Dietary Supplement Laboratory 

Quality Assurance Program: 

Exercise J Final Report

Melissa M. Phillips

Catherine A. Rimmer

Laura J. Wood

Mary Bedner

Kaitlyn D. Chieh

Rick L. Paul

http://dx.doi.org/10.6028/NIST.IR.7997

NISTIR 7997
Dietary Supplement Laboratory Quality Assurance Program:

Exercise F Final Report

Melissa M. Phillips

Catherine A. Rimmer

NISTIR 7997

Dietary Supplement Laboratory 

Quality Assurance Program: 

Exercise J Final Report

Melissa M. Phillips

Catherine A. Rimmer

Laura J. Wood

Mary Bedner

Kaitlyn D. Chieh

Rick L. Paul

Chemical Sciences Division

Material Measurement Laboratory

http://dx.doi.org/10.6028/NIST.IR.7997

April 2014

U.S. Department of Commerce

Penny Pritzker, Secretary

National Institute of Standards and Technology

Patrick D. Gallagher, Director
ii

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 (Method Comparison Data Summary View) ........................4

Sample/Control Comparison View (Sample/Sample Comparison View)...............5

Composition View ...................................................................................................5

Bias View.................................................................................................................5

NUTRITIONAL ELEMENTS (Ca, Mg, Zn) IN NATURAL AND ENHANCED WATERS 6

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

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

Natural Water...........................................................................................................6

Enhanced Water.......................................................................................................6

Study Results ......................................................................................................................6

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

Table 1. Individual data summary table (NIST) for nutritional elements in water............8

Table 2. Data summary table for calcium in natural and enhanced waters........................9

Table 3. Data summary table for magnesium in natural and enhanced waters................10

Table 4. Data summary table for zinc in natural and enhanced waters ...........................11

Figure 2. Calcium in enhanced water ..............................................................................13

Figure 3. Magnesium in SRM 1643e Trace Elements in Water .....................................14

Figure 4. Magnesium in enhanced water ........................................................................15

Figure 5. Zinc in SRM 1643e Trace Elements in Water .................................................16

Figure 6. Zinc in enhanced water ....................................................................................17

Figure 7. Calcium in SRM 1643e Trace Elements in Water ..........................................18

Figure 8. Calcium in enhanced water ..............................................................................19

Figure 9. Magnesium in SRM 1643e Trace Elements in Water .....................................20

Figure 10. Magnesium in enhanced water .......................................................................21

Figure 11. Zinc in SRM 1643e Trace Elements in Water ...............................................22

Figure 12. Zinc in enhanced water ..................................................................................23

Figure 13. Calcium in SRM 1643e Trace Elements in Water and enhanced water ........24

Figure 14. Magnesium in SRM 1643e Trace Elements in Water and enhanced water ...25

Figure 15. Zinc in SRM 1643e Trace Elements in Water and enhanced water ..............26

TOXIC ELEMENTS (As) IN ST. JOHN’S WORT DIETARY SUPPLEMENTS ...............27

Overview Sample...............................................................................................................27

St. John’s Wort Aerial Parts...................................................................................27

St. John’s Wort Methanol Extract..........................................................................27

Study Results 27

Technical Recommendations ............................................................................................28
iii

Table 5. Individual data summary table (NIST) for arsenic in St. John’s Wort dietary 

supplements............................................................................................................29

Table 6. Data summary table for arsenic in St. John’s Wort dietary supplements ..........30

Figure 16. Arsenic in candidate SRM 3264 Hypericum perforatum L. (St. John’s Wort) 

Methanol Extract (data summary view – digestion method).................................31

Figure 17. Arsenic in candidate SRM 3262 Hypericum perforatum L. (St. John’s Wort) 

Aerial Parts (data summary view – digestion method)..........................................32

Figure 18. Arsenic in candidate SRM 3262 Hypericum perforatum L. (St. John’s Wort)

Aerial Parts and candidate SRM 3264 Hypericum perforatum L. (St. John’s Wort) 

Methanol Extract (sample/sample comparison view)............................................33

WATER-SOLUBLE VITAMINS (B5, B6) IN DIETARY SUPPLEMENTS..........................34

Study Overview .................................................................................................................34

Sample Information ...........................................................................................................34

Multivitamin/Multielement Tablets.......................................................................34

Enhanced Water.....................................................................................................34

Study Results .....................................................................................................................35

Technical Recommendations.............................................................................................35

Table 7. Individual data summary table (NIST) for water-soluble vitamins in dietary 

supplements............................................................................................................18

Table 8. Data summary table for vitamin B5 (pantothenic acid) in dietary supplements 19

Table 9. Data summary table for vitamin B6 (pyridoxine hydrochloride) in dietary 

supplements............................................................................................................20

Figure 19. Pantothenic acid in SRM 3280 Multivitamin/Multielement Tablets .............39

Figure 20. Pantothenic acid in enhanced water................................................................40

Figure 21. Pyridoxine hydrochloride in SRM 3280 Multivitamin/Multielement Tablets 41

Figure 22. Pyridoxine hydrochloride in enhanced water ................................................42

Figure 23. Pantothenic acid in SRM 3280 Multivitamin/Multielement Tablets and 

enhanced water.......................................................................................................43

Figure 24. Pyridoxine hydrochloride in SRM 3280 Multivitamin/Multielement Tablets

and enhanced water ...............................................................................................44

VITAMIN E IN DIETARY SUPPLEMENTS ..........................................................................45

Study Overview .................................................................................................................45

Sample Information ..........................................................................................................45

Multivitamin/Multielement Tablets.......................................................................45

Enhanced Water.....................................................................................................45

Study Results .....................................................................................................................45

Technical Recommendations.............................................................................................46

Table 10. Individual data summary table (NIST) for α-tocopherol acetate in dietary 

supplements............................................................................................................47

Table 11. Data summary table for α-tocopherol acetate in dietary supplements.............48

Figure 25. α-Tocopherol acetate in SRM 3280 Multivitamin/Multielement Tablets (data 

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

Figure 26. α-Tocopherol acetate in enhanced water (data summary view) .....................50

Figure 27. α-Tocopherol acetate in SRM 3280 Multivitamin/Multielement Tablets and 

enhanced water (sample/control comparison view)...............................................51
iv

FATTY ACIDS IN BOTANICAL AND FISH OIL DIETARY SUPPLEMENTS................52

Study Overview .................................................................................................................52

Sample Information ...........................................................................................................52

Evening Primrose Oil52

Fish Oil...................................................................................................................52

Study Results .....................................................................................................................52

Technical Recommendations.............................................................................................53

Table 12. Individual data summary table (NIST) for fatty acids in botanical and fish oil 

dietary supplements ...............................................................................................54

Table 13. Data summary table for linoleic acid in botanical and fish oil dietary 

supplements............................................................................................................55

Table 14. Data summary table for α-linolenic acid in botanical and fish oil dietary 

supplements............................................................................................................56

Table 15. Data summary table for γ-linolenic acid in botanical and fish oil dietary 

supplements............................................................................................................57

Table 16. Data summary table for arachidonic acid in botanical and fish oil dietary 

supplements............................................................................................................58

Table 17. Data summary table for EPA in botanical and fish oil dietary supplements ...59

Table 18. Data summary table for DHA in botanical and fish oil dietary supplements ..60

Figure 28. Linoleic acid in SRM 3274-2 Evening Primrose Oil (data summary view) ..61

Figure 29. Linoleic acid in SRM 3275-2 Fish Oil (data summary view) ........................62

Figure 30. α-Linolenic acid in SRM 3274-2 Evening Primrose Oil (data summary view)63

Figure 31. α-Linolenic acid in SRM 3275-2 Fish Oil (data summary view)...................64

Figure 32. γ-Linolenic acid in SRM 3274-2 Evening Primrose Oil (data summary view)65

Figure 33. γ-Linolenic acid in SRM 3275-2 Fish Oil (data summary view) ...................66

Figure 34. Arachidonic acid in SRM 3275-2 Fish Oil (data summary view)..................67

Figure 35. EPA in SRM 3275-2 Fish Oil (data summary view)......................................68

Figure 36. DHA in SRM 3275-2 Fish Oil (data summary view).....................................69

Figure 37. Linoleic acid in SRM 3274-2 Evening Primrose Oil and SRM 3275-2 Fish 

Oil-2 (sample/control comparison view) ...............................................................70

Figure 38. α-Linolenic acid in SRM 3274-2 Evening Primrose Oil and SRM 3275-2 Fish 

Oil-2 (sample/control comparison view) ...............................................................71

Figure 39. γ-Linolenic acid in SRM 3274-2 Evening Primrose Oil and SRM 3275-2 Fish 

Oil-2 (sample/control comparison view) ...............................................................72

AFLATOXINS IN PEANUT BUTTER.....................................................................................73

Study Overview .................................................................................................................73

Sample Information ...........................................................................................................73

Peanut Butter ........................................................................................................73

Peanut Butter Samples...........................................................................................73

Study Results .....................................................................................................................73

Technical Recommendations.............................................................................................74

Table 19. Individual data summary table (NIST) for aflatoxins in peanut butter............75

Table 20. Data summary table for aflatoxin B1 in peanut butter.....................................76

Table 21. Data summary table for aflatoxin B2 in peanut butter.....................................76

Table 22. Data summary table for aflatoxin G1 in peanut butter.....................................77

Table 23. Data summary table for aflatoxin G2 in peanut butter.....................................77

Table 24. Data summary table for total aflatoxins in peanut butter.................................78
v

Figure 40. Aflatoxin B1 in SRM 2387 Peanut Butter (data summary view)...................79

Figure 41. Aflatoxin B2 in SRM 2387 Peanut Butter (data summary view)...................80

Figure 42. Total aflatoxins in SRM 2387 Peanut Butter (data summary view)...............81

Figure 43. Total aflatoxins in peanut butter sample 1 (low) (composition view)............82

Figure 44. Total aflatoxins in peanut butter sample 2 (medium) (composition view).....83

Figure 45. Total aflatoxins in peanut butter sample 3 (high) (composition view)...........84

Figure 46. Aflatoxins in peanut butter sample 1 (low) (bias view) .................................85

Figure 47. Aflatoxins in peanut butter sample 3 (medium) (bias view) ..........................86

Figure 48. Aflatoxins in peanut butter sample 3 (high) (bias view) ................................87

ISOFLAVONES IN SOY PRODUCTS .....................................................................................88

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

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

Soy Protein Isolate .............................................................................................................88

Soy Flour............................................................................................................................88

Study Results .....................................................................................................................89

Technical Recommendations.............................................................................................90

Table 25. Individual data summary table (NIST) for isoflavones in soy dietary 

supplements............................................................................................................92

Table 26. Data summary table for daidzin in soy dietary supplements ...........................93

Table 27. Data summary table for glycitin in soy dietary supplements...........................94

Table 28. Data summary table for genistin in soy dietary supplements ..........................95

Table 29. Data summary table for daidzein in soy dietary supplements..........................96

Table 30. Data summary table for glycitein in soy dietary supplements.........................97

Table 31. Data summary table for genistein in soy dietary supplements.........................98

Table 32. Data summary table for total isoflavones in soy dietary supplements.............99

Figure 49. Daidzin in SRM 3236 Soy Protein Isolate (data summary view, sample 

preparation comparison) ......................................................................................100

Figure 50. Daidzin in SRM 3234 Soy Flour (data summary view, sample preparation 

comparison)..........................................................................................................101

Figure 51. Glycitin in SRM 3236 Soy Protein Isolate (data summary view, sample 

preparation comparison) ......................................................................................102

Figure 52. Glycitin in SRM 3234 Soy Flour (data summary view, sample preparation 

comparison)..........................................................................................................103

Figure 53. Genistin in SRM 3236 Soy Protein Isolate (data summary view, sample 

preparation comparison) ......................................................................................104

Figure 54. Genistin in SRM 3234 Soy Flour (data summary view, sample preparation 

comparison)..........................................................................................................105

Figure 55. Daidzein in SRM 3236 Soy Protein Isolate (data summary view, sample 

preparation comparison) ......................................................................................106

Figure 56. Daidzein in SRM 3234 Soy Flour (data summary view, sample preparation 

comparison)..........................................................................................................107

Figure 57. Glycitein in SRM 3236 Soy Protein Isolate (data summary view, sample 

preparation comparison) ......................................................................................108

Figure 58. Glycitein in SRM 3234 Soy Flour (data summary view, sample preparation 

comparison)..........................................................................................................109

Figure 59. Genistein in SRM 3236 Soy Protein Isolate (data summary view, sample 

preparation comparison) ......................................................................................110
vi

Figure 60. Genistein in SRM 3234 Soy Flour (data summary view, sample preparation 

comparison)..........................................................................................................111

Figure 61. Total isoflavones in SRM 3236 Soy Protein Isolate (data summary view, 

sample preparation comparison)..........................................................................112

Figure 62. Total isoflavones in SRM 3234 Soy Flour (data summary view, sample 

preparation comparison) ......................................................................................113

Figure 63. Daidzin in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

(sample/control comparison view).......................................................................114

Figure 64. Glycitin in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

(sample/control comparison view).......................................................................115

Figure 65. Genistin in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

(sample/control comparison view).......................................................................116

Figure 66. Daidzein in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

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

Figure 67. Glycitein in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

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

Figure 68. Genistein in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

(sample/control comparison view).......................................................................119

Figure 69. Total isoflavones in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

(sample/control comparison view).......................................................................120

BOTANICAL IDENTITY OF PURE AND ADULTERATED GINKGO BILOBA ..........121

Study Overview ...............................................................................................................121

Sample Information .........................................................................................................121

Gingko biloba Extract..........................................................................................121

Gingko biloba Leaf ..............................................................................................121

Study Results ...................................................................................................................121

Technical Recommendations...........................................................................................121

Table 33. Individual data summary table (NIST) for botanical identity of pure and 

adulterated Ginkgo biloba....................................................................................123

Table 34. Data summary table for Ginkgo biloba extracts (yes/no) ..............................124

Table 35. Data summary table for Ginkgo biloba extracts (mass percentage). ............125

Table 36. Data summary table for Ginkgo biloba leaves (yes/no).................................126

Table 37. Data summary table for Ginkgo biloba leaves (mass percentage).................127

Figure 70. Adulterated Ginkgo biloba extract ................................................................128

Figure 71. Adulterated Ginkgo biloba leaves .................................................................129
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

including the dietary supplement current good manufacturing practices (cGMPs). Exercise J of 

this program offered the opportunity for laboratories to assess their in-house measurements of 

nutritional elements (Ca, Mg, and Zn), contaminants (arsenic and aflatoxins), water-soluble 

vitamins (vitamins B5 and B6), fat-soluble vitamins (vitamin E), fatty acids, isoflavones, and 

botanical authenticity/identification 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 and worldwide 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 Federal

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. In addition the DSHEA authorized the establishment of the Office of 

Dietary Supplements at the National Institutes of Health (NIH ODS). 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 AOAC Official Method of Analysis would benefit the 

dietary supplement community.

NIST has experience in the administration 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 wide range of matrices and analytes under the “dietary supplement” umbrella means 

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

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

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 tenth exercise of the DSQAP, Exercise J. Fifty-eight

laboratories responded to the call for participants distributed in April 2013. Samples were 

shipped to participants in June 2013, and results were returned to NIST by September 2013. 

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

February 2014.

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*:

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

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 

 3 ISO 13528:2005(E), Statistical methods for use in proficiency testing by interlaboratory comparisons, Annex C.
4

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 

value 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 or by measurements obtained from collaborating laboratories. 

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 samples in 

which a NIST certified or reference value is not available, the analytes are measured at NIST 

using an appropriate method. The NIST-assessed value represents the mean of at least three 

replicates. For materials acquired from another proficiency testing program, the consensus value 

and uncertainty from the completed round is used as the target range.

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 data reported by 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 (Method Comparison 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. Where appropriate, 

two consensus means may be calculated for the same sample if bimodality is identified in the 

data. In this case, two consensus means and ranges will be displayed in the data summary view. 

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. In the case in which a method comparison is relevant, different colored data points 

may be used to indicate laboratories that used a specific approach to sample preparation, 

analytical method, or quantitation.
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). The axes of this graph are centered about the consensus mean values 

for each sample or control, to a limit of zero and twice the consensus mean. Depending on the 

variability in the data, the axes may be scaled proportionally to better display the individual data 

points for each laboratory. In some cases, when the consensus and target ranges have limited 

overlap, the solid red box may only appear partially on the graph. If the variability in the data is 

high (greater than 100 % RSD), the dotted blue box may also only appear partially on the graph. 

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.

Composition View

In this view, used for the aflatoxins in peanut butter study, total composition of the sample is 

plotted as a function of the measurement of individual components. This view allows 

comparison of data in which limited statistical information is available as a result of low 

participation and/or reporting of one data point per sample. This view is also useful in 

comparison of methods in which a total composition is reported by some laboratories, but 

individual components are reported by other laboratories. One program goal is to allow 

laboratories to demonstrate laboratory performance, regardless of the analytical approach.

Bias View

In this view, used for the aflatoxins in peanut butter study, the laboratory ZNIST-score is overlaid 

for each component, and outlying ZNIST-scores are highlighted in the marginally different range 

(2 < |Z| < 3) in orange, and in the significantly different range (|Z| > 3) in red. This view 

demonstrates visually which components result in poor ZNIST-scores, and allows comparisons 

between problematic areas as a function of sample type. In an overall composition analysis, this 

view allows a laboratory to rapidly identify which analyte is resulting in erroneous results.
6

NUTRITIONAL ELEMENTS (Ca, Mg, Zn) IN NATURAL AND ENHANCED WATERS

Study Overview 

In this study, participants were provided with SRM 1643e Trace Elements in Water and 

commercially available enhanced water. Participants were asked to use in-house analytical 

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

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

Sample Information 

Natural Water. Participants were provided with one polyethylene bottle containing 125 mL of 

acidified water. Nitric acid is present at a concentration of approximately 0.8 mol/L to stabilize 

the trace elements. Before use, participants were instructed to thoroughly mix the contents of the 

bottle, and a sample size of at least 1.0 mL was recommended. Participants were asked to store 

the material at controlled room temperature, 10 °C to 30 °C, to prepare three samples, and to

report three values from the single bottle provided. Approximate analyte levels were not 

reported to participants prior to the study. The certified values in SRM 1643e Trace Elements in 

Water were determined at NIST using inductively coupled plasma mass spectrometry (ICP-MS)

and inductively coupled plasma optical emission spectrometry (ICP-OES). The certified values 

and uncertainties for Ca, Mg, and Zn are outlined in the table below.

Enhanced Water. Participants were provided with one 600 mL bottle of commercially available 

enhanced water. Before use, participants were instructed to thoroughly mix the contents of the 

bottle, and a sample size of at least 1.0 mL was recommended. Participants were asked to store 

the material at controlled room temperature, 10 °C to 30 °C, to prepare three samples, and to 

report three values from the single bottle provided. Approximate analyte levels were not 

reported to participants prior to the study. Certified values are not available for this material;

NIST provided values for Ca, Mg, and Zn based on triplicate analysis using ICP-OES using 

standard additions as the method of quantitation. The NIST values in enhanced water are 

reported in the table below with an estimated relative uncertainty of 5 %.

Analyte

Certified Mass Fraction

in SRM 1643e (μg/g)

Estimated Mass Fraction

in Commercial Enhanced Water (μg/g)

Ca 31.5 ± 1.1 201 ± 10

Mg 7.84 ± 0.096 86.4 ± 4.3

Zn 0.0765 ± 0.0021 6.79 ± 0.34

Study Results 

• Thirty-five laboratories enrolled in this exercise and received samples. Twenty-nine

laboratories reported results for calcium and magnesium (83 % participation). Twentysix to 29 laboratories reported results for zinc (74 % to 83 % participation). 

• The consensus means for calcium and magnesium in both materials and for zinc in the 

enhanced water were within the target range with an acceptable variability (5 % to 14 % 

relative standard deviation (RSD)). 

• The consensus mean for zinc in SRM 1643e was above the target range with a high

variability (59 % RSD). 

• Zinc in the natural water is two orders of magnitude lower than that of the enhanced 

water, making this material near the method detection limit (MDL) or limit of 
7

quantitation (LOQ) for some participants. Measuring near the MDL or LOQ can give 

erroneous values, either high or low. Figure 15 demonstrates this by showing the 

consensus zone more tightly surrounding the target zone for enhanced water than for the 

natural water.

• A majority of the laboratories reported using either open-beaker digestion (34 %) or 

microwave digestion (41 %) for sample preparation. The remaining laboratories reported 

using hot block digestion, dilution, or other methods (14 %). 

• A majority of the laboratories reported using either ICP-OES (55 %) or ICP-MS (41 %) 

as their analytical method. One laboratory reported using atomic absorption spectroscopy 

(AAS). 

Technical Recommendations 

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

• Both water samples should have been straightforward to digest; no trends were observed 

based on either the sample preparation method or analytical method used.

• The most likely source of error in this study is related to construction of the calibration

curves. To avoid calibration problems, be sure to

o Include the lowest and highest expected solution concentrations, plus one or two 

intermediate concentration points in the calibration curve.

o Ensure that the calibration curve is linear and surrounds expected sample 

concentrations following digestion and/or dilution. Samples should not go beyond 

the linear range of the calibration curve resulting in extrapolation of calibration 

curves leading to false values.

o Use a sufficient number of blanks to accurately determine MDL and LOQ.

• Run a quality control sample (either in-house or a commercially available reference 

material) of known concentration to ensure that your method is performing as expected.

• Double-check all calculations; miscalculations and reporting of wrong units were a cause 

for some errors.
8

Table 1. Individual data summary table (NIST) for nutritional elements in water.

Lab Code: NIST

Analyte Sample Units xi si Zcomm ZNIST N x* s* xNIST U 95

Ca Natural Water μg/g 31.5 1.1 0.0 0.0 29 31.4 3.2 31.5 1.1

Ca Enhanced Water μg/g 201 3 -0.3 0.0 29 204 10 201 10

Mg Natural Water μg/g 7.84 0.10 -0.2 0.0 29 7.92 0.45 7.84 0.10

Mg Enhanced Water μg/g 86.4 0.1 0.0 0.0 29 86.4 5.1 86.4 4.3

Zn Natural Water μg/g 0.0765 0.0021 -0.6 0.0 26 0.1160 0.0679 0.0765 0.0021

Zn Enhanced Water μg/g 6.79 0.02 -0.3 0.0 29 7.09 0.99 6.79 0.34

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U95 ±95% confidence interval

Zcomm Z-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

Exercise J - May 2013 - Nutritional Elements

1. Your Results 2. Community Results 3. Target

National Institute of Standards & Technology
9

Table 2. Data summary table for calcium in natural and enhanced waters.

Lab A B C Avg SD A B C Avg SD

NIST 31.5 1.1 198 202 203 201 3

J002 4.2 6.4 2.6 4.4 1.9 11 17 15 14 3

J006 31.0 30.9 31.1 31.0 0.1 229 223 227 226 3

J007 29.2 28.9 28.8 29.0 0.2 194 196 196 195 1

J008 30.8 28.9 29.9 29.9 1.0 190 190 191 190 1

J009

J014 34.4 34.0 33.2 33.9 0.6 211 205 210 209 3

J016 30.9 30.7 32.0 31.2 0.7 203 203 197 201 3

J017 28.7 28.2 28.3 28.4 0.3 210 208 210 209 1

J018

J019 2679.3 2475.3 2497.2 2550.6 112.0 240 229 253 241 12

J020 44.8 43.8 42.9 43.8 1.0 215 219 215 216 2

J021

J022 32.8 30.4 31.2 31.5 1.2 195 190 201 195 5

J024 34.8 35.0 34.8 34.9 0.1 219 230 216 222 7

J025 55.4 55.2 54.4 55.0 0.5 199 200 204 201 3

J026 29.1 28.5 31.1 29.6 1.3 187 202 201 197 8

J029 31.4 31.4 30.5 31.1 0.5 204 205 209 206 3

J031 30.0 30.0 30.0 30.0 0.0 204 204 202 203 1

J033

J034 24.4 24.7 24.9 24.7 0.3 209 208 207 208 1

J036 31.5 32.7 34.1 32.8 1.3 199 195 200 198 3

J037 28.7 27.9 28.0 28.2 0.4 204 203 206 205 2

J038 28.4 25.7 27.1 27.1 1.3 122 140 218 160 51

J039

J041

J047 32.6 32.5 32.5 32.5 0.1 203 204 204 204 1

J048 32.1 32.3 32.2 32.2 0.1 203 203 204 203 1

J051 29.7 30.0 29.8 29.8 0.1 201 201 202 201 0

J052 44.8 46.5 39.0 43.4 3.9 270 272 242 262 17

J053 31.9 32.1 30.6 31.5 0.8 198 192 206 199 7

J054 28.1 28.9 28.3 28.4 0.4 196 204 195 198 5

J055 33.2 32.5 32.6 32.8 0.4 199 199 199 199 0

J056 32.0 31.0 31.0 31.3 0.6 210 200 210 207 6

J057 30.9 29.7 29.6 30.1 0.7 201 204 203 202 2

J060 31.5 31.7 31.6 31.6 0.1 207 206 206 206 1

 Consensus Mean 31.4 Consensus Mean 204

 Consensus Standard Deviation 3.2 Consensus Standard Deviation 10

 Maximum 2550.6 Maximum 262

 Minimum 4.4 Minimum 14

 N 29 N 29

Calcium

SRM 1643e Trace Elements in Water (μg/g) Enhanced Water (μg/g)

Community 

Results Individual Results
10

Table 3. Data summary table for magnesium in natural and enhanced waters.

Lab A B C Avg SD A B C Avg SD

NIST 7.84 0.10 86.4 86.6 86.4 86.4 0.1

J002 6.61 6.35 6.80 6.59 0.22 72.9 71.8 72.7 72.5 0.6

J006 7.82 7.84 7.80 7.82 0.02 84.6 87.3 85.7 85.9 1.4

J007 7.64 7.52 7.60 7.59 0.06 86.3 86.8 88.7 87.3 1.3

J008 7.93 7.67 7.82 7.81 0.13 81.3 82.1 82.0 81.8 0.4

J009

J014 7.69 7.90 7.89 7.83 0.12 91.2 89.1 90.5 90.3 1.1

J016 8.33 8.14 8.24 8.24 0.10 91.5 91.7 90.0 91.1 0.9

J017 8.00 7.90 7.89 7.93 0.06 90.8 89.6 90.3 90.2 0.6

J018

J019 835.20 812.00 819.10 822.10 11.89 107.4 94.0 90.7 97.4 8.8

J020 12.60 12.60 12.20 12.47 0.23 91.5 82.6 92.8 89.0 5.6

J021

J022 8.33 7.85 8.34 8.17 0.28 94.3 95.1 92.2 93.8 1.5

J024 8.72 8.62 8.60 8.65 0.06 89.3 97.0 88.4 91.6 4.7

J025 7.70 7.70 7.60 7.67 0.06 85.3 86.2 87.8 86.4 1.3

J026 7.31 7.17 7.84 7.44 0.35 80.1 85.3 85.0 83.5 2.9

J029 7.87 7.72 7.37 7.65 0.26 83.1 84.5 85.6 84.4 1.3

J031 8.00 8.00 8.00 8.00 0.00 86.0 86.0 85.0 85.7 0.6

J033

J034 7.72 7.80 7.83 7.78 0.06 82.5 81.8 82.1 82.1 0.4

J036 8.16 8.58 10.80 9.18 1.42 89.1 87.9 88.6 88.5 0.6

J037 7.70 7.48 7.51 7.56 0.12 89.5 88.4 89.5 89.1 0.7

J038 7.06 6.41 6.80 6.76 0.33 44.6 55.2 90.9 63.6 24.2

J039

J041

J047 8.39 8.49 8.39 8.42 0.06 88.8 89.0 89.6 89.1 0.4

J048 8.00 8.10 8.05 8.05 0.05 82.5 82.3 82.1 82.3 0.2

J051 7.73 7.82 7.76 7.77 0.04 87.5 87.1 87.8 87.5 0.4

J052 8.06 8.07 8.11 8.08 0.02 75.7 75.7 75.5 75.6 0.1

J053 7.94 8.20 7.68 7.94 0.26 85.7 82.0 80.8 82.8 2.6

J054 7.54 7.52 7.61 7.56 0.05 86.3 88.2 89.1 87.9 1.4

J055 7.51 7.30 7.29 7.37 0.12 82.3 82.2 82.1 82.2 0.1

J056 8.20 7.80 8.20 8.07 0.23 95.0 92.0 94.0 93.7 1.5

J057 8.04 7.88 7.97 7.96 0.08 86.1 85.6 85.6 85.8 0.3

J060 7.30 8.10 8.07 7.82 0.45 86.6 86.6 86.9 86.7 0.2

 Consensus Mean 7.92 Consensus Mean 86.4

 Consensus Standard Deviation 0.45 Consensus Standard Deviation 5.1

 Maximum 822.10 Maximum 97.4

 Minimum 6.59 Minimum 63.6

 N 29 N 29

Magnesium

SRM 1643e Trace Elements in Water (μg/g) Enhanced Water (μg/g)

Individual Results Community Results
11

Table 4. Data summary table for zinc in natural and enhanced waters.

Lab A B C Avg SD A B C Avg SD

NIST 0.0765 0.0021 6.77 6.82 6.79 6.79 0.02

J002 842.5982 812.8184 1099.6997 918.3721 157.7387 824.04 842.77 862.72 843.18 19.34

J006 0.0817 0.0797 0.0396 0.0670 0.0238 8.19 8.63 8.27 8.37 0.23

J007 0.0710 0.0710 0.0700 0.0707 0.0006 6.40 6.43 6.55 6.46 0.08

J008 0.1558 0.1578 0.1698 0.1611 0.0076 6.32 6.27 6.34 6.31 0.04

J009

J014 0.0647 0.0660 0.0647 0.0651 0.0008 5.84 5.83 5.98 5.88 0.08

J016 0.0893 0.0896 0.0848 0.0879 0.0027 6.71 6.71 6.70 6.71 0.01

J017 0.0800 0.0755 0.0764 0.0773 0.0024 6.88 7.02 6.93 6.94 0.07

J018

J019 13.0000 11.7000 11.8000 12.1667 0.7234 16.30 15.10 15.70 0.85

J020 0.3400 0.2000 0.1300 0.2233 0.1069 6.80 6.60 6.50 6.63 0.15

J021

J022 0.0770 0.0710 0.0750 0.0743 0.0031 6.07 6.24 6.21 6.17 0.09

J024 0.0992 0.0948 0.1130 0.1023 0.0095 6.76 7.73 6.88 7.12 0.53

J025 1.5691 1.5741 1.5496 1.5643 0.0129 6.52 6.90 6.54 6.65 0.21

J026 0.1868 0.1776 0.1813 0.1819 0.0046 10.53 8.90 8.41 9.28 1.11

J029 0.0850 0.0820 0.0900 0.0857 0.0040 6.93 7.07 6.67 6.89 0.20

J031 7.00 7.00 7.00 7.00 0.00

J033

J034 6.97 6.91 6.91 6.93 0.03

J036 7.11 7.16 7.17 7.15 0.03

J037 0.0711 0.0714 0.0710 0.0712 0.0002 6.53 6.42 6.50 6.48 0.06

J038 0.3300 0.1800 0.1800 0.2300 0.0866 8.40 8.51 9.06 8.66 0.35

J039

J041

J047 0.0770 0.0760 0.0780 0.0770 0.0010 6.79 6.82 6.86 6.82 0.04

J048 0.0750 0.0760 0.0770 0.0760 0.0010 6.94 7.02 7.00 6.99 0.04

J051 0.0776 0.0773 0.0778 0.0776 0.0002 6.70 6.69 6.69 6.69 0.01

J052 0.0610 0.0610 0.0620 0.0613 0.0006 10.37 10.57 10.61 10.52 0.13

J053 0.0920 0.0860 0.0970 0.0917 0.0055 5.91 6.06 6.46 6.14 0.28

J054 0.1454 0.1453 0.1454 0.1454 0.0001 5.94 6.20 6.31 6.15 0.19

J055 0.0998 0.0976 0.0977 0.0983 0.0012 7.83 7.97 7.90 7.90 0.07

J056 0.0640 0.0640 0.0640 0.0640 0.0000 6.20 6.00 6.00 6.07 0.12

J057 0.0835 0.0775 0.0795 0.0802 0.0031 7.03 7.07 7.01 7.03 0.03

J060 0.0660 0.0660 0.0660 0.0660 0.0000 6.59 6.57 6.58 6.58 0.01

 Consensus Mean 0.1160 Consensus Mean 7.09

 Consensus Standard Deviation 0.0679 Consensus Standard Deviation 0.99

 Maximum 918.3721 Maximum 843.18

 Minimum 0.0613 Minimum 5.88

 N 26 N 29

Zinc

SRM 1643e Trace Elements in Water (μg/g) Enhanced Water (μg/g)

Individual Results Community Results
12

Figure 1. Calcium in SRM 1643e Trace Elements in Water (data summary view – digestion

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

standard deviation (error bars). The color of the data point represents the sample preparation 

(digestion) procedure employed. 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).
13

Figure 2. Calcium in enhanced water (data summary view – digestion method). In this view, 

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

bars). The color of the data point represents the sample preparation (digestion) procedure 

employed. 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 in triplicate by ICP-OES, bounded by an estimated 

relative uncertainty of 5 %.
14

Figure 3. Magnesium in SRM 1643e Trace Elements in Water (data summary view – digestion

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

standard deviation (error bars). The color of the data point represents the sample preparation 

(digestion) procedure employed. 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).
15

Figure 4. Magnesium in enhanced water (data summary view – digestion method). In this view, 

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

bars). The color of the data point represents the sample preparation (digestion) procedure 

employed. 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 in triplicate by ICP-OES, bounded by an estimated 

relative uncertainty of 5 %.
16

Figure 5. Zinc in SRM 1643e Trace Elements in Water (data summary view – digestion

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

standard deviation (error bars). The color of the data point represents the sample preparation 

(digestion) procedure employed. 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).
17

Figure 6. Zinc in enhanced water (data summary view – digestion method). In this view, 

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

bars). The color of the data point represents the sample preparation (digestion) procedure 

employed. 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 in triplicate by ICP-OES, bounded by an estimated 

relative uncertainty of 5 %.
18

Figure 7. Calcium in SRM 1643e Trace Elements in Water (data summary view – instrumental

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

standard deviation (error bars). The color of the data point represents the instrumental method 

employed. 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).
19

Figure 8. Calcium in enhanced water (data summary view – instrumental method). In this view, 

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

bars). The color of the data point represents the instrumental method employed. 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 in triplicate by ICP-OES, bounded by an estimated relative uncertainty of 5 %.
20

Figure 9. Magnesium in SRM 1643e Trace Elements in Water (data summary view –

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

laboratory standard deviation (error bars). The color of the data point represents the instrumental 

method employed. 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).
21

Figure 10. Magnesium in enhanced water (data summary view – instrumental method). In this 

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

(error bars). The color of the data point represents the instrumental method employed. 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 in triplicate by ICP-OES, bounded by an estimated relative uncertainty 

of 5 %.
22

Figure 11. Zinc in SRM 1643e Trace Elements in Water (data summary view – instrumental

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

standard deviation (error bars). The color of the data point represents the instrumental method 

employed. 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).
23

Figure 12. Zinc in enhanced water (data summary view – instrumental method). In this view, 

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

bars). The color of the data point represents the instrumental method employed. 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 in triplicate by ICP-OES, bounded by an estimated relative uncertainty of 5 %.
24

Figure 13. Calcium in SRM 1643e Trace Elements in Water and enhanced water 

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

(SRM 1643e Trace Elements in Water) with a certified value for the analyte are compared to the 

results for an unknown (enhanced water). The solid red box represents 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).
25

Figure 14. Magnesium in SRM 1643e Trace Elements in Water and enhanced water 

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

(SRM 1643e Trace Elements in Water) with a certified value for the analyte are compared to the 

results for an unknown (enhanced water). The solid red box represents 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).
26

Figure 15. Zinc in SRM 1643e Trace Elements in Water and enhanced water (sample/control 

comparison view). In this view, the individual laboratory results for the control (SRM 1643e

Trace Elements in Water) with a certified value for the analyte are compared to the results for an 

unknown (enhanced water). The solid red box represents the target zone for the control (x-axis) 

and unknown (y-axis). The dotted blue box represents the consensus zone for the control (xaxis) and the unknown sample (y-axis).
27

TOXIC ELEMENTS (As) IN ST. JOHN’S WORT DIETARY SUPPLEMENTS 

Study Overview 

In this study, participants were provided with two NIST candidate SRMs, SRM 3262 Hypericum 

perforatum L. (St. John’s Wort) Aerial Parts and SRM 3264 Hypericum perforatum L. (St. 

John’s Wort) Methanol Extract. Participants were asked to use in-house analytical methods to 

determine the mass fractions of arsenic (As) in each of the matrices and report values on an asreceived basis. 

Sample Information 

St. John’s Wort Aerial Parts. Participants were provided with three packets, each containing 

approximately 3 g of St. John’s Wort aerial parts. The plant was ground, homogenized, and 

heat-sealed inside 4 mil polyethylene bags, which were then sealed inside nitrogen-flushed 

aluminized plastic bags along with two packets of silica gel. Before use, participants were 

instructed to thoroughly mix the contents of each packet and use a sample size of at least 1.0 g. 

Participants were asked to store the material at controlled room temperature, 10 °C to 30 °C, and 

to prepare one sample and report one value from the each of the packets provided. Approximate 

analyte levels were not reported to participants prior to the study. The target value for arsenic in 

SRM 3262, (145 ± 8) ng/g, was determined at NIST using ICP-MS and instrumental neutron 

activation analysis (INAA).

St. John’s Wort Methanol Extract. Participants were provided with three packets, each

containing approximately 1.6 g of a methanol extract of St. John’s Wort. The extract was 

ground, homogenized, and heat-sealed inside 4 mil polyethylene bags, which were then sealed 

inside nitrogen-flushed aluminized plastic bags along with two packets of silica gel. Before use, 

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

of at least 1.0 g. Participants were asked to store the material at controlled room temperature, 

10 °C to 30 °C, and to prepare one sample and report one value from the each of the packets 

provided. Approximate analyte levels were not reported to participants prior to the study. The 

target value for arsenic in SRM 3264, (34.8 ± 3.0) ng/g, was determined at NIST using ICP-MS.

Study Results

• Thirty-four laboratories enrolled in this exercise and received samples. Twenty-seven

laboratories reported results for arsenic in St. John’s Wort aerial parts (79 % 

participation). Twenty-six laboratories reported results for arsenic in St. John’s Wort 

methanol extract (76 % participation).

• The consensus means for arsenic were within the target range with high variability (24 %

and 34 % relative standard deviation (RSD) for the aerial parts and methanol extract, 

respectively).

• The greater number of low values reported for the aerial parts, compared to the extract, is 

most likely a result of incomplete digestions.

• The extremely high values reported for the extract material is most likely a result of 

incorrect calibration curves or dilutions of samples.

• A majority of the laboratories reported using microwave digestion (63 %) for sample 

preparation. Open beaker digestion (26 %) and hot block digestion (7 %) were also 

reported as methods of sample preparation.

• All of the laboratories reported using ICP-MS as their analytical method for analysis. 
28

Technical Recommendations 

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

• While twice as many laboratories reported using microwave digestion for sample 

preparation than other methods reported, results did not vary based on the sample 

preparation method used.

• Arsenic is volatile, so care must be taken to not lose arsenic during sample preparation.

• With a vigorous microwave digestion (high heat) organoarsenic should all be converted 

to As+5 and any subsequent heating will not result in loss of As.

• Plant materials can be difficult to digest without the use of HF. 

• Arsenic in the methanol extract is at a very low level and may be close to detection limits. 

o Use a good calibration curve with low concentrations to help with accuracy.

o Use a sufficient number of blanks so an accurate method detection limit and limit 

of quantitation can be determined.

• Run a quality control sample of known concentration to ensure your method is 

performing as expected.

• Double-check all calculations for any errors.
29

Table 5. Individual data summary table (NIST) for arsenic in St. John’s Wort dietary supplements.

Lab Code: NIST

Analyte Sample Units xi si Zcomm ZNIST N x* s* xNIST U 95

As SJW Extract ng/g 34.8 3.0 -0.2 0.0 26 38.0 13.0 34.8 3.0

As SJW Aerial Parts ng/g 145 8 0.0 0.0 27 146 35 145 8

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U 95 ±95% confidence interval

Zcomm Z-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

Exercise J - May 2013 - Toxic Elements

1. Your Results 2. Community Results 3. Target

National Institute of Standards & Technology
30

Table 6. Data summary table for arsenic in St. John’s Wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 34.8 3.0 145 8

J002 29.9 23.5 30.3 27.9 3.8 123 128 118 123 5

J006 27.4 43.5 26.4 32.4 9.6 140 153 154 149 8

J007 42.0 38.8 46.8 42.5 4.0 136 122 116 125 10

J008 76.5 73.0 66.8 72.1 4.9 197 216 214 209 10

J009

J010 19.0 20.0 18.0 19.0 1.0 91 96 101 96 5

J012 32.0 33.4 40.3 35.2 4.4 131 133 165 143 19

J013 34.2 34.3 44.8 37.8 6.1 138 150 162 150 12

J014 23.2 19.6 20.6 21.1 1.9 132 133 144 136 7

J017 144 154 147 148 5

J018

J019

J020 39.6 38.5 37.3 38.5 1.2 143 128 128 133 9

J021

J022 54.4 59.8 53.7 56.0 3.3 209 192 213 205 11

J024 46.4 30.3 48.5 41.7 10.0 130 130 143 134 8

J025 19.6 22.4 14.6 18.9 4.0 95 91 114 100 12

J026 35.5 32.6 34.1 2.0 176 190 183 183 7

J029 46.2 50.8 51.6 49.5 2.9 168 135 134 146 19

J030 40.0 40.0 40.0 40.0 0.0 170 190 170 177 12

J031 114.0 117.0 121.0 117.3 3.5 124 116 113 118 6

J033 37.3 38.8 38.8 38.3 0.9 165 169 174 169 5

J035

J036 34.0 33.0 34.0 33.7 0.6 130 129 132 130 2

J037 34.0 46.0 32.0 37.3 7.6 109 131 120 120 11

J038

J041

J046 42.6 46.1 44.8 44.5 1.8 201 190 181 191 10

J047 31.0 33.0 31.0 31.7 1.2 122 132 135 130 7

J052 32.5 38.5 22.0 31.0 8.4 115 120 113 116 4

J053 23.0 25.0 22.0 23.3 1.5 121 124 119 121 3

J055 34.7 35.5 35.1 35.1 0.4 120 119 112 117 5

J056 62.0 68.0 60.0 63.3 4.2 180 170 150 167 15

J057 149.0 153.0 149.0 150.3 2.3 240 218 211 223 15

 Consensus Mean 38.0 Consensus Mean 146

 Consensus Standard Deviation 13.0 Consensus Standard Deviation 35

 Maximum 150.3 Maximum 223

 Minimum 18.9 Minimum 96

 N 26 N 27

Arsenic

SRM 3264 St. John's Wort Extract (ng/g) SRM 3262 St. John's Wort Aerial Parts (ng/g)

Individual Results Community Results
31

Figure 16. Arsenic in candidate SRM 3264 Hypericum perforatum L. (St. John’s Wort) 

Methanol Extract (data summary view – digestion method). In this view, individual laboratory 

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

identified by digestion method in this graph. 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 ICP-MS, bounded by an 

uncertainty estimated as twice the standard deviation.
32

Figure 17. Arsenic in candidate SRM 3262 Hypericum perforatum L. (St. John’s Wort) Aerial 

Parts (data summary view – digestion method). In this view, individual laboratory data are 

plotted with the individual laboratory standard deviation (error bars). The color of the data point 

represents the sample preparation (digestion) procedure employed. 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 mean of NIST 

value determined by ICP-MS and INAA, bounded by an uncertainty estimated as twice the 

combined standard deviation 
33

Figure 18. Arsenic in candidate SRM 3262 Hypericum perforatum L. (St. John’s Wort) Aerial 

Parts and candidate SRM 3264 Hypericum perforatum L. (St. John’s Wort) Methanol Extract 

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

(St. John’s Wort aerial parts) are compared to the results for a second sample (St. John’s Wort 

extract). The solid red box represents the target zone for the two samples, St. John’s Wort 

extract (x-axis) and St. John’s Wort aerial parts (y-axis). The dotted blue box represents the 

consensus zone for St. John’s Wort extract (x-axis) and St. John’s Wort aerial parts (y-axis).
34

WATER-SOLUBLE VITAMINS (B5, B6) IN DIETARY SUPPLEMENTS

Study Overview 

In this study, participants were provided with one NIST SRM, SRM 3280 Multivitamin/

Multielement Tablets, and one bottle of commercially available enhanced water. Participants 

were asked to use in-house analytical methods to determine the mass fractions of vitamins B5

and B6 in each of the matrices and report values on an as-received basis. Participants were asked 

to report the vitamin B5 and B6 content as pantothenic acid and pyridoxine hydrochloride, 

respectively.

Sample Information 

Multivitamin/Multielement Tablets. Participants were provided with one bottle containing 30 

multivitamin/multielement tablets. Before use, participants were instructed to grind all 30 

tablets, mix the resulting powder thoroughly, and use a sample size of at least 0.25 g. 

Participants were asked to store the material at controlled room temperature, 10 °C to 30 °C,

prepare three samples, and report three values from the single bottle provided. Approximate 

analyte levels were not reported to participants prior to the study. The NIST certified values and 

uncertainties for vitamin B5 in SRM 3280 were determined by isotope dilution liquid 

chromatography with mass spectrometric detection (ID-LC/MS) following solvent extraction, in 

combination with data from numerous collaborating laboratories. The NIST certified values and 

uncertainties for vitamin B6 in SRM 3280 were determined by LC with absorbance detection 

(LC/abs) and ID-LC/MS following solvent extraction, in combination with data from numerous 

collaborating laboratories. The certified values are reported in the table below, both on a drymass basis and after correction for moisture of the material (1.37 %).

Certified Mass Fraction 

in SRM 3280 (mg/g)

Certified Mass Fraction

in SRM 3280 (mg/g)

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

Pantothenic Acid (B5) 7.30 ± 0.96 7.20 ± 0.95

Pyridoxine Hydrochloride (B6) 1.81 ± 0.17 1.79 ± 0.17

Enhanced Water. Participants were provided with one 600 mL bottle of commercially available 

enhanced water. Before use, participants were instructed to thoroughly mix the contents of the 

bottle, and a sample size of at least 1.0 mL was recommended. Participants were asked to store 

the material at controlled room temperature, 10 °C to 30 °C, prepare three samples, and report 

three values from the single bottle provided. Approximate analyte levels were not reported to 

participants prior to the study. Certified values are not available for this material; NIST provided 

values for vitamins B5 and B6 based on triplicate analysis using ID-LC/MS. The NIST values in 

enhanced water are reported in the table below with an estimated uncertainty of 5 %. 

Analyte

Estimated Mass Fraction 

in Commercial Enhanced Water (μg/g)

Pantothenic Acid 39.3 ± 0.1

Pyridoxine Hydrochloride 6.53 ± 0.02
35

Study Results

• Thirty-three laboratories enrolled in this exercise and received samples. Twenty-three

laboratories reported results for vitamin B5 in SRM 3280 (70% participation) and 25

laboratories reported results for vitamin B6 in SRM 3280 (76% participation). Twentytwo laboratories reported results for both vitamins B5 and B6 in enhanced water (67 %

participation). 

• The consensus mean was within the target range for vitamins B5 and B6 in SRM 3280 and 

for vitamin B6 in the enhanced water. The variability in these measurements was 

excellent, with 10 % RSD for both vitamins in the multivitamin sample and 17 % RSD 

for vitamin B6 in the enhanced water.

• The consensus mean for vitamin B5 in the enhanced water was slightly below the target 

range, and the variability was slightly higher at 23 % RSD.

• A majority of the laboratories reported using solvent extraction (88 %) as the sample 

preparation method. Laboratories also reported using acid hydrolysis (8 %). 

• A majority of the laboratories reported using LC/Abs (88 %) as their instrumental method 

for analysis. Microbiological assay (8 %), LC/MS (4 %), and Direct Analysis in Real 

Time Mass Spectrometry (DART-MS) (4 %) were also reported.

• The level of vitamin B5 in enhanced water sample was 200 times lower than that in the 

multivitamin tablet, which may have resulted in analytical challenges. Laboratories using 

LC/Abs may have had difficulty detecting pantothenic acid accurately.

Technical Recommendations 

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

• In general, the results for this study were very good, and no analytical method was 

identified as being exceptionally good or problematic.

• When using LC-absorbance for a molecule like pantothenic acid without a chromophore, 

care must be taken to remove matrix interferences. Spiking of the sample used during 

method development may also be helpful to be sure that small chromatographic peaks are 

correctly identified.
36

Table 7. Individual data summary table (NIST) for water-soluble vitamins in dietary supplements.

Lab Code: NIST

Analyte Sample Units xi si Zcomm ZNIST N x* s* xNIST U 95

B5 Multivitamin mg/g 7.20 0.95 -0.5 0.0 23 7.58 0.76 7.20 0.95

B5 Enhanced Water μg/g 39.3 0.1 0.6 0.0 22 34.5 8.0 39.3 2.0

B6 Multivitamin mg/g 1.79 0.17 0.3 0.0 25 1.73 0.18 1.79 0.17

B6 Enhanced Water μg/g 6.53 0.02 -0.2 0.0 22 6.72 1.17 6.53 0.33

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U95 ±95% confidence interval

Zcomm Z-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

Exercise J - May 2013 - Water-Soluble Vitamins

1. Your Results 2. Community Results 3. Target

National Institute of Standards & Technology
37

Table 8. Data summary table for vitamin B5 (pantothenic acid) in dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 7.20 0.95 39.1 39.4 39.3 39.3 0.1

J002

J003

J004

J008 9.67 9.33 9.11 9.37 0.28 41.1 38.2 40.1 39.8 1.5

J011 8.92 8.92 8.97 8.94 0.03 39.6 39.4 39.5 39.5 0.1

J015 8.01 8.09 7.75 7.95 0.18 6.6 6.5 6.5 6.5 0.0

J016

J017 7.49 7.48 7.63 7.53 0.08 35.5 42.1 35.0 37.5 4.0

J018 8.00 8.00 8.00 8.00 0.00 49.0 50.0 50.0 49.7 0.6

J020 6.79 6.51 6.65 6.65 0.14 27.1 27.0 26.9 27.0 0.1

J021 3.84 4.05 3.79 3.89 0.14 14.1 14.0 14.1 14.0 0.1

J022 7.50 7.40 7.40 7.43 0.06 29.3 29.2 29.3 29.3 0.1

J025 7.40 7.50 7.40 7.43 0.06 35.0 33.0 35.0 34.3 1.2

J028

J029 7.26 7.47 7.47 7.40 0.12 37.8 38.9 38.8 38.5 0.6

J030 7.59 7.58 7.75 7.64 0.10 36.5 39.0 38.0 37.8 1.3

J031 7.53 7.66 7.65 7.61 0.07 34.7 34.6 33.9 34.4 0.4

J034 7.14 6.87 6.76 6.92 0.20 33.2 35.4 35.4 34.6 1.3

J036 7.24 1.79 7.23 5.42 3.14 36.2 36.1 36.1 36.1 0.0

J037

J038 8.26 7.93 7.89 8.03 0.20

J039

J042 7.95 7.90 7.34 7.73 0.34 30.9 35.6 36.9 34.5 3.2

J043 8.23 7.99 8.12 8.12 0.12 43.0 42.4 46.6 44.0 2.3

J044 7.30 7.20 7.19 7.23 0.06 32.9 31.1 36.2 33.4 2.6

J045 6.80 6.80 6.80 6.80 0.00 34300.0 35200.0 34700.0 34733.3 450.9

J047 7.13 7.13 7.30 7.19 0.10 34.9 36.0 35.8 35.6 0.6

J052

J053

J054 11.40 11.20 11.70 11.43 0.25 20.1 20.3 20.1 20.2 0.1

J057 8.45 7.99 7.95 8.13 0.28

J058 7.57 7.54 7.63 7.58 0.05 27.0 26.4 26.9 26.8 0.3

J059 35.2 34.2 35.0 34.8 0.6

 Consensus Mean 7.58 Consensus Mean 34.5

 Consensus Standard Deviation 0.76 Consensus Standard Deviation 8.0

 Maximum 11.43 Maximum 34733.3

 Minimum 3.89 Minimum 6.5

 N 23 N 22

Community 

Results

SRM 3280 Multivitamin/Multielement Tablets (mg/g) Enhanced Water (μg/g)

Pantothenic Acid

Individual Results
38

Table 9. Data summary table for vitamin B6 (pyridoxine hydrochloride) in dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 1.79 0.17 6.51 6.52 6.55 6.53 0.02

J002

J003

J004

J008 1.79 1.57 1.49 1.62 0.16 6.63 6.56 6.59 6.59 0.04

J011 2.15 2.12 2.10 2.12 0.03 6.58 6.52 6.65 6.58 0.07

J015 1.95 1.95 1.97 1.96 0.01 7.10 7.13 7.13 7.12 0.02

J016

J017 1.65 1.62 1.67 1.65 0.03 7.61 7.43 7.60 7.55 0.10

J018 1.54 1.57 1.51 1.54 0.03 7.00 7.00 7.00 7.00 0.00

J020 1.80 1.79 1.77 1.79 0.02 6.28 6.33 6.28 6.30 0.03

J021 1.74 1.75 1.82 1.77 0.04 8.24 8.20 8.13 8.19 0.06

J022 1.84 1.82 1.81 1.82 0.02 7.00 7.00 7.00 7.00 0.00

J025 1.96 1.92 1.94 1.94 0.02 6.70 6.80 6.70 6.73 0.06

J028

J029 1.87 1.92 1.86 1.88 0.03 5.45 5.23 5.30 5.33 0.11

J030 1.73 1.89 1.84 1.82 0.08 6.59 6.55 6.66 6.60 0.06

J031 1.75 1.81 1.78 1.78 0.03 7.05 6.65 6.45 6.72 0.31

J034 1.51 1.50 1.43 1.48 0.04 5.15 4.91 4.96 5.01 0.13

J036 1.71 1.64 1.73 1.69 0.05 6.18 6.18 6.42 6.26 0.14

J037

J038 1.87 1.84 1.81 1.84 0.03

J039

J042 1.83 1.81 1.81 1.82 0.01 5.57 5.55 5.55 5.56 0.01

J042 1.70 1.69 1.72 1.70 0.02

J044 1.53 1.49 1.48 1.50 0.03 5.70 5.97 5.62 5.76 0.18

J045 1.64 1.69 1.74 1.69 0.05 6800.80 6814.40 6814.80 6810.00 7.97

J047 1.65 1.66 1.72 1.68 0.04 6.50 6.50 6.50 6.50 0.00

J052 1.83 1.80 1.82 0.02

J053 1.35 1.37 1.30 1.34 0.03 5.10 5.31 5.21 0.15

J054 1.27 1.25 1.29 1.27 0.02 31.24 32.12 30.13 31.16 1.00

J057 1.71 1.67 1.67 1.68 0.02

J058 1.89 1.85 1.86 1.87 0.02 8.76 8.72 8.74 8.74 0.02

J059 6.56 6.19 6.48 6.41 0.19

 Consensus Mean 1.73 Consensus Mean 6.72

 Consensus Standard Deviation 0.18 Consensus Standard Deviation 1.17

 Maximum 2.12 Maximum 6810.00

 Minimum 1.27 Minimum 5.01

 N 25 N 22

Pyridoxine Hydrochloride

SRM 3280 Multivitamin/Multielement Tablets (mg/g) Enhanced Water (μg/g)

Individual Results Community Results
39

Figure 19. Pantothenic acid in SRM 3280 Multivitamin/Multielement Tablets (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).
40

Figure 20. Pantothenic acid in enhanced water (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 in triplicate by ID-LC/MS, bounded by an estimated relative uncertainty 

of 5 %.
41

Figure 21. Pyridoxine hydrochloride in SRM 3280 Multivitamin/Multielement Tablets (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).
42

Figure 22. Pyridoxine hydrochloride in enhanced water (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 in triplicate by ID-LC/MS, bounded by an estimated relative 

uncertainty of 5 %.
43

Figure 23. Pantothenic acid in SRM 3280 Multivitamin/Multielement Tablets and enhanced 

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

control (SRM 3280) with a certified value for the analyte are compared to the results for an 

unknown (enhanced water). The solid red box represents 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).
44

Figure 24. Pyridoxine hydrochloride in SRM 3280 Multivitamin/Multielement Tablets and 

enhanced water (sample/control comparison view). In this view, the individual laboratory results 

for the control (SRM 3280) with a certified value for the analyte are compared to the results for 

an unknown (enhanced water). The solid red box represents the target zone for the control (xaxis) and unknown sample (y-axis). The dotted blue box represents the consensus zone for the 

control (x-axis) and the unknown sample (y-axis).
45

VITAMIN E IN DIETARY SUPPLEMENTS

Study Overview 

In this study, participants were provided with one NIST SRM, SRM 3280 Multivitamin/ 

Multielement Tablets, and one bottle of commercially available enhanced water. Participants 

were asked to use in-house analytical methods to determine the mass fraction of vitamin E (as αtocopherol acetate) in each of the matrices and report values on an as-received basis.

Sample Information 

Multivitamin/Multielement Tablets. Participants were provided with one bottle containing 30 

multivitamin/multielement tablets. Before use, participants were instructed to grind all 30 

tablets, mix the resulting powder thoroughly, and use a sample size of at least 0.6 g. Participants 

were asked to store the material at controlled room temperature, 10 °C to 30 °C, prepare three 

samples, and report three values from the single bottle provided. Approximate analyte levels 

were not reported to participants prior to the study. The NIST certified value and uncertainty for 

vitamin E (as α-tocopherol acetate) in SRM 3280 was determined by LC/abs and LC/MS 

following solvent extraction, in combination with data from numerous collaborating laboratories. 

The certified value is reported in the table below as α-tocopherol equivalents and as α-tocopherol 

acetate, both on a dry-mass basis and after correction for moisture of the material (1.37 %).

Certified Mass Fraction

in SRM 3280 (mg/g)

Certified Mass Fraction

in SRM 3280 (mg/g)

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

α-tocopherol equivalents 21.4 ± 3.5 21.1 ± 3.5

α-tocopherol acetate 23.5 ± 3.8 23.2 ± 3.8

Enhanced Water. Participants were provided with one 600 mL bottle of commercially available 

enhanced water. Before use, participants were instructed to thoroughly mix the contents of the 

bottle, and a sample size of at least 1.0 mL was recommended. Participants were asked to store 

the material at controlled room temperature, 10 °C to 30 °C, prepare three samples, and report 

three values from the single bottle provided. Approximate analyte levels were not reported to 

participants prior to the study. Certified values are not available for this material.

Study Results

• Thirty-two laboratories enrolled in this exercise and received samples. Twenty 

laboratories reported results for vitamin E in SRM 3280 (62 % participation) and 18

laboratories reported results for vitamin E in enhanced water (56 % participation). 

• The consensus mean was within the target range for vitamin E in SRM 3280 with 

acceptable variability (9 % RSD).

• The consensus variability was also acceptable for vitamin E in enhanced water (21 % 

RSD), a sample with a concentration 1000 times lower than that in the control material.

• A majority of the laboratories reported using solvent extraction (85 %) as the sample 

preparation method. Some laboratories also reported using saponification (15 %).

• A majority of the laboratories reported using LC/Abs (85 %) as their instrumental method 

for analysis. Liquid chromatography/fluorescence (LC/FL) was also reported by some

laboratories (15 %).
46

Technical Recommendations 

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

• When using saponification for sample preparation, it is important to determine that the 

saponification reaction is complete. Additionally, if an internal standard approach to 

calibration is used, the internal standard must be stable under the sample preparation 

conditions.

• Absorbance and fluorescence detection are both valuable tools for the determination of 

tocopherols. Fluorescence detection is especially sensitive and selective for α-, β-, γ-, 

and δ-tocopherol, however it is not very sensitive for the detection of α-tocopherol 

acetate. In this study, if the samples were saponified and the analyte was converted to αtocopherol, fluorescence is an appropriate choice for detection.

• In general, the results reported for this study were very good, but there is a slight 

indication of calibration error in the sample/sample view. α-tocopherol acetate and αtocopherol calibrant concentrations should be determined spectrophotometrically with a 

liquid chromatography purity correction. Also, be certain that the correct molar 

absorptivity is used. 

• If laboratories would like further guidance on the use of calibrants traceable to molar 

absorptivity (rather than mass), please indicate the need. NIST staff will be happy to 

provide additional information and/or training as appropriate.
47

Table 10. Individual data summary table (NIST) for α-tocopherol acetate in dietary supplements.

Lab Code: NIST

Analyte Sample Units xi si Zcomm ZNIST N x* s* xNIST U 95

Vitamin E Multivitamin mg/g 23.2 3.8 0.1 0.0 20 23.0 2.2 23.2 3.8

Vitamin E Enhanced Water μg/g 18 23.8 4.9

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U 95 ±95% confidence interval

Zcomm Z-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

Exercise J - May 2013 - Vitamin E

1. Your Results 2. Community Results 3. Target

National Institute of Standards & Technology
48

Table 11. Data summary table for α-tocopherol acetate in dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 23.2 3.8

J002

J004

J008 22.4 22.2 23.3 22.6 0.6 17.6 18.6 18.6 18.3 0.6

J011 18.9 18.1 18.6 18.5 0.4

J015 21.5 21.1 20.7 21.1 0.4 27.5 23.7 23.5 24.9 2.3

J016 22.1 21.8 20.3 21.4 1.0 22.1 21.6 21.3 21.6 0.4

J017 23.3 24.7 26.9 25.0 1.8 23.2 30.0 27.0 26.7 3.4

J018

J020 36.0 34.0 35.0 35.0 1.0 19.6 21.6 21.3 20.8 1.1

J021 22.5 22.4 22.6 22.5 0.1 13.1 13.4 13.7 13.4 0.3

J022 23.2 23.8 24.0 23.7 0.4 25.1 26.2 25.7 25.7 0.6

J023

J025 26.5 29.0 28.8 28.1 1.4 109.0 48.0 53.0 70.0 33.9

J029 23.4 23.2 23.0 23.2 0.2 25.5 24.8 25.5 25.3 0.4

J030

J031 24.6 24.3 24.4 24.4 0.2

J036 23.9 23.8 23.9 23.9 0.1 25.8 27.4 27.5 26.9 0.9

J037

J038 26.1 26.0 25.1 25.7 0.5 28.6 30.6 27.4 28.9 1.6

J039

J042 21.1 21.2 21.2 21.1 0.0 26.0 24.4 28.0 26.1 1.8

J043 20.6 21.2 21.0 20.9 0.3 19.2 21.1 18.5 19.6 1.3

J044 22.9 23.6 23.1 23.2 0.4 26.4 19.5 22.2 22.7 3.5

J045

J047

J048 20.1 21.6 20.9 1.1 21.1 20.2 20.7 0.6

J052

J053 20.3 23.1 21.7 2.0 25.0 28.5 26.7 2.5

J054 24.2 24.1 24.6 24.3 0.3 30.0 27.1 29.3 28.8 1.5

J057 20.7 20.7

J058 21.9 21.2 22.8 22.0 0.8 19.1 15.7 16.4 17.1 1.8

J059

 Consensus Mean 23.0 Consensus Mean 23.8

 Consensus Standard Deviation 2.2 Consensus Standard Deviation 4.9

 Maximum 35.0 Maximum 70.0

 Minimum 18.5 Minimum 13.40

 N 20 N 18

Community 

Results Individual Results

Tocopherol Acetate

SRM 3280 Multivitamin/Multielement Tablets (mg/g) Enhanced Water (μg/g)
49

Figure 25. α-Tocopherol acetate in SRM 3280 Multivitamin/Multielement Tablets (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).
50

Figure 26. α-Tocopherol acetate in enhanced water (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.
51

Figure 27. α-Tocopherol acetate in SRM 3280 Multivitamin/Multielement Tablets and 

enhanced water (sample/control comparison view). In this view, the individual laboratory results 

for the control (multivitamin/multielement tablets) with a certified value for the analyte are 

compared to the results for an unknown (enhanced water). The solid red box represents the 

target zone for the control (x-axis). The dotted blue box represents the consensus zone for the 

control (x-axis) and the unknown sample (y-axis).
52

FATTY ACIDS IN BOTANICAL AND FISH OIL DIETARY SUPPLEMENTS

Study Overview 

In this study, participants were provided with two NIST SRMs, SRM 3274-2 Evening Primrose 

(Oenothera biennis) Oil and SRM 3275-2 Anchovy Oil. Participants were asked to use in-house 

analytical methods to determine the mass fractions of fatty acid methyl esters (FAMEs) in each 

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

Sample Information 

Evening Primrose Oil. Participants were provided with three ampoules, each containing 1.2 mL 

of evening primrose oil. The oil contained approximately 190 mg/L tert-butylhydroquinone 

(TBHQ) as an antioxidant and was packaged in amber glass ampoules under argon. Before use, 

participants were instructed to mix each ampoule thoroughly and a sample size of at least 0.5 g 

was recommended. Participants were asked to prepare one sample and report as many analytes 

as possible from each ampoule provided, and to store the oil under refrigeration at 0 °C to 4 °C. 

Approximate analyte levels were not reported to participants prior to the study. The NIST 

certified and reference values and uncertainties in SRM 3274-2 were determined by by gas 

chromatography-flame ionization detection (GC-FID) and gas chromatography/mass 

spectrometry (GC/MS) following multiple methods of hydrolysis and derivatization, and are 

summarized in the table below.

Fish Oil. Participants were provided with three ampoules, each containing 1.2 mL of anchovy 

oil. The oil contained mixed natural tocopherols at a minimum of 1 mg/g as an antioxidant and 

was packaged in amber glass ampoules under argon. Before use, participants were instructed to 

mix each ampoule thoroughly and a sample size of at least 0.5 g was recommended. Participants 

were asked to prepare one sample and report as many analytes as possible from each ampoule 

provided, and to store the oil under refrigeration at 0 °C to 4 °C. Approximate analyte levels 

were not reported to participants prior to the study. The NIST certified and reference values and 

uncertainties in SRM 3275-2 were determined by GC-FID and GC/MS following multiple 

methods of hydrolysis and derivatization, and are summarized in the table below.

Analyte

Certified Mass Fraction 

in SRM 3274-2 (mg/g as FAME)

Certified Mass Fraction 

in SRM 3275-2 (mg/g as FAME)

Linoleic Acid 745 ± 24 3.00 ± 0.42

α- Linoleic Acid 1.61 ± 0.12 1.42 ± 0.12

γ- Linoleic Acid 100.4 ± 4.1 0.507 ± 0.043

Arachidonic Acid 0.0221 ± 0.0020 22.9 ± 1.0

EPA 394 ± 17

DHA 187 ± 8

Study Results

• Twenty-one laboratories enrolled in this exercise and received samples. Thirteen 

laboratories reported data for at least one FAME (62 % participation). 

• A majority of the laboratories reported using derivatization/methylation as the sample 

preparation method (92 %). One laboratory also reported using hydrolysis as the sample 

preparation method.
53

• A majority of the laboratories reported using GC-FID (85 %) as their method for 

analysis. One laboratory reported using GC-MS and one laboratory reported using 

LC/absorbance.

• The consensus means for linoleic acid (LA) in both oils, α–linolenic acid (ALA) in the 

botanical oil, and EPA and DHA in the fish oil were within the target ranges with 

acceptable variabilities (5 % to 21 % RSD).

• The consensus mean for γ-linolenic acid (GLA) in the botanical oil was below the target 

range with low variability (5 % RSD).

• The consensus means for ALA, GLA, and AA in the fish oil were above the target range. 

The variabilities for ALA and GLA were high (31 % and 69 % RSD, respectively), while 

the variability for AA was acceptable (16 % RSD).

Technical Recommendations 

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

• With a small number of laboratories reporting data for these fatty acids, and a majority 

reporting use of the same or very similar methods, drawing extensive technical 

conclusions is difficult.

• Participants were asked to report concentrations for fatty acids as fatty acid methyl esters 

(FAMEs). In this case, FAMEs should be used as calibrants or non-esterified fatty acids 

should be carried through the entire sample preparation procedure (hydrolysis and 

derivatization) to improve quantitation.

• Knowledge of calibrant response when carried through the derivatization procedure is 

necessary. For example, at NIST, calibrants for EPA and DPA give response factors of 

1.3 and 1.6, respectively, corresponding to 30 % or 60 % low bias in the quantitation of 

these compounds if not considered.

• Similarly, for those laboratories using GC-MS, quantitation for some compounds may be 

inaccurate as a result of non-unity response factors from EI fragmentation.
54

Table 12. Individual data summary table (NIST) for fatty acids in botanical and fish oil dietary supplements.

Lab Code: NIST

Analyte Sample Units xi si Zcomm ZNIST N x* s* xNIST U 95

Linoleic Acid Primrose mg/g 745 24 1.0 0.0 10 710 34 745 24

Linoleic Acid Fish mg/g 3.00 0.42 -0.2 0.0 7 3.13 0.57 3.00 0.42

α-Linolenic Acid Primrose mg/g 1.61 0.12 0.2 0.0 8 1.54 0.33 1.61 0.12

α-Linolenic Acid Fish mg/g 1.42 0.12 -0.8 0.0 7 1.89 0.59 1.42 0.12

γ-Linolenic Acid Primrose mg/g 100 4 1.9 0.0 11 91 5 100 4

γ-Linolenic Acid Fish mg/g 0.507 0.043 -0.9 0.0 5 1.350 0.930 0.507 0.043

Arachidonic Acid Primrose mg/g 0.0221 0.0020 0.0 1 0.0221 0.0020

Arachidonic Acid Fish mg/g 22.9 1.0 -0.6 0.0 9 25.1 4.0 22.9 1.0

EPA Primrose mg/g 1

EPA Fish mg/g 394 17 0.2 0.0 13 385 49 394 17

DHA Primrose mg/g 0

DHA Fish mg/g 187 8 -0.1 0.0 13 188 16 187 8

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U95 ±95% confidence interval

Zcomm Z-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

Exercise J - May 2013 - Fatty Acids

1. Your Results 2. Community Results 3. Target

National Institute of Standards & Technology
55

Table 13. Data summary table for linoleic acid in botanical and fish oil dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 745 24 3.00 0.42

J002

J004

J007

J009

J011 668 663 657 663 6

J016

J019 755 756 756 755 1 3.59 3.18 3.04 3.27 0.29

J020

J023

J025

J027 739 738 739 739 1 3.28 3.30 3.28 3.29 0.01

J029 717 718 718 718 1 3.05 3.03 3.07 3.05 0.02

J031 714 714 719 716 3

J036 714 696 710 707 9 2.35 2.28 2.25 2.29 0.05

J038

J040 706 706 706 706 0 5.00 5.00 5.00 5.00 0.00

J043 726 709 714 716 9

J053 632 630 635 632 3 3.06 2.95 2.89 2.97 0.09

J054

J057 719 720 719 719 0 3.06 3.07 3.08 3.07 0.01

J059

 Consensus Mean 710 Consensus Mean 3.13

 Consensus Standard Deviation 34 Consensus Standard Deviation 0.57

 Maximum 755 Maximum 5.00

 Minimum 632 Minimum 2.29

 N 10 N 7

Linoleic Acid

SRM 3274-2 Evening Primrose Oil (mg/g) SRM 3275-2 Fish Oil (mg/g)

Individual Results Community Results
56

Table 14. Data summary table for α-linolenic acid in botanical and fish oil dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 1.61 0.12 1.42 0.12

J002

J004

J007

J009

J011

J016

J019 1.25 1.23 1.30 1.26 0.04 1.69 1.38 1.46 1.51 0.16

J020

J023

J025

J027 1.69 1.68 1.70 1.69 0.01 1.87 1.82 1.81 1.83 0.04

J029 1.59 1.56 1.62 1.59 0.03 1.42 1.45 1.43 1.43 0.02

J031

J036 1.60 1.54 1.59 1.58 0.03 2.57 2.53 2.50 2.53 0.04

J038

J040 2.00 2.00 2.00 2.00 0.00 2.00 2.00 2.00 2.00 0.00

J043 1.14 1.21 1.01 1.12 0.10

J053 1.36 1.32 1.39 1.36 0.04 1.30 1.33 1.30 1.31 0.02

J054

J057 1.76 1.76 1.75 1.76 0.01 2.59 2.61 2.62 2.61 0.02

J059

 Consensus Mean 1.54 Consensus Mean 1.89

 Consensus Standard Deviation 0.33 Consensus Standard Deviation 0.59

 Maximum 2.00 Maximum 2.61

 Minimum 1.12 Minimum 1.31

 N 8 N 7

α-Linolenic Acid

SRM 3274-2 Evening Primrose Oil (mg/g) SRM 3275-2 Fish Oil (mg/g)

Individual Results Community Results
57

Table 15. Data summary table for γ-linolenic acid in botanical and fish oil dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 100.4 4.1 0.507 0.043

J002

J004

J007

J009

J011 73.9 74.0 73.4 73.8 0.3

J016

J019 89.7 90.3 90.7 90.2 0.5

J020 89.0 89.0 89.0 89.0 0.0

J023

J025

J027 97.4 97.1 97.2 97.2 0.1 0.429 0.442 0.450 0.440 0.011

J029 93.0 93.0 93.0 93.0 0.0

J031 92.5 92.6 93.2 92.8 0.4

J036 92.5 90.2 91.9 91.5 1.2 0.899 0.905 0.881 0.895 0.012

J038

J040 91.0 91.0 91.0 91.0 0.0 1.000 1.000 1.000 1.000 0.000

J043 102.5 90.8 93.2 95.5 6.2

J053 80.4 79.3 80.0 79.9 0.6 2.126 2.000 2.090 2.072 0.065

J054

J057 94.4 94.4 94.4 94.4 0.0 2.350 2.360 2.360 2.357 0.006

J059

 Consensus Mean 91.1 Consensus Mean 1.353

 Consensus Standard Deviation 4.9 Consensus Standard Deviation 0.930

 Maximum 97.2 Maximum 2.357

 Minimum 73.8 Minimum 0.440

 N 11 N 5

γ-Linolenic Acid

SRM 3274-2 Evening Primrose Oil (mg/g) SRM 3275-2 Fish Oil (mg/g)

Individual Results Community Results
58

Table 16. Data summary table for arachidonic acid in botanical and fish oil dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 0.0221 0.0020 22.9 1.0

J002

J004

J007

J009

J011

J016

J019 24.8 24.4 23.3 24.1 0.8

J020

J023

J025

J027 26.9 26.6 26.6 26.7 0.2

J029 24.9 25.0 25.4 25.1 0.3

J031 24.4 24.2 24.1 24.2 0.2

J036

J038

J040 29.0 29.0 29.0 29.0 0.0

J043 20.6 16.1 21.4 19.4 2.9

J053 21.8 22.0 22.2 22.0 0.2

J054 105.0000 98.0000 119.0000 107.3333 10.6927 38.0 44.0 31.0 37.7 6.5

J057 24.1 24.1 24.1 24.1 0.0

J059

 Consensus Mean Consensus Mean 25.1

 Consensus Standard Deviation Consensus Standard Deviation 4.0

 Maximum Maximum 37.7

 Minimum Minimum 19.4

 N 1 N 9

Arachidonic Acid

SRM 3274-2 Evening Primrose Oil (mg/g) SRM 3275-2 Fish Oil (mg/g)

Individual Results Community Results
59

Table 17. Data summary table for EPA in botanical and fish oil dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 394 17

J002

J004

J007

J009

J011 354 359 362 358 4

J016

J019 1.70 1.12 1.06 1.29 0.35 71 72 74 72 2

J020 368 370 375 371 4

J023 443 445 453 447 5

J025

J027 412 408 408 409 3

J029 384 386 385 385 1

J031 386 385 384 385 1

J036 387 385 380 384 4

J038

J040 467 457 459 461 5

J043 374 364 389 375 12

J053 403 412 410 408 5

J054 174 209 205 196 19

J057 400 400 400 400 0

J059

 Consensus Mean Consensus Mean 385

 Consensus Standard Deviation Consensus Standard Deviation 49

 Maximum Maximum 461

 Minimum Minimum 72

 N 1 N 13

EPA

SRM 3274-2 Evening Primrose Oil (mg/g) SRM 3275-2 Fish Oil (mg/g)

Individual Results Community Results
60

Table 18. Data summary table for DHA in botanical and fish oil dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 187 8

J002

J004

J007

J009

J011 151 152 153 152 1

J016

J019 189 204 208 200 10

J020 175 176 178 176 2

J023 208 209 213 210 3

J025

J027 195 193 193 193 1

J029 182 181 182 182 1

J031 187 186 186 186 1

J036 195 194 187 192 4

J038

J040 212 208 209 210 2

J043 199 181 187 189 9

J053 164 165 165 165 1

J054 175 206 202 194 17

J057 185 186 185 185 0

J059

 Consensus Mean Consensus Mean 188

 Consensus Standard Deviation Consensus Standard Deviation 16

 Maximum Maximum 210

 Minimum Minimum 152

 N 0 N 13

DHA

SRM 3274-2 Evening Primrose Oil (mg/g) SRM 3275-2 Fish Oil (mg/g)

Individual Results Community Results
61

Figure 28. Linoleic acid in SRM 3274-2 Evening Primrose 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).
62

Figure 29. Linoleic acid in SRM 3275-2 Fish 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).
63

Figure 30. α-Linolenic acid in SRM 3274-2 Evening Primrose 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).
64

Figure 31. α-Linolenic acid in SRM 3275-2 Fish 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).
65

Figure 32. γ-Linolenic acid in SRM 3274-2 Evening Primrose 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).
66

Figure 33. γ-Linolenic acid in SRM 3275-2 Fish 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).
67

Figure 34. Arachidonic acid in SRM 3275-2 Fish 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).
68

Figure 35. EPA in SRM 3275-2 Fish 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).
69

Figure 36. DHA in SRM 3275-2 Fish 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).
70

Figure 37. Linoleic acid in SRM 3274-2 Evening Primrose Oil and SRM 3275-2 Fish Oil-2 

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

(botanical oil) with a certified value for the analyte are compared to the results for an unknown 

(fish oil). The solid red box represents 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).
71

Figure 38. α-Linolenic acid in SRM 3274-2 Evening Primrose Oil and SRM 3275-2 Fish Oil-2 

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

(botanical oil) with a certified value for the analyte are compared to the results for an unknown 

(fish oil). The solid red box represents 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).
72

Figure 39. γ-Linolenic acid in SRM 3274-2 Evening Primrose Oil and SRM 3275-2 Fish Oil-2 

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

(botanical oil) with a certified value for the analyte are compared to the results for an unknown 

(fish oil). The solid red box represents 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).
73

AFLATOXINS IN PEANUT BUTTER

Study Overview 

In this study, participants were provided with one NIST SRM, SRM 2387 Peanut Butter, and 

three peanut butter samples with low, medium, and high levels of aflatoxins. Participants were 

asked to use in-house analytical methods to determine the mass fractions of aflatoxins in each of 

the matrices and report values on an as-received basis. Participants were asked to report as many 

analytes as possible.

Sample Information 

Peanut Butter. Participants were provided with one jar containing 170 g of peanut butter. 

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

size of at least 50 g was recommended. Participants were asked to store the material under 

refrigeration, 0 °C to 4 °C, and prepare three samples and report three values for as many 

analytes as possible from the single jar provided. Approximate analyte levels were not reported 

to participants prior to the study. NIST reference values and uncertainties, determined by 

collaborating laboratories, are outlined in the table below.

Analyte

Reference Mass Fraction 

in SRM 2387 (ng/g)

Aflatoxin B1 4.2 ± 0.9

Aflatoxin B2 0.7 ± 0.3

Total Aflatoxins 5.0 ± 0.5

Peanut Butter Samples. Participants were provided with three 50 g jars of peanut butter labeled 

1, 2, and 3. Before use, participants were instructed to thoroughly mix the contents of each jar, 

and a sample size of at least 50 g was recommended. Participants were asked to store the 

material under refrigeration, 0 °C to 4 °C, and prepare one sample and report one value for as 

many analytes as possible from each jar provided. Approximate analyte levels were not reported

to participants prior to the study. NIST target values and uncertainties, determined by 

collaborating laboratories, are outlined in the table below.

Analyte

Estimated Mass 

Fraction in Peanut 

Butter 1 (Low) (ng/g)

Estimated Mass 

Fraction in Peanut 

Butter 2 (Med) (ng/g)

Estimated Mass 

Fraction in Peanut 

Butter 3 (High) (ng/g)

Aflatoxin B1 3.70 ± 0.63 7.17 ± 2.85 10.95 ± 2.22

Aflatoxin B2 1.01 ± 0.09 1.46 ± 1.08 2.16 ± 0.49

Aflatoxin G1 0.68 ± 0.29 1.81 ± 0.49

Aflatoxin G2 0.44 ± 0.62 0.43 ± 0.25

Total Aflatoxins 5.23 ± 2.22 9.95 ± 1.43 14.77 ± 2.85

Study Results

• Ten laboratories enrolled in this exercise and received samples. Five laboratories 

reported results for at least one aflatoxin per sample (50 % participation).

• The consensus means for aflatoxins B1 and B2 and total aflatoxins were within the target 

range with high variability (26 % to 36 % relative standard deviation (RSD)).
74

• In the low-level sample, consensus means for aflatoxin B1 and total aflatoxins were

within the target range, while the consensus mean for aflatoxin B2 was slightly below the 

target range. All three analytes were measured with high variability (27 % to 38 % 

RSD).

• In the mid-level sample, consensus means for all analytes were within the target ranges, 

but with high variability (14 % to 31 % RSD).

• In the high-level sample, consensus means for all analytes were above the target ranges 

with high variability (33 % to 49 % RSD).

• Four of five laboratories reported using extraction for sample preparation (80 %). One 

laboratory reported using slurry blending sample preparation (20 %).

• Two laboratories reported using LC-fluorescence as their analytical method for analysis

(40 %). Three laboratories reported using LC/MS/MS to measure aflatoxins (60 %).

Technical Recommendations

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

• Because the data for this study was very limited (only 4 or 5 laboratories reporting data), 

drawing extensive technical conclusions is difficult. Similarly, the high level of betweenlaboratory variability may be exaggerated as a result of the low number of participants.

• No trends were identified indicating that a particular sample preparation method or 

instrumental technique provided more accurate results than another.

• Always check that the levels of the analytes in the samples are within the calibration 

range. The high-biased results for the high-level sample may result from extrapolation of 

the calibration curves beyond their linear ranges.
75

Table 19. Individual data summary table (NIST) for aflatoxins in peanut butter.

Lab Code: NIST

Analyte Sample Units xi si Zcomm ZNIST N x* s* xNIST U 95

Aflatoxin B1 PB Control ng/g 4.2 0.9 -0.1 0.0 4 4.4 1.6 4.2 0.9

Aflatoxin B1 PB Sample 1 ng/g 3.70 0.63 0.4 0.0 4 3.36 0.96 3.70 0.63

Aflatoxin B1 PB Sample 2 ng/g 7.17 3 -0.7 0.0 4 8.62 2.10 7.17 2.85

Aflatoxin B1 PB Sample 3 ng/g 11.00 2.22 -0.6 0.0 4 13.9 4.5 11.0 2.2

Aflatoxin B2 PB Control ng/g 0.7 0.3 -0.1 0.0 4 0.7 0.2 0.7 0.3

Aflatoxin B2 PB Sample 1 ng/g 1.01 0.09 0.4 0.0 4 0.88 0.33 1.01 0.09

Aflatoxin B2 PB Sample 2 ng/g 1.46 1.08 -0.4 0.0 4 1.69 0.53 1.46 1.08

Aflatoxin B2 PB Sample 3 ng/g 2.16 0.49 -0.8 0.0 4 3.59 1.77 2.16 0.49

Aflatoxin G1 PB Control ng/g 1

Aflatoxin G1 PB Sample 1 ng/g 0

Aflatoxin G1 PB Sample 2 ng/g 0.68 0.29 -0.8 0.0 3 0.77 0.11 0.68 0.29

Aflatoxin G1 PB Sample 3 ng/g 1.81 0.49 -0.7 0.0 3 2.29 0.66 1.81 0.49

Aflatoxin G2 PB Control ng/g 0

Aflatoxin G2 PB Sample 1 ng/g 0

Aflatoxin G2 PB Sample 2 ng/g 0.44 0.62 0.0 0 0.44 0.62

Aflatoxin G2 PB Sample 3 ng/g 0.43 0.25 < -4 0.0 2 0.52 0.00 0.43 0.25

Total Aflatoxins PB Control ng/g 5.0 0.5 -0.1 0.0 5 5.2 1.4 5.0 0.5

Total Aflatoxins PB Sample 1 ng/g 5.23 2.22 0.9 0.0 5 4.20 1.11 5.23 2.22

Total Aflatoxins PB Sample 2 ng/g 9.95 1.43 -0.5 0.0 5 10.90 2.02 9.95 1.43

Total Aflatoxins PB Sample 3 ng/g 14.80 2.85 -0.5 0.0 5 18.0 6.2 14.8 2.9

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U 95 ±95% confidence interval

Zcomm Z-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

Exercise J - May 2013 - Aflatoxins

1. Your Results 2. Community Results 3. Target

National Institute of Standards & Technology
76

Table 20. Data summary table for aflatoxin B1 in peanut butter.

Table 21. Data summary table for aflatoxin B2 in peanut butter.

Lab A B C Avg SD Lo Med Hi

NIST 4.2 0.9 3.70 ± 0.63 7.17 ± 2.85 11.0 ± 2.2

J002

J007

J008

J029 5.4 5.4 5.8 5.5 0.2 2.60 7.85 13.2

J030 2.2 2.0 3.6 2.6 0.9 2.67 7.04 9.6

J035

J036

J052 3.9 4.0 3.8 3.9 0.1 3.90 8.30 13.4

J053 5.4 5.6 5.3 5.4 0.2 4.25 11.29 19.2

J059

 Consensus Mean 4.4 3.36 8.62 13.9

 Consensus Standard Deviation 1.6 0.96 2.10 4.5

 Maximum 5.5 4.25 11.29 19.2

 Minimum 2.6 2.60 7.04 9.6

 N 4 4 4 4

Aflatoxin B1

SRM 2387 Peanut Butter (ng/g) Peanut Butter Samples (ng/g)

Individual Results Community Results

Lab A B C Avg SD Lo Med Hi

NIST 0.7 0.3 1.01 ± 0.09 1.46 ± 1.08 2.16 ± 0.49

J002

J007

J008

J029 0.9 0.9 1.0 1.0 0.0 0.70 1.18 2.48

J030 0.5 0.7 0.8 0.6 0.2 0.96 1.58 2.33

J035

J036

J052 0.6 0.5 0.6 0.6 0.1 0.60 2.30 5.70

J053 0.8 0.8 0.6 0.7 0.1 1.25 1.68 3.86

J059

 Consensus Mean 0.7 0.88 1.69 3.59

 Consensus Standard Deviation 0.2 0.33 0.53 1.77

 Maximum 1.0 1.25 2.30 5.70

 Minimum 0.6 0.60 1.18 2.33

 N 4 4 4 4

Aflatoxin B2

SRM 2387 Peanut Butter (ng/g) Peanut Butter Samples (ng/g)

Individual Results Community Results
77

Table 22. Data summary table for aflatoxin G1 in peanut butter.

Table 23. Data summary table for aflatoxin G2 in peanut butter.

Lab A B C Avg SD Lo Med Hi

NIST 0.68 ± 0.29 1.81 ± 0.49

J002

J007

J008

J029 0.66 2.14

J030 0.3 0.3 0.3 0.3 0.0 0.81 1.79

J035

J036

J052

J053 0.84 2.93

J059

 Consensus Mean 0.77 2.29

 Consensus Standard Deviation 0.11 0.66

 Maximum 0.84 2.93

 Minimum 0.66 1.79

 N 1 0 3 3

Aflatoxin G1

SRM 2387 Peanut Butter (ng/g) Peanut Butter Samples (ng/g)

Individual Results Community Results

Lab A B C Avg SD Lo Med Hi

NIST 0.44 ± 0.62 0.43 ± 0.25

J002

J007

J008

J029 0.52

J030 0.52

J035

J036

J052

J053

J059

 Consensus Mean 0.52

 Consensus Standard Deviation 0.00

 Maximum 0.52

 Minimum 0.52

 N 0 0 0 2

Aflatoxin G2

SRM 2387 Peanut Butter (ng/g) Peanut Butter Samples (ng/g)

Individual Results Community Results
78

Table 24. Data summary table for total aflatoxins in peanut butter.

Lab A B C Avg SD Lo Med Hi

NIST 5.0 0.5 5.23 ± 2.22 9.95 ± 1.43 14.8 ± 2.9

J002

J007

J008 5.5 6.0 4.3 5.3 0.9 3.80 11.00 12.0

J029 6.4 6.3 6.8 6.5 0.3 3.30 9.69 18.3

J030 2.9 3.0 4.7 3.5 1.0 3.63 9.43 14.3

J035

J036

J052 4.5 4.5 4.4 4.5 0.1 4.50 10.60 19.1

J053 6.2 6.4 5.9 6.2 0.3 5.77 13.89 26.3

J059

 Consensus Mean 5.2 4.20 10.92 18.0

 Consensus Standard Deviation 1.4 1.11 2.02 6.2

 Maximum 6.5 5.77 13.89 26.3

 Minimum 3.5 3.30 9.43 12.0

 N 5 5 5 5

Total Aflatoxins

SRM 2387 Peanut Butter (ng/g) Peanut Butter Samples (ng/g)

Individual Results Community Results
79

Figure 40. Aflatoxin B1 in SRM 2387 Peanut Butter (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).
80

Figure 41. Aflatoxin B2 in SRM 2387 Peanut Butter (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).
81

Figure 42. Total aflatoxins in SRM 2387 Peanut Butter (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).
82

Figure 43. Total aflatoxins in peanut butter sample 1 (low) (composition view). In this view, 

total composition of the sample is plotted as a function of the measurement of individual 

components. The estimated value for total aflatoxins in this sample is (5.23 ± 2.22) ng/g.
83

Figure 44. Total aflatoxins in peanut butter sample 2 (medium) (composition view). In this 

view, total composition of the sample is plotted as a function of the measurement of individual 

components. The estimated value for total aflatoxins in this sample is (9.95 ± 1.43) ng/g.
84

Figure 45. Total aflatoxins in peanut butter sample 3 (high) (composition view). In this view, 

total composition of the sample is plotted as a function of the measurement of individual 

components. The estimated value for total aflatoxins in this sample is (14.8 ± 2.9) ng/g.
85

Figure 46. Aflatoxins in peanut butter sample 1 (low) (bias view). In this view, the ZNIST-score 

for each individual component as well as total aflatoxins are plotted. Values determined to be 

marginally different than the NIST target value (2 < |Z| < 3) are colored in orange. Values 

determined to be significantly different than the NIST target value (|Z| > 3) are colored in red.
86

Figure 47. Aflatoxins in peanut butter sample 3 (medium) (bias view). In this view, the ZNISTscore for each individual component as well as total aflatoxins are plotted. Values determined to 

be marginally different than the NIST target value (2 < |Z| < 3) are colored in orange. Values 

determined to be significantly different than the NIST target value (|Z| > 3) are colored in red.
87

Figure 48. Aflatoxins in peanut butter sample 3 (high) (bias view). In this view, the ZNIST-score 

for each individual component as well as total aflatoxins are plotted. Values determined to be 

marginally different than the NIST target value (2 < |Z| < 3) are colored in orange. Values 

determined to be significantly different than the NIST target value (|Z| > 3) are colored in red.
88

ISOFLAVONES IN SOY PRODUCTS

Study Overview 

In this study, participants were provided with two NIST SRMs, SRM 3236 Soy Protein Isolate 

and SRM 3234 Soy Flour. Participants were asked to use in-house analytical methods to 

determine the mass fractions of isoflavones (daidzin, glycitin, genistin, daidzein, glycitein, and 

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

Sample Information 

Soy Protein Isolate. Participants were provided with three packets, each containing 10 g of soy 

protein isolate. Before use, participants were instructed to mix each packet thoroughly and a

sample size of at least 100 mg was recommended. Participants were asked to prepare one 

sample and report as many analytes as possible from each packet provided, and to store the 

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

participants prior to the study. The NIST certified values and uncertainties for isoflavones in 

SRM 3236 were determined using LC/abs and ID-LC/MS following solvent extraction and basic 

hydrolysis, and are reported in the table below both on a dry-mass basis and after correction for 

moisture of the material (4.83 %).

Analyte

Certified Mass Fraction

in SRM 3236 (μg/g)

(dry-mass basis)

Certified Mass Fraction

in SRM 3236 (μg/g)

(as-received basis)

Daidzin 174 ± 23 165 ± 22

Glycitin 31.37 ± 0.52 29.85 ± 0.49

Genistin 329 ± 10 313 ± 10

Daidzein 104.31 ± 0.48 99.27 ± 0.46

Glycitein 22.71 ± 0.19 21.61 ± 0.18

Genistein 183 ± 14 174 ± 13

Total Isoflavones 845 ± 29 804 ± 27

Soy Flour. Participants were provided one 50 g bottle of defatted soy flour. Before use, 

participants were instructed to mix the bottle thoroughly and a sample size of at least 100 mg 

was recommended. Participants were asked to prepare three samples and report as many 

analytes as possible from the bottle provided, and to store the material at room temperature, 

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

The NIST estimated values for isoflavones in SRM 3234 were determined using LC/abs and IDLC/MS following solvent extraction and basic hydrolysis, and are reported in the table below on 

an as-received basis with an estimated uncertainty calculated as the standard deviation between 

the means of values determined by each method.
89

Analyte

NIST Estimated Mass Fraction 

in SRM 3234 (mg/g)

(as-received basis)

Daidzin 1693 ± 427

Glycitin 241 ± 31

Genistin 2011 ± 387

Daidzein 13.5 ± 1.9

Glycitein

Genistein 14.9 ± 0.1

Total Isoflavones 3973 ± 578

Study Results

• Sixteen laboratories enrolled in this exercise and received samples. Eleven laboratories 

reported data for at least some of the isoflavones in the study (69 % participation). 

• Laboratories reported using either solvent extraction (64 %) or solvent extraction with

hydrolysis (36 %) as the sample preparation method.

• A majority of the laboratories reported using LC/abs (91 %) for isoflavone determination. 

One laboratory reported using DART-MS as their instrumental method.

• The consensus means were within the target ranges with acceptable between-laboratory 

variability for daidzein and genistein in the soy protein isolate (4 % RSD for both 

analytes).

• The consensus means were above the target ranges for glycitein in the soy protein isolate 

and daidzein and genistein in the soy flour with high between-laboratory variability (29 

% to 94 % RSD).

• A clear distinction was observed for the glycoside (daidzin, glycitin, and genistin) results 

in both materials between the laboratories using solvent extraction compared to those 

including a hydrolysis step in their sample preparation.

o Separate consensus means and ranges were calculated for each sample preparation 

approach. The variances of each approach were found to be statistically equivalent, 

yet the mean values determined by each method for each analyte below were 

determined to be statistically different.

o With the exception of glycitin in the soy protein isolate, the consensus means for all 

of the glycosides as well as total isoflavones determined using hydrolysis were within 

the target ranges in both materials with excellent between-laboratory variability (5 % 

to 14 % RSD).

o The consensus mean for glycitin in the soy protein isolate for laboratories using 

hydrolysis was above the target range with slightly higher between-laboratory 

variability (24 % RSD).

o For laboratories using an extraction approach (without hydrolysis), consensus means 

for all isoflavones were below the target ranges. Between-laboratory variability was 

higher than when hydrolysis was used (21 % to 58 % RSD), with the exception of 

total isoflavones in the soy protein isolate (7 % RSD).
90

Analyte Consensus for Laboratories 

Using Extraction (μg/g)

Consensus for Laboratories 

Using Extraction with 

Hydrolysis (μg/g)

Soy Protein Isolate

Daidzin 70 ± 18 172 ± 15

Glycitin 13.2 ± 5.6 32.8 ± 8.0

Genistin 105 ± 26 308 ± 14

Total Isoflavones 471 ± 31 750 ± 100

Soy Flour

Daidzin 520 ± 100 1780 ± 170

Glycitin 91 ± 35 276 ± 25

Genistin 530 ± 200 2200 ± 130

Total Isoflavones 1040 ± 600 4220 ± 420

Technical Recommendations 

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

• A clear distinction was observed for the glycoside (daidzin, glycitin, and genistin) results 

in both materials between the laboratories using solvent extraction compared to those 

including a hydrolysis step in their sample preparation.

o The hydrolysis step is utilized to convert the acetyl- and malonyl-glycosides of 

daidzin, glycitin, and genistin to their glycoside forms.

o If acetyl- and malonyl-glycosides are present, the use of hydrolysis in the sample 

preparation will result in determination of higher concentrations of the glycosides 

than if hydrolysis is not used.

o Because the hydrolysis step does not cleave the glycosides from the isoflavone 

molecules, no increase in concentration is observed for the aglycones (daidzein, 

glycitein, and genistein) when hydrolysis is utilized.

o Laboratories that do not use a hydrolysis step must take extra precautions to provide 

accurate results.

• Ensure that acetyl- and malonyl-glycosides are not being cleaved during the 

extraction process, or that the extraction process being utilized is robust enough to 

provide consistent results with expected deviations in laboratory practice 

(extraction time, temperature, solvent concentration, etc.).

• Ensure that the acetyl- and malonyl-glycosides are sufficiently stable so they do 

not degrade during the course of the sample preparation and chromatographic 

runs, leading to inconsistent results.

• Ensure that the separation method is adequate to resolve malonyl- and acetylglycosides from aglycones and glycosides so that coelutions do not cause falsely 

high results.

• The most accurate chromatographic peak identification and quantitation relies on 

pure reference standards. If standards are not available for every acetyl- and 

malonyl-glycoside, ensure that quantitation using response of another compound 

is appropriate.

o Because the NIST target values were determined using a hydrolysis step, laboratories 

that also utilized the hydrolysis step were in better agreement. However, laboratories 
91

using only extraction are not incorrect, and it is therefore important to specify method 

information when discussing and reporting isoflavone concentrations.

• A calibration issue may also be underlying the data for the glycosides (daidzin, glycitin, 

and genistin), as observed in the sample/control comparison views. The diagonal line 

observed is somewhat pronounced due to the effect of sample processing (described 

above), but for those laboratories using only solvent extraction, there may be some 

evidence of a calibration error.

o Be sure to confirm the purity of all reference standards by measuring moisture as well 

as checking chromatographic purity. NMR can provide additional information.

o The best approach is to use a reference standard for each isoflavone in the calibration.

o If an internal standard approach is used, confirm that there are no chromatographic 

coelutions with the internal standard by running a blank.
92

Table 25. Individual data summary table (NIST) for isoflavones in soy dietary supplements.

Lab Code: NIST

Analyte Sample Units xi si Zcomm ZNIST N x* s* xNIST U 95

Daidzin Isolate μg/g 165 22 0.9 0.0 10 111 62 165 22

Daidzin Flour μg/g 1693 427 0.9 0.0 10 1050 727 1693 427

Glycitin Isolate μg/g 29.9 0.5 0.9 0.0 9 19.2 11.4 29.9 0.5

Glycitin Flour μg/g 241 31 0.8 0.0 9 159 105 241 31

Genistin Isolate μg/g 313 10 1.1 0.0 10 189 118 313 10

Genistin Flour μg/g 2011 387 0.8 0.0 10 1220 968 2011 387

Daidzein Isolate μg/g 99.3 0.5 0.0 0.0 9 99.1 13.6 99.3 0.5

Daidzein Flour μg/g 13.5 1.9 -0.7 0.0 10 37.8 35.7 13.5 1.9

Glycitein Isolate μg/g 21.6 0.2 -0.4 0.0 9 24.9 8.1 21.6 0.2

Glycitein Flour μg/g 4 149.0 285.0

Genistein Isolate μg/g 174 13 0.9 0.0 10 155 21 174 13

Genistein Flour μg/g 14.9 0.1 -0.4 0.0 10 16.6 4.8 14.9 0.1

Total Isoflavones Isolate μg/g 804 27 1.2 0.0 10 585 176 804 27

Total Isoflavones Flour μg/g 3973 578 0.9 0.0 11 2300 1800 3973 578

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U 95 ±95% confidence interval

Zcomm Z-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

Exercise J - May 2013 - Isoflavones

1. Your Results 2. Community Results 3. Target

National Institute of Standards & Technology
93

Table 26. Data summary table for daidzin in soy dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 165 22 1693 427

J002

J004

J005

J011 162 163 155 160 4 1540 1590 1540 1557 29

J019 50 52 50 51 1 384 410 532 442 79

J029 204 174 178 185 16 1880 1870 1870 1873 6

J030 189 175 181 181 7 1804 1771 1797 1791 17

J031 76 78 77 77 1 606 606 610 607 2

J035

J036 95 96 87 93 5 807 808 811 809 2

J049 74 74 73 74 1 607 614 611 5

J053 69 70 73 71 2 506 532 519 18

J054 52 55 57 55 3 402 401 403 402 1

J057

J058 164 155 162 160 5 1885 1863 1907 1885 22

J059

 Consensus Mean 111 Consensus Mean 1049

 Consensus Standard Deviation 62 Consensus Standard Deviation 727

 Maximum 185 Maximum 1885

 Minimum 51 Minimum 402

 N 10 N 10

Community 

Results

Daidzin

Individual Results

SRM 3236 Soy Protein Isolate (μg/g) SRM 3234 Soy Flour (μg/g)
94

Table 27. Data summary table for glycitin in soy dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 29.9 0.5 241 31

J002

J004

J005

J011

J019 11.2 10.6 10.5 10.8 0.4 91 92 101 94 6

J029 31.4 28.5 28.8 29.6 1.6 258 259 257 258 1

J030 39.3 38.8 44.6 40.9 3.2 308 295 299 301 7

J031 16.0 17.1 18.3 17.1 1.2 106 106 107 106 1

J035

J036 17.1 17.5 15.1 16.6 1.3 153 154 150 152 2

J049 13.0 12.1 11.1 12.1 1.0 81 85 83 3

J053 17.0 16.5 20.2 17.9 2.0 128 125 126 3

J054 5.0 5.0 5.0 5.0 0.0 44 43 45 44 1

J057

J058 29.3 27.2 27.3 27.9 1.2 267 271 267 268 2

J059

 Consensus Mean 19.2 Consensus Mean 159

 Consensus Standard Deviation 11.4 Consensus Standard Deviation 105

 Maximum 40.9 Maximum 301

 Minimum 5.0 Minimum 44

 N 9 N 9

Glycitin

SRM 3236 Soy Protein Isolate (μg/g) SRM 3234 Soy Flour (μg/g)

Individual Results Community Results
95

Table 28. Data summary table for genistin in soy dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 313 10 2011 387

J002

J004

J005

J011 311 314 294 306 11 2110 2180 2100 2130 44

J019 77 80 71 76 5 317 358 102 259 138

J029 311 283 291 295 14 2080 2080 2080 2080 0

J030 327 324 323 325 2 2260 2225 2258 2248 20

J031 130 126 130 129 2 651 657 661 656 5

J035

J036 126 127 116 123 6 677 681 675 678 3

J049 134 136 136 135 1 750 766 758 11

J053 114 111 111 112 1 592 620 606 20

J054 84 88 91 88 4 464 463 465 464 1

J057

J058 308 302 308 306 4 2344 2302 2352 2333 27

J059

 Consensus Mean 189 Consensus Mean 1221

 Consensus Standard Deviation 118 Consensus Standard Deviation 968

 Maximum 325 Maximum 2333

 Minimum 76 Minimum 259

 N 10 N 10

Genisitn

SRM 3236 Soy Protein Isolate (μg/g) SRM 3234 Soy Flour (μg/g)

Individual Results Community Results
96

Table 29. Data summary table for daidzein in soy dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 99.3 0.5 13.5 1.9

J002

J004

J005

J011

J019 119.6 120.6 117.6 119.2 1.6 63.4 66.7 81.9 70.7 9.9

J029 79.7 83.8 87.3 83.6 3.8 15.6 15.0 15.4 15.3 0.3

J030 116.0 117.1 120.3 117.8 2.2 35.2 29.1 28.9 31.1 3.6

J031 91.9 92.0 92.1 92.0 0.1 16.5 13.2 13.9 14.5 1.7

J035

J036 98.3 98.3 98.6 98.4 0.2 17.0 16.9 16.6 16.8 0.2

J049 93.7 93.0 92.5 93.1 0.6 85.0 83.0 84.0 1.4

J053 100.2 104.6 98.1 101.0 3.3 27.6 26.5 27.1 0.8

J054 94.0 92.0 92.0 92.7 1.2 11.1 11.3 11.0 11.1 0.2

J057

J058 94.9 91.9 95.0 93.9 1.8 16.8 15.9 15.7 16.1 0.6

J059 103.0 109.0 102.0 104.7 3.8

 Consensus Mean 99.1 Consensus Mean 37.8

 Consensus Standard Deviation 13.6 Consensus Standard Deviation 35.7

 Maximum 119.2 Maximum 104.7

 Minimum 83.6 Minimum 11.1

 N 9 N 10

Daidzein

SRM 3236 Soy Protein Isolate (μg/g) SRM 3234 Soy Flour (μg/g)

Individual Results Community Results
97

Table 30. Data summary table for glycitein in soy dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 21.6 0.2

J002

J004

J005

J011

J019 18.7 19.0 18.7 18.8 0.2 1.5 1.7 2.7 2.0 0.6

J029 18.7 18.1 18.4 18.4 0.3

J030 28.4 20.4 25.8 24.9 4.1 10.8 10.2 10.6 10.5 0.3

J031 24.9 23.1 22.1 23.4 1.4

J035

J036 47.2 46.6 46.3 46.7 0.5

J049 21.6 22.2 21.5 21.8 0.4

J053 23.9 25.7 25.8 25.1 1.1

J054 35.7 36.6 36.3 36.2 0.5 524.6 524.1 524.9 524.5 0.4

J057

J058 17.8 19.3 19.0 18.7 0.8

J059 57.0 62.0 56.0 58.3 3.2

 Consensus Mean 24.9 Consensus Mean 148.8

 Consensus Standard Deviation 8.1 Consensus Standard Deviation 285.4

 Maximum 46.7 Maximum 524.5

 Minimum 18.4 Minimum 2.0

 N 9 N 4

Glycitein

SRM 3236 Soy Protein Isolate (μg/g) SRM 3234 Soy Flour (μg/g)

Individual Results Community Results
98

Table 31. Data summary table for genistein in soy dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 174 13 14.9 0.1

J002

J004

J005

J011 169 170 159 166 6

J019 163 164 160 162 2 11.4 10.6 12.4 11.5 0.9

J029 127 121 122 123 3 13.6 14.0 14.7 14.1 0.6

J030 157 152 158 156 3 25.1 22.8 23.4 23.8 1.2

J031 155 153 156 155 2 14.5 14.7 13.9 14.4 0.4

J035

J036 164 164 167 165 2 14.1 14.7 15.1 14.6 0.5

J049 162 162 162 162 0 16.0 15.8 15.9 0.1

J053 121 120 123 121 1 17.9 18.2 18.1 0.2

J054 150 153 157 153 3 12.7 12.5 12.9 12.7 0.2

J057

J058 185 185 180 183 3 34.3 35.5 30.5 33.4 2.6

J059 19.0 15.0 18.0 17.3 2.1

 Consensus Mean 155 Consensus Mean 16.6

 Consensus Standard Deviation 21 Consensus Standard Deviation 4.8

 Maximum 183 Maximum 33.4

 Minimum 121 Minimum 11.5

 N 10 N 10

Genistein

SRM 3236 Soy Protein Isolate (μg/g) SRM 3234 Soy Flour (μg/g)

Individual Results Community Results
99

Table 32. Data summary table for total isoflavones in soy dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 804 27 3973 578

J002

J004

J005

J011 642 647 608 632 21 3650 3770 3640 3687 72

J019 440 446 427 438 10 869 939 832 880 54

J029 772 708 726 735 33 4247 4238 4237 4241 6

J030 857 828 852 845 15 4443 4353 4416 4404 46

J031 494 489 495 493 3 1394 1397 1406 1399 6

J035

J036 548 550 530 543 11 1668 1675 1668 1670 4

J049 498 499 496 498 2 1539 1564 1551 18

J053 445 448 451 448 3 1271 1321 1296 35

J054 421 430 438 430 9 1459 1455 1462 1458 3

J057

J058 798 780 792 790 9 4548 4487 4572 4535 43

J059 179 186 176 180 5

 Consensus Mean 585 Consensus Mean 2300

 Consensus Standard Deviation 176 Consensus Standard Deviation 1797

 Maximum 845 Maximum 4535

 Minimum 430 Minimum 180

 N 10 N 11

SRM 3236 Soy Protein Isolate (μg/g) SRM 3234 Soy Flour (μg/g)

Individual Results Community Results

Total Isoflavones
100

Figure 49. Daidzin in SRM 3236 Soy Protein Isolate (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation

method employed. The black solid lines represent the consensus means for each sample 

preparation method, and the black dotted lines represent the consensus variability calculated as 

one standard deviation about the consensus mean for that sample preparation method. The gray 

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

NIST certified value bounded by twice its uncertainty (U95).
101

Figure 50. Daidzin in SRM 3234 Soy Flour (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. The black solid lines represents the consensus mean for each sample 

preparation method, and the black dotted lines represent the consensus variability calculated as 

one standard deviation about the consensus mean for that sample preparation method. The gray 

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

NIST value determined by LC/abs and ID-LC/MS, bounded by an estimated uncertainty 

calculated as the standard deviation between the means of values determined by each method.
102

Figure 51. Glycitin in SRM 3236 Soy Protein Isolate (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. The black solid lines represents the consensus mean for each sample 

preparation method, and the black dotted lines represent the consensus variability calculated as 

one standard deviation about the consensus mean for that sample preparation method. The gray 

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

NIST certified value bounded by twice its uncertainty (U95).
103

Figure 52. Glycitin in SRM 3234 Soy Flour (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. The black solid lines represents the consensus mean for each sample 

preparation method, and the black dotted lines represent the consensus variability calculated as 

one standard deviation about the consensus mean for that sample preparation method. The gray 

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

NIST value determined by LC/abs and ID-LC/MS, bounded by an estimated uncertainty 

calculated as the standard deviation between the means of values determined by each method.
104

Figure 53. Genistin in SRM 3236 Soy Protein Isolate (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. The black solid lines represents the consensus mean for each sample 

preparation method, and the black dotted lines represent the consensus variability calculated as 

one standard deviation about the consensus mean for that sample preparation method. The gray 

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

NIST certified value bounded by twice its uncertainty (U95).
105

Figure 54. Genistin in SRM 3234 Soy Flour (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. The black solid lines represents the consensus mean for each sample 

preparation method, and the black dotted lines represent the consensus variability calculated as 

one standard deviation about the consensus mean for that sample preparation method. The gray 

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

NIST value determined by LC/abs and ID-LC/MS, bounded by an estimated uncertainty 

calculated as the standard deviation between the means of values determined by each method.
106

Figure 55. Daidzein in SRM 3236 Soy Protein Isolate (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. 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).
107

Figure 56. Daidzein in SRM 3234 Soy Flour (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. 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 LC/abs and ID-LC/MS, bounded by an estimated 

uncertainty calculated as the standard deviation between the means of values determined by each 

method.
108

Figure 57. Glycitein in SRM 3236 Soy Protein Isolate (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. 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).
109

Figure 58. Glycitein in SRM 3234 Soy Flour (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. 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. 
110

Figure 59. Genistein in SRM 3236 Soy Protein Isolate (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. 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).
111

Figure 60. Genistein in SRM 3234 Soy Flour (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. 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 LC/abs and ID-LC/MS, bounded by an estimated 

uncertainty calculated as the standard deviation between the means of values determined by each 

method.
112

Figure 61. Total isoflavones in SRM 3236 Soy Protein Isolate (data summary view, sample 

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

laboratory standard deviation (error bars). The color of the data point represents the sample 

preparation method employed. The black solid lines represents the consensus mean for each 

sample preparation method, and the black dotted lines represent the consensus variability 

calculated as one standard deviation about the consensus mean for that sample preparation 

method. 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. Total isoflavones in SRM 3234 Soy Flour (data summary view, sample preparation 

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

standard deviation (error bars). The color of the data point represents the sample preparation 

method employed. The black solid lines represents the consensus mean for each sample 

preparation method, and the black dotted lines represent the consensus variability calculated as 

one standard deviation about the consensus mean for that sample preparation method. The gray 

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

NIST value determined by LC/abs and ID-LC/MS, bounded by an estimated uncertainty 

calculated as the standard deviation between the means of values determined by each method.
114

Figure 63. Daidzin in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour (sample/control 

comparison view). In this view, the individual laboratory results for the control (soy protein 

isolate) with a certified value for the analyte are compared to the results for an unknown (soy 

flour). The solid red box represents 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).
115

Figure 64. Glycitin in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour (sample/control 

comparison view). In this view, the individual laboratory results for the control (soy protein 

isolate) with a certified value for the analyte are compared to the results for an unknown (soy 

flour). The solid red box represents 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).
116

Figure 65. Genistin in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour (sample/control 

comparison view). In this view, the individual laboratory results for the control (soy protein 

isolate) with a certified value for the analyte are compared to the results for an unknown (soy 

flour). The solid red box represents 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).
117

Figure 66. Daidzein in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour (sample/control 

comparison view). In this view, the individual laboratory results for the control (soy protein 

isolate) with a certified value for the analyte are compared to the results for an unknown (soy 

flour). The solid red box represents 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).
118

Figure 67. Glycitein in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

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

(soy protein isolate) with a certified value for the analyte are compared to the results for an 

unknown (soy flour). The dotted blue box represents the consensus zone for the control (x-axis) 

and the unknown sample (y-axis).
119

Figure 68. Genistein in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

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

(soy protein isolate) with a certified value for the analyte are compared to the results for an 

unknown (soy flour). The solid red box represents the target zone for the control (x-axis) and 

unknown sample (y-axis). The dotted blue box represents the consensus zone for the control (xaxis) and the unknown sample (y-axis).
120

Figure 69. Total isoflavones in SRM 3236 Soy Protein Isolate and SRM 3234 Soy Flour 

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

(soy protein isolate) with a certified value for the analyte are compared to the results for an 

unknown (soy flour). The solid red box represents the target zone for the control (x-axis) and 

unknown sample (y-axis). The dotted blue box represents the consensus zone for the control (xaxis) and the unknown sample (y-axis).
121

BOTANICAL IDENTITY OF PURE AND ADULTERATED GINKGO BILOBA

Study Overview 

In this study, ten vials labeled Ginkgo biloba extract and ten vials labeled Ginkgo biloba leaf 

were provided to the participants.

Sample Information 

Gingko biloba Extract. Participants were provided with ten vials labeled Ginkgo biloba extract. 

Before use, participants were instructed to mix each vial thoroughly. Participants were asked to 

report “yes” for vials identified as Ginkgo biloba. If possible, reporting of the mass fraction 

(percentage) of Ginkgo biloba was requested. Participants were asked to report “no” for vials 

identified as not Ginkgo biloba. If possible, participants were asked to indicate the main 

constituent of the sample.

Gingko biloba Leaf. Participants were provided with ten vials labeled Ginkgo biloba leaf. 

Before use, participants were instructed to mix each vial thoroughly. Participants were asked to 

report “yes” for vials identified as Ginkgo biloba. If possible, reporting of the mass fraction 

(percentage) of Ginkgo biloba was requested. Participants were asked to report “no” for vials 

identified as not Ginkgo biloba. If possible, participants were asked to indicate the main 

constituent of the sample.

Study Results

• Twenty-five laboratories enrolled in this exercise and received samples. Nineteen

laboratories reported results for some portion of the study (76 % participation). 

• The Gingko biloba extract samples were adulterated with 0 %, 10 %, 25 %, 50 %, or

75 % green tea extract and 0 %, 10 %, 25 %, 50 %, or 75 % of microcellulose filler.

• Six laboratories attempted to identify the mass percentage of Gingko biloba extract in 

each sample.

• The Gingko biloba leaf samples were adulterated with 0 %, 10 %, 25 %, 50 %, or 75 % 

green tea leaves and 0 %, 10 %, 25 %, 50 %, or 75 % of microcellulose filler.

• Five laboratories attempted to identify the mass percentage of Gingko biloba leaves in 

each sample.

• Laboratories that used only thin-layer chromatography (TLC) for identity and detection 

of adulteration were better able to detect adulteration with other plants than with the 

microcellulose, which added no bands to the chromatogram.

• Laboratories were able to detect adulteration with green tea at lower levels in the plant 

material than in the extract material.

• Detection of adulteration with microcellulose was approximately the same in the plant 

material and the extract material.

Technical Recommendations 

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

• TLC is a good screening method for identity and for finding adulteration, provided the 

adulterant has characteristic chromatographic bands that are different from those of the 

plant material under investigation.

• Laboratories that performed multiple methods (e.g., TLC and microscopy) were able to 

provide quantitative results for the percent adulteration as well as information as to 

whether or not the sample was an adulterated Ginkgo product.
122

• In future studies, more specific questions will be asked about testing methods.

• Laboratories will be given specific instructions on whether to test for authenticity/identity 

or adulteration.
123

Table 33. Individual data summary table (NIST) for botanical identity of pure and adulterated Ginkgo biloba.

NIST

Adulterant Y/N % NY/N NYes NNo N% Avg SD Y/NNIST %NIST

L8 Ginkgo Leaves -- Yes 100% 19 17 2 3 98% 3% Yes 100%

L5 Ginkgo Leaves Green Tea Leaves Yes 90% 19 9 10 5 82% 6% Yes 90%

L9 Ginkgo Leaves Green Tea Leaves Yes 75% 19 7 12 4 69% 3% Yes 75%

L2 Ginkgo Leaves Green Tea Leaves Yes 50% 19 7 12 5 58% 29% Yes 50%

L6 Ginkgo Leaves Green Tea Leaves Yes 25% 19 8 11 5 28% 6% Yes 25%

L4 Ginkgo Leaves -- Yes 100% 19 17 2 4 84% 33% Yes 100%

L1 Ginkgo Leaves Microcellulose Yes 90% 19 16 3 4 89% 5% Yes 90%

L7 Ginkgo Leaves Microcellulose Yes 75% 19 16 3 3 77% 3% Yes 75%

L3 Ginkgo Leaves Microcellulose Yes 50% 19 15 4 4 53% 5% Yes 50%

L10 Ginkgo Leaves Microcellulose Yes 25% 19 15 4 3 18% 8% Yes 25%

E5 Ginkgo Extract -- Yes 100% 18 16 2 4 90% 20% Yes 100%

E1 Ginkgo Extract Green Tea Extract Yes 90% 18 14 4 4 85% 7% Yes 90%

E2 Ginkgo Extract Green Tea Extract Yes 75% 17 5 12 5 62% 15% Yes 75%

E4 Ginkgo Extract Green Tea Extract Yes 50% 17 6 11 6 38% 26% Yes 50%

E9 Ginkgo Extract Green Tea Extract Yes 25% 17 7 10 3 28% 41% Yes 25%

E7 Ginkgo Extract -- Yes 100% 18 17 1 4 98% 3% Yes 100%

E8 Ginkgo Extract Microcellulose Yes 90% 18 7 11 4 68% 22% Yes 90%

E3 Ginkgo Extract Microcellulose Yes 75% 17 14 3 4 73% 13% Yes 75%

E6 Ginkgo Extract Microcellulose Yes 50% 17 12 5 4 43% 10% Yes 50%

E10 Ginkgo Extract Microcellulose Yes 25% 17 12 5 2 10% 0% Yes 25%

Lab Code:

Sample

1. Your Results 2. Community Results 3. Target

National Institute of Standards & Technology

Exercise J - May 2013 - Botanical ID
124

Table 34. Data summary table for Ginkgo biloba extracts (yes/no).

Adulterant

Mass % Ginkgo 100% 90% 75% 50% 25% 100% 90% 75% 50% 25%

Lab/Sample E5 E1 E2 E4 E9 E7 E8 E3 E6 E10

J001 Yes Yes No No No Yes No Yes Yes Yes

J002 Yes Yes No No No Yes No Yes Yes Yes

J003 Yes No No No No Yes No No No No

J005

J008 Yes No No No No Yes Yes Yes No No

J009

J010 Yes Yes Yes Yes

J011

J015 Yes Yes No No No Yes No Yes Yes Yes

J018 no no no yes yes no no no no no

J020 Yes Yes No No No Yes No Yes Yes Yes

J027

J029 Yes Yes No No No Yes No Yes Yes No

J031 No Yes No No No Yes No Yes No Yes

J032 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

J035

J036 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

J038

J043 Yes Yes No No No Yes No Yes Yes Yes

J049 Yes Yes No No Yes Yes No Yes Yes Yes

J050

J052 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

J053

J057 Yes No No No No Yes No No No No

J059

N 17 17 16 16 16 17 17 16 16 16

Yes 15 13 4 5 6 16 6 13 11 11

No 2 4 12 11 10 1 11 3 5 5

% Yes 88% 76% 25% 31% 38% 94% 35% 81% 69% 69%

% No 12% 24% 75% 69% 63% 6% 65% 19% 31% 31%

Community 

Results Individual Results

Ginkgo biloba Extracts

Green Tea Extract Inert Filler
125

Table 35. Data summary table for Ginkgo biloba extracts (mass percentage).

Adulterant

Mass % Ginkgo 100% 90% 75% 50% 25% 100% 90% 75% 50% 25%

Lab/Sample E5 E1 E2 E4 E9 E7 E8 E3 E6 E10

J001

J002

J003 100% 80% 60% 30% 10% 100% 77% 60% 30% 10%

J005

J008

J009

J010

J011

J015

J018 0.10% 0.08%

J020

J027

J029

J031 60% 80% 50% 70% 90% 50%

J032 100% 85% 65% 35% 10-15% 95% 80% 65% 40% 10-15%

J035

J036

J038

J043

J049

J050

J052 12% 10% 8% 5% 2% 11% 10% 8% 5% 1%

J053

J057 100% 95% 50% 25% 75% 100% 80% 75% 50% 10%

J059

Individual Results

Green Tea Extract Inert Filler

Ginkgo biloba Extracts
126

Table 36. Data summary table for Ginkgo biloba leaves (yes/no).

Adulterant

Mass % Ginkgo 100% 90% 75% 50% 25% 100% 90% 75% 50% 25%

Lab/Sample L8 L5 L9 L2 L6 L4 L1 L7 L3 L10

J001 Yes No No No No Yes Yes Yes Yes Yes

J002 Yes No No No No Yes Yes Yes Yes Yes

J003 Yes No No No No Yes No No No No

J005

J008 Yes Yes Yes Yes Yes Yes Yes Yes No No

J009

J010 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

J011

J015 Yes No No No No Yes Yes Yes Yes Yes

J018 no no no no no no no no no no

J020 Yes No No No No Yes Yes Yes Yes Yes

J027

J029 Yes No No No No Yes Yes Yes Yes Yes

J031 No Yes No No Yes No Yes Yes Yes Yes

J032 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

J035

J036 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

J038

J043 Yes No No No No Yes Yes Yes Yes Yes

J049 Yes Yes No No No Yes Yes Yes Yes Yes

J050 Yes No No No No Yes Yes Yes Yes Yes

J052 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

J053

J057 Yes No No No No Yes No No No No

J059 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

N 19 19 19 19 19 19 19 19 19 19

Yes 17 9 7 7 8 17 16 16 15 15

No 2 10 12 12 11 2 3 3 4 4

% Yes 89% 47% 37% 37% 42% 89% 84% 84% 79% 79%

% No 11% 53% 63% 63% 58% 11% 16% 16% 21% 21%

Ginkgo biloba Leaves

Green Tea Leaves Inert Filler

Individual Results Community Results
127

Table 37. Data summary table for Ginkgo biloba leaves (mass percentage).

Adulterant

Mass % Ginkgo 100% 90% 75% 50% 25% 100% 90% 75% 50% 25%

Lab/Sample L8 L5 L9 L2 L6 L4 L1 L7 L3 L10

J001

J002

J003 100% 80% 70% 40% 30% 100% 85% 75% 50% 20%

J005

J008

J009

J010

J011

J015

J018

J020

J027

J029

J031 75% 110% 35% 35% 85% 60%

J032 95% 85% 65% 45% 20% 100% 92% 75% 50% 25%

J035

J036

J038

J043

J049

J050

J052 0.68% 0.68% 0.76% 0.59% 0.53% 0.69% 0.66% 0.50% 0.34% 0.28%

J053

J057 100% 80% 70% 50% 25% 100% 95% 80% 50% 10%

J059

Green Tea Leaves Inert Filler

Individual Results

Ginkgo biloba Leaves
128

Figure 70. Adulterated Ginkgo biloba extract. These two charts show the number of laboratories 

reporting Ginkgo extract as being adulterated. The samples in the top chart were adulterated 

with varying amounts of green tea extract. The samples in the bottom chart were adulterated with 

varying amounts of cellulose.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Ginkgo E5

(100%)

Ginkgo E1

(90%)

Ginkgo E2

(75%)

Ginkgo E4

(50%)

Ginkgo E9

(25%)

Number of Labs

Gingko Extract Adulterated with Green Tea Extract

ID as Ginkgo - NO

ID as Ginkgo - YES

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Ginkgo E7

(100%)

Ginkgo E8

(90%)

Ginkgo E3

(75%)

Ginkgo E6

(50%)

Ginkgo E10

(25%)

Number of Labs

Gingko Extract Adulterated with Cellulose

ID as Ginkgo - NO

ID as Ginkgo - YES
129

Figure 71. Adulterated Ginkgo biloba leaves. These two charts show the number of laboratories 

reporting Ginkgo leaves as being adulterated. The samples in the top chart were adulterated with 

varying amounts of green tea leaves. The samples in the bottom chart were adulterated with 

varying amounts of cellulose.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Ginkgo L8

(100%)

Ginkgo L5

(90%)

Ginkgo L9

(75%)

Ginkgo L2

(50%)

Ginkgo L6

(25%)

Number of Labs

Gingko Leaves Adulterated with Green Tea Leaves

ID as Ginkgo - NO

ID as Ginkgo - YES

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Ginkgo L4

(100%)

Ginkgo L1

(90%)

Ginkgo L7

(75%)

Ginkgo L3

(50%)

Ginkgo L10

(25%)

Number of Labs

Gingko Leaves Adulterated with Cellulose

ID as Ginkgo - NO

ID as Ginkgo - YES