FAST LC-MS/MS ANALYSIS OF PHENYTOIN IN DRIED BLOOD SPOT: an alternative to sample collection

BALDO M; HUNZICKER G; GAREIS N; HEIN G; ANGELI E; MURGUIA M

Rosario (Santa Fe)

Congreso; 4ta Reunión Internacional de Ciencias Farmacéuticas (RICiFa); 2016

Driedblood spot (DBS) sampling represents a convenient tool for sample collectionowing to minimally invasive blood drawing. Besides, DBS sampling relates to thepractical and ethical considerations associated with less invasive andstressful trials.The aimof this study was to develop a Liquid chromatography-tandem mass spectrometry (LC-MS/MS)method for the Phenytoin (PHT) quantification in DBS, in order to compare witha classic protein precipitation method in plasma, and to find a correlationbetween both matrices avoiding a block experimental design that uses a bignumber of animals.A C18 column (100 x 2.1 mm, 3 µ particle size) wasused for liquid chromatography. Elution was performed in isocratic mode using acetonitrile:ammonium acetate 3 mM (50:50, v/v) as mobile phase. The total run time was 4min and the PHT retention time was 1.8 min. Mass detector was a 5500 Q-Trapoperating with an electrospray ionization using the multiple reactionmonitoring. The following transitions were used m/z 251.1 > 102.0 and m/z251.1 > 208.0.Sample preparation involved two micro-methods: a) 30 µL blood was pipetted onto DBS paper,then was extracted with MeOH and diluted with water before injection (10 µL),and b) 100 µL of plasma was precipitatedwith acetonitrile and diluted with water before injection (10 µL). Methods werevalidated according to the international regulations.A relative comparative bioavailability study wasperformed in a single-dose, randomized-sequence, open-label, 2-way crossoverstudy, in 16 healthy rabbits.DBS method showed linearity (r >0.999) over theworking range (0.12?20.40 µg/mL) with accuracy (85-115%) and precision (VariationCoefficient, CV<15%) acceptable. Cmax, Tmax and AUC, between methods (DBS vsplasma) and between experimental designs (classic vs block), showed CV andRelative Errors <15%.A rapid and accurateLC-MS/MS method for PHT quantification on DBS was successfully developed,showing comparable results between blood and plasma matrices.