NISTIR 8141

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report of 

Participant Results: Summer

2014 Comparability Study

(Exercise 9)

Mary Bedner 

Susan S.-C. Tai 

This publication is available free of charge from:

http://dx.doi.org/10.6028/NIST.IR.8141
NISTIR 8141

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report of 

Participant Results: Summer

2014 Comparability Study

(Exercise 9)

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.8141

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 ninth exercise of

this program, the Summer 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) 2973 Vitamin D Metabolites in Frozen Human Serum (High Level) and 

VitDQAP-I (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 ninth study are reported along with a 

summary of the analytical methods used.

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1

OVERVIEW OF THE SUMMER 2014 COMPARABILITY STUDY

For the Summer 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-I, and Vial B was SRM 2973 Vitamin 

D Metabolites in Frozen Human Serum (High Level), 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 53 participants and 63 datasets (nine participants provided data from two or 

more methods) in the Summer 2014 comparability study. Twenty-three of the datasets originated 

from immunoassay (IA) techniques, including 15 from chemiluminescence immunoassay (CLIA), 

two from enzyme immunoassay (EIA), five from radioimmunoassay (RIA), and one from 

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

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

methods; of those, 35 were from LC with tandem mass spectrometric detection (LC-MSn), and five

were from LC with ultraviolet absorbance detection (LC-UV). A summary of the LC methods used 

by the participants may be found in Appendices A-2 and A-3. 

The raw data received from all participants 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 in 

addition to 25(OH)DTotal. The 25(OH)D2 concentration was below the detection limit for most 

reported methods in the control and study materials. Ten LC participants reported results for 3-epi25(OH)D3.

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 METHOD USED TO EVALUATE THE CONTROL AND 

STUDY MATERIALS

NIST used isotope dilution LC-MS/MS (ID-LC-MS/MS)[1] 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 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). 

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

reported with expanded uncertainties (U) that approximate a 95 % confidence interval. U

incorporates components for measurement variability and measurement uncertainty associated with

the density of the materials and the purity of the reference standards as well as a 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-epi25(OH)D3 are reported as described for VitDQAP-I (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 values for 25(OH)DTotal in VitDQAP-I (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 uncertainty (U) incorporates

measurement uncertainties for the two analytes.

For SRM 2973 (Vial B), the reported NIST value for 25(OH)D3 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 [2]. The NIST 

values for 25(OH)D2, 3-epi-25(OH)D3, and 25(OH)DTotal in SRM 2973 (Vial B) are reference 

values. A NIST reference value is a best estimate of the true value provided by NIST where all 

known or suspected sources of bias have not been fully investigated by NIST [2]. More detailed 

information about the characterization of SRM 2973 and the components of the expanded 

uncertainty may be found in the Certificate of Analysis, located on the NIST website [3]. 

 

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

2 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

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

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SUMMER 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-I (Vial A), SRM 2973 (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-I

(Vial A), SRM 2973 (Vial B), and SRM 968d L1 (Control). 

VitDQAP-I SRM 2973 SRM 968d L1

Lab Method Vial A Vial B Control

017 CLIA 33.8 41.8 14.1

026 LC-MS/MS 30.7 38.3 12.0

030a RIA 35.1 44.5 14.2

056a LC-MS/MS 33.4 42.7 12.8

056b LC-MS/MS 30.3 38.1 12.8

060 LC-MS/MS 28.0 37.0 12.0

110 LC-UV 32.5 43.3 12.6

116 LC-MS/MS 35.1 43.3 15.1

119 LC-MS/MS 30.5 37.6 n/r

150 LC-MS/MS 28.2 37.1 12.4

161b LC-MS/MS 33.0 41.0 13.0

180 RIA 30.4 36.5 15.1

187 LC-MS/MS 33.8 44.8 12.1

188 CLIA 42.9 50.2 12.3

189 LC-UV 39.4 48.7 8.6

194 LC-MS/MS 33.9 38.8 12.1

196 CLIA 29.8 37.1 13.2

197 LC-MS/MS 30.3 40.8 12.4

199 LC-MS/MS 31.4 39.6 13.3

200 RIA 23.0 27.8 12.7

204a CLIA 31.0 43.2 14.9

204b LC-MS/MS 30.8 41.5 12.7

209 LC-MS/MS 34.0 48.8 12.4

210a RIA 32.1 40.2 17.4

210b CLIA 30.9 44.8 n/r

211 LC-MS/MS 37.8 49.1 12.6

212 LC-MS/MS 31.9 42.9 12.8

213a CLIA 35.2 48.4 7.4

213b EIA 25.2 32.3 13.9

214a RIA 17.1 16.6 6.3

214b CLIA 29.4 38.1 13.0

214c LC-MS/MS 31.3 38.6 12.2

215 LC-MS/MS 34.8 40.8 14.0

216 LC-MS/MS 37.2 41.3 12.3

217 LC-MS/MS 39.2 48.8 16.4

218a CLIA 31.1 40.7 14.0

218b LC-MS/MS 38.1 42.1 14.7

220 LC-MS/MS 31.0 38.4 12.5

221a LC-MS/MS 30.1 35.3 14.9

221b LC-UV 34.3 42.7 38.5

222 CLIA 35.3 40.1 15.0

225 LC-MS/MS 32.9 44.6 13.4

228a LC-MS/MS 37.0 45.7 12.0

242 LC-MS/MS 30.0 36.9 12.8

243a LC-UV 29.8 37.0 12.9

243b LC-MS/MS 30.0 37.0 12.6

244 LC-MS/MS 34.0 40.0 16.0

249 LC-MS/MS 30.4 41.2 12.8

251 LC-MS/MS 37.0 54.0 n/r

253 LC-MS/MS 30.8 39.6 12.2

255 LC-MS/MS 33.4 41.0 12.9

256 CLIA 33.0 35.8 11.4

257 CLIA 30.5 42.0 5.9

258 CLIA 33.9 64.4 13.6

259 LC-MS/MS 34.4 45.2 10.3

261 CLIA 24.2 37.4 6.6

262 CLIA 27.5 39.1 20.2

263 CLIA 33.7 37.6 12.8

264 LC-MS/MS 41.6 39.8 14.0

265 LC-MS/MS 35.0 44.0 13.0

266a LC-UV 41.3 50.8 16.4

266b EIA 29.2 38.3 18.5

267 CLEIA 30.4 39.3 12.4

n/r = not reported or not determined

VitDQAP-I SRM 2973 SRM 968d L1

Vial A Vial B Control

N 63 63 60

Median 32.1 40.8 12.8

MADe 2.9 4.1 1.1

CV% 9.1 10 8.7

N 23 23 22

Median 30.9 39.3 13.4

MADe 4.2 4.0 2.0

CV% 14 10 15

N 40 40 38

Median 33.2 41.1 12.8

MADe 3.6 4.0 0.8

CV% 11 9.8 6.4

N 35 35 33

Median 33.0 41.0 12.8

MADe 3.3 3.9 0.7

CV% 9.9 9.4 5.8

NIST Value 32.0 40.1 12.5

U 0.8 0.8 0.4

n/r = not reported or not determined

All 

methods

IA 

methods 

LC 

methods LC-MS

n

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

SRM 2973 (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 approximate 95 % confidence 

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).

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

expanded 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-I (Vial A): Figure 1

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

one RIA).

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

, 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 slightly higher than 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 2973 (Vial B): Figure 2

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

two RIA).

 For the LC results, two reported values are outside the consensus variability range (one LCMSn

, one LC-UV). 

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

expanded uncertainty range (red lines).

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SRM 968d L1 (Control): Figure 3

 The consensus variability range is larger for the IA results than for the LC results. 

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

one EIA, one RIA).

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

, three LC-UV). 

 The consensus median value for the IA results is slightly 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 variability range for 

both IA and LC. 

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

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

060150

24243b221a056b197249119026204b253220214c199212

225161b056a255187194209244259215265116

228a251216

211218b

217

264

243a

110

221b

189

266a

261

262

214b196

257210b204a218a

256

263017258

213a222

188

213b

266b

214a

200

180

210a

030a

267

10

20

30

40

50

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380

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

CLIA EIA RIA ECLIA 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 approx. 95% confidence uncertainty 

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Figure 2. Participant and NIST results for 25(OH)DTotal in SRM 2973 (Vial B) 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 approx. 95% confidence uncertainty 

221a

24243b060150119056b026220214c194199253264244197215161b255249216204b218b056a212116265225187259228a

209217211

251

243a

221b110

189

266a

256

196261263214b

262

222218a

017257

204a

210b

213a

188

258

213b

266b

214a

200

180

210a

030a

267

5

15

25

35

45

55

65

75

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380

25(OH)DTotal in SRM 2973 "Vial B" (ng/mL)

CLIA EIA RIA CLEIA LC-MSn 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,

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 approx. 95% confidence uncertainty 

259

026060228a187194214c253216150197209220211243b204b056a212242249056b255161b265

199225

215264

218b221a116

244

217

189

110

243a

266a

257

261

213a

256

188

263214b196

258

218a017

204a222

262

213b

266b

214a

200

030a

180

210a

267

4

6

8

10

12

14

16

18

20

22

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380

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

CLIA EIA RIA ECLIA LC-M Sn LC-UV

38.5 221b

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

(Vial A) and SRM 2973 (Vial B), and b) SRM 2973 (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-I (Vial A) and SRM 2973 (Vial B): Figure 4 a

 IA results that are outside of the consensus range include numbers 188, 200, 213a, 214a, and 

258.

 LC results that are outside of the consensus range include numbers 251, 264, and 266. 

 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 2973 (Vial B) and SRM 968d L1 (Control): Figure 4 b

 IA results that outside of the consensus range include numbers 188, 200, 210a, 213a, 214a, 257, 

258, 261, 262, and 266b

 LC results that are outside of the consensus range include numbers 116, 189, 217, 218b, 221a, 

221b, 244, 259, and 266a

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

IA consensus box, illustrating that the LC results are in slightly better agreement 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-I (Vial A) and 

SRM 2973 (Vial B) and

b) SRM 2973 (Vial B) and SRM 

968d L1 (Control) for all methods. 

116

188

189

200

210a

213a

214a

217

218b 221a

244

257

258

259

261

262

266a

266b

5

10

15

20

10 20 30 40 50 60 70

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

25(OH)DTotal in SRM 2973 "Vial B" (ng/mL)

221b

LC methods

IA methods

188

200

213a

214a

251

258

264

266a

10

20

30

40

50

60

70

10 20 30 40 50

25(OH)DTotal in SRM 2973 "Vial B" (ng/mL)

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

IA methods

LC methods

a

b

38.5

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LC method results for 25(OH)D3, 25(OH)D2, and 3-epi-25(OH)D3

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.

Of the 41 LC participants in the Summer 2014 comparability study, all but one lab reported values 

for 25(OH)D3, and the study results for 25(OH)D3 in the study materials and the control are 

presented in Table 2. Neither the study materials nor the control contained a significant 

concentration of 25(OH)D2, but the study results for the 12 participants that reported results for this 

metabolite in at least one of the materials are presented in Table 3. 

Since VitDQAP-I (Vial A) and SRM 2973 (Vial B) contain relatively high concentrations of 

25(OH)D3 (Table 2), the 3-epi-25(OH)D3 metabolite, which frequently correlates with 25(OH)D3, 

is also measureable in these materials. Ten LC participants reported values for the 3-epi-25(OH)D3

metabolite in at least one of the materials, and the results are presented in Table 4. Because the

biological significance of 3-epi-25(OH)D3 remains uncertain, this metabolite is not included in the 

25(OH)DTotal concentration. Therefore, LC methods that do not chromatographically separate this 

diastereomer from 25(OH)D3 yield biased results for 25(OH)D3 and hence 25(OH)DTotal.

The community results are summarized at the bottom of each table (Table 2 - 4), for all LC 

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

the MADe, and the CV %. The NIST values and the expanded uncertainties are also presented at 

the bottom of each table.

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Table 2. Summary of LC participant data and NIST results for 25(OH)D3 (ng/mL) in VitDQAP-I 

(Vial A), SRM 2973 (Vial B), and SRM 968d L1 (Control).

VitDQAP-I SRM 2973 SRM 968d L1

Lab Method Vial A Vial B Control

026 LC-MS/MS 29.9 37.4 11.6

056a LC-MS/MS 32.7 41.7 12.6

056b LC-MS/MS 29.6 37.4 12.6

060 LC-MS/MS 27.4 36.4 11.8

116 LC-MS/MS 35.1 43.3 15.1

119 LC-MS/MS 30.5 37.6 n/r

139 LC-UV 46.2 33.7 12.7

150 LC-MS/MS 28.2 37.1 12.4

161b LC-MS/MS 33.0 41.0 13.0

187 LC-MS/MS 33.8 44.8 12.1

189 LC-UV 39.4 48.7 8.6

194 LC-MS/MS 33.9 38.8 12.1

197 LC-MS/MS 30.3 40.8 12.4

199 LC-MS/MS 31.4 39.6 13.3

204b LC-MS/MS 30.8 41.5 12.7

209 LC-MS/MS 34.0 48.8 12.4

211 LC-MS/MS 37.8 49.1 12.6

212 LC-MS/MS 31.9 42.9 12.8

214c LC-MS/MS 31.3 38.6 12.2

215 LC-MS/MS 34.8 40.8 14.0

216 LC-MS/MS 36.4 40.6 12.2

217 LC-MS/MS 39.2 48.8 16.4

218b LC-MS/MS 38.1 41.8 14.7

220 LC-MS/MS 31.0 38.4 12.5

221a LC-MS/MS 30.1 35.3 14.9

221b LC-UV 34.3 42.7 38.5

225 LC-MS/MS 32.9 44.6 13.4

228a LC-MS/MS 37.0 45.7 12.0

242 LC-MS/MS 29.5 36.5 12.8

243a LC-UV 29.5 36.6 12.7

243b LC-MS/MS 29.3 36.4 12.7

244 LC-MS/MS 34.0 40.0 16.0

249 LC-MS/MS 30.4 41.2 12.8

251 LC-MS/MS 37.0 54.0 n/r

253 LC-MS/MS 30.1 39.0 12.1

255 LC-MS/MS 32.7 40.3 12.7

259 LC-MS/MS 34.4 45.2 10.3

264 LC-MS/MS 41.0 39.2 13.9

265 LC-MS/MS 35.0 44.0 13.0

266a LC-UV 41.3 50.8 16.4

N 40 40 38

Median 33.0 40.8 12.7

MADe 3.9 4.7 0.9

CV% 12 12 6.7

N 35 35 33

Median 32.7 40.8 12.7

MADe 3.4 3.7 0.8

CV% 10 9.1 6.4

NIST Value 31.3 39.4 12.4

U 0.8 0.8 0.4

n/r = not reported or not determined

25(OH)D3 (ng/mL)

LC 

methods LC-MS

n

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14

Table 3. Summary of LC participant and 

NIST results for 25(OH)D2 (ng/mL) in 

VitDQAP-I (Vial A), SRM 2973 (Vial B), 

and SRM 968d L1 (Control).

Table 4. Summary of LC participant and 

NIST results for 3-epi-25(OH)D3 (ng/mL) 

in VitDQAP-I (Vial A), SRM 2973 (Vial 

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

VitDQAP-I SRM 2973 SRM 968d L1

Lab Method Vial A Vial B Control

026 LC-MS/MS 0.8 1.0 0.4

056a LC-MS/MS 0.8 1.0 0.2

056b LC-MS/MS 0.7 0.7 0.2

060 LC-MS/MS 0.6 0.6 0.2

216 LC-MS/MS 0.7 0.7 0.2

218b LC-MS/MS 0.0 0.3 0.0

242 LC-MS/MS 0.5 0.4 n/d

243a LC-UV 0.5 0.4 n/d

243b LC-MS/MS 0.5 0.4 n/d

253 LC-MS/MS 0.7 0.6 0.1

255 LC-MS/MS 0.7 0.7 0.2

264 LC-MS/MS 0.6 0.6 0.1

N 12 12 9

Median 0.7 0.6 0.2

MADe 0.1 0.2 0.1

CV% 22 31 37

N 11 11 9

Median 0.7 0.6 0.2

MADe 0.1 0.1 0.1

CV% 15 20 37

NIST Value 0.68 0.65 0.1*

U 0.06 0.02 ---

n/d = not detected; *estimated value

25(OH)D2 (ng/mL)

LC 

methods LC-MS

n

VitDQAP-I SRM 2973 SRM 968d L1

Lab Method Vial A Vial B Control

026 LC-MS/MS 2.7 2.4 0.7

056a LC-MS/MS 1.8 3.6 1.3

060 LC-MS/MS 1.8 2.4 0.7

150 LC-MS/MS 2.0 3.0 <2

216 LC-MS/MS 2.0 2.3 0.7

242 LC-MS/MS 1.5 1.7 0.6

243a LC-UV 1.5 1.7 0.7

243b LC-MS/MS 1.4 1.8 0.6

249 LC-MS/MS 1.5 1.8 0.5

253 LC-MS/MS 1.9 2.1 0.7

N 10 10 9

Median 1.8 2.2 0.70

MADe 0.4 0.6 0.07

CV% 20 29 11

N 9 9 8

Median 1.8 2.3 0.70

MADe 0.2 0.7 0.08

CV% 13 32 12

NIST Value 1.7 2.1 0.65

U 0.1 0.1 0.03

< X = less than a quantitation limit of X

LC-MS

n LC 

methods

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

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15

Discussion of the Summer 2014 Comparability Study Results

In the Summer 2014 study of the VitDQAP, 25(OH)D3 is the predominant vitamin D metabolite 

contributing to 25(OH)DTotal in the two study materials and the control material, which is consistent 

with most materials evaluated in the VitDQAP. However, the two study materials VitDQAP-I (Vial 

A) and SRM 2973 (Vial B) both contain relatively high levels of 25(OH)D3, with NIST values of 

31.3 ng/mL ± 0.8 ng/mL and 39.4 ng/mL ± 0.8 ng, mL, respectively. SRM 2973 (Vial B) contains

the highest concentration of 25(OH)D3 of any study materials evaluated in the VitDQAP to date. 

The consensus variabilities for 25(OH)DTotal determined by the IA and LC techniques differ for 

SRM 968d L1 (Control), with values of 15 % and 6.4 %, respectively. When the IA and LC results 

for SRM 968d L1 (Control) are compared to the results from the previous two studies of the 

VitDQAP, the consensus variability for the LC methods has remained remarkably consistent around 

6 % while the consensus variability for the IA methods has fluctuated between 6 % and 18 %, 

indicating that IA methods are in less control. However, the IA variability for the VitDQAP-I (Vial 

A) and SRM 2973 (Vial B) study materials of 14 % and 10 %, respectively, indicate that the IA 

method performance is relatively consistent for different materials/matrices and 25(OH)D3

concentrations. Conversely, the LC method variability of 11 % and 10 %, respectively, for the two 

study materials VitDQAP-I (Vial A) and SRM 2973 (Vial B) is somewhat higher than for SRM 

968d L1 (Control), but still exhibits general consistency with performance on the blinded study 

samples containing predominantly 25(OH)D3 evaluated in previous studies. 

The participant performance in the Summer 2014 study is generally consistent with performance in 

the eight prior studies, indicating that the relatively high levels of 25(OH)D3 and hence 

25(OH)DTotal in the two study samples do not pose a particular measurement challenge. To date, the 

most significant variability in the participant results arose from study samples that contain relatively 

high levels of the 25(OH)D2 and 3-epi-25(OH)D3 metabolites. 

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16

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

180 RIA Samples were extracted with acetonitrile B

188 CLIA n/r C

196 CLIA No sample preparation required A

200 RIA Samples were extracted B

204a CLIA n/r A

210a RIA Sample was extracted with acetonitrile B

210b CLIA n/r D

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

213b EIA Samples, calibrators, and controls processed per 

manufacturer's protocol E

214a RIA n/r F

214b CLIA n/r A

218a CLIA Direct analysis n/r

222 CLIA n/r C

256 CLIA n/r A

257 CLIA Sample was thawed at room temperature until analysis D

258 CLIA n/r G

261 CLIA No sample preparation required G

262 CLIA n/r H

263 EIA On board displacement I

266b EIA n/r E

267 CLEIA n/r J

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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17

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)

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

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

APCI

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

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

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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

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)

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 × 2.1 mm, 

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

13C-labeled 

compound

Proteins precipitated n/r n/r

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

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19

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

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

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 x 

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

Liquid-liquid extraction using 

hexane

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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20

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

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

264 nm

266a alpha calcidol

Proteins precipitated with 

acetonitrile and methanol, 

extracted with SPE, eluted with 

ethyl acetate, evaporated, and 

reconstituted in acetonitrile

C18 column (4.6 mm × 150 mm; 

2.5 μm); gradient with acetonitrile 

and methanol; flow 0.5 mL/min

265 nm

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

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21

Appendix B. Raw participant 

data and NIST results for 

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

25(OH)DTotal, and 3-epi25(OH)D3, and in VitDQAP-I

(Vial A), SRM 2973 (Vial B), 

and SRM 968d L1 (Control).

VitDQAP-I SRM 2973 SRM 968d L1 VitDQAP-I SRM 2973 SRM 968d L1 VitDQAP-I SRM 2973 SRM 968d L1 VitDQAP-I SRM 2973 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 33.8 41.8 14.1 n/a n/a n/a

026 LC-MS/MS 0.8 1.0 0.4 29.9 37.4 11.6 30.7 38.3 12.0 2.7 2.4 0.7

030a RIA n/a n/a n/a n/a n/a n/a 35.1 44.5 14.2 n/a n/a n/a

056a LC-MS/MS 0.8 1.0 0.2 32.7 41.7 12.6 33.4 42.7 12.8 1.8 3.6 1.3

056b LC-MS/MS 0.7 0.7 0.2 29.6 37.4 12.6 30.3 38.1 12.8 n/r n/r n/r

060 LC-MS/MS 0.6 0.6 0.2 27.4 36.4 11.8 28.0 37.0 12.0 1.8 2.4 0.7

110 LC-UV n/r n/r n/r n/r n/r n/r 32.5 43.3 12.6 n/r n/r n/r

116 LC-MS/MS <3.3 <3.3 <3.3 35.1 43.3 15.1 35.1 43.3 15.1 <4.0 <4.0 <4.0

119 LC-MS/MS n/d n/d n/d 30.5 37.6 n/r 30.5 37.6 n/r n/r n/r n/r

139 LC-UV n/r n/r n/r 46.2 33.7 12.7 n/r n/r n/r n/r n/r n/r

150 LC-MS/MS <2 <2 <2 28.2 37.1 12.4 28.2 37.1 12.4 2.0 3.0 <2

180 RIA n/a n/a n/a n/a n/a n/a 30.4 36.5 15.1 n/a n/a n/a

187 LC-MS/MS <1.5 <1.5 <1.5 33.8 44.8 12.1 33.8 44.8 12.1 n/r n/r n/r

188 CLIA n/a n/a n/a n/a n/a n/a 42.9 50.2 12.3 n/a n/a n/a

189 LC-UV 0.0 0.0 0.0 39.4 48.7 8.6 39.4 48.7 8.6 n/r n/r n/r

194 LC-MS/MS <7.0 <7.0 <7.0 33.9 38.8 12.1 33.9 38.8 12.1 n/r n/r n/r

196 CLIA n/a n/a n/a n/a n/a n/a 29.8 37.1 13.2 n/a n/a n/a

197 LC-MS/MS <5 <5 <5 30.3 40.8 12.4 30.3 40.8 12.4 n/r n/r n/r

199 LC-MS/MS <2.0 <2.0 <2.0 31.4 39.6 13.3 31.4 39.6 13.3 n/r n/r n/r

200 RIA n/a n/a n/a n/a n/a n/a 23.0 27.8 12.7 n/a n/a n/a

204a CLIA n/a n/a n/a n/a n/a n/a 31.0 43.2 14.9 n/a n/a n/a

204b LC-MS/MS n/d n/d n/d 30.8 41.5 12.7 30.8 41.5 12.7 n/d < 2.4 n/d

209 LC-MS/MS <1.0 <1.0 <1.0 34.0 48.8 12.4 34.0 48.8 12.4 n/r n/r n/r

210a RIA n/a n/a n/a n/a n/a n/a 32.1 40.2 17.4 n/a n/a n/a

210b CLIA n/a n/a n/a n/a n/a n/a 30.9 44.8 n/r n/a n/a n/a

211 LC-MS/MS 0.0 0.0 0.0 37.8 49.1 12.6 37.8 49.1 12.6 n/r n/r n/r

212 LC-MS/MS <2 <2 <2 31.9 42.9 12.8 31.9 42.9 12.8 n/r n/r n/r

213a CLIA n/a n/a n/a n/a n/a n/a 35.2 48.4 7.4 n/a n/a n/a

213b EIA n/a n/a n/a n/a n/a n/a 25.2 32.3 13.9 n/a n/a n/a

214a RIA n/a n/a n/a n/a n/a n/a 17.1 16.6 6.3 n/a n/a n/a

214b CLIA n/a n/a n/a n/a n/a n/a 29.4 38.1 13.0 n/a n/a n/a

214c LC-MS/MS <1.0 <1.0 <1.0 31.3 38.6 12.2 31.3 38.6 12.2 n/r n/r n/r

215 LC-MS/MS <2 <2 <2 34.8 40.8 14.0 34.8 40.8 14.0 n/r n/r n/r

216 LC-MS/MS 0.7 0.7 0.2 36.4 40.6 12.2 37.2 41.3 12.3 2.0 2.3 0.7

217 LC-MS/MS <2 <2 <2 39.2 48.8 16.4 39.2 48.8 16.4 n/r n/r n/r

218a CLIA n/a n/a n/a n/a n/a n/a 31.1 40.7 14.0 n/a n/a n/a

218b LC-MS/MS 0.0 0.3 0.0 38.1 41.8 14.7 38.1 42.1 14.7 n/r n/r n/r

220 LC-MS/MS n/d n/d n/d 31.0 38.4 12.5 31.0 38.4 12.5 n/r n/r n/r

221a LC-MS/MS <2.0 <2.0 <2.0 30.1 35.3 14.9 30.1 35.3 14.9 n/r n/r n/r

221b LC-UV 0.0 0.0 0.0 34.3 42.7 38.5 34.3 42.7 38.5 n/r n/r n/r

222 CLIA n/a n/a n/a n/a n/a n/a 35.3 40.1 15.0 n/a n/a n/a

225 LC-MS/MS <5 <5 <5.0 32.9 44.6 13.4 32.9 44.6 13.4 n/r n/r n/r

228a LC-MS/MS n/d n/d n/d 37.0 45.7 12.0 37.0 45.7 12.0 n/r n/r n/r

242 LC-MS/MS 0.5 0.4 n/d 29.5 36.5 12.8 30.0 36.9 12.8 1.5 1.7 0.6

243a LC-UV 0.5 0.4 n/d 29.5 36.6 12.7 29.8 37.0 12.9 1.5 1.7 0.7

243b LC-MS/MS 0.5 0.4 n/d 29.3 36.4 12.7 30.0 37.0 12.6 1.4 1.8 0.6

244 LC-MS/MS <5 <5 <5 34.0 40.0 16.0 34.0 40.0 16.0 n/r n/r n/r

249 LC-MS/MS <0.8 <0.8 <0.8 30.4 41.2 12.8 30.4 41.2 12.8 1.5 1.8 0.5

251 LC-MS/MS <4 <4 n/r 37.0 54.0 n/r 37.0 54.0 n/r n/r n/r n/r

253 LC-MS/MS 0.7 0.6 0.1 30.1 39.0 12.1 30.8 39.6 12.2 1.9 2.1 0.7

255 LC-MS/MS 0.7 0.7 0.2 32.7 40.3 12.7 33.4 41.0 12.9 n/r n/r n/r

256 CLIA n/a n/a n/a n/a n/a n/a 33.0 35.8 11.4 n/a n/a n/a

257 CLIA n/a n/a n/a n/a n/a n/a 30.5 42.0 5.9 n/a n/a n/a

258 CLIA n/a n/a n/a n/a n/a n/a 33.9 64.4 13.6 n/a n/a n/a

259 LC-MS/MS <2 <2 <2 34.4 45.2 10.3 34.4 45.2 10.3 n/r n/r n/r

261 CLIA n/a n/a n/a n/a n/a n/a 24.2 37.4 6.6 n/a n/a n/a

262 CLIA n/a n/a n/a n/a n/a n/a 27.5 39.1 20.2 n/a n/a n/a

263 CLIA n/a n/a n/a n/a n/a n/a 33.7 37.6 12.8 n/a n/a n/a

264 LC-MS/MS 0.6 0.6 0.1 41.0 39.2 13.9 41.6 39.8 14.0 n/r n/r n/r

265 LC-MS/MS 0.0 0.0 0.0 35.0 44.0 13.0 35.0 44.0 13.0 n/r n/r n/r

266a LC-UV 0.0 0.0 0.0 41.3 50.8 16.4 41.3 50.8 16.4 n/r n/r n/r

266b EIA n/a n/a n/a n/a n/a n/a 29.2 38.3 18.5 n/a n/a n/a

267 CLEIA n/a n/a n/a n/a n/a n/a 30.4 39.3 12.4 n/a n/a n/a

NIST Value 0.68 0.65 0.1* 31.3 39.4 12.4 32.0 40.1 12.5 1.7 2.1 0.65

U 0.06 0.02 --- 0.8 0.8 0.4 0.8 0.8 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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This publication is available free of charge from: http://dx.doi.org/10.6028/NIST.IR.8141