A rapid HPLC method with fluorometric detection for determination of plasma itraconazole and hydroxy‐itraconazole concentrations in cystic fibrosis children with allergic bronchopulmonary aspergillosis

The development and validation of a simple, rapid and selective high‐performance liquid chromatography (HPLC) method is described for the quantitation of itraconazole and hydroxy‐itraconazole in 100 µL of plasma from a paediatric population. The mobile phase of methanol (75% v/v) and water (25% v/v) was pumped at 1 mL/min through a C 18 Symmetry ™ (3.9 mm i.d. × 150 mm) cartridge. Using a protein‐precipitation method, 100 µL internal standard (IS) solution (R051012, 555 µg/L in acetonitrile) were added to 100 µL of plasma followed by 10 µL zinc sulphate solution (20% w/v). Itraconazole, hydroxy‐itraconazole and IS eluted at 4.7, 8.3 and 12.5 min, respectively and were detected ßuorometrically at 250 nm (excitation) and 380 nm (emission). Recoveries were 87.1–96.7%. Calibrations in drug‐free plasma were linear ( r 2 > 0.99) from 50 to 2000 µg/L, using 1/ c 2 ( c = concentration) weighting. Intraday and interday imprecision (CV%) was 4.8–17.3 and 6.3–16.6% for itraconazole, and 4.6–17.9 and 7.02–18.4% for hydroxy‐itraconazole. Inaccuracy was −7.1 to −14.7% for itraconazole and −0.1 to −9.7% for hydroxy‐itraconazole. The clinical application of this method was demonstrated by measurement of itraconazole and hydroxy‐itraconazole in plasma samples drawn from paediatric cystic fibrosis patients, who were prescribed itraconazole for treatment of allergic bronchopulmonary aspergillosis. Copyright © 2005 John Wiley & Sons, Ltd.