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DEVELOPMENT & VALIDATION OF A GC-MS QUANTITATIVE METHOD FOR PLASMA HYDROXYUREA Tayeb KETTANI, Alain KUMPS Laboratoire de Biochimie Médicale, Institut de Pharmacie, Université Libre de Bruxelles. BACKGROUND & AIM OF THE STUDY
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DEVELOPMENT & VALIDATION OF A GC-MS QUANTITATIVE METHOD FOR PLASMA HYDROXYUREA • Tayeb KETTANI, Alain KUMPS • Laboratoire de Biochimie Médicale, Institut de Pharmacie, Université Libre de Bruxelles • BACKGROUND & AIM OF THE STUDY • Hydroxyurea (HU), a drug used in the treatment of severe forms of sickle cell disease (SCD), shows an overall clinical benefit for patients • Large individual variations of haematological and clinical responses are however observed • HU pharmacokinetics after oral administration is not clearly established in SCD young patients • An original assay of plasma HU has been developed and evaluated in the aim of studying pharmacokinetics and for possible TDM in adult and children patients MATERIALS & METHODS GC Hewlett-Packard 5890A série II MSD Hewlett-Packard 5972 Sample preparation GC-MS 50 µL plasma sample or calibrators (spiked plasma) + 100 µl internal standard solution (methoxyurea) MS Controller 504 Gerstel injector temp.: 150, increased to 270°C 1 µl splitless injection helium 40 cm/s oven temperature: 80 (1 min → 136 (12°C/min) → 270 (35°C/min) (2.5 min) interphase temp.: 250°C deproteinisation + 1000 µL ethanol:hexane (1:1, v/v) vortex and centrifugation • MS settings : • electron impact, 70 eV, 2100 v • SIM mode • HU : m/z 277 (+ 292) • internal std.: m/z234 (+ 219) • 4.26 cycles/sec column: HP-5-MS(phényl(5%)-méthyl (95%)polysiloxane ) 30 m x 0.32 mm x 0.25 µm silylation taking up aqueous phase evaporation to dryness under N2 + 100 µl BSTFA:TMCS:pyridine (100:1:20, v/v) / 60°C for 30 min RESULTS IS full chromatogram (10 mg HU / l serum) m/z 277 HU IS HU extracted ion chromatogram (0.4 mg HU / l serum) HU extracted ion chromatogram (10 mg HU / l serum) m/z 292 Analytical goals : acceptable error < 15% or < 1 mg/L,at least one of these conditions being encountered PLASMA BLANKS • 8 plasma-blanks : 0,03 – 0,20 mg/l • endogenous HU at very low level probably present ! • estimation of limit of detection is irrelevant area ratio (HU/IS) LINEARITYassessed from ≤ 0.4 to ≥ 100 mg/l added, mg/L area ratio (HU/IS) PRECISION CV%, as a function of concentration found, mg/L PRECISION on 7 levels (1 – 25 mg/L), 5 days, 2 extracts per day (70 essays) added, mg/L • CONCLUSION • The analytical performances of this method largely encounter our a priori fixed analytical goals • The method is suitable for pharmacokinetic studies and possible therapeutic drug monitoring of HU in SCD