NISTIR 8142

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report of 

Participant Results: Winter 2015

Comparability Study

(Exercise 10)

Mary Bedner 

This publication is available free of charge from:

http://dx.doi.org/10.6028/NIST.IR.8142
 

NISTIR 8142

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report of 

Participant Results: Winter 2015

Comparability Study

(Exercise 10)

Mary Bedner

Chemical Sciences Division

Material Measurement Laboratory

This publication is available free of charge from:

http://dx.doi.org/10.6028/NIST.IR.8142

September, 2016

U.S. Department of Commerce 

Penny Pritzker, Secretary

National Institute of Standards and Technology 

Willie E. May, Under Secretary of Commerce for Standards and Technology and Director
i

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 tenth exercise of

this program, the Winter 2015 Comparability Study, were asked to use the methodology of their

choice to measure concentrations of 25-hydroxyvitamin D in pooled human serum control and 

study materials distributed by NIST. The study materials consisted of Standard Reference 

Material (SRM) 972a Vitamin D Metabolites in Frozen Human Serum Level 2 and VitDQAPIII (a material designed for the VitDQAP). SRM 968d Fat-Soluble Vitamins, Carotenoids, and 

Cholesterol in Human Serum Level 1 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 tenth study are reported along with a summary of the analytical methods 

used.

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1

OVERVIEW OF THE WINTER 2015 COMPARABILITY STUDY

For the Winter 2015 comparability study of the collaborative National Institute of Standards and 

Technology and National Institutes of Health (NIST/NIH) Vitamin D Metabolites Quality 

Assurance Program (VitDQAP), human serum control and study materials were distributed to 

participants for evaluation. SRM 968d Fat-Soluble Vitamins, Carotenoids, and Cholesterol in 

Human Serum Level 1 (SRM 968d L1) was provided as a control material for assay validation. For 

SRM 968d L1 (Control), the participants were provided the NIST target values within the data 

reporting sheet so that they could qualify their methods prior to analyzing the study samples. The 

study materials consisted of two vials, each containing a sample of pooled human serum. In this 

study, Vial A was SRM 972a Vitamin D Metabolites in Frozen Human Serum Level 2 (SRM 972a

L2), and Vial B was VitDQAP-III, both of which contain endogenous levels of the vitamin D 

metabolites. Participants were asked to determine 25-hydroxyvitamin D in each of the human 

serum control and study samples. Individual concentration values for 25-hydroxyvitamin D3

(25(OH)D3), 25-hydroxyvitamin D2 (25(OH)D2), and 3-epi-25-hydroxyvitamin D3 (3-epi25(OH)D3) were requested along with a total concentration of 25-hydroxyvitamin D (25(OH)DTotal

= 25(OH)D2 + 25(OH)D3). 

There were a total of 52 participants and 58 datasets (6 participants provided data from two 

methods) in the Winter 2015 comparability study. Eighteen (18) of the datasets originated from 

immunoassay (IA) techniques, including 12 from chemiluminescence immunoassay (CLIA), two 

from enzyme immunoassay (EIA), three from radioimmunoassay (RIA), and one from 

chemiluminescence enzyme immunoassay (CLEIA). Appendix A-1 summarizes the IA methods 

used by the participants. Forty (40) of the datasets originated from liquid chromatographic (LC) 

methods; of those, 35 were from LC with tandem mass spectrometric detection (LC-MS/MS), one 

was from LC-MS, and four were from LC with ultraviolet absorbance detection (LC-UV). The LCMS/MS and LC-MS methods are collectively referred to as LC-MSn

. A summary of the LC 

methods used by the participants may be found in Appendices A-2 and A-3. (Note: The 

methodological information provided on the data reporting sheet was used to update the list from

previous comparability studies. For prior participants that did not provide method details for the 

Winter 2015 study, the information in the appendices were not edited and may not be current.) 

The raw data received from all participants are summarized in Appendix B. The IA methods do 

not distinguish between 25(OH)D3 and 25(OH)D2, and hence IA participants reported single values 

for 25(OH)DTotal in the control and study materials. The LC methods measure the vitamin D 

metabolites separately, and the majority of the LC participants reported values for 25(OH)D3 and 

25(OH)D2 in addition to 25(OH)DTotal; eight LC participants also reported results for 3-epi25(OH)D3. One participant also reported values for 24(R), 25-dihydroxyvitamin D3

(24(R),25(OH)2D3) and 1α, 25-dihydroxyvitamin D3 (1α,25(OH)2D3), which is not represented in 

Appendix B.

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

description of the NIST methods is provided in the next section of this report.

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2

SUMMARY OF NIST METHODS USED TO EVALUATE THE CONTROL AND STUDY 

MATERIALS

NIST used isotope dilution LC-MS/MS (ID-LC-MS/MS)[1] or a combination of ID-LC-MS/MS 

and ID-LC-MS [2] procedures to determine the vitamin D metabolites in the control and study 

materials evaluated in this comparability study. The ID-LC-MS/MS approach is a reference 

measurement procedure (RMP) for 25(OH)D3 and 25(OH)D2 that is recognized by the Joint 

Committee for Traceability in Laboratory Medicine (JCTLM). 

For SRM 972a L2 (Vial A), NIST determined 25(OH)D3 and 25(OH)D2 using both ID-LC-MS and 

the ID-LC-MS/MS RMP. The results for 25(OH)D3, 25(OH)D2, 25(OH)DTotal, and 3-epi-25(OH)D3

are a combination of results from the two NIST methods as well as a third method from the Centers 

for Disease Control and Prevention (CDC) and are certified values. A NIST certified value is a 

value for which NIST has the highest confidence in its accuracy in that all known or suspected

sources of bias have been investigated or taken into account [3]. Detailed information about the 

characterization of SRM 972a and the components of the expanded uncertainty (U) may be found in 

the Certificate of Analysis, located on the NIST website [4]. 

The NIST values for 25(OH)D3, 25(OH)D2, and 3-epi-25(OH)D3 in VitDQAP-III (Vial B) and 

SRM 968d L1 (Control) were determined solely with the ID-LC-MS/MS method. For VitDQAP-III 

(Vial B), the NIST values for 25(OH)D3, 25(OH)D2, and 3-epi-25(OH)D3 are reported with 

expanded uncertainties that incorporate components for measurement variability and measurement 

uncertainty associated with the density of the materials and the purity of the reference standards. In 

addition, the measurements include an additional 1 % type B uncertainty for unknown systematic 

errors, which is consistent with the practice used at NIST for clinical measurements [1]. For SRM 

968d L1 (Control), the NIST values for 25(OH)D3 and 3-epi-25(OH)D3 are reported as described 

for VitDQAP-III (Vial B), but the value for 25(OH)D2 was well below the limit of quantitation and 

was estimated to be 0.1 ng/mL based on one measurement. 

The NIST values for 25(OH)DTotal in VitDQAP-III (Vial B) and SRM 968d L1 (Control) are the 

sum of the individual values for 25(OH)D3 and 25(OH)D2, and the expanded uncertainties

incorporate the measurement uncertainties for the two analytes.

 

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

2 Bedner, M.; Phinney, K.W.; J. Chromatogr. A 2012 1240, 132–139. 

3 May, W.; Parris, R.; Beck II, C.; Fassett, J.; Greenberg, R.; Guenther, F.; Kramer, G.; Wise, S.; Gills, T.; Colbert, J.; 

Gettings, R.; MacDonald, B.; NIST Special Publication 260-136 2000; http://www.nist.gov/srm/publications.cfm

4 http://www.nist.gov/srm/index.cfm

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WINTER 2015 COMPARABILITY STUDY RESULTS AND DISCUSSION

Results for 25(OH)DTotal

A summary of the individual participant data for total 25-hydroxyvitamin D (25(OH)DTotal) in 

SRM 972a L2 (Vial A), VitDQAP-III (Vial B), and SRM 968d L1 (Control) is provided in Table 1.

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

methods only, 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 %). 

Table 1 also presents the NIST results for 25(OH)DTotal in the control and the two study materials. 

 

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Table 1. Summary of participant and NIST results for 25(OH)DTotal (ng/mL) in SRM 972a L2

(Vial A), VitDQ AP-III (Vial B), and SRM 968d L1 (Control). 

SRM 972a L2 VitDQAP-III SRM 968d L1

Vial A Vial B Control

N 58 58 57

Median 18.9 32.9 12.8

MADe 1.7 3.7 0.9

CV% 9.1 11 6.9

N 18 18 18

Median 18.1 30.2 14.0

MADe 1.9 2.7 1.9

CV% 10 9.1 14

N 40 40 39

Median 19.3 33.7 12.7

MADe 1.8 2.6 0.7

CV% 9.3 7.7 5.8

N 36 36 35

Median 19.1 33.7 12.7

MADe 1.7 2.5 0.7

CV% 8.7 7.5 5.8

NIST Value 18.9 32.7 12.5

U 0.4 0.7 0.4

LC 

methods

All 

methods

IA 

methods LC-MS

n

SRM 972a L2 VitDQAP-III SRM 968d L1

Lab Method Vial A Vial B Control

026 LC-MS/MS 18.9 33.6 12.7

030a RIA 19.8 38.4 13.1

056a LC-MS/MS 18.8 32.3 12.7

056b LC-MS/MS 18.9 32.5 12.8

060 LC-MS/MS 17.5 27.7 13.2

110 LC-UV 16.2 21.9 12.4

116 LC-MS/MS 21.6 35.0 13.5

119 LC-MS/MS 19.4 40.5 11.5

150 LC-MS/MS 17.0 33.0 11.0

161b LC-MS/MS 18.5 36.6 13.1

180 RIA 17.8 29.4 13.3

187 LC-MS/MS 21.5 35.2 13.3

188 CLIA 26.6 40.6 15.0

189 LC-UV 20.6 38.0 10.6

194 LC-MS/MS 21.0 28.6 13.0

196 CLIA 18.5 29.9 15.2

197 LC-MS/MS 17.7 30.0 12.2

198a LC-MS/MS 22.5 37.7 12.8

198c CLIA 17.1 28.6 5.7

199 LC-MS/MS 20.6 35.3 13.6

204b LC-MS/MS 18.2 32.1 12.6

209 LC-MS/MS 20.6 36.6 14.1

211 LC-MS/MS 18.8 32.9 12.5

212 LC-MS/MS 18.1 32.9 12.3

214b CLIA 16.6 28.4 21.2

214c LC-MS/MS 19.4 32.9 12.2

215 LC-MS/MS 20.4 37.2 13.2

216 LC-MS/MS 19.5 33.7 12.6

217 LC-MS/MS 19.8 37.0 12.6

218a CLIA 17.6 31.8 13.5

221b LC-UV 19.3 30.6 14.8

221c LC-MS 19.3 25.2 13.6

225 LC-MS/MS 21.3 38.1 15.5

228a LC-MS/MS 17.9 31.2 12.4

231b CLIA 20.0 30.4 11.9

241 LC-MS/MS 17.7 33.4 11.3

243a LC-UV 25.3 34.5 12.5

243b LC-MS/MS 24.3 37.8 12.2

244 LC-MS/MS 17.0 35.0 12.1

249 LC-MS/MS 19.7 31.4 12.1

251 LC-MS/MS 22.0 40.0 n/r

253 LC-MS/MS 20.3 35.4 12.8

255 LC-MS/MS 18.8 32.8 13.2

256 CLIA 16.0 24.6 13.7

258 CLIA 20.9 25.5 17.9

259 LC-MS/MS 18.4 34.3 12.7

261 CLIA 17.3 23.0 14.4

262 CLIA 18.4 31.3 20.9

263 CLIA 18.6 35.0 12.6

267 CLEIA 17.8 32.1 12.6

268a RIA 16.5 24.8 13.3

268b EIA 21.1 41.4 21.8

269 LC-MS/MS 18.1 33.7 12.9

270 LC-MS/MS 18.5 26.6 9.3

271 LC-MS/MS 15.0 32.1 11.9

272 LC-MS/MS 19.4 35.4 12.7

273 EIA 17.7 31.8 14.6

274 CLIA 24.7 29.9 21.2

n/r = not reported or not determined

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For all participant datasets, the single reported values for 25(OH)DTotal in SRM 972a L2 (Vial A), 

VitDQAP-III (Vial B), and SRM 968d L1 (Control) are plotted in Figure 1, Figure 2, and

Figure 3, respectively. The results from immunoassay methods are displayed with open dark blue 

circles (○), and the results from the LC-based methods are displayed with open light blue circles 

(○). The results from the individual methods were sorted separately, as indicated by the x-axis 

labels. 

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

median and the consensus expanded uncertainty (2  MADe) were determined. For both of the 

major techniques (IA or LC) in each figure, the solid lines () and () represent the 

consensus median, and the dashed lines (- - - - -) and (- - - - -) represent the consensus expanded 

uncertainty interval (median ± 2  MADe). The laboratories with results that fall between the two 

dashed lines are within the consensus range for their technique (IA or LC).

The red lines () in each figure (Figures 1 – 3) represent the NIST value and its associated 

uncertainty (i.e., value ± U). NIST has confidence that the “true” value for each material lies within 

this interval. When these lines are not within the consensus ranges for each technique (IA or LC), 

then there may be method bias. 

Specific results for each of the three study materials are summarized below. Note that the 

assessment is based on the actual reported values, not the lines and symbols, which have been 

enlarged to show detail and the laboratory number.

SRM 972a L2 (Vial A): Figure 1

 For the IA results, two reported values are outside of the consensus range (both CLIA).

 For the LC results, three reported values are outside of the consensus range (two LC-MSn, one 

LC-UV).

 The consensus median value for the IA results is slightly lower than the NIST expanded 

uncertainty range (red lines). 

 The consensus median value for the LC results is comparable to the NIST expanded uncertainty 

range (red lines).

 The NIST expanded uncertainty range (red lines) falls within the consensus range for both IA 

and LC. 

VitDQAP-III (Vial B): Figure 2

 For the IA results, five reported values are outside the consensus range (three CLIA, one EIA, 

and one RIA).

 For the LC results, six reported values are outside the consensus range (five LC-MSn, one LCUV). 

 The consensus median value for the IA results is lower than the NIST expanded uncertainty 

range (red lines).

 The consensus median value for the LC results is slightly higher than the NIST expanded 

uncertainty range (red lines).

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 The NIST expanded uncertainty range (red lines) falls within the consensus ranges for both IA 

and LC. 

SRM 968d L1 (Control): Figure 3

 For the IA results, five reported values are outside of the consensus range (four CLIA, one EIA).

 For the LC results, five reported values are outside of the consensus range (three LC-MSn, two 

LC-UV). 

 The consensus median value for the IA results is higher than the NIST expanded uncertainty 

range (red lines).

 The consensus median value for the LC results is comparable to the NIST expanded uncertainty 

range (red lines).

 The NIST expanded uncertainty range (red lines) falls within the consensus range for both IA 

and LC. 

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Figure 1. Participant and NIST results for 25(OH)DTotal in SRM 972a L2 (Vial A) as determined by immunoassay (CLIA, EIA, RIA, 

and CLEIA) and LC (LC-MSnand LC-UV) methods. 

 IA method laboratory values 

 IA method consensus range encloses ± 2 MADe around consensus median

 LC method laboratory values

 LC method consensus range encloses ± 2 MADe around consensus median

 NIST value range encloses expanded uncertainty interval 

271

150244

060197241228a212269204b259270161b056a211255056b026

221c119214c272216249217

253215199209

194225187116

251

198a

243b

110

221b

189

243a

256

214b

198c261

218a

262196263

231b

258

274

188

273

268b

268a

180

030a

267

10

15

20

25

30

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

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

CLIA EIA RIA CLEIA LC-M Sn LC-UV

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Figure 2. Participant and NIST results for 25(OH)DTotal in VitDQAP-III (Vial B) as determined by immunoassay (CLIA, EIA, RIA, 

and CLEIA) and LC (LC-MSnand LC-UV) methods. 

221c

270

060

194

197

228a249

204b271056a056b25521121214c150

241026216269

259

116244187199253272

161b209

217215

198a243b225

251

119

110

221b

243a

189

261

256

258

214b198c

196274

231b

262

218a

263

188

273

268b

268a

180

030a

267

20

25

30

35

40

45

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

25(OH)DTotal in VitDQAP-III "Vial B" (ng/mL)

CLIA EIA RIA CLEIA LC-M Sn LC-UV

 IA method laboratory values 

 IA method consensus range encloses ± 2 MADe around consensus median

 LC method laboratory values

 LC method consensus range encloses ± 2 MADe around consensus median

 NIST value range encloses expanded uncertainty interval 

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Figure 3. Participant and NIST results for 25(OH)DTotal in SRM 968d Level 1 (Control) as determined by immunoassay (CLIA, EIA,

RIA, and CLEIA) and LC (LC-MSnand LC-UV) methods. 

270

150241119

271244249243b197214c212228a211204b217216056a259272026056b198a253269194161b060215255187116199221c

209

225

189

110243a

221b

198c

231b

263

218a256

261

188196

258

262

214b274

273

268b

030a180268a

267

5

10

15

20

25

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

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

CLIA EIA RIA CLEIA LC-M Sn LC-UV

 IA method laboratory values 

 IA method consensus range encloses ± 2 MADe around consensus median

 LC method laboratory values

 LC method consensus range encloses ± 2 MADe around consensus median

 NIST value range encloses expanded uncertainty interval 

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Figure 4 presents direct graphical comparisons of the 25(OH)DTotal results for a) SRM 972a L2 

(Vial A) and VitDQAP-III (Vial B), and b) VitDQAP-III (Vial B) and SRM 968d L1 (Control). 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 the Youden comparison plots are summarized below.

SRM 972a L2 (Vial A) and VitDQAP-III (Vial B): Figure 4 a

 IA results that are not included in the consensus ranges include numbers 030a, 188, 261, 268b, 

and 274

 LC results that are not included in the consensus ranges include numbers 060, 110, 119, 221c, 

243a, 243b, 251, 270, and 271

 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. 

VitDQAP-III (Vial B) and SRM 968d L1 (Control): Figure 4 b

 IA results that are not included in the consensus ranges include numbers 030a, 188, 198c, 214b, 

261, 262, 268b, and 274

 LC results that are not included in the consensus ranges include numbers 060, 110, 119, 150, 

189, 221b, 221c, 225, and 270

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

corner of the IA consensus box, illustrating that the LC results are in better agreement with the 

NIST results than are the IA results for these materials. 

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Figure 4. Youden comparison 

plot of the results for 

25(OH)DTotal in a) 972a L2 (Vial 

A) and VitDQAP-III (Vial B) and

b) VitDQAP-III (Vial B) and 

SRM 968d L1 (Control) for all 

methods. 

030a

060

110

119 188

221c

243a

243b

251

261

268b

270

271

274

20

25

30

35

40

10 15 20 25

25(OH)DTotal in VitDQAP-III "Vial B" (ng/mL)

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

IA methods

LC methods

060 030a

110

119

150

188

189

198c

214b

221b

221c

225

261

262

268b

270

274

5

10

15

20

20 25 30 35 40

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

25(OH)DTotal in VitDQAP-III "Vial B" (ng/mL)

LC methods

IA methods

a

b

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Discussion of Results for 25(OH)DTotal

In the Winter 2015 comparability study, both SRM 972a L2 (Vial A) and SRM 968d L1 (Control) 

contain predominantly 25(OH)D3. The CV %’s of 9.1 % and 6.9 % (all methods) for SRM 972a L2 

(Vial) A and SRM 968d L1 (Control), respectively, are consistent with participant performance for 

other materials containing predominantly 25(OH)D3 that were evaluated in previous comparability 

studies of the VitDQAP.

The VitDQAP-III material (Vial B) is different from SRM 972a L2 (Vial A) and SRM 968d L1 

(Control) because it contains measurable 25(OH)D2 in addition to 25(OH)D3. The metabolite 

25(OH)D2 represents 20 % of the 25(OH)DTotal concentration in VitDQAP-III (Vial A), based on the 

NIST values of 6.5 ng/mL ± 0.2 ng/mL for 25(OH)D2 and 32.7 ng/mL ± 0.7 ng/mL for 

25(OH)DTotal. When materials containing appreciable amounts of 25(OH)D2 (> 13 ng/mL) were 

evaluated in previous comparability studies of the VitDQAP, the results were bimodal, with the IA 

methods underrepresenting the 25(OH)DTotal concentration. In addition, the CV % (all methods) for 

those materials was relatively large (approximately 17 % to 28 %). The results for VitDQAP-III 

(Vial B), do not reveal those same trends: the CV % (all methods) is relatively low (11 %), and the 

IA method results overlap almost completely with the LC results. The difference in the observed 

results for the VitDQAP-III material (Vial B) is likely attributable to both the relatively high 

concentration of 25(OH)DTotal and the relatively low concentration of 25(OH)D2, which causes any 

effect from the 25(OH)D2 contribution to be lost in the overall variability of the results. However, 

the median IA result for VitDQAP-III (Vial B) is biased 7 % and 10 % lower than the NIST and the 

LC median results, respectively, which may be attributable to the nonequivalent response of many 

IA methods to 25(OH)D2.

The Winter 2015 exercise was the first to utilize study materials that were evaluated in previous 

comparability studies of the VitDQAP. VitDQAP-III (Vial B) was also evaluated in the Winter 

2014 comparability study, and SRM 972a L2 (Vial A) was previously evaluated in Winter 2012. 

Table 2 provides the program results for each of these two study materials for the labs participating 

in the current study. Using the results in Table 2, labs that participated in the prior studies could

assess their performance for these materials over time. In addition, it is informative to compare the 

summary statistics at the bottom of Table 2. For both materials, the median and CV % results are 

very consistent across both comparability studies in which the materials were evaluated, even 

though there are fewer labs and hence data points (N) for the prior studies.

Figure 5 presents direct graphical comparisons of the 25(OH)DTotal results for 25(OH)DTotal in a) 

VitDQAP-III (Vial B) in the present study (Winter 2015) and in a previous study (Winter 2014 –

Vial A) and b) SRM 972a L2 (Vial A) in the present study (Winter 2015) and in a previous study 

(Winter 2012 – Vial B and D). The features of the plots are the same as described for Figure 4. 

The clustering of results around the NIST value in both Figure 5 a and b illustrates that there are 

not consistent within-laboratory biases for VitDQAP-III and SRM 972a L2 over 1 and 3 years, 

respectively, and that the within-round variability is consistent with the over-time variability. While 

the vast majority of labs yield results that are within the consensus boxes for their techniques, the 

labs that fall outside are not in as good statistical control. For VitDQAP-III, these labs include 

030a, 060, 110, 119, 188, 251, 259, and 261 (Figure 5a), and for SRM 972a L2, these labs include

188, 189, 216, 228a, and 243a (Figure 5b). 

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This publication is available free of charge from: http://dx.doi.org/10.6028/NIST.IR.8142
13

Table 2. Summary of participant data for 25(OH)DTotal (ng/mL) in VitDQAP-III in the current 

study (Vial B) and a prior study (Winter 2014 – Vial A) as well as for SRM 972a L2 in the current 

study (Vial A) and a prior study (Winter 2012 – Vial B and D).

Winter 2015 Winter 2014 Winter 2015 Winter 2012

Vial B Vial B Vial A Vial B, D

N 58 45 58 27

Median 32.9 33.0 18.9 19.4

MADe 3.7 3.3 1.7 1.6

CV% 11 10 9 8.4

N 18 12 18 4

Median 30.2 31.7 18.1 18.3

MADe 2.7 3.2 1.9 1.6

CV% 9.1 10 10 8.5

N 40 33 40 23

Median 33.7 33.4 19.3 19.5

MADe 2.6 3.1 1.8 1.8

CV% 7.7 9 9.3 9.1

N 36 29 36 19

Median 33.7 33.7 19.1 19.5

MADe 2.5 2.5 1.7 1.6

CV% 7.5 7.5 8.7 8.4

NIST Value 32.7 32.7 18.9 18.9

U 0.7 0.7 0.4 0.4

All 

methods

IA 

methods 

LC 

methods LC-MS

n

VitDQAP-III SRM 972a L2 Winter 2015 Winter 2014 Winter 2015 Winter 2012

Lab Method Vial B Vial A Vial A Vial B, D

026 LC-MS/MS 33.6 33.7 18.9 20.9

030a RIA 38.4 40.5 19.8 X

056a LC-MS/MS 32.3 34.5 18.8 19.5

056b LC-MS/MS 32.5 34.0 18.9 X

060 LC-MS/MS 27.7 33.4 17.5 22.3

110 LC-UV 21.9 21.8 16.2 18.9

116 LC-MS/MS 35.0 34.2 21.6 18.3

119 LC-MS/MS 40.5 39.5 19.4 17.9

150 LC-MS/MS 33.0 31.2 17.0 X

161b LC-MS/MS 36.6 X 18.5 X

180 RIA 29.4 29.1 17.8 16.9

187 LC-MS/MS 35.2 30.3 21.5 X

188 CLIA 40.6 36.1 26.6 20.5

189 LC-UV 38.0 27.6 20.6 27.4

194 LC-MS/MS 28.6 33.0 21.0 19.1

196 CLIA 29.9 30.7 18.5 19.0

197 LC-MS/MS 30.0 34.2 17.7 19.0

198a LC-MS/MS 37.7 30.8 22.5 20.6

198c CLIA 28.6 29.1 17.1 X

199 LC-MS/MS 35.3 38.2 20.6 19.5

204b LC-MS/MS 32.1 33.8 18.2 X

209 LC-MS/MS 36.6 33.9 20.6 19.2

211 LC-MS/MS 32.9 36.0 18.8 18.7

212 LC-MS/MS 32.9 35.7 18.1 21.9

214b CLIA 28.4 31.2 16.6 X

214c LC-MS/MS 32.9 31.7 19.4 X

215 LC-MS/MS 37.2 37.2 20.4 20.2

216 LC-MS/MS 33.7 32.9 19.5 26.2

217 LC-MS/MS 37.0 X 19.8 19.4

218a CLIA 31.8 33.7 17.6 17.5

221b LC-UV 30.6 37.5 19.3 18.3

221c LC-MS 25.2 X 19.3 X

225 LC-MS/MS 38.1 31.0 21.3 23.0

228a LC-MS/MS 31.2 35.4 17.9 25.9

231b CLIA 30.4 X 20.0 X

241 LC-MS/MS 33.4 28.7 17.7 19.4

243a LC-UV 34.5 27.8 25.3 21.4

243b LC-MS/MS 37.8 28.2 24.3 X

244 LC-MS/MS 35.0 33.0 17.0 18.0

249 LC-MS/MS 31.4 33.3 19.7 X

251 LC-MS/MS 40.0 34.4 22.0 X

253 LC-MS/MS 35.4 35.5 20.3 X

255 LC-MS/MS 32.8 32.9 18.8 X

256 CLIA 24.6 30.4 16.0 X

258 CLIA 25.5 32.8 20.9 X

259 LC-MS/MS 34.3 26.1 18.4 X

261 CLIA 23.0 32.3 17.3 X

262 CLIA 31.3 34.1 18.4 X

263 CLIA 35.0 28.7 18.6 X

267 CLEIA 32.1 X 17.8 X

268a RIA 24.8 X 16.5 X

268b EIA 41.4 X 21.1 X

269 LC-MS/MS 33.7 X 18.1 X

270 LC-MS/MS 26.6 X 18.5 X

271 LC-MS/MS 32.1 X 15.0 X

272 LC-MS/MS 35.4 X 19.4 X

273 EIA 31.8 X 17.7 X

274 CLIA 29.9 X 24.7 X

X = did not participate in that study

VitDQAP-III SRM 972a L2

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14

Figure 5. Youden comparison 

plot of the results for 25(OH)DTotal

(ng/mL) in a) VitDQAP-III (Vial 

B) in the present study (Winter

2015) and in a previous study 

(Winter 2014 – Vial A) and b) 

SRM 972a L2 (Vial A) in the 

present study (Winter 2015) and in 

a previous study (Winter 2012 –

Vial B and D). 

188

189

216

228a

243a

12

16

20

24

28

12 16 20 24 28

25(OH)DTotal in SRM 972a L2 "Vial A" Winter 2015

25(OH)DTotal in SRM 972a L2 "Vial B, D" Winter 2012

IA methods

LC methods

030a

060

110

119

188

251

259

261

20

25

30

35

40

45

20 25 30 35 40 45

25(OH)DTotal in VitDQAP-III "Vial A" Winter 2014

25(OH)DTotal in VitDQAP-III "Vial B" Winter 2015

IA methods

LC methods

a

b

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15

LC method results for 25(OH)D3, 25(OH)D2, and 3-epi-25(OH)D3 in VitDQAP-III (Vial B)

Of the two major techniques IA and LC, only the LC methods can measure the individual vitamin D 

metabolites. Given that 25(OH)DTotal is the sum of 25(OH)D2 and 25(OH)D3, LC methods require 

accurate, unbiased measurements of both 25(OH)D2 and 25(OH)D3 to obtain the correct values for 

25(OH)DTotal. In the Winter 2015 comparability study, only VitDQAP-III (Vial B) contained a 

significant concentration of the 25(OH)D2 metabolite, and the results for the individual metabolites 

in that study material are detailed below. 

 

Of the 40 LC participants in the Winter 2015 comparability study, all reported values for 25(OH)D3

and all but two reported values for 25(OH)D2 in VitDQAP-III (Vial B). Since VitDQAP-III (Vial 

B) contains relatively high amounts of 25(OH)D3 (NIST value 26.2 ng/mL ± 0.6 ng/mL), the 3-epi25(OH)D3 metabolite is also measureable in this material. Eight LC participants reported values for 

the 3-epi-25(OH)D3 metabolite. The study results and the NIST values for 25(OH)D3, 25(OH)D2, 

and 3-epi-25(OH)D3 in VitDQAP-III (Vial B) are presented in Table 3.

The single reported values for 25(OH)D3 and 25(OH)D2 in VitDQAP-III (Vial B) are plotted in 

Figure 5 a and b, respectively. The results from LC-MSnand LC-UV were sorted separately, as 

indicated by the x-axis labels. In each plot, the consensus median is represented by the solid line

(), and the expanded uncertainty interval (2  MADe), is represented by the dashed lines 

(- - - -). The laboratories with results that fall between the two dashed lines are within the 

consensus variability range. 

The red lines () in Figures 5 a and b represent the NIST value and its associated uncertainty 

(i.e., value ± U). NIST has confidence that the “true” value for each metabolite lies within this 

interval. When these lines are not within the consensus range, then there may be method bias. 

Specific results for 25(OH)D3 and 25(OH)D2 in VitDQAP-III (Vial B) are summarized below:

25(OH)D3 in VitDQAP-III (Vial B): Figure 5 a

 Seven reported values are outside of the consensus variability range (five LC-MSn, two LCUV). 

 The consensus median value is slightly higher than the NIST expanded uncertainty range (red 

lines).

 The NIST expanded uncertainty range (red lines) falls within the consensus variability range.

25(OH)D2 in VitDQAP-III (Vial B): Figure 5 b

 Four reported values are outside of the consensus variability range, all from LC-MSn. 

 The consensus median value is in good agreement with the NIST expanded uncertainty range 

(red lines).

______________________________________________________________________________________________________ 

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16

Table 3. Summary of LC participant and NIST results for 25(OH)D3, 25(OH)D2, and 

3-epi-25(OH)D3 (ng/mL) in VitDQAP-III (Vial B).

25(OH)D3 25(OH)D2 3-epi-25(OH)D3

Lab Method Vial B Vial B Vial B

026 LC-MS/MS 26.9 6.8 2.2

056a LC-MS/MS 25.6 6.7 1.5

056b LC-MS/MS 25.9 6.5 n/r

060 LC-MS/MS 21.9 5.8 1.7

110 LC-UV 17.4 6.8 n/r

116 LC-MS/MS 28.7 6.3 <4.0

119 LC-MS/MS 25.1 15.4 n/r

150 LC-MS/MS 27.0 6.0 n/r

161b LC-MS/MS 28.8 7.8 n/r

187 LC-MS/MS 29.3 5.9 n/r

189 LC-UV 31.1 6.9 n/r

194 LC-MS/MS 28.6 <7.0 n/r

197 LC-MS/MS 24.3 5.7 n/r

198a LC-MS/MS 29.0 8.7 n/r

199 LC-MS/MS 29.4 5.9 n/r

204b LC-MS/MS 25.5 6.6 n/d

209 LC-MS/MS 29.7 6.9 n/r

211 LC-MS/MS 26.3 6.7 n/r

212 LC-MS/MS 28.0 8.1 n/r

214c LC-MS/MS 27.2 5.7 n/r

215 LC-MS/MS 28.4 8.8 n/r

216 LC-MS/MS 26.6 7.1 1.7

217 LC-MS/MS 28.0 9.0 n/r

221b LC-UV 25.4 5.2 n/r

221c LC-MS 25.2 0.0 n/r

225 LC-MS/MS 32.4 5.7 n/r

228a LC-MS/MS 24.6 6.6 2.3

241 LC-MS/MS 27.9 5.5 1.1

243a LC-UV 34.5 n/d n/d

243b LC-MS/MS 33.9 3.9 n/d

244 LC-MS/MS 27.0 8.0 n/r

249 LC-MS/MS 25.0 6.4 1.3

251 LC-MS/MS 33.0 7.0 n/r

253 LC-MS/MS 27.7 7.7 n/r

255 LC-MS/MS 26.5 6.3 n/r

259 LC-MS/MS 27.8 6.5 n/r

269 LC-MS/MS 23.9 9.8 n/r

270 LC-MS/MS 21.1 5.5 n/r

271 LC-MS/MS 23.8 8.3 n/r

272 LC-MS/MS 27.6 7.8 1.3

N 40 38 8

Median 27.1 6.7 1.6

MADe 2.5 1.3 0.4

CV% 9.3 20 28

N 36 35 8

Median 27.1 6.6 1.6

MADe 2.3 1.3 0.4

CV% 8.6 20 28

NIST Value 26.2 6.5 1.6

U 0.6 0.2 0.1

n/r = not reported or not determined; n/d = not detected

< x = less than a reported quantitation limit of x LC methods LC-MS

n

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17

Figure 5. Participant and NIST results for a) 25(OH)D3 and b) 25(OH)D2 in VitDQAP-III (Vial B). 

 

270

060

271269197228a249119221c204b056a056b211255216026150244214c272253259241217212215194116161b198a187199209

225

251

243b

110

221b

189

243a

10

15

20

25

30

35

40

70 75 80 85 90 95 100 105 110

25(OH)D3 in VitDQAP-III "Vial B" (ng/mL)

LC-M Sn LC-UV

221c

243b

270241197214c225060187199150255116249259056b204b228a211056a026209251216

253161b272244212271

198a215217

269

119

221b

110189

0

2

4

6

8

10

12

14

16

70 75 80 85 90 95 100 105 110

25(OH)D2 in VitDQAP-III "Vial B" (ng/mL)

LC-M Sn LC-UV

a

b

 LC method laboratory values

 LC method consensus range encloses ± 2 MADe around consensus median

 NIST value range encloses approx. 95% confidence uncertainty 

______________________________________________________________________________________________________ 

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18

Dihydroxyvitamin D3 Metabolites

The Winter 2015 comparability study is the first in which a participant, Lab 269, reported results for 

two dihydroxyvitamin D3 metabolites, 24, 25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1α, 25-

dihydroxyvitamin D3 (1α,25(OH)2D3), in each of the study materials. The results provided by 

participant 269 for these metabolites are provided in the following table:

Results reported by 

Lab 269

24,25(OH)2D3

(ng/mL)

1α,25(OH)2D3

(ng/mL)

SRM 972a L2 (Vial A) 1.40 ± 0.07 0.0405 ± 0.0095

VitDQAP-III (Vial B) 2.50 ± 0.16 0.0442 ± 0.0084

SRM 968d L1 (Control) 0.687 ± 0.032 0.0628 ± 0.0068

NIST has developed a candidate RMP for the determination of 24R,25(OH)2D3 and has provided 

reference values for this metabolite in SRM 972a. For SRM 972a L2 (Vial A), the NIST reference 

value is 1.41 ± 0.05 ng/mL (95 % confidence interval), which agrees well with Lab 269’s value. 

NIST has not developed a method for the 1α,25(OH)2D3 metabolite to date. 

Conclusions from the Winter 2015 Comparability Study of the VitDQAP

The Winter 2015 comparability study was the tenth exercise and marked the five-year point for the 

VitDQAP. Over those five years and ten studies, the participant performance was consistent for 

study materials that contain predominantly 25(OH)D3; the CV was in the range from 7 % to 19 %, 

and the median values were biased slightly high relative to the NIST values. In the Winter 2015

comparability study, both SRM 972a L2 (Vial A) and SRM 968d L1 (Control) also contained

predominantly 25(OH)D3. The median participant results (all methods) for these materials agreed

well with the NIST values but otherwise follow these longstanding trends. In addition, Winter 2015 

represented the second study in which SRM 972a L2 (Vial A) was evaluated in the VitDQAP. 

Table 2 and Figure 5 b contains the program results for this material in both studies and 

demonstrates the consistency of the participant results for SRM 972a L2.

When VitDQAP-III (Vial B) was evaluated in the Winter 2014 study, it was the first study material 

that had an ‘intermediate’ concentration of 25(OH)D2 (NIST value 6.5 ng/mL ± 0.2 ng/mL) in 

addition to a significant concentration of 25(OH)D3 (NIST value 26.3 ng/mL ± 0.7 ng/mL). The 

material was first selected for study because it was anticipated that the IA methods would 

underrepresent the 25(OH)DTotal concentration due to nonequivalent response to the 25(OH)D2

metabolite. To the contrary, in both the Winter 2014 and the current Winter 2015 studies the IA 

results overlapped almost completely with the LC results, and any effect from the 25(OH)D2

metabolite was lost in the overall variability of the results for the VitDQAP-III study material (Vial 

B). As when previously evaluated, the median IA result for VitDQAP-III (Vial B) was biased 

lower than the median LC and NIST results, which is the only indication of potential non-equivalent 

response to the 25(OH)D2 metabolite. The consistency of the participant results for VitDQAP-III 

(Vial B) are also evident from the results provided in Table 2 and Figure 5 a.

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19

Appendix A-1. Summary of immunoassay methods used as reported by the study participants.

Laboratory 

Number IA Method Sample Preparation Vendor/kit*

30a RIA Samples were extracted with acetonitrile A

180 RIA Samples were extracted with acetonitrile A

188 CLIA n/r B

196 CLIA No sample preparation required C

198c CLIA n/r n/r

214b CLIA n/r C

218a CLIA Direct analysis C

231b CLIA n/r B

256 CLIA n/r C

258 CLIA n/r D

261 CLIA No sample preparation required D

262 CLIA n/r E

263 EIA On board displacement F

267 CLEIA n/r G

268a RIA n/r H

268b EIA n/r I

273 EIA n/r n/r

274 CLIA n/r D

n/r = not reported

*NIST cannot endorse or recommend commercial products, therefore individual vendors/kits are indicated with a unique letter but not identified

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20

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

Laboratory 

Number

Internal 

Standard (IS) Sample Preparation Chromatographic Conditions Detection: MRM ions

26

25(OH)D2-d6 and 

25(OH)D3-d6

Liquid-liquid extraction method

PFP column (100 mm × 3.2 mm); 

isocratic elution with 82 % 

methanol/18 % water; 

flow 0.4 mL/min

25(OH)D3 401/365; 

25(OH)D2 413/355;

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

56a

25(OH)D2-d3; 

25(OH)D3-d6; 

3-epi-25(OH)D3-d3

Samples were extracted with 

hexane, evaporated, then 

reconstituted with 69 % methanol

PFP column (100 mm × 2.1 mm; 

1.9 μm); 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; 

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

3-epi-25(OH)D3-d3 386/368

56b n/r n/r n/r n/r

60 25(OH)D3

 -d6

IS was added, and then samples 

were extracted with acetonitrile, 

evaporated, and reconstituted 

with 90 % methanol/10 % water

PFP column (100 mm × 3.0 mm; 

2.6 μm); gradient with water, 

methanol and acetonitrile (0.05 % 

formic acid)

25(OH)D3 383/211; 

25(OH)D3-d6 389/211; 

25(OH)D2 413/355; 

3-epi-25(OH)D3 401/383

116 25(OH)D3

 -d6

Serum proteins were precipitated 

with methanol

Online SPE; reversed-phase 

column; isocratic elution with 

95 % methanol/5 % water; 

flow 0.6 mL/min

25(OH)D3 383/211; 

25(OH)D3-d6 389/211; 

25(OH)D2 395/269

119 25(OH)D3

 -d6

Samples were mixed with ethanol 

containing the IS, equilibrated, 

mixed, extracted with hexane, 

evaporated, and reconstituted in 

methanol

C18 column (150 mm × 3.0 mm; 

2.7 μm); Gradient with water and 

methanol (0.1 % formic acid)

Exact mass system

25(OH)D3 

383.32932/365.31897; 

25(OH)D3-d6 389.36658; 

25(OH)D2 

395.32946/377.31894

150

25(OH)D2-d6 and 

25(OH)D3-d3

Sample (200 μL) was mixed with 

IS solution, liquid-liquid extracted, 

centrifuged, supernatant 

evaporated, and reconstituted in 

mobile phase

PFP column (100 mm × 3.0 mm; 

2.6 μm); isocratic separation with 

74 % methanol/26 % water 

(2 mmol/L ammonium acetate, 

0.1 % formic acid); 

flow 0.5 mL/min

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

25(OH)D2 413/395, 413/365

161b 25(OH)D3

 -d6 Protein precipitation

Reversed-phase column (50 mm × 

2.1 mm; 2.6 μm); gradient with 

methanol and water (0.1 % formic 

acid); flow 0.5 mL/min

25(OH)D3 383/211;

25(OH)D2 395/269

187

deuterated 

standards for 

25(OH)D2 and 

25(OH)D3

SPE

C18 column (50 mm × 2.1 mm; 

3 μm); gradient with methanol and 

water

25(OH)D2 413/395; 

25(OH)D3 401/383

194 25(OH)D3

 -d6

Proteins precipitated with 

acetonitrile, top layer removed, 

evaporated, and reconstituted 

with methanol

C8 column (50 mm × 2 mm); 

isocratic elution with 70 % 

acetonitrile/ 30 % water; 

flow 0.7 mL/min

25(OH)D2 395/119; 

25(OH)D3 383/211

197 25(OH)D3

 -d6

Precipitating agent added (200 μL 

with 20 ng IS) to each serum 

sample (200 μL), calibrator and 

control sample followed by mixing, 

centrifugation, and analysis

C18 column (50 mm × 4.6 mm; 

5 μm); column temp 45 °C; gradient 

with water and methanol; 

flow 1.0 mL/min

n/r

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21

198a 25(OH)D3

 -d6

Proteins precipitated with 

methanol, followed by ZnSO4 

addition, hexane extraction, 

centrifugation, evaporation under 

N2, and reconstitution in methanol 

(0.1 % formic acid)

C18 column (50 mm 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-d6 407/389, 

407/371

199 proprietary proprietary proprietary proprietary

204b

25(OH)D2-d3; 

25(OH)D3-d6; 

3-epi-25(OH)D3-d3

Protein crash with 73 % methanol 

followed by liquid-liquid extraction 

with hexane, centrifugation, 

evaporation, and reconstitution in 

mobile phase

PFP column (100 mm × 2.1 mm; 

1.9 μm); column temperature 30 °C; 

isocratic elution with 73 % 

methanol/27 % water; 

flow 0.4 mL/min

APCI

25(OH)D3 383/365, 383/257;

25(OH)D2 395/377, 395/209;

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

383/257

209 25(OH)D3

 -d6

Proteins were precipitated with 

5 % ZnSO4 in methanol

C8 column (50 mm × 2 mm; 5 μm); 

gradient with water/methanol; flow 

0.7 mL/min

APCI

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

25(OH)D3-d6 389/211; 

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

211 25(OH)D3

-d6

Proteins precipitated with 

acetonitrile containing IS followed 

by centrifugation

Turbulent flow column (32 mm x 

4.6 mm; 3 μm)

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

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

395/209 (quant), 395/377 

(qual)

212 25(OH)D3

-d6

Serum (100 μL) proteins 

precipitated using 5 % 

methanol/95 % acetonitrile 

containing the IS (350 μL)

C8 column (50 mm × 2 mm; 

3 μm); gradient of 60 % to 98 % 

acetonitrile (0.1 % formic acid)

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

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

214c 25(OH)D3

-d6

Samples were extracted with 

hexane, centrifuged, evaporated, 

and filtered

Column (50 mm × 2.1 mm); isocratic 

elution with 85 % methanol/ 15 % 

water/ 0.1 % formic acid; 

flow 0.3 mL/min

25(OH)D3 401/383; 

25(OH)D3-d6 407/389; 

25(OH)D2 413/395

215 25(OH)D3

-d6

Protein precipitation with 

methanol/isopropanol and ZnSO4; 

supernatant extracted using SPE

C18 column (50 mm × 2.1 mm; 

2.6 μm) column; gradient with water 

(0.1 % formic acid, 5 mmol/L 

ammonium formate) and methanol 

(0.05 % formic acid)

ESI

25(OH)D3 401/383; 

25(OH)D2 413/395; 

25(OH)D3-d6 407/389

216

Derivatized 

deuterated 

standard

Samples extracted using liquidliquid extraction then labeled with 

a derivatization reagent

Reversed-phase column (150 mm × 

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

-d6

Protein precipitation with ZnSO4 in 

methanol followed by SPE

C8 column (50 mm × 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)

221c

25(OH)D2-d6 and 

25(OH)D3-d3

Protein crash with acetonitrile 

containing IS; SPE extraction; 

elution with methanol/acetonitrile 

solution; evaporation; 

reconstitution with acetonitrile

PFP column (50 mm × 3.0 mm; 

2.7 μm); elution with 

methanol/water/formic acid; column 

40 °C

LC-MS SIM

25(OH)D3 383; 

25(OH)D2 395; 

25(OH)D3-d6 389;

25(OH)D2-d6 401

225 25(OH)D3

-d6 Liquid-liquid extraction PFP column (100 mm × 2.1 mm); 

gradient with methanol/water

25(OH)D3 401/107; 

25(OH)D2 413/83

228a n/r n/r n/r n/r

241 25(OH)D3

-d6

Acetonitrile containing the IS 

(100 μL) added to sample 

(200 μL) to precipitate proteins, 

followed hexane extraction, 

centrifugation, evaporation, and 

reconstitution with 50 % methanol

PFP column (100 mm × 2.1 mm; 

2.6 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-d6 389/211

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22

243b 25(OH)D3

-d6

Samples (400 μL) were mixed with 

solution containing the IS (400 μL)

and the mobile phase (500 μL); 

samples were centrifuged; 

supernatant was diluted; portion 

(50 μL) was injected

PFP column (150 mm × 2 mm); 

isocratic separation with 85 % 

methanol/15 % water; 

flow 0.3 mL/min

25(OH)D3 383/257; 

25(OH)D2 395/269; 

25(OH)D3-d6 389/263;

244 25(OH)D3

-d6

Protein precipitation followed by 

filtration

CN column; mobile phase 

consisting of distilled water (formic 

acid) and methanol

25(OH)D3 383/211; 

25(OH)D3-d6 389/211; 

25(OH)D2 395/269

249

25(OH)D2-d3; 

25(OH)D3-d6; 

3-epi-25(OH)D3-d3

Serum was deproteinated with 

NaOH and 90 % acetonitrile/ 10 % 

methanol followed by SPE

PFP column (100 mm × 2.1 mm; 

1.8 μm); gradient separation with 

water (2 mmol/L ammonium 

acetate) and methanol; 

flow 0.35 mL/min

25(OH)D3 401/159; 

25(OH)D2 413/159

251

25(OH)D2-d3 and 

25(OH)D3-d3

Protein precipitation followed by 

SPE

Phenyl column (50 mm × 2.1 mm; 

1.7 μm); gradient with water and 

methanol (0.1 % formic acid, 

2 mmol/L ammonium acetate); flow 

0.45 mL/min

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

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

413/83 (quant), 413/355 

(qual); 25(OH)D3-d3 404/162; 

25(OH)D2-d3 416/358

253

25(OH)D2-d3 and 

25(OH)D3-d3

The sample was extracted, 

centrifuged, and derivatized

C18 column (150 mm × 2.1 mm); 

gradient separation with water and 

methanol; flow 0.4 mL/min

25(OH)D2 588; 

25(OH)D3 576

255 deuterium labeled 

compound

Samples were extracted and 

derivatized with 4-phenyl-1,2,4-

triazoline-3,5-dione

Reversed-phase column (50 mm × 

2.1 mm); gradient with methanol; 

flow 0.5 mL/min

25(OH)D3 607/298; 

25(OH)D2 619/298

259 25(OH)D3

-d6

Liquid-liquid extraction using 

hexane

C8 column; gradient with 

methanol/water/0.1 % formate; 

column temperature 40 °C

25(OH)D3 401/355; 

25(OH)D2 413/355; 

25(OH)D3-d6 407/371

269 25(OH)D3

-d6

Samples were spiked with IS(s), 

deprotonated with acetonitrile, 

filtered, dried, derivatized with 4-

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

overnight at 4°C, dried, 

reconstituted with 

cyclohexyldodecylurea, and 

filtered 

C18 column (100 mm × 2.1 mm; 

1.7 μM); gradient separation with 

0.1 % formic acid and water 

(10 %)/acetonitrile (90 %); 

flow 0.25 mL/min

24,25(OH)2D3-d6 580/298

24,25(OH)2D3 574/298

1a,25(OH)2D3-d6 580/314

1a,25(OH)2D3 574/314 

25(OH)D3-d6 564/298 

25(OH)D3 558/298

25(OH)D2 570/ 298

270 25(OH)D3

-d6

Samples were precipitated, 

centrifuged, evaporated, 

reconstituted, centrifuged, and 

upper layer injected

C18 column (300 mm × 4.6 mm; 

3.5 μM); isocratic separation with 

50 % water/ 50 % methanol; 

flow 1.0 mL/min

25(OH)D3 401/383; 

25(OH)D2 413/395; 

25(OH)D3-d6 407/389

271 25(OH)D3

-d6 Protein precipitation

C8 column (3 μm); gradient with 

water/acetonitrile/0.1 % formic acid; 

flow 0.7 mL/min

25(OH)D3 383/229; 

25(OH)D2 395/269

272 Isotopically labeled 

internal standards

Samples were precipitated and 

centrifuged before injection

Analytical column and trap column 

from a kit; separation using a binary 

gradient system and an additional 

isocratic pump

25(OH)D3 383/365, 383/299; 

IS (1): 386/257, 386/232; 

25(OH)D2 395/269, 395/251; 

3-epi-25(OH)D3 383/257, 

383/299;

3-epi-25(OH)D2 395/269, 

395/251; 

IS (2): 386/257, 386/232

C18 = octadecyl; C8 = octyl; PFP = pentafluorophenyl; SPE = solid phase extraction; CN = cyano; 

MRM = multiple reaction monitoring; quant/qual = quantitative/qualitative ions; n/r = not reported;

APPI = atmospheric pressure photoionization; APCI = atmospheric pressure chemical ionization; ESI = electrospray ionization

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23

Appendix A-3. Summary of LC-UV methods as reported by the study participants.

Laboratory 

Number

Internal 

Standard (IS) Sample Preparation Chromatographic Conditions Wavelength

110 n/a

Samples (500 μL) were mixed with 

ethanol (500 μL), extracted twice 

with hexane/methylene chloride 

(5:1), evaporated, and 

reconstituted

C18 column (2.1 mm × 100 mm; 

1.8 μm); gradient with 

acetonitrile/methanol (85:15) and 

isopropanol (100 %)

267 nm

189 unidentified Protein precipitation followed by 

SPE

Reversed-phase column (150 mm × 

4.6 mm); isocratic separation; flow 

0.7 mL/min

265 nm

221b laurophenone

Protein crash with acetonitrile 

solution containing IS, followed by 

SPE, elution with 

methanol/acetonitrile solution, 

evaporation, and reconstitution 

with acetonitrile

CN column (150 mm × 5 mm;

3.5 μm); elution with 

methanol/water/formic acid; column 

temperature 47 °C 

275 nm

243a dodecanophenone

Samples (400 μL) were mixed with 

solution containing the IS 

(400 μL), precipitation reagent 

was added (500 μL), and portion 

of upper layer (50 μL) was 

injected

C18 column (100 mm × 3 mm); 

isocratic elution with water and 

isobutanol; flow 1.2 mL/min; column 

temperature 25 °C

264 nm

C18 = octadecyl; SPE = solid phase extraction; CN = cyano; n/a = not applicable

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This publication is available free of charge from: http://dx.doi.org/10.6028/NIST.IR.8142
24

Appendix B. Raw 

participant data and 

NIST results for 

25(OH)D2, 25(OH)D3,

25(OH)DTotal, and 3-epi25(OH)D3 in SRM 972a 

L2 (Vial A), VitDQAPIII (Vial B), and SRM 

968d L1 (Control).

SRM 972a L2 VitDQAP-III SRM 968d L1 SRM 972a L2 VitDQAP-III SRM 968d L1 SRM 972a L2 VitDQAP-III SRM 968d L1 SRM 972a L2 VitDQAP-III SRM 968d L1

Lab Method Vial A Vial B Control Vial A Vial B Control Vial A Vial B Control Vial A Vial B Control

026 LC-MS/MS 0.7 6.8 0.3 18.2 26.9 12.4 18.9 33.6 12.7 1.5 2.2 0.6

030a RIA n/a n/a n/a n/a n/a n/a 19.8 38.4 13.1 n/a n/a n/a

056a LC-MS/MS 0.6 6.7 0.6 18.2 25.6 12.1 18.8 32.3 12.7 1.2 1.5 0.7

056b LC-MS/MS 0.8 6.5 <0.6 18.1 25.9 12.8 18.9 32.5 12.8 n/r n/r n/r

060 LC-MS/MS 0.9 5.8 0.2 16.6 21.9 13.0 17.5 27.7 13.2 1.3 1.7 0.9

110 LC-UV 3.5 6.8 n/d 12.8 17.4 12.4 16.2 21.9 12.4 n/r n/r n/r

116 LC-MS/MS <3.3 6.3 <3.3 21.6 28.7 13.5 21.6 35.0 13.5 <4.0 <4.0 <4.0

119 LC-MS/MS n/d 15.4 n/d 19.4 25.1 11.5 19.4 40.5 11.5 n/r n/r n/r

150 LC-MS/MS <2 6.0 <2 17.0 27.0 11.0 17.0 33.0 11.0 n/r n/r n/r

161b LC-MS/MS <4 7.8 <4 18.5 28.8 13.1 18.5 36.6 13.1 n/r n/r n/r

180 RIA n/a n/a n/a n/a n/a n/a 17.8 29.4 13.3 n/a n/a n/a

187 LC-MS/MS 0.0 5.9 0.0 21.5 29.3 13.3 21.5 35.2 13.3 n/r n/r n/r

188 CLIA n/a n/a n/a n/a n/a n/a 26.6 40.6 15.0 n/a n/a n/a

189 LC-UV 0.0 6.9 0.0 20.6 31.1 10.6 20.6 38.0 10.6 n/r n/r n/r

194 LC-MS/MS <7.0 <7.0 <7.0 21.0 28.6 13.0 21.0 28.6 13.0 n/r n/r n/r

196 CLIA n/a n/a n/a n/a n/a n/a 18.5 29.9 15.2 n/a n/a n/a

197 LC-MS/MS <5 5.7 <5 17.7 24.3 12.2 17.7 30.0 12.2 n/r n/r n/r

198a LC-MS/MS <5.0 8.7 <5.0 22.5 29.0 12.8 22.5 37.7 12.8 n/r n/r n/r

198c CLIA n/a n/a n/a n/a n/a n/a 17.1 28.6 5.7 n/a n/a n/a

199 LC-MS/MS <2.0 5.9 <2.0 20.6 29.4 13.6 20.6 35.3 13.6 n/r n/r n/r

204b LC-MS/MS n/d 6.6 n/d 18.2 25.5 12.6 18.2 32.1 12.6 n/d n/d n/d

209 LC-MS/MS <1.0 6.9 <1.0 20.6 29.7 14.1 20.6 36.6 14.1 n/r n/r n/r

211 LC-MS/MS 0.0 6.7 0.0 18.8 26.3 12.5 18.8 32.9 12.5 n/r n/r n/r

212 LC-MS/MS <2 8.1 <2 18.1 28.0 12.3 18.1 32.9 12.3 n/r n/r n/r

214b CLIA n/a n/a n/a n/a n/a n/a 16.6 28.4 21.2 n/a n/a n/a

214c LC-MS/MS <1.0 5.7 <1.0 19.4 27.2 12.2 19.4 32.9 12.2 n/r n/r n/r

215 LC-MS/MS <2 8.8 <2 20.4 28.4 13.2 20.4 37.2 13.2 n/r n/r n/r

216 LC-MS/MS 0.8 7.1 0.2 18.7 26.6 12.5 19.5 33.7 12.6 1.3 1.7 0.8

217 LC-MS/MS <0.8 9.0 <0.8 19.8 28.0 12.6 19.8 37.0 12.6 n/r n/r n/r

218a CLIA n/a n/a n/a n/a n/a n/a 17.6 31.8 13.5 n/a n/a n/a

221b LC-UV 0.0 5.2 0.0 19.3 25.4 14.8 19.3 30.6 14.8 n/r n/r n/r

221c LC-MS 0.0 0.0 0.0 19.3 25.2 13.6 19.3 25.2 13.6 n/r n/r n/r

225 LC-MS/MS <5 5.7 <5 21.3 32.4 15.5 21.3 38.1 15.5 n/r n/r n/r

228a LC-MS/MS n/d 6.6 n/d 17.9 24.6 12.4 17.9 31.2 12.4 1.8 2.3 0.75

231b CLIA n/a n/a n/a n/a n/a n/a 20.0 30.4 11.9 n/a n/a n/a

241 LC-MS/MS 0.7 5.5 0.3 17.0 27.9 11.0 17.7 33.4 11.3 0.7 1.1 0.7

243a LC-UV n/d n/d n/d 25.3 34.5 12.5 25.3 34.5 12.5 n/d n/d n/d

243b LC-MS/MS n/d 3.9 n/d 24.3 33.9 12.2 24.3 37.8 12.2 1.6 n/d n/d

244 LC-MS/MS 0.0 8.0 0.0 17.0 27.0 12.1 17.0 35.0 12.1 n/r n/r n/r

249 LC-MS/MS 0.0 6.4 0.0 19.7 25.0 12.1 19.7 31.4 12.1 1.6 1.3 0.5

251 LC-MS/MS <4 7.0 n/r 22.0 33.0 n/r 22.0 40.0 n/r n/r n/r n/r

253 LC-MS/MS 0.9 7.7 0.2 19.4 27.7 12.6 20.3 35.4 12.8 n/r n/r n/r

255 LC-MS/MS 0.9 6.3 0.1 18.0 26.5 13.1 18.8 32.8 13.2 n/r n/r n/r

256 CLIA n/a n/a n/a n/a n/a n/a 16.0 24.6 13.7 n/a n/a n/a

258 CLIA n/a n/a n/a n/a n/a n/a 20.9 25.5 17.9 n/a n/a n/a

259 LC-MS/MS n/d 6.5 n/d 18.4 27.8 12.7 18.4 34.3 12.7 n/r n/r n/r

261 CLIA n/a n/a n/a n/a n/a n/a 17.3 23.0 14.4 n/a n/a n/a

262 CLIA n/a n/a n/a n/a n/a n/a 18.4 31.3 20.9 n/a n/a n/a

263 CLIA n/a n/a n/a n/a n/a n/a 18.6 35.0 12.6 n/a n/a n/a

267 CLEIA n/a n/a n/a n/a n/a n/a 17.8 32.1 12.6 n/a n/a n/a

268a RIA n/a n/a n/a n/a n/a n/a 16.5 24.8 13.3 n/a n/a n/a

268b EIA n/a n/a n/a n/a n/a n/a 21.1 41.4 21.8 n/a n/a n/a

269 LC-MS/MS 2.8 9.8 2.1 15.3 23.9 10.8 18.1 33.7 12.9 n/r n/r n/r

270 LC-MS/MS 1.4 5.5 0.9 17.1 21.1 8.3 18.5 26.6 9.3 n/r n/r n/r

271 LC-MS/MS <4 8.3 <4 15.0 23.8 11.9 15.0 32.1 11.9 n/r n/r n/r

272 LC-MS/MS 0.6 7.8 0.0 18.8 27.6 12.7 19.4 35.4 12.7 1.5 1.3 0.9

273 EIA n/a n/a n/a n/a n/a n/a 17.7 31.8 14.6 n/a n/a n/a

274 CLIA n/a n/a n/a n/a n/a n/a 24.7 29.9 21.2 n/a n/a n/a

NIST Value 0.81 6.5 0.1* 18.1 26.2 12.4 18.9 32.7 12.5 1.3 1.6 0.7

U 0.06 0.2 --- 0.4 0.6 0.4 0.4 0.7 0.4 0.1 0.1 0.03

*estimated value (no uncertainty determined)

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

n/a = not applicable (for immunoassay methods); n/r = not reported or not determined; 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)

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