VDSCP: Reference Method Laboratory

The Vitamin D Reference Method Laboratory uses a unique, highly accurate and precise reference method. The method provides reference measurements for total 25-hydroxyvitamin D (sum of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) in serum.

CDC’s reference method for total 25-hydroxyvitamin D uses high-performance liquid chromatography coupled with tandem mass spectrometry. It separately quantitates 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The method has a high level of specificity and is not affected by other vitamin D isomers, such as C3-epi-25-hydroxyvitamin D3.

This reference method meets stringent analytical performance criteria (maximum allowable bias: ≤1.7%; maximum allowable imprecision: ±5%). This ensures that measurement results are highly certain and can be used to assess the measurement accuracy of clinical vitamin D tests.

The reference method is calibrated using certified reference material (SRM 2972a from the National Institute of Standards and Technologyexternal icon [NIST] ). Therefore, measurement results obtained with this reference method are traceable to the International System of Unitsexternal icon (SI) , in accordance with the International Organization for Standardization (ISO) standard for traceability in laboratory medicine.

CDC’s vitamin D reference method2 provides highly accurate measurements of total 25-hydroxyvitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3). The Joint Committee for Traceability in Laboratory Medicine (JCTLM) databaseexternal icon lists the method as a higher-order reference measurement procedure. As such , the reference method can be used to ascertain the accuracy of commercial assays and laboratory-developed tests.

This vitamin D reference method is comparable to other reference methods, such as those used by NIST and the University of Ghent.


2Mineva EM, Schleicher RL, Chaudhary-Webb M, Maw KL, Botelho JC, Vesper HW, Pfeiffer CM. 2015. A candidate reference measurement procedure for quantifying serum concentrations of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 using isotope-dilution liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 407(19):5615 –24.

SRM 2972aexternal icon consists of two separate solutions of the vitamin D metabolites 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in ethanol. It is intended primarily for use in calibration of instruments and techniques to test for these metabolites. These solutions are prepared with a high level of accuracy. The purity of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 used to prepare these solutions is well defined. The manufacturing of the solutions is highly controlled to ensure consistency among vials and stability of the material.

Traceability in laboratory medicine describes a process used to link a patient’s results to a reference material or reference method. For vitamin D, SRM 2972a is used to calibrate a reference method. The reference method assigns reference values to serum samples. These serum samples are then used to calibrate a routine vitamin D test used to measure patient samples. In this process, measurement results for patient samples are traceable to SRM 2972a.

Establishing traceability in laboratory medicine is important because it defines the point of reference to which patient measurements are linked. This helps to ensure measurements in patient care are comparable.

The Joint Committee on Traceability in Laboratory Medicineexternal icon (JCTLM) was created in 2002 through a declaration of cooperation between the International Bureau of Weights and Measures, the International Federation for Clinical Chemistry and Laboratory Medicine, and the International Laboratory Accreditation Cooperation. One of its main objectives is to promote traceability to appropriate measurement standards in ISO documents 17511, 17025, and 18153. JCTLM maintains a database with reference methods that meet the criteria mentioned in these ISO standards.

Page last reviewed: July 6, 2017