CCQM-K132: low-polarity analytes in a biological matrix: vitamin D metabolites in human serum
Authors: Wise Stephen A , Tai Susan S -C , Duewer David L , Bedner Mary , Camara Johanna E , Lippa Katrice A , Qinde Liu , Kang Dukjin , Kim Byungjoo , Quan Can , Shi Lianhua , Nammoonnoy Jintana , Vamathevan Veronica , Gören Ahmet Ceyhan , Bilsel khan , Yilmaz Hasibe 
Published: August 2017

Vitamin D is a fat-soluble vitamin that occurs primarily in two forms, vitamin D2 and vitamin D3. Vitamin D3 is produced naturally when skin is exposed to UV radiation, is naturally-occurring in foods (generally of animal origin), and is fortified in some foods and dietary supplements. Vitamin D2 occurs in food (generally plant sources) and until recently was the form most often used in dietary supplements. Vitamin D is metabolized in the body to produce several closely related, hydroxylated species (metabolites), with 25-hydroxyvitamin D3 [25(OH)D3] and 25-hydroxyvitamin D2 [25(OH)D2] as the most common metabolites measured in human serum. Concentrations of total vitamin D in human serum, calculated as the sum of 25(OH)D2 and 25(OH)D3, are typically in the 16 ng/g to 30 ng/g (40 nmol/L to 75 nmol/L) range, with 25(OH)D3 usually accounting for more than 90 % of the total. An epimer of 25(OH)D3, 3-epi-25(OH)D3, can be present at levels up to 10 % of 25(OH)D3 concentration.
Seven National Metrology Institutions participated in the Track C Key Comparison CCQM-K132 low-polarity analytes in a biological matrix: vitamin D metabolites in human serum. Participants were requested to evaluate the mass fractions, expressed in ng/g, of 25(OH)D3, 25(OH)D2, and 3-epi-25(OH)D3 in two human serum materials, termed Serum Pool I and Serum Pool II. Due to the known low levels of 3-epi-25(OH)D3 in both materials and the very low level of 25(OH)D2 in Serum Pool I, the study protocol stated that key comparison reference values (KCRVs) would be assigned only to 25(OH)D3 in both materials and 25(OH)D2 in Serum Pool II. Results for 3-epi-25(OH)D3 were requested to evaluate the separation technologies employed; 3-epi-25(OH)D3 needs to be chromatographically separated from 25(OH)D3 for proper quantification of 25(OH)D3. Results for 25(OH)D2 in Serum Pool I were requested to explore measurement performance at its low level. All participants used isotope dilution liquid chromatography with tandem mass spectrometry detection (ID LC-MS/MS) for the measurement of the vitamin D metabolites.
Successful participation in CCQM K132 demonstrates capabilities in analysis of low molecular mass (100 g/mol to 500 g/mol) and low-polarity (nonpolar, pKow < −2) analytes at the 1 ng/g to 500 ng/g mass fraction range in complex biological matrixes with core competencies for sample preparation and analysis using ID LC-MS/MS. This study extends the mass fraction capability range to 105 to 106 times lower than that demonstrated in previous CCQM Key Comparisons for cholesterol in serum, another nonpolar clinical analyte.

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To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/.
The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).