NISTIR 8133

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report of 

Participant Results: Winter

2014 Comparability Study

(Exercise 8)

Mary Bedner 

Susan S.-C. Tai 

This publication is available free of charge from:

http://dx.doi.org/10.6028/NIST.IR.8133
NISTIR 8133

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report of 

Participant Results: Winter

2014 Comparability Study

(Exercise 8)

Mary Bedner

Susan S.-C. Tai

Chemical Sciences Division

Material Measurement Laboratory

This publication is available free of charge from:

http://dx.doi.org/10.6028/NIST.IR.8133

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
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 eighth exercise of this 

program, the Winter 2014 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) 909c Human Serum and VitDQAP-III (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 eighth study are reported along with a 

summary of the analytical methods used.

i

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1

OVERVIEW OF THE WINTER 2014 COMPARABILITY STUDY

For the Winter 2014 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. Standard Reference Material (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 VitDQAP-III, and Vial B was SRM 909c Frozen 

Human Serum, 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-epi-25(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 58 participants and 71 datasets (12 participants provided data from two or 

more methods) in the Winter 2014 comparability study. Twenty-eight of the datasets originated 

from immunoassay (IA) techniques, including 19 from chemiluminescence immunoassay (CLIA), 

four from enzyme immunoassay (EIA), and five from radioimmunoassay (RIA). Appendix A-1

summarizes the IA methods used by the participants. Forty-three of the datasets originated from 

liquid chromatographic (LC) methods; of those, 37 were from LC with tandem mass spectrometric 

detection (LC-MSn), and six were from LC with ultraviolet absorbance detection (LC-UV). A 

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

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

not distinguish between 25(OH)D3 and 25(OH)D2, and 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; eleven LC participants also reported results for 3-epi25(OH)D3 in at least one of the study materials.

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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SUMMARY OF THE 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 (25(OH)D3, 25(OH)D2, and 

3-epi-25(OH)D3) in the control and study materials evaluated in this comparability study. The IDLC-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 909c (Vial B), NIST determined 25(OH)D3 using both ID-LC-MS and the ID-LC-MS/MS 

RMP. The result for 25(OH)D3 is a combination of results from the two methods and is a certified 

value. 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] and 

are reflected in the expanded uncertainty (U). Note that even though the NIST value for 25(OH)D3

is certified, this value does not currently appear on the Certificate of Analysis for SRM 909c. The 

25(OH)D2 concentration was below the quantitation limit (≈ 0.5 ng/mL) in SRM 909c, and thus the 

NIST value for 25(OH)DTotal includes only the certified result for 25(OH)D3. 

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

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

(Vial A), 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 A), 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 A) and SRM 968d L1 (Control) are the 

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

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

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WINTER 2014 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 

VitDQAP-III (Vial A), SRM 909c (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 VitDQAP-III

(Vial A), SRM 909c (Vial B), and SRM 968d L1 (Control). 

VitDQAP-III SRM 909c SRM 968d L1

Vial A Vial B Control

N 71 71 70

Median 32.3 21.2 12.8

MADe 3.1 4.3 1.4

CV% 9.5 20 11

N 28 28 28

Median 31.1 18.2 13.0

MADe 3.0 5.6 2.3

CV% 9.5 31 18

N 43 43 42

Median 33.4 21.7 12.8

MADe 3.6 3.3 0.7

CV% 11 15 5.8

N 37 37 36

Median 33.8 21.6 12.8

MADe 3.1 2.1 0.7

CV% 9.0 9.6 5.3

NIST Value 32.7 20.7 12.5

U 0.7 0.7 0.4

All 

methods

IA 

methods 

LC 

methods LC-MS

n

VitDQAP-III SRM 909c SRM 968d L1

Lab Method Vial A Vial B Control

017 CLIA 31.0 16.2 13.9

026 LC-MS/MS 33.7 21.7 13.4

030a RIA 40.5 24.8 15.3

030b LC-MS/MS 24.5 23.9 13.2

056a LC-MS/MS 34.5 20.3 12.8

056b LC-MS/MS 34.0 20.3 12.7

060 LC-MS/MS 33.4 22.0 13.0

110 LC-UV 21.8 79.6 12.8

116 LC-MS/MS 34.2 20.7 12.8

119 LC-MS/MS 39.5 27.3 15.6

139 LC-UV 24.7 51.9 13.1

150 LC-MS/MS 31.2 17.7 12.1

161 CLIA 30.6 14.4 11.8

180 RIA 29.1 19.5 14.9

185a LC-MS/MS 38.4 36.4 17.3

185b CLIA 16.4 33.3 14.5

187 LC-MS/MS 30.3 19.0 13.1

188 CLIA 36.1 18.4 14.1

189 LC-UV 27.6 38.2 16.5

193 EIA 33.9 22.3 18.6

194 LC-MS/MS 33.0 15.8 13.0

196 CLIA 30.7 15.1 14.1

197 LC-MS/MS 34.2 22.0 13.1

198a LC-MS/MS 30.8 18.5 12.6

198c CLIA 29.1 19.7 8.3

199 LC-MS/MS 38.2 29.6 13.7

200 RIA 27.7 18.0 13.8

204a CLIA 27.5 14.0 12.2

204b LC-MS/MS 33.8 20.2 12.8

209 LC-MS/MS 33.9 22.9 12.5

210a RIA 31.4 17.9 12.4

210b CLIA 38.7 25.0 9.1

211 LC-MS/MS 36.0 18.3 13.6

212 LC-MS/MS 35.7 22.3 12.3

213a CLIA 30.4 19.4 6.5

213b EIA 26.3 17.3 12.9

214a RIA 27.5 31.3 11.5

214b CLIA 31.2 14.4 13.0

214c LC-MS/MS 31.7 20.5 12.2

215 LC-MS/MS 37.2 21.2 13.6

216 LC-MS/MS 32.9 21.9 12.8

218a CLIA 33.7 14.5 14.5

218b LC-MS/MS 31.8 23.8 12.2

220 LC-MS/MS 36.0 23.0 15.0

221a LC-MS/MS 36.7 31.7 15.0

221b LC-UV 37.5 36.3 10.7

222 CLIA 35.2 23.5 11.1

225 LC-MS/MS 31.0 14.8 10.4

228a LC-MS/MS 35.4 21.5 12.7

231 LC-UV 30.2 30.8 8.5

241 LC-MS/MS 28.7 17.2 12.3

242 LC-MS/MS 31.1 18.7 12.2

243a LC-UV 27.8 19.6 11.5

243b LC-MS/MS 28.2 21.1 12.4

244 LC-MS/MS 33.0 25.0 13.0

247a CLIA 31.9 16.7 11.7

247b EIA 31.0 22.3 15.7

249 LC-MS/MS 33.3 21.7 12.6

251 LC-MS/MS 34.4 21.6 n/r

253 LC-MS/MS 35.5 21.0 13.1

255 LC-MS/MS 32.9 20.2 12.8

256 CLIA 30.4 17.3 18.5

257 CLIA 31.9 22.4 6.2

258 CLIA 32.8 17.5 11.4

259 LC-MS/MS 26.1 25.5 14.8

260 EIA 32.0 22.0 18.2

261 CLIA 32.3 16.2 10.3

262 CLIA 34.1 52.1 16.5

263 CLIA 28.7 13.4 12.5

264 LC-MS/MS 41.4 29.0 11.4

265 LC-MS/MS 39.0 24.0 14.0

n/r = not reported or not determined

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For all participant datasets, the single reported values for 25(OH)DTotal in VitDQAP-III (Vial A), 

SRM 909c (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 variability (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 expanded uncertainty interval (2 

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

consensus variability area for their technique (IA or LC).

For the IA data for SRM 909c (Vial B), the consensus variability based on MADe is an 

overestimation of the expanded uncertainty interval about the median (Figure 2). The large MADe 

is a result of the non-Gaussian data distribution that contributes to a relatively wide distribution of 

the central 50 % of the data. 

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.

VitDQAP-III (Vial A): Figure 1

 For the IA results, three reported values are outside of the consensus variability range (two

CLIA, one RIA).

 For the LC results, five reported values are outside of the consensus variability range (three LCMSn

, two 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 variability ranges 

for both IA and LC. 

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SRM 909c (Vial B): Figure 2

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

is not well-described with a MADe estimation as described above.

 For the IA results, three reported values are outside the consensus variability range (two CLIA, 

one RIA).

 For the LC results, 10 reported values are outside the consensus variability range, including five 

from LC-MSnand five of the six LC-UV results. 

 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 with the NIST expanded 

uncertainty range (red lines).

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

for both IA and LC. 

SRM 968d L1 (Control): Figure 3

 For the IA results, six reported values are outside of the consensus variability range (four CLIA, 

two EIA).

 For the LC results, nine reported values are outside of the consensus variability range (six LCMSn

, three LC-UV). 

 The consensus median values for both the IA and LC results are comparable with the NIST 

expanded uncertainty range (red lines).

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

both IA and LC. 

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Figure 1. Participant and NIST results for 25(OH)DTotal in VitDQAP-III (Vial A) as determined by immunoassay (CLIA, EIA, and 

RIA) 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 approx. 95% confidence uncertainty 

030b

259

243b241

187198a225242150214c218b

255216194244249060026204b209056b116197251056a

228a253212211220

221a215

199185a265119

264

110

139

189243a

231

221b

185b

204a

263198c

213a256161196017214b

247a257261258

218a262

222

188

210b

213b

247b

260

193

214a200

180

210a

030a

10

20

30

40

50

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

CLIA EIA RIA LC-M Sn LC-UV

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Figure 2. Participant and NIST results for 25(OH)DTotal in SRM 909c (Vial B) as determined by immunoassay (CLIA, EIA, and RIA) 

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 approx. 95% confidence uncertainty 

79.6

225

194

241150211198a242187

204b255056a056b214c116253243b215228a251026249216060197212209220218b030b265

244259

119

264199

221a

185a

243a

231

221b

189

139

263204a161214b218a196

017261247a256258188

213a198c

257

222

210b

185b

262

213b

260247b193

210a200

180

030a

214a

0

10

20

30

40

50

60

25(OH)DTotal in SRM 909c "Vial B" (ng/mL)

110

CLIA EIA RIA LC-M Sn LC-UV

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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,

and RIA) 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 approx. 95% confidence uncertainty 

225

264

150242214c218b21241243b209198a249056b228a056a116204b216255060194244187197253030b026211215199265

259220221a

119

185a

231

221b

243a

110139

189

257213a

198c

210b

261

222258247a161

204a263

214b

017188196

185b218a

262

256

213b

247b

260

193

214a

210a

200

180

030a

0

5

10

15

20

25

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

CLIA EIA RIA LC-M Sn LC-UV

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Figure 4 presents direct graphical comparisons of the 25(OH)DTotal results for a) VitDQAP-III 

(Vial A) and SRM 909c (Vial B), and b) SRM 909c (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.

VitDQAP-III (Vial A) and SRM 909c (Vial B): Figure 4 a

 IA results that are not included in the consensus ranges include numbers 030a, 185b, 210b, 

214a, and 262

 LC results that are not included in the consensus ranges include numbers 030b, 110, 139, 185a, 

189, 199, 221a, 221b, 225, 231, 259, and 264 

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

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

these materials. 

SRM 909c (Vial B) and SRM 968d L1 (Control): Figure 4 b

 IA results that are not included in the consensus ranges include numbers 185b, 193, 213a, 214a, 

256, 257, 260, and 262

 LC results that are not included in the consensus ranges include numbers 110, 119, 139, 185a, 

189, 199, 220, 221a, 221b, 225, 231, 259, and 264

 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. 

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Figure 4. Youden comparison 

plot of the results for 

25(OH)DTotal in a) VitDQAP-III 

(Vial A) and SRM 909c (Vial 

B) and b) 909c (Vial B) and 

SRM 968d L1 (Control) for all 

methods. 

110

030a 030b

139

185a

185b

189

199

210b

214a 221a

221b

225

231

259

262

264

0

10

20

30

40

50

10 20 30 40 50

25(OH)DTotal in SRM 909c "Vial B" (ng/mL)

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

IA methods

LC methods

119

139

185a

185b

189

193

199

213a

214a

220 221a

221b 225

231

256

257

259

260

262

264

5

10

15

20

0 10 20 30 40 50

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

25(OH)DTotal in SRM 909c "Vial B" (ng/mL)

110

LC methods

IA methods

a

b

79.6

79.6

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Discussion of Results for 25(OH)DTotal

The consensus variability of 11 % (all methods) for SRM 968d L1 (Control) is higher than the 7 % 

CV % obtained for this material in the last comparability study (Summer 2013), but it is 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 A) is different from SRM 909c (Vial B) and SRM 968d L1 

(Control) because it contains significant amounts of both 25(OH)D2 and 25(OH)D3 that contribute 

to 25(OH)DTotal. 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 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 A), do not reveal those same trends: the CV % (all methods) is 

relatively low (9.5 %), and the IA method results overlap almost completely with the LC results. 

The difference in the observed results for the VitDQAP-III material (Vial A) is most 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 A) is 

biased 5 % and 7 % lower than the NIST and the LC median results, respectively, which could be 

partially attributable to the nonequivalent response of many IA methods to 25(OH)D2.

Like SRM 968d L1, SRM 909c (Vial B) contains predominantly 25(OH)D3. However, the allmethod consensus variability of 20 % for SRM 909c is somewhat large for a material with a 

relatively high 25(OH)D3 concentration (NIST value 20.7 ng/mL ± 0.7 ng/mL). When SRM 909c 

was evaluated at NIST using the ID-LC/MS methodology, interferences from the matrix were 

observed near the retention times for both 25(OH)D3 and the labeled internal standard that required 

method modifications to avoid a potential bias [1]. However, when SRM 909c was evaluated at 

NIST using the ID-LC-MS/MS methodology, interferences were observed only at the retention time 

for 3-epi-25(OH)D3. Depending on the separation and detection parameters used by participants, 

the matrix peaks could interfere in the determination of 25(OH)D3 and/or 3-epi-25(OH)D3. The IA 

results had the largest variability (CV % of 31 %) for SRM 909c, but some of this is attributable to 

the non-normal distribution of the results; non-Gaussian distributions have also been observed for 

several materials previously evaluated in the VitDQAP. It is unclear if the IA methods are 

susceptible to the matrix interferences observed in SRM 909c or if these interferences contribute to 

the non-normal distribution. However, it is probable that the LC-UV methods were affected by the 

matrix interferences, as five of the six LC-UV results for 25(OH)DTotal were outliers and biased high

(Figure 2), indicating potential coelution of the matrix peaks with the peak for 25(OH)D3.

Both of the Youden plots in Figure 4 incorporate the results for SRM 909c (Vial B) and reveal a 

large number of outliers, more than has been typically observed in Youden comparisons in previous 

comparability studies. The large number of outliers is mostly attributable to the variability of the 

results for SRM 909c, further illustrating that there is something unique about the SRM 909c matrix 

that is influencing the results.

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13

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

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 2014 comparability study, only VitDQAP-III (Vial A) 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 43 LC participants in the Winter 2014 comparability study, all but one of the LC participants 

reported values for 25(OH)D3, and all but three reported values for 25(OH)D2 in VitDQAP-III (Vial 

A); the study results for 25(OH)D3 and 25(OH)D2 are presented in Table 2. Since VitDQAP-III 

(Vial A) contains appreciable amounts of 25(OH)D3 (NIST value 26.2 ng/mL ± 0.7 ng/mL), the 3-

epi-25(OH)D3 metabolite is also measureable in this material. Ten LC participants reported values 

for the 3-epi-25(OH)D3 metabolite, and the results are also presented in Table 2.

The community results are summarized at the bottom of Table 2 for all LC methods and for the LCMSn methods only. These summarized results include N, the median value, the MADe, and the 

CV %. Table 2 also presents the NIST values and the expanded uncertainties (U) for 25(OH)D3, 

25(OH)D2, and 3-epi-25(OH)D3 in VitDQAP-III (Vial A).

 

The single reported values for 25(OH)D3 and 25(OH)D2 in VitDQAP-III (Vial A) 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 A) are summarized below:

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

 Four reported values are outside of the consensus variability range (two LC-MSn, two LC-UV). 

 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 A): Figure 5 b

 Five reported values are outside of the consensus variability range including two from LC-MSn

and three of the four LC-UV results. 

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

(red lines).

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

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This publication is available free of charge from: http://dx.doi.org/10.6028/NIST.IR.8133
14

Table 2. 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 A).

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

Lab Method Vial A Vial A Vial A

026 LC-MS/MS 26.6 7.1 1.5

056a LC-MS/MS 27.8 6.7 0.5

056b LC-MS/MS 26.8 7.2 n/r

060 LC-MS/MS 27.4 6.0 1.1

110 LC-UV 18.6 <2 n/r

116 LC-MS/MS 28.8 5.4 <4.0

119 LC-MS/MS 34.3 5.2 n/r

139 LC-UV 24.7 n/d n/r

150 LC-MS/MS 25.1 6.1 <2

185a LC-MS/MS 30.5 7.9 n/r

187 LC-MS/MS 24.1 6.2 n/r

189 LC-UV 21.4 6.2 n/r

194 LC-MS/MS 25.8 7.2 n/r

197 LC-MS/MS 27.2 7.0 n/r

198a LC-MS/MS 24.5 6.3 n/r

199 LC-MS/MS 30.6 7.6 n/r

204b LC-MS/MS 27.8 6.0 <2.5

209 LC-MS/MS 27.2 6.7 n/r

211 LC-MS/MS 27.6 8.4 n/r

212 LC-MS/MS 27.2 8.5 n/r

214c LC-MS/MS 26.1 5.6 n/r

215 LC-MS/MS 29.2 8.0 n/r

216 LC-MS/MS 26.4 6.6 1.4

218b LC-MS/MS 24.6 7.2 n/r

220 LC-MS/MS 28.0 8.0 n/r

221a LC-MS/MS 28.6 8.1 n/r

221b LC-UV 26.8 10.7 n/r

225 LC-MS/MS 24.7 6.3 n/r

228a LC-MS/MS 28.9 6.5 n/r

231 LC-UV 30.2 0.0 n/r

241 LC-MS/MS 23.6 5.1 0.8

242 LC-MS/MS 25.3 5.8 1.3

243a LC-UV 23.7 4.1 1.5

243b LC-MS/MS 24.3 4.0 1.6

244 LC-MS/MS 27.0 6.0 n/r

249 LC-MS/MS 26.1 7.2 1.4

251 LC-MS/MS 28.4 6.0 n/r

253 LC-MS/MS 27.7 7.8 1.5

255 LC-MS/MS 27.3 5.6 n/r

259 LC-MS/MS 26.1 5.2 n/r

264 LC-MS/MS 30.0 11.4 n/r

265 LC-MS/MS 32.0 7.0 n/r

N 42 40 10

Median 27.1 6.5 1.4

MADe 2.4 1.0 0.1

CV% 8.8 16 11

N 36 36 9

Median 27.2 6.6 1.4

MADe 1.9 1.0 0.1

CV% 7.1 14 11

NIST Value 26.2 6.5 1.6

U 0.7 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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15

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

A). 

 

241

187243b198a218b225150242

194259214c249216026056b244197209212255060211253056a204b220251221a116228a215

264

185a199

265

119

110

189

243a

139

221b

231

15

20

25

30

35

40

42 52 62 72 82 92 102 112 122 132 142 152 162

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

LC-MSn LC-UV

243b

241119259116255214c242060204b244251150187198a225228a216209056a

197265026218b056b249194

199253185a215220221a

211212

264

231

243a

189

221b

0

2

4

6

8

10

12

14

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

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

LC-MSn

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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16

Conclusions from the Winter 2014 Comparability Study of the VitDQAP

In the seven previous comparability studies of the VitDQAP, 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 2014 comparability study, both SRM 909c (Vial B) and SRM 968d L1 (Control) also 

contain predominantly 25(OH)D3. Of these materials, SRM 968d (Control) follows the previouslyobserved trends. However, the results for SRM 909c (Vial B) exhibited larger variability than 

expected (all-method CV of 20 %) given the relatively high concentration of 25(OH)D3. In 

addition, the median IA result for 25(OH)DTotal in this material was biased 12 % lower than the 

NIST median value. It is unclear to what extent these observations are due to the potential matrix 

interferences that are present in SRM 909c (Vial B).

VitDQAP-III (Vial A) was the first study material evaluated in the VitDQAP 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). It was anticipated 

that the IA methods would underrepresent the 25(OH)DTotal concentration due to nonequivalent 

response to the 25(OH)D2 metabolite, which is the trend observed for other materials with relatively 

high concentrations of 25(OH)D2 that had been previously evaluated in the VitDQAP. To the 

contrary, 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 A). The median IA result for VitDQAP-III (Vial A) 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. However, the IA median result for SRM 909c (Vial B), which does not 

contain significant 25(OH)D2, was also biased somewhat low in this comparability study, further 

blurring any conclusions about the IA response to 25(OH)D2 in the VitDQAP-III (Vial A) material.

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17

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

Laboratory 

Number IA Method Sample Preparation Vendor/kit*

17 CLIA n/r A

30a RIA Samples were extracted with acetonitrile B

161 CLIA n/r A

180 RIA Samples were extracted with acetonitrile B

185b CLIA n/r A

188 CLIA n/r C

193 EIA n/r D

196 CLIA No sample preparation required A

198c CLIA n/r E

200 RIA Samples were extracted B

204a CLIA n/r A

210a RIA Sample was extracted with acetonitrile B

210b CLIA n/r E

213a CLIA Sample was thawed and gently mixed prior to analysis E

213b EIA Samples, calibrators, and controls processed per 

manufacturer's protocol D

214a RIA n/r F

214b CLIA n/r A

218a CLIA Direct analysis n/r

222 CLIA n/r C

247a CLIA No pretreatment was used C

247b EIA No pretreatment was used D

256 CLIA n/r A

257 CLIA Sample was thawed at room temperature until analysis E

258 CLIA n/r G

260 EIA n/r n/r

261 CLIA No sample preparation required G

262 CLIA n/r H

263 EIA On board displacement I

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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18

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

30b 25(OH)D3

 -d6

Samples were prepared with 

disposable pipette extraction

C18 column; isocratic elution with 

85 % acetonitrile/15 % methanol; 

flow 0.5 mL/min

25(OH)D3 383/211

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)

APPI

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)

25(OH)D3

 401/383; 

25(OH)D3

-d6 407/371 and 

407/389; 

25(OH)D2

 395/209 and 

395/251

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 reconstitued 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

185a n/r Liquid-liquid extraction PFP column (50 mm × 2.1 mm) n/r

187 n/r 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

198a 25(OH)D3

 -d6

Proteins precipitated with 

methanol, followed by hexane 

extraction, centrifugation, 

evaporation under N2

, and 

reconstitution in methanol with 0.1 

% formic acid

C18 column (50 mm × 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

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19

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 

deuteriated 

standard

Samples extracted using liquidliquid extraction then labeled with 

a derivatization reagent

Revered-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

218b

25(OH)D2-d3 and 

25(OH)D3-d3

Sample was extracted, filtered, 

centrifuged, etc.

Phenyl column (50 mm × 2.1 mm; 

1.7 μm); flow 0.45 mL/min

25(OH)D3 401; 

25(OH)D2 413

220

25(OH)D2-d3 and 

25(OH)D3-d6

Protein crash with 90 % methanol/ 

10 % ZnSO4 and then acetonitrile/ 

1 % formic acid; sample filtered; 

phospholipids removed with SPE

C18 column (20 mm × 2.1mm, 2.7 

μm); gradient with water and 

acetonitrile; flow 1 mL/min; column 

40 °C

MRM with dehydrated 

precursor and product ions

221a 25(OH)D3

-d6

Protein crash with 1% methanol in 

acetonitrile containing the IS

CN column (50 mm × 3.0 mm; 

1.8 μm); methanol/water gradient at 

50 °C

APCI

25(OH)D3 383/211; 

25(OH)D3-d6 389/211; 

25(OH)D2 395/209

225 25(OH)D3

-d6 Liquid-liquid extraction

C8 column (50 mm × 2.1 mm; 

1.7 μm); gradient with 

methanol/water; flow 0.4 mL/min

25(OH)D3 401/107; 

25(OH)D2 413/83

228a D8-labeled 

compound Proteins precipitated n/r n/r

241 25(OH)D3

-d6

Acetonitrile containing the IS (100 

μL) added to sample (200 μL) to 

precipate proteins, followed by 

extraction with hexane, 

centrifugation, removal of 

supernatant, evaporation, and 

reconstitution in methanol solution

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)

APCI

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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20

242 25(OH)D3

-d6

Water with 0.1 % formic acid 

(500 μL) and the IS (400 μL) were 

added to the sample (400 μL), 

followed by centrifugation and 

dilution of supernatant with water

PFP column (150 mm × 2 mm; 

3 μm); isocratic elution with 18 % 

water/ 82 % methanol/ 0.1 % formic 

acid; flow 0.35 mL/min

APCI

25(OH)D3 383/257; 

25(OH)D2 395/269; 

25(OH)D3-d6 389/263;

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

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

243b 25(OH)D3

-d6

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

PFP column (150 mm × 2 mm); 

isocratic separation with 85 % 

methanol/15 % water; flow 0.3 mL/m

25(OH)D3 383/257; 

25(OH)D3-d6 389/269; 

25(OH)D2 395/269

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); 

isocratic separation with 77.5 % 

methanol/22.5 % water; 

flow 0.2 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 deuterium labeled 

25(OH)D3

n/r C8 column; gradient with 

methanol/water/0.1 % formate n/r

264 clozapine

Samples were extracted with tertbutyl methyl ether, evaporated, 

and derivatized with 4-phenyl1,2,4-triazoline-3,5-dione

Reversed-phase column (150 mm × 

4.6 mm); isocratic separation with 

85 % methanol/15 % water/0.3 % 

formic acid; flow 1.4 mL/min

n/r

265 n/r n/r n/r n/r

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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21

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

139 proprietary The sample was extracted, 

centrifuged, and injected

Reversed-phase column heated to 

40 °C, isocratic separation with 

proprietary mobile phase; flow 

1 mL/min

264 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

231 1-alpha-hydroxyvitamin D3

Samples were extracted with 

hexane/dichloromethane, 

evaporated, and reconstituted 

with mobile phase (phosphate 

buffer/acetonitrile)

Reversed-phase column (250 mm × 

4.5 mm; 5μm), isocratic separation 

with 14 % phosphate buffer, 86 % 

acetonitrile; flow 1.2 mL/min

265 nm

243a laurophenone

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

Reversed-phase column (150 mm × 

3 mm); isocratic separation with 

63 % acetonitrile/37 % water; flow 

1 mL/min

264 nm

n/a = not applicable; SPE = solid phase extraction

______________________________________________________________________________________________________ 

This publication is available free of charge from: http://dx.doi.org/10.6028/NIST.IR.8133
22

Appendix B. Raw participant data and 

NIST results for 25(OH)D2, 25(OH)D3,

25(OH)DTotal, and 3-epi-25(OH)D3, in 

VitDQAP-III (Vial A), SRM 909c

(Vial B), and SRM 968d L1 (Control).

VitDQAP-III SRM 909c SRM 968d L1 VitDQAP-III SRM 909c SRM 968d L1 VitDQAP-III SRM 909c SRM 968d L1 VitDQAP-III SRM 909c 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

017 CLIA n/a n/a n/a n/a n/a n/a 31.0 16.2 13.9 n/a n/a n/a

026 LC-MS/MS 7.1 0.4 0.2 26.6 21.3 13.2 33.7 21.7 13.4 1.5 1.7 1.0

030a RIA n/a n/a n/a n/a n/a n/a 40.5 24.8 15.3 n/a n/a n/a

030b LC-MS/MS n/r n/r n/r n/r n/r n/r 24.5 23.9 13.2 n/r n/r n/r

056a LC-MS/MS 6.7 0.6 0.6 27.8 19.7 12.2 34.5 20.3 12.8 0.5 0.5 0.5

056b LC-MS/MS 7.2 0.3 0.1 26.8 20.0 12.6 34.0 20.3 12.7 n/r n/r n/r

060 LC-MS/MS 6.0 0.9 0.2 27.4 21.1 12.8 33.4 22.0 13.0 1.1 0.9 0.8

110 LC-UV <2 <2 <2 18.6 76.0 11.9 21.8 79.6 12.8 n/r n/r n/r

116 LC-MS/MS 5.4 <3.3 <3.3 28.8 20.7 12.8 34.2 20.7 12.8 <4.0 <4.0 <4.0

119 LC-MS/MS 5.2 n/d n/d 34.3 27.3 15.6 39.5 27.3 15.6 n/r n/r n/r

139 LC-UV n/d n/d n/d 24.7 51.9 13.1 24.7 51.9 13.1 n/r n/r n/r

150 LC-MS/MS 6.1 <2 <2 25.1 17.7 12.1 31.2 17.7 12.1 <2 <2 <2

161 CLIA n/a n/a n/a n/a n/a n/a 30.6 14.4 11.8 n/a n/a n/a

180 RIA n/a n/a n/a n/a n/a n/a 29.1 19.5 14.9 n/a n/a n/a

185a LC-MS/MS 7.9 0.0 0.0 30.5 36.4 17.3 38.4 36.4 17.3 n/r 7.0 n/r

185b CLIA n/a n/a n/a n/a n/a n/a 16.4 33.3 14.5 n/a n/a n/a

187 LC-MS/MS 6.2 0.0 0.0 24.1 19.0 13.1 30.3 19.0 13.1 n/r n/r n/r

188 CLIA n/a n/a n/a n/a n/a n/a 36.1 18.4 14.1 n/a n/a n/a

189 LC-UV 6.2 23.4 0.0 21.4 14.8 16.5 27.6 38.2 16.5 n/r n/r n/r

193 EIA n/a n/a n/a n/a n/a n/a 33.9 22.3 18.6 n/a n/a n/a

194 LC-MS/MS 7.2 <7.0 <7.0 25.8 15.8 13.0 33.0 15.8 13.0 n/r n/r n/r

196 CLIA n/a n/a n/a n/a n/a n/a 30.7 15.1 14.1 n/a n/a n/a

197 LC-MS/MS 7.0 <5 0.1 27.2 22.0 13.0 34.2 22.0 13.1 n/r n/r n/r

198a LC-MS/MS 6.3 <5 <5 24.5 18.5 12.6 30.8 18.5 12.6 n/r n/r n/r

198c CLIA n/a n/a n/a n/a n/a n/a 29.1 19.7 8.3 n/a n/a n/a

199 LC-MS/MS 7.6 2.1 <2 30.6 27.5 13.7 38.2 29.6 13.7 n/r n/r n/r

200 RIA n/a n/a n/a n/a n/a n/a 27.7 18.0 13.8 n/a n/a n/a

204a CLIA n/a n/a n/a n/a n/a n/a 27.5 14.0 12.2 n/a n/a n/a

204b LC-MS/MS 6.0 n/d n/d 27.8 20.2 12.8 33.8 20.2 12.8 <2.5 <2.5 <2.5

209 LC-MS/MS 6.7 <1.0 <1.0 27.2 22.9 12.5 33.9 22.9 12.5 n/r n/r n/r

210a RIA n/a n/a n/a n/a n/a n/a 31.4 17.9 12.4 n/a n/a n/a

210b CLIA n/a n/a n/a n/a n/a n/a 38.7 25.0 9.1 n/a n/a n/a

211 LC-MS/MS 8.4 <5 <5 27.6 18.3 13.6 36.0 18.3 13.6 n/r n/r n/r

212 LC-MS/MS 8.5 0.0 0.0 27.2 22.3 12.3 35.7 22.3 12.3 n/r n/r n/r

213a CLIA n/a n/a n/a n/a n/a n/a 30.4 19.4 6.5 n/a n/a n/a

213b EIA n/a n/a n/a n/a n/a n/a 26.3 17.3 12.9 n/a n/a n/a

214a RIA n/a n/a n/a n/a n/a n/a 27.5 31.3 11.5 n/a n/a n/a

214b CLIA n/a n/a n/a n/a n/a n/a 31.2 14.4 13.0 n/a n/a n/a

214c LC-MS/MS 5.6 <0.5 <0.5 26.1 20.5 12.2 31.7 20.5 12.2 n/r n/r n/r

215 LC-MS/MS 8.0 <2 <2 29.2 21.2 13.6 37.2 21.2 13.6 n/r n/r n/r

216 LC-MS/MS 6.6 0.3 0.1 26.4 21.5 12.7 32.9 21.9 12.8 1.4 1.1 0.6

218a CLIA n/a n/a n/a n/a n/a n/a 33.7 14.5 14.5 n/a n/a n/a

218b LC-MS/MS 7.2 0.1 0.0 24.6 23.7 12.2 31.8 23.8 12.2 n/r n/r n/r

220 LC-MS/MS 8.0 n/d n/d 28.0 23.0 15.0 36.0 23.0 15.0 n/r n/r n/r

221a LC-MS/MS 8.1 <5.0 <5.0 28.6 31.7 15.0 36.7 31.7 15.0 n/r n/r n/r

221b LC-UV 10.7 15.1 <5.0 26.8 21.2 10.7 37.5 36.3 10.7 n/r n/r n/r

222 CLIA n/a n/a n/a n/a n/a n/a 35.2 23.5 11.1 n/a n/a n/a

225 LC-MS/MS 6.3 <5 <5 24.7 14.8 10.4 31.0 14.8 10.4 n/r n/r n/r

228a LC-MS/MS 6.5 n/d n/d 28.9 21.5 12.7 35.4 21.5 12.7 n/r n/r n/r

231 LC-UV 0.0 0.0 0.0 30.2 30.8 8.5 30.2 30.8 8.5 n/r n/r n/r

241 LC-MS/MS 5.1 0.2 0.1 23.6 17.0 12.2 28.7 17.2 12.3 0.8 0.4 0.6

242 LC-MS/MS 5.8 n/d n/d 25.3 18.7 12.2 31.1 18.7 12.2 1.3 2.2 0.6

243a LC-UV 4.1 n/d n/d 23.7 19.6 11.5 27.8 19.6 11.5 1.5 2.4 n/d

243b LC-MS/MS 4.0 n/d n/d 24.3 21.1 12.4 28.2 21.1 12.4 1.6 2.5 n/d

244 LC-MS/MS 6.0 <5 <5 27.0 25.0 13.0 33.0 25.0 13.0 n/r n/r n/r

247a CLIA n/a n/a n/a n/a n/a n/a 31.9 16.7 11.7 n/a n/a n/a

247b EIA n/a n/a n/a n/a n/a n/a 31.0 22.3 15.7 n/a n/a n/a

249 LC-MS/MS 7.2 0.3 0.2 26.1 21.4 12.4 33.3 21.7 12.6 1.4 1.4 0.4

251 LC-MS/MS 6.0 <4 n/r 28.4 21.6 n/r 34.4 21.6 n/r n/r n/r n/r

253 LC-MS/MS 7.8 0.3 0.1 27.7 20.7 13.0 35.5 21.0 13.1 1.5 1.1 0.6

255 LC-MS/MS 5.6 0.3 0.1 27.3 19.9 12.7 32.9 20.2 12.8 n/r n/r n/r

256 CLIA n/a n/a n/a n/a n/a n/a 30.4 17.3 18.5 n/a n/a n/a

257 CLIA n/a n/a n/a n/a n/a n/a 31.9 22.4 6.2 n/a n/a n/a

258 CLIA n/a n/a n/a n/a n/a n/a 32.8 17.5 11.4 n/a n/a n/a

259 LC-MS/MS 5.2 <2 <2 26.1 20.3 14.8 26.1 25.5 14.8 n/r n/r n/r

260 EIA n/a n/a n/a n/a n/a n/a 32.0 22.0 18.2 n/a n/a n/a

261 CLIA n/a n/a n/a n/a n/a n/a 32.3 16.2 10.3 n/a n/a n/a

262 CLIA n/a n/a n/a n/a n/a n/a 34.1 52.1 16.5 n/a n/a n/a

263 CLIA n/a n/a n/a n/a n/a n/a 28.7 13.4 12.5 n/a n/a n/a

264 LC-MS/MS 11.4 0.7 0.1 30.0 28.3 11.3 41.4 29.0 11.4 n/r n/r n/r

265 LC-MS/MS 7.0 0.0 0.0 32.0 24.0 14.0 39.0 24.0 14.0 n/r n/r n/r

NIST Value 6.49 n/r 0.10 26.2 20.7 12.4 32.7 20.7 12.5 1.6 n/r 0.65

U 0.17 n/r --- 0.6 0.7 0.4 0.7 0.7 0.4 0.1 n/r 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)

____________________________________________________________________________________________

This publication is available free of charge from: http://dx.doi.org/10.6028/NIST.IR.8133