Mass fragmentographic assay for 25-hydroxyvitamin D in plasma without derivatization: Enhanced sensitivity for metabolites of vitamins D2 and D3 after pre-column dehydration

A mass fragmentographic method for the measurement in plasma of underivatized, dehydrated 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 was developed. Quantitative dehydration of vitamin D metabolites was achieved prior to gas chromatography using a new high-temperature injection system and a small precolumn packed with aluminium powder in the injection port, followed by mass fragmentography on an inexpensive bench-top mass spectrometer. Plasma (2 ml) was incubated with hexadeuteriated 25-hydroxyvitamin D3 prior to acetonitrile extraction and purification using cartridges pre-packed with microparticulate silica using reversed- and normal-phase solvent systems. After purification, capillary gas chromatography mass spectrometry was carried out following dehydration of the secosteroids on the aluminium power pre-column at 400°C. High-intensity dehydrated molecular ions were produced which were used for selected ion monitoring. The assay sensitivity for 25-hydroxyvitamin D was approximately 1 ng ml−1. The intra-assay variation was less than 7% and the recovery of added standard was quantitative. It is suggested that this method may be used to provide target values for much needed quality control schemes to monitor assays routinely used in the estimation of 25-hydroxyvitamin D. The behaviour of a number of other hydroxylated vitamin D metabolites, when injected without derivatization on to a gas chromatographic column, was also investigated.