cholecalciferol and 4-phenyl-1-2-4-triazoline-3-5-dione

A method for analysing vitamin D(3) (VD3, cholecalciferol) has been established and validated. This method is rapid and cost effective and is intended for use in quality control in the manufacture of fortified infant formulae and milk powders. Milk or reconstituted milk powder was solubilised in methanol and extracted in one step into isooctane, which was separated by centrifugation. A portion of the isooctane layer was then transferred, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione was added to derivatise VD3. The analyte was then re-extracted into a small volume of acetonitrile and analysed by reverse-phase chromatography. Detection was by triple quadrupole mass spectrometer using a selective transition, m/z 560 → 298. An internal standard, deuterium-labelled VD3, was used to correct for losses in extraction and any variation in derivatisation and ionisation efficiencies. The method has been subjected to a single-laboratory validation and has been found to be linear, highly selective and accurate with respect to National Institute of Standards and Technology Standard Reference Material 1849, analyte spiking experiments and comparison with an LC-UV-based method. The repeatability standard deviation was 4.23 %. Significantly for routine laboratories, the method returns results within 2 h, generates minimal waste and minimises health and safety concerns to the analyst.

Topics: Animals; Chemistry Techniques, Analytical; Cholecalciferol; Chromatography, Liquid; Humans; Infant Formula; Milk; Tandem Mass Spectrometry; Triazoles

The structural specificity of vitamin D derivatization by PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) was probed using synthetic analogues and ion trap mass spectrometry. EB 1089, a vitamin D(3) analogue which contains a second site for Diels--Alder cycloaddition on its side-chain, allowed the examination of derivatization modes and comparisons of ion fragment structures. The origins of a PTAD-vitamin D(3) ion fragment, commonly used in metabolite characterization and quantitation of vitamin D(3) analogues (m/z 314), were established; ion trap mass spectrometry revealed that the PTAD comprises a portion of this diagnostic fragment, and is not lost by a retro-Diels--Alder step. Furthermore, the unique structure of the EB 1089 side-chain also permits facile determination of its side-chain metabolism. Use of PTAD derivatization and detection of metabolite-specific ion fragments identify hydroxylation at the end of the EB 1089 sidechain. It is believed that the results from these studies provide a clearer understanding of the mass spectrometry of triazolinedione derivatives, not only in the specific case of EB 1089, but also in their application to other vitamin D compounds.

Topics: Calcitriol; Cholecalciferol; Hydroxylation; Molecular Structure; Spectrometry, Mass, Electrospray Ionization; Triazoles

A mass spectrometric method for the detection of vitamin D3 metabolites is described. This method involves the derivatization of the metabolites by cycloaddition with 4-phenyl-1,2,4-triazoline-3,5-dione, followed by their characterization by continuous-flow fast atom bombardment (CF-FAB) tandem mass spectrometry (MS-MS) and high-performance liquid chromatography (HPLC). Using HPLC, this derivatization has been shown to increase the UV detectability of 25-hydroxyvitamin D3 by about 5-fold. The FAB spectra of the adducts are dominated by peaks corresponding to a protonated molecule and a fragment ion derived in part from the loss of the side chain. Multiple reaction monitoring (MRM) of this transition by MS-MS may be utilized for trace level analysis of vitamin D metabolites. Sample introduction by flow injection yields detection limits in the low nanogram to high picogram range, whereas the use of on-line capillary LC has been found to decrease the detection limits to the low picogram level.

Topics: Cholecalciferol; Chromatography, High Pressure Liquid; Spectrometry, Mass, Fast Atom Bombardment; Spectrophotometry, Ultraviolet; Triazoles