Vitamin D is a fat-soluble steroid vitamin, of which 25-(OH)-vitamin D, the major circulating form produced in the liver, is being further metabolized in the proximal tubule of the kidney to 1α,25-dihydroxyvitamin D (calcitriol), which is the most biologically active form of vitamin D. The influence of 1,25(OH)2-vitamin D on calcium and phosphate metabolism, as well as bone metabolism, is very well researched. The innate immune system is stimulated and the risk of upper respiratory infections is decreased by 1,25(OH)2-vitamin D2/3, however severe vitamin D deficiency is the leading cause of nutritional rickets. Poor vitamin D status is strongly linked to colon cancer and dialyses patients are monitored to appraise their kidney function. Robust routine measurements of 1,25(OH)2-vitamin D2/3 by mass spectrometry can be challenging due to other isobar vitamin D species, low circulating blood concentration of the targeted metabolites (5-100 pg/mL) as well as poor ionization. Moreover, currently no commercial solution is offered combining an easy to use sample preparation and derivatization in one assay.
The 1,25-(OH)2-vitamin D2/D3 IDKenhance® LC-MS/MS kit is for the LC-MS/MS based quantification of 1,25-(OH)2-vitamin D2/D3 from 500 µl of serum and plasma. Samples were mixed with two isotopically labelled internal standards and worked up by using immunoaffinity enrichment with single filter tubes (ImmuTube®) or 96-well filter plates (ImmuPlate®). After evaporation of the eluate a fast derivatization step was applied, followed by a quick second evaporation. Thereafter, samples were reconstituted and injected into the LC-MS/MS system with a total cycle time 4.5 min. LC-MS/MS systems used for analysis were an Nexera 2 LC (Shimadzu) coupled to a QTRAP 5500 (AB Sciex) and an Acquity I-class UPLC coupled to a Xevo TQ-XS (Waters). Data analysis was performed with Skyline (184.108.40.2066).
The IDKenhance® derivatization showed a mean signal enhancement in peak areas of factor ~15 in comparison to underivatized 1,25(OH)2-vitamin D2/3. Results showed baseline separation of 1,25(OH)2-vitamin D2/3 from isobar metabolites (25,26-(OH)2-vitamin D3; 24,25-(OH)2-vitamin D2/3; 23,25-(OH)2-vitamin D3). The analysis of a broad calibration range covering the physiological range (5-250 pg/mL) using six calibrators as well as 3 quality controls (LQC: 17 pg/mL; MQC: 40 pg/mL; HQC: 100 pg/mL) showed robust linear regression with R² >0.99 and good precision (< 15/20%). The low Limit of Quantification (LLOQ) of 5 pg/mL was achieved (CV <20%), which supports accurate determination of extremely deficient patients. Validation of the assay was performed according to the requirements published by the FDA.
The sensitivity and the robustness of the assay was significantly improved, when at the same time the LC-MS/MS run time for measurement of 1,25-(OH)2-vitamin D3/D2 was reduced to ~ 4.5 min. This LC-MS/MS kit is registered under IVDD and subject for registration under IVDR. The use of a 96-well filter plates (ImmuPlate®) and the reduced LC-MS/MS cycle time of 4.5 min pushed the sample through significantly. Due to derivatization the ionization of both analytes was enhanced, peak areas were increased by a factor of 15 and signal to noise ratio was significantly improved. In conclusion, this new developed assay is the first commercially available kit which offers a complete solution for the routine analysis of 1,25(OH)2-vitamin D2/3 using LC-MS/MS methodology.