This post is part of a series on bone markers standardization from the Committee on Bone Metabolism of the International Federation of Clinical Chemistry.;

Requests to assess vitamin D status by measuring 1α,25(OH)2D (or calcitriol) are still reaching medical laboratories on a daily basis. Although 1α,25(OH)2D is the biologically active form of vitamin D, the circulating concentration is tightly regulated by the enzyme 1α-hydroxylase and provides little information on the supply with its direct precursor 25(OH)D. Measurement of 1α,25(OH)2D is only recommended in the following circumstances (1):

  1. disorders of 1α-hydroxylation (like in type 1 vitamin D dependent rickets or hypophosphatemic rickets) where serum levels are expected to decrease,
  2. disorders of the vitamin D receptor (like in type 2 vitamin D dependent rickets) where serum levels are increased,
  3. extrarenal hydroxylation (like in sarcoidosis, tuberculosis, rheumatoid arthritis, inflammatory bowel disease and lymphoproliferative disease) where serum levels are also expected to increase.

Of note, chronic kidney disease (CKD) is a disease where renal 1α-hydroxylation is decreased, but monitoring calcitriol levels in CKD patients is not recommended (2). Accurate measurement of calcitriol is challenging. Indeed, it circulates in picomolar concentrations the blood, has a very short half-life (approximately 4-6 hours), and is very lipophilic. Different methods are available on the market. The older ones are manual or semi-automated RIA or ELISA assays that use polyclonal sheep anti-1α,25(ΟΗ)2D, an extraction with organic solvents and separation by chromatography. Some assays also use immunoaffinity to isolate 1α,25(ΟΗ)2D from the sample before quantification by RIA or ELISA. In recent years, several fully automated and semi-automated methods that include a manual extraction step appeared on the market. Fully automated methods represent a major improvement as they allow the easy measurement of 1α,25(ΟΗ)2D on widely available standard analyzers with increased sensitivity and specificity.

Liquid chromatography coupled with mass spectrometry (LC-MS/MS) methods have also been developed to quantify 1α,25(ΟΗ)2D. Such methods provide superior sensitivity and specificity and allow the simultaneous quantification of other vitamin D metabolites (3). However, measuring calcitriol by LC-MS/MS is far from being an easy task. Indeed, ionization of the molecule is very low and there is a risk of interference from other metabolites, such as isobars, which possess the same molecular weight but differ in the position of hydroxylation (e.g. 24,25(OH)2D), epimers (1α,25(ΟΗ)2-3epi-D and 24,25(OH)2-3epi-D) (4) and enantiomers (1β,25(ΟΗ)2D) (5) can also impact the quantitation of 1α,25(ΟΗ)2D by LC-MS/MS.

In contrast to 25(OH)D and 24,25(OH)2D, universally accepted reference methods and standard reference materials for 1α,25(ΟΗ)2D are still lacking. Consequently, significant differences between labs and between methods exist, which limits the comparability of results and hampers the implementation of universal reference intervals. Therefore, the standardization of 1α,25(ΟΗ)2D measurement is one of the priorities of the IFCC C-BM Committee.


  1. Makris K, Sempos C, Cavalier E. The measurement of vitamin D metabolites: part I—metabolism of vitamin D and the measurement of 25-hydroxyvitamin D. Hormones. Hormones; 2020;
  2. KDIGO 2017 Clinical Practice Guideline Update for the Diagnosis, Evaluation, Prevention, and Treatment of Chronic Kidney Disease–Mineral and Bone Disorder (CKD-MBD). Kidney Int Suppl. 2017;7:1–59.
  3. Bikle DD. Vitamin D Assays. Front Horm Res [Internet]. S. Karger AG; 2018 [cited 2020 Dec 15];50:14–30. Available from:
  4. Kamao M, Tatematsu S, Reddy GS, Hatakeyama S, Sugiura M, Ohashi N, et al. Isolation, identification and biological activity of 24R,25-dihydroxy-3-epi-vitamin D3: a novel metabolite of 24R,25-dihydroxyvitamin D3 produced in rat osteosarcoma cells (UMR 106). J Nutr Sci Vitaminol (Tokyo). 2001;47:108–15.
  5. Pauwels S, Jans I, Billen J, Heijboer A, Verstuyf A, Carmeliet G, et al. 1β,25-Dihydroxyvitamin D3: A new vitamin D metabolite in human serum. J Steroid Biochem Mol Biol [Internet]. Elsevier Ltd; 2017 [cited 2020 Dec 15];173:341–8. Available from: