NISTIR 7892

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Winter 2011 

Comparability Study (Exercise 3)

Mary Bedner

Katrice A. Lippa

http://dx.doi.org/10.6028/NIST.IR.7892
NISTIR 7892

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Winter 2011 

Comparability Study (Exercise 3)

Mary Bedner

Katrice A. Lippa

Chemical Sciences Division

Material Measurement Laboratory

http://dx.doi.org/10.6028/NIST.IR.7892

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) 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 third exercise of this program, the 

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

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

study materials consisted of SRM 972 Vitamin D in Human Serum (Level 3) and SRM 968d FatSoluble 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, 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 third exercise are reported along with a summary of the analytical 

methods used. 
2

OVERVIEW OF THE WINTER 2011 COMPARABILITY STUDY

For the Winter 2011 Comparability Study (Exercise 3) 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 three samples (vials A, B, and C) of human 

serum (study materials). In this exercise, vial A was SRM 972 Vitamin D in Human Serum Level 3 

(SRM 972 L3), which is a blended human serum pool with an endogenous 25(OH)D3 level but an 

augmented 25(OH)D2 level. Vials B and C 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 that contains endogenous vitamin D metabolite levels. 

In the Winter 2011 exercise, there were a total of 33 participants and 35 datasets (two participants

provided data for two different methods). Fourteen of the datasets originated from immunoassay 

(IA) techniques, including four 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-one of the datasets originated from liquid 

chromatographic (LC) methods; of those, 16 were from LC with tandem mass spectrometric 

detection (LC-MS/MS) and five were from LC with ultraviolet absorbance detection (LC-UV). A 

summary of the LC methods used by the participants may be found in Appendices A-2 and A-3. 

The raw data received from all participants is summarized in Appendix B. For SRM 972 L3 and 

SRM 968d L1, the immunoassay methods reported values for 25(OH)DTotal only, whereas LC-based 

methods reported values for 25(OH)D2, 25(OH)D3 and 25(OH)DTotal. The LC participant results 

reveal that SRM 972 L3 contains significant levels of both 25(OH)D3 and 25(OH)D2 that contribute 

to 25(OH)DTotal. Conversely, SRM 968d L1 contains very low levels of 25(OH)D2 with only two 

the LC participants reporting values (ranging from 0.18 ng/mL to 1.80 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. 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 SRM 968d L1 was below the quantitation limit (≈ 0.5 ng/mL) for the NIST method.
3

WINTER 2011 EXERCISE RESULTS AND DISCUSSION

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

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

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

the SRM 2972 control solutions is provided in Table 1.

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. 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%). Table 1 also presents the NIST 

certified values with expanded uncertainties corresponding to 95% confidence.
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.

 n/r = not reported

SRM 2972 SRM 2972

Lab Method Value Lab Method Value

030 RIA 252.5 1.3 030 RIA 348.0

032 LC-UV 261.0 3.0 032 LC-UV 300.9

056 LC-MS/MS 239.6 2.0 056 LC-MS/MS 333.4

062 RIA n/r 1.3 062 RIA 329.8

110 LC-UV 218.0 3.0 110 LC-UV 326.0

116 LC-MS/MS 236.8 2.0 116 LC-MS/MS 390.7

139 LC-UV 262.2 3.0 139 LC-UV 325.8

150 LC-MS/MS 253.0 2.0 150 LC-MS/MS 398.0

160b CLIA 125.0 1.1 160b CLIA 225.0

183a LC-MS/MS 185.0 2.0 183a LC-MS/MS 329.0

183b CLIA 191.0 1.1 183b CLIA 262.0

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

194 LC-MS/MS 230.0 2.0 194 LC-MS/MS 336.5

195 LC-MS/MS 235.0 2.0 195 LC-MS/MS 332.0

196 CLIA 199.5 1.1 196 CLIA 477.5

197 LC-MS/MS 205.0 2.0 197 LC-MS/MS 290.0

198a LC-MS/MS 250.7 2.0 198a LC-MS/MS 337.0

199 LC-MS/MS 233.0 2.0 199 LC-MS/MS 331.0

200 RIA 187.5 1.3 200 RIA 280.6

201 EIA 171.0 1.2 201 EIA 341.0

202 LC-MS/MS 228.2 2.0 202 LC-MS/MS 345.2

205 LC-MS/MS 265.4 2.0 205 LC-MS/MS 337.4

209 LC-MS/MS 237.6 2.0 209 LC-MS/MS 318.6

210 RIA 142.9 1.3 210 RIA 269.2

211 LC-MS/MS 311.5 2.0 211 LC-MS/MS 423.0

212 LC-MS/MS 242.3 2.0 212 LC-MS/MS 333.7

N 25 N 26

Median 235.0 Median 332.7

MADe 26.7 MADe 15.4

CV% 11.4 CV% 4.6

N 7 N 8

Median 187.5 236 Median 305.2

MADe 24.5 139 MADe 58.5

CV% 13.0 139 CV% 19.2

N 18 N 18

Median 238.1 271 Median 333.6

MADe 16.7 205 MADe 9.0

CV% 7.0 205 CV% 2.7

N 15 N 15

Median 237.6 Median 334.8

MADe 11.3 MADe 5.6

CV% 4.7 CV% 1.7

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

IA 

methods 

LC 

methods

LCMS/MS
5

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

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

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

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

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

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

Consensus statistics were not calculated for the UV results because of the limited number of 

datasets (N = 3). 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.
6

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

LC-UV methods. The grey-shaded bars represent the ranges bound by the NIST certified values 

with ± U95 expanded uncertainty. 
7

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

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

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

2. Conversely, laboratory results that fall outside of (or on the edge of) the blue consensus 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 160b, 210, 211) that are near the Youden line but are clearly 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.

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 

032

116 150

160b

183b

196

197

200

201

210

211

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)
8

25(OH)D in SRM 972 L3 and SRM 968d L1

A summary of the individual participant data for 25(OH)DTotal in samples SRM 972 L3 and SRM 

968d L1 (vial A and vials B & C, respectively) is provided in Table 2. The summarized data 

include the 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-MS/MS methods only. These 

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

MADe, and the CV%.

Table 2 also presents the NIST results for the two study materials. For SRM 972 L3, the NIST 

result is the sum of the certified values for 25(OH)D3 and 25(OH)D2, and the 95% confidence limit 

(U95) was approximated using the individual uncertainties reported for the two analytes in the 

Certificate of Analysis.1 For SRM 968d L1, the NIST value for 25(OH)D3 was obtained using an 

LC-MS/MS reference measurement procedure2 recognized by the Joint Committee for Traceability 

in Laboratory Medicine (JCTLM), and the U95 confidence interval includes components for both 

measurement variability (N = 8) and measurement uncertainty associated with the density. The 

25(OH)D2 was below the quantitation limit (≈ 0.5 ng/mL) in SRM 968d L1 for the NIST method.

 1 https://www-s.nist.gov/srmors/view_cert.cfm?srm=972 2 Tai, S. S.-C., Bedner, M. and Phinney, K.W. Anal. Chem. 2010 82, 1942-1948.
9

Table 2. Summary of participant data for 25(OH)DTotal (ng/mL) in SRM 972 L3 and SRM 968d 

L1. 

SRM 972 L3

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

017 CLIA 38.1 17.0 18.3 17.7 0.9 5.2

026 LC-MS/MS 43.7 12.4 12.5 12.5 0.1 0.6

030 RIA 41.8 19.5 18.5 19.0 0.7 3.7

032 LC-UV 49.6 11.1 10.8 11.0 0.2 1.9

056 LC-MS/MS 46.2 11.7 12.9 12.3 0.9 7.1

062 RIA 25.5 12.3 12.5 12.4 0.1 1.1

110 LC-UV 16.4 14.3 10.6 12.5 2.6 21

116 LC-MS/MS 47.0 12.2 13.3 12.8 0.8 6.1

139 LC-UV 44.7 13.7 12.6 13.1 0.8 6.0

150 LC-MS/MS 49.0 16.0 15.0 15.5 0.7 4.6

160b CLIA 31.0 13.0 12.0 12.5 0.7 5.7

180 RIA 29.2 13.1 13.0 13.0 0.1 0.8

183a LC-MS/MS 55.0 15.0 16.0 15.5 0.7 4.6

183b CLIA 25.7 13.1 14.5 13.8 1.0 7.2

185 LC-MS/MS 49.3 12.6 12.7 12.6 0.0 0.2

188 CLIA 23.8 14.6 15.2 14.9 0.4 2.8

189 LC-UV 44.2 9.1 9.6 9.4 0.4 3.8

194 LC-MS/MS 46.4 14.7 12.0 13.4 1.9 14

195 LC-MS/MS 52.9 16.0 15.5 15.8 0.4 2.2

196 CLIA 34.8 16.2 16.9 16.6 0.5 3.0

197 LC-MS/MS 41.0 11.0 10.0 10.5 0.7 6.7

198a LC-MS/MS 47.1 14.7 14.6 14.7 0.1 0.5

198b EIA 33.0 15.0 16.0 15.5 0.7 4.6

199 LC-MS/MS 41.6 11.5 11.6 11.6 0.1 0.6

200 RIA 32.8 14.0 14.0 14.0 0.0 0.0

201 EIA 22.4 14.6 15.2 14.9 0.4 2.8

202 LC-MS/MS 53.0 14.6 14.3 14.5 0.2 1.5

205 LC-MS/MS 50.4 13.8 13.4 13.6 0.3 2.2

207 LC-UV 37.6 26.3 13.0 19.7 9.4 48

209 LC-MS/MS 39.2 14.4 13.8 14.1 0.4 3.0

210 RIA 34.1 12.3 15.3 13.8 2.1 15

211 LC-MS/MS 61.3 15.2 16.4 15.8 0.8 5.4

212 LC-MS/MS 46.7 13.9 13.7 13.8 0.1 1.0

213 EIA 26.0 17.6 14.6 16.1 2.1 13

214 CLIA 28.5 13.5 13.6 13.6 0.1 0.5

N 35 35 35 35

Median 41.6 14.0 13.7 13.8

MADe 11.5 1.8 1.9 2.0

CV% 27.5 13 14.1 14.5

N 14 14 14 14

Median 30.1 14.3 14.9 14.5

MADe 6.3 1.9 1.8 1.8

CV% 20.8 13 12.0 13

N 21 21 21 21

Median 46.7 13.9 13.0 13.4 55.6 16.6

MADe 4.4 1.9 1.9 1.6 37.8 10.1

CV% 9.5 13.9 14.8 12.2

N 16 16 16 16

Median 47.1 14.2 13.5 13.7

MADe 4.9 1.9 1.6 1.7

CV% 10.5 13.5 11.5 12.6

NIST Value 44.90 12.38 12.38 12.38 47.2

U 95 2.28 0.28 0.28 0.28 42.6

All 

methods IA methods 

SRM 968d L1 SRM 968d L1 Combined

LC 

methods

LCMS/MS
10

For all participant datasets, the single reported values for 25(OH)DTotal in SRM 972 L3 and the 

mean values with error bars (representing the lab mean value ± 2 × SD) for 25(OH)DTotal in SRM 

968d L1 are plotted in Figure 3. The results from immunoassay methods are displayed with closed 

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

segregated by MS/MS detection (■) and UV detection (). For the IA and LC 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 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 972 L3

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

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

• The consensus median value for the IA results is 36% lower than the median value for all LC 

methods.

• The consensus median value for the LC results falls within the NIST expanded uncertainty 

range (grey-shaded bar).

• There is no overlap between the NIST value and the consensus range for IA.

SRM 968d L1

• For both the IA and LC results, all but three of the mean value data points are within the 

consensus variability range.

• The consensus median for the IA results is 8% higher than the consensus median value for the 

LC results. 

• Both the IA and LC consensus median values are higher than the NIST value. 

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

The IA and LC techniques perform differently for SRM 972 L3, which contains native levels of 

25(OH)D3 but an augmented 25(OH)D2 level. The augmented 25(OH)D2 is not bound in the serum 

in the same manner as the native 25(OH)D3 and 25(OH)D2, which leads to an underrepresentation 

on the 25(OH)DTotal with the IA methods. The NIST value for SRM 972 L3 was obtained with a 

combination of LC-MS and LC-MS/MS methods, which explains the better agreement with the 

participant LC results.
11

Figure 3. 25(OH)DTotal levels in SRM 972 L3 and SRM 968d L1 as determined by immunoassay, 

LC-MS/MS and LC-UV methods. The grey-shaded bars represent the ranges bound by the NIST 

values with ± estimated U95 uncertainty.
12

A direct comparison of results for 25(OH)DTotal between SRM 972 L3 and SRM 968d L1 is 

provided in the Youden plot in Figure 4. Because of the bimodal results obtained for SRM 972 L3, 

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 ranges are not included in the blue consensus box and are highlighted with their 

laboratory code numbers (numbers 110, 207, 30, 189, 211).

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 (12.38/44.90) for SRM 968d L1 and SRM 972 L3 across the magnitude of the yand x-axis, respectively. The Youden line runs directly through the LC consensus box because the 

NIST values were obtained with LC methods as previously mentioned. 

Figure 4. Comparison of results for 25(OH)DTotal for all methods. Data that fall outside the 

consensus boxes are labeled with their laboratory number. 

 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 

030

110

189

207

211

5

10

15

20

25

5 15 25 35 45 55 65

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

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

IA methods

LC methods
13

A summary of the individual participant data obtained with LC methods for 25(OH)D3 in samples 

SRM 972 L3 and SRM 968d L1 (vial A and vials B & C, respectively) is provided in Figure 5,

which also includes a Youden plot of the results. Laboratory results that are within the consensus 

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

outside of (or on the edge of) the blue consensus box are highlighted with their laboratory code 

numbers, which includes three laboratories that use LC-UV (110, 189, 207) and one that uses LCMS/MS (211). 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 (12.38/18.50) for 25(OH)D3 across the magnitude of the y- and x-axis, respectively. 

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

was detected but not quantitated in either material. The 3-epi-25(OH)D3 coelutes with 25(OH)D3

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

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

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

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

the 25(OH)D3 values reported by the rest of the LC participants 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.
14

 LC method laboratory values

 LC method consensus box encloses ± 2 MADe around consensus medians

 NIST values with corresponding Youden line 

110 189

207

211

5

10

15

20

25

5 15 25 35

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

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

LC methods

Figure 5. Summary of participant data obtained with LC methods for 25(OH)D3 (ng/mL) in SRM 

972 L3 and SRM 968d L1 and the resulting Youden comparison plot. Data that fall outside the 

consensus boxes are labeled with their laboratory number.

SRM 972 L3

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

026 LC-MS/MS 19.6 12.4 12.5 12.5 0.1 0.6

032 LC-UV 18.2 11.1 10.8 11.0 0.2 1.9

056 LC-MS/MS 19.8 11.7 12.9 12.3 0.9 7.1

110 LC-UV 7.9 11.5 7.4 9.5 2.9 31

116 LC-MS/MS 20.7 12.2 13.3 12.8 0.8 6.1

139 LC-UV 16.8 13.7 12.6 13.1 0.8 6.0

150 LC-MS/MS 22.0 16.0 15.0 15.5 0.7 4.6

183a LC-MS/MS 22.0 15.0 16.0 15.5 0.7 4.6

185 LC-MS/MS 20.4 12.6 12.7 12.6 0.0 0.2

189 LC-UV 24.8 9.1 9.6 9.4 0.4 3.8

194 LC-MS/MS 18.7 14.7 12.0 13.4 1.9 14

195 LC-MS/MS 23.6 16.0 15.5 15.8 0.4 2.2

197 LC-MS/MS 17.0 11.0 10.0 10.5 0.7 6.7

198a LC-MS/MS 18.0 14.7 14.6 14.7 0.1 0.5

199 LC-MS/MS 16.8 11.5 11.6 11.6 0.1 0.6

202 LC-MS/MS 20.5 14.6 14.3 14.5 0.2 1.5

205 LC-MS/MS 20.9 13.6 13.2 13.4 0.3 2.2

207 LC-UV 17.0 26.3 13.0 19.6 9.4 48

209 LC-MS/MS 18.3 14.4 13.8 14.1 0.4 3.0

211 LC-MS/MS 27.9 15.2 16.4 15.8 0.8 5.4

212 LC-MS/MS 19.1 13.9 13.7 13.8 0.1 1.0

N 21 21 21 21

Median 19.6 13.7 13.0 13.4

MADe 2.4 2.2 1.9 1.9

CV% 12.1 15.9 14.8 14.4

N 16 16 16 16

Median 20.1 14.2 13.5 13.6

MADe 2.3 1.9 1.6 1.6

CV% 11.6 13.5 11.5 12.0

NIST Value 18.50 12.38 12.38 12.38

U 95 1.10 0.28 0.28 0.28

SRM 968d L1 SRM 968d L1 Combined

LC 

methods

LCMS/MS
15

Correlation of 25(OH)D in SRM 972 L3 and SRM 968d L1 with Clinical Ranges

As indicated in Table 2, the NIST values for 25(OH)DTotal in SRM 972 L3 and SRM 968d L1 are 

44.90 ng/mL ± 2.28 ng/mL and 12.38 ng/mL ± 0.28 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 concentration in SRM 972 L3 is consistent 

with “adequate” levels of vitamin D, but SRM 968d L1 is in the “inadequate” range. The median 

participant results (for all methods) of 41.6 ng/mL and 13.8 ng/mL for SRM 972 L3 and SRM 968d 

L1, respectively, also indicate that the concentrations in these materials are consistent with 

“adequate” and “inadequate” 25(OH)D, respectively. However, the range of 25(OH)D values 

reported by program participants of 16.4 ng/mL to 61.3 ng/mL and 9.4 ng/mL to 19.7 ng/mL for 

SRM 972 L3 and SRM 968d L1, respectively, resulted in an overall program CV% ranging from 

15% to 28% (Table 2).

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.

A graphical representation of the single reported values for 25(OH)DTotal in SRM 972 L3 and the 

mean values with error bars (representing the lab mean value ± 2 × SD) for 25(OH)DTotal in SRM 

968d L1 overlaid with the clinical ranges for 25(OH)D from Table 3 is presented in Figure 6. The 

majority of the participant results for SRM 972 L3 indicate this study material has adequate 

25(OH)D, but the reported results range from inadequate to potentially adverse levels of 25(OH)D. 

For SRM 968d L1, the range of reported values is smaller but includes values that are in the 

deficient as well as inadequate regions. 

As indicated in previous reports, 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. 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. The significant difference in results between the IA and LC 

results for SRM 972 L3 indicates that study materials with strictly native levels of 25(OH)D will be 

critical to help meet these goals in future exercises.
16

Figure 6. 25(OH)DTotal levels in SRM 972 L3 and SRM 968d L1 superimposed over clinically 

relevant serum 25-hydroxyvitamin D (25(OH)DTotal) concentration levels as reported by NIH 

(Table 2). 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.

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; sample was extracted n/r

160b CLIA Samples were thawed, swirled, and analyzed. n/r

180 RIA Samples were prepared per vendor sample extraction protocol I

125 detection using 

Gamma counter

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

188 EIA n/r n/r

196 CLIA The human serum samples were analyzed neat n/r

198b EIA n/r n/r

200 RIA Sample was extracted n/r

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

210 RIA n/r n/r

213 EIA n/r n/r

214 CLIA n/r n/r

n/r = not reported

OD = optical density
18

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 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 and 

25(OH)D3-d 6

Samples were extracted with 

hexane, evaporated, then 

reconstituted with 69% methanol

PFP column (100 x 2.1 mm; 1.9 

μm); isocratic elution; flow 0.4 

mL/min

25(OH)D3 383/365; 

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

25(OH)D2 395/377; 

25(OH)D2-d 3 398/380

116 25(OH)D3 -d 6

Serum proteins were precipitated 

with methanol

LC column; isocratic separation with 

95% methanol, 5% water; flow 0.6 

mL/min; online SPE

25(OH)D3 383/211; 

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

25(OH)D2 395/269

150 Deuterated stable 

isotope compounds

The samples were liquid-liguid 

extracted, centrifuged, separated, 

evaporated, and reconstituted in 

mobile phase

LC column (100 x 2.1 mm); Isocratic 

separation with 74% methanol, 26% 

water; flow 0.5 mL/min

2 MRM transitions per 

analyte

183a 25(OH)D3 -d 6

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 

NH4C2H3O2

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

185 25(OH)D2-d 6 and 

25(OH)D3-d 6

n/r C18 (50 x 2 mm); methanol/water 

gradient

25(OH)D2 413/355; 

25(OH)D3 401/365

194 25(OH)D3 -d 6

Proteins precipitated with 

acetonitrile, top layer aspirated 

then evaporated under nitrogen 

and reconstituted in methanol

C8 column (50 x 2 mm); isocratic 

elution with 98% acetonitrile, 2% 

water

25(OH)D3 383/211; 

25(OH)D2 395/119

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); 

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 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-d 6 407/389, 

407/371

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

202 d 6-labeled 

compound Sample was extracted

C18 column (50 x 2.1 mm); 

Gradient with 10% acetonitrile 

(0.1% formic acid), 90% methanol; 

flow 0.3 mL/min

n/r

205 25(OH)D2-d 3 and 

25(OH)D3-d 3

Proprietary method Proprietary conditions n/r
19

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

209 25(OH)D3 -d 6

Proteins in the samples (100 μL) 

were precipitated with 5% ZnSO4 

in methanol containing IS (200 

μL), followed by an incubation 

period, centrifugation, and 

analysis of the supernatant (10 

μL).

C8 column (50 x 2 mm; 5 μm); 

gradient with water/methanol; flow 

0.7 mL/min

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

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

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

211 25(OH)D3 -d 6

Extraction with acetonitrile 

containing IS followed by 

centrifugation

Column (33 x 4.6 mm; 3 μm); 

turboflow with methanol/water 

gradient

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

383/257 (qual); 25(OH)D2 

395/377 (quant), 395/209 

(qual)

212 25(OH)D3 -d 6

IS (100 μL) added to sample (200 

μL) then extracted with hexane (1 

mL). Hexane (700 μL) evaporated 

and reconstituted with 1:1 

methanol:water (150 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

PFP = pentafluorophenyl

MRM = multiple reaction monitoring

SPE = solid phase extraction

quant = quantitative ions

qual = qualitative ions

n/r = not reported

Participant 

Number

Internal Standard 

(IS) Sample Preparation Chromatographic Conditions Wavelength

32 Proprietary Samples were extracted with 

filtration

C18 column (300 x 3.9 mm; 4 μm); 

proprietary mobile phase; flow 

0.7mL/min

265 nm

110 n/r n/r n/r n/r

139 Proprietary

The sample was extracted, 

centrifuged and injected directly 

onto LC column

Required reagents, column, 

controls and calibrators supplied in 

"kit" form

264 nm

189 Obtained from kit 

supplier

Proteins were precipitated, 

analytes were extracted using 

SPE cartridges

LC (150 x 4.6 mm) column; isocratic 

separation with commercial mobile 

phase; flow 0.7mL/min

265 nm

207 n/r n/r n/r n/r

n/r = not reported

SPE = solid phase extraction
20

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

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

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

017 CLIA n/a n/a n/a n/a n/a n/a 38.1 17.0 18.3 n/r n/r

026 LC-MS/MS 24.1 <1.0 <1.0 19.6 12.4 12.5 43.7 12.4 12.5 n/r n/r

030 RIA n/a n/a n/a n/a n/a n/a 41.8 19.5 18.5 252.5 348.0

032 LC-UV 31.4 n/d n/d 18.2 11.1 10.8 49.6 11.1 10.8 261.0 300.9

056 LC-MS/MS 26.4 n/d n/d 19.8 11.7 12.9 46.2 11.7 12.9 239.6 333.4

062 RIA n/a n/a n/a n/a n/a n/a 25.5 12.3 12.5 n/r 329.8

110 LC-UV 7.3 1.4 1.8 7.9 11.5 7.4 16.4 14.3 10.6 218.0 326.0

116 LC-MS/MS 26.3 < 3.3 < 3.3 20.7 12.2 13.3 47.0 12.2 13.3 236.8 390.7

139 LC-UV 27.9 n/d n/d 16.8 13.7 12.6 44.7 13.7 12.6 262.2 325.8

150 LC-MS/MS 27.0 <2 <2 22.0 16.0 15.0 49.0 16.0 15.0 253.0 398.0

160b CLIA n/a n/a n/a n/a n/a n/a 31.0 13.0 12.0 125.0 225.0

180 RIA n/a n/a n/a n/a n/a n/a 29.2 13.1 13.0 n/r n/r

183a LC-MS/MS 33.0 <4 <4 22.0 15.0 16.0 55.0 15.0 16.0 185.0 329.0

183b CLIA n/a n/a n/a n/a n/a n/a 25.7 13.1 14.5 191.0 262.0

185 LC-MS/MS 28.9 n/d n/d 20.4 12.6 12.7 49.3 12.6 12.7 238.6 334.8

188 CLIA n/a n/a n/a n/a n/a n/a 23.8 14.6 15.2 n/r n/r

189 LC-UV 19.4 n/d n/d 24.8 9.1 9.6 44.2 9.1 9.6 n/r n/r

194 LC-MS/MS 27.7 <7.0 <7 18.7 14.7 12.0 46.4 14.7 12.0 230.0 336.5

195 LC-MS/MS 29.3 <4.0 <4.0 23.6 16.0 15.5 52.9 16.0 15.5 235.0 332.0

196 CLIA n/a n/a n/a n/a n/a n/a 34.8 16.2 16.9 199.5 477.5

197 LC-MS/MS 24.0 <5 <5 17.0 11.0 10.0 41.0 11.0 10.0 205.0 290.0

198a LC-MS/MS 29.1 <5.0 <5.0 18.0 14.7 14.6 47.1 14.7 14.6 250.7 337.0

198b EIA n/a n/a n/a n/a n/a n/a 33.0 15.0 16.0 n/r n/r

199 LC-MS/MS 24.8 <4 <4 16.8 11.5 11.6 41.6 11.5 11.6 233.0 331.0

200 RIA n/a n/a n/a n/a n/a n/a 32.8 14.0 14.0 187.5 280.6

201 EIA n/a n/a n/a n/a n/a n/a 22.4 14.6 15.2 171.0 341.0

202 LC-MS/MS 32.5 n/d n/d 20.5 14.6 14.3 53.0 14.6 14.3 228.2 345.2

205 LC-MS/MS 29.5 0.2 0.2 20.9 13.6 13.2 50.4 13.8 13.4 265.4 337.4

207 LC-UV 20.6 n/d n/d 17.0 26.3 13.0 37.6 26.3 13.0 n/r n/r

209 LC-MS/MS 21.0 <1.0 <1.0 18.3 14.4 13.8 39.2 14.4 13.8 237.6 318.6

210 RIA n/a n/a n/a n/a n/a n/a 34.1 12.3 15.3 142.9 269.2

211 LC-MS/MS 33.4 n/d n/d 27.9 15.2 16.4 61.3 15.2 16.4 311.5 423.0

212 LC-MS/MS 27.6 n/d n/d 19.1 13.9 13.7 46.7 13.9 13.7 242.3 333.7

213 EIA n/a n/a n/a n/a n/a n/a 26.0 17.6 14.6 n/r n/r

214 CLIA n/a n/a n/a n/a n/a n/a 28.5 13.5 13.6 n/r n/r

NIST Value 26.40 <0.5 <0.5 18.50 12.38 12.38 44.90 12.38 12.38 238.6 334.8

U 95 2.00 0.0 0.0 1.10 0.28 0.28 2.28 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)