NIST/NIH Vitamin D Metabolites 

Quality Assurance Program

Report of Participant Results:

Winter 2012 Comparability Study 

(Exercise 5)

Mary Bedner

Katrice A. Lippa

Susan S.-C. Tai

http://dx.doi.org/10.6028/NIST.IR.7894

NISTIR 7894
NISTIR 7894

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program

Report of Participant Results:

Winter 2012 Comparability Study 

(Exercise 5)

Mary Bedner

Katrice A. Lippa

Susan S.-C. Tai

Chemical Sciences Division

Material Measurement Laboratory

http://dx.doi.org/10.6028/NIST.IR.7894

U.S. Department of Commerce

Rebecca M. Blank, Acting Secretary

National Institute of Standards and Technology

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

March 2013
ii

ABSTRACT

The National Institute of Standards and Technology (NIST) has 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 fifth exercise of this program, the 

Winter 2012 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 1950 Metabolites in Human Plasma, SRM 972a Vitamin D 

Metabolites in Human Serum (Level 2), 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 study, and laboratories were identified by code 

numbers known only to them. The results from this fifth study are reported along with a summary 

of the analytical methods used. 
1

OVERVIEW OF THE WINTER 2012 COMPARABILITY STUDY

For the Winter 2012 Comparability Study of VitDQAP (Exercise 5), 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 plasma or serum (study materials). In this study, vial 

A was SRM 1950 Metabolites in Human Plasma, vials B and D were duplicate samples of SRM 

972a Vitamin D Metabolites in Human Serum Level 2 (SRM 972a L2), and vial C was SRM 968d 

Fat-Soluble Vitamins, Carotenoids and Cholesterol in Human Serum Level 1 (SRM 968d L1). All 

materials consisted of blended human plasma or serum pools with endogenous 25(OH)D levels. 

There were a total of 51 participants and 57 datasets (six participants provided data for two different 

methods) in the Winter 2012 study. Seventeen of the datasets originated from immunoassay (IA) 

techniques, including three from enzyme immunoassay (EIA), eight from chemiluminescence 

immunoassay (CLIA), and six from radioimmunoassay (RIA). Appendix A-1 summarizes the

immunoassay methods used by the participants. Forty of the datasets originated from liquid 

chromatographic (LC) methods; of those, 32 were from LC with tandem mass spectrometric 

detection (LC-MS/MS), one was from LC-MS (orbitrap), and seven 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 are summarized in Appendix B. All datasets from the 

immunoassay methods reported single values for 25(OH)DTotal in SRM 1950, SRM 972a L2, and 

SRM 968d L1. LC participants provided values for 25(OH)D2, 25(OH)D3, as well as 25(OH)DTotal

in SRM 1950, SRM 972a L2, and SRM 968d L1. 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.

SRM 1950, SRM 972a L2, and SRM 968d L1 contain low levels of 25(OH)D2 (reported participant 

values ranging from 0.2 ng/mL to 1.7 ng/mL), and most of the LC labs indicated this analyte was 

below their quantitation limit of <1 ng/mL to <7 ng/mL. Therefore, the 25(OH)DTotal values

reported in Appendix B are the same as the 25(OH)D3 values in the serum and plasma materials for 

the majority of LC participants. 

Appendix B also provides the summarized results from the National Institute of Standards and 

Technology (NIST) for each of the serum materials. The 25(OH)D2 in SRM 968d L1 was below 

the quantitation limit (≈ 0.5 ng/mL) for the NIST method. 
2

WINTER 2012 COMPARABILITY STUDY 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 57 datasets received for the Winter 

2012 study, only 36 reported values for the ethanolic controls; of those, three were from 

immunoassay methods and 33 were from LC methods. 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%). The consensus results using robust statistics (i.e., median and MADe) were not 

calculated for the data from the IA methods because of the limited number of data reported.

The control materials were characterized at NIST using both gravimetry and LC-MS. Table 1

presents the NIST certified values with expanded uncertainties corresponding to 95% confidence

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

the data reporting sheet so that they could qualify their methods prior to analyzing the study 

samples. 
3

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.

SRM 2972 SRM 2972

25(OH)D2 25(OH)D3

Lab Method Value Lab Method Value

056 LC-MS/MS 230.6 2.0 056 LC-MS/MS 342.4

060 LC-MS/MS n/r 2.0 060 LC-MS/MS 372.0

062 RIA n/r 1.3 062 RIA 323.1

110 LC-UV 237.1 3.0 110 LC-UV 334.1

116 LC-MS/MS 245.1 2.0 116 LC-MS/MS 334.6

119 LC-MS 182.4 2.0 119 LC-MS 343.4

124 LC-MS/MS 47.0 2.0 124 LC-MS/MS 123.0

139 LC-UV 239.7 3.0 139 LC-UV 328.5

160a LC-MS/MS 170.0 2.0 160a LC-MS/MS 281.0

184 LC-MS/MS 240.5 2.0 184 LC-MS/MS 336.5

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

186 LC-MS/MS 238.0 2.0 186 LC-MS/MS 335.0

194 LC-MS/MS 240.5 2.0 194 LC-MS/MS 330.5

195 LC-MS/MS 241.0 2.0 195 LC-MS/MS 333.0

196 CLIA 198.6 1.1 196 CLIA 298.8

197 LC-MS/MS 234.0 2.0 197 LC-MS/MS 336.0

198a LC-MS/MS 207.4 2.0 198a LC-MS/MS 327.1

199 LC-MS/MS 245.0 2.0 199 LC-MS/MS 349.0

200 RIA 278.1 1.3 200 RIA 386.9

202 LC-MS/MS 241.5 2.0 202 LC-MS/MS 339.0

209 LC-MS/MS 248.7 2.0 209 LC-MS/MS 336.7

210a RIA 230.0 1.3 210a RIA 294.3

211 LC-MS/MS 240.4 2.0 211 LC-MS/MS 328.1

212 LC-MS/MS 231.5 2.0 212 LC-MS/MS 330.9

216 LC-MS/MS 258.0 2.0 216 LC-MS/MS 333.0

217 LC-MS/MS 233.8 2.0 217 LC-MS/MS 358.1

218b LC-MS/MS 240.9 2.0 218b LC-MS/MS 333.9

221a LC-MS/MS 249.7 2.0 221a LC-MS/MS 357.8

223 LC-MS/MS 241.2 2.0 223 LC-MS/MS 313.2

225 LC-MS/MS 235.9 2.0 225 LC-MS/MS 284.1

228a LC-MS/MS 240.0 2.0 228a LC-MS/MS 334.0

231 LC-UV 268.7 3.0 231 LC-UV 287.3

234 LC-MS/MS 246.0 2.0 234 LC-MS/MS 300.0

241 LC-MS/MS 212.0 2.0 241 LC-MS/MS 302.5

242 LC-MS/MS 245.0 2.0 242 LC-MS/MS 332.0

243 LC-UV 245.9 3.0 243 LC-UV 325.6

244 LC-MS/MS 222.0 2.0 244 LC-MS/MS 337.0

245 LC-UV 240.8 3.0 245 LC-UV 335.5

N 36 N 38

Median 240.2 Median 333.5

MADe 8.5 MADe 8.8

CV% 3.5 CV% 2.6

N 33 N 34

Median 240.4 254 Median 334.0

MADe 7.0 226 MADe 7.8

CV% 2.9 226 CV% 2.3

N 28 N 29

Median 240.2 Median 334.0

MADe 8.0 MADe 7.4

CV% 3.3 CV% 2.2

NIST Value 238.6 NIST Value 334.0

U 95 3.9 U 95 5.2

All 

methods

LC 

methods LC-MSn
4

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 (), and the results from the LC-based methods are displayed with 

closed black squares (■).

From the single reported values for all LC datasets, the consensus median and the consensus 

variability (2 × MADe) were determined (reported in Table 1). In Figure 1, the solid lines () 

represent the consensus median and the dashed lines (- - - - -) represent the approximate 95% 

confidence interval (2 × MADe) for the LC datasets; the laboratories with results that fall between 

the two dashed lines are within the consensus variability. 

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)). The 

consensus median value for the LC methods lies within the NIST expanded uncertainty range for 

both 25(OH)D2 and 25(OH)D3. 
5

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.
6

25(OH)D in SRM 1950, SRM 972a L2, and SRM 968d L1

A summary of the individual participant data for 25(OH)DTotal in samples SRM 1950 (vial A), SRM 

972a L2 (vials B & D), and SRM 968d L1 (vial C) is provided in Table 2. The summarized data 

also include the mean, standard deviation (SD), and percent relative standard deviation (%rSD) of 

the two reported values for SRM 972a L2.

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 N, the median value, the MADe, and the CV%.

Table 2 also presents the NIST results with approximated 95% confidence limits (U95) obtained for 

the three study materials.

For SRM 1950, 25(OH)DTotal is the sum of the NIST certified and reference values for 25(OH)D3

and 25(OH)D2, respectively, and the 95% confidence limit (U95) was approximated using the 

individual uncertainties reported for the two analytes. Details about the NIST methods and 

measurements are reported in the Certificate of Analysis for SRM 1950a. 

For SRM 972a L2, the NIST result for 25(OH)DTotal is the sum of the certified values for 25(OH)D3

and 25(OH)D2, and the 95% confidence limit (U95) incorporates the uncertainties for the two 

analytes. For SRM 968d L1, the NIST value for 25(OH)D3 was obtained using an LC-MS/MS 

reference measurement procedureb 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 SRM 968d L1 and was not included in 

the results for 25(OH)DTotal.

 a https://www-s.nist.gov/srmors/view_cert.cfm?srm=1950

b Tai, S. S.-C., Bedner, M. and Phinney, K.W. Anal. Chem. 2010 82, 1942-1948.
7

Table 2. Summary of participant data for 25(OH)DTotal (ng/mL) in SRM 1950, SRM 972a L2, and 

SRM 968d L1. SRM 1950 SRM 972a L2 SRM 968d L1 SRM 972a L2

Lab Method Vial A Vial B Vial C Vial D Mean SD %RSD

017 CLIA 24.5 19.2 13.7 17.9 18.5 0.9 5.0

026 LC-MS/MS 29.1 20.6 13.9 21.1 20.9 0.4 1.7

056 LC-MS/MS 25.3 19.2 13.1 19.7 19.5 0.4 1.8

060 LC-MS/MS 33.9 22.4 15.1 22.1 22.3 0.2 1.0

062 RIA 28.7 20.6 13.3 21.2 20.9 0.4 2.0

086a CLIA 32.9 21.6 14.5 23.3 22.5 1.2 5.4

086b RIA 33.0 24.0 17.0 28.0 26.0 2.8 11

110 LC-UV 24.4 19.9 38.9 17.9 18.9 1.4 7.5

116 LC-MS/MS 26.1 18.2 13.5 18.4 18.3 0.2 1.0

119 LC-MS 25.5 18.0 12.9 17.8 17.9 0.1 0.8

124 LC-MS/MS 26.3 18.7 15.5 20.3 19.5 1.1 5.8

139 LC-UV 29.6 23.0 22.1 21.8 22.4 0.8 3.8

160a LC-MS/MS 25.3 18.6 12.0 18.3 18.5 0.2 1.1

161 CLIA 26.7 19.9 17.0 18.5 19.2 1.0 5.2

180 RIA 25.1 16.1 12.1 17.6 16.9 1.1 6.5

184 LC-MS/MS 28.7 20.1 13.6 17.5 18.8 1.8 9.8

185a LC-MS/MS 27.0 19.3 14.7 32.8 26.1 9.5 37

185b CLIA 33.3 21.5 13.8 20.2 20.9 0.9 4.4

186 LC-MS/MS 13.0 8.0 7.0 12.0 10.0 2.8 28

188 CLIA 31.5 21.8 16.2 19.1 20.5 1.9 9.3

189 LC-UV 30.9 28.4 10.0 26.4 27.4 1.4 5.2

191 RIA 27.5 18.5 14.4 19.0 18.8 0.3 1.8

194 LC-MS/MS 26.5 20.7 11.4 17.4 19.1 2.3 12

195 LC-MS/MS 26.3 18.7 11.8 18.3 18.5 0.3 1.5

196 CLIA 28.0 18.5 15.3 19.4 19.0 0.6 3.4

197 LC-MS/MS 28.0 19.0 14.0 19.0 19.0 0.0 0.0

198a LC-MS/MS 29.9 20.7 14.4 20.5 20.6 0.1 0.7

198b EIA 31.6 20.3 15.3 20.2 20.3 0.1 0.3

199 LC-MS/MS 23.0 20.3 13.1 18.7 19.5 1.1 5.8

200 RIA 25.8 18.9 14.6 18.8 18.9 0.1 0.4

201 EIA 33.4 21.7 16.0 22.8 22.3 0.8 3.5

202 LC-MS/MS 27.2 21.9 14.1 20.6 21.3 0.9 4.3

209 LC-MS/MS 26.7 20.6 13.7 17.8 19.2 2.0 10

210a RIA 26.8 22.1 15.1 19.6 20.8 1.8 8.7

210b CLIA 30.4 18.9 15.1 20.2 19.6 0.9 4.7

211 LC-MS/MS 26.9 18.7 13.1 18.7 18.7 0.0 0.0

212 LC-MS/MS 30.8 22.8 14.5 21.0 21.9 1.3 5.8

215 LC-MS/MS 24.0 20.4 12.0 20.0 20.2 0.3 1.4

216 LC-MS/MS 33.5 25.6 17.9 26.7 26.2 0.8 3.0

217 LC-MS/MS 24.8 19.6 13.6 19.2 19.4 0.3 1.5

218a CLIA 26.4 17.7 14.6 17.2 17.5 0.4 2.0

218b LC-MS/MS 27.2 24.9 18.3 27.2 26.1 1.6 6.2

219 LC-MS/MS 26.2 19.8 13.4 19.5 19.7 0.2 1.1

220 LC-MS/MS 28.0 22.0 15.0 21.0 21.5 0.7 3.3

221a LC-MS/MS 26.1 17.5 12.8 19.8 18.7 1.6 8.7

221b LC-UV 25.3 15.9 51.0 20.6 18.3 3.3 18

223 LC-MS/MS 24.8 18.6 13.3 18.3 18.5 0.2 1.1

225 LC-MS/MS 32.4 24.8 20.1 21.1 23.0 2.6 11.4

228a LC-MS/MS 28.8 24.6 15.6 27.1 25.9 1.8 6.8

231 LC-UV 30.6 20.8 45.7 21.0 20.9 0.1 0.7

234 LC-MS/MS 25.2 18.9 13.7 18.0 18.5 0.6 3.4

236 CLIA 27.8 17.4 12.5 17.9 17.7 0.4 2.0

241 LC-MS/MS 25.8 19.5 12.5 19.2 19.4 0.2 1.1

242 LC-MS/MS 28.3 20.5 14.5 20.6 20.6 0.1 0.3

243 LC-UV 28.8 21.2 14.8 21.5 21.4 0.2 1.0

244 LC-MS/MS 27.0 18.0 14.0 18.0 18.0 0.0 0.0

245 LC-UV 38.5 33.2 15.9 27.2 30.2 4.2 13.9

N 57 57 57 57 57

Median 27.2 20.1 14.4 19.7 19.5

MADe 2.8 2.2 1.6 2.1 1.8

CV% 10 11 11 11 9.3

N 17 17 17 17 17

Median 28.0 19.9 14.6 19.4 19.6

MADe 3.6 2.4 1.2 1.3 1.9

CV% 13 12 8 6.9 9.8

N 40 40 40 40 40

Median 27.0 20.2 14.0 19.9 19.5

MADe 2.5 2.2 1.6 2.3 1.7

CV% 9.4 11 11 12 8.8

N 33 33 33 33 33

Median 26.7 19.8 13.7 19.5 19.5

MADe 2.1 1.6 1.2 1.8 1.5

CV% 7.8 8.2 8.7 9.1 7.6

NIST Value 25.3 18.9 12.4 18.9 18.9

U 95 0.8 0.4 0.3 0.4 0.4

All 

methods

IA 

methods 

LC 

methods LC-MSn

SRM 972a L2 Combined
8

For all participant datasets, the single reported values for 25(OH)DTotal in SRM 1950 and SRM 968d 

L1, and the average reported values (± 2 SD) for SRM 972a L2 are plotted in Figure 2. The results 

from immunoassay methods are displayed with closed red circles (), and the results from the LCbased methods are displayed with closed black squares (■). Each figure also has a legend that 

indicates which individual methods were used to obtain the reported values: CLIA, EIA, RIA, LCMSn

, or LC-UV.

From the average 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 2). For each of the 

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

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

For the IA data for material SRM 1950, 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

(Figure 2). 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 SRM 1950.

For the LC datasets for SRM 1950 and for both the LC and IA datasets for SRM 972a L2 and SRM 

968d L1, the laboratories with results that fall between the two dashed 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 2 are summarized below.

SRM 1950

• For the IA results, the data appear to be non-normally distributed, and the consensus variability 

is not well-described with a MADe estimation.

• 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 and overlaps the IA data range

SRM 972a L2

• For the IA results, all but one dataset are within the consensus variability range when the 

average results are considered.

• For the LC results, all but seven datasets are within the consensus variability range when the 

average results are considered.

• The consensus median values are comparable for both the IA results and LC results and are

slightly higher than the NIST expanded uncertainty range (grey-shaded bar).
9

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

ranges for both IA and LC.

SRM 968d L1

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

• For the LC results, nine datasets are outside of the consensus variability range (four LC-MSn

, 

five LC-UV).

• 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 and overlaps the consensus variability range for IA.

Overall, the results for the three study materials are consistent, with the majority of the participant 

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

high for the three materials, ranging from 9.3% to 11% when all methods are considered (Table 2). 

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

studies of the 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-forpurpose” 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 detected in all study materials but quantitated in SRM 972a L2 only (1.29 ng/mL ±

0.06 ng/mL). 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 that ranged from ≈ 3% to ≈ 6% of 25(OH)DTotal 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-epi-25(OH)D3. It is unclear how the presence of 3-epi-25(OH)D3 affects the 25(OH)DTotal

for immunoassay results.
10

Figure 2. Plots of the single reported values for 25(OH)DTotal in SRM 1950 and SRM 968d L1, and 

the average reported values (± 2 SD) for SRM 972a L2 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.
11

Figure 2 (cont’d). Plots of the single reported values for 25(OH)DTotal in SRM 1950 and SRM 

968d L1, and the average reported values (± 2 SD) for SRM 972a L2 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.
12

Figure 3 is a direct graphical comparison of the 25(OH)D results for the duplicate samples of

SRM 972a L2 (vial B and vial D). For the first Youden plot (Panel A), there are two blue 

consensus boxes, 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.

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

are not included in the consensus ranges and are highlighted with their laboratory code numbers 

(numbers 186, 180, 225, 245, 189, 216, 218b, 228, 086b and 185a). The NIST value for this 

material (18.9 ng/mL) is denoted with a red diamond symbol (), and the Youden line (y=x) 

centered on the NIST value is illustrated by a red line () across the magnitude of the y- and xaxis, respectively. The Youden line runs through both the IA and LC consensus boxes for these 

materials.

For the second Youden plot (Panel B), the results for SRM 972a L2 are evaluated with respect to a 

10% range relative to the NIST value for this material (18.9 ng/mL). Laboratory results that fall 

outside of this range are indicative of non-repeatable measurement performance for this material 

and are highlighted with their laboratory code numbers (numbers 186, 221b, 194, 209, 184, 225, 

245, 086b and 185a). In general, the combined results for vial B and vial D from these laboratories 

had a relative standard deviation ≥ 10% (Table 2). The relative distance of the individual 

laboratory results from the Youden line (y=x) is also indicative of the relative level of imprecision 

between the two results. 
13

.

Figure 3. Youden comparison plot of the 

results for 25(OH)DTotal in SRM 972a L2

“Vial B” and “Vial D” for all methods

Panel A: Data that fall outside the 

consensus boxes are labeled with their 

laboratory number.

Panel B: Data for the two replicates that 

are more than 10% discrepant are labeled 

with their laboratory number.

A

B

 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 

086b

180

185a

186

189

216

218b

225 228

245

5

10

15

20

25

30

35

40

5 10 15 20 25 30 35 40

25(OH)DTotal in SRM 972a L2 "Vial B" (ng/mL)

25(OH)DTotal in SRM 972a L2 "Vial D" (ng/mL)

IA methods

LC methods

 IA method laboratory values 

 LC method laboratory values

 NIST value with corresponding y = x and ± 10% lines 

086b

184 185a

186

194

209

221b

225

245

5

10

15

20

25

30

35

40

5 10 15 20 25 30 35 40

25(OH)DTotal in SRM 972a L2 "Vial B" (ng/mL)

25(OH)DTotal in SRM 972a L2 "Vial D" (ng/mL)
14

Correlation of 25(OH)D in SRM 1950, SRM 972a L2, and W012-3 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 single reported values for 25(OH)DTotal in SRM 1950 and SRM 

968d L1 and the mean values with error bars (representing the lab mean value ± 2 × SD) for 

25(OH)DTotal in SRM 972a L2 overlaid with the clinical ranges from Table 3 are presented in 

Figure 4. Specific results as assessed from Figure 4 are summarized below:

SRM 1950

• All but one of the participant results are in the adequate 25(OH)D concentration range.

• The NIST value (25.3 ng/mL ± 0.8 ng/mL) is in the adequate 25(OH)D concentration range.

SRM 972a L2

• The participant results are almost equally split between the inadequate and adequate 

25(OH)D concentration ranges.

• The NIST value (18.9 ng/mL ± 0.4 ng/mL) is in the inadequate 25(OH)D concentration 

range.

SRM 968d L1

• The range of participant results for SRM 968d L1 is larger than for the other materials.

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

several also reported deficient and adequate concentration values.

• The NIST value (12.4 ng/mL ± 0.3 ng/mL) is in the inadequate 25(OH)D concentration 

range.

The consensus CV% of the participant results from all methods was ≈ 10 % 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. 
15

Figure 4. 25(OH)DTotal levels in SRM 1950, SRM 972a L2, 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. The error bars represent 2 × SD of the duplicate results.
16

Figure 4 (cont’d) 25(OH)DTotal levels in SRM 1950, SRM 972a L2, 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. 
17

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

Laboratory 

Number IA Method Sample Preparation Detection

17 CLIA n/r n/r

62 RIA 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

180 RIA Samples prepared per manufacturer's instructions I

125 detection

185b CLIA n/r n/r

188 EIA None n/r

191 RIA Samples were prepared as per kit protocol I

125 detection using Gamma 

counter

196 CLIA The human serum samples were analyzed neat; calibration 

solutions were diluted 1:4 in a diluent mix and analyzed. n/r

198b EIA n/r n/r

200 RIA Sample was extracted n/r

201 EIA n/r n/r

210a RIA Sample was extracted with acetonitrile n/r

210b CLIA n/r n/r

218a CLIA Direct analysis n/r

236 CLIA Centrifuged n/r

n/r = not reported
18

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

60 25(OH)D3 -d 6

Serum proteins were precipitated 

with acetonitrile containing the IS, 

followed by centrifugation and 

injection of the supernatant

n/r

25(OH)D3 383/211; 

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

25(OH)D2 395/269

116 25(OH)D3 -d 6

Serum proteins were precipitated, 

followed by centrifugation and 

injection of the supernatant

2-dimensional LC-MS/MS

25(OH)D3 383/211; 

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

25(OH)D2 395/269

119 25(OH)D3 -d 6

IS was added and serum (150 μL) 

proteins were precipitiated with 

methanol (150 μL), followed by 

hexane extraction (1.5 mL), 

evaporation, and reconstitution 

with methanol (150 μL)

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 

25(OH)D3 401.33824; 

25(OH)D3-d 6 407.37907; 

25(OH)D2 413.33929

124 25(OH)D2-d 6 and 

25(OH)D3-d 6

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

160 25(OH)D2-d 6 and 

25(OH)D3-d 6

IS added to sample, followed by 

centrifugation, evaporation, 

reconstitution, mixing, and 

centrifugation (all using filter 

plate)

C18 column (50 x 2.1 mm); gradient 

with methanol/water; flow 0.7 

mL/min

MS/MS at m/z 413.3/337.3

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 mM 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

185a 25(OH)D2-d 6 and 

25(OH)D3-d 6

Liquid-liquid extraction; 40 μL 

sample

C18 column; methanol/water 

gradient MRM

186 25(OH)D3 -d 6

Sample was deproteinized in 

acetone, extracted with 3 volumes 

of hexanes, evaporated, and 

reconstituted in methanol/water

C18 column (50 x 2.1 mm; 1.7μm)

25(OH)D3 401/383 (quant), 

401/159 (qual); 25(OH)D3-d 6 

407/159

194 25(OH)D3 -d 6

Proteins precipitated with 

acetonitrile, top layer removed, 

evaporated, and reconstituted 

with methanol

C8 column (50 x 2mm) 25(OH)D2 395.3/119.0; 

25(OH)D3 383.4/211.3

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
19

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 n/r 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

209 25(OH)D3 -d 6

Proteins were precipitated with 

ZnSO4 in methanol

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)

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 IS

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 and ZnSO4; 

supernatant extracted using solid 

phase extraction

C18 column (50 x 2.1mm; 2.6 μm) 

column; gradient with water (0.1% 

formic acid, 5 mmol/L ammonium 

formate) and methanol (0.05% 

formic acid)

25(OH)D3 401/383; 

25(OH)D2 413/395; 

25(OH)D3-d 6 407/389

216 25(OH)D2-d 3 and 

25(OH)D3-d 6

Samples extracted using liquidliquid extraction then labeled with 

a derivatization reagent

C18 column (200 x 2.1 mm); 

gradient from 25% water (0.05% 

formic acid) to 50% acetonitrile 

(0.05% formic acid); flow 0.2 

mL/min

n/r

217 25(OH)D3-d 6

Protein precipitation with ZnSO4 in 

methanol followed by solid phase 

extraction

C8 column (50 x 2.1 mm; 1.7 μm); 

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)

218b 25(OH)D2-d 3 and 

25(OH)D3-d 3

Sample was extracted, filtered, 

centrifuged, etc.

Phenyl column (50 x 2.1 mm; 1.7 

μm); flow 0.45 mL/min

25(OH)D3 401; 

25(OH)D2 413

219 25(OH)D3-d 6

Samples were protein crashed in 

conjunction with internal standard 

addition, vortexed, centrifuged

Automated 2-dimensional 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); sample filtered 

then phospholipids removed with 

solid phase extraction

C18 column (20 x 2.1mm, 2.7μm); 

gradient with water and acetonitrile; 

flow 1 mL/min; column 40 °C

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

221a 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

223 25(OH)D2-d 3 and 

25(OH)D3-d 6

Protein precipitation with 

methanol followed by liquid-liquid 

extraction with cyclohexane:ethyl 

acetate (9:1); supernatant 

evaporated then derivatized with 4-

phenyl-1,2,4-triazoline-3,5-dione 

in acetonitrile

C18 column (100 x 2.1 mm; 1.7 

μm); gradient with water, 

acetonitrile, 0.01% formic acid

25(OH)D3 558/298; 

25(OH)D3-d 6 564/298; 

25(OH)D2 570/298; 

25(OH)D2-d 3 573/301
20

225 25(OH)D3-d 6 Extracted with hexane

C8 column (50 x 2.1 mm; 1.7 μm); 

gradient with water/methanol; flow 

0.4 mL/min

n/r

228 25(OH)D3-d 6

Proteins precipitated with 

isopropanol:acetonitrile:methanol 

(200:720:80), followed by 

centrifugation and removal of 

supernatant

C18 (30 x 3.0 mm); gradient with 

water (ammonium acetate/formic 

acid) amd methanol; flow 0.8 

mL/min

25(OH)D3 401; 

25(OH)D2 413

234 25(OH)D3-d 6

The samples are protein crashed 

using acetonitrile and separated 

from the protein

A turbo column is used for cleanup 

followed by a C18 analytical 

column; water and methanol mobile 

phase

25(OH)D3 383/365; 

25(OH)D2 395/209; 

25(OH)D3-d 6 389/211

241 25(OH)D3-d 6

Acetonitrile containing the IS (100 

μL) added to sample (50 μL) to 

precipate proteins, followed by 

mixing, sonication, and 

centrifugation

C8 column (50 x 2 mm; 3 μm); 

gradient starting with 50% 

methanol (0.1% formic acid), 50% 

water (0.1% formic acid)

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

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

395/119 (quant), 395/211 

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

242 25(OH)D3-d 6

Ethanol containing the IS (75 μL)

and acetonitrile (500 μL) added to 

sample (400 μL) to precipate 

proteins, followed by extraction 

with heptane, evaporation, and 

reconsitution in methanol

Reversed-phase column (150 x 2 

mm); gradient with 

acetonitrile/water; flow 0.35 mL/min

25(OH)D3 401/383; 

25(OH)D2 413/395; 

25(OH)D3-d 6 407/389

244 25(OH)D3-d 6

Protein precipitation followed by 

filtration

CN column; mobile phase 

consisting of distilled water/formic 

acid and methanol

25(OH)D2 395/269; 

25(OH)D3 383/211

MRM = multiple reaction monitoring

quant = quantitative ions

qual = qualitative ions

PFP = pentafluorophenyl

CN = cyano

n/r = not reported
21

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

Laboratory 

Number

Internal Standard 

(IS) Sample Preparation Chromatographic Conditions Wavelength

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%)

268 nm

139 Proprietary The sample was extracted, 

centrifuged and injected

Reversed-phase column, isocratic 

separation with proprietary mobile 

phase; flow 1 mL/min

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 

0.7 mL/min

265 nm

221b 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; 47 °C

275 nm

231 1-alpha(OH)D3

Samples were extracted with 

hexane/dichloromethane, 

evaporated and reconstituted with 

mobile phase (phosphate 

buffer/acetonitrile)

Reversed-phase column (250 x 4.5 

mm; 5μm), isocratic separation with 

14% phosphate buffer, 86% 

acetonitrile; flow 1.2 mL/min

265 nm

243 Laurophenone

Reagent 1 containing the 

ethanolic IS (400 μL) added to 

sample (400 μL), followed by 

vortexing the precipitation reagent 

(500 μL) and sampling of the 

supernatant

Reversed-phase column (150 x 3 

mm); isocratic separation with 65% 

acetonitrile, 35% water; flow 1 

mL/min

264 nm

245 Proprietary

Precipant added to sample, 

followed by addition of IS, mixing, 

and centrifugation.

Flow 1 mL/min 264 nm

n/r = not reported

CN = cyano
22

Appendix B. Raw participant data and NIST results for 25(OH)D2, 25(OH)D3 and 25(OH)DTotal in SRM 1950, SRM 972a L2, 

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

SRM 1950 SRM 972a L2 SRM 968d L1 SRM 972a L2 SRM 1950 SRM 972a L2 SRM 968d L1 SRM 972a L2 SRM 1950 SRM 972a L2 SRM 968d L1 SRM 972a L2

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 24.5 19.2 13.7 17.9 n/r n/r

026 LC-MS/MS <1.0 1.0 <1.0 <1.0 29.1 19.6 13.9 21.1 29.1 20.6 13.9 21.1 n/r n/r

056 LC-MS/MS 0.5 0.7 n/d 0.9 24.9 18.5 13.1 18.8 25.3 19.2 13.1 19.7 230.6 342.4

060 LC-MS/MS < 2 < 2 < 2 < 2 33.9 22.4 15.1 22.1 33.9 22.4 15.1 22.1 n/r 372.0

062 RIA n/a n/a n/a n/a n/a n/a n/a n/a 28.7 20.6 13.3 21.2 n/r 323.1

086a CLIA n/a n/a n/a n/a n/a n/a n/a n/a 32.9 21.6 14.5 23.3 n/r n/r

086b RIA n/a n/a n/a n/a n/a n/a n/a n/a 33.0 24.0 17.0 28.0 n/r n/r

110 LC-UV <4 <4 <4 <4 22.2 18.1 34.7 16.1 24.4 19.9 38.9 17.9 237.1 334.1

116 LC-MS/MS < 3.3 < 3.3 < 3.3 < 3.3 26.1 18.2 13.5 18.4 26.1 18.2 13.5 18.4 245.1 334.6

119 LC-MS n/d n/d n/d n/d 25.5 18.0 12.9 17.8 25.5 18.0 12.9 17.8 182.4 343.4

124 LC-MS/MS <4.0 <4.0 <4.0 <4.0 26.3 18.7 15.5 20.3 26.3 18.7 15.5 20.3 47.0 123.0

139 LC-UV n/a n/a n/a n/a 29.6 23.0 22.1 21.8 29.6 23.0 22.1 21.8 239.7 328.5

160a LC-MS/MS <1.0 <1.0 <1.0 <1.0 25.3 18.6 12.0 18.3 25.3 18.6 12.0 18.3 170.0 281.0

161 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 26.7 19.9 17.0 18.5 n/r n/r

180 RIA n/a n/a n/a n/a n/a n/a n/a n/a 25.1 16.1 12.1 17.6 n/r n/r

184 LC-MS/MS <1.0 <1.0 <1.0 <1.0 28.7 20.1 13.6 17.5 28.7 20.1 13.6 17.5 240.5 336.5

185a LC-MS/MS n/d n/d n/d n/d 27.0 19.3 14.7 32.8 27.0 19.3 14.7 32.8 238.6 334.8

185b CLIA n/a n/a n/a n/a n/a n/a n/a n/a 33.3 21.5 13.8 20.2 n/r n/r

186 LC-MS/MS n/d n/d n/d n/d 13.0 8.0 7.0 12.0 13.0 8.0 7.0 12.0 238.0 335.0

188 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 31.5 21.8 16.2 19.1 n/r n/r

189 LC-UV n/d n/d n/d n/d 30.9 28.4 10.0 26.4 30.9 28.4 10.0 26.4 n/r n/r

191 RIA n/a n/a n/a n/a n/a n/a n/a n/a 27.5 18.5 14.4 19.0 n/r n/r

194 LC-MS/MS <7.0 <7.0 <7.0 <7.0 26.5 20.7 11.4 17.4 26.5 20.7 11.4 17.4 240.5 330.5

195 LC-MS/MS n/d n/d n/d n/d 26.3 18.7 11.8 18.3 26.3 18.7 11.8 18.3 241.0 333.0

196 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 28.0 18.5 15.3 19.4 198.6 298.8

197 LC-MS/MS <5 <5 <5 <5 28.0 19.0 14.0 19.0 28.0 19.0 14.0 19.0 234.0 336.0

198a LC-MS/MS <5.0 <5.0 <5.0 <5.0 29.9 20.7 14.4 20.5 29.9 20.7 14.4 20.5 207.4 327.1

198b EIA n/a n/a n/a n/a n/a n/a n/a n/a 31.6 20.3 15.3 20.2 n/r n/r

199 LC-MS/MS <2 <2 <2 <2 23.0 20.2 13.1 18.7 23.0 20.3 13.1 18.7 245.0 349.0

200 RIA n/a n/a n/a n/a n/a n/a n/a n/a 25.8 18.9 14.6 18.8 278.1 386.9

201 EIA n/a n/a n/a n/a n/a n/a n/a n/a 33.4 21.7 16.0 22.8 n/r n/r

202 LC-MS/MS n/d n/d n/d n/d 27.2 21.9 14.1 20.6 27.2 21.9 14.1 20.6 241.5 339.0

209 LC-MS/MS <1.0 <1.0 <1.0 <1.0 26.7 20.6 13.7 17.8 26.7 20.6 13.7 17.8 248.7 336.7

210a RIA n/a n/a n/a n/a n/a n/a n/a n/a 26.8 22.1 15.1 19.6 230.0 294.3

210b CLIA n/a n/a n/a n/a n/a n/a n/a n/a 30.4 18.9 15.1 20.2 n/r n/r

211 LC-MS/MS n/d n/d n/d n/d 26.9 18.7 13.1 18.7 26.9 18.7 13.1 18.7 240.4 328.1

212 LC-MS/MS n/d <4 n/d <4 30.8 22.8 14.5 21.0 30.8 22.8 14.5 21.0 231.5 330.9

215 LC-MS/MS n/d 0.4 n/d 0.4 24.0 20.0 12.0 19.6 24.0 20.4 12.0 20.0 n/r n/r

216 LC-MS/MS 0.4 0.7 0.2 1.0 33.1 24.9 17.7 25.7 33.5 25.6 17.9 26.7 258.0 333.0

217 LC-MS/MS < 2 < 2 < 2 < 2 24.8 19.6 13.6 19.2 24.8 19.6 13.6 19.2 233.8 358.1

218a CLIA n/a n/a n/a n/a n/a n/a n/a n/a 26.4 17.7 14.6 17.2 n/r n/r

218b LC-MS/MS n/d n/d n/d n/d 27.2 24.9 18.3 27.2 27.2 24.9 18.3 27.2 240.9 333.9

219 LC-MS/MS <4.0 <4.0 <4.0 <4.0 26.2 19.8 13.4 19.5 26.2 19.8 13.4 19.5 n/r n/r

220 LC-MS/MS <5.0 <5.0 <5.0 <5.0 28.0 22.0 15.0 21.0 28.0 22.0 15.0 21.0 n/r n/r

221a LC-MS/MS n/d n/d n/d n/d 26.1 17.5 12.8 19.8 26.1 17.5 12.8 19.8 249.7 357.8

221b LC-UV n/d n/d n/d n/d 25.3 15.9 51.0 20.6 25.3 15.9 51.0 20.6 n/r n/r

223 LC-MS/MS <5 <5 <5 <5 24.8 18.6 13.3 18.3 24.8 18.6 13.3 18.3 241.2 313.2

225 LC-MS/MS <5.0 <5.0 <5.0 <5.0 32.4 24.8 20.1 21.1 32.4 24.8 20.1 21.1 235.9 284.1

228a LC-MS/MS 0.8 1.5 0.4 1.7 28.1 23.1 15.2 25.4 28.8 24.6 15.6 27.1 240.0 334.0

231 LC-UV n/d n/d 33.0 n/d 30.6 20.8 12.7 21.0 30.6 20.8 45.7 21.0 268.7 287.3

234 LC-MS/MS <3 <3 <3 <3 25.2 18.9 13.7 18.0 25.2 18.9 13.7 18.0 246.0 300.0

236 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 27.8 17.4 12.5 17.9 n/r n/r

241 LC-MS/MS 0.4 0.9 n/d 0.9 25.4 18.6 12.5 18.3 25.8 19.5 12.5 19.2 212.0 302.5

242 LC-MS/MS n/d n/d n/d n/d 28.3 20.5 14.5 20.6 28.3 20.5 14.5 20.6 245.0 332.0

243 LC-UV n/d n/d n/d n/d 28.8 21.2 14.8 21.5 28.8 21.2 14.8 21.5 245.9 325.6

244 LC-MS/MS <5 <5 <5 <5 27.0 18.0 14.0 18.0 27.0 18.0 14.0 18.0 222.0 337.0

245 LC-UV n/d n/d n/d n/d 38.5 33.2 15.9 27.2 38.5 33.2 15.9 27.2 240.8 335.5

NIST Value 0.52 0.81 <0.5 0.81 24.8 18.1 12.4 18.1 25.3 18.9 12.4 18.9 238.6 334.8

U 95 0.17 0.06 0.00 0.06 0.8 0.4 0.3 0.4 0.8 0.4 0.3 0.4 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)