NISTIR 7890

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Winter 2010 

Comparability Study (Exercise 1)

Mary Bedner

Katrice A. Lippa

http://dx.doi.org/10.6028/NIST.IR.7890
NISTIR 7890

NIST/NIH Vitamin D Metabolites 

Quality Assurance Program Report 

of Participant Results: Winter 2010 

Comparability Study (Exercise 1)

Mary Bedner

Katrice A. Lippa 

Chemical Sciences Division

Material Measurement Laboratory

http://dx.doi.org/10.6028/NIST.IR.7890

December 2012

U.S. Department of Commerce 

Rebecca Blank, Acting Secretary

National Institute of Standards and Technology 

Patrick D. Gallagher, Under Secretary of Commerce for Standards and Technology and Director 
1

ABSTRACT

The National Institute of Standards and Technology (NIST) has established a Vitamin D 

Metabolites Quality Assurance Program in collaboration with the National Institutes of Health 

(NIH) Office of Dietary Supplements (ODS). For this first pilot exercise (Winter 2010 

Comparability Study), participants were asked to use the methodology of their choice to measure 

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

study material consisted of triplicate samples of SRM 1950 Metabolites in Human Plasma. SRM 

2972, which is comprised of separate ethanolic calibration solutions with known concentrations 

of 25(OH)D2 and 25(OH)D3, was provided as a control material. Participants provided their 

data to NIST, where it was compiled and evaluated for trueness relative to the NIST value, 

within-laboratory precision, and concordance within the participant community. A report of 

results was provided to all participants of the exercise, and laboratories were identified by code 

numbers known only to them. The results from this first pilot exercise are reported along with a 

summary of the analytical methods used. 
2

OVERVIEW OF WINTER 2010 COMPARABILITY STUDY

For the Winter 2010 Comparability Study (Exercise 1) of the NIST/NIH Vitamin D Metabolites 

Quality Assurance Program (VitDQAP), control and human serum study samples were 

distributed to participants for evaluation. SRM 2972, which is comprised of separate ethanolic 

solutions with known concentrations of 25-hydroxyvitamin D2 (25(OH)D2) and 25-

hydroxyvitamin D3 (25(OH)D3), was provided as a control material for assay calibration or 

verification. Participants were asked to provide single results for each of these solutions. In 

addition, participants were asked to determine 25(OH)D2, 25(OH)D3, and a total concentration 

of 25-hydroxyvitamin D (25(OH)DTotal = 25(OH)D2 + 25(OH)D3) in each of three vials A, B, 

and C, which were triplicate samples of SRM 1950 Metabolites in Human Plasma.

In the Winter 2010 exercise, there were a total of 16 participants and 17 datasets (one participant 

provided data for two different methods). Ten of these datasets originated from liquid 

chromatographic (LC) methods; of those, 9 were from LC with tandem mass spectrometric 

detection (LC-MS/MS) and 1 was from LC with ultraviolet absorbance detection (LC-UV). 

Seven datasets originated from immunoassay (IA) techniques, including 3 from enzyme 

immunoassay (EIA), 1 from chemiluminescence immunoassay (CLIA), and 3 from 

radioimmunoassay (RIA). Appendices A-1 and A-2 summarize the immunoassay and LC 

methods, respectively, used by the participants.

The raw data received from all participants is summarized in Appendix B. The data include the 

results for 25(OH)D2, 25(OH)D3 and 25(OH)DTotal in the three vials (A, B and C) of SRM 1950 

(study sample) and for 25(OH)D2 and 25(OH)D3 in the ethanolic calibration solutions. Only the 

LC-based methods could distinguish between 25(OH)D2 and 25(OH)D3 in the SRM 1950 

samples. All 10 of the LC-based datasets reported values for 25(OH)D3, but only two reported 

values for 25(OH)D2. The amount of 25(OH)D2 in SRM 1950 was very low and below the 

detection limit for most of the LC-based methods. The seven datasets from the immunoassay 

methods could not distinguish between 25(OH)D2 and 25(OH)D3 and reported the 25(OH)DTotal

in SRM 1950 (study sample). However, both LC and immunoassay datasets provided individual 

values for 25(OH)D2 and 25(OH)D3 in the ethanolic controls because the analytes were in 

separate solutions.

Appendix B also provides the summarized NIST results for both SRM 1950 (study material) and 

SRM 2972 (controls).
3

WINTER 2010 EXERCISE RESULTS AND DISCUSSION

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

A summary of the individual participant data for 25(OH)D2 and 25(OH)D3 in the SRM 2972 

control solutions is provided in Table 1. The community results are summarized at the bottom 

of the table for all reported methods and the LC methods only. The community results include 

the total number of quantitative values reported (N), the median value, the MADe (the median 

absolute deviation estimate, a robust estimate of the standard deviation), and the percent 

coefficient of variation (CV%). Consensus statistics were not calculated for the data from the IA 

methods because of the limited number of data reported (N = 3). Table 1 also presents the NIST 

certified values with expanded uncertainties corresponding to 95% confidence.

For the ethanolic control solutions (SRM 2972), the single data values for 25(OH)D2 and 

25(OH)D3 reported by each individual laboratory are plotted in Figures 1a and 1b, respectively. 

The two primary methods of analysis (LC and immunoassay) are displayed separately with 

closed () and open () circles, respectively. For each of these graphs, the black solid line 

() represents the consensus median, and the black dotted lines (- - - - -) represent the 

consensus variability (2 × MADe). The laboratories with results that fall between the two dotted 

lines are within the consensus variability area. The NIST-assessed values of 238.6 ng/mL ± 3.9 

ng/mL and 334.8 ng/mL ± 5.2 ng/mL for 25(OH)D2 and 25(OH)D3, respectively, for this control 

material (SRM 2972) are provided by red squares () (with error bars representing ± U95). 
4

Table 1. Summary of participant data for 25(OH)D2 (ng/mL) and 25(OH)D3 (ng/mL) in the 

SRM 2972 control solutions.

SRM 2972 SRM 2972

Lab Method Value Lab Method Value

056 LC-MS/MS 211.3 ## 056 LC-MS/MS 260.4

060 LC-MS/MS 235.0 ## 060 LC-MS/MS 324.2

062 RIA 175.7 ## 062 RIA 340.3

160 LC-MS/MS 237.0 ## 160 LC-MS/MS 329.0

182 LC-MS/MS 262.0 ## 182 LC-MS/MS 368.0

183a LC-MS/MS 161.2 ## 183a LC-MS/MS 353.8

183b CLIA 205.0 ## 183b CLIA 291.0

184 LC-MS/MS 223.0 ## 184 LC-MS/MS 351.0

185a LC-MS/MS 201.0 ## 185a LC-MS/MS 354.4

186 LC-MS/MS 240.6 ## 186 LC-MS/MS 333.8

187 LC-MS/MS 214.0 ## 187 LC-MS/MS 347.0

191 RIA 309.6 ## 191 RIA 319.8

N 12 N 12

Median 218.5 Median 337.1

MADe 26.7 MADe 22.8

CV% 12.2 CV% 6.8

N 9 N 9

Median 223.0 ## Median 347.0

MADe 20.8 ## MADe 19.6

CV% 9.3 ## CV% 5.6

NIST Value 238.6 NIST Value 334.0

U 95 3.9 U 95 5.2

25(OH)D2 (ng/mL) 25(OH)D3 (ng/mL)

All 

methods

LC 

methods
5

Figure 1. Winter 2010 exercise results for (a) 25(OH)D2 and (b) 25(OH)D3 in the ethanolic 

control (SRM 2972).

(a) 25(OH)D2

(b) 25(OH)D3
6

25(OH)D in the study material SRM 1950

A summary of the individual participant data for 25(OH)DTotal in SRM 1950 is provided in 

Table 2. The summarized data include the mean, standard deviation (SD), and relative percent 

standard deviation (%rSD) of the three reported values (vials A, B, and C) for SRM 1950.

The community results are summarized at the bottom of the table for all reported methods, the 

IA methods only, the LC methods only and the LC-MS/MS methods only. These summarized 

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

and the percent coefficient of variation.

The table also presents the sum of the NIST certified and reference values for 25(OH)D3 and 

25(OH)D2, respectively, for SRM 1950 (study sample) with approximated 95% confidence 

limits (U95). NIST results for SRM 1950 were obtained using data from both LC-MS and 

LC-MS/MS techniques; details about the methods and measurements are reported in the 

Certificate of Analysis for SRM 19501

. 

For each participant who used an LC method, the calculated average (mean) value and error bars 

(representing the lab mean value ± 2 × SD) for 25(OH)D3 in SRM 1950 (study sample) are 

plotted in Figure 2. The data for 25(OH)D2 are not provided in graphical form because only two 

laboratories reported values that were above their detection limits. When the error bars for each 

data point are considered, all laboratory data are within the consensus variability (2 × MADe). 

For all participant datasets, the calculated average (mean) values and error bars (representing the 

lab mean value ± 2 × SD) for 25(OH)DTotal in SRM 1950 (study sample) are plotted in Figure 3. 

From the mean values for all datasets, the consensus median and the consensus variability (2 ×

MADe) were determined. When the error bars for each data point are considered, all laboratory 

data are within the consensus variability. Both primary methods of analysis (LC, immunoassay) 

provide similar mean values and uncertainties for SRM 1950 (study sample).

The precision (as %rSD) for the three replicate analyses of SRM 1950 (study sample) ranged 

from 1% to 10% for the individual laboratories, where over 2/3 of the laboratories that 

participated had method precision under 5%. When all reported data for SRM 1950 (study 

sample) were considered, the consensus variability was ≈ 14% and the consensus median was 

biased ≈ 7% higher than the NIST-assessed value for this material. A goal for this program is to 

determine the cause of any bias and to achieve better agreement between the consensus median 

value and the NIST-assessed value. While the precision of most individual laboratories was 

acceptable, another program goal is to reduce the consensus variability to better represent the 

community’s measurement capability while also recognizing that a ‘fit-for-purpose’ variabilitylevel may exist. One approach to meet this goal is to strive for within-laboratory precision of 

less than 5% for all participants. 

 1 https://www-s.nist.gov/srmors/view_cert.cfm?srm=1950
7

Table 2. Summary of participant data for 25(OH)DTotal (ng/mL) in SRM 1950. 

SRM 1950 SRM 1950 SRM 1950

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

056 LC-MS/MS 24.9 24.2 24.3 24.5 0.4 1.5

060 LC-MS/MS 26.5 26.7 27.1 26.8 0.3 1.1

062 RIA 22.6 24.3 23.4 23.4 0.9 3.6

160 LC-MS/MS 26.3 29.2 27.3 27.6 1.5 5.3

180 RIA 25.4 27.0 25.5 26.0 0.9 3.5

182 LC-MS/MS 24.0 26.0 29.0 26.3 2.5 9.6

183a LC-MS/MS 31.3 29.8 29.3 30.1 1.0 3.5

183b CLIA 30.0 29.1 29.8 29.6 0.5 1.6

184 LC-MS/MS 23.8 28.3 27.1 26.4 2.3 8.8

185a LC-MS/MS 26.4 26.7 27.0 26.7 0.3 1.3

186 LC-MS/MS 27.6 25.3 23.5 25.5 2.0 8.0

187 LC-MS/MS 25.5 24.8 26.2 25.5 0.7 2.7

188 CLIA 28.0 28.0 25.0 27.0 1.7 6.4

189 LC-UV 29.8 29.3 28.6 29.2 0.6 2.1

190 EIA 32.5 30.4 30.4 31.1 1.2 3.9

191 RIA 29.9 30.4 30.1 30.1 0.2 0.8

192 EIA 23.5 23.4 24.2 23.7 0.4 1.7

N 17 17 17 17

Median 26.4 27.0 27.1 26.7

MADe 3.5 3.2 3.1 1.8

CV% 13.2 12 11 7

N 7 7 7 7

Median 28.0 28.0 25.5 27.0

MADe 3.8 3.5 3.0 4.6

CV% 13.7 13 12 17

N 10 10 10 10

Median 26.3 26.7 27.1 26.6

MADe 2.0 2.6 1.8 1.6

CV% 7.6 9.7 6.6 5.9

N 9 9 9 9

Median 26.3 26.7 27.1 26.4

MADe 1.9 2.4 1.3 1.3

CV% 7.3 8.9 4.9 5.1

NIST Value 25.30 25.30 25.30 25.30

U 95 0.79 0.79 0.79 0.79

All 

methods

IA 

methods 

LC 

methods

LCMS/MS

SRM 1950 Combined
8

Figure 2. Winter 2010 exercise results for 25(OH)D3 using only LC methods in SRM 1950 

(study sample).

Figure 3. Winter 2010 exercise results for 25(OH)DTotal in SRM 1950 (study sample).
9

Appendix A-1. Summary of immunoassay methods used by VitDQAP Winter 2010 participants 

Participant 

Number Immunoassay Sample Preparation Detection

62 RIA Samples were extracted, centrifuged Gamma counter

180 RIA Samples were prepared per vendor's sample extraction 

protocol

 I125 detection using gamma 

counter

183b CLIA n/r n/r

188 EIA n/r n/r

190 EIA Calibrators, controls and test specimens were all diluted with 

biotin-labeled 25(OH)D and analyzed in duplicate Microplate reader

191 RIA n/r n/r

192 EIA n/r

Software was used to convert the 

absorbance values obtained at 

450 nm to get the concentration

n/r = not reported
10

Appendix A-2. Summary of LC methods used by VitDQAP Winter 2010 participants

Participant 

Number

Internal Standard 

(IS) Sample Preparation Chromatographic Conditions Detection

56 25(OH)D2-d3 and 

25(OH)D3-d6

Samples were extracted with 

acetonitrile, centrifuged, then 

filtered

Online SPE with C8 column (2.1 x 

30 mm); separation on C18 column 

(2.1 x 50 mm); step gradient with 

methanol/water; flow 0.5 mL/min

MS/MS MRM: 25(OH)D3 m/z 

401/383; 25(OH)D3-d6 m/z 

407/389; 25(OH)D2 m/z 

413/395; 25(OH)D2-d3 m/z 

416/398

60 25(OH)D3 -d6

IS solution was added to plasma 

(150 μL), and proteins were 

precipiated by addition of a ternary 

extraction solvent. Sample 

cetrifuged, supernatant transfered 

to new vial, dried, and dissolved in 

mobile phase

C18 column (3.0 x 150 mm); A: 

0.05% formic acid in water, B: 

0.05% formic acid in 

methanol/acetonitrile (80/20, 

volume fraction); isocratic elution 

with 92% B from 0-2 min, step 

gradient to 100% B (2-8 min), 

equilibration (8-13) min; flow 0.55 

mL/min

MS/MS (positive) MRM: 

25(OH)D3 m/z 383/211; 

25(OH)D3-d6 m/z 389/211; 

25(OH)D2 m/z 395/270

160 25(OH)D2-d6 and 

25(OH)D3-d6

Samples were extracted in 

acetonitrile, centrifuged and filtered

2-Dimensional separation CN 

column (2.1x 50mm) (1st 

Dimension), C18 column (2.1x 

50mm) (2nd Dimension); gradient 

with 10 mmol/L formic acid in water 

and 10 mmol/L formic acid in 

methanol

MS/MS MRM: 25(OH)D3 m/z 

401/365; 25(OH)D3-d6 m/z 

407/371; 25(OH)D2 m/z 

413/337; 25(OH)D2-d6 m/z 

419/337

182 25(OH)D3 -d6

Proteins were precipitated with 

acetonitrile/methanol (3:1) and IS 

directly in 96 well plate. Plate was 

covered, mixed manually, and 

centrifuged

C18 column (2.1 x 50 mm); gradient 

elution from 60%-100% methanol 

with formic acid and ammonium 

acetate modifiers

MS/MS MRM: 25(OH)D3 m/z 

401/365 (quant), m/z 

401/383 (qual); 25(OH)D2 

m/z 413/355 (quant), m/z 

413/271 (qual)

183a 25(OH)D3 -d6

IS (25 μL) was added to plasma 

(150 μL), followed by protein 

precipiation and extraction with 0.1 

mol/L ZnSO4 (150 μL), methanol 

(300 μL), and hexane (750 μL); 

extract dried and dissolved with 

70% methanol, 30% water with 2 

mmol/L ammonium acetate

C8 column (2.1 X 50 mm); isocratic 

elution with 73% methanol/ 27% 

water; flow 0.4 mL/min

MS/MS MRM: 25(OH)D3 m/z 

401/159, 401/383; 25(OH)D2 

m/z 413/82, 413/395

184 25(OH)D3 -d6

Plasma (200 μL) extracted with 

acetonitrile and IS (700 μL); mixed, 

centrifuged, and filtered

C18 column (100 x 2.1 mm; 5μm); 

linear gradient from 60% B to 98% 

B over 2 min (A: 0.1% formic acid in 

water, B: methanol with 0.1% formic 

acid and 5 mmol/L ammonium 

acetate)

MS/MS (APCI) MRM: 

25(OH)D3 m/z 383/257; 

25(OH)D3-d6 m/z 389/263; 

25(OH)D2 m/z 395/209

185 n/r n/r n/r MS/MS

186 25(OH)D3 -d6

Proteins were removed by addition 

of ZnSO4, followed by methanol 

extraction and centrifugation; 

analytes were liquid/liquid extracted 

with three volumes of hexane, 

dried, and dissolved in 

methanol/water (65:35)

Phenyl column (2.1 x 100; 1.7um); 

40°C; gradient: 65-85 % B over 3 

min (A: 0.1% formic acid in water; B: 

0.1% formic acid in methanol); flow 

0.45 mL/min

MS/MS MRM: 25(OH)D3 m/z 

401/159; 25(OH)D2 m/z 

413/395

187 n/r n/r n/r MS/MS

189 Obtained from kit 

supplier

Proteins were precipitated, samples 

centrifuged, analytes in the 

supernatant were extracted using 

SPE cartridges

Commercially obtained reagent set 

and column; isocratic elution; flow 

0.7mL/min 

UV at 265 nm

SPE = solid phase extraction

MRM = multiple reaction monitoring

CN = cyano phase

quant = quantitative ions

qual = qualitative ions

n/r = not reported
11

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

SRM 1950 SRM 1950 SRM 1950 SRM 1950 SRM 1950 SRM 1950 SRM 1950 SRM 1950 SRM 1950

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

056 LC-MS/MS 0.5 0.4 <0.4 24.4 23.8 24.3 24.9 24.2 24.3 211.3 260.4

060 LC-MS/MS <2 < 2 < 2 26.5 26.7 27.1 26.5 26.7 27.1 235.0 324.2

062 RIA n/a n/a n/a n/a n/a n/a 22.6 24.3 23.4 175.7 340.3

160 LC-MS/MS <1.0 <1.0 <1.0 26.3 29.2 27.3 26.3 29.2 27.3 237.0 329.0

180 RIA n/a n/a n/a n/a n/a n/a 25.4 27.0 25.5 n/r n/r

182 LC-MS/MS <2 <2 <2 24.0 26.0 29.0 24.0 26.0 29.0 262.0 368.0

183a LC-MS/MS <4 <4 <4 31.3 29.8 29.3 31.3 29.8 29.3 161.2 353.8

183b CLIA n/a n/a n/a n/a n/a n/a 30.0 29.1 29.8 205.0 291.0

184 LC-MS/MS <1.0 <1.0 <1.0 23.8 28.3 27.1 23.8 28.3 27.1 223.0 351.0

185a LC-MS/MS n/d n/d n/d 26.4 26.7 27.0 26.4 26.7 27.0 201.0 354.4

186 LC-MS/MS n/r n/r n/r 27.6 25.3 23.5 27.6 25.3 23.5 240.6 333.8

187 LC-MS/MS 0.7 0.8 0.7 24.8 24.0 25.5 25.5 24.8 26.2 214.0 347.0

188 CLIA n/a n/a n/a n/a n/a n/a 28.0 28.0 25.0 n/r n/r

189 LC-UV n/d n/d n/d 29.8 29.3 28.6 29.8 29.3 28.6 n/r n/r

190 EIA n/a n/a n/a n/a n/a n/a 32.5 30.4 30.4 n/r n/r

191 RIA n/a n/a n/a n/a n/a n/a 29.9 30.4 30.1 309.6 319.8

192 EIA n/a n/a n/a n/a n/a n/a 23.5 23.4 24.2 n/r n/r

NIST Value 0.52 0.52 0.52 24.78 24.78 24.78 25.30 25.30 25.30 238.6 334.8

U 95 0.17 0.17 0.17 0.77 0.77 0.77 0.79 0.79 0.79 3.9 5.2

SRM 2972

25(OH)D2/D3 (ng/mL)

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

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