American Association for Clinical Chemistry
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December 2009 Clinical Laboratory News: Vitamin D Testing

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December 2009: Volume 35, Number 12

Vitamin D Testing
How Will We Get it Right?
By Rosemary L. Schleicher, PhD, and Christine M. Pfeiffer, PhD

During the last 10 years, researchers have made a number of exciting discoveries about vitamin D. The prohormone is thought to play a role in a host of conditions, including certain cancers, type 1 diabetes, multiple sclerosis, tuberculosis, Alzheimer’s disease, psoriasis, and all-cause mortality. With all the media attention on vitamin D’s purported superpowers, the demand for vitamin D testing has soared and physicians and patients are now paying serious attention to vitamin D status.

Most people are aware that vitamin D can be obtained by exposing skin to sunlight, eating certain foods, or taking vitamin supplements. However, vitamin D itself is biologically inert and must undergo two hydroxylation reactions to be activated. The first step is catalyzed by a liver enzyme, producing 25-hydroxyvitamin D (25(OH)D), a longer-lived metabolite that is a suitable marker of vitamin D status (1). For the past three decades, clinicians have relied primarily on immunoassay measurements of this metabolite to determine a patient’s vitamin D status.

Today, new technology has emerged for measuring vitamin D status, including high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (LC-MS/MS). Our lab at the Centers for Disease Control and Prevention (CDC) has measured 25(OH)D for the National Health and Nutrition Examination Surveys from NHANES III (1988–1994) through NHANES 2005–2006. Here we present our experience with the analytical quality of a commercially available vitamin D immunoassay and a newer analytical method developed at CDC, as well as a description of our experience with a newly available standard reference material.

History of Vitamin D Assays

The first method for measuring 25(OH)D was a competitive protein-binding assay that used a rat vitamin D binding protein and 3H-labeled 25(OH)D3 (2). This assay was subsequently replaced with a simpler radioimmunoassay (RIA) using 3H and later 125I-labeled 25(OH)D (3). For nearly as long as competitive binding assays have been used in clinical labs, research scientists have used chemistry-based methods such as HPLC with UV detection or gas or liquid chromatography coupled to mass spectrometry (MS) to measure or confirm serum concentrations of 25(OH)D (4–6).

Chromatographic methods are less susceptible to sample matrix effects than immunoassays (7). In particular, LC-MS/MS first resolves compounds chromatographically and then detects them at specific masses. This methodology provides a high probability that a molecule of interest will be correctly identified and quantitated because it is detected as a specific transition from one mass fragment to another. In other words, it is a highly specific method. Furthermore, in isotope dilution LC-MS/MS, a stable isotope-labeled analog of the compound of interest is added during the first step of sample preparation and carried throughout the assay, correcting for any potential analyte loss and behaving virtually identically to the compound of interest during chromatography and detection.

In contrast, specificity is less assured when analytes are measured using immunoassays that rely upon binding to an antibody. Cross-reactivity with nonspecific compounds is a well known issue in immunoassays. Even though extensive cross-reactivity testing may be performed for validation of immunoassays, it is never exhaustive.

Based on data from the largest survey of its kind, Vitamin D External Quality Assessment Scheme (DEQAS,, most participating labs use immunoassays to measure 25(OH)D (Figure 1, above). While the number of DEQAS participants has nearly doubled in the past 2 years, the number using LC-MS/MS has remained at 9%–10%. The four most commonly used tests in the summer 2009 DEQAS exercise were DiaSorin Liaison Total (36%), IDS enzyme-linked immunoassay (19%), automated IDS enzyme-linked immunoassay (11%), and LC-MS/MS (10%) (Figure 2, below).

NHANES Studies with a Commercial 25(OH)D Assay

The Nutrition Laboratory at CDC has measured 25(OH)D in more than 60,000 NHANES specimens using the DiaSorin RIA. Typically, CDC uses chromatography-based analytical methods to measure nutritional indicators; therefore, the use of an RIA to assess vitamin D status has been an exception to the rule. Generally, unless assay features such as accuracy, precision, or specificity are in doubt, CDC chemists and epidemiologists are reluctant to change assay methods because each change complicates the assessment of long-term population trends for NHANES and requires extensive cross-over studies to relate the new method to the old.

In 2004, however, the CDC laboratory began to develop an LC-MS/MS method for measuring 25(OH)D. CDC pursued this initiative because we found that the 25(OH)D immunoassay lacked the precision necessary for our studies. Furthermore, in the late 1990s, the manufacturer made changes to the assay that affected performance and therefore the results of our long-term study. In the reformulated assay, DiaSorin incorporated a higher affinity antibody that improved precision and sensitivity by reducing non-specific binding.

As shown in later studies, the reformulated DiaSorin assay used during NHANES 2000–2006 measured approximately 12% lower than the original assay used for NHANES 1988–1994 (8, 9). One explanation for this result is that the higher specificity of the new antibody measured fewer cross reactants. Furthermore, we observed certain shifts in QC values as a result of reagent lot variation, complicating interpretation of time trend data for NHANES (10). Understandably, from time-to-time manufacturers need to change lot numbers for various components of their kits such as calibrators, tracers, or antibodies. These changes should not cause systematic shifts. However, we noticed several times during analyses of the NHANES 2000–2006 specimens that the assay performed predominantly on one side or the other of the mean for a certain period.

Based on an 11.3% intra-individual coefficient of variation (CV) for 25(OH)D (11), quality goals for analytical imprecision, should be: 2.8% (optimal); 5.6% (desirable); and 8.5% (minimal) (12). Because manufacturer QC materials are characterized by wide QC limits (18%–20%), we generated and characterized large batches of in-house QC materials that lasted for several years and produced narrower QC limits (9%–14%). But with the assay shifts noted above, the overall CV for 25(OH)D for NHANES 2000–2006 was 13%–15%. This amount of imprecision does not meet the minimal quality goal for good precision; therefore, for our long-term study of vitamin D status, we decided to pursue an alternative analytical method.

Developing an LC-MS/MS Assay

Developing an assay for 25(OH)D was not a straightforward task for the CDC laboratory. The method of Vogeser et al. (13) was a convenient starting point because we had access to a MicroMass Quattro LC TMS system as used in this candidate reference method. But from the beginning, we were unable to confirm that mass to charge ratio (m/z) 159 is the major product ion for 25(OH)D3. Instead, we found m/z 383 to be the major product ion, which represents loss of water as shown by others (14, 15). In addition, we wanted to measure 25(OH)D2, which was not addressed by Vogeser et al. (13). As we explored various aspects of the method while optimizing the instrument, we noted that the matrix in which the calibrators were prepared had a major impact on detector response, a finding generally not reported in other publications as an analytical issue (16).

For comparison purposes, we prepared calibrators in 85% methanol, 4% albumin in PBS, or serum. Over the range of 10–100 ng/mL, the signal intensities for both analytes in 85% methanol were about 40% lower than those in serum and 15% lower than those in 4% albumin. The lowest concentration calibrators for 25(OH)D2 and 25(OH)D3 were not detectable in 85% methanol. Although the internal standard compensated for the differences among different matrices, lower responses would be expected to decrease assay precision and accuracy, particularly at low analyte concentrations. Calibration solutions prepared in a serum-like matrix would be optimal, but the use of serum or plasma containing endogenous 25(OH)D requires a calibration correction that adds error to the calibration curve. We therefore opted to use 4% albumin in PBS to provide a background-free protein matrix.

Throughout our calibration efforts, the hydrophobic nature of 25(OH)D was a concern. We were worried about adsorption of the analyte to the walls of tubes and therefore designed an experiment in which we looked at repeated transfer of solutions into fresh containers (16). Contrary to our expectation, we found a positive interference at m/z 383 for 25(OH)D3 when a certain brand of polypropylene container was used. The interference nearly doubled the signal in the medium QC pool. Adsorptive losses were nil.

After several years of work, we finally settled on a chromatographic separation method that we believed would help ensure consistent results for the NHANES analyses (16).

Although this method is less than ideal, it does have improved precision compared with the DiaSorin RIA (CV ≤11% and ≤16% for 25(OH)D3 and 25(OH)D2, respectively).

Further Improvements

Although most specimens gave reproducible results using our first LC-MS/MS method, low concentrations of 25(OH)D2 in some specimens were not reproducibly measurable due to a baseline interference peak eluting in the proximity of 25(OH)D2. In an effort to further improve the assay, we set out to obtain better chromatographic resolution along with automated sample processing (Table 1, below). We recently concluded validation of a new automated method in which the chromatographic separation of 25(OH)D2 and 25(OH)D3 from interfering peaks is optimal, yet quick (10 minutes). The method uses a 96-well plate format and can easily handle 80 patient samples per day by using a robot to facilitate reproducible pipetting. Other improvements include testing smaller volumes of serum (100 μL) and including isotopically labeled 25(OH)D2 as a second internal standard. Table 2 (below) lists the performance characteristics of this assay method.

Table 1
Features of LC-MS/MS Method for 25(OH)D2 and 25(OH)D3


96-well plate


Thermo Quantum Ultra

25(OH)D2 & 25(OH)D3 calibration ranges

6–125 and 13–250 nmol/L

Internal standards (deuterium-labeled)

D6-25(OH)D3 & D3-25(OH)D2

Sample preparation

Hamilton Starlet robot

Serum volume

100 μL

Mobile phase

Gradient – methanol/water

Run time

10 minutes


80 patient samples per day

Improvements include better chromatographic resolution, automated sample processing, smaller volume of serum per test, and addition of a second internal standard, isotopically labeled 25(OH)D2.

Table 2
Performance Characteristics of LC-MS/MS Method for Measuring 25(OH)D


25(OH)D2 separately quantitated from 25(OH)D3, but 25(OH)D3 not separated from 3-epi-25(OH)D3

(using NIST SRM 972)

25(OH)D3 (+1% to +4%);
25(OH)D2 (–8% to +30%)

Recovery (± SD)

25(OH)D3 (95% ± 3%); 25(OH)D2 (97% ± 3%)

Precision (CV)

25(OH)D3 (5%–8%); 25(OH)D2 (6%–10% at ≥7.5 nmol/L)


25(OH)D3 (<1 nmol/L); 25(OH)D2 (<1 nmol/L)

However, this is not the end of the story. When we set out to develop an LC-MS/MS method for monitoring 25(OH)D2 and 25(OH)D3 in NHANES, separating and measuring the C3-epimer of 25(OH)D3 was not an objective because this compound was reported to be present only in infants less than 1 year old (14), an age group not monitored for 25(OH)D in NHANES. More recently, however, the National Institute of Standards and Technology (NIST) confirmed the presence of the epimer in serum materials from adults using their LC-MS/MS reference method. This epimer, not captured by the DiaSorin RIA (14, 17), adds bias to conventional chromatographic assays because it is not chromatographically resolved from 25(OH)D3 using the typical C18 HPLC columns.

Cyanopropyl-bonded HPLC columns and longer run times may be necessary to ensure a baseline separation of the epimer from 25(OH)D3 (18). To achieve bias-free monitoring of the vitamin D status of the U.S. population, we revised our LC-MS/MS method to separate the epimer from 25(OH)D3 and are now in the process of validating the method.

Vitamin D Reference Materials: NIST’s Role

Labs also need a reference material to ensure accurate 25(OH)D assessments. In 2005, the National Institutes of Health, Office of Dietary Supplements contracted with NIST to prepare matrix-based standard reference materials (SRM) for 25(OH)D (19). SRM 972 was released in July 2009 and is now available to labs. Only one of the materials is native serum (level 1). The others are either diluted with horse serum (level 2), or spiked with 25(OH)D2 (level 3) or 3-epi-25(OH)D3 (level 4).

The CDC Nutrition Laboratory worked with NIST to measure 25(OH)D in these materials using our automated LC-MS/MS method before a second internal standard (D3-25(OH)D2) was added to the procedure. To test the materials, we prepared duplicate preparations per vial, two vials per day over 4 days. Our LC-MS/MS values were 1%–4% higher than NIST values for 25(OH)D3 using their LC-MS/MS reference method procedure, which has an analytical coefficient of variation of 2%–3%. The agreement between CDC and NIST values for 25(OH)D2 was good for levels 3 and 4—6% and 11% higher, respectively—but worse for levels 1 and 2—8% lower and 30% higher, respectively—where 25(OH)D2 values were low (<5 nmol/L). NIST has incorporated the CDC LC-MS/MS data into the certificate of analysis for SRM 972 (see the NIST website).

Relating Immunoassay Values to LC/MS-MS Values

The long history of working with the DiaSorin RIA and NHANES samples prompted us to investigate the relationship of those values to the LC-MS/MS method. Therefore, we also characterized 25(OH)D values in SRM 972 with the DiaSorin RIA using the same protocol (duplicate measurements using eight vials over 4 days) for levels 1, 2, and 3; level 4 was not available at the time (Table 3). Levels 1 and 2 were within 12% of the expected values when 25(OH)D2 and 25(OH)D3 were summed together. Level 3, which was spiked with 25(OH)D2, showed about two-thirds of the target value, possibly because the vitamin D metabolite was spiked.

Table 3
Method Comparison with SRM 972


NIST certificate of analysis values from LC-MS/MS (nmol/L)

CDC DiaSorin RIA (nmol/L)



Total 25(OH)D

Total 25(OH)D


59.6 ± 2.1

1.46 ± 0.49


60.5 ± 4.9


30.8 ± 1.5

4.14 ± 0.19


39.0 ± 4.5


46.2 ± 2.8

64.1 ± 4.8


72.4 ± 8.0

Levels 1, 2, and 3 using the DiaSorin RIA compared with NIST-certified (blue) or reference (green ) values. Means ± U95 (expanded uncertainty) for NIST data and mean ± SD for CDC data are displayed.

These data raise the issue of commutability of the reference materials, defined as the equivalence of the mathematical relationships between different measurement procedures for a reference material and for representative unaltered samples from healthy and diseased individuals (20). Commutability is assay-specific and therefore not all levels of SRM 972 are likely or need to be commutable for every assay. Formal studies using the different methods are underway to measure 25(OH)D in SRM 972 to identify which levels are commutable for which assay methods.

Getting It Right

The ultimate goal of the CDC Nutrition Laboratory is to obtain accurate 25(OH)D values. Consequently, precise methods and traceable reference materials are essential to establishing an individual’s vitamin D status. The availability of standard reference materials is a key element of getting vitamin D assessments right.

How will we use SRM materials to improve 25(OH)D testing? The CDC Nutrition Laboratory will incorporate these materials into value assignment of daily calibration materials and into calibration verification procedures performed at least semiannually. When preparing in-house calibration materials, we will use SRM 972 to assign values to the new lot.

NIST also plans to release SRM 2972, a set of two materials containing 25(OH)D2 or 25(OH)D3 at known concentrations diluted in solvent. These materials are suitable for further dilution to use in linearity testing and for calibration verification. Ultimately, the availability of these tools will allow the CDC Nutrition Laboratory to be able to produce reliable estimates of vitamin D levels in the U.S. population.

For clinical labs, using standard reference materials and regularly participating in a proficiency testing program are essential to keeping laboratory assays on target.


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Rosemary L. Schleicher, PhD, is a research chemist in the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Ga.



Christine M. Pfeiffer, PhD, is branch chief of the Nutritional Biomarkers Branch in the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Ga.



Acknowledgement: The authors wish to acknowledge Mary Frances Picciano from the National Institutes of Health Office of Dietary Supplements for supporting the development of standard reference materials for 25(OH)D testing and Clifford L. Johnson from the CDC, National Center for Health Statistics for continued support to monitor 25(OH)D levels in NHANES. Drs. Les McCoy and Huiping Chen and Mses. Madhu Chaudhary-Webb and Donna LaVoie are key personnel in the CDC Nutrition Laboratory responsible for much of the work described in this article.

DISCLOSURE STATEMENT: The authors have nothing to disclose.