/g)

Analyte (dry-mass basis) (as-received basis)

Ca 838 ± 20 0.813 ± 0.019

Cu 71.6 ± 1.6 0.0695 ± 0.0016

Mn 18.5 ± 0.2 0.0180 ± 0.0002

Ca supplement. Participants were provided with one packet containing approximately 10 g of 

calcium dietary supplement powder. The calcium supplements were purchased commercially, 

then ground, sieved, and heat-sealed inside nitrogen-flushed 0.1 mm (4 mil) polyethylene bags, 

which were then sealed inside aluminized plastic bags with 2 packets of silica gel. Before use, 

participants were instructed to thoroughly mix the contents of the packet and use a sample size of 

at least 0.25 g. Participants were asked to report three values from the single packet provided 

and store the material at controlled room temperature, 10 °C to 30 °C. Approximate analyte 

levels were not reported prior to the study. NIST values in candidate SRM 3532 will be certified 

using X-ray fluorescence (XRF) and inductively coupled plasma optical emission spectrometry 

(ICP-OES) following microwave digestion using standard additions as the method of 

quantitation. The preliminary NIST values in candidate SRM 3532, estimated from the mean of 

these two methods of analysis, are reported in the table below with an estimated uncertainty of 

5 %.

Estimated Certified Mass Fraction (mg/g)

Analyte (as-received basis)

Ca 170 ± 8.5

Cu 0.270 ± 0.014

Mn 0.530 ± 0.027
7

Study Results

• Forty-nine laboratories enrolled in this exercise and received samples. Thirty-seven

laboratories reported results for calcium (76 % participation), 38 laboratories reported 

results for copper (78 % participation), and 34 laboratories reported results for

manganese (71 % participation). 

• The consensus means for calcium and copper in the dietary supplement were within the 

target range with acceptable variability (6 % and 14 % relative standard deviation (RSD), 

respectively). The consensus mean for manganese in the dietary supplement was within 

but near the low end of the target range, again with acceptable variability (11 % RSD).

• The consensus mean for calcium in the oyster tissue was within but at the high end of the 

target range, while the consensus mean for copper was within but near the low end of the 

target range. Again, both had acceptable variability (15 % and 9 % RSD, respectively). 

The consensus mean for manganese in the oyster tissue was within the target range with 

acceptable variability (9 % RSD). 

• A majority of the laboratories reported using either open-beaker digestion (29 % to 41 %) 

or microwave digestion (32 % to 41 %) for sample preparation. Some laboratories used 

hot block digestion (15 % to 16 %). Other laboratories reported using dry ashing or

partial digestion within plastic bottles.

• A majority of the laboratories reported using either ICP-OES (46 %) or ICP-MS (41 %) 

as their analytical method. Other laboratories reported using atomic absorption 

spectroscopy (AAS), titrimetry, or total reflection X-ray fluorescence (TXRF). 

Technical Recommendations

The following recommendations are based on results obtained by the participants in this study. 

• While there seemed to be only a slight difference in results between open-beaker 

digestions and microwave digestions, it did appear that the open-beaker digestions were 

slightly more effective, with more results in the consensus range. Open-beaker digestions

work well for these three elements since they are neither easily volatilized nor found as 

contaminants in most laboratories. Participants would be able to digest materials until 

they could actually see that the material was fully dissolved.

• When using ICP-OES for value assignment, there are usually several wavelengths 

available for each analyte. Using several wavelengths for each analyte helps in the 

determination of interferences or background shifts due to matrix effects at any one 

wavelength.

• With both ICP-OES and ICP-MS, it is important to check the calibration curve for 

linearity. 

• With ICP-OES, some elements will only be linear within a specific range. 

Solution concentrations need to fall within that linear range. 

• With ICP-MS, many instruments run in pulse mode, which is more sensitive. If 

the calibration curve goes outside of the dynamic range for pulse mode then the 

instrument will use both the pulse and analog mode. The ICP-MS must be 

calibrated for both modes in this case. It is often easier and more accurate to have 

a narrower range of calibration points, making sure the calibration curve is linear 

in the pulse mode.
8

• More accurate measurements can be achieved by making sure the sample concentrations 

fall within the middle of the calibration curve.

• Double-check all calculations; this is a cause for many errors. 
9

Table 1. Individual data table (NIST) for nutritional elements in foods and dietary supplements.
10

Table 2. Data summary table for calcium in foods and dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 0.813 0.019 170 8.5

H802

H803

H804 0.760 0.716 0.748 0.741 0.023 165 168 170 168 2.8

H805 0.816 0.782 0.775 0.791 0.022 178 180 179 179 1.0

H806 0.928 0.893 0.877 0.899 0.026 182 180 179 180 1.7

H807 0.887 0.846 0.885 0.873 0.023 181 188 186 185 4.0

H808 0.778 0.753 0.754 0.762 0.014 172 156 158 162 8.7

H809

H810 0.832 0.821 0.815 0.823 0.009 165 164 163 164 1.0

H811 1.626 1.627 1.610 1.621 0.010 178 178 180 179 0.9

H812 1.010 1.000 1.000 1.003 0.006 179 181 180 180 1.0

H814 3.570 3.480 3.520 3.523 0.045 167 168 171 169 2.1

H816 4.370 5.080 3.490 4.313 0.797 163 165 154 161 5.8

H817 0.810 0.770 0.790 0.790 0.020 153 156 167 159 7.3

H819 0.824 0.938 0.798 0.853 0.074 163 163 159 162 2.3

H820 1.071 0.793 0.888 0.918 0.141 164 166 166 165 1.3

H821 0.870 0.880 0.870 0.873 0.006 159 160 159 159 0.6

H822 1.120 1.040 1.040 1.067 0.046 164 165 164 164 0.6

H825 0.840 0.840 0.860 0.847 0.012 170 170 170 170 0.0

H828

H829

H831 0.830 0.820 0.830 0.827 0.006 177 172 160 169 8.9

H833 0.880 0.860 0.860 0.867 0.012 177 177 174 176 1.9

H836 0.787 0.855 0.752 0.798 0.052 168 170 170 170 1.4

H840 0.878 0.874 0.863 0.872 0.008 179 164 176 173 7.6

H843

H844 0.780 0.773 0.775 0.776 0.003 169 171 170 170 1.0

H847 0.727 0.678 0.724 0.710 0.027 169 169 169 169 0.2

H848 0.751 0.764 0.757 0.757 0.007 168 169 170 169 0.8

H849 0.855 0.867 0.873 0.865 0.009 169 174 172 3.6

H850 0.833 0.846 0.841 0.840 0.007 173 172 171 172 0.8

H852 0.857 0.852 0.860 0.857 0.004 184 189 183 185 3.4

H853 0.813 0.822 0.786 0.807 0.018 214 216 212 214 2.3

H854

H857 0.573 0.585 0.577 0.578 0.006 192 181 177 184 7.8

H858 1.441 1.420 1.365 1.409 0.039 267 268 269 268 0.7

H861

H862 1.001 0.917 1.026 0.981 0.057 162 167 159 163 3.9

H863 1.800 1.760 1.600 1.720 0.106 173 174 174 173 0.7

H864

H866

H867 0.680 0.620 0.667 0.656 0.031 71 70 88 76 10.0

H868 0.666 0.646 0.690 0.667 0.022 119 117 119 118 1.2

H869

H870 0.864 0.770 0.910 0.848 0.071 188 105 188 160 47.9

H871 0.670 0.620 0.660 0.650 0.026 167 166 165 166 0.7

H873

H874 0.800 0.800 0.800 0.800 0.000 186 186

H875 0.830 0.770 0.800 0.800 0.030 152 151 138 147 8.1

 Consensus Mean 0.852 Consensus Mean 170

 Consensus Standard Deviation 0.137 Consensus Standard Deviation 10.6

 Maximum 4.313 Maximum 268

 Minimum 0.578 Minimum 76

 N 37 N 36

Calcium

SRM 1566b Oyster Tissue (mg/g) SRM 3532 Calcium Dietary Supplement (mg/g)

Community 

Results Individual Results
11

Table 3. Data summary table for copper in foods and dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 0.0695 0.0016 0.270 0.014

H802

H803

H804 0.0619 0.0595 0.0618 0.0611 0.0014 0.245 0.245 0.242 0.244 0.002

H805 0.0676 0.0667 0.0674 0.0672 0.0005 0.274 0.286 0.278 0.279 0.006

H806 0.0749 0.0743 0.0744 0.0745 0.0003 0.311 0.314 0.317 0.314 0.003

H807 0.0657 0.0660 0.0676 0.0664 0.0010 0.256 0.255 0.258 0.256 0.002

H808 0.0640 0.0654 0.0642 0.0645 0.0008 0.235 0.233 0.237 0.235 0.002

H809

H810 0.0707 0.0704 0.0702 0.0704 0.0003 0.277 0.272 0.272 0.274 0.003

H811 0.0600 0.0600 0.0596 0.0599 0.0002 0.211 0.212 0.215 0.213 0.002

H812 0.0760 0.0770 0.0750 0.0760 0.0010 0.299 0.301 0.300 0.300 0.001

H814 0.0800 0.0740 0.0780 0.0773 0.0031 0.331 0.326 0.322 0.326 0.005

H816 0.0650 0.0650 0.0650 0.0650 0.0000 0.270 0.280 0.280 0.277 0.006

H817 0.0700 0.0700 0.0700 0.0700 0.0000 0.270 0.260 0.270 0.267 0.006

H819 0.0728 0.0827 0.0713 0.0756 0.0062 0.309 0.305 0.301 0.305 0.004

H820 0.0647 0.0637 0.0655 0.0646 0.0009 0.314 0.320 0.315 0.316 0.003

H821 0.0790 0.0770 0.0780 0.0780 0.0010 0.245 0.253 0.246 0.248 0.004

H822 0.0714 0.0718 0.0683 0.0705 0.0019 0.245 0.266 0.259 0.257 0.011

H825 0.0710 0.0720 0.0740 0.0723 0.0015 0.220 0.220 0.220 0.220 0.000

H828

H829

H831 0.0700 0.0700 0.0700 0.0700 0.0000 0.280 0.280 0.280 0.280 0.000

H833 0.0700 0.0700 0.0700 0.0700 0.0000 0.270 0.270 0.270 0.270 0.000

H836 0.0674 0.0692 0.0663 0.0676 0.0015 0.276 0.273 0.277 0.275 0.002

H840 0.0759 0.0759 0.0190 0.0569 0.0329 0.295 0.288 0.286 0.290 0.005

H843

H844 0.0721 0.0719 0.0720 0.0720 0.0001 0.307 0.311 0.315 0.311 0.004

H847 0.0610 0.0620 0.0600 0.0610 0.0010 0.359 0.375 0.370 0.368 0.008

H848 0.0550 0.0550 0.0570 0.0557 0.0012 0.308 0.327 0.329 0.321 0.012

H849 0.0729 0.0744 0.0749 0.0741 0.0010 0.273 0.266 0.270 0.005

H850 0.0726 0.0731 0.0732 0.0730 0.0003 0.298 0.299 0.300 0.299 0.001

H852 0.0701 0.0690 0.0700 0.0697 0.0006 0.267 0.270 0.267 0.268 0.002

H853 0.0653 0.0642 0.0619 0.0638 0.0017 0.324 0.327 0.326 0.326 0.002

H854

H857 0.0700 0.0710 0.0690 0.0700 0.0010 0.280 0.269 0.264 0.271 0.008

H858 0.0664 0.0677 0.0665 0.0668 0.0007 0.193 0.192 0.193 0.193 0.001

H861

H862 0.0700 0.0700 0.0696 0.0699 0.0002 0.302 0.304 0.314 0.307 0.006

H863 0.0531 0.0531 0.0575 0.0546 0.0025 0.308 0.298 0.292 0.299 0.008

H864

H866

H867 0.0679 0.0735 0.0648 0.0687 0.0044 0.237 0.171 0.323 0.244 0.076

H868 0.0640 0.0641 0.0626 0.0636 0.0008 0.231 0.245 0.239 0.238 0.007

H869

H870 0.0516 0.0551 0.0538 0.0535 0.0018 0.149 0.143 0.120 0.137 0.015

H871 0.0590 0.0610 0.0620 0.0607 0.0015 0.260 0.260 0.230 0.250 0.017

H873 0.0671 0.0579 0.0589 0.0613 0.0050 0.202 0.205 0.210 0.206 0.004

H874 0.0672 0.0657 0.0669 0.0666 0.0008 0.242 0.242

H875 0.0640 0.0660 0.0640 0.0647 0.0012 0.290 0.280 0.290 0.287 0.006

 Consensus Mean 0.0673 Consensus Mean 0.273

 Consensus Standard Deviation 0.0065 Consensus Standard Deviation 0.040

 Maximum 0.0780 Maximum 0.368

 Minimum 0.0535 Minimum 0.137

 N 38 N 37

Copper

SRM 1566b Oyster Tissue (mg/g) SRM 3532 Calcium Dietary Supplement (mg/g)

Individual Results Community Results
12

Table 4. Data summary table for manganese in foods and dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 0.0180 0.0002 0.530 0.027

H802

H803

H804 0.0178 0.0169 0.0173 0.0173 0.0004 0.470 0.506 0.465 0.480 0.022

H805 0.0179 0.0178 0.0180 0.0179 0.0001 0.469 0.451 0.446 0.455 0.012

H806 0.0190 0.0183 0.0183 0.0185 0.0004 0.463 0.455 0.455 0.458 0.005

H807 0.0160 0.0163 0.0159 0.0161 0.0002 0.535 0.503 0.522 0.520 0.016

H808 0.0158 0.0164 0.0159 0.0160 0.0003 0.462 0.473 0.461 0.465 0.007

H809

H810 0.0181 0.0181 0.0179 0.0180 0.0001 0.533 0.500 0.498 0.510 0.020

H811 0.0220 0.0209 0.0206 0.0212 0.0007 0.503 0.495 0.489 0.495 0.007

H812

H814 0.0840 0.0820 0.0820 0.0827 0.0012 0.529 0.522 0.535 0.529 0.007

H816 0.0450 0.0200 0.0200 0.0283 0.0144 0.520 0.500 0.610 0.543 0.059

H817 0.0199 0.0195 0.0194 0.0196 0.0003 0.409 0.430 0.433 0.424 0.013

H819 0.0187 0.0213 0.0184 0.0195 0.0016 0.482 0.513 0.453 0.483 0.030

H820 0.470 0.460 0.478 0.470 0.009

H821 0.0200 0.0200 0.0200 0.0200 0.0000 0.475 0.475 0.452 0.467 0.013

H822

H825 0.0180 0.0180 0.0180 0.0180 0.0000 0.500 0.500 0.530 0.510 0.017

H828

H829

H831 0.0200 0.0200 0.0200 0.0200 0.0000 0.440 0.430 0.430 0.433 0.006

H833 0.0200 0.0200 0.0200 0.0200 0.0000 0.440 0.430 0.440 0.437 0.006

H836 0.0169 0.0163 0.0160 0.0164 0.0005 0.434 0.446 0.439 0.440 0.006

H840 0.0190 0.0190 0.0187 0.0189 0.0002 0.430 0.423 0.403 0.419 0.014

H843

H844 0.0175 0.0175 0.0175 0.0175 0.0000 0.479 0.478 0.496 0.484 0.010

H847 0.0160 0.0160 0.0160 0.0160 0.0000 0.573 0.521 0.558 0.551 0.027

H848 0.0160 0.0170 0.0170 0.0167 0.0006 0.565 0.614 0.638 0.606 0.037

H849 0.0187 0.0190 0.0189 0.0189 0.0002 0.475 0.486 0.481 0.008

H850 0.0181 0.0183 0.0182 0.0182 0.0001 0.542 0.510 0.490 0.514 0.026

H852 0.0183 0.0182 0.0183 0.0183 0.0001 0.525 0.540 0.548 0.538 0.012

H853 0.0154 0.0151 0.0145 0.0150 0.0004 0.522 0.586 0.567 0.558 0.033

H854

H857 0.0190 0.0190 0.0180 0.0187 0.0006 0.523 0.535 0.559 0.539 0.018

H858 0.0189 0.0188 0.0183 0.0187 0.0003 0.524 0.527 0.584 0.545 0.034

H861

H862 0.0190 0.0185 0.0188 0.0188 0.0003 0.452 0.518 0.488 0.486 0.033

H863

H864

H866

H867 0.0132 0.0185 0.0198 0.0172 0.0035 0.391 0.312 0.234 0.312 0.079

H868 0.0168 0.0170 0.0166 0.0168 0.0002 0.490 0.509 0.511 0.503 0.012

H869

H870 0.0183 0.0178 0.0224 0.0195 0.0025 0.640 0.594 0.706 0.647 0.056

H871 0.0160 0.0160 0.0160 0.0160 0.0000 0.440 0.480 0.450 0.457 0.021

H873 0.0189 0.0169 0.0171 0.0176 0.0011 0.410 0.425 0.436 0.423 0.013

H874 0.0181 0.0175 0.0178 0.0178 0.0003 0.415 0.415

H875 0.0160 0.0170 0.0170 0.0167 0.0006 0.430 0.420 0.420 0.423 0.006

 Consensus Mean 0.0182 Consensus Mean 0.488

 Consensus Standard Deviation 0.0017 Consensus Standard Deviation 0.054

 Maximum 0.0827 Maximum 0.647

 Minimum 0.0150 Minimum 0.312

 N 34 N 34

Manganese

SRM 1566b Oyster Tissue (mg/g) SRM 3532 Calcium Dietary Supplement (mg/g)

Individual Results Community Results
13

Figure 1. Calcium in SRM 1566b Oyster Tissue (data summary view). In this view, individual 

laboratory data are plotted with the individual laboratory standard deviation (error bars). The 

black solid line represents the consensus mean, and the black dotted lines represent the 

consensus variability calculated as one standard deviation about the consensus mean. The gray 

shaded region represents the target zone for “acceptable” performance, which encompasses the 

NIST certified value bounded by twice its uncertainty (U95).
14

Figure 2. Calcium in candidate SRM 3532 Calcium Dietary Supplement (data summary view). 

In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). Data points that are unfilled represent laboratories that only reported a 

single value for that analyte and therefore were not included in the consensus mean. The black 

solid line represents the consensus mean, and the black dotted lines represent the consensus 

variability calculated as one standard deviation about the consensus mean. The gray shaded 

region represents the target zone for “acceptable” performance, which encompasses an 

approximation of the NIST certified value bounded by an approximated uncertainty of 5 %. The 

approximate certified value is the mean of results from ICP-OES and XRF.
15

Figure 3. Copper in SRM 1566b Oyster Tissue (data summary view). In this view, individual 

laboratory data are plotted with the individual laboratory standard deviation (error bars). The 

black solid line represents the consensus mean, and the black dotted lines represent the 

consensus variability calculated as one standard deviation about the consensus mean. The gray 

shaded region represents the target zone for “acceptable” performance, which encompasses the 

NIST certified value bounded by twice its uncertainty (U95).
16

Figure 4. Copper in candidate SRM 3532 Calcium Dietary Supplement (data summary view). 

In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). Data points that are unfilled represent laboratories that only reported a 

single value for that analyte and therefore were not included in the consensus mean. The black 

solid line represents the consensus mean, and the black dotted lines represent the consensus 

variability calculated as one standard deviation about the consensus mean. The gray shaded 

region represents the target zone for “acceptable” performance, which encompasses an 

approximation of the NIST certified value bounded by an approximated uncertainty of 5 %. The 

approximate certified value is the mean of results from ICP-OES and XRF.
17

Figure 5. Manganese in SRM 1566b Oyster Tissue (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
18

Figure 6. Manganese in candidate SRM 3532 Calcium Dietary Supplement (data summary 

view). In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). Data points that are unfilled represent laboratories that only reported a 

single value for that analyte and therefore were not included in the consensus mean. The black 

solid line represents the consensus mean, and the black dotted lines represent the consensus 

variability calculated as one standard deviation about the consensus mean. The gray shaded 

region represents the target zone for “acceptable” performance, which encompasses an 

approximation of the NIST certified value bounded by an approximated uncertainty of 5 %. The 

approximate certified value is the mean of results from ICP-OES and XRF.
19

Figure 7. Calcium in SRM 1566b Oyster Tissue and candidate SRM 3532 Calcium Dietary 

Supplement (sample/sample comparison view). In this view, the individual laboratory results for 

one sample (SRM 1566b Oyster Tissue) with a certified value for the analyte are compared to the 

results for a second sample (candidate SRM 3532 Calcium Dietary Supplement). The error bars 

represent the individual laboratory standard deviation. The solid red lines represent the target 

zone for the control (x-axis) and unknown (y-axis). The dotted blue box represents the 

consensus zone for the control (x-axis) and the unknown sample (y-axis). 
20

Figure 8. Copper in SRM 1566b Oyster Tissue and candidate SRM 3532 Calcium Dietary 

Supplement (sample/sample comparison view). In this view, the individual laboratory results for 

one sample (SRM 1566b Oyster Tissue) with a certified value for the analyte are compared to the 

results for a second sample (candidate SRM 3532 Calcium Dietary Supplement). The error bars 

represent the individual laboratory standard deviation. The solid red lines represent the target 

zone for the control (x-axis) and unknown (y-axis). The dotted blue box represents the 

consensus zone for the control (x-axis) and the unknown sample (y-axis).
21

Figure 9. Manganese in SRM 1566b Oyster Tissue and candidate SRM 3532 Calcium Dietary 

Supplement (sample/sample comparison view). In this view, the individual laboratory results for 

one sample (SRM 1566b Oyster Tissue) with a certified value for the analyte are compared to the 

results for a second sample (candidate SRM 3532 Calcium Dietary Supplement). The error bars 

represent the individual laboratory standard deviation. The solid red lines represent the target 

zone for the control (x-axis) and unknown (y-axis). The dotted blue box represents the 

consensus zone for the control (x-axis) and the unknown sample (y-axis). 
22

PAHs IN GREEN TEA

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 1647e PAH Solution and 

SRM 3254 Camellia sinensis (Green Tea) Leaves. Participants were asked to use in-house 

analytical methods to determine the mass fractions of ten polycyclic aromatic hydrocarbons 

(PAHs) – naphthalene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, 

benz[a]anthracene, chrysene, triphenylene, and benzo[a]pyrene) – in each of the matrices and 

report values on an as-received basis.

Sample Information

Neat solution. Participants were provided with three ampoules, each containing approximately 

1.2 mL of an acetonitrile solution of 16 PAHs. The solution was prepared gravimetrically from 

individual compounds and aliquotted into 2 mL amber glass ampoules, which were purged with 

argon prior to adding the solution. Before use, participants were instructed to mix thoroughly the 

contents of the ampoule. Participants were asked to report a single value from each ampoule and 

store the material at controlled room temperature, 10 °C to 30 °C. Approximate analyte levels 

were not reported prior to the study. NIST certified values in SRM 1647e were based on the 

gravimetric preparation with purity assessment of the neat PAHs and analysis using liquid 

chromatography (LC) with absorbance detection. The certified values and uncertainties for each 

PAH in SRM 1647e are reported in the table below.

Green tea. Participants were provided with three packets, each containing approximately 3 g of 

green tea (Camellia sinensis) leaves. The green tea leaves were ground, sieved, and heat-sealed 

inside nitrogen-flushed 0.1 mm (4 mil) polyethylene bags, which were then sealed inside 

aluminized plastic bags with 2 packets of silica gel. Before use, participants were instructed to

thoroughly mix the contents of the packet and use a sample size of at least 0.3 g. Participants 

were asked to report a single value from each packet and store the material at controlled room 

temperature, 10 °C to 30 °C. Approximate analyte levels were not reported prior to the study.

Values in SRM 3254 were determined by gas chromatography (GC) with MS detection 

following pressurized-fluid extraction. The estimated values are based on an average and 

standard deviation of single measurements from three packets and are provided on an as-received 

basis in the table below.

Certified Mass Fraction NIST-Determined Mass Fraction

Analyte in SRM 1647e (mg/kg) in SRM 3254 (ng/g)

Naphthalene 25.48 ± 0.58 48.1 ± 4.0

Fluorene 6.09 ± 0.14 12.7 ± 2.6

Phenanthrene 4.52 ± 0.11 102 ± 13

Anthracene 1.01 ± 0.02 4.22 ± 0.69

Fluoranthene 9.73 ± 0.21 47.4 ± 5.1

Pyrene 10.88 ± 0.22 27.7 ± 1.5

Benz[a]anthracene 5.25 ± 0.11 4.24 ± 0.25

Chrysene 4.62 ± 0.10

Chrysene + Triphenylene 15.9 ± 0.4

Benzo[a]pyrene 6.25 ± 0.15
23

Study Results

• Twelve laboratories enrolled in this exercise and received samples, and six laboratories 

reported results for at least some of the PAHs (50 % participation).

• The consensus means for all PAHs in the neat solution were lower than the target range 

with high variability (35 % to over 100 % RSD).

• The consensus means for all PAHs in the green tea were higher than the target range with 

high variability (53 % to over 100 % RSD).

• Two laboratories (40 %) reported using pressurized-fluid extraction for sample 

preparation, two laboratories (40 %) reported using direct injection, and one laboratory 

(20 %) reported using Soxhlet extraction.

• Three laboratories (60 %) used GC-MS as their analytical method. Two laboratories 

(40 %) reported using GC with flame ionization detection (FID).

• Three laboratories (60 %) reported using an internal standard approach to calibration, and 

two laboratories (40 %) reported using an external standard approach to calibration.

Technical Recommendations

While more data is needed to draw strong conclusions about results of this study, the following 

recommendations are based on results that were obtained by the participants in this study. 

• Low values obtained for the neat solution could be the result of improper calibration.

• Low values obtained for the neat solution could also be the result of excessive sample 

preparation. The neat solution only required dilution prior to injection.

• High results for the green tea sample could be a result of improper calibration. This is

especially true if the differences between certified values and measured values for the 

control (SRM 1647e) were used to calculate a correction factor. Correction factors 

frequently lead to biased results due to differences in matrix effects.
24

Table 5. Individual data table (NIST) for PAHs in green tea.
25

Table 6. Data summary table for naphthalene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 25480 580 48.1 4.0

H803

H810

H813

H818 12968 12199 12308 12492 416 115 330 270 238 111

H819 21700 20400 20300 20800 781

H821

H843

H847 3288 3580 3759 3542 238

H848

H862

H865

H873 23310 23920 23930 23720 355 660 720 690 690 30

 Consensus Mean 15138 Consensus Mean 464

 Consensus Standard Deviation 10293 Consensus Standard Deviation 362

 Maximum 23720 Maximum 690

 Minimum 3542 Minimum 238

 N 4 N 2

Naphthalene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Community 

Results Individual Results
26

Table 7. Data summary table for fluorene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 6090 140 12.7 2.6

H803

H810

H813

H818 3636 3497 3442 3525 100 90 90 100 93.3 5.8

H819 5080 5100 5260 5147 99

H821

H843

H847 328 357 363 349 19 534 446 499 493.1 44.4

H848 464 339 379 394 64 445 424 408 425.7 18.6

H862

H865

H873 4920 5570 4910 5133 378 240 280 270 263.3 20.8

 Consensus Mean 2910 Consensus Mean 319

 Consensus Standard Deviation 2732 Consensus Standard Deviation 203

 Maximum 5147 Maximum 493

 Minimum 349 Minimum 93

 N 5 N 4

Fluorene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results
27

Table 8. Data summary table for phenanthrene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 4520 110 102.0 13.0

H803

H810

H813

H818 3054 2836 2821 2903 130 95 105 95 98.3 5.8

H819 4130 4020 4200 4117 91

H821

H843

H847 261 258 259 259 2 76 120 143 113.0 34.0

H848 331 255 268 285 40 122 109 119 116.7 6.8

H862

H865

H873 4010 4060 4190 4087 93 390 440 440 423.3 28.9

 Consensus Mean 2330 Consensus Mean 188

 Consensus Standard Deviation 2202 Consensus Standard Deviation 178

 Maximum 4117 Maximum 423

 Minimum 259 Minimum 98

 N 5 N 4

Phenanthrene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results
28

Table 9. Data summary table for anthracene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 1010 20 4.2 0.7

H803

H810

H813

H818 793 800 668 754 74 ND ND ND

H819 923 897 949 923 26

H821

H843

H847 53 52 50 52 2 124.4 23.0 28.0 58.5 57.2

H848 65 52 50 56 8 30.8 29.4 29.7 30.0 0.7

H862

H865

H873 890 990 1020 967 68 80.0 70.0 90.0 80.0 10.0

 Consensus Mean 550 Consensus Mean 56.1

 Consensus Standard Deviation 522 Consensus Standard Deviation 28.5

 Maximum 967 Maximum 80.0

 Minimum 52 Minimum 30.0

 N 5 N 3

Anthracene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results
29

Table 10. Data summary table for fluoranthene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 9730 210 47.4 5.1

H803

H810

H813

H818 5742 4965 5035 5247 430 40.0 45.0 40.0 42 3

H819 9200 9460 10100 9587 463

H821

H843

H847

H848

H862

H865

H873 9950 9300 9030 9427 473 260.0 250.0 240.0 250 10

 Consensus Mean 8087 Consensus Mean 146

 Consensus Standard Deviation 2790 Consensus Standard Deviation 167

 Maximum 9587 Maximum 250

 Minimum 5247 Minimum 41.7

 N 3 N 2

Fluoranthene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results
30

Table 11. Data summary table for pyrene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 10880 220 27.7 1.5

H803

H810

H813

H818 6301 5478 5517 5765 465 25.0 30.0 30.0 28.3 2.9

H819 10600 10800 11400 10933 416

H821

H843

H847 644 620 570 611 38

H848 753 583 609 648 92

H862

H865

H873 10370 10680 10380 10477 176 180.0 160.0 160.0 166.7 11.5

 Consensus Mean 5687 Consensus Mean 97.5

 Consensus Standard Deviation 5716 Consensus Standard Deviation 111

 Maximum 10933 Maximum 167

 Minimum 611 Minimum 28.3

 N 5 N 2

Pyrene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results
31

Table 12. Data summary table for benz[a]anthracene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 5250 110 4.2 0.3

H803

H810

H813

H818 2890 2580 2587 2686 177 15.0 ND 10.0 12.5 3.5

H819 5330 5620 5900 5617 285

H821

H843

H847 334 270 228 277 53 66.4 79.0 99.0 81.5 16.4

H848 352 272 271 298 46 63.7 72.5 56.2 64.1 8.2

H862

H865 3731 3852 3855 3813 71 2.8 2.9 3.0 2.9 0.1

H873 5320 4640 5110 5023 348 90.0 80.0 90.0 86.7 5.8

 Consensus Mean 2952 Consensus Mean 49.5

 Consensus Standard Deviation 2606 Consensus Standard Deviation 44.5

 Maximum 5617 Maximum 86.7

 Minimum 277 Minimum 2.9

 N 6 N 5

Benz(a)anthracene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results
32

Table 13. Data summary table for chrysene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 4620 100

H803

H810

H813

H818 2929 2735 2852 2839 98 10.0 ND 10.0 10.0 0.0

H819 4330 4540 4460 4443 106

H821

H843

H847 299 251 207 253 46 39.5 46.0 68.0 51.2 14.9

H848 331 252 258 280 44 149.1 228.0 206.0 194.4 40.7

H862

H865 3470 3551 3661 3561 96 9.4 9.4 10.0 9.6 0.4

H873 6110 5570 4890 5523 611 90.0 80.0 90.0 86.7 5.8

 Consensus Mean 2816 Consensus Mean 70.4

 Consensus Standard Deviation 2460 Consensus Standard Deviation 86.6

 Maximum 5523 Maximum 194

 Minimum 253 Minimum 9.6

 N 6 N 5

Chrysene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results
33

Table 14. Data summary table for triphenylene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST

H803

H810

H813

H818 2929 2735 2852 2839 98 10.0 ND 10.0 10.00 0.00

H819

H821

H843

H847

H848

H862

H865

H873

 Consensus Mean Consensus Mean

 Consensus Standard Deviation Consensus Standard Deviation

 Maximum Maximum

 Minimum Minimum

 N 1 N 1

Triphenylene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results
34

Table 15. Data summary table for chrysene + triphenylene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 4620 100 15.9 0.4

H803

H810

H813

H818 5859 5470 5703 5677 196 20.0 20.0 20.0 0.0

H819 4330 4540 4460 4443 106

H821

H843

H847 299 251 207 253 46 39.5 46.0 68.0 51.2 14.9

H848 331 252 258 280 44 149.1 228.0 206.0 194.4 40.7

H862

H865 3470 3551 3661 3561 96 9.4 9.4 10.0 9.6 0.4

H873 6110 5570 4890 5523 611 90.0 80.0 90.0 86.7 5.8

 Consensus Mean 3290 Consensus Mean 72.4

 Consensus Standard Deviation 2795 Consensus Standard Deviation 84.5

 Maximum 5677 Maximum 194

 Minimum 253 Minimum 9.6

 N 6 N 5

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results

Chrysene + Triphenylene
35

Table 16. Data summary table for benzo[a]pyrene in green tea.

Lab A B C Avg SD A B C Avg SD

NIST 6250 150

H803

H810

H813

H818 4033 3629 3800 3820 203 10.0 10.0 10.0 10.0 0.0

H819 6020 6740 6980 6580 500

H821

H843

H847 497 378 330 402 86

H848

H862

H865 4722 5041 5057 4940 189 1.8 2.2 2.2 2.1 0.2

H873 5910 5860 5480 5750 235

 Consensus Mean 4298 Consensus Mean 6.0

 Consensus Standard Deviation 2727 Consensus Standard Deviation 6.4

 Maximum 6580 Maximum 10.0

 Minimum 402 Minimum 2.1

 N 5 N 2

Benzo(a)pyrene

SRM 1647e PAH Solution (ng/g) SRM 3254 Green Tea (ng/g)

Individual Results Community Results
36

Figure 10. Naphthalene in SRM 1647e PAH Solution (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
37

Figure 11. Naphthalene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary 

view). In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST value determined by GC-MS, bounded by twice the standard deviation 

observed for three measurements.
38

Figure 12. Fluorene in SRM 1647e PAH Solution (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
39

Figure 13. Fluorene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary view). 

In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST value determined by GC-MS, bounded by twice the standard deviation 

observed for three measurements.
40

Figure 14. Phenanthrene in SRM 1647e PAH Solution (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
41

Figure 15. Phenanthrene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary 

view). In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST value determined by GC-MS, bounded by twice the standard deviation 

observed for three measurements.
42

Figure 16. Anthracene in SRM 1647e PAH Solution (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
43

Figure 17. Anthracene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary 

view). In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST value determined by GC-MS, bounded by twice the standard deviation 

observed for three measurements.
44

Figure 18. Fluoranthene in SRM 1647e PAH Solution (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
45

Figure 19. Fluoranthene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary 

view). In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST value determined by GC-MS, bounded by twice the standard deviation 

observed for three measurements.
46

Figure 20. Pyrene in SRM 1647e PAH Solution (data summary view). In this view, individual 

laboratory data are plotted with the individual laboratory standard deviation (error bars). The 

black solid line represents the consensus mean, and the black dotted lines represent the 

consensus variability calculated as one standard deviation about the consensus mean. The gray 

shaded region represents the target zone for “acceptable” performance, which encompasses the 

NIST certified value bounded by twice its uncertainty (U95).
47

Figure 21. Pyrene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary view). 

In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST value determined by GC-MS, bounded by twice the standard deviation 

observed for three measurements.
48

Figure 22. Benz[a]anthracene in SRM 1647e PAH Solution (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
49

Figure 23. Benz[a]anthracene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data 

summary view). In this view, individual laboratory data are plotted with the individual 

laboratory standard deviation (error bars). The black solid line represents the consensus mean, 

and the black dotted lines represent the consensus variability calculated as one standard deviation 

about the consensus mean. The gray shaded region represents the target zone for “acceptable” 

performance, which encompasses the NIST value determined by GC-MS, bounded by twice the 

standard deviation observed for three measurements.
50

Figure 24. Chrysene in SRM 1647e PAH Solution (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
51

Figure 25. Chrysene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary view). 

In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean.
52

Figure 26. Sum of chrysene and triphenylene in SRM 1647e PAH Solution (data summary 

view). In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
53

Figure 27. Sum of chrysene and triphenylene in SRM 3254 Camellia sinensis (Green Tea) 

Leaves (data summary view). In this view, individual laboratory data are plotted with the 

individual laboratory standard deviation (error bars). The black solid line represents the 

consensus mean, and the black dotted lines represent the consensus variability calculated as one 

standard deviation about the consensus mean. The gray shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST value determined by GC-MS, 

bounded by twice the standard deviation observed for three measurements.
54

Figure 28. Benzo[a]pyrene in SRM 1647e PAH Solution (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
55

Figure 29. Benzo[a]pyrene in SRM 3254 Camellia sinensis (Green Tea) Leaves (data summary 

view). In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. 
56

Figure 30. Phenanthrene in SRM 1647e PAH Solution and SRM 3254 Camellia sinensis (Green 

Tea) Leaves (sample/control comparison view). In this view, the individual laboratory results 

for the control (SRM 1647e PAH Solution) with a certified value for the analyte are compared to 

the results for an unknown (SRM 3254 Camellia sinensis (Green Tea) Leaves). The error bars 

represent the individual laboratory standard deviation. The solid red lines represent the target 

zone for the control (x-axis) and unknown sample (y-axis). The dotted blue box represents the 

consensus zone for the control (x-axis) and the unknown sample (y-axis). 
57

Figure 31. Anthracene in SRM 1647e PAH Solution and SRM 3254 Camellia sinensis (Green 

Tea) Leaves (sample/control comparison view). In this view, the individual laboratory results 

for the control (SRM 1647e PAH Solution) with a certified value for the analyte are compared to 

the results for an unknown (SRM 3254 Camellia sinensis (Green Tea) Leaves). The error bars 

represent the individual laboratory standard deviation. The solid red lines represent the target 

zone for the control (x-axis) and unknown sample (y-axis). The dotted blue box represents the 

consensus zone for the control (x-axis) and the unknown sample (y-axis). 
58

Figure 32. Benz[a]anthracene in SRM 1647e PAH Solution and SRM 3254 Camellia sinensis

(Green Tea) Leaves (sample/control comparison view). In this view, the individual laboratory 

results for the control (SRM 1647e PAH Solution) with a certified value for the analyte are 

compared to the results for an unknown (SRM 3254 Camellia sinensis (Green Tea) Leaves). The 

error bars represent the individual laboratory standard deviation. The solid red lines represent 

the target zone for the control (x-axis) and unknown sample (y-axis). The dotted blue box

represents the consensus zone for the control (x-axis) and the unknown sample (y-axis). 
59

Figure 33. Chrysene in SRM 1647e PAH Solution and SRM 3254 Camellia sinensis (Green 

Tea) Leaves (sample/control comparison view). In this view, the individual laboratory results 

for the control (SRM 1647e PAH Solution) with a certified value for the analyte are compared to 

the results for an unknown (SRM 3254 Camellia sinensis (Green Tea) Leaves). The error bars 

represent the individual laboratory standard deviation. The dotted blue box represents the 

consensus zone for the control (x-axis) and the unknown sample (y-axis). 
60

Figure 34. Sum of chrysene and triphenylene in SRM 1647e PAH Solution and SRM 3254 

Camellia sinensis (Green Tea) Leaves (sample/control comparison view). In this view, the 

individual laboratory results for the control (SRM 1647e PAH Solution) with a certified value for 

the analyte are compared to the results for an unknown (SRM 3254 Camellia sinensis (Green 

Tea) Leaves). The error bars represent the individual laboratory standard deviation. The solid 

red lines represent the target zone for the control (x-axis) and unknown sample (y-axis). The

dotted blue box represents the consensus zone for the control (x-axis) and the unknown sample 

(y-axis). 
61

CHOLINE IN FOODS

Study Overview

In this study, participants were provided with two NIST SRMs, candidate SRM 1845a Whole 

Egg Powder and candidate SRM 3234 Soy Flour, neither of which has been fortified with 

choline. Participants were asked to use in-house analytical methods to determine the mass 

fractions of total choline in each of the matrices and report values on an as-received basis. 

Participants were not asked to report the choline content in any particular form; NIST values are 

reported as the choline ion.

Sample Information

Whole egg powder. Participants were provided with three vials, each containing approximately 

1.5 g of unfortified whole egg powder from a single production lot. The material is a freeflowing, fine powder prepared from USDA-inspected whole eggs. Before use, participants were 

instructed to thoroughly mix and homogenize the contents of the vial and use a sample size of at 

least 100 mg. Participants were asked to report a single value from each vial provided and to 

store the egg powder at controlled room temperature, 10 °C to 30 °C. Approximate analyte 

levels were not reported prior to the study. The NIST certified value for choline in candidate 

SRM 1845a will be determined using microwave acid digestion followed by ID-LC-MS in 

combination with data from external collaborating laboratories. An estimation of the certified 

value of the choline ion, (14. 71 ± 0.33) mg/g, is provided as the mean and standard deviation of 

duplicate ID-LC-MS measurements from 10 packets.

Soy Flour. Participants were provided with three vials, each containing approximately 1.5 g of 

defatted soy flour. Before use, participants were instructed to thoroughly mix and homogenize 

the contents of the vial and use a sample size of at least 400 mg. Participants were asked to 

report a single value from each vial provided and to store the egg powder at controlled room 

temperature, 10 °C to 30 °C. Approximate analyte levels were not reported prior to the study. 

The NIST certified value for choline in candidate SRM 3234 will be determined using 

microwave acid digestion followed by isotope dilution LC-MS in combination with data from 

external collaborating laboratories. An estimation of the certified value of the choline ion, 

(2.663 ± 0.023) mg/g, is provided as the mean and standard deviation of duplicate ID-LC-MS 

measurements from 12 packets.

Study Results

• Thirteen laboratories enrolled in this exercise and received samples, and seven 

laboratories reported results for the egg powder and soy flour (54 % participation).

• For both materials, the consensus ranges were very wide and were higher than the NIST 

target range (Figures 35 and 36).

• The dispersion of the data could be a result of challenges in completely extracting and 

hydrolyzing the samples.

• In the soy flour, five of the seven laboratories (71 %) reported values that were 

reasonably close to the target range. The remaining two laboratories reported values 

that were significantly higher than the target range (5 times higher and almost 100 

times higher). This could indicate an interference in the analytical method (titration 
62

and spectrophotometry) caused by matrix components. More information is needed 

about the analytical methods to draw more conclusive inferences.

• In the egg powder, four of the seven laboratories (57 %) reported values that were 

reasonably close to the target range. Two of the remaining laboratories reported 

values that were significantly lower than the target range (2 times lower and 500 

times lower). This could indicate incomplete extraction (both laboratories reported 

using solvent extraction). Another laboratory reported a value that was 5 times higher 

than the target value. This laboratory also reported very high values for the soy flour, 

indicating a possible calibration error.

• Laboratories that reported low values for the egg powder did not report low values for 

the soy flour. This indicates that the egg powder may contain more choline esters that 

require hydrolysis prior to analytical determination.

• The NIST values were determined using microwave acid digestion. As a result, the 

NIST target ranges and the consensus means may not overlap when participating 

laboratories use less extensive extraction procedures. This may result in a 

discrepancy between laboratories (such as NIST) determining the “total” choline 

content and laboratories determining “free” choline content.

• In general, the instrumental method used did not correlate with any trend in the data. 

In this case, variability in the data is more likely related to sample preparation than to 

instrumental method. A larger data set and more information from participants is 

necessary to draw any strong correlations between method and result.

Technical Recommendations

The following are recommendations based on results obtained by the participants in this study. 

• The literature indicates that some proportion of the total choline is present in these 

matrices as choline esters that require acidic or basic hydrolysis to release choline ion. 

Participants should be clear as to what form of choline is reported and whether a sample 

preparation procedure will yield total or free choline.

• No analytical method was identified as being exceptionally good or problematic. For 

these types of samples, the extraction method seems to be more critical than the 

instrumental methods used by participants.

• Participants were not asked to report choline results in any specific molecular form. The 

NIST estimation of the certified value is reported as the choline ion. Conversion to the 

choline hydroxide form would increase the values by 16 %. Two laboratories reported 

values 13.5 % and 17.7 % greater than the NIST value for the soy flour, which could be 

explained by a difference in the reported form. However, these same two laboratories 

reported values for the egg powder that were only 8.2 % and 4.1 % greater than the NIST 

value, respectively. While a small error due to inconsistent reporting of results is 

possible, it does not completely explain the outlying results.
63

Table 17. Individual data table (NIST) for choline in foods.
64

Table 18. Data summary table for choline in foods.

Lab A B C Avg SD A B C Avg SD

NIST 2.66 0.02 14.7 0.3

H801

H803

H810 3.1 3.1 2.9 3.02 0.1 15.7 15.7 16.3 15.9 0.3

H816 129 130 133 131 2.5 76.1 78.2 81.2 78.5 2.6

H821

H824

H826 2.4 2.4 2.4 2.41 0.02 14.8 14.8 14.7 14.8 0.1

H829

H845 2.0 2.0 2.2 2.07 0.08 13.4 13.2 13.6 13.4 0.2

H846

H860 3.1 3.1 3.1 3.13 0.02 15.1 15.4 15.3 15.3 0.1

H862 12.2 12.3 11.3 11.93 0.53 6.7 6.6 7.2 6.8 0.3

H870 2.2 1.8 1.8 1.95 0.25 0.028 0.028 0.028 0.028 0.000

 Consensus Mean 5.59 Consensus Mean 13.2

 Consensus Standard Deviation 6.05 Consensus Standard Deviation 9.1

 Maximum 131 Maximum 78.5

 Minimum 1.95 Minimum 0.028

 N 7 N 7

Individual Results

SRM 3234 Soy Flour (mg/g)

Community 

Results

SRM 1845a Whole Egg Powder (mg/g)

Choline
65

Figure 35. Choline in candidate SRM 3234 Soy Flour (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST value determined by ID-LC-MS bounded by twice the standard deviation observed for 

24 measurements.
66

Figure 36. Choline in candidate SRM 1845a Whole Egg Powder (data summary view). In this 

view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST value determined by ID-LC-MS bounded by twice the standard deviation 

observed for 20 measurements.
67

Figure 37. Choline in SRM 3234 Soy Flour and candidate SRM 1845a Whole Egg Powder

(sample/sample comparison view). In this view, the individual laboratory results for one sample

(candidate SRM 1845a Whole Egg Powder) with a certified value for the analyte are compared 

to the results for a second sample (candidate SRM 3234 Soy Flour). The error bars represent the 

individual laboratory standard deviation. The solid red lines represent the target zone for the 

control (x-axis) and the unknown sample (y-axis). The dotted blue box represents the consensus 

zone for the control (x-axis) and the unknown sample (y-axis). 
68

Figure 38. Expanded view of choline in SRM 3234 Soy Flour and candidate SRM 1845a Whole 

Egg Powder (sample/sample comparison view). In this view, the individual laboratory results 

for one sample (candidate SRM 1845a Whole Egg Powder) with a certified value for the analyte

are compared to the results for a second sample (candidate SRM 3234 Soy Flour). The error bars 

represent the individual laboratory standard deviation. The solid red lines represent the target 

zone for the control (x-axis) and the unknown sample (y-axis).
69

TOCOPHEROLS IN FOODS

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 3276 Carrot Extract in Oil 

and candidate SRM 1845a Whole Egg Powder. Participants were asked to use in-house 

analytical methods to determine the mass fractions of four tocopherols (α-tocopherol, βtocopherol, γ-tocopherol, and δ-tocopherol) as well as the total amount of tocopherols in each of 

the matrices and report values on an as-received basis.

Sample Information

Carrot extract. Participants were provided with three ampoules, each containing approximately 

1 mL of carrot extract in oil. The carrot extract in oil was mixed with butylated hydroxytoluene 

(BHT, approximately 670 μg/g) and ampouled under argon. Before use, participants were 

instructed to mix thoroughly the contents of the ampoule and use a sample size of at least 50 mg. 

Participants were asked to report a single value from each ampoule and store the extract at 

controlled room temperature, 10 °C to 30 °C. Approximate analyte levels were not reported

prior to the study. The NIST certified values and uncertainties in SRM 3276 were determined by 

LC-fluorescence following solvent extraction and are reported in the table below.

Egg powder. Participants were provided with three vials, each containing approximately 1.5 g of 

whole egg powder. The material is a free-flowing, fine powder prepared from USDA-inspected 

whole eggs. Before use, participants were instructed to mix thoroughly the contents of the vial

and use a sample size of at least 0.5 g. Participants were asked to report a single value from each

vial and store the material at controlled room temperature, 10 °C to 30 °C. Approximate analyte 

levels were not reported prior to the study. The NIST certified values and uncertainties in 

candidate SRM 1845a Whole Egg Powder will be determined by a combination of LCfluorescence data and data from external collaborating laboratories. An estimation of the values 

and uncertainties is provided in the table below as the mean and standard deviation of duplicate 

measurements performed by three (γ-tocopherol) or six (α-tocopherol) external collaborating 

laboratories. All laboratories used saponification in the sample preparation and liquid 

chromatography with either absorbance or fluorescence detection to measure the tocopherols in 

candidate SRM 1845a.

Certified Mass Fraction NIST-Determined Mass Fraction

Analyte in SRM 3276 (μg/g) in Candidate SRM 1845a (μg/g)

α-tocopherol 36.0 ± 7.0

β-tocopherol

γ-tocopherol 373 ± 34 12.0 ± 5.6

δ-tocopherol 443 ± 64

Study Results

• Forty-one laboratories enrolled in this exercise and received samples, and 20 laboratories 

reported results for at least some of the tocopherols (49 % participation).

• NIST target values are available for γ-tocopherol and δ-tocopherol in the carrot oil 

sample.
70

• The consensus mean for γ-tocopherol was within the target range, while the 

consensus mean for δ-tocopherol was slightly above the target range.

• The consensus ranges were quite wide for both compounds in the carrot extract (33 % 

and 70 % RSD, respectively).

• NIST target values are available for α-tocopherol and γ-tocopherol in the egg powder 

sample. The consensus means for both compounds were within the target range, and the 

consensus ranges were both quite wide (48 % and 60 % RSD, respectively).

• Results for total tocopherols were calculated as the sum of all four tocopherol values 

reported for each sample.

• In the carrot oil, the consensus mean was lower than the target range with a wide 

uncertainty (125 % RSD).

• In the egg powder, the consensus mean was within the target range, but had a wide 

uncertainty (59 % RSD).

• Many laboratories reported values for tocopherols not known to be present at 

quantifiable levels in the materials.

• Eleven laboratories (55 %) reported using saponification followed by extraction, while 

eight laboratories (40 %) reported using solvent extraction to prepare samples. One 

laboratory (5 %) reported using derivatization in the sample preparation.

• A majority of laboratories (75 %) used LC-absorbance for analysis. Three laboratories 

(15 %) reported using LC-fluorescence, one laboratory (5 %) reported using LC-MS, and 

one laboratory (5 %) reported using GC-MS.

• A majority of laboratories (88 %) reported using an internal standard approach to 

calibration. Two laboratories (12 %) reported using a standard addition approach to 

calibration.

Technical Recommendations

The following recommendations are based on results obtained by the participants in this study. 

• A calibration error is possible, based on the sample/control comparison graphs, but more 

data for the entire sample set is needed to conclusively determine the source of error.

• Spiking studies or subjecting calibrant materials to the same preparation procedure as the 

samples (extraction, hydrolysis, etc.) can help to identify if tocopherols are being 

degraded during sample preparation.

• Tocopherol calibrant mass fraction should always be determined spectrophotometrically.

• If saponification is used for the sample preparation and an internal standard approach is 

taken to quantitation, it is imperative that laboratories check the stability of the internal 

standard.
71

Table 19. Individual data table (NIST) for tocopherols in foods.
72

Table 20. Data summary table for α–tocopherol in foods.

Lab A B C Avg SD A B C Avg SD

NIST 36.0 7.0

H801

H803

H805

H807

H809

H810 63.4 62.4 66.2 64.0 2.0

H812 25.6 22.3 25.0 24.3 1.8 66.0 78.0 62.0 68.7 8.3

H814 53.1 55.0 50.0 52.7 2.5

H815 44.5 46.2 46.1 45.6 0.9 47.1 48.9 49.6 48.5 1.3

H816 0.6 0.7 0.7 0.0 0.7 0.8 0.6 0.7 0.1

H820

H821

H823 20.5 21.0 20.0 20.5 0.5 63.1 62.2 63.7 63.0 0.8

H824 6560 6440 6470 6490 62 31.1 27.8 30.1 29.7 1.7

H826 14.0 14.0 16.0 14.7 1.2 56.0 61.0 63.0 60.0 3.6

H828

H829

H830

H832

H834

H835

H839

H842

H843

H846

H847 3.9 3.8 6.7 4.8 1.7 27.6 22.1 32.3 27.3 5.1

H848 4.1 3.9 6.8 4.9 1.7 25.8 20.6 30.2 25.5 4.8

H850

H852

H853 69.7 66.1 65.2 67.0 2.4

H855 41.9 41.9 73.1 107.0 83.0 87.7 17.4

H856

H857 8.1 7.3 25.4 13.6 10.2 18.5 22.5 17.2 19.4 2.8

H858

H861

H862 69.4 72.2 68.4 70.0 2.0

H870 40.5 40.1 38.5 39.7 1.1

H871

H872

H873 15.9 16.7 16.0 16.2 0.4 56.3 59.2 55.7 57.1 1.9

H874

 Consensus Mean 19.1 Consensus Mean 49.5

 Consensus Standard Deviation 17.9 Consensus Standard Deviation 24.0

 Maximum 6490 Maximum 87.7

 Minimum 0.7 Minimum 0.70

 N 10 N 16

Community 

Results Individual Results

α-tocopherol

SRM 3276 Carrot Extract in Oil (μg/g) SRM 1845a Whole Egg Powder (μg/g)
73

Table 21. Data summary table for β–tocopherol in foods.

Lab A B C Avg SD A B C Avg SD

NIST

H801

H803

H805

H807

H809

H810

H812

H814

H815 3.81 4.90 4.44 4.38 0.55

H816

H820

H821

H823 7.44 6.91 6.73 7.03 0.37 0.54 0.62 0.62 0.59 0.04

H824

H826 8.00 6.00 6.00 6.67 1.15

H828

H829

H830

H832

H834

H835

H839

H842

H843

H846

H847

H848

H850

H852

H853

H855

H856

H857

H858

H861

H862

H870

H871

H872

H873

H874

 Consensus Mean 6.02 Consensus Mean

 Consensus Standard Deviation 1.63 Consensus Standard Deviation

 Maximum 7.03 Maximum

 Minimum 4.38 Minimum

 N 3 N 1

β-tocopherol

SRM 3276 Carrot Extract in Oil (μg/g) SRM 1845a Whole Egg Powder (μg/g)

Individual Results Community Results
74

Table 22. Data summary table for γ–tocopherol in foods.

Lab A B C Avg SD A B C Avg SD

NIST 443 64 12.0 5.6

H801

H803

H805

H807

H809

H810 342 334 336 337 4 19.8 20.7 24.4 21.6 2.4

H812

H814

H815 201 216 198 205 10 4.7 6.0 6.4 5.7 0.9

H816

H820

H821

H823 467 452 456 459 7 23.3 23.2 23.6 23.4 0.2

H824

H826 380 396 382 386 9 20.0 22.0 22.0 21.3 1.2

H828

H829

H830

H832

H834

H835

H839

H842

H843

H846

H847

H848

H850

H852

H853 477 468 494 479 13 29.5 29.8 32.1 30.5 1.4

H855 391 396 399 395 4 19.0 18.9 18.8 18.9 0.1

H856

H857 15 20 20 18 3 0.6 0.6 0.3 0.5 0.2

H858

H861

H862 467 459 460 462 4

H870 420 450 400 423 25 18.5 19.6 19.1 19.1 0.6

H871

H872

H873

H874

 Consensus Mean 370 Consensus Mean 17.8

 Consensus Standard Deviation 123 Consensus Standard Deviation 10.7

 Maximum 479 Maximum 30.5

 Minimum 18 Minimum 0.52

 N 9 N 8

Community 

Results

γ-tocopherol

SRM 3276 Carrot Extract in Oil (μg/g) SRM 1845a Whole Egg Powder (μg/g)

Individual Results
75

Table 23. Data summary table for δ–tocopherol in foods.

Lab A B C Avg SD A B C Avg SD

NIST 373 34

H801

H803

H805

H807

H809

H810 264 262 261 262 2

H812

H814

H815 102 93 99 98 4

H816

H820

H821

H823 390 386 388 388 2 1.11 1.06 1.02 1.06 0.04

H824

H826 331 350 338 340 10

H828

H829

H830

H832

H834

H835

H839

H842

H843

H846

H847

H848

H850

H852

H853 900 892 918 903 14

H855 440 445 445 443 3

H856

H857

H858

H861

H862 331 327 326 328 3

H870 2010 2120 2020 2050 61 27.00 34.50 21.90 27.80 6.34

H871

H872

H873

H874

 Consensus Mean 472 Consensus Mean 14.43

 Consensus Standard Deviation 361 Consensus Standard Deviation 21.44

 Maximum 2050 Maximum 27.80

 Minimum 98 Minimum 1.06

 N 8 N 2

δ-tocopherol

SRM 3276 Carrot Extract in Oil (μg/g) SRM 1845a Whole Egg Powder (μg/g)

Individual Results Community Results
76

Table 24. Data summary table for total tocopherol in foods.

Lab A B C Avg SD A B C Avg SD

NIST 816 72 48.0 9.0

H801

H803

H805

H807

H809

H810 606 596 597 600 6 83.2 83.1 90.6 85.6 4.3

H812 25.6 22.3 25.0 24.3 1.8 66.0 78.0 62.0 68.7 8.3

H814 0.00 0.00 0.00 0.00 0.00 53.1 55.0 50.0 52.7 2.5

H815 351 360 347 353 7 51.8 54.9 56.0 54.2 2.2

H816 0.00 0.64 0.66 0.43 0.38 0.73 0.77 0.59 0.70 0.09

H820

H821

H823 884 867 871 874 9 88.1 87.0 89.0 88.0 1.0

H824 6560 6440 6470 6490 62 31.1 27.8 30.1 29.7 1.7

H826 733 766 742 747 17 76.0 83.0 85.0 81.3 4.7

H828

H829

H830

H832

H834 21.5 21.0 19.8 20.8 0.9 60.6 61.4 53.8 58.6 4.2

H835 17.2 18.2 18.0 17.8 0.5 62.0 56.2 62.9 60.4 3.6

H839 18.7 20.5 20.0 19.7 0.9 56.2 53.7 55.2 55.0 1.3

H842

H843

H846

H847 3.92 3.80 6.74 4.82 1.66 27.6 22.1 32.3 27.3 5.1

H848 4.09 3.89 6.84 4.94 1.65 25.8 20.6 30.2 25.5 4.8

H850

H852

H853 1378 1359 1412 1383 27 99.2 95.9 97.3 97.5 1.7

H855 831 841 886 853 29 92.1 126.0 102.0 106.7 17.4

H856

H857 22.8 27.4 45.7 32.0 12.1 19.1 23.1 17.5 19.9 2.9

H858

H861

H862 798 786 786 790 7 69.4 72.2 68.4 70.0 2.0

H870 2430 2570 2420 2473 84 86.0 94.2 79.5 86.6 7.4

H871

H872

H873 15.9 16.7 16.0 16.2 0.4 56.3 59.2 55.7 57.1 1.9

H874

 Consensus Mean 433 Consensus Mean 59.9

 Consensus Standard Deviation 572 Consensus Standard Deviation 30.9

 Maximum 6490 Maximum 106.7

 Minimum 0.00 Minimum 0.70

 N 19 N 19

SRM 1845a Whole Egg Powder (μg/g)

Total tocopherols

SRM 3276 Carrot Extract in Oil (μg/g)

Individual Results Community Results
77

Figure 39. α-Tocopherol in SRM 3276 Carrot Extract in Oil (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). Data points that are unfilled represent laboratories that only reported a single value for 

that analyte and therefore were not included in the consensus mean. The black solid line 

represents the consensus mean, and the black dotted lines represent the consensus variability 

calculated as one standard deviation about the consensus mean.
78

Figure 40. α-Tocopherol in candidate SRM 1845a Whole Egg Powder (data summary view). In 

this view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses an approximation of the NIST certified value based on LC-absorbance and LCfluorescence data from six external collaborating laboratories, bounded by twice the standard 

deviation observed for 10 total measurements.
79

Figure 41. β-Tocopherol in SRM 3276 Carrot Extract in Oil (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean.
80

Figure 42. γ-Tocopherol in SRM 3276 Carrot Extract in Oil (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
81

Figure 43. γ-Tocopherol in candidate SRM 1845a Whole Egg Powder (data summary view). In 

this view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses an approximation of the NIST certified value based on LC-absorbance and LCfluorescence data from three external collaborating laboratories, bounded by twice the standard 

deviation observed for 5 total measurements.
82

Figure 44. δ-Tocopherol in SRM 3276 Carrot Extract in Oil (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
83

Figure 45. Total tocopherols in SRM 3276 Carrot Extract in Oil (data summary view). In this 

view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST value bounded by twice its uncertainty (U95), calculated as a combination 

of the certified values for γ-tocopherol and δ-tocopherol.
84

Figure 46. Total tocopherols in candidate SRM 1845a Whole Egg Powder (data summary 

view). In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses an approximation of the NIST certified value based on LC-absorbance and LCfluorescence data from six external collaborating laboratories, bounded by twice the standard 

deviation observed for 10 total measurements, calculated as a combination of the values for αtocopherol and γ-tocopherol.
85

Figure 47. α-Tocopherol in candidate SRM 1845a Whole Egg Powder and SRM 3276 Carrot 

Extract in Oil (sample/control comparison view). In this view, the individual laboratory results 

for the control (SRM 3276 Carrot Extract in Oil) with a certified value for the analyte are 

compared to the results for an unknown (candidate SRM 1845a Whole Egg Powder). The error 

bars represent the individual laboratory standard deviation. The dotted blue box represents the 

consensus zone for the control (x-axis) and the unknown sample (y-axis). 
86

Figure 48. γ-Tocopherol in candidate SRM 1845a Whole Egg Powder and SRM 3276 Carrot 

Extract in Oil (sample/control comparison view). In this view, the individual laboratory results 

for the control (SRM 3276 Carrot Extract in Oil) with a certified value for the analyte are 

compared to the results for an unknown (candidate SRM 1845a Whole Egg Powder). The error 

bars represent the individual laboratory standard deviation. The solid red lines represent the 

target zone for the control (x-axis) and the unknown sample (y-axis). The dotted blue box

represents the consensus zone for the control (x-axis) and the unknown sample (y-axis). 
87

Figure 49. Total tocopherols in candidate SRM 1845a Whole Egg Powder and SRM 3276 

Carrot Extract in Oil (sample/control comparison view). In this view, the individual laboratory 

results for the control (SRM 3276 Carrot Extract in Oil) with a certified value for the analyte are 

compared to the results for an unknown (candidate SRM 1845a Whole Egg Powder). The error 

bars represent the individual laboratory standard deviation. The solid red lines represent the 

target zone for the control (x-axis) and the unknown samples (y-axis). The dotted blue box

represents the consensus zone for the control (x-axis) and the unknown sample (y-axis). 
88

FATTY ACIDS IN BOTANICAL OILS

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 3274-3 Flaxseed (Linium 

usitatissimum) Oil and SRM 3274-4 Perilla (Perilla frutescens) Oil. Participants were asked to 

use in-house analytical methods to determine the mass fractions of four fatty acids (linoleic acid, 

α-linolenic acid, γ-linolenic acid, and arachidonic acid) in each of the matrices and report values 

on an as-received basis. Participants were not instructed to report values for fatty acids in a 

certain form; NIST values are reported as triglycerides.

Sample Information

Flaxseed oil. Participants were provided with three ampoules, each containing approximately 

1.2 mL of flaxseed oil from a single lot. The oil contained approximately 190 mg/L tertbutylhydroquinone (TBHQ) as an antioxidant and was packaged in amber glass ampoules under 

argon. Before use, participants were instructed to thoroughly mix the contents of the ampoule 

and use a sample size of at least 0.5 g. Participants were asked to report a single value from each 

ampoule and store the flaxseed oil in a refrigerator at 0 °C to 4 °C. Approximate analyte levels 

were not reported prior to the study. The NIST certified values and uncertainties in SRM 3274-3 

were determined by GC-FID and GC/MS following multiple methods of hydrolysis and 

derivatization, and are summarized in the table below.

Perilla oil. Participants were provided with three ampoules, each containing approximately 

1.2 mL of perilla oil from a single lot. The oil contained approximately 190 mg/L TBHQ as an 

antioxidant and was packaged in amber glass ampoules under argon. Before use, participants 

were instructed to mix thoroughly the contents of the ampoule and use a sample size of at least 

0.5 g. Participants were asked to report a single value from each ampoule and store the perilla 

oil in a refrigerator at 0 °C to 4 °C. Approximate analyte levels were not reported prior to the 

study. The NIST certified values and uncertainties in SRM 3274-4 were determined by GC-FID 

and GC/MS following multiple methods of hydrolysis and derivatization, and are summarized in 

the table below.

Certified Mass Fraction Certified Mass Fraction

Analyte in SRM 3274-3 (mg/g) in SRM 3274-4 (mg/g)

Linoleic acid 171 ± 11 160 ± 14

α-Linolenic acid 579 ± 30 629 ± 28

γ-Linolenic acid 1.55 ± 0.25* 2.08 ± 0.48*

Arachidonic acid 0.633 ± 0.029

*reference value

Study Results

• Thirty-seven laboratories enrolled in this exercise and received samples, and 20 

laboratories reported results for at least some of the fatty acids (54 % participation).

• The consensus mean for linoleic acid was lower than the target range in both study 

materials, and the consensus mean for α-linolenic acid was within the target range in both 

study materials. The consensus ranges were reasonable (less than the size of the NIST 

target range) for both compounds in both study materials.
89

• Figures 58 and 59 indicate a possible calibration issue with these compounds. 

Laboratories that reported high values for flaxseed oil also reported high values for 

perilla oil. The same is true for laboratories reporting low values.

• Five laboratories (25 %) reported using an external standard calibration approach, 

while 12 laboratories (60 %) reported using an internal standard calibration approach. 

When compared, the two approaches give similar results for these compounds.

• The consensus mean for γ-linolenic acid was well within the target range for both study 

materials. For perilla oil, the consensus range was also contained within the NIST target 

range.

• Not many laboratories were able to measure arachidonic acid (five laboratories for 

flaxseed oil and three laboratories for perilla oil). A certified value is only available in 

the flaxseed oil, and the consensus mean was higher and the consensus range 

significantly wider than the target range.

• Almost all laboratories (95 %) used a hydrolysis and derivatization procedure for sample 

preparation followed by GC-FID as their analytical method. One laboratory reported 

using GC-MS.

Technical Recommendations

The following recommendations are based on results obtained by the participants in this study. 

• The trend observed in the sample/control comparison graph is indicative of a calibration 

error in the determination of linoleic acid and α–linolenic acid.

• Spiking studies or subjecting calibrant materials to the same preparation procedure as the 

samples (extraction, hydrolysis, derivatization, etc.) can help to identify if fatty acids are 

being degraded during sample preparation.

• Participants were not asked to report fatty acid results in any specific molecular form. 

The NIST certified values are reported as triglycerides. Conversion of fatty acid results 

between triglycerides and fatty acid methyl esters (FAMEs) free fatty acids would only 

result in a maximum of 5 % error. While a small error due to inconsistent reporting of 

results is possible, it does not completely explain the outlying results.
90

Table 25. Individual data table (NIST) for fatty acids in botanical oils.
91

Table 26. Data summary table for linoleic acid in botanical oils.

Lab A B C Avg SD A B C Avg SD

NIST 171 11.0 160 14.0

H801

H803

H805 129 129 128 129 0.6 109 112 112 111 1.7

H809

H810 162 164 162 163 1.2 140 140 139 140 0.6

H815 181 183 182 182 1.2 156 157 157 157 0.4

H817 168 168 168 168 0.1 144 144 144 144 0.1

H818 180 143 167 163 18.8 142 138 140 140 2.4

H819

H820 130 138 135 134 4.3 94 89 90 91 2.9

H821

H823 145 144 144 144 0.4 126 126 126 126 0.4

H824 158 158 159 158 0.6 137 136 136 136 0.6

H825 160 160 159 160 0.6 136 136 136 136 0.0

H827

H828

H829

H835 170 170 171 170 0.1 147 147 147 147 0.2

H838 169 169 169 169 0.0 146 144 146 145 1.2

H839 171 170 171 171 0.6 148 147 148 148 0.2

H841 157 162 164 161 3.5 140 140 140 140 0.5

H842

H843

H846

H850 78 77 77 77 0.4 68 68 66 67 1.0

H852

H854

H855 157 155 155 156 1.0 133 132 135 133 1.5

H857 169 175 179 174 5.0 149 155 146 150 4.6

H858

H861

H862 163 163 163 163 0.3 140 138 139 139 1.1

H864

H867 159 163 161 161 1.9 148 141 146 145 3.6

H872 127 148 145 140 11.4 81 124 140 115 30.6

H873 164 169 168 167 2.6 140 143 140 141 1.7

H874

 Consensus Mean 159 Consensus Mean 137

 Consensus Standard Deviation 15 Consensus Standard Deviation 13

 Maximum 182 Maximum 157

 Minimum 77 Minimum 67

 N 20 N 20

Linoleic Acid

SRM 3274-3 Flaxseed Oil (mg/g) SRM 3274-4 Perilla Oil (mg/g)

Individual Results Community Results
92

Table 27. Data summary table for α-linolenic acid in botanical oils.

Lab A B C Avg SD A B C Avg SD

NIST 579 30.0 629 28.0

H801

H803

H805 394 400 391 395 4.6 400 415 403 406 7.9

H809

H810 539 547 541 542 4.2 569 571 565 568 3.1

H815 497 504 501 501 3.4 528 531 529 529 1.5

H817 561 562 563 562 0.8 592 592 592 592 0.3

H818 599 476 555 543 62.1 582 561 570 571 10.2

H819

H820 502 531 519 517 14.7 461 439 442 447 11.8

H821

H823 493 490 491 491 1.5 523 525 525 524 1.4

H824 531 530 532 531 1.0 561 560 556 559 2.6

H825 548 548 547 548 1 573 573 573 573 0.0

H827

H828

H829

H835 575 574 575 575 0.3 603 604 604 604 0.5

H838 571 574 577 574 3.0 609 608 601 606 4.4

H839 578 575 578 577 1.6 607 609 609 608 1.1

H841 523 537 548 536 12.4 567 571 574 571 3.3

H842

H843

H846

H850 265 264 263 264 0.9 285 282 278 282 3.7

H852

H854

H855 533 529 528 530 2.5 555 551 558 555 3.9

H857 559 579 596 578 18.5 605 632 589 609 21.7

H858

H861

H862 590 595 597 594 3.5 620 622 612 618 5.4

H864

H867 492 506 495 497 7.3 578 542 553 558 18.5

H872 461 513 510 495 29.2 355 532 599 495 126.1

H873 554 561 555 557 3.8 566 585 572 574 9.7

H874

 Consensus Mean 535 Consensus Mean 558

 Consensus Standard Deviation 42 Consensus Standard Deviation 51

 Maximum 594 Maximum 618

 Minimum 264 Minimum 282

 N 20 N 20

α-Linolenic Acid

SRM 3274-3 Flaxseed Oil (mg/g) SRM 3274-4 Perilla Oil (mg/g)

Individual Results Community Results
93

Table 28. Data summary table for γ-linolenic acid in botanical oils.

Lab A B C Avg SD A B C Avg SD

NIST 1.55 0.25 2.08 0.48

H801

H803

H805

H809

H810 2.00 1.96 1.95 1.97 0.03

H815 1.70 1.70 1.70 1.70 0.00 1.80 1.90 1.80 1.83 0.06

H817 1.83 1.81 1.87 1.84 0.03 1.82 1.82 1.83 1.82 0.01

H818 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

H819

H820

H821

H823 2.03 2.00 2.04 2.02 0.02

H824 2.00 2.00 2.00 2.00 0.00 4.00 4.00 4.00 4.00 0.00

H825 <1 <1 <1 2.20 2.20 2.20 2.20 0.00

H827

H828

H829

H835 2.10 2.20 2.10 2.13 0.06

H838 2.00 2.00 2.00 2.00 0.00

H839 2.10 2.10 2.10 2.10 0.00

H841 1.90 2.08 2.32 2.10 0.21

H842

H843

H846

H850

H852

H854

H855 2.00 2.00 2.00 2.00 0.00

H857

H858

H861

H862 1.70 1.70 1.80 1.73 0.06

H864

H867 0.89 0.95 1.06 0.97 0.09 0.93 1.02 1.09 1.01 0.08

H872

H873 2.29 2.11 2.02 2.14 0.14

H874

 Consensus Mean 1.63 Consensus Mean 2.00

 Consensus Standard Deviation 0.52 Consensus Standard Deviation 0.20

 Maximum 2.00 Maximum 4.00

 Minimum 0.97 Minimum 1.01

 N 4 N 14

γ-Linolenic Acid

SRM 3274-3 Flaxseed Oil (mg/g) SRM 3274-4 Perilla Oil (mg/g)

Individual Results Community Results
94

Table 29. Data summary table for arachidonic acid in botanical oils.

Lab A B C Avg SD A B C Avg SD

NIST 0.633 0.029

H801

H803

H805

H809

H810

H815

H817 1.120 1.150 1.110 1.127 0.021 0.230 0.220 0.210 0.220 0.010

H818 1.000 <0.01 <0.01 1.000 <0.01 <0.01 <0.01

H819

H820

H821

H823 1.026 1.047 1.075 1.049 0.025 1.186 1.116 1.364 1.222 0.128

H824

H825 <1 <1 <1 <1 <1 <1

H827

H828

H829

H835

H838

H839

H841

H842

H843

H846

H850

H852

H854

H855

H857

H858

H861

H862

H864

H867 0.319 0.344 0.400 0.354 0.041

H872

H873 0.730 0.710 0.720 0.720 0.010 0.790 0.720 0.750 0.753 0.035

H874

 Consensus Mean 0.850 Consensus Mean 0.732

 Consensus Standard Deviation 0.359 Consensus Standard Deviation 0.569

 Maximum 1.127 Maximum 1.222

 Minimum 0.354 Minimum 0.220

 N 4 N 3

Arachidonic Acid

SRM 3274-3 Flaxseed Oil (mg/g) SRM 3274-4 Perilla Oil (mg/g)

Individual Results Community Results
95

Figure 50. Linoleic Acid [C18:2, n-6] in SRM 3274-3 Flaxseed Oil (data summary view). In 

this view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
96

Figure 51. Linoleic Acid [C18:2, n-6] in SRM 3274-4 Perilla Oil (data summary view). In this 

view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
97

Figure 52. α-Linolenic Acid [C18:3, n-3] in SRM 3274-3 Flaxseed Oil (data summary view). 

In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). The black solid line represents the consensus mean, and the black dotted 

lines represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
98

Figure 53. α-Linolenic Acid [C18:3, n-3] in SRM 3274-4 Perilla Oil (data summary view). In 

this view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
99

Figure 54. γ-Linolenic Acid [C18:3, n-6] in SRM 3274-3 Flaxseed Oil (data summary view). In 

this view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST reference value bounded by twice its uncertainty (U95).
100

Figure 55. γ-Linolenic Acid [C18:3, n-6] in SRM 3274-4 Perilla Oil (data summary view). In 

this view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST reference value bounded by twice its uncertainty (U95).
101

Figure 56. Arachidonic Acid [C20:4, n-6] in SRM 3274-3 Flaxseed Oil (data summary view). 

In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). Data points that are unfilled represent laboratories that only reported a 

single value for that analyte and therefore were not included in the consensus mean. The black 

solid line represents the consensus mean, and the black dotted lines represent the consensus 

variability calculated as one standard deviation about the consensus mean. The gray shaded 

region represents the target zone for “acceptable” performance, which encompasses the NIST 

certified value bounded by twice its uncertainty (U95).
102

Figure 57. Arachidonic Acid [C20:4, n-6] in SRM 3274-4 Perilla Oil (data summary view). In 

this view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean.
103

Figure 58. Linoleic acid in SRM 3274-3 Flaxseed Oil and SRM 3274-4 Perilla Oil

(sample/control comparison view). In this view, the individual laboratory results for the control 

(SRM 3274-3 Flaxseed Oil) with a certified value for the analyte are compared to the results for 

an unknown (SRM 3274-4 Perilla Oil). The error bars represent the individual laboratory 

standard deviation. The solid red lines represent the target zone for the control (x-axis) and the 

unknown sample (y-axis). The dotted blue box represents the consensus zone for the control (xaxis) and the unknown sample (y-axis). 
104

Figure 59. α-Linolenic acid in SRM 3274-3 Flaxseed Oil and SRM 3274-4 Perilla Oil

(sample/control comparison view). In this view, the individual laboratory results for the control 

(SRM 3274-3 Flaxseed Oil) with a certified value for the analyte are compared to the results for 

an unknown (SRM 3274-4 Perilla Oil). The error bars represent the individual laboratory 

standard deviation. The solid red lines represent the target zone for the control (x-axis) and the 

unknown sample (y-axis). The dotted blue box represents the consensus zone for the control (xaxis) and the unknown sample (y-axis). 
105

PHYTOSTEROLS IN SAW PALMETTO

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 3251 Serenoa repens

Extract and SRM 3250 Serenoa repens (Fruit). Participants were asked to use in-house 

analytical methods to determine the mass fractions of three phytosterols (campesterol, βsitosterol, and stigmasterol) in each of the matrices and report values on an as-received basis.

Sample Information

Saw palmetto extract. Participants were provided with three ampoules, each containing 

approximately 1 mL of a carbon dioxide extract of saw palmetto (Serenoa repens). The oil was 

packaged in amber glass ampoules under argon. Before use, participants were instructed to 

thoroughly mix the contents of the ampoule and use a sample size of at least 125 mg. 

Participants were asked to report a single value from each ampoule and store the extract at 

controlled room temperature, 10 °C to 30 °C. Approximate analyte levels were not reported 

prior to the study. The NIST certified values in SRM 3251 were determined by GC-FID 

(following hydrolysis and derivatization) and LC-MS (following hydrolysis). The certified 

values and their associated uncertainties are provided on an as-received basis in the table below.

Saw palmetto berries. Participants were provided with three packets, each containing 

approximately 6 g of saw palmetto (Serenoa repens) fruit. The ground saw palmetto berries 

were heat-sealed inside nitrogen-flushed 0.1 mm (4 mil) polyethylene bags, which were then 

sealed inside aluminized plastic bags with 2 packets of silica gel. Before use, participants were 

instructed to thoroughly mix the contents of the packet and use a sample size of at least 0.5 g. 

Participants were asked to report a single value from each packet and store the material at 

controlled room temperature, 10 °C to 30 °C. Approximate analyte levels were not reported 

prior to the study. The NIST certified values in SRM 3250 were determined by GC-FID 

(following extraction, hydrolysis and derivatization) and LC-MS (following extraction and 

hydrolysis). The certified values and their associated uncertainties, corrected for the moisture 

content of the material (6.42 %), are provided on an as-received basis in the table below.

Certified Mass Fraction

Certified Mass Fraction in SRM 3250 (mg/g)

Analyte in SRM 3251 (mg/g) (as-received basis)

Campesterol 0.533 ± 0.031 0.1100 ± 0.0023

β-Sitosterol 1.666 ± 0.064 0.425 ± 0.017

Stigmasterol 0.247 ± 0.040 0.0446 ± 0.0019

Study Results

• Twenty-four laboratories enrolled in this exercise and received samples, and eight 

laboratories reported results (33 % participation).

• The consensus means for campesterol, β-sitosterol, and stigmasterol in the extract were 

within the target range, but the consensus ranges were quite wide for all three (33 % to 

over 100 % RSD).
106

• The consensus means for campesterol and β-sitosterol in the ground berries were well

below the target range, while the consensus mean for stigmasterol was within the target 

range. The consensus ranges were quite wide for all three (40 % to 50 % RSD).

• The sample/control comparison graphs (Figures 66-68) indicate a possible calibration 

error. Laboratories that reported high values for the extract also reported high values for 

the berries. The same is true for laboratories reporting low values.

• Half of the laboratories reported using a hydrolysis approach for sample preparation. 

Two laboratories (25 %) reported using solvent extraction, and one laboratory reported 

using a shaking/sonication extraction (13 %). Laboratories using solvent extraction 

reported values at or below the target value.

• Almost all laboratories (88 %) used GC-FID as their analytical method. One laboratory 

reported using GC-MS. The laboratory using GC-MS reported values that were below 

the target and consensus means.

• Half of the laboratories reported using an internal standard approach to calibration, and 

these laboratories consistently reported values at or above the target range in the extract 

material. Three laboratories (38 %) reported using an external standard approach to 

calibration, and these laboratories consistently reported values at or below the target 

range in the extract material. One laboratory (13 %) reported using a standard addition 

approach to calibration.

Technical Recommendations

The following recommendations are based on results obtained by the participants in this study. 

• A calibration error is apparent in the sample/control comparison graphs. Calibrant 

materials should be subjected to the same preparation procedure as the samples 

(derivatization, hydrolysis, etc.). 

• When sample preparation is extensive, an internal standard approach may be required to 

improve accuracy and precision.

• If an internal standard approach is used, it is best to add the internal standard at the 

earliest possible point (i.e. prior to extraction, saponification, and/or derivatization)
107

Table 30. Individual data table (NIST) for phytosterols in saw palmetto.
108

Table 31. Data summary table for campesterol in saw palmetto.

Lab A B C Avg SD A B C Avg SD

NIST 0.533 0.031 0.110 0.002

H801

H803

H805 0.480 0.479 0.468 0.476 0.007 0.070 0.072 0.073 0.072 0.002

H810 0.602 0.609 0.617 0.609 0.008 0.104 0.093 0.090 0.096 0.007

H813

H814

H815 0.489 0.488 0.493 0.490 0.003 0.067 0.066 0.062 0.065 0.003

H816

H821

H823 0.659 0.678 0.631 0.656 0.024 0.070 0.093 0.094 0.086 0.014

H824 0.880 0.890 0.890 0.887 0.006 0.150 0.160 0.150 0.153 0.006

H828

H835

H837

H845

H851

H852

H854

H858

H859

H862 0.070 0.060 0.070 0.067 0.006

H865 1.092 0.850 0.554 0.832 0.269 0.063 0.065 0.113 0.080 0.028

H870

H872 0.225 0.213 0.216 0.218 0.006 0.015 0.031 0.032 0.026 0.010

 Consensus Mean 0.529 Consensus Mean 0.080

 Consensus Standard Deviation 0.320 Consensus Standard Deviation 0.037

 Maximum 0.887 Maximum 0.153

 Minimum 0.067 Minimum 0.026

 N 8 N 7

Community 

Results

Campesterol

Individual Results

SRM 3251 Saw Palmetto Extract (mg/g) SRM 3250 Saw Palmetto Fruit (mg/g)
109

Table 32. Data summary table for β-sitosterol in saw palmetto.

Lab A B C Avg SD A B C Avg SD

NIST 1.67 0.06 0.425 0.017

H801

H803

H805 1.61 1.60 1.57 1.59 0.02 0.260 0.274 0.277 0.270 0.009

H810 1.78 1.97 1.69 1.81 0.14 0.307 0.360 0.330 0.332 0.027

H813

H814

H815 1.51 1.53 1.51 1.51 0.01 0.258 0.268 0.261 0.262 0.005

H816

H821

H823 1.74 1.61 1.71 1.69 0.07 0.259 0.270 0.267 0.265 0.006

H824 2.12 2.13 2.15 2.13 0.02 0.380 0.400 0.390 0.390 0.010

H828

H835

H837

H845

H851

H852

H854

H858

H859

H862 0.26 0.25 0.26 0.26 0.01 0.100 0.110 0.120 0.110 0.010

H865 1.79 1.83 2.54 2.06 0.42 0.323 0.308 0.232 0.288 0.049

H870

H872 0.99 0.96 0.93 0.96 0.03 0.077 0.161 0.171 0.136 0.052

 Consensus Mean 1.56 Consensus Mean 0.257

 Consensus Standard Deviation 0.56 Consensus Standard Deviation 0.106

 Maximum 2.13 Maximum 0.390

 Minimum 0.26 Minimum 0.110

 N 8 N 8

β-Sitosterol

SRM 3251 Saw Palmetto Extract (mg/g) SRM 3250 Saw Palmetto Fruit (mg/g)

Individual Results Community Results
110

Table 33. Data summary table for stigmasterol in saw palmetto.

Lab A B C Avg SD A B C Avg SD

NIST 0.247 0.040 0.0446 0.0019

H801

H803

H805 0.259 0.259 0.244 0.254 0.009 0.0380 0.0380 0.0390 0.0383 0.0006

H810 0.297 0.308 0.304 0.303 0.006 0.0600 0.0540 0.0530 0.0557 0.0038

H813

H814

H815 0.174 0.176 0.176 0.175 0.001 0.0280 0.0290 0.0276 0.0282 0.0007

H816

H821

H823 0.215 0.186 0.237 0.213 0.026 0.0440 0.0330 0.0680 0.0483 0.0179

H824 0.290 0.290 0.290 0.290 0.000 0.0500 0.0500 0.0500 0.0500 0.0000

H828

H835

H837

H845

H851

H852

H854

H858

H859

H862 0.030 0.030 0.030 0.030 0.000

H865 0.586 0.592 0.719 0.632 0.075 0.0920 0.0870 0.0750 0.0847 0.0087

H870

H872 0.137 0.130 0.124 0.130 0.007 0.0079 0.0190 0.0198 0.0156 0.0067

 Consensus Mean 0.229 Consensus Mean 0.0453

 Consensus Standard Deviation 0.327 Consensus Standard Deviation 0.0237

 Maximum 0.632 Maximum 0.0847

 Minimum 0.030 Minimum 0.0156

 N 8 N 7

Stigmasterol

SRM 3251 Saw Palmetto Extract (mg/g) SRM 3250 Saw Palmetto Fruit (mg/g)

Individual Results Community Results
111

Figure 60. Campesterol in SRM 3251 Serenoa repens Extract (data summary view). In this 

view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
112

Figure 61. Campesterol in SRM 3250 Serenoa repens (Fruit) (data summary view). In this 

view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
113

Figure 62. β-Sitosterol in SRM 3251 Serenoa repens Extract (data summary view). In this 

view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
114

Figure 63. β-Sitosterol in SRM 3250 Serenoa repens (Fruit) (data summary view). In this view, 

individual laboratory data are plotted with the individual laboratory standard deviation (error 

bars). The black solid line represents the consensus mean, and the black dotted lines represent 

the consensus variability calculated as one standard deviation about the consensus mean. The 

gray shaded region represents the target zone for “acceptable” performance, which encompasses 

the NIST certified value bounded by twice its uncertainty (U95).
115

Figure 64. Stigmasterol in SRM 3251 Serenoa repens Extract (data summary view). In this 

view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
116

Figure 65. Stigmasterol in SRM 3250 Serenoa repens (Fruit) (data summary view). In this 

view, individual laboratory data are plotted with the individual laboratory standard deviation

(error bars). The black solid line represents the consensus mean, and the black dotted lines 

represent the consensus variability calculated as one standard deviation about the consensus 

mean. The gray shaded region represents the target zone for “acceptable” performance, which 

encompasses the NIST certified value bounded by twice its uncertainty (U95).
117

Figure 66. Campesterol in SRM 3250 Serenoa repens (Fruit) and SRM 3251 Serenoa repens

Extract (sample/control comparison view). In this view, the individual laboratory results for the

control (SRM 3251 Serenoa repens Extract) with a certified value for the analyte are compared 

to the results for an unknown (SRM 3250 Serenoa repens (Fruit)). The error bars represent the 

individual laboratory standard deviation. The solid red lines represent the target zone for the 

control (x-axis) and the unknown sample (y-axis). The dotted blue box represents the consensus 

zone for the control (x-axis) and the unknown sample (y-axis). 
118

Figure 67. β-sitosterol in SRM 3250 Serenoa repens (Fruit) and SRM 3251 Serenoa repens

Extract (sample/control comparison view). In this view, the individual laboratory results for the

control (SRM 3251 Serenoa repens Extract) with a certified value for the analyte are compared 

to the results for an unknown (SRM 3250 Serenoa repens (Fruit)). The error bars represent the 

individual laboratory standard deviation. The solid red lines represent the target zone for the 

control (x-axis) and the unknown sample (y-axis). The dotted blue box represents the consensus 

zone for the control (x-axis) and the unknown sample (y-axis). 
119

Figure 68. Stigmasterol in SRM 3250 Serenoa repens (Fruit) and SRM 3251 Serenoa repens

Extract (sample/control comparison view). In this view, the individual laboratory results for the

control (SRM 3251 Serenoa repens Extract) with a certified value for the analyte are compared 

to the results for an unknown (SRM 3250 Serenoa repens (Fruit)). The error bars represent the 

individual laboratory standard deviation. The solid red lines represent the target zone for the 

control (x-axis) and the unknown sample (y-axis). The dotted blue box represents the consensus 

zone for the control (x-axis) and the unknown sample (y-axis).