NISTIR 7893

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Summer 

2011 Comparability Study 

(Exercise 4)

Katrice A. Lippa

Mary Bedner

Susan S.-C. Tai

http://dx.doi.org/10.6028/NIST.IR.7893
NISTIR 7893

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Summer 

2011 Comparability Study 

(Exercise 4)

Katrice A. Lippa

Mary Bedner

Susan S.-C. Tai

Chemical Sciences Division

Material Measurement Laboratory

http://dx.doi.org/10.6028/NIST.IR.7893

December 2012

U.S. Department of Commerce 

Rebecca Blank, Acting Secretary

National Institute of Standards and Technology 

Patrick D. Gallagher, Under Secretary of Commerce for Standards and Technology and Director 
1

ABSTRACT

The National Institute of Standards and Technology (NIST) recently established a Vitamin D 

Metabolites Quality Assurance Program (VitDQAP) in collaboration with the National Institutes of 

Health (NIH) Office of Dietary Supplements. Participants in the fourth exercise of this program, 

the Summer 2011 Comparability Study, were asked to use the methodology of their choice to 

measure concentrations of 25-hydroxyvitamin D in control and study materials distributed by NIST. 

The study materials consisted of SRM 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in 

Human Serum (Level 1, Level 2, and Level 3) and SRM 968d Fat-Soluble Vitamins, Carotenoids 

and Cholesterol in Human Serum (Level 1). SRM 2972, which is comprised of separate ethanolic 

calibration solutions with known concentrations of 25(OH)D2 and 25(OH)D3, was provided as a 

control material. Participants provided their data to NIST, where it was compiled and evaluated for 

trueness relative to the NIST value and concordance within the participant community. A report of 

results was provided to all participants of the exercise, and laboratories were identified by code 

numbers known only to them. The results from this fourth exercise are reported along with a 

summary of the analytical methods used. 
2

OVERVIEW OF THE SUMMER 2011 COMPARABILITY STUDY

For the Summer 2011 Comparability Study (Exercise 4) of VitDQAP, control and human serum 

study samples were distributed to participants for evaluation. SRM 2972, which is comprised of 

separate ethanolic solutions with known concentrations of 25-hydroxyvitamin D2 (25(OH)D2) and

25-hydroxyvitamin D3 (25(OH)D3), was provided as a control material for assay calibration or 

verification. Participants were asked to provide single results for each of these solutions. In 

addition, participants were asked to determine concentration values for 25(OH)D2, 25(OH)D3, and 

a total concentration of 25-hydroxyvitamin D (25(OH)DTotal = 25(OH)D2 + 25(OH)D3) for each of 

four samples (vials A, B, C, and D) of human serum (study materials). In this exercise, vials A, B, 

and C were SRM 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum Level 

1 (SRM 968e L1), Level 2 (SRM 968e L2), and Level 3 (SRM 968e L3), respectively. Vial D was 

SRM 968d Fat-Soluble Vitamins, Carotenoids and Cholesterol in Human Serum Level 1 (SRM 

968d L1). All materials consisted of blended human serum pools with endogenous 25(OH)D levels. 

There were a total of 41 participants and 45 datasets (four participants provided data for two 

different methods) in the Summer 2011 exercise. Seventeen of the datasets originated from 

immunoassay (IA) techniques, including three from enzyme immunoassay (EIA), nine from 

chemiluminescence immunoassay (CLIA), and five from radioimmunoassay (RIA). Appendix A-1

summarizes the immunoassay methods used by the participants. Twenty-eight of the datasets 

originated from liquid chromatographic (LC) methods; of those, 22 were from LC with tandem 

mass spectrometric detection (LC-MS/MS), one was from LC-MS (orbitrap), and five were from 

LC with ultraviolet absorbance detection (LC-UV). A summary of the LC methods used by the 

participants may be found in Appendices A-2 and A-3. From here, LC-MS/MS and LC-MS are 

collectively referred to as LC-MSn.

The raw data received from all participants is summarized in Appendix B. For all study materials, 

the immunoassay methods reported values for 25(OH)DTotal only, whereas LC participants provided 

values for 25(OH)D2, 25(OH)D3, and 25(OH)DTotal. However, all four serum materials contain 

very low levels of 25(OH)D2 (participant reported values ranging from 0.1 ng/mL to 0.4 ng/mL), 

and most of the LC labs indicated this analyte was below their quantitation limit of <1 ng/mL to <5 

ng/mL. Therefore, the 25(OH)DTotal values reported in Appendix B are the same as the 25(OH)D3

values in the serum materials for the majority of LC participants. Both LC and immunoassay 

datasets provided individual values for 25(OH)D2 and 25(OH)D3 in the ethanolic controls because 

the analytes were in separate solutions.

Appendix B also provides the summarized NIST results for each of the serum materials. The 

25(OH)D2 was below the quantitation limit (≈ 0.5 ng/mL) in all materials for the NIST method.
3

SUMMER 2011 EXERCISE RESULTS AND DISCUSSION

25(OH)D2 and 25(OH)D3 in the control solutions (SRM 2972)

Participants were asked to analyze the control materials to qualify their assays prior to measuring 

the study materials. A summary of the individual participant data for 25(OH)D2 and 25(OH)D3 in 

the SRM 2972 control solutions is provided in Table 1. Of the 45 datasets received for the Summer 

2011 exercise, only 26 reported values for the both 25(OH)D2 and 25(OH)D3 ethanolic controls; of 

those, four were from immunoassay methods and 22 were from LC methods. Three additional 

participants provided values for only the 25(OH)D3 ethanolic controls. Overall, the control 

solutions appeared more compatible with the LC methods, and several of the immunoassay 

participants reported that the calibration solutions were not compatible with their method and did 

not provide values. 

The community results are summarized at the bottom of Table 1 for all reported methods, the LC 

methods only, and the LC-MSn methods only. The community results include the total number of 

quantitative values reported (N), the median value for each analyte, the MADe (the median absolute 

deviation estimate, a robust estimate of the standard deviation), and the percent coefficient of 

variation (CV%). Consensus statistics were not calculated for the data from the IA methods 

because of the limited number of data reported (N = 4). Table 1 also presents the NIST certified 

values with expanded uncertainties corresponding to 95% confidence.
4

Table 1. Summary of participant data and community results for 25(OH)D2 (ng/mL) and 

25(OH)D3 (ng/mL) in the SRM 2972 control solutions.

n/r = not reported

SRM 2972 SRM 2972

Lab Method Value Lab Method Value

032 LC-UV 261.5 3.0 032 LC-UV 344.0

056 LC-MS/MS 227.3 2.0 056 LC-MS/MS 321.3

110 LC-UV 233.4 3.0 110 LC-UV 331.7

116 LC-MS/MS 241.8 2.0 116 LC-MS/MS 331.0

119 LC-MS 243.2 2.0 119 LC-MS 352.6

128 LC-MS/MS n/r 2.0 128 LC-MS/MS 333.5

139 LC-UV 241.5 3.0 139 LC-UV 330.5

184 LC-MS/MS 236.5 2.0 184 LC-MS/MS 338.5

185 LC-MS/MS 238.6 2.0 185 LC-MS/MS 334.8

186 LC-MS/MS n/r 2.0 186 LC-MS/MS 320.0

187 LC-MS/MS 235.0 2.0 187 LC-MS/MS 337.0

188 CLIA 401.5 1.1 188 CLIA 381.5

189 LC-UV n/r 3.0 189 LC-UV 280.9

195 LC-MS/MS 242.0 2.0 195 LC-MS/MS 330.0

196 CLIA 210.4 1.1 196 CLIA 452.0

197 LC-MS/MS 234.3 2.0 197 LC-MS/MS 342.5

198a LC-MS/MS 245.5 2.0 198a LC-MS/MS 308.6

199 LC-MS/MS 233.0 2.0 199 LC-MS/MS 329.0

200 RIA 216.0 1.3 200 RIA 319.1

202 LC-MS/MS 241.0 2.0 202 LC-MS/MS 334.5

209 LC-MS/MS 240.6 2.0 209 LC-MS/MS 332.8

210a RIA 209.4 1.3 210a RIA 299.1

211 LC-MS/MS 238.0 2.0 211 LC-MS/MS 303.8

212 LC-MS/MS 248.8 2.0 212 LC-MS/MS 341.7

215 LC-MS/MS 226.7 2.0 215 LC-MS/MS 386.2

217 LC-MS/MS 220.8 2.0 217 LC-MS/MS 394.3

220 LC-MS/MS 232.4 2.0 220 LC-MS/MS 324.4

221a LC-MS/MS 253.0 2.0 221a LC-MS/MS 365.0

221b LC-UV 177.0 3.0 221b LC-UV 214.0

N 26 N 29

Median 237.3 Median 332.8

MADe 8.0 MADe 16.6

CV% 3.4 CV% 5.0

N 22 N 25

Median 238.3 253 Median 332.8

MADe 7.3 224 MADe 13.2

CV% 3.0 224 CV% 4.0

N 18 N 20

Median 238.3 Median 334.0

MADe 6.6 MADe 12.0

CV% 2.8 CV% 3.6

NIST Value 238.6 NIST Value 334.0

U 95 3.9 U 95 5.2

25(OH)D3 (ng/mL)

All 

methods

LC 

methods LC-MSn

25(OH)D2 (ng/mL)
5

For all participant datasets, the single data values reported for 25(OH)D2 and 25(OH)D3 in the 

control solutions, SRM 2972, are plotted in Figure 1. The results from immunoassay methods are 

displayed with closed red circles (). The results from the LC-based methods are displayed with 

black squares and are segregated by MSn detection (■) and UV detection ().

From the single reported values for all datasets for a given technique (IA or LC), the consensus 

median and the consensus variability (2 × MADe) were determined (reported in Table 1). For each 

of the techniques within both graphs, the solid lines () represent the consensus median and the 

dotted lines (- - - - -) represent the consensus variability (2 × MADe).

The laboratories with results that fall between the two dotted lines are within the consensus 

variability area for their technique (IA or LC). For the results from both techniques (IA or LC), the 

consensus variability is larger for 25(OH)D3 than for 25(OH)D2, but the highest variability was 

obtained for the IA results for 25(OH)D3. 

The grey-shaded bar in Figure 1 represents the interval in which NIST believes the “true value” 

exists for these solutions (i.e., NIST value ± approximately 95% confidence intervals (U95)). 

Participants were provided these values both on the SRM 2972 shipping package and within the 

data reporting sheet. The consensus median value for the LC methods lies within the NIST 

expanded uncertainty range for both 25(OH)D2 and 25(OH)D3. The consensus median value for 

the IA methods lies outside the NIST expanded uncertainty range for both 25(OH)D2 and 

25(OH)D3, indicating a potential method bias. 
6

Figure 1. 25(OH)D2 and 25(OH)D3 values in SRM 2972 for immunoassay and LC methods. The 

grey-shaded bars represent the ranges bound by the NIST certified values with ± U95 expanded 

uncertainty.
7

A direct comparison of results for 25(OH)D2 and 25(OH)D3 in the SRM 2972 control solutions is 

provided in the Youden plot in Figure 2. Laboratory results that are within the consensus range for 

both the 25(OH)D2 and 25(OH)D3 ethanolic controls are within the blue consensus box in Figure 

2. Conversely, laboratory results that fall outside of (or on the edge of) the blue consensus boxes 

are highlighted with their laboratory code numbers (221b, 210a, 200, 196, 217, 215, 32). The NIST 

values are denoted with a red diamond symbol (). The Youden line centered on the NIST values 

is illustrated by a red line () that represents the relative ratio of the NIST values (334.8/238.6) 

for 25(OH)D3 and 25(OH)D2 across the magnitude of the y- and x-axis, respectively.

Participant data that are near the Youden line but are clearly above or below the consensus box 

(number 221b) may suggest that these measurements are biased high or low due to a calibration 

error. However, correlation with the Youden line may be complicated for the control solutions 

because separate calibration solutions are likely prepared for measurement of 25(OH)D2 and 

25(OH)D3, particularly for LC-based methods.

Figure 2. Youden comparison of the results for 25(OH)D2 and 25(OH)D3 in the SRM 2972 control 

solutions.

 Laboratory values 

 All method consensus box encloses ± 2 MADe around consensus median

 NIST values with corresponding Youden line 

032

196

200

210a

215

217

221b

200

300

400

500

160 200 240 280

25(OH)D3 in SRM 2972 (ng/mL)

25(OH)D2 in SRM 2972 (ng/mL)
8

25(OH)D in SRM 968e L1, SRM 968e L2, SRM 968e L3, and SRM 968d L1

A summary of the individual participant data for 25(OH)DTotal in samples SRM 968e L1, SRM 

968e L2, SRM 968e L3, and SRM 968d L1 (vials A, B, C, and D, respectively) is provided in Table 

2. 

The community results are summarized at the bottom of the table for all reported methods, the 

immunoassay methods only, the LC methods only, and the LC-MSn methods only. These 

summarized results include the total number of quantitative values reported, the median value, the 

MADe, and the CV%.

Table 2 also presents the NIST results for the four study materials. For SRM 968e L1, L2, and L3, 

the NIST results are the certified values for 25(OH)D3 with an expanded uncertainty corresponding 

to the 95% confidence interval (U95)

1

. For SRM 968d L1, the NIST value for 25(OH)D3 was 

obtained using an LC-MS/MS reference measurement procedure2 recognized by the Joint 

Committee for Traceability in Laboratory Medicine (JCTLM), and the U95 confidence interval 

includes components for both measurement variability (N = 8) and measurement uncertainty 

associated with the density. The 25(OH)D2 was below the quantitation limit (≈ 0.5 ng/mL) in all 

materials and was not included in the results for 25(OH)DTotal.

 1 https://www-s.nist.gov/srmors/view_cert.cfm?srm=968E 2 Tai, S. S.-C., Bedner, M. and Phinney, K.W. Anal. Chem. 2010 82, 1942-1948.
9

Table 2. Summary of participant data for 25(OH)DTotal (ng/mL) in SRM 968e L1, SRM 968e L2, 

SRM 968e L3, and SRM 968d L1. 

 n/r = not reported

SRM 968e L1 SRM 968e L2 SRM 968e L3 SRM 968d L1

Lab Method Vial A Vial B Vial C Vial D

017 CLIA 8.1 15.1 26.5 15.7

026 LC-MS/MS 7.9 14.4 20.1 16.6

032 LC-UV 7.0 12.5 20.4 11.5

056 LC-MS/MS 7.2 12.3 21.8 13.2

086a CLIA 8.0 16.0 28.0 17.0

086b RIA 11.0 19.0 29.0 19.0

110 LC-UV 6.5 9.1 13.0 13.6

116 LC-MS/MS 9.5 15.6 24.0 14.3

119 LC-MS 9.4 14.8 21.8 14.6

124 LC-MS/MS 10.1 14.8 24.4 13.9

128 LC-MS/MS 11.2 17.8 27.2 18.1

139 LC-UV 10.1 17.1 25.3 17.9

161 CLIA 6.9 15.0 28.5 17.4

184 LC-MS/MS 6.5 12.3 19.5 13.6

185 LC-MS/MS 8.8 16.1 24.6 14.8

186 LC-MS/MS 9.0 12.0 26.0 16.0

187 LC-MS/MS 7.5 14.4 21.9 13.4

188 CLIA 11.8 20.3 27.8 15.2

189 LC-UV 12.3 16.6 30.4 n/r

191 RIA 7.7 11.3 18.1 14.0

192 EIA 7.5 17.9 27.1 20.1

195 LC-MS/MS 7.3 13.3 21.1 12.7

196 CLIA 7.6 14.9 25.2 15.9

197 LC-MS/MS 7.4 13.7 21.4 13.8

198a LC-MS/MS 9.6 17.2 22.9 15.2

198b EIA 9.0 17.0 34.0 16.0

199 LC-MS/MS 9.1 13.0 20.5 13.2

200 RIA 7.0 12.2 18.0 13.9

202 LC-MS/MS 7.8 14.1 23.1 13.6

209 LC-MS/MS 7.6 14.3 20.8 12.8

210a RIA 12.9 19.7 23.5 18.9

210b CLIA 9.4 16.1 30.4 17.7

211 LC-MS/MS 5.9 11.7 16.5 10.7

212 LC-MS/MS 8.1 13.4 22.9 13.4

213 EIA 9.3 14.0 18.6 17.7

214a RIA 7.9 14.4 24.7 13.5

214b CLIA 7.9 16.4 27.2 15.6

215 LC-MS/MS 8.2 15.0 21.2 14.1

217 LC-MS/MS 8.4 14.7 21.9 14.1

218 CLIA 5.5 15.1 25.2 14.1

219 LC-MS/MS 6.9 14.7 18.6 12.3

220 LC-MS/MS 7.5 13.8 20.0 13.1

221a LC-MS/MS 7.3 13.3 21.7 12.3

221b LC-UV 6.3 12.2 22.4 n/r

222 CLIA 11.1 20.3 31.1 15.6

N 45 45 45 43

Median 7.9 14.7 22.9 14.1

MADe 1.5 2.1 3.7 1.9

CV% 18.8 14 16 13.7

N 17 17 17 17

Median 8.0 16.0 27.1 15.9

MADe 1.5 2.4 2.8 2.7

CV% 18.5 15 10 16.8

N 28 28 28 26

Median 7.9 14.2 21.8 13.6

MADe 1.4 1.6 2.0 1.1

CV% 17.9 11.0 9.2 8.2

N 23 23 23 23

Median 7.9 14.3 21.8 13.6

MADe 1.1 1.3 1.9 1.0

CV% 14.1 9.3 8.8 7.6

NIST Value 7.09 12.90 19.90 12.38

U 95 0.14 0.30 0.40 0.28

All 

methods

IA 

methods 

LC 

methods LC-MSn
10

For all participant datasets, the single reported values for 25(OH)DTotal in SRM 968e L1, SRM 968e 

L2, SRM 968e L3, and SRM 968d L1 are plotted in Figure 3. The results from immunoassay 

methods are displayed with closed red circles (). The results from the LC-based methods are 

displayed with black squares and are segregated by MSn detection (■) and UV detection (). Each 

figure also has a legend that indicates which individual methods were used to obtain the reported 

values: CLIA, EIA, RIA, LC-MSn, or LC-UV.

For each of the techniques within both graphs, the solid lines () represent the consensus median 

and the dotted lines (- - - - -) represent the consensus variability (2 × MADe). The laboratories with 

results that fall between the two dotted lines are within the consensus variability area for their 

technique (IA or LC). The grey-shaded bar for each figure represents the NIST value and its 

associated uncertainty (i.e., value ± U95). NIST believes that the “true” value for each material lies 

within this interval. When this bar is not within the consensus range, then there may be method 

bias. 

Specific results as assessed from Figure 3 are summarized below.

SRM 968e L1

• For the IA results, all but three datasets are within the consensus variability range.

• For the LC results, all but two datasets are within the consensus variability range.

• The consensus median value is nearly identical for the IA and LC results.

• The consensus median value for both IA and LC results are higher than the NIST expanded 

uncertainty range (grey-shaded bar).

• The NIST expanded uncertainty range (grey-shaded bar) falls within the consensus variability 

ranges for both IA and LC.

SRM 968e L2

• For the IA results, all datasets are within the consensus variability range.

• For the LC results, all but two datasets are within the consensus variability range.

• The consensus variability range is larger for the IA results than the LC results.

• The consensus median value for the IA results is higher than the consensus median value for the 

LC results; both LC and IA median values are higher than the NIST expanded uncertainty range 

(grey-shaded bar).

• The NIST expanded uncertainty range (grey-shaded bar) falls within the consensus variability 

ranges for both IA and LC.

SRM 968e L3

• For the IA results, all but four datasets are within the consensus variability range.

• For the LC results, all but four datasets are within the consensus variability range.
11

• The consensus median value for the IA results is higher than the consensus median value for the 

LC results; both LC and IA median values are higher than the NIST expanded uncertainty range 

(grey-shaded bar).

• The NIST expanded uncertainty range (grey-shaded bar) falls within the consensus variability 

range for LC, but not for IA.

SRM 968d L1

For the IA data for SRM 968d L1, the consensus variability based on MADe is an overestimation of 

the 95% confidence limits about the median. The non-Gaussian data distribution contributes to a 

relatively wide range for the central 50% of this data, resulting in a large MADe. Since the 

consensus variability is not well-described with a MADe estimation, a meaningful assessment of the 

consensus range, the outlying results, and the agreement with the NIST value is hindered for the IA 

results.

• For the LC results, all but five datasets are within the consensus variability range.

• The consensus median value for the IA results is higher than the consensus median value for the 

LC results; both LC and IA median values are higher than the NIST expanded uncertainty range 

(grey-shaded bar).

• The NIST expanded uncertainty range (grey-shaded bar) falls within the consensus variability 

range for LC.

Overall, the results for the four study materials are very consistent, with the participant values 

always higher than the NIST value. In addition, the consensus variability is similar but relatively 

high for the four materials, ranging from 14% to 19% for all methods (Table 2). Similar trends 

have also been observed for many of the study materials evaluated in previous exercises of 

VitDQAP. A goal of the program is to achieve better agreement between the participant consensus 

median value and the NIST value and to better understand the sources of bias between the results. 

In addition, a major goal of VitDQAP is to reduce the consensus variability to better represent the 

community’s measurement capability while also recognizing that a “fit-for-purpose” variability 

level may exist. 

It is notable that the NIST method separates 25(OH)D3 and its 3-epimer, 3-epi-25(OH)D3, which 

was not quantitated in the study materials. The 3-epi-25(OH)D3 coelutes with 25(OH)D3 using 

typical chromatographic columns (C8, C18) and is detected by the same multiple reaction 

monitoring (MRM) ions in MS/MS and absorbance wavelength in UV, leading to a potential bias 

for LC-based methods. One of the LC-MS/MS participants (number 56) noted using a method that 

separates 3-epi-25(OH)D3 and provided values for this analyte in the study materials. However, the 

25(OH)D3 values reported by LC participants that use C8 and C18 columns represent the sum of 

25(OH)D3 and 3-epi-25(OH)D3, and 25(OH)DTotal also includes a contribution from 3-epi25(OH)D3. It is unclear how the presence of 3-epi-25(OH)D3 affects the 25(OH)DTotal for 

immunoassay results.
12

Figure 3. 25(OH)DTotal levels in SRM 968e L1, SRM 968e L2, SRM 968e L3 and SRM 968d L1

as determined by immunoassay (CLIA, EIA and RIA)and LC (LC-MSn and LC-UV) methods. The 

grey-shaded bars represent the ranges bound by the NIST values with ± estimated U95 uncertainty.
13

Figure 3 (cont’d). 25(OH)DTotal levels in SRM 968e L1, SRM 968e L2, SRM 968e L3 and SRM 

968d L1 as determined by immunoassay (CLIA, EIA and RIA) and LC (LC-MSn and LC-UV) 

methods. The grey-shaded bars represent the ranges bound by the NIST values with ± estimated U95

uncertainty.
14

A direct comparison of the results for SRM 968e L1 and SRM 968e L3, which represent the lowest 

and highest 25(OH)DTotal levels in the Summer 2011 study samples, respectively, is provided in the 

Youden plot in Figure 4. Also in this figure is a Youden plot comparing the results for SRM 968e 

L2 and SRM 968d L1, which both have comparable 25(OH)DTotal levels. There are two blue 

consensus boxes in each plot, one for IA methods and one for LC methods (as indicated). 

Laboratory results that are within the consensus range for both study materials are within the blue 

consensus boxes (in each plot). Conversely, laboratory results that fall outside of (or on the edge 

of) either of the consensus ranges are not included in the blue consensus boxes and are highlighted 

with their laboratory code numbers.

The NIST values for these materials are denoted with a red diamond symbol (). The Youden 

lines centered on the NIST values are illustrated by a red line (), which represents the relative 

ratio of the NIST values (19.90/7.09 for SRM 968e L3/SRM 968e L1 and 12.38/12.90 for SRM 

968d L1/SRM 968e L2) across the magnitude of the y- and x-axis, respectively. The Youden lines 

run through both the IA and LC consensus boxes for these materials. 

Since the four study materials for the Summer 2011 exercise had very low levels of 25(OH)D2, the 

results for 25(OH)DTotal and 25(OH)D3 are the same for the majority of the LC participants. 

Therefore, the Youden plots comparing the results for 25(OH)D3 obtained by the LC participants 

are nearly identical to the plots in Figure 4 and are not presented separately in this report.
15

Figure 4. Youden comparison plots of the results for 25(OH)DTotal in SRM 968e L1 and SRM 

968e L3 and in SRM 968e L2 and SRM 968d L1 for all methods. Data that fall outside the 

consensus boxes are labeled with their laboratory number.

110

128

186

188

189

191

198b

200

210a

211

213b

5

10

15

20

25

30

35

40

2 4 6 8 10 12 14 16

25(OH)DTotal in SRM 968e L3 "Vial C" (ng/mL)

25(OH)DTotal in SRM 968e L1 "Vial A" (ng/mL)

IA methods

LC methods

 IA method laboratory values 

 IA method consensus box encloses ± 2 MADe around consensus medians

 LC method laboratory values

 LC method consensus box encloses ± 2 MADe around consensus medians

 NIST values with corresponding Youden line 

026

110

128 139

186

211

5

10

15

20

25

5 10 15 20 25

25(OH)DTotal in SRM 968d L1"Vial D" (ng/mL)

25(OH)DTotal in SRM 968e L2 "Vial B" (ng/mL)

IA methods

LC methods
16

Correlation of 25(OH)D in SRM 968e L1, SRM 968e L2, SRM 968e L3, and SRM 968d L1 

with Clinical Ranges

The current guidance regarding 25(OH)D concentrations and human health (obtained from the NIH 

website) is presented in Table 3. 

Table 3. Serum 25-Hydroxyvitamin D [25(OH)D] Concentrations and Health [1]

ng/mL nmol/L Health Status

<12 <30 Associated with vitamin D deficiency, leading to rickets 

in infants and children and osteomalacia in adults

12–20 30-50 Generally considered inadequate for bone and overall 

health in healthy individuals

≥ 20 ≥ 50 Generally considered adequate for bone and overall 

health in healthy individuals

>50 >125 Emerging evidence links potentially adverse effects to 

such high levels, particularly >150 nmol/L (>60 ng/mL)

Table from http://ods.od.nih.gov/factsheets/vitamind#h4

[1] Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC: National Academy 

Press, 2010.

Graphical representations of the participant and NIST results for SRM 968e L1, SRM 968e L2, 

SRM 968e L3, and SRM 968d L1 overlaid with the clinical ranges for 25(OH)D from Table 3 are 

presented in Figure 5. 

Specific results as assessed from Figure 5 are summarized below:

SRM 968e L1

• The majority of the participant results are in the deficient 25(OH)D concentration range, but 

two are in the inadequate range.

• The NIST value (7.09 ng/mL ± 0.14 ng/mL) is in the deficient 25(OH)D concentration 

range.

SRM 968e L2

• The majority of the participant results are in the inadequate 25(OH)D concentration range, 

but some reported deficient as well as adequate concentration values.

• The NIST value (12.90 ng/mL ± 0.30 ng/mL) is in the inadequate 25(OH)D concentration 

range.

SRM 968e L3

• The range of participant results for SRM 968e L3 is larger than for the other materials.

• The majority of participant results are in the adequate 25(OH)D concentration range, but 

several also reported inadequate concentration values.

• The NIST value (19.90 ng/mL ± 0.40 ng/mL) is in the inadequate 25(OH)D concentration 

range.
17

SRM 968d L1

• The majority of the participant results are in the inadequate 25(OH)D concentration range, 

but some reported deficient as well as adequate concentration values.

• The NIST value (12.38 ng/mL ± 0.28 ng/mL) is in the inadequate 25(OH)D concentration 

range.

The consensus CV% of the participant results from all methods ranged from 14% to 19% for the 

study materials (Table 2). Large consensus variability has implications regarding the accuracy of 

25(OH)D measurements for the diagnosis of vitamin D status, particularly given the narrow ranges 

associated with vitamin D deficiency and inadequacy. 
18

Figure 5. 25(OH)DTotal levels in SRM 968e L1, SRM 968e L2, SRM 968e L3, and SRM 968d L1 

superimposed over clinically-relevant serum 25-hydroxyvitamin D (25(OH)DTotal) concentration

levels as reported by NIH (Table 3). The grey-shaded bars represent the ranges bound by the NIST 

values with ± estimated U95 uncertainty. 
19

Figure 5 (cont’d) 25(OH)DTotal levels in SRM 968e L1, SRM 968e L2, SRM 968e L3 and SRM 

968d L1 superimposed over clinically-relevant serum 25-hydroxyvitamin D (25(OH)DTotal) 

concentration levels as reported by NIH (Table 3). The grey-shaded bars represent the ranges bound 

by the NIST values with ± estimated U95 uncertainty. 
20

Appendix A-1. Summary of immunoassay methods used by participants. 

Laboratory 

Number IA Method Sample Preparation Detection

17 CLIA n/r n/r

86a CLIA n/r n/r

86b RIA n/r n/r

161 CLIA

Sample incubated for 30 min with anti-25(OH)D antibodies 

attached to paramagnetic particles in a buffer that dissociates 

25(OH)D from binding proteins. Magnetic separation and 

washing removes unbound reagents. Trigger reagent used to 

initiate the chemiluminescent reaction.

Relative light units (from 

luminometer) are compared 

to a stored master curve to 

determine the concentration 

of 25(OH)D

188 CLIA

Samples were thawed, swirled, and analyzed. Controls were 

diluted with phosphate-buffered saline (PBS) (100μL control + 

400 μL PBS).

n/r

191 RIA Samples were prepared as per kit protocol I

125 detection using Gamma 

counter

192 EIA n/r OD reading at 450 nm

196 CLIA The human serum samples were analyzed neat; calibration 

solutions were diluted 1:4 in water and analyzed. n/r

198b EIA n/r n/r

200 RIA Sample was extracted n/r

210a RIA Sample was extracted n/r

210b CLIA n/r n/r

213b EIA n/r n/r

214a RIA Sample was extracted n/r

214b CLIA n/r n/r

218a CLIA n/r n/r

222 CLIA n/r n/r

OD = optical density

n/r = not reported
21

Appendix A-2. Summary of LC-MSn methods reported by participants. 

Laboratory 

Number

Internal Standard 

(IS) Sample Preparation Chromatographic Conditions Detection: MRM ions

26 25(OH)D2-d 6 and 

25(OH)D3-d 6

Liquid-liquid extraction method

C18 column (50 x 2.1 mm); isocratic 

separation with 95% methanol, 5% 

water; flow 0.2 mL/min

25(OH)D2 413/355; 

25(OH)D3 401/365

56

25(OH)D2-d 3; 

25(OH)D3-d 6; 3-epi25(OH)D2-d 3

Samples were extracted with 

hexane, evaporated, then 

reconstituted with 69% methanol

PFP column (100 x 2.1 mm; 1.9 

μm); isocratic elution; flow 0.4 

mL/min

25(OH)D3 383/365; 

25(OH)D3-d 6 389/371; 

25(OH)D2 395/377; 

25(OH)D2-d 3 398/380

116 25(OH)D3 -d 6

Serum proteins were precipitated 

with methanol

LC column; isocratic separation with 

95% methanol, 5% water; flow 0.6 

mL/min; online SPE

25(OH)D3 383/211; 

25(OH)D3-d 6 389/211 ; 

25(OH)D2 395/269

119 25(OH)D3 -d 6

Serum (150 μL) proteins were 

precipitiated with methanol 

containing IS (200 μL), followed 

by hexane extraction (1 mL), 

evaporation, and reconstitution 

with methanol

C18 column (150 x 3.0 mm, 2.7 

μm); gradient with methanol and 

water (0.1% formic acid); flow 0.65 

mL/min

Orbitrap MS

Detection at [M+H]+

: 

25(OH)D3 401.34141; 

25(OH)D3-d 6 407.37907; 

25(OH)D2 413.34141

124

deuterated 

25(OH)D2 and 

deuterated 

25(OH)D3

Solid-phase extraction

Phenyl column (50 x 2.1 mm; 

1.7μm), gradient with 

methanol/water (both with 

ammonium acetate and formic acid)

n/r

128 n/r n/r n/r n/r

184 25(OH)D3 -d 6

Serum (200 μL) treated with 

acetonitrile containing IS (700 

μL); mixed, centrifuged, and 

filtered 

C18 column (100 x 2.1mm; 5μm); 

Linear gradient from 40% A (0.1% 

formic acid in water) and 60% B 

(0.1% formic acid/5 mmol/L 

ammonium acetate in methanol) 

98% B in 2 min

25(OH)D3 383/257; 

25(OH)D3-d 6 389/263; 

25(OH)D2 395/209

185 25(OH)D2-d 6 and 

25(OH)D3-d 6

Liquid-liquid extraction; 100 μL 

sample

C18 column; methanol/water 

gradient MRM

186 25(OH)D3 -d 6

Deproteinized with cold methanol; 

liquid/liquid extraction with hexane

C18 column (50 x 2.1 mm; 1.7μm), 

45° C, methanol/water gradient

25(OH)D3 401/159 (quant), 

401/383 (qual); 25(OH)D3-d 6 

407/159

187 n/r n/r n/r n/r

195 25(OH)D2-d 3 and 

25(OH)D3-d 6

Samples extracted then 

derivatized

LC column (30 x 2.1 mm); gradient 

with methanol/water n/r

197 25(OH)D3 -d 6

Precipitating agent added (200 μL 

with 20 ng IS) to each serum (200 

μL), calibrator and control sample 

followed by mixing, centrifugation, 

and analysis

C18 column (50 x 4.6 mm; 5 μm); 

flow 1.0 mL/min; column temp 45°C; 

gradient with water and methanol

n/r

198a 25(OH)D3 -d 6

Proteins precipitated with 

methanol, followed by hexane 

extraction, centrifugation, 

evaporation under N2, and 

reconstitution in methanol (0.1% 

formic acid)

C18 column (50 x 2.1 mm; 3.5 μm); 

isocratic elution with 85% methanol 

(0.1% formic acid); flow 0.5 mL/min

25(OH)D3 401/383, 401/365; 

25(OH)D2 413/395, 413/355; 

25(OH)D3-d 6 407/389, 

407/371

199 25(OH)D3-d 6 n/r n/r n/r

202 d 6-labeled 

compound Sample was extracted

C18 column (50 x 2.1 mm); gradient 

with 10 % acetonitrile (0.1% formic 

acid), 90% methanol; flow 0.3 

mL/min

n/r
22

209 25(OH)D3 -d 6

Proteins were precipitated with 

ZnSO4, followed by centrifugation 

and analysis

C8 column (50 x 2 mm; 5 μm); 

gradient with water/methanol; flow 

0.7 mL/min

25(OH)D3 383/229,383/211; 

25(OH)D3-d 6 389/211; 

25(OH)D2 395/269, 395/119

211 25(OH)D3-d 6

Extraction with acetonitrile 

containing IS followed by 

centrifugation

Column (33 x 4.6 mm; 3 μm); 

turboflow with methanol/water 

gradient

25(OH)D3 383/365 (quant), 

383/357 (qual); 25(OH)D2 

395/377 (quant), 395/209 

(qual)

212 25(OH)D3-d 6

Serum (100 μL) precipitated with 

5:95 methanol:acetonitrile (350 

μL) containing the deuterated 

internal standard.

C8 column (50 x 2mm; 3 μm); 

gradient starting with 60% 

acetonitrile (0.1% formic acid), 40% 

water (0.1% formic acid)

25(OH)D3 383/229,383/211; 

25(OH)D3-d 6 389/211; 

25(OH)D2 395/269, 395/119

215 25(OH)D3-d 6

Protein precipitation with 

methanol/isopropanol followed by 

liquid/liquid extraction with hexane

C18 column ( 50 x 2.1mm; 2.6 μm); 

gradient from 85% to 100% 

methanol; flow 0.25 mL/min

n/r

217 25(OH)D3-d 6

Protein precipitation with ZnSO4 in 

methanol followed by SPE 

extraction

C8 column (50 x 2.1 mm; 1.7μm); 

gradient of 70% to 98% methanol 

(with 0.1% formic acid); flow 0.4 

mL/min

25(OH)D3 401/159 (quant), 

401/383 (qual); 25(OH)D2 

413/88 (quant), 413/395 

(qual)

219 25(OH)D3-d 6

Samples were protein crashed in 

conjunction with internal standard 

addition, vortexed, centrifuged

Automated 2-D system

25(OH)D3 401/365; 

25(OH)D2 413/355; 

25(OH)D3-d 6 407/371

220 25(OH)D2-d 3 and 

25(OH)D3-d 6

Protein crash with 90% methanol, 

10% ZnSO4 and then acetonitrile 

(1% formic acid). Precipitated 

sample is passed through an SPE 

plate.

C18 column (20 x 2.1mm, 2.7μm); 

flow 1 mL/min

25(OH)D3 383/211 (quant), 

383/229 (qual); 25(OH)D2 

395/119 (quant), 395/269 

(qual); 25(OH)D3-d 6 389/211; 

25(OH)D2-d 3 398/272

221 25(OH)D3-d 6

Protein crash with 1% methanol in 

acetonitrile containing IS

CN column (50 x 3.0 mm; 1.8 μm); 

methanol/water gradient at 50 °C

25(OH)D3 383/211; 

25(OH)D3-d 6 389/211; 

25(OH)D2 395/209

MRM = multiple reaction monitoring

PFP = pentafluorophenyl

quant = quantitative ions

qual = qualitative ions

SPE = solid phase extraction

n/r = not reported
23

Appendix A-3. Summary of LC-UV methods used by participants.

Laboratory 

Number

Internal Standard 

(IS) Sample Preparation Chromatographic Conditions Wavelength

32 Proprietary

Samples were extracted with 

filtration; The controls were 

evaporated with a known ratio of 

internal standard, reconstituted, 

and injected

C18 column (300 x 3.9 mm; 4 μm); 

proprietary mobile phase; flow 

0.7mL/min

265 nm

110 n/r

Samples were extracted twice with 

hexane/methylene chloride (5:1), 

evaporated and reconstituted

Ultra-fast LC; gradient with 

acetonitrile/methanol (85:15) and 

isopropanol (100%)

n/r

139 Proprietary

The sample was extracted, 

centrifuged and injected directly 

onto LC column

Required reagents, column, 

controls and calibrators supplied in 

"kit" form.

264 nm

189 Added before 

extraction

Proteins were disrupted and 

precipitated; analytes were 

extracted using solid-phase 

extraction

LC column (150 x 4.6 mm); isocratic 

separation with commercial mobile 

phase; flow 0.7mL/min

265 nm

221 Laurophenone

Protein crash with acetonitrile 

(contaning IS), followed by 

extraction on C-18 sorbent, 

elution with methanol/acetonitrile, 

evaporation, and reconstitution 

with acetonitrile

CN column (150 x 4.6 mm; 3.5 μm); 

methanol/water/formic acid mobile 

phase; 50 °C

275 nm

n/r = not reported
24

Appendix B. Raw participant data for 25(OH)D2, 25(OH)D3 and 25(OH)DTotal in SRM 968e L1, SRM 968e L2, SRM 968e L3, 

and SRM 968d L1 and the control solutions, SRM 2972.

SRM 968e L1 SRM 968e L2 SRM 968e L3 SRM 968d L1 SRM 968e L1 SRM 968e L2 SRM 968e L3 SRM 968d L1 SRM 968e L1 SRM 968e L2 SRM 968e L3 SRM 968d L1

Lab Method Vial A Vial B Vial C Vial D Vial A Vial B Vial C Vial D Vial A Vial B Vial C Vial D 25(OH)D2 25(OH)D3

017 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 8.1 15.1 26.5 15.7 n/r n/r

026 LC-MS/MS <1.0 <1.0 <1.0 1.0 7.9 14.4 20.1 15.6 7.9 14.4 20.1 16.6 n/r n/r

032 LC-UV <5 <5 <5 <5 7.0 12.5 20.4 11.5 7.0 12.5 20.4 11.5 261.5 344.0

056 LC-MS/MS <1.2 <1.2 <1.2 <1.2 7.2 12.3 21.8 13.2 7.2 12.3 21.8 13.2 227.3 321.3

086a CLIA n/a n/a n/a n/a n/a n/a n/a n/a 8.0 16.0 28.0 17.0 n/r n/r

086b RIA n/a n/a n/a n/a n/a n/a n/a n/a 11.0 19.0 29.0 19.0 n/r n/r

110 LC-UV <4.0 <4.0 <4.0 <4.0 6.5 9.1 13.0 13.6 6.5 9.1 13.0 13.6 233.4 331.7

116 LC-MS/MS < 3.3 < 3.3 < 3.3 < 3.3 9.5 15.6 24.0 14.3 9.5 15.6 24.0 14.3 241.8 331.0

119 LC-MS < 1.0 < 1.0 < 1.0 < 1.0 9.4 14.8 21.8 14.6 9.4 14.8 21.8 14.6 243.2 352.6

124 LC-MS/MS <4.0 <4.0 <4.0 <4.0 10.1 14.8 24.4 13.9 10.1 14.8 24.4 13.9 n/r n/r

128 LC-MS/MS n/d n/d n/d n/d 11.2 17.8 27.2 18.1 11.2 17.8 27.2 18.1 n/r 333.5

139 LC-UV n/d n/d n/d n/d 10.1 17.1 25.3 17.9 10.1 17.1 25.3 17.9 241.5 330.5

161 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 6.9 15.0 28.5 17.4 n/r n/r

184 LC-MS/MS <1.0 <1.0 <1.0 <1.0 6.5 12.3 19.5 13.6 6.5 12.3 19.5 13.6 236.5 338.5

185 LC-MS/MS n/d n/d n/d n/d 8.8 16.1 24.6 14.8 8.8 16.1 24.6 14.8 238.6 334.8

186 LC-MS/MS n/d n/d n/d n/d 9.0 12.0 26.0 16.0 9.0 12.0 26.0 16.0 n/r 320.0

187 LC-MS/MS n/d n/d n/d n/d 7.5 14.4 21.9 13.4 7.5 14.4 21.9 13.4 235.0 337.0

188 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 11.8 20.3 27.8 15.2 401.5 381.5

189 LC-UV n/d n/d n/d n/d 12.3 16.6 30.4 n/r 12.3 16.6 30.4 n/r n/r 280.9

191 RIA n/a n/a n/a n/a n/a n/a n/a n/a 7.7 11.3 18.1 14.0 n/r n/r

192 EIA n/a n/a n/a n/a n/a n/a n/a n/a 7.5 17.9 27.1 20.1 n/r n/r

195 LC-MS/MS n/d n/d n/d n/d 7.3 13.3 21.1 12.7 7.3 13.3 21.1 12.7 242.0 330.0

196 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 7.6 14.9 25.2 15.9 210.4 452.0

197 LC-MS/MS <5 <5 <5 <5 7.4 13.7 21.4 13.8 7.4 13.7 21.4 13.8 234.3 342.5

198a LC-MS/MS <5.0 <5.0 <5.0 <5.0 9.6 17.2 22.9 15.2 9.6 17.2 22.9 15.2 245.5 308.6

198b EIA n/a n/a n/a n/a n/a n/a n/a n/a 9.0 17.0 34.0 16.0 n/r n/r

199 LC-MS/MS <4 <4 <4 <4 9.1 13.0 20.5 13.2 9.1 13.0 20.5 13.2 233.0 329.0

200 RIA n/a n/a n/a n/a n/a n/a n/a n/a 7.0 12.2 18.0 13.9 216.0 319.1

202 LC-MS/MS n/d n/d n/d n/d 7.8 14.1 23.1 13.6 7.8 14.1 23.1 13.6 241.0 334.5

209 LC-MS/MS <1.0 <1.0 <1.0 <1.0 7.6 14.3 20.8 12.8 7.6 14.3 20.8 12.8 240.6 332.8

210a RIA n/a n/a n/a n/a n/a n/a n/a n/a 12.9 19.7 23.5 18.9 209.4 299.1

210b CLIA n/a n/a n/a n/a n/a n/a n/a n/a 9.4 16.1 30.4 17.7 n/r n/r

211 LC-MS/MS n/d n/d n/d n/d 5.9 11.7 16.5 10.7 5.9 11.7 16.5 10.7 238.0 303.8

212 LC-MS/MS n/d n/d n/d n/d 8.1 13.4 22.9 13.4 8.1 13.4 22.9 13.4 248.8 341.7

213 EIA n/a n/a n/a n/a n/a n/a n/a n/a 9.3 14.0 18.6 17.7 n/r n/r

214a RIA n/a n/a n/a n/a n/a n/a n/a n/a 7.9 14.4 24.7 13.5 n/r n/r

214b CLIA n/a n/a n/a n/a n/a n/a n/a n/a 7.9 16.4 27.2 15.6 n/r n/r

215 LC-MS/MS 0.3 0.4 0.2 0.1 7.9 14.6 21.0 14.0 8.2 15.0 21.2 14.1 226.7 386.2

217 LC-MS/MS 0.4 0.3 0.3 0.1 8.0 14.4 21.6 14.0 8.4 14.7 21.9 14.1 220.8 394.3

218 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 5.5 15.1 25.2 14.1 n/r n/r

219 LC-MS/MS <4.0 <4.0 <4.0 <4.0 6.9 14.7 18.6 12.3 6.9 14.7 18.6 12.3 n/r n/r

220 LC-MS/MS <5.0 <5.0 <5.0 <5.0 7.5 13.8 20.0 13.1 7.5 13.8 20.0 13.1 232.4 324.4

221a LC-MS/MS n/d n/d n/d n/d 7.3 13.3 21.7 12.3 7.3 13.3 21.7 12.3 253.0 365.0

221b LC-UV n/d n/d n/d n/d 6.3 12.2 22.4 n/r 6.3 12.2 22.4 n/r 177.0 214.0

222 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 11.1 20.3 31.1 15.6 n/r n/r

NIST Value <0.5 <0.5 <0.5 <0.5 7.09 12.90 19.90 12.38 7.09 12.90 19.90 12.38 238.6 334.8

U 95 0.0 0.0 0.0 0.0 0.14 0.30 0.40 0.28 0.14 0.30 0.40 0.28 3.9 5.2

SRM 2972

25(OH)D2/D3 (ng/mL)

*n/a = not applicable (for immunoassay methods); n/r = not reported; n/d = not detected; < X = less than a reported quantitation limit of X

25(OH)D3 (ng/mL) 25(OH)DTotal 25(OH)D (ng/mL) 2 (ng/mL)