Vitamin D3 metabolites quantification; an example of where fast chromatography can lead to matrix effects.

Today, mass spectrometry (MS) is commonly found in many analytical laboratories. Coupling MS to LC is regarded as an important achievement nowadays since it enables the analysis of non-volatile compounds which make up the vast majority of molecules. For most applications the quantification measurements made by MS are characterized by high sensitivity and high specificity. The latter feature enables the simplification of sample preparation before instrumental analysis. LC-MS/MS (liquid chromatography-tandem mass spectrometry) is having a big impact in clinical laboratories where up to now most of the quantitative measurements are performed by immuno-assays (IAs). For some applications like steroid analysis, IAs have demonstrated a lack of specificity and therefore there is an advantage in switching to LC-MS/MS.

Measurement of Vitamin D

Amongst the large number of papers published by various authors on steroid analysis, we have recently presented a paper [1] dealing with the measurement of the 1,25 di-hydroxy-vitamin D3 (“DHVD3”), a secosteroid

which presents several challenging features like a low propensity to be ionized. a very low concentration in plasma, and no straightforward and specific derivatization step for enhancing poor instrumental sensitivity.

By using a highly sensitive tandem mass spectrometer and a sophisticated two-dimension chromatography (2D-LC) we have been able to quantify such a challenging molecule at concentrations down to 15 pg/mL with an injection corresponding to 30 μL of the original plasma and with the sample pre-treatment limited to a protein precipitation step....