NISTIR 7891

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Summer 

2010 Comparability Study 

(Exercise 2)

Katrice A. Lippa

Mary Bedner

Susan S.-C. Tai

http://dx.doi.org/10.6028/NIST.IR.7891
NISTIR 7891

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Summer 

2010 Comparability Study 

(Exercise 2)

Katrice A. Lippa

Mary Bedner

Susan S.-C. Tai

Chemical Sciences Division

Material Measurement Laboratory

http://dx.doi.org/10.6028/NIST.IR.7891

December 2012

U.S. Department of Commerce 

Rebecca Blank, Acting Secretary

National Institute of Standards and Technology 

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

ABSTRACT

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

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

of Health (NIH) Office of Dietary Supplements. Participants in the second exercise of this 

program, the Summer 2010 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 968d Fat-Soluble Vitamins, 

Carotenoids and Cholesterol in Human Serum (Level 1 and Level 2). 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, within-laboratory 

precision, and concordance within the participant community. A report of results was provided 

to all participants of the exercise, and laboratories were identified by code numbers known only 

to them. The results from this second exercise are reported along with a summary of the 

analytical methods used. 
2

OVERVIEW OF SUMMER 2010 COMPARABILITY STUDY

For the Summer 2010 Comparability Study (Exercise 2) of the NIST/NIH Vitamin D 

Metabolites Quality Assurance Program (VitDQAP), control and human serum study samples 

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

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

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

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

addition, participants were asked to determine 25(OH)D2, 25(OH)D3, and a total concentration 

of 25-hydroxyvitamin D (25(OH)DTotal = 25(OH)D2 + 25(OH)D3) in each of four samples (vials 

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

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

Level 1 (SRM 968d L1), which is a blended human serum pool with endogenous vitamin D 

levels. Vials C and D were duplicate samples of SRM 968d Level 2 (SRM 968d L2), which is a 

blended human serum pool that contains endogenous vitamin D levels but has augmented αtocopherol (vitamin E) and β-carotene (vitamin A) levels. 

In the Summer 2010 exercise, there were a total of 37 participants and 39 datasets (two

participants provided data for two different methods). Sixteen of the datasets originated from 

immunoassay (IA) techniques, including six from enzyme immunoassay (EIA), five from 

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

A-1 summarizes the immunoassay methods used by the participants. Twenty-three of the 

datasets originated from liquid chromatographic (LC) methods; of those, 17 were from LC with 

tandem mass spectrometric detection (LC-MS/MS), five were from LC with ultraviolet 

absorbance detection (LC-UV), and one was from LC with electrochemical detection (LC-EC). 

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 is summarized in Appendix B. For SRM 968d L1 

and SRM 968d L2, the immunoassay methods reported values for 25(OH)DTotal only, whereas 

LC-based methods reported values for 25(OH)DTotal, 25(OH)D2, and 25(OH)D3. Both materials 

contained very low levels of 25(OH)D2 with only four and seven of the LC participants reporting 

values in SRM 968d L1 and SRM 968d L2, respectively. Two of these participants reported 

25(OH)D2 values at low levels ranging from 0.04 ng/mL to 3.60 ng/mL, but most labs indicated 

this analyte was below their quantitation limit of <1 ng/mL to <7 ng/mL. For the majority of the 

participants using LC, the 25(OH)DTotal data reported in Appendix B is the same as their 

reported data for 25(OH)D3 for SRM 968d L1 and SRM 968d L2. Both LC and immunoassay 

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

because the analytes were in separate solutions.

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

25(OH)D2 in both SRM 968d L1 and SRM 968d L2 was below the quantitation limit (≈ 0.5 

ng/mL) for the NIST method.
3

SUMMER 2010 EXERCISE RESULTS AND DISCUSSION

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

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. The community results are summarized at the bottom 

of the table for all reported methods, the immunoassay methods only, LC methods only, and the 

LC-MS/MS methods only. The community results include the total number of quantitative 

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

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

(CV%). Consensus statistics were not calculated for the data from the UV and EC methods 

because of the limited number of data reported (4 and 1 values, respectively). Table 1 also 

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

confidence.

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

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

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

with black squares and are segregated by MS/MS detection (■) and UV/EC detection (). From 

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

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

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

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

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

technique (IA or LC). The graphs reveal that the consensus variability range for the participants 

who reported results using IA methods is quite large for both analytes. Several other IA 

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

not provide values. Overall, the control solutions appeared more compatible with the LC 

methods, which exhibited less consensus variability. The NIST certified value is provided by a 

grey-shaded bar that represents the value and its associated uncertainty (± U95); these “target” 

values were provided to participants in the reporting sheet. 

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

is provided in the Youden plot in Figure 2. Laboratories that are within the consensus range for 

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

Figure 2. Conversely, laboratories that fall outside of (or on the edge of) the blue consensus box 

are highlighted with their laboratory code numbers. The NIST values are denoted with a red 

diamond symbol (). The Youden line centered on the NIST values is illustrated by a red line 

() that represents the relative ratio of the NIST values (334.8/238.6) for 25(OH)D3 and 

25(OH)D2 across the magnitude of the y- and x-axis, respectively. Participant data (numbers 

193, 175, 139, 198a, 191, 196) that are near the Youden line but are above or below the

consensus box may suggest that these measurements are biased high or low due to a calibration 

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

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

25(OH)D3, particularly for LC-based methods. 
4

Table 1. Summary of participant data 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 239.9 1.3 030 RIA 341.9

032 LC-UV 215.0 3.0 032 LC-UV 333.0

056 LC-MS/MS 255.3 2.0 056 LC-MS/MS 334.8

062 RIA 245.0 1.3 062 RIA 575.9

110 LC-UV 247.0 3.0 110 LC-UV 315.0

116 LC-MS/MS 237.3 2.0 116 LC-MS/MS 364.6

139 LC-UV 328.9 3.0 139 LC-UV 368.5

150 LC-MS/MS 218.0 2.0 150 LC-MS/MS 291.0

169 LC-ECD 241.9 4.0 169 LC-ECD 339.5

175 CLIA 169.5 1.1 175 CLIA 254.0

182 LC-MS/MS 235.2 2.0 182 LC-MS/MS 326.6

183a LC-MS/MS 199.0 2.0 183a LC-MS/MS 326.0

184 LC-MS/MS 230.2 2.0 184 LC-MS/MS 325.0

187 LC-MS/MS 210.0 2.0 187 LC-MS/MS 331.0

191 RIA 303.3 1.3 191 RIA 465.8

193 EIA 123.6 1.2 193 EIA 191.2

194 LC-MS/MS 242.5 2.0 194 LC-MS/MS 346.0

195 LC-MS/MS 237.0 2.0 195 LC-MS/MS 330.0

196 CLIA 364.2 1.1 196 CLIA 562.0

197 LC-MS/MS 247.5 2.0 197 LC-MS/MS 337.5

198a LC-MS/MS 286.7 2.0 198a LC-MS/MS 406.4

199 LC-MS/MS 244.0 2.0 199 LC-MS/MS 336.0

200 RIA 245.0 1.3 200 RIA 373.0

202 LC-MS/MS 234.9 2.0 202 LC-MS/MS 352.8

203 LC-UV 206.8 3.0 203 LC-UV 377.6

204 CLIA 189.0 1.1 204 CLIA 337.0

N 26 N 26

Median 238.6 Median 337.3

MADe 19.0 MADe 20.6

CV% 8.0 CV% 6.1

N 8 N 8

Median 242.4 Median 357.5

MADe 84.7 412 MADe 157.0

CV% 34.9 73 CV% 43.9

N 18 N 18

Median 237.2 Median 335.4

MADe 15.0 267 MADe 14.7

CV% 6.3 207 CV% 4.4

N 13 N 13

Median 237.0 Median 334.8

MADe 10.4 MADe 13.0

CV% 4.4 CV% 3.9

NIST Value 238.6 NIST Value 334.0

U 95 3.9 U 95 5.2

25(OH)D3 (ng/mL)

All 

methods

IA 

methods 

LC 

methods

LCMS/MS

25(OH)D2 (ng/mL)
5

Figure 1. 25(OH)D2 and 25(OH)D3 values in SRM 2972 for immunoassay, LC-MS/MS and 

LC-UV or LC-EC methods. The grey-shaded bars represent the ranges bound by the NIST 

certified values with ± U95 expanded uncertainty.
6

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

solutions.

 Laboratory values 

 All method consensus box encloses ± 2 MADe around consensus median

 NIST values with corresponding Youden line 

062

139

150

175

183a

191

193

196

198a

204

150

250

350

450

550

100 200 300 400

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

25(OH)D2 in SRM 2972 (ng/mL)
7

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

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

SRM 968d L2 (vials A&B and vials C&D, respectively) is provided in Table 2. The 

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

deviation (%rSD) of the two reported values for SRM 968d L1 or SRM 968d 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-MS/MS methods only. These 

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

the MADe, and the percent coefficient of variation. Consensus statistics were not calculated for 

the data from the UV and EC methods because of the limited number of data reported (5 and 1 

values, respectively). 

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

SRM 968d L1 and SRM 968d L2. The NIST values for 25(OH)D3 were obtained using an 

LC-MS/MS reference measurement procedure1 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 both 

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

 1 Tai, S. S.-C., Bedner, M. and Phinney, K.W. Anal. Chem. 2010 82, 1942-1948.
8

Table 2: Summary of participant data for 25(OH)DTotal (ng/mL) in SRM 968d L1 and SRM 

968d L2. 

n/d = not detected; n/a = not applicable

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

017 CLIA 11.4 12.5 8.8 9.2 12.0 0.8 6.5 9.0 0.2 2.7

026 LC-MS/MS 13.1 11.1 9.8 11.6 12.1 1.4 11.7 10.7 1.3 11.9

030 RIA 13.6 13.7 9.2 8.9 13.7 0.1 0.5 9.1 0.2 2.3

032 LC-UV 13.5 14.6 18.4 18.5 14.1 0.8 5.5 18.5 0.1 0.4

056 LC-MS/MS 13.7 11.0 11.3 11.0 12.4 1.9 15.5 11.2 0.2 1.9

062 RIA 14.2 14.1 16.9 17.2 14.2 0.1 0.5 17.1 0.2 1.2

110 LC-UV 7.6 9.9 7.0 6.8 8.8 1.6 18.6 6.9 0.1 2.0

116 LC-MS/MS 13.3 12.6 13.1 10.7 13.0 0.5 3.8 11.9 1.7 14.3

128 LC-MS/MS 12.6 12.1 12.7 12.2 12.4 0.4 2.9 12.5 0.4 2.8

139 LC-UV 9.2 9.1 n/d 94.8 9.2 0.1 0.8 94.8 n/a n/a

141 EIA 9.7 12.1 4.1 5.5 10.9 1.7 15.6 4.8 1.0 20.6

150 LC-MS/MS 15.0 15.1 12.2 11.3 15.1 0.1 0.5 11.8 0.6 5.4

169 LC-ECD 19.9 20.1 19.6 19.0 20.0 0.1 0.7 19.3 0.4 2.2

175 CLIA 15.7 15.8 11.1 11.3 15.8 0.1 0.4 11.2 0.1 1.3

180 RIA 17.3 16.6 14.1 14.3 17.0 0.5 3.0 14.2 0.1 0.9

182 LC-MS/MS 11.7 11.5 12.0 12.3 11.6 0.1 1.2 12.2 0.2 1.7

183a LC-MS/MS 13.5 14.9 12.0 12.8 14.2 1.0 7.0 12.4 0.6 4.6

183b CLIA 14.2 14.3 9.8 10.0 14.3 0.1 0.5 9.9 0.1 1.4

184 LC-MS/MS 12.5 12.7 23.9 22.5 12.6 0.1 1.1 23.2 1.0 4.3

185 LC-MS/MS 14.7 14.8 12.6 12.7 14.7 0.1 0.5 12.6 0.0 0.3

186 LC-MS/MS 18.6 19.0 15.8 13.0 18.8 0.3 1.5 14.4 2.0 13.7

187 LC-MS/MS 14.1 12.9 11.9 12.3 13.5 0.8 6.3 12.1 0.3 2.4

188 CLIA 14.6 13.6 10.6 10.5 14.1 0.7 5.0 10.6 0.1 0.7

189 LC-UV 15.9 12.0 11.5 12.6 14.0 2.8 19.8 12.1 0.8 6.5

191 RIA 14.3 14.3 9.8 9.8 14.3 0.0 0.0 9.8 0.0 0.0

193 EIA 16.4 16.4 11.6 12.1 16.4 0.0 0.0 11.9 0.4 3.0

194 LC-MS/MS 12.5 13.7 10.9 11.9 13.1 0.8 6.5 11.4 0.7 6.2

195 LC-MS/MS 12.5 12.6 14.4 12.1 12.6 0.1 0.6 13.3 1.6 12.3

196 CLIA 13.9 13.8 9.7 9.9 13.9 0.1 0.5 9.8 0.1 1.3

197 LC-MS/MS 16.0 15.0 12.0 13.0 15.5 0.7 4.6 12.5 0.7 5.7

198a LC-MS/MS 12.6 15.8 11.2 8.5 14.2 2.3 15.9 9.9 1.9 19.4

198b EIA 15.0 16.0 11.0 10.0 15.5 0.7 4.6 10.5 0.7 6.7

199 LC-MS/MS 11.3 12.8 10.8 11.8 12.0 1.0 8.5 11.3 0.7 6.0

200 RIA 13.9 13.9 10.3 10.0 13.9 0.0 0.0 10.2 0.2 2.1

201 EIA 18.5 17.1 12.8 12.6 17.8 1.0 5.6 12.7 0.1 1.1

202 LC-MS/MS 13.6 12.8 11.6 11.2 13.2 0.6 4.5 11.4 0.3 2.4

203 LC-UV 17.2 17.5 17.1 18.0 17.3 0.2 0.9 17.6 0.6 3.6

204 CLIA 13.1 13.2 10.2 9.7 13.2 0.1 0.5 10.0 0.4 3.6

206 EIA 16.4 16.2 11.7 11.5 16.3 0.1 0.5 11.6 0.2 1.5

N 39 39 38 39 39 39

Median 13.9 13.8 11.6 11.8 14.0 11.8

MADe 1.9 1.9 1.9 1.9 2.1 1.9

CV% 13.9 14 16.0 16 14.9 15.8

N 16 16 16 16 16 16

Median 14.3 14.2 10.5 10.0 14.2 10.3

MADe 1.4 1.9 1.4 1.4 1.7 1.6 14

CV% 9.9 14 13.6 14 12 15 7

N 23 23 22 23 23 23

Median 13.5 12.8 12.0 12.3 13.2 12.2

MADe 1.5 2.6 1.4 1.1 1.5 1.3

CV% 11.0 20.6 11.7 9 11.2 10.7

N 17 17 17 17 17 17

Median 13.3 12.8 12.0 12.1 13.1 12.1

MADe 1.2 1.9 1.0 1.0 1.5 0.9

CV% 8.9 14.8 8.6 9 11.3 7.7

NIST Value 12.38 12.38 10.37 10.37 12.38 10.37

U 95 0.28 0.28 0.23 0.23 0.28 0.23 11

LC 

methods

LCMS/MS All methods IA methods 

SRM 968d L1 SRM 968d L2 SRM 968d L1 Combined SRM 968d L2 Combined
9

For all participant datasets, the mean values and error bars (representing ± 2 × SD) for 

25(OH)DTotal in SRM 968d L1 and SRM 968d L2 are plotted in Figure 3. The results from 

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

methods are displayed with black squares and are segregated by MS/MS detection (■) and 

UV/EC detection (). From the mean 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 dotted lines (- - - - -) represent the consensus variability (2 × MADe). 

Note that consensus statistics were not calculated for the UV and EC results because of the 

limited number of datasets. The laboratories with results that fall between the two dotted lines 

are within the consensus variability area for their technique (IA or LC). The NIST value for 

these materials is provided by a grey-shaded bar that represents the value and its associated 

uncertainty (± U95). 

Specific results as assessed from Figure 3 are summarized below.

SRM 968d L1

• For the immunoassay results, all laboratory data are within the consensus variability when 

the error bars for each data point are considered.

• For the LC-MS/MS results, all but one of the datasets are within the consensus variability. 

• The LC-UV and LC-EC techniques provide more variable results for 25(OH)D, with only 

three of the six data points falling in the consensus range when the error bars are considered.

• The median value for the IA results is slightly higher than the median value for the LCmethods.

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

• The NIST value is included within the consensus ranges for both IA and LC methods.

SRM 968d L2

• The IA results exhibit an asymmetrical distribution towards higher levels of 25(OH)D. 

• For the LC-MS/MS results, all but one of the datasets are within the consensus variability.

• The LC-UV and LC-EC techniques provide more variable results for 25(OH)D, with only 

one dataset in the consensus range.

• The consensus median for the LC method results is higher than the IA consensus median 

(opposite of what was observed for SRM 968d L1). 

• The LC consensus median is higher than the NIST value, whereas the consensus median for 

the IA results is directly comparable with the NIST value. 

• The NIST value is included within the consensus ranges for both IA and LC methods. 
10

Figure 3. 25(OH)DTotal levels in SRM 968d L1 and SRM 968d L2 as determined by 

immunoassay, LC-MS/MS, and LC-UV or LC-EC methods. Data that extend beyond the y-axis 

scale are denoted by an arrow. The grey-shaded bars represent the ranges bound by the NIST 

values with ± estimated U95 uncertainty. 
11

A direct comparison of results for 25(OH)DTotal between SRM 968d L1 and SRM 968d L2 is 

provided in the Youden plot in Figure 4. 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 in Figure 4. Conversely, 

laboratories that fall outside of (or on the edge of) either of the consensus ranges are not included 

in the blue consensus boxes and are highlighted with their laboratory code numbers. The NIST 

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

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

of the NIST values (10.37/12.38) for SRM 968d L2 and SRM 968d L1 across the magnitude of 

the y- and x-axis, respectively. Participant data (numbers 110, 180, 201, 186, 203, 169) that are 

near the Youden line but are above or below the consensus box suggest that these measurements 

may be biased high or low due to a calibration error. Laboratories that fall into this category 

may want to investigate how their calibrants are prepared, the purity of the calibrant material(s), 

and/or the method used to evaluate calibrant concentration (e.g., gravimetry vs. UVspectrophotometry). Several laboratories (numbers 62, 32, 184 and 139 (data not observable on 

y-axis scale)) provided results that were relatively consistent with consensus for the SRM 968d 

L1 material but not for SRM 968d L2 material. 

Figure 4. Comparison of results for 25(OH)DTotal for all methods. 

 IA method laboratory values 

 IA method consensus box encloses ± 2 MADe around consensus medians

 LC method laboratory values

 LC method consensus box encloses ± 2 MADe around consensus medians

 NIST values with corresponding Youden line 

032

062

110

169

180

184

186

201

203

5

10

15

20

25

5 10 15 20 25

25(OH)DTotal in SRM 968d L2 "Vials C&D" (ng/mL)

25(OH)DTotal in SRM 968d L1 "Vials A&B" (ng/mL)

IA methods

LC methods

139
12

As indicated in Figure 3 and Table 2, the NIST values for SRM 968d L1 and SRM 968d L2 are 

12.38 ng/mL ± 0.28 ng/mL, and 10.37 ng/mL ± 0.23 ng/mL, respectively. According to the 

current guidance regarding 25(OH)D levels and human health (obtained from the NIH website 

and presented in Table 3), the NIST data indicate that the concentrations in both materials are 

consistent with “inadequate” levels of vitamin D, but SRM 968d L2 may also be consistent with 

“deficient” 25(OH)D. SRM 968d L1 and SRM 968d L2 were selected because they are in the 

clinically-important, narrow range in which accurate and reliable measurements of 25(OH)D are 

important. The median participant results (for all methods) of 14.0 ng/mL and 11.8 ng/mL for 

SRM 968d L1 and SRM 968d L2, respectively, also indicate that the concentrations in these 

materials are consistent with “inadequate” 25(OH)D. However, the range of 25(OH)D values 

reported by program participants of 8.8 ng/mL to 20.0 ng/mL and 4.8 ng/mL to 94.8 ng/mL for 

SRM 968d L1 and SRM 968d L2, respectively, resulted in an overall program CV% of ≈ 16% 

(Table 2). Because the concentration ranges that distinguish inadequate and adequate levels of 

25(OH)D are narrow (Table 3), relatively precise within-laboratory measurements are necessary. 

One of the goals of the program is to reduce the consensus variability to better represent the 

community’s measurement capability while also recognizing that a “fit-for-purpose” variabilitylevel may exist. Another 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. 

One source of bias in the LC-MS/MS measurements is contribution from 3-epi-25(OH)D3, 

which coelutes with 25(OH)D3 using typical chromatographic columns (C8, C18) and responds 

to the same multiple reaction monitoring (MRM) conditions. The NIST method separates 

25(OH)D3 and 3-epi-25(OH)D3, which was detected but not quantitated in either material. This 

bias was estimated to be approximately 6% and 7% at NIST for SRM 968d L1 and SRM 968d 

L2, respectively, which might explain the difference observed for SRM 968d L1 but does not 

fully account for the difference observed for SRM 968d L2 (LC-MS/MS median 7% and 17% 

higher than the NIST value for SRM 968d L1 and SRM 968d L2, respectively). One of the 

LC-MS/MS participants (Lab 56) also noted the presence of 3-epi-25(OH)D3 in both study 

materials. It is unclear how the presence of 3-epi-25(OH)D3 affects the immunoassay results.
13

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

ng/mL nmol/L Health Status

<10-11 <25-27.5 Associated with vitamin D deficiency, leading to rickets 

in infants and children and osteomalacia in adults [1,2]

<10-15 <25-37.5 Generally considered inadequate for bone and overall 

health in healthy individuals [1,2]

≥15 ≥37.5 Generally considered adequate for bone and overall 

health in healthy individuals [1]

Consistently 

>200

Consistently >500 Considered potentially toxic, leading to hypercalcemia 

and hyperphosphatemia, although human data are 

limited. In an animal model, concentrations ≤400 ng/mL 

(≤1,000 nmol/L) demonstrated no toxicity [3,4].

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

[1] Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes: Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. 

Washington, DC: National Academy Press, 1997

[2] Scientific Advisory Committee on Nutrition. Update on Vitamin D. Position Statement by the Scientific Advisory Committee on Nutrition. 

London: The Stationery Office, Limited, 2007

[3] Jones G. Pharmacokinetics of vitamin D toxicity. Am J Clin Nutr 2008;88:582S-6S. [PubMed abstract]

[4] Shepard RM, DeLuca HF. Plasma concentrations of vitamin D3 and its metabolites in the rat as influenced by vitamin D3 or 245-

hydroxyvitamin D3 intakes. Arch Biochem Biophys 1980;202:43-53. [PubMed abstract]
14

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

Participant 

Number IA Method Sample Preparation Detection

17 CLIA n/r n/r

30 RIA 50 μL sample was extracted with 500 μL acetonitrile n/r

62 RIA RIA kit Gamma counter

141 EIA

Sample diluted 1:20 followed by 25(OH)D releasing reagent; 

ELISA plate precoated with 25(OH)D - sample added; free 

25(OH)D on plate competes to bind with antibody; a 

peroxidase conjugate added

OD reading at 450nm; color is 

inversely proportional to 

25(OH)D in sample

175 CLIA

1st incubation (10 min): 25-OH-D is dissociated from its 

binding protein and binds to the specific antibody on the solid 

phase; tracer (vitamin D linked to an isoluminol derivative) is 

added 10 min later. 2nd incubation: unbound material 

removed with a wash cycle; starter reagents added; a flash 

chemiluminescent reaction initated

Photomultiplier measures 

relative light units, which is 

inversely proportional to the 

concentration of 25(OH)D 

present in calibrators, controls or 

samples. 

180 RIA Samples were prepared per vendor's sample extraction 

protocol

 I125 detection using gamma 

counter

183b CLIA None; calibrators diluted to get samples in analytical range n/r

188 EIA n/r n/r

191 RIA n/r n/r

193 EIA Samples centrifuged OD reading

196 CLIA The human serum samples were thawed and analyzed n/r

198b EIA n/r n/r

200 RIA Sample was extracted n/r

201 EIA Sample was thawed and swirled prior to analysis OD reading at 450 nm

204 CLIA Sample was well-mixed prior to analysis n/r

206 EIA n/r n/r

OD = optical density

n/r = not reported
15

Appendix A-2. Summary of LC-MS/MS methods used by participants. 

Participant 

Number

Internal Standard 

(IS) Sample Preparation Chromatographic Conditions Detection: MRM ions

26

Deuterated 

25(OH)D2 and 

25(OH)D3

Sample was extracted, 

evaporated, and reconstituted 

with methanol/water

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-d3 and 

25(OH)D3-d6

Liquid-liquid extraction PFP column; isocratic elution; flow 

0.4 mL/min

25(OH)D3 383/365; 

25(OH)D3-d6 389/371; 

25(OH)D2 395/377; 

25(OH)D2-d3 398/380

116 25(OH)D3 -d6

Serum proteins were precipitated 

with methanol

LC column; Isocratic separation with 

95% methanol, 5% water; flow 0.6 

mL/min; Online SPE

25(OH)D3 383/211; 

25(OH)D3-d6 389/211 ; 

25(OH)D2 395/269

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

150 25(OH)D2-d6 and 

25(OH)D3-d3

The samples were liquid-liguid 

extracted, centrifuged, separated, 

evaporated under nitrogen, 

reconstituted in mobile phase, and 

analyzed

LC column (100 x 2.1 mm); Isocratic 

separation with 74% methanol, 26% 

water; flow 0.5 mL/min

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

25(OH)D3-d3 404/386; 

25(OH)D2 413/355, 413/361; 

25(OH)D2-d6 419/401

182 25(OH)D3 -d6

Proteins were precipitated with 

acetonitrile and IS directly in 96 

well plate

C18 column (50 x 2 mm); Gradient 

from 60% to 100% methanol

25(OH)D3 401/365 (quant), 

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

413/355 (quant), 413/271 

(qual); 25(OH)D3-d6 407/371 

(quant), 407/389 (qual)

183a 25(OH)D3 -d6

IS (25 μL) was added to sample 

(150 μL), followed by protein 

precipiation and extraction with 

0.1 mol/L ZnSO4 (150 μL), 

methanol (300 μL), and hexane 

(750 μL); extract dried and 

dissolved with 70% methanol, 

30% water with 2 mmol/L 

ammonium acetate

C8 column (50 x 2.1 mm); isocratic 

elution with 73% methanol, 27% 

water; flow 0.4 mL/min

25(OH)D3 401/159, 401/383; 

25(OH)D2 413/82, 413/395

184 25(OH)D3 -d6

Serum (200 μL) extracted with 

acetonitrile and IS (700 μL); 

mixed, centrifuged, and filtered

C18 column (100 x 2.1mm; 5μm); 

linear gradient from 60% B to 98% 

B over 2 min (A: 0.1% formic acid in 

water, B: methanol with 0.1% formic 

acid and 5 mmol/L ammonium 

acetate)

25(OH)D3 383/257; 

25(OH)D3-d6 389/263; 

25(OH)D2 395/209 (APCI)

185 25(OH)D2-d6 and 

25(OH)D3-d6

Liquid-Liquid extraction C18 (50 x 2 mm); flow 0.4 mL/min; 

methanol gradient (3.6 min) n/r

186 25(OH)D3 -d6

Protein precipitation with 

methanol, liquid-liquid extraction 

with hexane followed by 

centrifugation, evaporation and 

reconstitution in mobile phase

Phenyl LC column (50 x 2.1 mm; 

1.7μm); temp. 35° C 25(OH)D3 383/159 (ESI)

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

194 25(OH)D3 -d6

Proteins precipitated, followed by 

centrifugation, evaporation of top 

layer, and reconstitution

C8 column (50 x 2 mm); isocratic 

elution with 70% acetonitrile/ 30% 

water; flow 0.7 mL/min

25(OH)D3 383/211; 

25(OH)D2 395/119

195 25(OH)D2-d3 and 

25(OH)D3-d6

Samples extracted then 

derivatized

LC column (30 x 2.1 mm); gradient 

with methanol/water n/r
16

197 25(OH)D3 -d6

Precipitating agent added (200 μL 

with 20 ng IS) to each serum (200 

μL), calibrator and control sample 

followed by mixing, centrifugation, 

and analysis

C18 column (50 x 4.6 mm; 5 μm); 

flow 1.0 mL/min; column temp 45°C; 

gradient with water and methanol

n/r

198a 25(OH)D3 -d6

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 um); 

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-d6 407/389, 

407/371

199 25(OH)D2-d3 and 

25(OH)D3-d6

Sample (100 μL) extracted with 

acetonitrile (200 μL) containing 

IS, followed by mixing (15 s), 

equilibration (30 min @ 4°C), 

mixing (15 s), centrifugation (10 

min), analysis of supernatant

C18 column (50 x 2.1mm, 5 μm); 

gradient with 0.1% formic acid in 

water and 0.1% formic acid in 

methanol; flow 0.7mL/min

25(OH)D3 383/211, 383/257, 

383/365; 25(OH)D3-d6 

389/211, 389/263, 389/371; 

25(OH)D2 395/209, 395/211, 

395/269, 395/377; 25(OH)D2-

d3 398/211, 398/230, 

398/272, 398/380

202 D6-labeled 

compound Sample was extracted

C18 column (50 x 2.1 mm); 

Gradient with 10% acetonitrile 

(containing 0.1% formic acid), 90% 

methanol; flow 0.3 mL/min

n/r

MRM= multiple reaction monitoring

PFP = pentafluorophenyl phase

SPE = solid phase extraction

quant = quantitative ions

qual = qualitative ions

ESI = electrospray ionization

APCI = atmospheric pressure chemical ionization

n/r = not reported
17

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

Participant 

Number

Internal Standard 

(IS) Sample Preparation Chromatographic Conditions Detection

32 Proprietary Samples were extracted with 

filtration

C18 column (300 x 3.9 mm; 4 μm); 

proprietary mobile phase; flow 

0.7mL/min

UV at 265 nm

110 None

Ethanol (0.3-0.5 mL) used to 

precipiate proteins in serum 

(0.3–0.5 mL); sample extracted 

with two volumes of hexanedichloromethane, evaporated, 

then dissolved

LC C18 column (100 x 2.1 mm; 1.8 

μm); C18 column (220 x 4.6 mm; 5 

μm); Column temp 35°C and 21°C; 

gradient: from 5% B to 50% B from 

0.8 min to 2 min, hold until 3.5 min 

(A: 85% acetonitrile, 15% methanol, 

B: isopropanol); flow 0.5 ml/min

UV at 267 nm

139 Proprietary

Samples extracted by 

precipitation, centrifuged, and an 

aliquot injected directly

Column and mobile phase are 

proprietary UV at 264 nm

169 Proprietary SPE Column and mobile phase are 

proprietary

EC at 450 mv (screening 

electrode) and 750 mv 

(measuring electrode)

189 Obtained from kit 

supplier

Proteins were precipitated, 

samples centrifuged, analytes in 

the supernatant were extracted 

using SPE cartridges

LC column (4.6 x150 mm); isocratic 

separation with commercial mobile 

phase; flow 0.7mL/min

UV at 265 nm

203 n/r

Serum (0.5 mL) precipitated with 

methanol/isopropanol solution, 

and then extracted with hexane

LC column (150 x 4.6 mm); isocratic 

separation with methanol/TFA ; flow 

0.6 mL/min 

UV at 265 nm

SPE = solid phase extraction

TFA = trifluoroacetic acid

n/r = not reported
18

Appendix B. Raw participant data for 25(OH)D2, 25(OH)D3 and 25(OH)DTotal.

SRM 968d L1 SRM 968d L1 SRM 968d L2 SRM 968d L2 SRM 968d L1 SRM 968d L1 SRM 968d L2 SRM 968d L2 SRM 968d L1 SRM 968d L1 SRM 968d L2 SRM 968d 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 11.4 12.5 8.8 9.2 n/r n/r

026 LC-MS/MS < 1.0 < 1.0 < 1.0 < 1.0 13.1 11.1 9.8 11.6 13.1 11.1 9.8 11.6 n/r n/r

030 RIA n/a n/a n/a n/a n/a n/a n/a n/a 13.6 13.7 9.2 8.9 239.9 341.9

032 LC-UV n/d n/d 3.5 3.6 13.5 14.6 14.9 14.9 13.5 14.6 18.4 18.5 215.0 333.0

056 LC-MS/MS 0.3 n/d 0.6 0.6 13.4 11.0 10.7 10.4 13.7 11.0 11.3 11.0 255.3 334.8

062 RIA n/a n/a n/a n/a n/a n/a n/a n/a 14.2 14.1 16.9 17.2 245.0 575.9

110 LC-UV n/d n/d n/d n/d 7.6 9.9 7.0 6.8 7.6 9.9 7.0 6.8 247.0 315.0

116 LC-MS/MS < 3.3 < 3.3 < 3.3 < 3.3 13.3 12.6 13.1 10.7 13.3 12.6 13.1 10.7 237.3 364.6

128 LC-MS/MS n/d n/d n/d n/d 12.6 12.1 12.7 12.2 12.6 12.1 12.7 12.2 n/r n/r

139 LC-UV n/r n/r n/r n/r 9.2 9.1 n/d 94.8 9.2 9.1 n/d 94.8 328.9 368.5

141 EIA n/a n/a n/a n/a n/a n/a n/a n/a 9.7 12.1 4.1 5.5 n/r n/r

150 LC-MS/MS n/d n/d n/d n/d 15.0 15.1 12.2 11.3 15.0 15.1 12.2 11.3 218.0 291.0

169 LC-ECD 7.0 7.6 9.6 9.1 12.9 12.5 10.0 9.9 19.9 20.1 19.6 19.0 241.9 339.5

175 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 15.7 15.8 11.1 11.3 169.5 254.0

180 RIA n/a n/a n/a n/a n/a n/a n/a n/a 17.3 16.6 14.1 14.3 n/r n/r

182 LC-MS/MS 0.6 0.7 1.3 1.4 11.1 10.8 10.7 10.9 11.7 11.5 12.0 12.3 235.2 326.6

183a LC-MS/MS < 4 < 4 < 4 < 4 13.5 14.9 12.0 12.8 13.5 14.9 12.0 12.8 199.0 326.0

183b CLIA n/a n/a n/a n/a n/a n/a n/a n/a 14.2 14.3 9.8 10.0 n/r n/r

184 LC-MS/MS < 1.0 < 1.0 1.1 < 1.0 12.5 12.7 22.8 22.5 12.5 12.7 23.9 22.5 230.2 325.0

185 LC-MS/MS n/d n/d n/d n/d 14.7 14.8 12.6 12.7 14.7 14.8 12.6 12.7 n/r n/r

186 LC-MS/MS n/r n/r n/r n/r 18.6 19.0 15.8 13.0 18.6 19.0 15.8 13.0 n/r n/r

187 LC-MS/MS n/d n/d 1.0 0.9 14.1 12.9 10.9 11.4 14.1 12.9 11.9 12.3 210.0 331.0

188 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 14.6 13.6 10.6 10.5 n/r n/r

189 LC-UV n/d n/d n/d n/d 15.9 12.0 11.5 12.6 15.9 12.0 11.5 12.6 n/r n/r

191 RIA n/a n/a n/a n/a n/a n/a n/a n/a 14.3 14.3 9.8 9.8 303.3 465.8

193 EIA n/a n/a n/a n/a n/a n/a n/a n/a 16.4 16.4 11.6 12.1 123.6 191.2

194 LC-MS/MS < 7 < 7 < 7 < 7 12.5 13.7 10.9 11.9 12.5 13.7 10.9 11.9 242.5 346.0

195 LC-MS/MS < 4.0 < 4.0 < 4.0 < 4.0 12.5 12.6 14.4 12.1 12.5 12.6 14.4 12.1 237.0 330.0

196 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 13.9 13.8 9.7 9.9 364.2 562.0

197 LC-MS/MS < 5 < 5 < 5 < 5 16.0 15.0 12.0 13.0 16.0 15.0 12.0 13.0 247.5 337.5

198a LC-MS/MS < 5 < 5 < 5 < 5 12.6 15.8 11.2 8.5 12.6 15.8 11.2 8.5 286.7 406.4

198b EIA n/a n/a n/a n/a n/a n/a n/a n/a 15.0 16.0 11.0 10.0 n/r n/r

199 LC-MS/MS 0.04 0.05 0.4 0.4 11.3 12.7 10.4 11.4 11.3 12.8 10.8 11.8 244.0 336.0

200 RIA n/a n/a n/a n/a n/a n/a n/a n/a 13.9 13.9 10.3 10.0 245.0 373.0

201 EIA n/a n/a n/a n/a n/a n/a n/a n/a 18.5 17.1 12.8 12.6 n/r n/r

202 LC-MS/MS n/d n/d n/d n/d 13.6 12.8 11.6 11.2 13.6 12.8 11.6 11.2 234.9 352.8

203 LC-UV n/d n/d n/d n/d 17.2 17.5 17.1 18.0 17.2 17.5 17.1 18.0 206.8 377.6

204 CLIA n/a n/a n/a n/a n/a n/a n/a n/a 13.1 13.2 10.2 9.7 189.0 337.0

206 EIA n/a n/a n/a n/a n/a n/a n/a n/a 16.4 16.2 11.7 11.5 n/r n/r

NIST Value <0.5 <0.5 <0.5 <0.5 12.38 12.38 10.37 10.37 12.38 12.38 10.37 10.37 238.6 334.8

U 95 0.0 0.0 0.0 0.0 0.28 0.28 0.23 0.23 0.28 0.28 0.23 0.23 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)