NISTIR 7895

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Summer

2012 Comparability Study

(Exercise 6)

Katrice A. Lippa

Mary Bedner

http://dx.doi.org/10.6028/NIST.IR.7895
NISTIR 7895

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Summer

2012 Comparability Study

(Exercise 6)

Katrice A. Lippa

Mary Bedner 

Chemical Sciences Division

Material Measurement Laboratory

http://dx.doi.org/10.6028/NIST.IR.7895

March 2013

U.S. Department of Commerce 

Rebecca Blank, Acting Secretary

National Institute of Standards and Technology 

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

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 sixth exercise of this program, 

the Summer 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 968e Fat-Soluble Vitamins, Carotenoids, and 

Cholesterol in Human Serum Level 1 and SRM 972a Vitamin D Metabolites in Human Serum 

Level 1 and Level 3. 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 sixth study are reported along with a summary of the analytical 

methods used.
1

OVERVIEW OF THE SUMMER 2012 COMPARABILITY STUDY

For the Summer 2012 Comparability Study (Exercise 6) 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 results for single measurements of each of these 

solutions. In addition, participants were asked to determine 25-hydroxyvitamin D in four samples 

of human serum (study materials). Individual concentration values for 25(OH)D2, 25(OH)D3, and 

3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3) were requested along with a total concentration of 

25-hydroxyvitamin D (25(OH)DTotal = 25(OH)D2 + 25(OH)D3) for each of four samples A, B, C, 

and D. In this study, A, B, C, and D were all blended human serum pools with endogenous 

25(OH)D levels. Vial A was SRM 972a Vitamin D Metabolites in Human Serum Level 1 (SRM 

972a L1), and vial B was SRM 972a Level 3 (SRM 972a L3). Vials C and D were duplicate 

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

(SRM 968d L1).

There were a total of 48 participants and 56 datasets (eight participants provided data for two 

different methods) in the Summer 2012 study. Eighteen of the datasets originated from 

immunoassay (IA) techniques, including three from enzyme immunoassay (EIA), 11 from 

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

summarizes the IA methods used by the participants. Thirty-eight of the datasets originated from 

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

detection (LC-MSn), and six 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. 

The raw data received from all participants are summarized in Appendices B-1 and B-2. All 

datasets from the immunoassay methods reported single values for 25(OH)DTotal in SRM 972a L1, 

SRM 972a L3, and SRM 968d L1. LC participants provided values for 25(OH)D2, 25(OH)D3, and 

3-epi-25(OH)D3 as well as 25(OH)DTotal in SRM 972a L1, SRM 972a L3, 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.

Both SRM 972a L1 and SRM 968d L1 contain low levels of 25(OH)D2 (reported values ranging 

from 0.2 ng/mL to 0.7 ng/mL), and most of the LC labs indicated this analyte was below their 

quantitation limit of <1 ng/mL to <7 ng/mL. Conversely, SRM 972a L3 has a high level of 

25(OH)D2, and all but two of the LC participants reported values; in addition, one LC participant 

reported that the 25(OH)D2 was below their detection limit of 4 ng/mL. In addition, two LC 

participants provided values for 3-epi-25(OH)D3 in all three materials, one participant provided a 

value for SRM 972a L3 only, and two labs indicated that this analyte was below their quantitation 

limit of 2 ng/mL and 7 ng/mL in all three materials (Appendix B-2). 

Appendices B-1 and B-2 also provide 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; 3-epi-25(OH)D3 was detected 

in SRM 968d L1 but not quantitated.
2

SUMMER 2012 COMPARABILITY STUDY RESULTS AND DISCUSSION

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

For the Summer 2012 study, the control solutions were only provided to participants who requested 

them on their enrollment forms. A summary of the participant data for 25(OH)D2 and 25(OH)D3 in 

the SRM 2972 control solutions is provided in Table 1. The majority of the datasets received for 

the Summer 2012 study were from LC methods, and many participants using IA methods did not 

request the calibration solutions because of compatibility issues with their assays. 

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 community results were not calculated for the data from the IA methods 

because of the limited number of data reported (N = 2).

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

Lab Method Value Lab Method Value

030 RIA 257.0 1.3 030 RIA 408.5

056 LC-MS/MS 246.7 2.0 056 LC-MS/MS 346.3

060 LC-MS/MS 210.1 2.0 060 LC-MS/MS 348.1

110 LC-UV 239.2 3.0 110 LC-UV 335.2

128 LC-MS/MS n/r 2.0 128 LC-MS/MS 332.0

139 LC-UV 256.0 3.0 139 LC-UV 335.0

183b CLIA 262.4 1.1 183b CLIA 336.8

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

194 LC-MS/MS 240.5 2.0 194 LC-MS/MS 335.0

197 LC-MS/MS 238.0 2.0 197 LC-MS/MS 343.0

198a LC-MS/MS 228.2 2.0 198a LC-MS/MS 324.1

199 LC-MS/MS 249.4 2.0 199 LC-MS/MS 329.9

202 LC-MS/MS 246.0 2.0 202 LC-MS/MS 345.0

211 LC-MS/MS 229.0 2.0 211 LC-MS/MS 280.0

216 LC-MS/MS 229.9 2.0 216 LC-MS/MS 343.3

218b LC-MS/MS 239.1 2.0 218b LC-MS/MS 336.0

228a LC-MS/MS 217.3 2.0 228a LC-MS/MS 331.5

231 LC-UV 245.3 3.0 231 LC-UV 294.5

242 LC-MS/MS 239.4 2.0 242 LC-MS/MS 333.4

243 LC-UV 240.6 3.0 243 LC-UV 331.7

244 LC-MS/MS 215.0 2.0 244 LC-MS/MS 331.0

248 LC-MS/MS 238.6 2.0 248 LC-MS/MS 334.8

249 LC-MS/MS 238.4 2.0 249 LC-MS/MS 324.2

250 LC-MS/MS 281.6 2.0 250 LC-MS/MS 382.0

253 LC-MS/MS 235.5 2.0 253 LC-MS/MS 307.0

N 24 N 25

Median 239.2 Median 334.8

MADe 10.7 MADe 7.3

CV% 4.5 CV% 2.2

N 22 N 23

Median 238.9 Median 334.8

MADe 10.1 MADe 7.3

CV% 4.2 CV% 2.2

N 18 N 19

Median 238.5 Median 334.8

MADe 11.6 MADe 12.2

CV% 4.9 CV% 3.6

NIST Value 238.6 NIST Value 334.0

U 95 3.9 U 95 5.2

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

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 certified values with ± U95 expanded uncertainty). 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 972a L1, SRM 972a L3, and SRM 968d L1

A summary of the individual participant data for 25(OH)DTotal in SRM 972a L1 (vial A), SRM 972a 

L3 (vial B), and SRM 968d L1 (vials C and D) is provided in Table 2. The summarized data also 

include the average (mean), standard deviation (SD), and percent relative standard deviation 

(%rSD) of the two reported values for SRM 968d L1.

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 for the three study materials. For SRM 972a L1 and SRM 

972a L3, the NIST result is the sum of the certified values for 25(OH)D3 and 25(OH)D2 with the 

corresponding 95% confidence limits (U95). For SRM 968d L1, the NIST value for 25(OH)D3 was 

obtained using an LC-MS/MS reference measurement procedurea recognized by the JCTLM 

(N = 8), and the U95 confidence interval includes components for both measurement variability 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 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 972a L1, SRM 972a L3, 

and SRM 968d L1. SRM 972a L1 SRM 972a L3 SRM 968d L1 SRM 968d L1

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

017 CLIA 28.1 27.7 14.0 14.7 14.4 0.5 3.4

026 LC-MS/MS 31.0 33.5 12.7 13.4 13.1 0.5 3.8

030 RIA 32.4 26.7 15.8 14.4 15.1 1.0 6.6

056 LC-MS/MS 30.9 34.8 12.6 12.3 12.5 0.2 1.7

060 LC-MS/MS 37.1 42.2 15.3 14.6 15.0 0.5 3.3

086a CLIA 27.4 26.8 14.4 15.7 15.1 0.9 6.1

086b RIA 32.0 40.0 17.0 17.0 17.0 0.0 0.0

110 LC-UV 21.5 23.3 14.3 17.9 16.1 2.5 16

116 LC-MS/MS 35.1 37.9 15.4 15.5 15.5 0.1 0.5

128 LC-MS/MS 37.0 24.1 15.6 14.1 14.9 1.1 7.1

139 LC-UV 32.0 34.7 13.0 13.6 13.3 0.4 3.2

180 RIA 32.8 34.8 13.7 16.0 14.8 1.6 11

183b CLIA 28.0 25.0 13.8 13.9 13.9 0.1 0.5

185a LC-MS/MS 32.1 38.0 14.4 12.3 13.4 1.5 11.1

187 LC-MS/MS 32.7 33.1 13.1 12.9 13.0 0.1 1.1

188 CLIA 31.4 29.4 12.9 14.1 13.5 0.9 6.5

189 LC-UV 37.7 14.0 12.0 10.4 11.2 1.1 10

194 LC-MS/MS 32.3 34.1 12.9 13.1 13.0 0.1 1.1

196 CLIA 31.6 29.2 14.5 15.6 15.1 0.8 5.2

197 LC-MS/MS 32.0 36.3 13.8 14.3 14.1 0.4 2.5

198a LC-MS/MS 29.8 31.2 12.9 15.2 14.1 1.6 12

198b EIA 28.2 27.6 12.2 14.1 13.2 1.3 10

199 LC-MS/MS 30.7 33.7 12.7 12.6 12.7 0.1 0.6

202 LC-MS/MS 33.4 35.3 12.9 12.7 12.8 0.1 1.1

209 LC-MS/MS 31.7 39.6 11.9 12.8 12.4 0.6 5.2

210a RIA 31.4 31.3 12.6 12.8 12.7 0.1 1.2

210b CLIA 26.8 26.4 13.2 13.7 13.5 0.4 2.6

211 LC-MS/MS 31.5 34.1 12.3 11.7 12.0 0.4 3.5

212 LC-MS/MS 35.4 44.2 14.7 15.1 14.9 0.3 1.9

213a CLIA 27.5 25.0 3.7 3.7 3.7 0.0 0.8

213b EIA 28.2 23.8 12.9 12.2 12.5 0.5 4.3

215 LC-MS/MS 32.4 32.4 12.0 13.6 12.8 1.1 8.8

216 LC-MS/MS 30.5 33.4 13.2 12.8 13.0 0.2 1.9

217 LC-MS/MS 30.5 34.1 12.4 12.6 12.5 0.1 1.1

218a CLIA 29.8 29.2 13.7 14.7 14.2 0.7 5.0

218b LC-MS/MS 30.4 41.0 14.2 14.5 14.4 0.2 1.5

219 LC-MS/MS 30.0 33.6 12.4 12.7 12.6 0.2 1.7

220 LC-MS/MS 35.0 37.0 14.0 14.0 14.0 0.0 0.0

221a LC-MS/MS 27.9 31.0 11.6 12.9 12.3 0.9 7.5

221b LC-UV 24.4 28.0 25.0 24.7 24.9 0.2 0.9

222 CLIA 33.1 35.3 13.5 12.3 12.9 0.8 6.6

228a LC-MS/MS 45.2 51.4 17.3 17.9 17.6 0.4 2.4

228b CLIA 30.5 26.7 3.2 3.1 3.2 0.0 1.1

231 LC-UV 26.6 36.9 14.8 14.0 14.4 0.6 3.9

234 LC-MS/MS 34.5 39.2 13.4 14.7 14.1 0.9 6.5

241 LC-MS/MS 30.5 33.7 13.2 12.6 12.9 0.4 3.3

242 LC-MS/MS 28.9 37.8 13.4 12.4 12.9 0.7 5.3

243 LC-UV 30.1 38.8 13.6 12.3 13.0 0.9 7.0

244 LC-MS/MS 29.0 34.0 12.0 11.0 11.5 0.7 6.1

247a CLIA 38.1 37.1 17.7 14.2 16.0 2.5 16

247b EIA 31.3 30.5 16.3 16.0 16.1 0.2 1.4

248 LC-MS/MS 31.7 41.1 14.0 14.2 14.1 0.1 1.0

249 LC-MS/MS 30.0 35.0 13.7 11.4 12.6 1.6 13

250 LC-MS/MS 37.0 39.2 13.1 15.2 14.2 1.5 10

251 LC-MS/MS 31.5 33.6 15.2 14.7 15.0 0.4 2.4

253 LC-MS/MS 31.0 35.7 12.9 13.3 13.1 0.3 2.2

N 56 56 56 56 56

Median 31.3 33.9 13.4 13.8 13.4

MADe 2.1 5.9 1.2 1.6 1.3

CV% 6.6 17 8.9 12 9.5

N 18 18 18 18 18

Median 30.9 28.5 13.7 14.2 14.0

MADe 3.1 3.1 1.2 2.1 1.6

CV% 9.9 11 8.7 15 11

N 38 38 38 38 38

Median 31.5 34.8 13.2 13.4 13.1

MADe 2.2 3.4 1.2 1.4 1.3

CV% 6.8 9.8 9.0 11 9.9

N 32 32 32 32 32

Median 31.6 34.9 13.1 13.2 13.0

MADe 1.7 2.9 1.1 1.3 1.1

CV% 5.4 8.3 8.3 10 8.3

NIST Value 29.3 33.2 12.4 12.4 12.4

U 95 1.1 0.6 0.3 0.3 0.3

All 

methods

IA 

methods 

SRM 968d L1 Combined

LC 

methods LC-MSn
8

For all participant datasets, the single reported values for 25(OH)DTotal in SRM 972a L1 and 

SRM 972a L3 and the average reported values (± 2 SD) for SRM 968d L1 are plotted in Figure 2. 

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 (■). Each figure also has a 

legend that indicates which individual methods were used to obtain the reported values: IA, 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 approximate 95% confidence intervals (2 × MADe). 

For the IA data for SRM 972a L1, the consensus variability based on MADe is an overestimation of 

the 95% confidence limits about the median (Figure 2). This stems from the non-Gaussian data

distribution that contributes to a relatively wide distribution of central 50% of this data, resulting in 

a large MADe. 

For the LC datasets for SRM 972a L1 and for both the LC and IA datasets for SRM 972a L3and 

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 972a L1

• 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, eight datasets are outside of the consensus variability range (four LC-MSn

, 

four LC-UV).

• The consensus median value for the LC results is slightly higher than the consensus median 

value for the IA results; both LC and IA median values are ≈ 5% higher than the NIST 

expanded uncertainty range (grey-shaded bar).

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

ranges both for LC and IA results. 

SRM 972a L3

• For the IA results, four datasets are outside the consensus variability range.

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

, two 

LC-UV).

• The consensus median value for the IA results is considerably lower (≈ 25%) than the consensus 

median value for the LC results; the IA median value is ≈ 15% lower than the NIST expanded 

uncertainty range (grey-shaded bar) whereas the LC median value is ≈ 5% higher than the NIST 

expanded uncertainty range.
9

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

ranges for both IA and LC results.

SRM 968d L1

• For the IA results, two mean values are outside of the consensus variability range.

• For the LC results, three mean values are outside of the consensus variability range (one LCMSn

, two LC-UV).

• The consensus median value for the IA results is marginally higher (≈ 5%) 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 both LC and IA.

For SRM 972a L1 and SRM 968d L1, the majority of the participant values for 25(OH)DTotal are 

higher than the NIST value, regardless of technique used (IA or LC) (Figure 2). In addition, the 

consensus variability is similar for those two materials (≈ 7% to 10%) when the results from all 

methods are considered (Table 2). However, for SRM 972a L3, the majority of the LC results are 

higher than the NIST value, whereas the majority of the IA results are lower than the NIST value 

(Figure 2); the consensus variability is 17% for SRM 972a L3 when the results for all methods are 

considered (Table 2). The difference in results for SRM 972a L1 and SRM 968d L1 versus SRM 

972a L3 is most likely attributable to the large contribution of 25(OH)D2 to 25(OH)DTotal in the 

latter material, with the IA methods underrepresenting 25(OH)D2.

For SRM 972a L1, the 3-epi-25(OH)D3 is also a significant vitamin D metabolite with a 

concentration of 1.84 ng/mL ± 0.08 ng/mL, corresponding to ≈ 6% of the 25(OH)D3 concentration 

of 28.8 ng/mL ± 1.1 ng/mL (NIST values). Three of the LC-MS participants also reported values 

for 3-epi-25(OH)D3 (1.5 ng/mL, 1.9 ng/mL and 2.1 ng/mL) in SRM 972a L1 (Appendix B-2). 

Likewise, the 3-epi-25(OH)D3 also has a significant concentration of 1.18 ng/mL ± 0.13 ng/mL in 

SRM 972a L3, or ≈ 6% of the 25(OH)D3 concentration of 19.8 ± 0.5 ng/mL (NIST values), and two 

LC labs also provided values for this metabolite (0.7 ng/mL and 1.4 ng/mL). For the methods 

reported by many LC participants (Appendix A-2, A-3), the 3-epi-25(OH)D3 coelutes with 

25(OH)D3 and is detected by the same multiple reaction monitoring (MRM) ions in MS/MS and 

absorbance wavelength in UV, leading to a positive bias in the 25(OH)DTotal results. It is unclear 

how the presence of 3-epi-25(OH)D3 affects the 25(OH)DTotal for immunoassay results. Given that 

the consensus median values for the LC and IA methods are generally ≈7% to ≈10% higher than the 

NIST value, it is likely that some of this bias is attributable to contribution from 3-epi-25(OH)D3.
10

Figure 2. 25(OH)DTotal levels in SRM 972a L1, SRM 972a L3, and SRM 968d L1 as determined

by immunoassay (IA) 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). 25(OH)DTotal levels in SRM 972a L1, SRM 972a L3, and SRM 968d L1 as 

determined by immunoassay (IA) and LC (LC-MSn and LC-UV) methods. 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. 
12

Figure 3 presents direct graphical comparisons of the 25(OH)DTotal results for the studied serum 

materials: A) SRM 972a L3 and SRM 972a L1; B) SRM 968d L1 and SRM 972a L1 and C) SRM 

968d L1 and SRM 972a L3. In each plot, 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. In each plot, The NIST values for the

materials are 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-axis and x-axis,

respectively.

Specific results as assessed from Figure 3 are summarized below.

SRM 972a L3 and SRM 972a L1 (Figure 3A)

• Laboratory results that are not included in the consensus ranges include numbers 060, 086b, 

110, 128, 180, 189, 212, 221b, 222, 231, 247a, and 250.

• The Youden line runs through both the IA and LC consensus boxes for these materials.

• Most of the IA results for both materials are lower than the results from the LC methods, and the 

IA and LC consensus boxes partially overlap but exhibit significant separation from each other. 

SRM 968d L1 and SRM 972a L1 (Figure 3B)

• Laboratory results that are not included in the consensus ranges include numbers 060, 110, 128, 

189, 213a, 221b, 228a, 228b, 231, 247a, 250.

• The Youden line runs through the center of both the IA and LC consensus boxes, illustrating 

that both the IA and LC results are in agreement with each other and with the NIST results for 

these materials. 

SRM 968d L1 and SRM 972a L3 (Figure 3C)

• Laboratory results that are not included in the consensus ranges include numbers 060, 086b, 

110, 128, 180, 189, 212, 213a, 221b, 222, 228a, 228b, 247a.

• The Youden line runs through the center of the LC consensus box and through the bottom 

corner of the IA consensus box for these materials, illustrating that the LC results are in better 

agreement with the NIST results for these materials. 

• In general, the IA results tend to be lower than the LC results for material SRM 972a L3, 

leading to consensus boxes that partially overlap but exhibit significant separation from each 

other. 

The Youden plots involving SRM 972a L3 reveal separation of the IA and LC consensus boxes, 

further illustrating the difference in results for the two techniques for the material with high native 

levels of 25(OH)D2.
13

 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 

060

086b

110

128

180

189

212

221b

222

231 247a

250

10

20

30

40

50

10 20 30 40 50

25(OH)DTotal in SRM 972a L3 "Vial B" (ng/mL)

25(OH)DTotal in SRM 972a L1 "Vial A" (ng/mL)

IA methods

LC methods

Figure 3. Youden comparison plot of the results for 25(OH)DTotal in SRM 972a L3 (Vial B) and 

SRM 972a L1 (Vial A) for all methods

A
14

Figure 3 (cont’d). Youden comparison plot of the results for 25(OH)DTotal in SRM 968d L1 (Vials 

C,D) and SRM 972a L1 (Vial A) for all methods

 IA method laboratory mean values ± 2 SD (y-axis only) 

 IA method consensus box encloses ± 2 MADe around consensus medians

 LC method laboratory mean values ± 2 SD (y-axis only)

 LC method consensus box encloses ± 2 MADe around consensus medians

 NIST values with corresponding Youden line 

060

110

128

189

213a

221b

228a

228b

231

247a

250

0

5

10

15

20

25

10 20 30 40 50

25(OH)DTotal in SRM 968d L1 "Vials C,D" (ng/mL)

25(OH)DTotal in SRM 972a L1 "Vial A" (ng/mL)

IA methods

LC methods

B
15

Figure 3 (cont’d). Youden comparison plot of the results for 25(OH)DTotal in SRM 968d L1 (Vials 

C,D) and SRM 972a L3 (Vial B) for all methods

 IA method laboratory mean values ± 2 SD (y-axis only) 

 IA method consensus box encloses ± 2 MADe around consensus medians

 LC method laboratory mean values ± 2 SD (y-axis only) 

 LC method consensus box encloses ± 2 MADe around consensus medians

 NIST values with corresponding Youden line 

060

086b

110

128 180

189

212

213a

221b

222

228a

228b

247a

0

5

10

15

20

25

10 20 30 40 50

25(OH)DTotal in SRM 968d L1 "Vials C,D" (ng/mL)

25(OH)DTotal in SRM 972a L3 "Vial B" (ng/mL)

IA methods

LC methods

C
16

25(OH)D2 and 25(OH)D3 in SRM 972a L3 (LC methods only)

Of the two major techniques IA and LC, only the LC techniques can independently measure the 

25(OH)D2 and 25(OH)D3 components of 25(OH)DTotal. SRM 972a L3 contains appreciable 

concentrations of both metabolites, and a summary of the individual LC participant data for 

25(OH)D2 and 25(OH)D3 in SRM 972a L3 (vial B) is provided in Table 3. 

The community results are summarized at the bottom of the table for all LC methods and for the 

LC-MSn methods only. These summarized results include N, the median value, the MADe, and the

CV%. For the participant results, the consensus variability is much larger for 25(OH)D2 (17%) than 

it is for 25(OH)D3 (6%). The source of the difference in measurement uncertainty for the two 

metabolites is unclear.

Table 3 also presents the NIST certified values for 25(OH)D3 and 25(OH)D2 and the 95% 

confidence limits (U95) in SRM 972a L3. 
17

Table 3. Summary of LC participant data and community results for 25(OH)D2 (ng/mL) and 

25(OH)D3 (ng/mL) in SRM 972a L3 (Vial B).

25(OH)D2 25(OH)D3

SRM 972a L3 SRM 972a L3

Lab Method Vial B Vial B

026 LC-MS/MS 13.4 20.1

056 LC-MS/MS 14.7 20.1

060 LC-MS/MS 17.3 24.9

110 LC-UV <4 23.3

116 LC-MS/MS 15.5 22.4

128 LC-MS/MS n/r 24.1

185a LC-MS/MS 17.2 20.8

187 LC-MS/MS 12.5 20.6

187 LC-MS/MS 12.5 20.6

189 LC-UV n/d 14.0

197 LC-MS/MS 15.0 21.3

198a LC-MS/MS 12.5 18.7

198a LC-MS/MS 12.5 18.7

199 LC-MS/MS 13.2 20.5

202 LC-MS/MS 13.5 21.8

211 LC-MS/MS 14.1 20.0

212 LC-MS/MS 22.4 21.8

215 LC-MS/MS 12.0 20.4

216 LC-MS/MS 13.7 19.7

217 LC-MS/MS 14.1 20.0

217 LC-MS/MS 14.1 20.0

218b LC-MS/MS 16.7 24.3

219 LC-MS/MS 12.7 20.9

220 LC-MS/MS 14.0 23.0

221a LC-MS/MS 12.4 18.6

221b LC-UV 11.1 16.9

228a LC-MS/MS 20.8 30.6

231 LC-UV 15.6 21.3

234 LC-MS/MS 18.9 20.3

241 LC-MS/MS 13.3 20.4

242 LC-MS/MS 17.9 19.9

243 LC-UV 18.6 20.2

248 LC-MS/MS 16.7 24.4

249 LC-MS/MS 15.9 19.1

250 LC-MS/MS 16.4 22.8

251 LC-MS/MS 13.6 20.0

253 LC-MS/MS 14.8 20.9

N 34 37

Median 14.1 20.5

MADe 2.4 1.2

CV% 17 5.8

N 31 32

Median 14.1 20.6

MADe 2.4 1.1

CV% 17 5.2

NIST Value 13.3 19.8

U 95 0.3 0.5

LC-MSn LC methods
18

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

SRM 972a L3 are plotted in Figure 4 and are displayed with white triangles () and grey triangles 

(), respectively. The values for 25(OH)DTotal represent the sum of 25(OH)D2 and 25(OH)D3 and 

are plotted as red triangles (). In Figure 4, the red solid lines () represent the consensus 

median and the red dashed lines (- - - - -) represent the approximate 95% confidence interval (2 ×

MADe) for all LC method results. 

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

variability range. For SRM 972a L3, there are three outlying LC results (one LC-MSn, two LC-UV) 

that underestimated the 25(OH)DTotal either because they did not measure or did not detect the 

25(OH)D2. For the LC methods, independent, accurate measurements of both 25(OH)D2 and 

25(OH)D3 are required to obtain accurate values for 25(OH)DTotal.

The grey-shaded bars in Figure 4 represent the interval in which NIST believes the “true value” 

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

median results for both 25(OH)D2 and 25(OH)D3 are higher than the NIST expanded uncertainty 

range; however, the NIST value falls within the consensus range for the LC methods for both 

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

Figure 4. 25(OH)D2, 25(OH)D3 and 25(OH)DTotal levels in SRM 972a L3 as determined by LC 

(LC-MSn and LC-UV) methods. The grey-shaded bars represent the ranges bound by the NIST 

values with ± estimated U95 uncertainty.
19

Correlation of 25(OH)D in SRM 972a L1, SRM 972a 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 4. 

Table 4. 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 972a L1, SRM 972a L3, and 

SRM 968d L1 overlaid with the clinical ranges for 25(OH)D from Table 4 are presented in 

Figure 5. Specific results as assessed from Figure 5 are summarized below:

SRM 972a L1

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

or reported values is large (from 21.5 ng/mL to 45.2 ng/mL).

• The NIST value (29.3 ng/mL ± 1.1 ng/mL) is in the adequate 25(OH)D concentration range.

SRM 972a L3

• The majority of the participant results are in the adequate range, but there is one result in 

each of the inadequate and potentially adverse ranges; the range of participant results is 

large (from 14.0 ng/mL to 51.4 ng/mL).

• The NIST value (33.2 ng/mL ± 0.6 ng/mL) is in the adequate 25(OH)D concentration range. 

SRM 968d L1

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

results in the deficient and adequate concentration ranges were also reported.

• 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 SRM 972a L1 and 

968d L1 and 17% for SRM 972a L3 (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. 
20

Figure 5. 25(OH)DTotal levels in SRM 972a L1, SRM 972a 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. 
21

Figure 5 (cont’d) 25(OH)DTotal levels in SRM 972a L1, SRM 972a 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. 
22

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

Laboratory 

Number IA Method Sample Preparation Detection

17 CLIA n/r n/r

30 RIA Sample was extracted with acetonitrile (50 μL sample + 500 μL 

acetonitrile); controls measured using water as the diluent

Gamma counter with data 

reduction software

86a CLIA n/r n/r

86b RIA n/r n/r

180 RIA Samples extracted with acetonitrile I

125 detection

183b CLIA n/r n/r

188 CLIA None n/r

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

210a RIA Sample was extracted with acetonitrile n/r

210b CLIA n/r n/r

213a EIA Sample was thawed and gently mixed prior to analysis n/r

213b CLIA Sample was thawed and gently mixed prior to analysis n/r

218a CLIA Direct analysis n/r

222 CLIA n/r n/r

228b CLIA n/r n/r

247a CLIA Sample was thawed, mixed well and used in the assay n/r

247b EIA Sample was thawed, mixed well and used in the assay UV at 450 nm with a 

reference filter at 630 nm

n/r = not reported
23

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

PFP column (100 x 3.2 mm); 

isocratic separation with 82% 

methanol, 18% water; flow 0.4 

mL/min

25(OH)D2 413/355; 

25(OH)D3 401/365; 

3-epi-25(OH)D3 401/365

56

25(OH)D2-d 3; 

25(OH)D3-d 6; 

3-epi-25(OH)D3-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; 

3-epi-25(OH)D3 383/365

60 25(OH)D3 -d 6

IS was added and serum (150 μL) 

proteins were precipitated with 

ternary solvent, followed by 

centrifugation, evaporation, and 

reconstitution

C18 column (150 x 3.0 mm); 

gradient with water, methanol and 

acetonitrile (0.05% formic acid); 

flow 0.55 mL/min

25(OH)D3 383/211; 

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

25(OH)D2 395/270

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

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

185a 25(OH)D2-d 6 and 

25(OH)D3-d 6

Liquid-liquid extraction; 40 μL 

sample

C18 column; methanol/water 

gradient MRM

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

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

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); 

column temp 45°C; gradient with 

water and methanol; flow 1.0 

mL/min

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 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 water (10 mmol/L ammonium 

formate) and methanol (0.1% formic 

acid); flow 0.4 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

Proteins precipitated with 

acetonitrile containing IS followed 

by centrifugation

Column (33 x 4.6 mm; 3 μm)

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

383/257 (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 

containing the IS (350 μL)

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
24

 

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 

413/83 (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 Online SPE n/r n/r

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

n/r

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

228a n/r Proteins precipitated followed by 

centrifugation 

C18 column; gradient with water 

and methanol

25(OH)D3 401; 

25(OH)D2 413

234 25(OH)D3-d 6

The samples are protein crashed 

using acetonitrile

A turbo column is used for cleanup 

followed by a C18 analytical 

column; isocratic separation with 

water and methanol

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

248 25(OH)D2-d 3 and 

25(OH)D3-d 3

IS was added and the sample was 

precipitated with acetonitrile, 

centrifuged, and injected

C18 column (100 x 2.1 mm; 2.5 

μm); gradient with water and 

methanol, each containing 2 mmol/L 

ammonium acetate

25(OH)D3 383/257 (quant), 

383/365 (qual); 25(OH)D2 

395/377 (quant), 395/269 

(qual); 25(OH)D3-d 3 386/257 

(quant), 386/368 (qual); 

25(OH)D2-d3 398/380 

(quant), 398/271 (qual)

249 25(OH)D2-d 3 and 

25(OH)D3-d 6

Proteins precipitated followed by 

centrifugation and SPE

PFP column (100 x 2.1 mm; 1.8 

μm); gradient separation with water 

(2 mmol/L ammonium acetate, 0.1% 

formic acid) and methanol (0.3% 

formic acid); flow 0.3 mL/min

25(OH)D3 401/159; 

25(OH)D3-d 6 407/159; 

25(OH)D2 413/159; 

25(OH)D2-d 3 416/83
25

250 25(OH)D2-d 3 and 

25(OH)D3-d 3

Protein crash followed by SPE

Phenyl column (50 x 2.1 mm); 

gradient with water and methanol, 

each containing 2 mmol/L 

ammonium acetate and 0.1% formic 

acid; flow 0.45 mL/min

25(OH)D3 401/159, 401/365; 

25(OH)D2 413/355, 413/83

251 25(OH)D2-d 3 and 

25(OH)D3-d 3

Protein precipitation followed by 

SPE

Phenyl column (50 x 2.1 mm; 1.7 

μm); gradient with water and 

methanol, each containing 2 mmol/L 

ammonium acetate and 0.1% formic 

acid; flow 0.45 mL/min

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

401/365 (qual); 25(OH)D2 

413/355 (quant), 413/83 

(qual); 25(OH)D3-d 3 

404/162; 25(OH)D2-d3 

416/358

253 d -labeled isotope

The sample was extracted with 

acetonitrile/trifluroacetic acid and 

then centrifuged

C18 column (50 x 2.1 mm); isocratic 

separation with water and methanol; 

flow 0.4 mL/min

25(OH)D2 619/298; 

25(OH)D3 607/298

MRM = multiple reaction monitoring

PFP = pentafluorophenyl

n/r = not reported

quant = quantitative ions

qual = qualitative ions

SPE = solid phase extraction

CN = cyano
26

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

Reversed-phase 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 1alpha(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

n/r = not reported

CN = cyano
27

Appendix B-1. Raw participant data and NIST results for 25(OH)D2, 25(OH)D3 and 25(OH)DTotal in SRM 972a L1, SRM 972a 

L3,and SRM 968d L1 and the control solutions, SRM 2972.

SRM 972a L1 SRM 972a L3 SRM 968d L1 SRM 968d L1 SRM 972a L1 SRM 972a L3 SRM 968d L1 SRM 968d L1 SRM 972a L1 SRM 972a L3 SRM 968d L1 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 28.1 27.7 14.0 14.7 n/r n/r

026 LC-MS/MS <1.0 13.4 <1.0 <1.0 31.0 20.1 12.7 13.4 31.0 33.5 12.7 13.4 n/r n/r

030 RIA n/a n/a n/a n/a n/a n/a n/a n/a 32.4 26.7 15.8 14.4 257.0 408.5

056 LC-MS/MS 0.7 14.7 n/d n/d 30.2 20.1 12.6 12.3 30.9 34.8 12.6 12.3 246.7 346.3

060 LC-MS/MS < 2.00 17.3 n/d n/d 37.1 24.9 15.3 14.6 37.1 42.2 15.3 14.6 210.1 348.1

086a CLIA n/a n/a n/a n/a n/a n/a n/a n/a 27.4 26.8 14.4 15.7 n/r n/r

086b RIA n/a n/a n/a n/a n/a n/a n/a n/a 32.0 40.0 17.0 17.0 n/r n/r

110 LC-UV <4 <4 <4 <4 19.9 23.3 14.3 15.3 21.5 23.3 14.3 17.9 239.2 335.2

116 LC-MS/MS < 3.3 15.5 < 3.3 < 3.3 35.1 22.4 15.4 15.5 35.1 37.9 15.4 15.5 n/r n/r

128 LC-MS/MS n/r n/r n/r n/r 37.0 24.1 15.6 14.1 37.0 24.1 15.6 14.1 n/r 332.0

139 LC-UV n/d 12.2 n/d n/d 32.0 22.5 13.0 13.6 32.0 34.7 13.0 13.6 256.0 335.0

180 RIA n/a n/a n/a n/a n/a n/a n/a n/a 32.8 34.8 13.7 16.0 n/r n/r

183b CLIA n/a n/a n/a n/a n/a n/a n/a n/a 28.0 25.0 13.8 13.9 262.4 336.8

185a LC-MS/MS n/d 17.2 2.1 n/d 32.1 20.8 12.3 12.3 32.1 38.0 14.4 12.3 238.6 334.8

187 LC-MS/MS n/d 12.5 n/d n/d 32.7 20.6 13.1 12.9 32.7 33.1 13.1 12.9 n/r n/r

188 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 31.4 29.4 12.9 14.1 n/r n/r

189 LC-UV n/d n/d n/d n/d 37.7 14.0 12.0 10.4 37.7 14.0 12.0 10.4 n/r n/r

194 LC-MS/MS <7 14.1 <7 <7 32.3 20.0 12.9 13.1 32.3 34.1 12.9 13.1 240.5 335.0

196 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 31.6 29.2 14.5 15.6 n/r n/r

197 LC-MS/MS <5 15.0 <5 <5 32.0 21.3 13.8 14.3 32.0 36.3 13.8 14.3 238.0 343.0

198a LC-MS/MS <5 12.5 <5 <5 29.8 18.7 12.9 15.2 29.8 31.2 12.9 15.2 228.2 324.1

198b EIA n/a n/a n/a n/a n/a n/a n/a n/a 28.2 27.6 12.2 14.1 n/r n/r

199 LC-MS/MS < 2 13.2 < 2 < 2 30.7 20.5 12.7 12.6 30.7 33.7 12.7 12.6 249.4 329.9

202 LC-MS/MS n/d 13.5 n/d n/d 33.4 21.8 12.9 12.7 33.4 35.3 12.9 12.7 246.0 345.0

209 LC-MS/MS <1.0 15.3 <1.0 <1.0 31.7 24.3 11.9 12.8 31.7 39.6 11.9 12.8 n/r n/r

210a RIA n/a n/a n/a n/a n/a n/a n/a n/a 31.4 31.3 12.6 12.8 n/r n/r

210b CLIA n/a n/a n/a n/a n/a n/a n/a n/a 26.8 26.4 13.2 13.7 n/r n/r

211 LC-MS/MS n/d 14.1 n/d n/d 31.5 20.0 12.3 11.7 31.5 34.1 12.3 11.7 229.0 280.0

212 LC-MS/MS n/d 22.4 n/d n/d 35.4 21.8 14.7 15.1 35.4 44.2 14.7 15.1 n/r n/r

213a CLIA n/a n/a n/a n/a n/a n/a n/a n/a 27.5 25.0 3.7 3.7 n/r n/r

213b EIA n/a n/a n/a n/a n/a n/a n/a n/a 28.2 23.8 12.9 12.2 n/r n/r

215 LC-MS/MS 0.4 12.0 n/d n/d 32.0 20.4 12.0 13.6 32.4 32.4 12.0 13.6 n/r n/r

216 LC-MS/MS 0.5 13.7 0.2 0.2 30.0 19.7 13.0 12.6 30.5 33.4 13.2 12.8 229.9 343.3

217 LC-MS/MS < 2 14.1 < 2 < 2 30.5 20.0 12.4 12.6 30.5 34.1 12.4 12.6 n/r n/r

218a CLIA n/a n/a n/a n/a n/a n/a n/a n/a 29.8 29.2 13.7 14.7 n/r n/r

218b LC-MS/MS n/d 16.7 n/d n/d 30.4 24.3 14.2 14.5 30.4 41.0 14.2 14.5 239.1 336.0

219 LC-MS/MS <4.0 12.7 <4.0 <4.0 30.0 20.9 12.4 12.7 30.0 33.6 12.4 12.7 n/r n/r

220 LC-MS/MS <5 14.0 <5 <5 35.0 23.0 14.0 14.0 35.0 37.0 14.0 14.0 n/r n/r

221a LC-MS/MS n/d 12.4 n/d n/d 27.9 18.6 11.6 12.9 27.9 31.0 11.6 12.9 n/r n/r

221b LC-UV n/d 11.1 n/d n/d 24.4 16.9 25.0 24.7 24.4 28.0 25.0 24.7 n/r n/r

222 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 33.1 35.3 13.5 12.3 n/r n/r

228a LC-MS/MS n/d 20.8 n/d n/d 45.2 30.6 17.3 17.9 45.2 51.4 17.3 17.9 217.3 331.5

228b CLIA n/a n/a n/a n/a n/a n/a n/a n/a 30.5 26.7 3.2 3.1 n/r n/r

231 LC-UV n/d 15.6 n/d n/d 26.6 21.3 14.8 14.0 26.6 36.9 14.8 14.0 245.3 294.5

234 LC-MS/MS < 3.0 18.9 < 3.0 < 3.0 34.5 20.3 13.4 14.7 34.5 39.2 13.4 14.7 n/r n/r

241 LC-MS/MS n/d 13.3 n/d n/d 30.5 20.4 13.2 12.6 30.5 33.7 13.2 12.6 n/r n/r

242 LC-MS/MS n/d 17.9 n/d n/d 28.9 19.9 13.4 12.4 28.9 37.8 13.4 12.4 239.4 333.4

243 LC-UV n/d 18.6 n/d n/d 30.1 20.2 13.6 12.3 30.1 38.8 13.6 12.3 240.6 331.7

244 LC-MS/MS <5 13.0 <5 <5 29.0 21.0 12.0 11.0 29.0 34.0 12.0 11.0 215.0 331.0

247a CLIA n/a n/a n/a n/a n/a n/a n/a n/a 38.1 37.1 17.7 14.2 n/r n/r

247b EIA n/a n/a n/a n/a n/a n/a n/a n/a 31.3 30.5 16.3 16.0 n/r n/r

248 LC-MS/MS < 3 16.7 < 3 <3 31.7 24.4 14.0 14.2 31.7 41.1 14.0 14.2 238.6 334.8

249 LC-MS/MS n/d 15.9 n/d n/d 30.0 19.1 13.7 11.4 30.0 35.0 13.7 11.4 238.4 324.2

250 LC-MS/MS <2.4 16.4 <2.4 <2.4 37.0 22.8 13.1 15.2 37.0 39.2 13.1 15.2 281.6 382.0

251 LC-MS/MS <4 13.6 <4 <4 31.5 20.0 15.2 14.7 31.5 33.6 15.2 14.7 n/r n/r

253 LC-MS/MS n/d 14.8 n/d n/d 31.0 20.9 12.9 13.3 31.0 35.7 12.9 13.3 235.5 307.0

NIST Value 0.54 13.30 <0.5 <0.5 18.80 19.80 12.38 12.38 29.30 33.20 12.38 12.38 238.6 334.0

U 95 0.06 0.30 0.00 0.00 1.10 0.50 0.28 0.28 1.10 0.60 0.28 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)
28

Appendix B-2. Raw participant data and NIST results for 3-epi-25(OH)D3 in SRM 972a L1, SRM 972a L3, and SRM 968d L1

SRM 972a L1 SRM 972a L3 SRM 968d L1 SRM 968d L1

Lab Method Vial A Vial B Vial C Vial D

017 CLIA n/r n/r n/r n/r

026 LC-MS/MS 1.5 0.7 0.3 0.4

030 RIA n/r n/r n/r n/r

056 LC-MS/MS 1.9 1.2 0.6 0.7

060 LC-MS/MS n/r n/r n/r n/r

086a CLIA n/r n/r n/r n/r

086b RIA n/r n/r n/r n/r

110 LC-UV n/r n/r n/r n/r

116 LC-MS/MS n/r n/r n/r n/r

128 LC-MS/MS n/r n/r n/r n/r

139 LC-UV n/r n/r n/r n/r

180 RIA n/r n/r n/r n/r

183b CLIA n/r n/r n/r n/r

185a LC-MS/MS n/r n/r n/r n/r

187 LC-MS/MS n/r n/r n/r n/r

188 CLIA n/r n/r n/r n/r

189 LC-UV n/r n/r n/r n/r

194 LC-MS/MS <7 <7 <7 <7

196 CLIA n/r n/r n/r n/r

197 LC-MS/MS n/r n/r n/r n/r

198a LC-MS/MS n/r n/r n/r n/r

198b EIA n/r n/r n/r n/r

199 LC-MS/MS n/r n/r n/r n/r

202 LC-MS/MS n/r n/r n/r n/r

209 LC-MS/MS n/r n/r n/r n/r

210a RIA n/r n/r n/r n/r

210b CLIA n/r n/r n/r n/r

211 LC-MS/MS n/r n/r n/r n/r

212 LC-MS/MS n/r n/r n/r n/r

213a CLIA n/r n/r n/r n/r

213b EIA n/r n/r n/r n/r

215 LC-MS/MS n/r n/r n/r n/r

216 LC-MS/MS n/r n/r n/r n/r

217 LC-MS/MS <2 <2 <2 <2

218a CLIA n/r n/r n/r n/r

218b LC-MS/MS n/r n/r n/r n/r

219 LC-MS/MS n/r n/r n/r n/r

220 LC-MS/MS n/r n/r n/r n/r

221a LC-MS/MS n/r n/r n/r n/r

221b LC-UV n/r n/r n/r n/r

222 CLIA n/r n/r n/r n/r

228a LC-MS/MS n/r n/r n/r n/r

228b CLIA n/r n/r n/r n/r

231 LC-UV n/r n/r n/r n/r

234 LC-MS/MS n/r n/r n/r n/r

241 LC-MS/MS n/r n/r n/r n/r

242 LC-MS/MS n/r n/r n/r n/r

243 LC-UV n/r n/r n/r n/r

244 LC-MS/MS n/r n/r n/r n/r

247a CLIA n/r n/r n/r n/r

247b EIA n/r n/r n/r n/r

248 LC-MS/MS n/r n/r n/r n/r

249 LC-MS/MS 2.1 n/r n/r n/r

250 LC-MS/MS n/r n/r n/r n/r

251 LC-MS/MS n/r n/r n/r n/r

253 LC-MS/MS n/r n/r n/r n/r

NIST Value 1.84 1.18 <0.5 <0.5

U 95 0.08 0.13 0.00 0.00

*n/a = not applicable (for immunoassay methods); n/r = not reported; < X = less than a 

reported quantitation limit of X

epi-25(OH)D3 (ng/mL)