Quantification of serum 25-hydroxyvitamin D: a comparison among immunoassay, HPLC-UV, and HPLC-MS
A. Saba1, R. Bozic2, G. Viccica1, M. Maccheroni3, L. Cianferotti1, G. Chiellini1, A. Raffaelli4, R. Zucchi1 & C. Marcocci1
Vitamin D deficiency is widespread among all age and ethnic groups. Serum 25-hydroxyvitamin D [25(OH)D] is the most reliable marker of vitamin D status. Adequate levels of serum 25(OH)D are necessary to sustain the pleiotropic effects of vitamin D, either skeletal (classical) or extra-skeletal (non-classical). Concentration levels ≧50 nmol/L (20 ng/ml) are required for optimal musculoskeletal health. However, levels above 75 nmol/L (30 ng/ml) may be necessary to maximize musculoskeletal benefits and take advantage of the extra-skeletal effects of vitamin D. Traditional assays based on immunoassay display a substantial intra- and inter-assay variability (up to 30%) around the cut off limits, which may cause misclassification of individual vitamin D status. Conversely, Mass Spectrometry coupled to High Performances Liquid Chromatography (HPLC-MS) offers a high quantification accuracy. This is mainly due to its high selectivity, which reduces the contribution of interfering compounds to the final results.
Herein we report the results of measuring serum 25OHD using three different kits from DiaSorin (Liaison), Eureka (HPLC-UV), and PerkinElmer (HPLC-MS). Due to the features of MS, PerkinElmer method/kit was considered as the gold standard. It involves a quick sample preparation, consisting just in the addition of stable-isotope-labeled internal standards (ISs) and protein precipitation. Quantification was performed by a tandem quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source, in positive ion mode and multiple reaction monitoring (MRM). Specific ISs allowed to quantify separately 25(OH)D2 and 25(OH)D3.
The analysis of about 200 blood samples, either from healthy volunteers (controls) or patients with endocrine pathologies, showed the real advantages of LC-MS/MS with respect to the other methods, consisting of a better accuracy due to the absence of cross-reactivity and the possibility of separately detect 25(OH)D3 and 25(OH)D2. The comparison results on real sample will be reported.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.