U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Centers for Disease Control and Prevention

National Center for Health Statistics

Abstract

Background—Serum concentrations of 25-hydroxyvitamin D (25OHD) were 

measured for the National Health and Nutrition Examination Survey (NHANES) 

over the 1988–2006 period using a radioimmunoassay (RIA). In 2010, the Centers 

for Disease Control and Prevention (CDC) reissued RIA-harmonized 25OHD for 

NHANES 2004 and 2006, and advised users to adjust the original RIA data from 

1988–1994 by using an equation. Beginning with NHANES 2007–2008, a liquid 

chromatography–tandem mass spectrometry (LC–MS/MS) method measured 25OHD.

Methods—A method comparison (bridging) study was designed to convert 

original RIA 25OHD to LC–MS/MS-equivalents. This report compares the predictive 

ability of a competitor regression model (Model 2) to the equations that CDC publicly 

released in 2015 (Model 1). The models differ by time period variable and use of 

transformations. 

Results—The two models provided similar adjusted R2

 (Model 1: 88.9%, Model 

2: 90.4%) and root mean square error of prediction (plus or minus 9 to 10 nanomoles 

per liter [nmol/L]). Applying these models to NHANES 1988–2006 RIA 25OHD, the 

Pearson correlation of LC–MS/MS-equivalent concentrations was 0.99; the median 

difference between models was 0 nmol/L (interquartile range: –2.8 to 1 nmol/L). In 

contrast to declining RIA-harmonized 25OHD, both models showed little change 

in LC–MS/MS-predicted 25OHD over the 1988–2006 period. For 2001–2006, both 

models predicted similar prevalences of 25OHD less than 30 nmol/L, which were 

lower than the prevalence estimates based on RIA-harmonized data. Mean weighted 

LC–MS/MS-equivalent concentrations based on either model were about 3 nmol/L 

lower for the 1988–1994 survey and about 3 nmol/L higher for the 2001–2006 

surveys, effectively smoothing out temporal trends observed using the harmonized 

RIA data.

Conclusions—Given minimal differences between models, final selection was 

based on public availability of the regression data. The bridging equations provide a 

way to use the previous RIA results to obtain LC–MS/MS-equivalent concentrations 

and evaluate temporal trends in vitamin D status.

Keywords: vitamin D • regression equation • laboratory comparison • NHANES

Introduction

When important clinical methods 

change, laboratories perform comparison 

(bridging) studies to relate the old and 

new methods. These bridging efforts 

provide information useful for the 

continuity of care of individual patients 

and for evaluating trends in monitoring 

public health. Generally, results from 

the old method are converted to newmethod equivalents. In the current study, 

the old method was a radioimmunoassay 

(RIA) for the vitamin D status indicator, 

which is serum concentrations of 

25-hydroxyvitamin D (25OHD); the 

new method is a liquid chromatography–

tandem mass spectrometry (LC–MS/MS) 

method traceable to Standard Reference 

Materials (SRM) from the National 

Institute of Standards and Technology 

(NIST).

A reformulation by the RIA 

manufacturer had identified a major shift 

in values (–12%) in kits available around 

2001, and subtle drifts in values (plus 

or minus 5%–10%) on two occasions 

in 2004 and 2006, which were likely 

due to kit lot-to-lot variability (1). To 

compensate for these RIA shifts and 

drifts, the National Health and Nutrition 

Examination Survey (NHANES) had 

used stored specimens and long-term 

Number 93  April 25, 2016

National Health Statistics Reports

A Method-bridging Study for Serum 25-hydroxyvitamin D 

to Standardize Historical Radioimmunoassay Data to 

Liquid Chromatography–Tandem Mass Spectrometry 

by Rosemary L. Schleicher, Ph.D., and Maya R. Sternberg, Ph.D., National Center for Environmental Health; David A. Lacher, M.D., 

National Center for Health Statistics; Christopher T. Sempos, Ph.D., National Institutes of Health; Anne C. Looker, Ph.D., National 

Center for Health Statistics; Ramon A. Durazo-Arvizu, Ph.D., and Elizabeth A. Yetley, Ph.D., National Institutes of Health; Madhulika 

Chaudhary-Webb, M.S., Khin L. Maw, M.S., and Christine M. Pfeiffer, Ph.D., National Center for Environmental Health; and Clifford L. 

Johnson, M.S.P.H., National Center for Health Statistics
Page 2 National Health Statistics Reports  Number 93  April 25, 2016

quality control materials to harmonize 

the 25OHD results (1). Consequently, 

although the true values of serum 

specimens may not be known with a 

specified degree of assurance, specimens 

tested at any given time should produce 

approximately the same results, using 

adjustments, despite kit changes. This 

concept was endorsed by a roundtable 

of experts in vitamin D (1) that was 

convened to consider the best way 

to address the problem of reagent kit 

variability, which was confounding the 

interpretation of population trend data. 

However, these experts also agreed that 

an interim harmonization based on RIA 

results needed to be replaced as early 

as possible by a long-term objective to 

standardize the 25OHD RIA results to 

LC–MS/MS, allowing a more accurate 

interpretation of long-term trends.

To achieve the long-term objective, a 

subset of serum samples from NHANES 

III (1988–1994) and NHANES 2001–

2006 that were originally tested for 

25OHD using RIA were retested using an 

LC–MS/MS method that was traceable to 

SRM. These data were used to develop 

regression equations relating the old 

and new methods so that LC–MS/MSequivalent 25OHD could be predicted for 

any RIA results from these surveys.

Methods

Background on NHANES

The serum specimens and 25OHD 

data in this study came from NHANES 

III and NHANES 2001–2006. NHANES 

is a cross-sectional survey of the civilian 

noninstitutionalized population of the 

United States, conducted by the Centers 

for Disease Control and Prevention’s 

(CDC) National Center for Health 

Statistics (NCHS). Sampling design and 

data collection methods are available (2). 

Survey participants were interviewed at 

home and had blood collected at mobile 

examination centers. Participation rates 

for each survey are detailed elsewhere (3).

Laboratory measurements of 

25OHD using RIA and

LC–MS/MS

For the surveys conducted between 

1988 and 2006, RIA (DiaSorin, 

Stillwater, Minn.) was used to measure 

total 25OHD (in duplicate) (4). A fully 

validated LC–MS/MS method (5), 

traceable to NIST reference materials, 

was used to measure 25-hydroxyvitamin 

D2 (25OHD2), 25-hydroxyvitamin 

D3 (25OHD3), and the C3 epimer of 

25OHD3 for selected stored specimens 

for those NHANES participants during 

1988–2006 having available RIA data for 

the bridging study (in duplicate). Once 

the bridging study was nearly finished, 

the same assay was used to measure 

25OHD for all eligible participants in 

NHANES 2007–2010 (in singlicate). 

For the LC–MS/MS method, total 

25OHD was defined as the sum of 

25OHD3 and 25OHD2, excluding the 

C3 epimer of 25OHD3, about which 

less is known. The bias of the LC–MS/

MS method relative to NIST SRM 

during the course of the bridging study 

was modest (3% or less) for 25OHD3

and 25OHD2 at concentrations greater 

than 2 nanomoles per liter (nmol/L). 

Furthermore, the average bias of CDC’s 

LC–MS/MS method for a set of 50 

individual donor serum samples—

derived from an interlaboratory 

comparison study performed around 

the time of the bridging study—was 

1.4% (95% confidence interval [CI]: 

0.9%–1.8%). The serum samples were 

from the first interlaboratory comparison 

study sponsored by the Vitamin D 

Standardization Program (6) relative to 

two independent reference measurement 

procedures, carried out by NIST and the 

University of Ghent (7,8).

Quality assurance for 

bridging study

To achieve imprecision and bias 

goals for the LC–MS/MS assay (9), 

the 25OHD assay performance was 

continually monitored using quality 

control (QC) pools, reference materials, 

and external quality assurance program 

feedback. Calibration was performed 

using in-house prepared stock solutions. 

Value transfer from NIST SRM 2972 

(ethanol matrix) was performed with 

each new lot of calibrators using either 

direct (CDC calibrators assigned using 

SRM 2972 calibrators) or indirect (CDC 

calibrators adjusted using SRM 2972 

and SRM 972) value transfer procedures. 

Each run contained standard reference 

materials from NIST (10), namely, four 

levels of SRM 972 (serum matrix) and 

three levels of serum bench QC pools, 

which were analyzed at the beginning 

and end of the run. Decision rules for 

out-of-control runs were Westgardtype but more flexible to allow for 

multiple QC pools (11). For 25OHD3, 

low pools with mean concentrations 

of 28.5–29.7 nmol/L, medium pools 

with mean concentrations of 52.6–63.7 

nmol/L, and high pools with mean 

concentrations of 86.0–92.2 nmol/L had 

coefficients of variation (CV) of 4% or 

less. For 25OHD2, low pools with mean 

concentrations of 5.0–13.9 nmol/L had 

CV 6%–8%, medium pools with mean 

concentrations of 10.8–38.9 nmol/L had 

CV 4%–5%, and high pools with mean 

concentrations of 21.6–62.7 nmol/L had 

CV 6%.

A number of additional quality 

assurance activities were conducted 

to confirm the validity of the bridging 

study results. The stability of 25OHD in 

long-term storage and under accelerated 

degradation conditions was established 

during the design phase of this study, 

because plans called for using serum 

that was in storage for up to 23 years for 

NHANES III, for which pristine (i.e., 

frozen once, never thawed) specimens 

were not available. For NHANES 2001–

2006, residual serum stored at –70°C and 

freeze-thawed multiple times, or pristine 

serum stored in liquid nitrogen fumes 

at –130°C, were available. The 25OHD 

results from 35 NHANES specimens 

stored at –70°C for 10 years, which may 

have undergone multiple freeze-thaws for 

RIA testing, were compared with serum 

specimens stored at –130°C from the 

same 35 participants and never thawed 

until LC–MS/MS testing. There was 95% 

power for a 0.05-level equivalence test 

with a sample size of 35 to test that the 

difference of the mean responses was 

within equivalence bounds (–2.0 nmol/L, 

2.0 nmol/L), assuming no difference 

between surplus and pristine samples, 

a correlation of 0.98, and a common 
National Health Statistics Reports  Number 93  April 25, 2016 Page 3

standard deviation of 16 nmol/L. No 

significant difference was found in 

25OHD3 concentrations. Both storage 

conditions were found to be essentially 

the same (mean paired 25OHD3

difference: –0.74 [95% CI: –2.17 to 0.68] 

nmol/L; p = 0.30).

Using QC pools, 25OHD metabolites 

were shown to be stable in serum stored 

at 37°C for at least 8 days, and four 

freeze-thaw cycles were not associated 

with loss of 25OHD (12). Using three 

QC pools used by the laboratory during 

1994–1995 and the final prediction 

model used to standardize NHANES 

data, the predicted LC–MS/MS results 

for these pools were shown to be within 

one root mean square error of prediction 

(rMSEP) of the LC–MS/MS results that 

were measured 17 years later in 2012, 

affirming that 25OHD is stable over long 

periods.

Because NHANES 1988–1994 

specimens were collected in serum 

separator tubes, and NHANES 2001–

2006 specimens were collected in red-top 

serum vacuum tubes, matrix equivalency 

needed to be established. Serum prepared 

from serum separator and red-top tubes 

from 27 blood donors were tested for 

25OHD. Over 95% power was achieved 

for a 0.05-level equivalence test with 

a sample size of 27 to test that the 

difference of the mean responses was 

within equivalence bounds (–2.0 nmol/L, 

2.0 nmol/L), assuming no difference 

between two matrices, a correlation of 

0.98, and a common standard deviation of 

8.5 nmol/L. No significant difference was 

found in 25OHD3 concentrations. Both 

matrices were found to be essentially the 

same (mean paired 25OHD3 difference: 

–0.01 [95% CI: –1.33 to 1.32] nmol/L;

p = 0.99).

To determine the stability of the LC–

MS/MS assay, more than 100 specimens 

were retested from NHANES 2005–2008, 

covering the period of the bridging study 

and the time period in which NHANES 

2007–2008 samples were tested. 

Specifically, all specimens with adequate 

volume were retested from the 2005–

2006 bridging study (n = 52; 25OHD 

range: 12–152 nmol/L); Bland-Altman 

bias of this set, retested in 2014 compared 

with the original testing in 2011, was not 

significant (difference –0.1%; 95% CI: 

–2.2 to 2.0%; p = 0.92). Specimens from 

NHANES 2007–2008 were also retested 

(n = 73; 25OHD range: 20–140 nmol/L); 

Bland-Altman bias of the retest in 2014 

compared with the original testing in 

2012 also was not significant (difference 

–0.1%; 95% CI: –1.4 to 1.3%; p = 0.88).

Bridging study design

The basic design of the method 

comparison was reviewed and evaluated 

by a panel of experts (1). The Clinical 

and Laboratory Standards Institute 

(Wayne, PA) method evaluation protocol, 

EP09–A2, was used as a guideline. The 

majority of the NHANES III samples 

(90%) were measured using the original 

RIA assay of 1995–1996; NHANES 

samples from 2000–2006 were assayed 

by use of the reformulated RIA assay 

within 12 months of collection. No 

specimens were collected for 25OHD 

measurements in 1999; because data 

confidentiality requirements placed 

participant 25OHD data from year 2000 

in restricted access at the NCHS Research 

Data Center, the survey period 1999–

2000 was not included in this analysis.

For NHANES III, the data were 

sorted from lowest to highest 25OHD, 

ranked into quartiles, and then sorted by 

date; overall, approximately 100–200 

specimens per quartile were selected 

to provide a representative range of 

dates and concentrations. NHANES III 

specimens were randomized for LC–MS/

MS testing. The majority of specimens 

were selected from 1995 and 1996 (dates 

of analysis), because the majority of RIA 

analyses were performed during these 

years (e.g., 1994 [19%], 1995 [35%], 

1996 [44%], and 1997 [2%]). Due to low 

numbers, 1997 results were combined 

with those from 1996 for generating 

regression equations. Fourteen specimens 

per run were selected for analysis of 

25OHD metabolites, but sometimes 

fewer (12 or 13 specimens) were tested 

due to insufficient quantity. NHANES III 

analysis was complete after 43 analytical 

runs.

Historical RIA data from NHANES 

2001–2006 were sorted into survey year 

(2001, 2002, 2003, 2004, 2005, and 

2006). Within each survey year, RIA 

data were sorted by lowest to highest 

25OHD. The sorted data were divided 

into quartiles. Within quartiles, the data 

were sorted by month of laboratory 

measurement. For NHANES 2001–2006, 

25–40 specimens per quartile per year 

were selected to provide a representative 

range of dates and concentrations; 

these were randomized for testing. 

Four specimens per year were selected 

for analysis of 25OHD metabolites in 

each run, but sometimes fewer than 

four specimens were tested because of 

insufficient specimen quantity. After 

25 analytical runs, another selection of 

specimens was carried out, both to obtain 

enough specimens due to insufficient 

quantity and to enhance the number of 

specimens measured at the tails of the 

distribution. The method comparison data 

set was not limited by age and included 

sera from participants under age 12 years. 

NHANES 2001–2006 and NHANES III 

specimens were analyzed together. In 

total, 57 analytical runs were performed 

to complete the method comparison 

study.

One of the major issues to address 

was how to model the kit-to-kit 

variability of RIA. Although the date 

of RIA measurement could be used, 

such exact dates and years are not 

part of publicly released data sets. If 

the proposed statistical model used 

measurement year, the yearly regression 

equations would not be publicly released 

due to confidentiality concerns, and 

NCHS would need to calculate the 

predicted new-method results, withhold 

the regression equations, and re-release 

the data. Therefore, the alternative of 

providing regression equations for each 

survey period was proposed. This would 

allow data users to access the regression 

equations used in the re-released data. 

However, because the assay fluctuations 

were most closely captured by using the 

calendar year of RIA measurement, the 

need for 1-year regression equations was 

evaluated against statistical models that 

used the survey period. Other features 

were considered, including the need for 

a transformation to address regression 

assumptions such as linearity, normality, 

and homogeneity; the need to address 

measurement error related to the RIA 

assay; and whether other covariates 

beyond RIA values and the time of 

measurement should be considered.
Page 4 National Health Statistics Reports  Number 93  April 25, 2016

Model development

The data, based on 1,448 subjects 

from NHANES 1988–2006 selected 

as part of the crossover data set, were 

used to develop regression equations to 

predict NHANES participants’ LC–MS/

MS-equivalent concentrations from 

their previously measured RIA values. 

The dependent variable was the sum of 

25OHD2 and 25OHD3 in nmol/L units 

based on LC–MS/MS. When 25OHD2

was less than the limit of detection 

(LOD), the value for 25OHD2 was 

calculated as LOD divided by the square 

root of 2. The primary independent 

variable was the original RIA result 

in nanograms per milliliter (ng/mL) 

multiplied by 2.496 to convert to nmol/L. 

Several statistical issues that pertain 

to any linear regression model such as 

linearity, constant variance, normality, 

and lack of measurement error in the 

covariate(s) were evaluated. Graphical 

diagnostics were used to assess model 

adequacy, as well as to identify any 

potentially influential data points. 

Potential outliers were rechecked using 

LC–MS/MS, but they were verified. 

Historically, it is known that the 

distribution of 25OHD is slightly skewed 

right with some nonconstant variance. 

Statistical approaches to address these 

types of violations of a linear regression 

model included the use of a square root 

transformation, weighted least squares, or 

piecewise regression. Ultimately, a square 

root transformation, or a combination of 

piecewise regression and ordinary least 

squares regression, was selected for the 

final models. 

Because it was known that the 

manufacturer reformulated the RIA and 

that lot-to-lot variability in the assay kits 

was noted on two separate occasions 

during the 2001–2006 survey periods, 

a covariate closely aligned with the 

time of RIA analysis was to be used 

in developing the models. The choice 

between using survey period (publicly 

available) and year of RIA analysis 

(more closely related to the date of RIA 

measurement but not publicly available) 

to mimic the timing of any assay 

fluctuations as a covariate in the models 

was considered. Two competing models 

were selected for further evaluation.

Model 1 is described in an online 

analytical note for NHANES III 

(1988–1994), NHANES 2001–2006, and 

NHANES 2007–2010 as a data advisory 

 

Figure 1. Serum 25-hydroxyvitamin D concentrations measured by LC–MS/MS and predicted from Model 1 using different regression 

approaches for bridging study data set, stratified by survey period: National Health and Nutrition Examination Survey, 1988–1994 and 

2001–2006
National Health Statistics Reports  Number 93  April 25, 2016 Page 5

informing researchers that previously 

released nonstandardized NHANES 

25OHD data have been converted to 

standardized 25OHD and re-released 

(13). A comparison of linear, Deming, 

and piecewise regression equations for 

each survey cycle (Figure 1) shows 

a large difference between the linear 

and piecewise fit for NHANES III, but 

negligible differences for the 2001–2006 

period. Therefore, Model 1 used a 

piecewise linear regression for NHANES 

III to account for some nonlinearity, and a 

separate ordinary least squares regression 

for each subsequent survey cycle. Model 

2 used a square root transformation on 

both the dependent variable LC–MS/MS 

25OHD and the independent variable RIA 

25OHD to address the mild concentration 

dependence and nonlinearity. Models 1 

and 2 can each be formulated into single 

multiple linear regression (MLR) models 

using indicator variables and properly 

defined interactions. This facilitates a 

direct comparison of model diagnostics 

and adjusted R-squared. MLR Model 

1 consisted of the primary independent 

variable (RIA 25OHD) and a set of 

three indicator variables (X1–X3) for 

each survey cycle (2001–2002, 2003–

2004, and 2005–2006). The reference 

survey period was selected arbitrarily 

as NHANES III, so X1 = X2 = X3 = 0. 

Interactions were included between 

each of the three indicator variables 

and RIA to allow for a unique slope for 

each survey cycle. To account for the 

piecewise regression for NHANES III, an 

indicator variable was created that was 1 

when RIA 25OHD was greater than 102.2 

nmol/L, and 0 otherwise; an interaction 

between this indicator variable and RIA 

25OHD allowed for a separate slope 

when RIA was greater than 102.2 nmol/L 

during NHANES III (1988–1994). Model 

1 did not use any transformation for 

either the dependent variable (LC–MS/

MS 25OHD) or the primary independent 

variable (RIA 25OHD). MLR Model 2 

consisted of a square root transformation 

for the primary independent variable 

(RIA 25OHD) and a set of eight indicator 

variables (X1–X8) for each year of 

RIA measurement (1994, 1995, 1996, 

2001, 2002, 2003, 2004, and 2005). The 

reference year was selected arbitrarily 

as 2006, such that X1 = X2 = ... X8 = 0. 

Interactions were included between each 

of the eight indicator variables and RIA 

25OHD to allow for separate slopes for 

each year of RIA measurement. 

To compare the predictive ability 

of each model, the mean square error of 

prediction (MSEP) was estimated using 

fivefold cross validation. MSEP is the 

average squared difference between the 

observed LC–MS/MS value and the 

LC–MS/MS-equivalent prediction from 

a specified model. To estimate the MSEP 

using fivefold cross validation, a stratified 

sample of 80% of the original bridging 

data set was randomly selected. The 

strata were the year of RIA measurement 

to ensure 80% representation in any 

given year. The 80% sample was used as 

a training data set to estimate the model 

parameters, and the remaining 20% was 

used as a validation data set to calculate 

the MSEP. This was repeated five times, 

and the five MSEPs were averaged; the 

square root of the MSEP (rMSEP) was 

used to judge the models. The smaller the 

rMSEP, the better the predictive ability of 

a given model.

To examine the difference between 

the two models in terms of their impact 

on the assessment of temporal trends 

in the U.S. population, the unweighted 

and weighted arithmetic means, the 5th 

and 95th percentiles, and the prevalence 

of less than 30 nmol/L of LC–MS/MSequivalent total 25OHD were calculated 

for each NHANES period (1988–1994, 

2001–2002, 2003–2004, and 2005–2006), 

and results were compared with the 

original RIA and harmonized RIA 

results. NHANES examination weights 

were used for the weighted means and 

percentiles, and variance estimates based 

on Taylor series linearization (14) were 

used for the 95% CI.

Statistical analyses were performed 

using SAS (version 9.3, SAS Institute 

Inc., Cary, N.C.) and SUDAAN (version 

11.0.1, RTI International, Research 

Triangle Park, N.C.).

Results

The individual simple linear 

regression equations for each survey for 

Model 1 and for each year of RIA testing 

for Model 2 are shown in Table A. Model 

1 was chosen as the final model and was 

used to provide LC–MS/MS-equivalent 

25OHD for the historical NHANES 

(1988–2006) that were originally tested 

using RIA (13). A graphical comparison 

of the predicted LC–MS/MS-equivalent 

and actual LC–MS/MS-measured 

25OHD for each of the two models in 

Table A. Individual regression models for predicting serum concentrations of LC–MS/MSequivalent 25-hydroxyvitamin D from original radioimmunoassay 25-hydroxyvitamin D, 

using a bridging data set: National Health and Nutrition Examination Survey, 1988–1994 and 

2001–2006

Regression model and

time period Equation

Model 1

Survey period:

1988–1994 . . . . . . . . . . . . . . . . If RIAoriginal ≤ 102, then LC–MS/MSequivalent = 1.57548 + 0.8429 • RIAoriginal

If RIAoriginal > 102, then LC–MS/MSequivalent = 59.2296 + 0.2788 • RIAoriginal

2001–2002 . . . . . . . . . . . . . . . . LC–MS/MSequivalent = 6.43435 + 0.95212 • RIAoriginal

2003–2004 . . . . . . . . . . . . . . . . LC–MS/MSequivalent = 1.72786 + 0.98284 • RIAoriginal

2005–2006 . . . . . . . . . . . . . . . . LC–MS/MSequivalent = 8.36753 + 0.97012 • RIAoriginal

Model 2 

Year of RIA analysis:

1994 . . . . . . . . . . . . . . . . . . . . . SQRT(LC–MS/MSequivalent) = 0.94761 + 0.80717 • SQRT(RIAoriginal)

1995 . . . . . . . . . . . . . . . . . . . . . SQRT(LC–MS/MSequivalent) = 1.16953 + 0.7708 • SQRT(RIAoriginal)

1996 . . . . . . . . . . . . . . . . . . . . . SQRT(LC–MS/MSequivalent) = 0.35182 + 0.85957 • SQRT(RIAoriginal)

2001 . . . . . . . . . . . . . . . . . . . . . SQRT(LC–MS/MSequivalent) = 0.6371 + 0.91432 • SQRT(RIAoriginal)

2002 . . . . . . . . . . . . . . . . . . . . . SQRT(LC–MS/MSequivalent) = 0.67272 + 0.96489 • SQRT(RIAoriginal)

2003 . . . . . . . . . . . . . . . . . . . . . SQRT(LC–MS/MSequivalent) = 0.10961 + 1.01678 • SQRT(RIAoriginal)

2004 . . . . . . . . . . . . . . . . . . . . . SQRT(LC–MS/MSequivalent) = 0.47863 + 0.92234 • SQRT(RIAoriginal)

2005 . . . . . . . . . . . . . . . . . . . . . SQRT(LC–MS/MSequivalent) = 0.17074 + 0.9854 • SQRT(RIAoriginal)

2006 . . . . . . . . . . . . . . . . . . . . . SQRT(LC–MS/MSequivalent) = 0.90722 + 0.97113 • SQRT(RIAoriginal)

NOTES: All units are in nanomole per liter. LC–MS/MS is liquid chromatography–tandem mass spectrometry. RIA is 

radioimmunoassay. SQRT is square root. 

SOURCE: CDC/NCHS, National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006.
Page 6 National Health Statistics Reports  Number 93  April 25, 2016

the bridging data set showed similar 

predictions for each model (Figure 2). 

For the bridging data set, the median 

difference between the predicted values 

(Model 2 – Model 1) was less than 

1 nmol/L (median = –0.77 nmol/L; 

interquartile range [IQR]

= –3.4 to 2.3 nmol/L), and overall 

Pearson correlation was 0.99. A 

comparison of the model diagnostics, 

using multiple linear regression models, 

showed that Model 1 had slightly 

less preferable visual features, with 

the histogram of residuals being a 

little less normally distributed, and 

residuals showing a slight concentration 

dependence at the high end (Figure 3). 

The residuals from Model 2 were more 

closely distributed normally without 

concentration-dependence; the estimated 

R-squared from the two models were 

similar (Model 1, 88.9%, compared with 

Model 2, 90.3%). 

The estimated rMSEP for the 

bridging study data set was plus or minus 

9 to 10 nmol/L for both models, although 

it tended to be slightly smaller for Model 

2 (Table B). However, when applied to a 

small independent validation data set (a 

set of 66 samples from NHANES 2001–

2006, for which only singlicate LC–MS/

MS 25OHD data were available), Model 

1 had a slightly smaller rMSEP. In 

addition to the two models described 

in this report, a number of different 

statistical models were evaluated. These 

models included approaches based 

on weighted regression and Deming 

regression, with the ratio of the error 

Figure 2. Serum 25-hydroxyvitamin D concentrations predicted from models 1 and 2 compared with measured LC–MS/MS concentrations in 

bridging study data set, stratified by survey period: National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006

 
National Health Statistics Reports  Number 93  April 25, 2016 Page 7

variances either assumed to be 1 or a 

value estimated from CDC laboratory

QC results. However, the rMSEP were 

much higher for these than for the two 

models described above and, therefore, 

these models are not described.

The equations from Model 1 and 

Model 2 were each used to predict 

LC–MS/MS-equivalent concentrations 

based on the original NHANES RIA 

25OHD from each survey (1988–1994, 

2001–2002, 2003–2004, and 2005–

2006). The estimated distributions of 

the predictions made from each model 

were similar, except for the right tail 

of 1988–1994 (Figure 4). Overall, the 

Pearson correlation between LC–MS/

MS-equivalent concentrations for 

models 1 and 2 across all of the surveys 

was 0.99, and the median difference of 

the predicted LC–MS/MS-equivalent 

concentrations between these models 

(Model 2 – Model 1) was 0 nmol/L

(IQR = –2.8 to 1 nmol/L). 

Unweighted mean 25OHD based on 

the original RIA and harmonized RIA 

concentrations, and the predictions from 

models 1 and 2, showed that the predicted 

LC–MS/MS-equivalent data from models 

1 and 2 differed by less than 2 nmol/L 

when based on multiyear survey data 

(Table C). For NHANES 2001–2006, 

the differences between the unweighted 

mean predictions for models 1 and 2 were 

more apparent when looking at the 1-year 

rather than the 2-year mean predictions, 

ostensibly due to the effect of averaging 

over 2 years (Table C). As expected, the 

average RIA-harmonized values were 

lower in 1988–1994 and 2003–2004, 

but higher in 2005–2006, than the 

original RIA 25OHD values due to the 

harmonization efforts. Although both 

models 1 and 2 provided lower mean 

estimates for 1988–1994 than the original 

RIA, these standardized estimates were 

also lower than the harmonized RIA 

values. In contrast, during the entire 

Figure 3. Summary of regression diagnostics from two multiple linear regression models for predicting serum concentrations of LC–MS/

MS-equivalent 25-hydroxyvitamin D from original radioimmunoassay 25-hydroxyvitamin D using a bridging data set: National Health and 

Nutrition Examination Survey, 1988–1994 and 2001–2006

 
Page 8 National Health Statistics Reports  Number 93  April 25, 2016

2001–2006 period, models 1 and 2 

provided higher unweighted means than 

the original RIA and harmonized RIA 

means (Table C).

Weighted predicted means using 

models 1 and 2 showed small differences 

(2 nmol/L or less) across demographic 

subgroups in the different surveys

(Table 1). The demographic subgroup 

that deviated the most when comparing 

the weighted mean predictions between 

Model 1 and Model 2 was the

non-Hispanic black population. For 

example, in 2001–2002, the Model 1 

weighted mean was 1.1 nmol/L higher for 

non-Hispanic black persons than Model 

2, but for other demographic subgroups 

Model 1 was approximately 0.9 nmol/L 

lower than Model 2. Similarly, for 

2005–2006, an approximately 0.3 nmol/L 

difference was noted between Model 

1 and Model 2 for other demographic 

subgroups, but Model 1 averaged 2.9 

nmol/L higher than Model 2 for nonHispanic black persons (Table 1). 

The differences in the two models’ 

predictions of the LC–MS/MS-estimated 

tails of the distributions of 25OHD 

varied across the demographic subgroups 

depending on the survey cycle (Table 2). 

The estimated 95th percentile from each 

model differed the most for the 1988–

1994 survey. For example, the estimated 

95th percentile for Model 1 was 10 

nmol/L lower for youth aged 12–19 years 

compared with Model 2, whereas the 95th 

percentile for Model 1 was 4.9 nmol/L 

higher for non-Hispanic black persons 

compared with the Model 2 estimate. The 

estimated 5th percentiles were relatively 

similar between the models across the 

demographic subgroups, with a less than 

1 nmol/L difference from 1988 to 2004. 

However, for 2005–2006, the estimated 

5th percentiles from Model 1 were 

approximately 3 nmol/L higher across all 

subgroups compared with Model 2. 

When using each of the prediction 

models to generate LC–MS/MSstandardized data, neither model revealed 

much change in the prevalence of 

25OHD less than 30 nmol/L (Table 3) 

over the 1988–2006 period. In contrast, 

based on the RIA-harmonized data, 

the prevalence of 25OHD less than 30 

nmol/L was higher in the 2001–2006 

surveys compared with the 1988–1994 

survey. Compared with the RIAharmonized data, the mean weighted LC–

MS/MS-equivalent concentrations from 

either model were approximately

3 nmol/L lower for the 1988–1994 period 

and approximately 3 nmol/L higher for 

the 2001–2006 period; this effectively 

smoothed out any temporal trends in the 

means observed with the harmonized and 

the original RIA data (Figure 5).

Conclusions

The objective of this study was to 

standardize previously obtained 25OHD 

RIA results from NHANES 1988–2006 

to LC–MS/MS to allow a more accurate 

interpretation of long-term trends in 

vitamin D status in the United States 

over this 18-year time span. Starting 

with NHANES 2007–2008, the new 

Table B. Root mean square error of prediction from fivefold cross-validation testing for 

bridging study data set and independent data set

Data set Model 1 Model 2 

NHANES survey period: nmol/L

1988–1994 . . . . . . . . . . . . . . . . . . . . . . . 9.4 9.5

2001–2002 . . . . . . . . . . . . . . . . . . . . . . . 9.1 8.2

2003–2004 . . . . . . . . . . . . . . . . . . . . . . . 9.7 9.3

2005–2006 . . . . . . . . . . . . . . . . . . . . . . . 10.6 9.5

Independent data set . . . . . . . . . . . . . . . . . 7.9 8.1

NOTES: NHANES is National Health and Nutrition Examination Survey, and nmol/L is nanomole per liter. Model 1, used for 

generating publicly released liquid chromatography–tandem mass spectrometry (LC–MS/MS)-equivalent data, is based on 

NHANES data. Model 2 is based on the year in which radioimmunoassay (RIA) data were generated (date of RIA testing is 

available from the NCHS Research Data Center). RIA data, collected from a set of 66 samples from NHANES 2001–2006 for 

which only singlicate LC–MS/MS 25-hydroxyvitamin D concentration data were available, were standardized using models 1 or 2, 

and the root mean square error of prediction was calculated for each model. 

SOURCE: CDC/NCHS, National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006.

Table C. Serum concentrations of 25-hydroxyvitamin D measured using original radioimmunoassay, or harmonized using stored survey 

specimens or quality-control pool data, or standardized using regression models, for persons aged 12 years and over, by survey period, 

phase, or year: National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006

Time period Sample size RIA–original RIA–harmonized

LC–MS/MS-standardized

Model 1 Model 2

n nmol/L

1988–1994 . . . . . . . . . . . . . . . . . . 18,851 64.6 57.0 55.0 53.3

Phase 1: 1988–1991 . . . . . . . . 8,977 65.7 58.0 55.9 54.9

Phase 2: 1991–1994 . . . . . . . . 9,874 63.6 56.2 54.1 51.9

2001–2002 . . . . . . . . . . . . . . . . . . 6,816 53.4 53.3 57.2 57.8

2003–2004. . . . . . . . . . . . . . . . . . 6,553 56.6 54.2 57.4 57.2

2005–2006. . . . . . . . . . . . . . . . . . 6,480 48.7 52.5 55.6 55.0

2001 . . . . . . . . . . . . . . . . . . . . . . . 3,507 53.9 53.9 57.7 56.9

2002. . . . . . . . . . . . . . . . . . . . . . . 3,309 52.8 52.8 56.7 58.7

2003. . . . . . . . . . . . . . . . . . . . . . . 3,278 52.7 51.8 53.5 55.8

2004. . . . . . . . . . . . . . . . . . . . . . . 3,275 60.5 56.6 61.2 58.6

2005. . . . . . . . . . . . . . . . . . . . . . . 3,241 51.0 52.4 57.9 53.8

2006. . . . . . . . . . . . . . . . . . . . . . . 3,239 46.3 52.6 53.4 56.3

NOTES: Values are unweighted arithmetic means. RIA is radioimmunoassay; nmol/L is nanomole per liter; and LC–MS/MS is liquid chromatography–tandem mass spectrometry. Two models were 

used to estimate LC–MS/MS-equivalent values from RIA-original data: Model 1 uses regression equations for each survey period, and Model 2 uses regression equations for each calendar year in 

which RIA data were tested. 

SOURCE: CDC/NCHS, National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006.
National Health Statistics Reports  Number 93  April 25, 2016 Page 9

standardized LC–MS/MS method 

developed by CDC was used on a routine 

basis to measure 25OHD (15). Providing 

continuity when methods change is 

important for facilitating interpretation 

of the data. Standardized data are the 

best possible data to use for this purpose, 

because they are anchored to reference 

materials with specified limits on 

accuracy.

This report summarizes some of 

the technical details about how various 

models were evaluated for predicting 

LC–MS/MS-equivalent concentrations 

of 25OHD from RIA results. In the 

past, when a method was changed 

in NHANES, a single crossover 

study would typically be undertaken 

Figure 4. Estimated kernel density curves of LC–MS/MS-standardized serum 25-hydroxyvitamin D concentrations for regression models 

1 and 2 for persons aged 12 years and over, stratified by survey period: National Health and Nutrition Examination Survey, 1988–1994 and 

2001–2006

 
Page 10 National Health Statistics Reports  Number 93  April 25, 2016

and a regression equation would be 

derived to facilitate temporal trend 

comparisons using the old method. 

However, this crossover study was not 

as straightforward because the RIA kits 

that were used were not harmonized 

to produce comparable results over 

the 1988–2006 period. A number of 

different models were investigated, but 

the two models with the smallest rMSEP 

described in this report produced very 

similar results. One model used the 

survey periods (1988–1994, 2001–2002, 

2003–2004, and 2005–2006) to derive 

separate equations; the other was based 

on the exact year of RIA measurement 

(1994, 1995, 1996, 2001, 2002, 2003, 

2004, 2005, and 2006). Because the first 

model was not dependent on nonpublicuse data, this model was used to estimate 

vitamin D status for NHANES III and 

NHANES 2001–2006 data. These 

updated data were publicly released in 

2015 with a corresponding Analytical 

Note (13).

Recognizing the limitations of using 

regression models to bridge the change 

in laboratory methods is important. First, 

these data are not actually measured 

LC–MS/MS values, but rather predicted 

from a regression model and, therefore, 

are inherently less variable than what 

would be expected had all the values 

actually been measured by LC–MS/MS. 

Secondly, the bridging data were not a 

random sample but were intentionally 

sampled across the full range of RIA 

concentrations and dates of RIA analyses. 

Therefore, the estimated relationship 

between LC–MS/MS and RIA using 

these models no longer reflects the 

relationship of these two variables in 

the underlying population; instead it 

resembles how laboratories typically 

perform method comparisons. Thirdly, 

regression is designed to predict the 

mean and is not optimal for the tails of 

the distribution, whereas the tails are 

important for estimating those at risk for 

deficiency or excess vitamin D intake. 

Fourthly, immunoassays are relatively 

nonspecific, often showing substantial 

positive and negative interferences, so 

the RIA data used for predicting LC–MS/

MS-equivalent concentrations were less 

than ideal. Retesting all of the samples 

was not possible logistically because 

of the number involved (greater than 

38,000). 

Assay standardization is an 

essential process in the development 

of evidence-based clinical and public 

health guidelines for the assessment of 

vitamin D deficiency. Using a laboratory 

method that is traceable to international 

reference materials, a study was designed 

to standardize historical 25OHD RIA 

data from NHANES to LC–MS/MSequivalents for 1988–2006. These 

predicted 25OHD data were compared 

with directly measured NHANES 

2007–2010 LC–MS/MS 25OHD (3). 

Together, these data provide new insight 

into the long-term vitamin D status of the 

U.S. population.

References

1. Yetley EA, Pfeiffer CM, Schleicher RL, 

Phinney KW, Lacher DA, Christakos 

S, et al. NHANES monitoring of serum 

25-hydroxyvitamin D: A roundtable 

summary. J Nutr 140(11):2030S–45S. 

2010.

2. National Center for Health Statistics. 

About the National Health and Nutrition 

Examination Survey. Available from: 

http://www.cdc.gov/nchs/nhanes/about_

nhanes.htm.

3. Schleicher RL, Sternberg MR, Lacher DA, 

Sempos CT, Looker AC, Durazo-Arvizu 

RA, et al. Temporal trends in the vitamin 

D status of the U.S. population from 

1988 to 2010 using standardized serum 

concentrations of 25-hydroxyvitamin D. 

2016 (unpublished report). 

4. Pfeiffer C. Laboratory procedure manual: 

25-hydroxyvitamin D in serum, NHANES 

2003–2004. Atlanta, GA: National Center 

for Environmental Health. Available from: 

http://www.cdc.gov/nchs/data/nhanes/

nhanes_03_04/l06vid_c_met_Vitamin_D.

pdf.

5. Schleicher RL, Encisco SE, ChaudharyWebb M, Paliakov E, McCoy LF, Pfeiffer 

CM. Isotope dilution ultra performance 

liquid chromatography–tandem mass 

spectrometry method for simultaneous 

measurement of 25-hydroxyvitamin D2, 

25-hydroxyvitamin D3 and 3-epi-25-

hydroxyvitamin D3 in human serum. Clin 

Chim Acta 412(17–18):1594–9. 2011.

6. Sempos CT, Vesper HW, Phinney KW, 

Thienpont LM, Coates PM. Vitamin 

D status as an international issue: 

National surveys and the problem of 

standardization. Scand J Clin Lab Invest 

243 Suppl:32–40. 2012.

Figure 5. Original radioimmunoassay, harmonized radioimmunoassay, and standardized 

LC-MS/MS serum 25-hydroxyvitamin D concentrations for persons aged 12 years and over, 

stratified by survey period: National Health and Nutrition Examination Survey, 1988–1994 

and 2001–2006

 
National Health Statistics Reports

 Number 93 April 25, 2016 Page 11

7. Tai SS, Bedner M, Phinney KW. 

Development of a candidate reference 

measurement procedure for the 

determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in 

human serum using isotope-dilution 

liquid chromatography–tandem mass 

spectrometry. Anal Chem 82(5):1942–8. 

2010.

8. Stepman HC, Vanderroost A, Van 

Uytfanghe K, Thienpont LM. Candidate 

reference measurement procedures 

for serum 25-hydroxyvitamin D

3 and 

25-hydroxyvitamin D

2 by using isotopedilution liquid chromatography–tandem 

mass spectrometry. Clin Chem 57(3):441–

8. 2011.

9. Stöckl D, Sluss PM, Thienpont LM. 

Specifications for trueness and precision 

of a reference measurement system for 

serum/plasma 25-hydroxyvitamin D 

analysis. Clin Chim Acta 408(1–2):8–13. 

2009.

10. Phinney KW, Bedner M, Tai SS, 

Vamathevan VV, Sander LC, Sharpless 

KE, et al. Development and certification 

of a standard reference material for 

vitamin D metabolites in human serum. 

Anal Chem 84(2):956–62. 2012.

11. Caudill SP, Schleicher RL, Pirkle JL. 

Multi-rule quality control for the agerelated eye disease study. Stat Med 

27(20):4094–106. 2008.

12. Encisco SE, Sternberg M, ChaudharyWebb M, Paliakov E, Schleicher RL. 

Effects of sample matrices, extreme 

storage temperatures, and repeated 

freeze/thaw cycles on integrity of 

25-hydroxyvitamin D. Clin Chem 

57(S10):A91. 2011.

13. National Center for Health Statistics. 

National Health and Nutrition 

Examination Survey: Analytical note for 

25-hydroxyvitamin D data analysis using 

NHANES III (1988–1994), NHANES 

2001–2006, and NHANES 2007–2010 

(October 2015). Available from: http://

wwwn.cdc.gov/Nchs/Nhanes/VitaminD/

AnalyticalNote.aspx?h=http://wwwn.

cdc.gov/Nchs/Nhanes/2009-2010/VID_F.

htm&t=VID_F%20Doc.

14. Cochran WG. Sampling techniques. 3rd 

ed. Hoboken, NJ: Wiley. 1977.

15. Schleicher RL, Looker AC, Yetley 

EA, Lacher DA, Sempos CT, Taylor 

CL, et al. Nationally representative 

estimates of serum 25-hydroxyvitamin 

D metabolite concentrations measured 

by liquid chromatography–tandem mass 

spectrometry in the U.S. population: 

National Health and Nutrition 

Examination Survey 2007–2010. J Nutr. 

In press 2016. 
Page 12 National Health Statistics Reports  Number 93  April 25, 2016

Table 1. Original radioimmunoassay, harmonized radioimmunoassay, and standardized LC-MS/MS serum 25-hydroxyvitamin D 

concentrations for persons aged 12 years and over, by demographic subgroup and survey period: National Health and Nutrition 

Examination Survey, 1988–1994 and 2001–2006

Group 1988–1994 2001–2002 2003–2004 2005–2006

Total1 nmol/L (confidence interval)

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.4 (72.7–76.2) 58.6 (56.7–60.6) 62.1 (58.5–65.6) 54.2 (51.8–56.7)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65.3 (63.8–66.8) 58.6 (56.7–60.6) 59.5 (56.7–62.2) 58.3 (55.4–61.1)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . 62.3 (61.1–63.5) 62.2 (60.4–64.1) 62.7 (59.3–66.2) 61.0 (58.6–63.4)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . . 60.6 (59.3–61.9) 63.0 (60.6–65.4) 62.4 (59.9–65.0) 60.6 (57.6–63.7)

Age (years)

12–19:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80.0 (76.6–83.5) 59.4 (57.1–61.7) 63.3 (58.7–67.8) 55.2 (51.6–58.8)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70.0 (67.1–73.0) 59.4 (57.1–61.7) 60.7 (56.7–64.7) 58.9 (55.1–62.8)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . 66.2 (64.1–68.4) 63.0 (60.8–65.2) 63.9 (59.4–68.4) 61.9 (58.5–65.4)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . 64.8 (62.2–67.4) 63.8 (61.1–66.6) 63.7 (59.8–67.7) 61.2 (57.1–65.3)

20–39:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77.6 (75.2–80.1) 59.2 (56.9–61.4) 62.3 (58.3–66.3) 55.8 (52.7–59.0)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68.0 (66.0–70.1) 59.2 (56.9–61.4) 59.6 (56.3–62.8) 59.8 (56.3–63.3)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . 64.4 (62.8–66.0) 62.8 (60.6–64.9) 62.9 (59.0–66.9) 62.5 (59.5–65.6)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . 63.0 (61.2–64.7) 63.4 (60.6–66.2) 62.7 (59.6–65.8) 62.1 (58.4–65.8)

40–59:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.1 (69.2–73.0) 58.8 (56.2–61.3) 61.5 (57.2–65.8) 53.3 (50.8–55.9)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.5 (60.9–64.1) 58.8 (56.2–61.3) 59.0 (55.7–62.3) 57.2 (54.2–60.2)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . 60.1 (58.7–61.5) 62.4 (59.9–64.8) 62.2 (58.0–66.4) 60.1 (57.7–62.6)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . 58.2 (56.8–59.6) 63.2 (60.4–66.0) 61.9 (58.7–65.0) 59.6 (56.3–62.9)

60 and over:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68.6 (67.3–70.0) 56.7 (54.1–59.3) 61.8 (59.3–64.4) 52.6 (50.1–55.2)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60.4 (59.3–61.5) 56.7 (54.1–59.3) 59.4 (57.3–61.5) 57.1 (54.4–59.8)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . 58.4 (57.4–59.5) 60.4 (58.0–62.9) 62.5 (60.0–65.0) 59.4 (57.0–61.9)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . 56.3 (55.3–57.3) 61.2 (58.2–64.2) 62.1 (60.4–63.8) 59.8 (56.8–62.7)

Sex

Male:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78.7 (76.7–80.6) 59.6 (57.6–61.7) 62.4 (58.7–66.1) 54.3 (52.1–56.5)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68.9 (67.2–70.6) 59.6 (57.6–61.7) 59.8 (56.8–62.8) 58.3 (55.6–61.0)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . 65.6 (64.3–66.9) 63.2 (61.3–65.1) 63.1 (59.4–66.7) 61.1 (58.9–63.2)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . 63.8 (62.3–65.2) 64.1 (61.6–66.6) 62.8 (60.0–65.6) 60.7 (57.7–63.8)

Female:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70.5 (68.6–72.4) 57.7 (55.3–60.0) 61.7 (58.3–65.1) 54.2 (51.3–57.1)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.0 (60.4–63.6) 57.7 (55.3–60.0) 59.2 (56.6–61.8) 58.2 (55.0–61.3)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . 59.2 (57.9–60.6) 61.3 (59.1–63.5) 62.4 (59.0–65.8) 60.9 (58.2–63.7)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . 57.7 (56.3–59.1) 62.0 (59.4–64.7) 62.1 (59.7–64.5) 60.5 (57.2–63.9)

Race and Hispanic origin

Mexican American:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.7 (61.8–65.6) 51.1 (47.8–54.4) 53.4 (49.9–57.0) 44.1 (40.0–48.2)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56.3 (54.7–57.8) 51.1 (47.8–54.4) 50.8 (47.9–53.8) 47.9 (43.3–52.6)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . 54.7 (53.3–56.2) 55.0 (51.9–58.2) 54.2 (50.7–57.7) 51.2 (47.2–55.1)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . 52.8 (51.5–54.1) 56.6 (52.9–60.3) 54.3 (51.0–57.6) 50.9 (46.1–55.8)

Non-Hispanic black:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49.2 (47.1–51.3) 34.6 (33.3–35.8) 39.9 (36.6–43.1) 34.3 (32.1–36.6)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.0 (42.3–45.8) 34.6 (33.3–35.8) 38.8 (35.7–41.8) 36.4 (34.0–38.8)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . 42.8 (41.1–44.6) 39.3 (38.2–40.5) 40.9 (37.7–44.1) 41.7 (39.5–43.9)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . 41.8 (40.0–43.5) 38.2 (36.2–40.2) 41.2 (38.3–44.0) 38.8 (35.9–41.8)

Non-Hispanic white:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80.3 (78.3–82.2) 64.0 (61.7–66.2) 67.8 (64.3–71.4) 59.6 (57.5–61.8)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70.2 (68.6–71.9) 64.0 (61.7–66.2) 65.0 (62.7–67.3) 64.0 (61.9–66.1)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . 66.7 (65.4–68.0) 67.3 (65.2–69.4) 68.4 (64.9–71.9) 66.2 (64.1–68.3)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . 64.9 (63.5–66.4) 68.3 (65.8–70.7) 67.9 (65.6–70.3) 66.3 (63.9–68.7)

1

“Other” race and Hispanic-origin group not shown but included in total estimates.

NOTES: Values are weighted arithmetic means (95% confidence interval) that are not age-standardized. LC–MS/MS is liquid chromatography–tandem mass spectrometry; nmol/L is nanomole per 

liter; and RIA is radioimmunoassay. RIA-original is original RIA results; RIA-harmonized is results adjusted to minimize reformulation and lot-to-lot variability in RIA kits. Regression Model 1 (using 

equations for each survey) and Model 2 (using equations for each calendar year in which RIA data were tested) were used to generate LC–MS/MS-standardized data. 
National Health Statistics Reports  Number 93  April 25, 2016 Page 13 Table 2. LC–MS/MS-equivalent tail percentiles for serum 25-hydroxyvitamin D for persons aged 12 years and over, by demographic subgroup and survey period: National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006 Group 5th percentile 95th percentile 1988–1994 2001–2002 2003–2004 2005–2006 1988–1994 2001–2002 2003–2004 2005–2006 Total1 nmol/L (confidence interval) RIA-original . . . . . . . . . . . . . . . . . . . . . 32.0 (30.8–33.1) 21.9 (20.4–23.6) 23.5 (20.2–26.2) 19.8 (18.4–21.2) 127 (124–133) 96.3 (92.7–103) 103 (97.7–112) 89.2 (86.5–93.0) RIA-harmonized . . . . . . . . . . . . . . . . . . 29.6 (28.6–30.5) 21.9 (20.4–23.6) 23.5 (20.1–25.9) 21.3 (19.5–23.0) 110 (107–114) 96.3 (92.7–103) 97.2 (92.9–104) 95.3 (91.8–99.3) LC–MS/MS-standardized (Model 1) . . 28.6 (27.6–29.5) 27.4 (25.8–28.9) 24.8 (21.6–27.5) 27.6 (26.2–29.0) 94.8 (93.6–96.2) 98.1 (94.7–104) 103 (97.8–112) 94.9 (92.3–98.6) LC–MS/MS-standardized (Model 2) . . 28.7 (27.3–30.0) 27.1 (24.6–28.3) 26.1 (23.6–28.5) 25.0 (22.2–27.6) 99.5 (96.7–104) 101 (98.0–107) 102 (98.0–107) 97.4 (94.0–102) Age (years) 12–19: RIA-original . . . . . . . . . . . . . . . . . . . 35.6 (32.2–39.3) 24.5 (20.8–27.2) 24.9 (21.2–28.0) 19.7 (17.4–21.8) 137 (129–152) 95.9 (91.7–102) 108 (98.5–123) 91.4 (84.8–102) RIA-harmonized . . . . . . . . . . . . . . . . 32.6 (29.7–35.7) 24.5 (20.8–27.2) 24.8 (21.0–27.3) 21.2 (17.6–23.9) 118 (112–131) 95.9 (91.7–102) 101 (93.9–112) 96.1 (88.5–111) LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . . . . . . 31.6 (28.7–34.7) 29.7 (26.3–32.3) 26.2 (22.5–29.2) 27.5 (25.2–29.5) 97.3 (95.3–102) 97.7 (93.8–104) 108 (98.5–123) 97.1 (90.6–107) LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . . . . . . . 31.7 (28.9–34.1) 28.3 (24.6–32.4) 26.9 (23.7–30.1) 25.0 (19.0–28.5) 107 (102–118) 101 (96.3–109) 104 (98.4–116) 97.8 (91.2–112) 20–39: RIA–original . . . . . . . . . . . . . . . . . . . 32.4 (30.1–33.7) 21.2 (19.4–23.1) 22.6 (19.2–25.6) 19.9 (18.4–21.5) 135 (126–140) 102 (94.6–110) 109 (99.5–119) 94.9 (91.4–100) RIA–harmonized . . . . . . . . . . . . . . . 29.9 (28.0–31.0) 21.2 (19.4–23.1) 22.5 (19.0–25.4) 21.1 (19.0–23.3) 116 (109–121) 102 (94.6–110) 104 (95.7–110) 99.0 (95.3–112) LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . . . . . . 28.9 (27.0–30.0) 26.7 (24.9–28.4) 23.9 (20.6–26.9) 27.7 (26.2–29.2) 96.8 (94.4–98.3) 103 (96.5–111) 109 (99.5–118) 100 (97–106) LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . . . . . . . 28.8 (26.8–30.5) 24.9 (22.3–27.8) 24.5 (21.4–27.6) 24.5 (20.2–28.3) 105 (99.3–109) 105 (101–113) 107 (101–115) 102 (97.4–113) 40–59: RIA–original 31.3 (29.5–32.6) 21.4 (18.7–24.4) 23.7 (18.8–27.4) 19.3 (17.1–21.2) 123 (117–126) 94.7 (90.4–104) 99.4 (92.3–115) 87.1 (84.9–91.3) RIA–harmonized . . . . . . . . . . . . . . . 28.9 (27.4–30.0) 21.4 (18.7–24.4) 23.6 (18.7–26.9) 20.8 (18.8–22.8) 106 (101–109) 94.7 (90.4–104) 94.3 (88.9–102) 93.9 (88.8–99.8) LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . . . . . . 27.9 (26.4–29.0) 26.9 (24.2–29.7) 25.0 (20.2–28.7) 27.1 (25.0–29.0) 93.4 (91.9–94.5) 96.6 (92.5–105) 99.5 (92.4–115) 92.9 (90.7–96.9) LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . . . . . . . 27.9 (26.9–29.1) 27.0 (22.2–28.8) 26.2 (21.3–29.5) 24.5 (22.5–27.1) 95.1 (92.7–99.2) 101 (95.2–107) 98.2 (93.2–107) 96.3 (91.1–102) 60 and over: RIA–original . . . . . . . . . . . . . . . . . . . 31.3 (29.8–32.6) 23.1 (21.1–24.4) 24.2 (21.8–26.1) 20.6 (18.6–22.7) 115 (113–119) 91.7 (88.3–94.8) 100 (95.1–106) 83.6 (79.9–89.3) RIA–harmonized . . . . . . . . . . . . . . . 29.0 (27.7–30.1) 23.1 (21.1–24.4) 24.2 (21.8–25.8) 22.3 (20.4–24.3) 99.3 (97.5–103) 91.7 (88.3–94.8) 93.9 (90.0–102) 90.3 (87.4–93.7) LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . . . . . . 28.0 (26.7–29.1) 28.4 (26.5–29.6) 25.5 (23.1–27.4) 28.4 (26.4–30.4) 91.2 (90.6–92.3) 93.7 (90.5–96.7) 100 (95.2–106) 89.4 (85.9–95.0) LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . . . . . . . 28.2 (26.7–29.4) 27.1 (24.6–29.8) 26.4 (23.8–28.6) 26.7 (23.0–29.7) 90.8 (89.1–92.8) 95.9 (92.3–101) 98.0 (94.7–105) 92.5 (89.3–96.1) Sex Male: RIA–original . . . . . . . . . . . . . . . . . . . 36.1 (34.6–37.9) 25.2 (22.8–26.6) 27.2 (22.8–29.9) 21.5 (19.4–23.4) 130 (126–136) 95.2 (91.1–102) 96.8 (92.4–104) 86.3 (83.0–90.8) RIA–harmonized . . . . . . . . . . . . . . . 33.0 (31.8–34.5) 25.2 (22.8–26.6) 26.5 (22.6–29.0) 23.2 (20.8–25.2) 112 (108–118) 95.2 (91.1–102) 91.7 (88.5–97.5) 92.6 (87.6–98.5) LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . . . . . . 32.0 (30.8–33.5) 30.4 (28.2–31.7) 28.5 (24.1–31.1) 29.2 (27.2–31.0) 95.5 (94.2–97.3) 97.1 (93.1–104) 96.9 (92.5–104) 92.1 (88.8–96.5) LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . . . . . . . 32.1 (30.7–33.4) 29.3 (27.3–31.5) 29.4 (25.6–32.0) 27.4 (24.2–29.2) 101 (98.3–106) 101 (95.8–108) 96.0 (93.0–101) 94.7 (89.6–101) Female: RIA-original . . . . . . . . . . . . . . . . . . . 29.4 (27.8–31.0) 20.0 (17.9–21.8) 20.8 (18.9–23.0) 18.7 (17.1–20.2) 124 (121–130) 98.3 (93.1–106) 110 (102–119) 92.4 (87.8–96.6) RIA-harmonized . . . . . . . . . . . . . . . . 27.3 (26.0–28.7) 20.0 (17.9–21.8) 20.7 (18.8–23.0) 20.1 (18.2–21.9) 107 (105–112) 98.3 (93.1–106) 104 (97.5–110) 96.8 (92.9–107) LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . . . . . . 26.3 (25.0–27.7) 25.5 (23.5–27.2) 22.1 (20.3–24.3) 26.5 (25.0–28.0) 93.9 (93.0–95.5) 100 (95.1–108) 110 (102–118) 98.0 (93.6–102) LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . . . . . . . 26.6 (25.1–27.9) 24.7 (21.5–27.2) 23.7 (21.2–25.3) 23.7 (20.7–26.7) 97.6 (94.4–102) 102 (98.3–111) 107 (103–114) 99.0 (94.7–109) See footnotes at end of table.
Page 14 National Health Statistics Reports  Number 93  April 25, 2016 Table 2. LC–MS/MS-equivalent tail percentiles for serum 25-hydroxyvitamin D for persons aged 12 years and over, by demographic subgroup and survey period: National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006—Con. Group 5th percentile 95th percentile 1988–1994 2001–2002 2003–2004 2005–2006 1988–1994 2001–2002 2003–2004 2005–2006 Race and Hispanic origin nmol/L (confidence interval) Mexican American: RIA-original . . . . . . . . . . . . . . . . . . . 30.3 (28.6–32.0) 22.2 (21.1–23.3) 21.8 (19.8–23.8) 17.9 (14.8–20.2) 104 (102–108) 81.8 (77.6–91.1) 85.0 (81.8–89.2) 70.4 (66.2–78.4) RIA-harmonized . . . . . . . . . . . . . . . . 28.1 (26.7–29.6) 22.2 (21.1–23.3) 21.8 (19.8–23.8) 19.4 (17.3–21.1) 90.6 (88.4–93.8) 81.8 (77.6–91.1) 80.2 (76.7–83.8) 76.3 (71.4–84.6) LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . . . . . . 27.1 (25.7–28.6) 27.6 (26.5–28.6) 23.1 (21.2–25.1) 25.7 (22.7–28.0) 88.3 (87.4–89.4) 84.3 (80.3–93.1) 85.3 (82.1–89.4) 76.7 (72.6–84.4) LC–MS/MS–standardized (Model 2) . . . . . . . . . . . . . . . . . . . . . 27.4 (25.2–28.9) 27.4 (24.8–29.8) 24.0 (21.6–26.1) 23.1 (18.7–26.2) 83.6 (81.0–86.1) 88.3 (83.8–97.8) 84.9 (81.7–88.7) 78.8 (73.4–113) Non-Hispanic black: RIA-original . . . . . . . . . . . . . . . . . . . 21.6 (20.1–22.7) 12.9 (9.28–14.0) 14.1 (12.6–15.4) 12.7 (11.6–13.3) 87.7 (85.5–90.9) 64.9 (62.3–67.5) 71.1 (65.5–79.0) 62.1 (58.9–69.6) RIA-harmonized . . . . . . . . . . . . . . . . 20.8 (19.5–21.7) 12.9 (9.28–14.0) 13.9 (11.5–15.3) 13.7 (12.8–14.5) 76.5 (74.7–79.2) 64.9 (62.3–67.5) 67.4 (63.6–73.7) 65.4 (62.3–70.4) LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . . . . . . 19.8 (18.5–20.7) 18.7 (15.3–19.8) 15.6 (14.1–16.8) 20.7 (19.6–21.3) 75.5 (73.7–78.2) 68.2 (65.7–70.7) 71.6 (66.1–79.4) 68.6 (65.5–75.9) LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . . . . . . . 20.2 (18.8–21.2) 16.8 (14.1–19.1) 16.2 (14.4–17.8) 15.9 (15.8–16.5) 70.6 (69.0–72.9) 68.8 (64.0–73.8) 71.7 (68.9–76.5) 68.5 (65.4–72.6) Non-Hispanic white: RIA-original . . . . . . . . . . . . . . . . . . . 38.1 (36.5–39.4) 29.3 (27.6–30.7) 30.4 (26.8–33.9) 27.0 (24.8–29.0) 133 (129–138) 101 (95.9–108) 110 (102–118) 93.5 (90.3–96.7) RIA-harmonized . . . . . . . . . . . . . . . . 34.7 (33.3–35.8) 29.3 (27.6–30.7) 29.6 (27.5–32.0) 30.5 (26.7–31.8) 115 (111–119) 101 (95.9–108) 103 (97.3–109) 98.4 (95.7–106) LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . . . . . . . . . 33.7 (32.3–34.8) 34.4 (32.7–35.7) 31.6 (28.1–35.0) 34.5 (32.4–36.5) 96.3 (95.1–97.7) 103 (97.7–109) 109 (102–117) 99.1 (96.0–102) LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . . . . . . . . . 33.5 (32.1–34.9) 33.5 (31.6–35.4) 32.4 (29.0–36.1) 33.3 (31.6–35.0) 104 (100–108) 106 (101–111) 107 (102–115) 101 (97.6–107)

1“Other” race and Hispanic origin group not shown but included in total estimates. 

NOTES: LC–MS/MS is liquid chromatography–tandem mass spectrometry; nmol/L is nanomole per liter; and RIA is radioimmunoassay. Values are weighted percentiles; data were standardized to LC–MS/MS-equivalents by using Model 1, for which regression equations were generated for each survey period (1988–1994, 2001–2002, 2003–2004, and 2005–2006), or Model 2, for which regression equations were generated for each calendar year in which RIA data were tested (1994, 1995, 1996, 2001, 2002, 2003, 2004, 2005, and 2006). SOURCE: CDC/NCHS, National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006.
National Health Statistics Reports  Number 93  April 25, 2016 Page 15

Table 3. Prevalence of serum 25-hydroxyvitamin D concentrations less than 30 nmol/L from original radioimmunoassay, harmonized 

radioimmunoassay, and standardized LC-MS/MS concentrations for persons aged 12 years and over, by demographic subgroup and survey 

period: National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006

Group 1988–1994 2001–2002 2003–2004 2005–2006

Total1 Percent (confidence interval)

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 (3.2–4.5) 9.0 (7.3–11) 7.5 (5.3–10) 12 (9.2–15)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 (4.5–6.1) 9.0 (7.3–11) 8.1 (5.9–11) 10 (7.8–13)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . . 6.0 (5.2–6.9) 5.4 (4.1–7.0) 7.5 (5.3–10) 5.2 (3.8–6.9)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . . 5.9 (4.9–7.1) 7.0 (5.2–9.4) 7.5 (5.3–10) 7.5 (5.4–10)

Age (years)

12–19: 

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 (2.0–4.0) 7.0 (4.4–11) 6.5 (4.2–9.8) 12 (8.1–17)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 (2.9–5.2) 7.0 (4.4–11) 7.0 (4.6–11) 10 (6.8–14)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . 4.2 (3.2–5.4) 4.1 (2.6–6.4) 6.5 (4.2–9.8) 5.3 (3.3–8.2)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . 4.2 (3.1–5.6) 5.7 (3.2–9.7) 6.5 (4.2–9.8) 7.8 (4.7–13)

20–39:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 (2.8–4.8) 9.5 (7.7–12) 8.2 (5.7–12) 12 (8.7–16)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 (3.9–6.4) 9.5 (7.7–12) 9.2 (6.6–13) 9.6 (6.9–13)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . 6.0 (5.0–7.2) 6.0 (4.6–7.7) 8.2 (5.7–12) 5.1 (3.7–7.1)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . 5.7 (4.3–7.4) 7.6 (5.6–10) 8.2 (5.7–12) 7.5 (5.0–11)

40–59:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 (3.4–5.1) 9.4 (7.2–12) 7.1 (4.5–11) 12 (9.2–16)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8 (4.9–6.8) 9.4 (7.2–12) 7.4 (4.8–11) 11 (8.3–14)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . 6.6 (5.6–7.9) 5.7 (4.0–8.1) 7.1 (4.5–11) 5.5 (3.9–7.8)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . 6.5 (5.4–7.8) 7.3 (5.3–10) 7.1 (4.5–11) 8.2 (6.0–11)

60 and over:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 (3.3–5.0) 8.8 (6.7–11) 7.6 (5.6–10) 11 (8.6–14)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7 (4.8–6.7) 8.8 (6.7–11) 8.3 (6.1–11) 9.3 (7.2–12)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . 6.4 (5.5–7.5) 4.4 (3.3–5.9) 7.6 (5.6–10) 4.6 (3.3–6.4)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . 6.6 (5.4–8.1) 6.2 (4.2–9.1) 7.6 (5.6–10) 6.3 (4.5–8.8)

Sex

Male:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 (1.9–2.7) 6.8 (5.4–8.5) 5.1 (3.2–8.1) 10 (7.5–13)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 (2.7–3.8) 6.8 (5.4–8.5) 5.7 (3.8–8.7) 8.6 (6.5–11)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . 3.7 (3.1–4.3) 3.8 (3.0–4.7) 5.1 (3.2–8.1) 4.1 (2.9–5.6)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . 3.6 (2.9–4.5) 5.1 (3.7–7.1) 5.1 (3.2–8.1) 6.2 (4.3–8.8)

Female:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 (4.2–6.3) 11 (8.8–14) 9.7 (7.2–13) 13 (10–17)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 (6.0–8.4) 11 (8.8–14) 10.4 (7.8–14) 12 (8.7–15)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . 8.2 (7.1–9.5) 6.9 (4.9–9.5) 9.7 (7.2–13) 6.2 (4.5–8.5)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . 8.0 (6.6–9.7) 8.8 (6.4–12) 9.7 (7.2–13) 8.8 (6.2–12)

Race and Hispanic origin

Mexican American:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 (3.7–5.8) 10 (7.7–13) 9.3 (6.5–13) 20 (14–27)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 (5.2–7.9) 10 (7.7–13) 11 (8.1–15) 17 (11–24)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . 7.3 (6.1–8.8) 5.3 (3.8–7.2) 9.3 (6.5–13) 7.7 (4.3–13)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . 7.5 (5.8–9.6) 6.2 (4.0–9.5) 9.3 (6.5–13) 11 (6.8–18)

Non-Hispanic black:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 (14–20) 42 (39–46) 30 (23–37) 43 (37–49)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 (19–26) 42 (39–46) 31 (24–38) 40 (35–46)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . 24 (21–28) 28 (25–33) 30 (23–37) 22 (18–27)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . 24 (20–29) 36 (30–42) 30 (23–37) 34 (27–42)

Non-Hispanic white:

RIA-original . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7 (1.3–2.1) 3.9 (3.0–5.1) 3.5 (2.5–4.9) 5.3 (3.8–7.3)

RIA-harmonized . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 (1.9–3.0) 3.9 (3.0–5.1) 3.8 (2.8–5.2) 4.3 (3.3–5.5)

LC–MS/MS-standardized (Model 1) . . . . . . . . . . . . 2.8 (2.4–3.5) 2.2 (1.6–2.9) 3.5 (2.5–4.9) 2.1 (1.6–2.8)

LC–MS/MS-standardized (Model 2) . . . . . . . . . . . . 2.8 (2.2–3.6) 2.8 (2.1–3.8) 3.5 (2.5–4.9) 2.8 (2.1–3.6)

1

“Other” race and Hispanic-origin group not shown but included in total estimates. 

NOTES: LC–MS/MS is liquid chromatography–tandem mass spectrometry; nmol/L is nanomole per liter; and RIA is radioimmunoassay. Values are weighted prevalences (95% confidence interval) 

that were not age-standardized. RIA-original is original RIA results; RIA-harmonized is results adjusted to minimize reformulation and lot-to-lot variability in RIA kits. Regression Model 1 (based on 

survey period) and Model 2 (based on the year in which the original RIA data were generated) were used to generate LC–MS/MS-standardized data. 

SOURCE: CDC/NCHS, National Health and Nutrition Examination Survey, 1988–1994 and 2001–2006.
National Health Statistics Reports  Number 93  April 25, 2016

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Suggested citation

Schleicher RL, Sternberg MR, Lacher 

DA, et. al. A method-bridging study for 

serum 25-hydroxyvitamin D to standardize 

historical radioimmunoassay data to liquid 

chromatography–tandem mass spectrometry. 

National health statistics reports; no 93. 

Hyattsville, MD: National Center for Health 

Statistics. 2016.

Copyright information

All material appearing in this report is in the 

public domain and may be reproduced or 

copied without permission; citation as to 

source, however, is appreciated.

National Center for Health Statistics

Charles J. Rothwell, M.S., M.B.A., Director

Nathaniel Schenker, Ph.D., Deputy Director

Jennifer H. Madans, Ph.D., Associate Director 

for Science

Division of Health Care Statistics

Kathryn S. Porter, M.D., M.S., Director

Ryne Paulose-Ram, Ph.D., Associate Director 

for Science

Acknowledgments 

The authors gratefully acknowledge the contribution of laboratory members Sara Encisco and Paul Yoon of CDC’s National Center for 

Environmental Health. We also thank Dr. Paul Coates, NIH Office of Dietary Supplements, for his valuable scientific input and support.