An on-line, hyphenated technique based on AMD-FDIC-MS for separating and quantitatively determining biomarkers of Lysosomal Storage Diseases in human fluids
Analytical techniques for determining several neutrals sphingolipids which are biomarkers of lysosomal storage diseases are required for their diagnostics and treatment monitoring. For this purpose, a hyphenated technique has been developed, based on sequential coupling of Automated Multiple Development (AMD), Fluorescence Detection by Intensity Changes (FDIC) and Mass Spectrometry (MS).
Different gradient separation strategies using AMD have been proposed depending on which biomarkers are considered. Primuline-postimpregnated silica gel plates have been used for FDIC and for further analyte quantification. Then, an on-line, direct transfer of the corresponding biomarker peak has been performed from the primuline-impregnated plate to a mass spectrometer through an elution-based interface. Peak identification has been done by ESI and APCI which provide useful complementary information. Primuline has been demonstrated to be compatible with MS, and does not interfere in the MS identification of studied metabolites.
Sphingomyelin (SM), a biomarker of Niemann-Pick disease, has been determined in human plasma from two healthy volunteers, under the following conditions. SM was separated from the other metabolites in 19 min. using 2-step AMD gradient (from 80:20 to 50:50, v:v) over 60 mm total developing distance. FDIC was carried out with primuline (200 ppm; λexc= 365 nm). SM quantification from FDIC signals was performed using nonlinear calibration by the standard addition method ([SM]= 280, 289 ppm; RSD<6%). After extracting SM peak using the elution-based interface, it was identified by ESI and APCI-MS.
Issues on molecular mechanism of the fluorescent response of biomarkers on primuline-impregnated plates, analytical conditions for determining other low-concentration metabolites in asma, and strategies aiming their double quantification in plasma by both FDIC and API-MS will