NISTIR 8000

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Summer

2013 Comparability Study

(Exercise 7)

Mary Bedner

Katrice A. Lippa

Susan S.-C. Tai

This publication is available free of charge from:

http://dx.doi.org/10.6028/NIST.IR.8000
NISTIR 8000

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Summer

2013 Comparability Study

(Exercise 7)

Mary Bedner

Katrice A. Lippa

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.8000

February 2015

U.S. Department of Commerce 

Penny Pritzker, Secretary

National Institute of Standards and Technology 

Willie May, Acting Under Secretary of Commerce for Standards and Technology and Director 
ii

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 seventh

exercise of this program, the Summer 2013 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 SRM 972a Vitamin D Metabolites in Human Serum Level 4 and 

VitDQAP-II (a material designed for the VitDQAP). SRM 968e 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 

seventh study are reported along with a summary of the analytical methods used.
1

OVERVIEW OF THE SUMMER 2013 COMPARABILITY STUDY

For the Summer 2013 comparability study of the VitDQAP, pooled human serum control and 

study samples were distributed to participants for evaluation. SRM 968d Fat-Soluble Vitamins,

Carotenoids, and Cholesterol in Human Serum Level 1 (SRM 968d L1) was provided as a 

control material for assay calibration or verification. The blinded study samples consisted of two 

vials, Vial A and Vial B. Vial A was VitDQAP-II, which contains only endogenous 25-

hydroxyvitamin D (25(OH)D) levels. Vial B was SRM 972a Vitamin D Metabolites in Human 

Serum Level 4 (SRM 972a L4), which contains endogenous levels of 25-hydroxyvitamin D2

(25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3) but was fortified with 3-epi-25-

hydroxyvitamin D3 (3-epi-25(OH)D3). Participants were asked to provide individual 

concentration values for 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3 along with a total 

concentration of 25(OH)D (25(OH)DTotal = 25(OH)D2 + 25(OH)D3) for the control and each 

study sample.

There were a total of 44 participants and 49 datasets (five participants provided data from two 

different methods) in the Summer 2013 comparability study. Sixteen of the datasets originated 

from immunoassay (IA) techniques, including 13 from chemiluminescence immunoassay 

(CLIA) and three from radioimmunoassay (RIA). Note that none of the participants used 

enzyme immunoassay (EIA) in this study. Appendix A-1 summarizes the IA methods used by 

the participants. Thirty-three of the datasets originated from liquid chromatographic (LC) 

methods; of those, 30 were from LC with tandem mass spectrometric detection (LC-MSn), and 

three 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. All datasets from 

the immunoassay methods reported a single value for 25(OH)DTotal whereas LC participants 

provided values for 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3 as well as 25(OH)DTotal in 

VitDQAP-II (Vial A), SRM 972a L4 (Vial B), and SRM 968d L1 (Control). 

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

The control material (SRM 968d L1) and the two study samples (SRM 972a L4 and VitDQAPII) all contain low levels of 25(OH)D2, thus 25(OH)D3 represents the predominant metabolite 

contributing to 25(OH)DTotal. However, the two study materials, VitDQAP-II (Vial A) and SRM 

972a L4 (Vial B), both contain measurable amounts of 3-epi-25(OH)D3.
2

SUMMER 2013 COMPARABILITY STUDY RESULTS AND DISCUSSION

25(OH)DTotal in VitDQAP-II (Vial A), SRM 972a L4 (Vial B), and SRM 968d L1 (Control)

A summary of the individual participant data for 25(OH)DTotal in VitDQAP-II (Vial A), SRM 

972a L4 (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 the three study materials. For SRM 972a L4 (Vial B), 

the NIST result is the sum of the certified values for 25(OH)D3 and 25(OH)D2 with the 

corresponding 95% confidence limits (U95). 

The NIST values for 25(OH)D3 in VitDQAP-II (N = 8) and SRM 968d L1 (N = 5) were obtained 

using an LC-MS/MS reference measurement procedure (RMP)1recognized by the Joint 

Committee for Traceability in Laboratory Medicine (JCTLM). The NIST value for 25(OH)D2

was also obtained using the RMP for VitDQAP-II (N = 5), but for SRM 968d L1 the value was 

well below the limit of quantitation and was estimated to be 0.1 ng/mL (N = 1). The NIST 

values for 25(OH)DTotal in VitDQAP-II (Vial A) and SRM 968d L1 (Control) reported in 

Table 1 are the sum of the individual values for 25(OH)D3 and 25(OH)D2, and the 95% 

confidence limit (U95) incorporates the uncertainties for the two analytes and includes 

components for measurement variability and measurement uncertainty associated with the 

density of the materials.

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. 

 

1 Tai, S. S.-C., Bedner, M. and Phinney, K.W. Anal. Chem. 2010 82, 1942-1948.
3

Table 1. Summary of participant data for 

25(OH)DTotal (ng/mL) in VitDQAP-II (Vial A), 

SRM 972a L4 (Vial B), and SRM 968d L1 

(Control). 

VitDQAP-II SRM 972a L4 SRM 968d L1

Lab Method Vial A Vial B Control

017 CLIA 36.8 27.0 13.0

030 RIA 33.6 26.3 12.8

056 LC-MS/MS 36.4 27.5 12.3

060 LC-MS/MS 39.4 30.9 12.8

086a CLIA 40.7 30.7 14.0

110 LC-UV 30.1 44.7 13.2

116 LC-MS/MS 36.7 24.4 16.5

119 LC-MS/MS 41.7 65.3 15.1

128 LC-MS/MS 24.8 33.5 12.3

139 LC-UV 44.2 64.4 14.7

183b CLIA 37.0 29.2 13.5

187 LC-MS/MS 39.6 59.7 12.5

188 CLIA 47.0 35.2 13.6

194 LC-MS/MS 43.4 64.5 12.5

196 CLIA 40.9 29.6 14.6

197 LC-MS/MS 33.9 46.7 12.8

198a LC-MS/MS 49.7 56.6 11.4

198c CLIA 40.8 26.6 15.4

199 LC-MS/MS 41.5 71.0 12.7

200 RIA 30.8 22.9 12.8

209 LC-MS/MS 42.4 49.7 13.0

210a RIA 38.5 34.5 8.5

210b CLIA 40.8 39.8 < 3.0

211 LC-MS/MS 42.0 58.0 15.3

213a CLIA 49.2 49.8 9.0

214b CLIA 39.6 28.8 13.1

214c LC-MS/MS 36.1 53.7 12.1

215 LC-MS/MS 40.4 57.2 13.9

216 LC-MS/MS 38.2 29.3 12.6

217 LC-MS/MS 37.2 54.0 12.8

218a CLIA 37.5 28.6 12.8

218b LC-MS/MS 42.3 42.7 13.1

220 LC-MS/MS 39.0 59.0 13.0

221a LC-MS/MS 35.5 25.1 16.9

222 CLIA 51.6 34.8 12.4

225 LC-MS/MS 44.6 65.9 11.2

228a LC-MS/MS 34.6 51.6 12.4

231 LC-UV 41.3 56.4 n/r

241 LC-MS/MS 43.3 68.0 14.9

242 LC-MS/MS 35.1 30.5 11.9

244 LC-MS/MS 36.5 43.1 12.5

247a CLIA 50.0 34.1 13.1

248 LC-MS/MS 43.0 55.0 14.0

249 LC-MS/MS 36.4 29.1 12.4

250 LC-MS/MS 44.3 67.7 13.9

253 LC-MS/MS 41.7 33.0 14.1

254a LC-MS/MS 40.5 59.5 12.9

254b CLIA 37.5 29.6 12.5

255 LC-MS/MS 50.1 60.4 16.4

N 49 49 47

Median 40.4 42.7 12.9

MADe 4.7 20.2 0.9

CV% 11.7 47 6.9

N 16 16 15

Median 40.2 29.6 13.0

MADe 4.3 4.7 0.7

CV% 10.7 16 5.7

N 33 33 32

Median 40.4 54.0 12.9

MADe 4.7 15.6 0.8

CV% 11.7 28.8 6.3

N 30 30 30

Median 40.0 53.9 12.8

MADe 4.9 16.2 0.7

CV% 12.2 30.1 5.8

NIST Value 37.5 30.0 12.5

U 95 1.0 1.0 0.3

All 

methods

IA 

methods 

LC 

methods LC-MS

n
4

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

SRM 972a L4 (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 (○). For the LC results in all three figures, the majority of the data points are from LCMSn methods. However, the LC-UV results were sorted separately and are plotted at the right 

end of the LC results as labeled.

From the single reported values for all LC 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 

uncertainty (i.e., value ± U95). NIST believes that the “true” value for each material lies within 

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

bias. 

Specific results for each of the three study materials are summarized below:

VitDQAP-II (Vial A): Figure 1

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

CLIA, one RIA).

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

LC-MSn, one LC-UV).

 The consensus median values for both the LC and IA results agree well with each other but 

are slightly higher than the NIST expanded uncertainty range (red lines).

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

both for LC and IA results. 

SRM 972a L4 (Vial B): Figure 2

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

CLIA).

 For the LC results, the consensus variability range is very large, and there are no outliers. 

 The consensus median value for the IA results agrees well with the NIST expanded 

uncertainty range (red lines).

 The consensus median value for the LC results is considerably higher (80%) than both the IA 

median value and 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 results.

SRM 968d L1 (Control): Figure 3

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

CLIA).
5

 For the LC results, eight reported values are outside of the consensus variability range (seven 

LC-MSn, one LC-UV).

 The consensus median value for the IA results is comparable to the consensus median value 

for the LC results; both LC and IA median values are slightly higher than the NIST expanded 

uncertainty range (red lines).

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

for both LC and IA.

The consensus variability of ≈ 7% to 12% (all methods) for SRM 968d L1 (Control) and 

VitDQAP-II (Vial A) is consistent with participant performance for other materials containing 

predominantly 25(OH)D3 that were evaluated in previous comparability studies of the VitDQAP.

For SRM 972a L4 (Vial B), the LC results are bimodal, where nine reported results agree well 

with both the NIST value and the reported IA results, but the majority of the LC results (73%) 

are biased high (Figure 2). The bimodal results contribute to the large consensus variability 

(47%) for this material when the results for all methods are considered (Table 1). SRM 972a L4 

(Vial B) was fortified with 3-epi-25(OH)D3, and the NIST-certified value for this vitamin D 

metabolite is 26.4 ng/mL ± 2.1 ng/mL. The biological significance of 3-epi-25(OH)D3 is 

uncertain, and this metabolite is not included in the 25(OH)DTotal concentration. Therefore, LC

methods that do not chromatographically separate the 3-epi-25(OH)D3 yield biased results for 

25(OH)D3 and hence 25(OH)DTotal because the 3-epi-25(OH)D3 and the 25(OH)D3 are 

diastereomers that are detected by the same multiple reaction monitoring (MRM) ions in MS/MS 

and absorbance wavelength in UV. Since the majority of the reported LC methods do not 

separate the 25(OH)D3 and 3-epi-25(OH)D3 (Appendix A-2, A-3), the median LC value of 54.0 

ng/mL for 25(OH)DTotal in SRM 972a L4 is biased 80% higher than the NIST value of 30.0 

ng/mL ± 1.0 ng/mL. The majority of the IA methods, on the other hand, do not have crossreactivity with the 3-epi-25(OH)D3 metabolite and yield an unbiased median result of 29.6 

ng/mL for 25(OH)DTotal in SRM 972a L4.

VitDQAP-II (Vial A) also has a significant 3-epi-25(OH)D3 concentration of 3.4 ng/mL ± 0.1

ng/mL, or ≈ 9% of the 25(OH)D3 concentration of 37.0 ng/mL ± 0.4 ng/mL (NIST values). In 

theory, bimodal results should have also been obtained for this material, but the 9% bias is 

indistinguishable in the overall method variability of 12% for the LC results.

Of the nine LC participants that used methods that separate the 3-epi-25(OH)D3, seven reported 

values for this metabolite in the control and study materials. The LC method results for 

25(OH)D3 and 3-epi-25(OH)D3 are presented and discussed in detail later in this report.
6

Figure 1. 25(OH)DTotal levels in VitDQAP-II (Vial A) as determined by immunoassay (CLIA and RIA) and LC (LC-MSnand LCUV) methods. The red lines represent the ranges bound by the NIST values with  estimated U95 uncertainty.

128

197228a242221a214c056249244116217216220060187215254a199253119211218b209248241194250225

198a255

110

231

139

200

030

017183b218a254b210a214b086a198c210b196

188

213a247a

222

10

20

30

40

50

60

70

80

01234567810911123141516171819202122234252627282930313233345363738394041424344456474849505152535455567585960616263646566678697071727374757677789081828384858687888990192939495969798100 99101 102 103 104 105 106 107 108 109 110 1111231415161718192021 122 123 14 125 126 127 128 129 130 131 132 133 134 15 136 137 138 139 140 141 142 143 144 145 16 147 148 149 150 151 152 153 154 155 156 17 158 159 160 161 162 163 164 165 166 167 18 169 170 171 172 173 174 175 176 177 178 19 10 181 182 183 184 185 186 187 188 189 190 11 192 193 194 195 196 197 198 199 200 201 22 203 204 205 206 207 208 209 210 211 212 23 214 215 216 217 218 219 220 2212223425262728293031 232 233 234 25 236 237 238 239 240 241 242 243 244 245

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

IA 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 
7

Figure 2. 25(OH)DTotal levels in SRM 972a L4 (Vial B) as determined by immunoassay (CLIA and RIA) and LC (LC-MSnand LCUV) methods. The red lines represent the ranges bound by the NIST values with  estimated U95 uncertainty.

 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 

116221a

056

249216242060

253128

218b244

197

209

228a

214c217248

198a215211220254a187255

194119225

250241

199

110

231

139

200

030198c017

218a214b183b196254b086a

247a210a222188

210b

213a

5

15

25

35

45

55

65

75

85

01234567810911123141516171819202122234252627282930313233345363738394041424344456474849505152535455567585960616263646566678697071727374757677789081828384858687888990192939495969798100 99101 102 103 104 105 106 107 108 109 110 1111231415161718192021 122 123 14 125 126 127 128 129 130 131 132 133 134 15 136 137 138 139 140 141 142 143 144 145 16 147 148 149 150 151 152 153 154 155 156 17 158 159 160 161 162 163 164 165 166 167 18 169 170 171 172 173 174 175 176 177 178 19 10 181 182 183 184 185 186 187 188 189 190 11 192 193 194 195 196 197 198 199 200 201 22 203 204 205 206 207 208 209 210 211 212 23 214 215 216 217 218 219 220 2212223425262728293031 232 233 234 25 236 237 238 239 240 241 242 243 244 245

25(OH)DTotal in SRM 972a L4 "Vial B" (ng/mL)

IA LC-M Sn LC-UV
8

Figure 3. 25(OH)DTotal levels in SRM 968d L1 (Control) as determined by immunoassay (CLIA and RIA) and LC (LC-MSnand LCUV) methods. The red lines represent the ranges bound by the NIST values with  estimated U95 uncertainty.

 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

198a

242

214c

056128228a249187194244216199060197217254a209220218b

215250248253

241

119

211

255116

221a

110

139

222254b

218a030200

017214b247a

183b188

086a

196

198c

10

11

12

13

14

15

16

17

18

01234567810911123141516171819202122234252627282930313233345363738394041424344456474849505152535455567585960616263646566678697071727374757677789081828384858687888990192939495969798100 99101 102 103 104 105 106 107 108 109 110 1111231415161718192021 122 123 14 125 126 127 128 129 130 131 132 133 134 15 136 137 138 139 140 141 142 143 144 145 16 147 148 149 150 151 152 153 154 155 156 17 158 159 160 161 162 163 164 165 166 167 18 169 170 171 172 173 174 175 176 177 178 19 10 181 182 183 184 185 186 187 188 189 190 11 192 193 194 195 196 197 198 199 200 201 22 203 204 205 206 207 208 209 210 211 212 23 214 215 216 217 218 219 220 2212223425262728293031 232 233 234 25 236 237 238 239 240 241 242 243 244 245

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

IA LC-M Sn LC-UV
9

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

(Vial A) and SRM 972a L4 (Vial B), and B) SRM 972a L4 (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-II (Vial A) and SRM 972a L4 (Vial B): Figure 4 A

 Laboratory results that are not included in the consensus ranges include numbers 110, 128, 

200, 210b, 213a, 222, and 247a

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

corner of the LC consensus box for these materials, illustrating that the IA results are in 

better agreement with the NIST results for these materials. 

 Most of the LC results for SRM 972a L4 are higher than both the Youden line and the IA 

consensus box; however, the LC box overlaps both the Youden line and the IA consensus 

box because the LC consensus box is very large in the SRM 972a L4 dimension (y-axis). 

SRM 972a L4 (Vial B) and SRM 968d L1 (Control): Figure 4 B

 Laboratory results that are not included in the consensus ranges include numbers 116, 119, 

139, 196, 198c, 210a, 211, 213a, 221a, 241, and 255

 The Youden line runs through the center of the IA consensus box and through the left corner 

of the LC consensus box for these materials, illustrating that the IA results are in better 

agreement with the NIST results for these materials. 

 Most of the LC results for SRM 972a L4 are higher than both the Youden line and the IA 

consensus box; however, the LC box overlaps both the Youden line and the IA consensus 

box because the LC consensus box is very large in the SRM 972a L4 dimension (x-axis). 

Both of these Youden plots involving SRM 972a L4 (Vial B) reveal separation of the IA results 

and the majority of the LC results, further illustrating the difference in results for the two 

techniques for the material with high levels of 3-epi-25(OH)D3.
10

Figure 4. Youden comparison plot 

of the results for 25(OH)DTotal in

A) VitDQAP-II (Vial A) and SRM 

972a L4 (Vial B) and

B) 972a L4 (Vial B) and SRM 968d 

L1 (Control) for all methods

 IA method laboratory values 

 IA method consensus box encloses ± 2 MADe around consensus medians

 LC method laboratory values

 LC method consensus box encloses ± 2 MADe around consensus medians

 NIST values with corresponding Youden line 

A

B

110

128

200

210b

213a

222 247a

255

0

10

20

30

40

50

60

70

80

90

10 20 30 40 50 60

25(OH)DTotal in SRM 972a L4 "Vial B" (ng/mL)

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

116

119

196 139

198c

210a

211

213a

221a

241

255

5

10

15

20

25

0 10 20 30 40 50 60 70 80 90

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

25(OH)DTotal in SRM 972a L4 "Vial B" (ng/mL)
11

25(OH)D3 and 3-epi-25(OH)D3 in VitDQAP-II (Vial A), SRM 972a L4 (Vial B), and SRM 

968d L1 (Control): LC methods only

Of the two major techniques IA and LC, only the LC methods can independently measure the 

25(OH)D2 and 25(OH)D3 components of 25(OH)DTotal, and therefore LC methods require 

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

for 25(OH)DTotal. The 25(OH)D2 metabolite does not contribute significantly to the 25(OH)DTotal

in either of the two study materials or the control. However, both VitDQAP-II (Vial A) and 

SRM 972a L4 (Vial B) contain significant concentrations of the 3-epi-25(OH)D3 metabolite. 

Therefore, the 3-epi-25(OH)D3 needs to be separated from 25(OH)D3 to avoid a significant 

measurement bias.

In the Summer 2013 comparability study, all but one of the LC participants reported values for 

25(OH)D3, and the reported values are summarized in Table 2. 

Of the 33 LC participants, nine used methods that separated the 3-epi-25(OH)D3 interference 

from 25(OH)D3 (Appendix A-2, A-3). Of those nine, seven participants reported values for 3-

epi-25(OH)D3 in the study samples and in the control, and the results are summarized in Table 3.

For both Table 2 and Table 3, the community results are summarized at the bottom of the table 

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

median value, the MADe, and the CV%. Table 2 and Table 3 also present the NIST values and 

the 95% confidence limits (U95) for 25(OH)D3 and 3-epi-25(OH)D3, respectively, in the study 

and control materials. 

For the participant results for SRM 972a L4 (Vial B), the consensus variability is large for 

25(OH)D3 (30%). The source of the measurement uncertainty for 25(OH)D3 is the wideranging, bimodal results from labs that separate the 3-epi-25(OH)D3 and those that do not. 

However, the results for the seven participants that measure 3-epi-25(OH)D3 exhibit relatively 

low variability for VitDQAP-II (Vial A) and SRM 972a L4 (Vial B) with a CV ≈ 8%.
12

Table 2. Summary of LC participant data and community results for 25(OH)D3 (ng/mL) in the 

study samples and control.

VitDQAP-II SRM 972a L4 SRM 968d L1

Lab Method Vial A Vial B Control

056 LC-MS/MS 35.8 26.8 12.1

060 LC-MS/MS 39.4 30.9 12.8

110 LC-UV 30.1 44.7 13.2

116 LC-MS/MS 36.7 24.4 16.5

119 LC-MS/MS 41.7 65.3 15.1

128 LC-MS/MS 24.8 33.5 12.3

187 LC-MS/MS 39.6 59.7 12.5

194 LC-MS/MS 43.4 64.5 12.5

197 LC-MS/MS 33.9 46.7 12.8

198a LC-MS/MS 49.7 56.6 11.4

199 LC-MS/MS 41.5 71.0 12.7

209 LC-MS/MS 42.4 49.7 13.0

211 LC-MS/MS 42.0 58.0 15.3

214c LC-MS/MS 36.1 53.7 12.1

215 LC-MS/MS 40.4 56.8 13.9

216 LC-MS/MS 37.8 28.9 12.5

217 LC-MS/MS 37.2 54.0 12.8

218b LC-MS/MS 42.3 42.7 13.1

220 LC-MS/MS 39.0 59.0 13.0

221a LC-MS/MS 35.5 25.1 16.9

225 LC-MS/MS 44.6 65.9 11.2

228a LC-MS/MS 34.6 51.6 12.4

231 LC-UV 41.3 56.4 n/r

241 LC-MS/MS 43.3 68.0 14.9

242 LC-MS/MS 35.1 30.5 11.9

244 LC-MS/MS 36.5 43.1 12.5

248 LC-MS/MS 42.7 55.4 14.1

249 LC-MS/MS 36.4 29.1 12.4

250 LC-MS/MS 44.3 67.7 13.9

253 LC-MS/MS 41.2 32.5 13.9

254a LC-MS/MS 40.3 59.4 12.9

255 LC-MS/MS 49.5 59.9 16.1

N 32 32 31

Median 40.0 53.9 12.8

MADe 4.9 15.9 0.7

CV% 12.2 29.5 5.8

N 29 29 29

Median 40.3 54.0 12.8

MADe 4.6 16.8 0.7

CV% 11.4 31.0 5.8

NIST Value 37.0 29.4 12.4

U 95 0.4 0.9 0.3

LC 

methods LC-MS

n
13

Table 3. Summary of LC participant data and community results for 3-epi-25(OH)D3 (ng/mL) 

in the study samples and control.

VitDQAP-II SRM 972a L4 SRM 968d L1

Lab Method Vial A Vial B Control

056 LC-MS/MS 3.5 24.3 1.8

060 LC-MS/MS 3.3 28.7 0.7

116 LC-MS/MS 5.8 27.3 <4.0

216 LC-MS/MS 3.1 27.0 0.6

242 LC-MS/MS 2.6 20.7 0.6

249 LC-MS/MS 3.2 28.5 0.4

253 LC-MS/MS 3.4 25.8 0.7

N 7 7 6

Median 3.3 27.0 0.7

MADe 0.3 2.2 0.1

CV% 7.6 8.2 13.6

N 7 7 6

Median 3.3 27.0 0.7

MADe 0.3 2.2 0.1

CV% 7.6 8.2 13.6

NIST Value 3.4 26.4 0.66

U 95 0.1 2.1 0.02

LC 

methods LC-MS

n
14

Figure 5 and Figure 6 present direct graphical comparisons of the LC results for 25(OH)D3 and 

3-epi-25(OH)D3, respectively, for A) VitDQAP-II (Vial A) and SRM 972a L4 (Vial B), and B) 

SRM 972a L4 (Vial B) and SRM 968d L1 (Control). In each plot, there is one blue consensus 

box for the LC results. Laboratory results that are within the consensus range for both study 

materials are within the blue consensus box. Conversely, laboratory results that fall outside of 

(or on the edge of) the consensus box are not included in the consensus range 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.

25(OH)D3 in VitDQAP-II (Vial A) and SRM 972a L4 (Vial B): Figure 5 A

 Laboratory results that are not included in the consensus range include numbers 110 and 128.

 The Youden line runs through the cluster of LC results that separate the 25(OH)D3 and 3-epi25(OH)D3, illustrating the better agreement of these results with the NIST values for these 

materials. 

 Most of the LC results for SRM 972a L4 are higher than the Youden line; however, the 

Youden line overlaps the bottom of the LC consensus box, which is very large in the SRM 

297a L4 dimension (y-axis). 

25(OH)D3 in SRM 972a L4 (Vial B) and SRM 968d L1 (Control): Figure 5 B

 Laboratory results that are not included in the consensus range include numbers 116, 119, 

211, 221a, 225, 241, and 255.

 The Youden line runs through the cluster of the LC results that separate the 25(OH)D3 and 3-

epi-25(OH)D3, illustrating the better agreement of these results with the NIST values for 

these materials. 

 Most of the LC results for SRM 972a L4 are higher than the Youden line; however, the 

Youden line overlaps the left corner of the LC consensus box, which is very large in the 

SRM 297a L4 dimension (x-axis). 

3-epi-25(OH)D3 in VitDQAP-II (Vial A) and SRM 972a L4 (Vial B): Figure 6 A

 Laboratory results that are not included in the consensus range include numbers 116 and 242.

 The Youden line runs through the center of the consensus box, indicating good agreement of 

the LC results with the NIST values for these materials. 

3-epi-25(OH)D3 in SRM 972a L4 (Vial B) and SRM 968d L1 (Control): Figure 6 B

 Only three of the laboratory results are included in the consensus range for these materials 

because of the variability of the results for SRM 968d L1 (y-axis).

 The Youden line runs through the center of the consensus box, indicating good agreement of 

the LC results with the NIST values for these materials even with the higher variability of 

results for SRM 968d L1 (y-axis).
15

Figure 5. Youden comparison plot of the results for 25(OH)D3 in A) VitDQAP-II (Vial A) and 

SRM 972a L4 (Vial B) and B) SRM 972a L4 (Vial B) and SRM 968d L1 (Control) for LC 

methods.

 LC method laboratory values

 LC method consensus box encloses ± 2 MADe around consensus medians

 NIST values with corresponding Youden line 

A

B

110

128

0

10

20

30

40

50

60

70

80

90

10 20 30 40 50 60

25(OH)D3 in SRM 972a L4 "Vial B" (ng/mL)

25(OH)D3in VitDQAP-II "Vial A" (ng/mL)

116

119 211

221a

225

241

255

5

10

15

20

25

0 10 20 30 40 50 60 70 80 90

25(OH)D3 in SRM 968d L1 "Control" (ng/mL)

25(OH)D3in SRM 972a L4 "Vial B" (ng/mL)
16

Figure 6. Youden comparison plot of the results for 3-epi-25(OH)D3 in A) VitDQAP-II (Vial 

A) and SRM 972a L4 (Vial B) and B) SRM 972a L4 (Vial B) and SRM 968d L1 (Control) for 

LC methods.

 LC method laboratory values

 LC method consensus box encloses ± 2 MADe around consensus medians

 NIST values with corresponding Youden line 

A

B

116

242

15

20

25

30

35

1 2 3 4 5 6

3-epi-25(OH)D3 in SRM 972a L4 "Vial B" (ng/mL)

3-epi-25(OH)D3in VitDQAP-II "Vial A" (ng/mL)

056

116

242

249

0.0

0.5

1.0

1.5

2.0

15 20 25 30 35

3-epi-25(OH)D3 in SRM 968d L1 "Control" (ng/mL)

3-epi-25(OH)D3in SRM 972a L4 "Vial B" (ng/mL)
17

Conclusions from the Summer 2013 Comparability Study of the VitDQAP

In the six 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 Summer 2013 comparability study, both VitDQAP-II (Vial A) and SRM 968d L1 (Control) 

also contain predominantly 25(OH)D3. The participant results for VitDQAP-II (Vial A) and 

SRM 968d L1 (Control) had a CV of 12% and 7%, respectively, and the all-method median 

values were biased slightly high relative to the NIST values, indicating the performance for these 

two materials is comparable to similar materials previously evaluated in the VitDQAP. 

For SRM 972a L4 (Vial B), however, which contained similar concentrations of both 25(OH)D3

and 3-epi-25(OH)D3, the results were not comparable. The majority of the LC methods did not 

separate the 3-epi-25(OH)D3 interference from the 25(OH)D3 measurement and obtained results 

for 25(OH)DTotal that were biased significantly high, which led to the largest all-method CV 

(47%) to date for any material evaluated in the VitDQAP. While VitDQAP-II (Vial A) also 

contained a measureable amount of 3-epi-25(OH)D3 and should have produced similar results, 

the 3-epi-25(OH)D3 was low enough (≈ 3.4 ng/mL) that the bias was not observable in the 

overall method CV of 12% for that study material. Participants that use LC methods are 

encouraged to utilize chromatographic conditions and columns that separate the 3-epi-25(OH)D3

interference to eliminate this potential measurement bias.
18

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

Laboratory 

Number IA Method Sample Preparation Vendor/kit*

17 CLIA n/r A

30 RIA Samples were extracted with acetonitrile D

86a CLIA n/r A

183b CLIA n/r A

188 CLIA None B

196 CLIA The human serum samples were analyzed neat A

198c CLIA n/r n/r

200 RIA n/r D

210a RIA Sample was extracted with acetonitrile D

210b CLIA n/r C

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

214b CLIA n/r n/r

218a CLIA Direct analysis n/r

222 CLIA n/r B

247a CLIA Sample was thawed, mixed well and used in the assay B

254b CLIA n/r A

n/r = not reported

*NIST cannot endorse or recommend commercial products, so individual vendors/kits are indicated with a unique letter but not identified
19

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

Laboratory 

Number

Internal 

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

56

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 x 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

60 25(OH)D3

 -d6

IS was added, and then samples 

were extracted with acetonitrile, 

evaporated, and reconstituted 

with 80% methanol/20% water

PFP column (100 x 3.0 mm; 2.6 

μm); gradient with water, methanol 

and acetonitrile (0.05% formic acid)

25(OH)D3 383/211; 

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 mixed with ethanol 

containing the IS, equilibrated, 

mixed, extracted with hexane, 

evaporated, and reconstituted in 

mobile phase

C18 column (150 x 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

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

187 n/r SPE C18 column (50 x 2.1 mm; 3 μm)

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 x 2mm); 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 (200 

μL), calibrator and control sample 

followed by mixing, centrifugation, 

and analysis

C18 column (50 x 4.6 mm; 5 μm); 

column temp 45°C; gradient with 

water and methanol; flow 1.0 

mL/min

n/r

198a 25(OH)D3

 -d6

Proteins precipitated with 

methanol, followed by hexane 

extraction, centrifugation, 

evaporation under N2, and 

reconstitution in methanol (0.1% 

formic acid)

C18 column (50 x 2.1 mm; 3.5 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 n/r n/r n/r n/r

209 25(OH)D3

 -d6

Proteins were precipitated with 

ZnSO4 in methanol

C8 column (50 x 2 mm; 5 μm); 

gradient with water/methanol; flow 

0.7 mL/min

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

25(OH)D3-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 x 4.6 

mm; 3 μm)

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

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

395/209 (quant), 395/377 

(qual)

214c 25(OH)D3

-d6

Samples were extracted with 

hexane, centrifuged, evaporated, 

and filtered

Column (50 x 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 x 2.1mm; 2.6 μm) 

column; gradient with water (0.1% 

formic acid, 5 mmol/L ammonium 

formate) and methanol (0.05% 

formic acid)

25(OH)D3 401/383; 

25(OH)D2 413/395; 

25(OH)D3-d6 407/389
20

216

Derivatized 

deuteriated 

standard

Samples extracted using liquidliquid extraction then labeled with 

a derivatization reagent

Revered-phase column (150 x 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 x 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 x 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 

then phospholipids removed with 

SPE

C18 column (20 x 2.1mm, 2.7μm); 

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 IS

CN column (50 x 3.0 mm; 1.8 μm); 

methanol/water gradient at 50 °C

25(OH)D3 383/211; 

25(OH)D3-d6 389/211; 

25(OH)D2 395/209

225 n/r n/r n/r n/r

228a D8-labeled 

compound Proteins precipitated n/r n/r

241 25(OH)D3

-d6

Acetonitrile containing the IS (100 

μL) added to sample (50 μL) to 

precipate proteins, followed by 

mixing, sonication, and 

centrifugation

C8 column (50 x 2 mm; 3 μm); 

gradient starting with 50% methanol 

(0.1% formic acid), 50% water 

(0.1% formic acid)

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

383/229 (qual); 25(OH)D2 

395/119 (quant), 395/211 

(qual); 25(OH)D3-d6 389/211

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 x 2 mm; 3 μm); 

isocratic elution with 18% water/ 

82% methanol/ 0.1% formic acid; 

flow 0.35 mL/min

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)D2 395/269; 

25(OH)D3 383/211

248

25(OH)D2-d3 and 

25(OH)D3-d3

Serum is precipitated with 

methanol/ZnSO4 containing the IS 

and then with acetonitrile, 

centrifuged, and injected

Reversed-phase column (75 x 2.1 

mm; 2.5 μm); gradient with water 

and methanol; flow 0.55 mL/min

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

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

25(OH)D3-d3 386/257, 

386/368; 

25(OH)D2-d3 398/380, 

398/272

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 x 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

250 n/r Protein crash followed by SPE

Phenyl column (50 x 2.1 mm); 

gradient with 15% water and 85% 

methanol; flow 0.45 mL/min

MRM

253

25(OH)D2-d3 and 

25(OH)D3-d3

The sample was extracted, 

centrifuged, and derivatized

C18 column (150 x 2.1 mm); 

isocratic separation with 22.5% 

water/ 77.5% methanol; flow 0.2 

mL/min

25(OH)D2 588; 

25(OH)D3 576

254a 25(OH)D3

-d6

IS was added to each sample 

(200 μL) and mixed; acetontrile 

was added, followed by mixing, 

centrifugation, and injection

C8 column (50 x 2.0 mm; 3 μm); 

elution with water and acetonitrile, 

each containing 0.1% formic acid

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

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

25(OH)D3-d6 389/211

255 deuterium labeled 

compound

Samples were extracted and 

derivatized with 4-phenyl-l ,2,4-

triazoline-3,5-dione

Reversed-phase column (50 x 2.1 

mm); gradient with methanol; flow 

0.5 mL/min

25(OH)D3 607/298; 

25(OH)D2 619/298

MRM = multiple reaction monitoring; PFP = pentafluorophenyl; SPE = solid phase extraction; n/r = not reported; CN = cyano; quant/qual = quantitative/qualitative ions
21

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

231 1alpha(OH)D3

Samples were extracted with 

hexane/dichloromethane, 

evaporated and reconstituted with 

mobile phase (phosphate 

buffer/acetonitrile)

Reversed-phase column (250 x 4.5 

mm; 5μm), isocratic separation with 

14% phosphate buffer, 86% 

acetonitrile; flow 1.2 mL/min

265 nm

n/a = not applicable
22

Appendix B-1. Raw participant data and NIST results for 25(OH)D2, 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)DTotal in VitDQAP-II 

(Vial A), SRM 972a L4 (Vial B), and SRM 968d L1 (Control).
23

VitDQAP-II SRM 972a L4 SRM 968d L1 VitDQAP-II SRM 972a L4 SRM 968d L1 VitDQAP-II SRM 972a L4 SRM 968d L1 VitDQAP-II SRM 972a L4 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 36.8 27.0 13.0 n/r n/r n/r

030 RIA n/a n/a n/a n/a n/a n/a 33.6 26.3 12.8 n/r n/r n/r

056 LC-MS/MS 0.6 0.7 0.2 35.8 26.8 12.1 36.4 27.5 12.3 3.5 24.3 1.8

060 LC-MS/MS <0.5 <0.5 <0.5 39.4 30.9 12.8 39.4 30.9 12.8 3.3 28.7 0.7

086a CLIA n/a n/a n/a n/a n/a n/a 40.7 30.7 14.0 n/r n/r n/r

110 LC-UV <2 <2 <2 30.1 44.7 13.2 30.1 44.7 13.2 n/r n/r n/r

116 LC-MS/MS <3.3 <3.3 <3.3 36.7 24.4 16.5 36.7 24.4 16.5 5.8 27.3 <4.0

119 LC-MS/MS n/r n/r n/r 56.0 87.0 20.0 56.0 87.0 20.0 n/r n/r n/r

128 LC-MS/MS n/a n/a n/a 24.8 33.5 12.3 24.8 33.5 12.3 n/r n/r n/r

139 LC-UV n/r n/r n/r n/r n/r n/r 44.2 64.4 14.7 n/r n/r n/r

183b CLIA n/a n/a n/a n/a n/a n/a 37.0 29.2 13.5 n/r n/r n/r

187 LC-MS/MS < 1.5 < 1.5 < 1.5 39.6 59.7 12.5 39.6 59.7 12.5 n/r n/r n/r

188 CLIA n/a n/a n/a n/a n/a n/a 47.0 35.2 13.6 n/r n/r n/r

194 LC-MS/MS <7 <7 <7 43.4 64.5 12.5 43.4 64.5 12.5 n/r n/r n/r

196 CLIA n/a n/a n/a n/a n/a n/a 40.9 29.6 14.6 n/r n/r n/r

197 LC-MS/MS <0.5 <0.5 <0.5 33.9 46.7 12.8 33.9 46.7 12.8 n/r n/r n/r

198a LC-MS/MS <5.0 <5.0 <5.0 49.7 56.6 11.4 49.7 56.6 11.4 n/r n/r n/r

198c CLIA n/a n/a n/a n/a n/a n/a 40.8 26.6 15.4 n/r n/r n/r

199 LC-MS/MS < 2 < 2 < 2 41.5 71.0 12.7 41.5 71.0 12.7 n/r n/r n/r

200 RIA n/a n/a n/a n/a n/a n/a 30.8 22.9 12.8 n/r n/r n/r

209 LC-MS/MS <1.0 <1.0 <1.0 42.4 49.7 13.0 42.4 49.7 13.0 n/r n/r n/r

210a RIA n/a n/a n/a n/a n/a n/a 38.5 34.5 8.5 n/r n/r n/r

210b CLIA n/a n/a n/a n/a n/a n/a 40.8 39.8 < 3.0 n/r n/r n/r

211 LC-MS/MS 0.0 0.0 0.0 42.0 58.0 15.3 42.0 58.0 15.3 n/r n/r n/r

213a CLIA n/a n/a n/a n/a n/a n/a 49.2 49.8 9.0 n/r n/r n/r

214b CLIA n/a n/a n/a n/a n/a n/a 39.6 28.8 13.1 n/r n/r n/r

214c LC-MS/MS <1.0 <1.0 <1.0 36.1 53.7 12.1 36.1 53.7 12.1 n/r n/r n/r

215 LC-MS/MS 0.0 0.4 0.0 40.4 56.8 13.9 40.4 57.2 13.9 n/r n/r n/r

216 LC-MS/MS 0.4 0.4 0.1 37.8 28.9 12.5 38.2 29.3 12.6 3.1 27.0 0.6

217 LC-MS/MS <2 <2 <2 37.2 54.0 12.8 37.2 54.0 12.8 n/r n/r n/r

218a CLIA n/a n/a n/a n/a n/a n/a 37.5 28.6 12.8 n/r n/r n/r

218b LC-MS/MS 0.0 0.0 0.0 42.3 42.7 13.1 42.3 42.7 13.1 n/r n/r n/r

220 LC-MS/MS <5.0 <5.0 <5.0 39.0 59.0 13.0 39.0 59.0 13.0 n/r n/r n/r

221a LC-MS/MS 0.0 0.0 0.0 35.5 25.1 16.9 35.5 25.1 16.9 n/r n/r n/r

222 CLIA n/a n/a n/a n/a n/a n/a 51.6 34.8 12.4 n/r n/r n/r

225 LC-MS/MS <5.0 <5.0 <5.0 44.6 65.9 11.2 44.6 65.9 11.2 n/r n/r n/r

228a LC-MS/MS n/d n/d n/d 34.6 51.6 12.4 34.6 51.6 12.4 n/r n/r n/r

231 LC-UV n/d n/d n/d 41.3 56.4 n/r 41.3 56.4 n/r n/r n/r n/r

241 LC-MS/MS < 0.5 < 0.5 n/d 43.3 68.0 14.9 43.3 68.0 14.9 n/r n/r n/r

242 LC-MS/MS n/d n/d n/d 35.1 30.5 11.9 35.1 30.5 11.9 2.6 20.7 0.6

244 LC-MS/MS <5 <5 <5 36.5 43.1 12.5 36.5 43.1 12.5 n/r n/r n/r

247a CLIA n/a n/a n/a n/a n/a n/a 50.0 34.1 13.1 n/r n/r n/r

248 LC-MS/MS <3.0 <3.0 <3.0 42.7 55.4 14.1 43.0 55.0 14.0 n/r n/r n/r

249 LC-MS/MS <0.8 <0.8 <0.8 36.4 29.1 12.4 36.4 29.1 12.4 3.2 28.5 0.4

250 LC-MS/MS < 2.5 < 2.5 < 2.5 44.3 67.7 13.9 44.3 67.7 13.9 n/r n/r n/r

253 LC-MS/MS 0.5 0.5 0.2 41.2 32.5 13.9 41.7 33.0 14.1 3.4 25.8 0.7

254a LC-MS/MS 0.1 0.1 0.0 40.3 59.4 12.9 40.5 59.5 12.9 n/r n/r n/r

254b CLIA n/a n/a n/a n/a n/a n/a 37.5 29.6 12.5 n/r n/r n/r

255 LC-MS/MS 0.6 0.5 0.3 49.5 59.9 16.1 50.1 60.4 16.4 n/r n/r n/r

NIST Value 0.44 0.55 0.1* 37.0 29.4 12.4 37.5 30.0 12.5 3.4 26.4 0.66

U 95 0.04 0.10 --- 0.4 0.9 0.3 1.0 1.0 0.3 0.1 2.1 0.02

*estimated value (no uncertainty determined)

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

n/a = not applicable (for immunoassay methods); n/r = not reported; n/d = not detected; < X = less than a reported quantitation limit of X

25(OH)D3 (ng/mL) 25(OH)DTotal 25(OH)D (ng/mL) 2 (ng/mL)