Enantioselective analysis of N-hydroxymexiletine glucuronide in human plasma for pharmacokinetic studies

Enzymatic hydrolysis with beta-glucuronidase/sulfatase was used for the ena ntioselective determination of N-hydroxymexiletine glucuronide in plasma fo r pharmacokinetic studies. N-Hydroxymexiletine glucuronide was determined a s the quantity of mexiletine released by hydrolysis (difference between the enantiomeric concentrations of mexiletine obtained with and without hydrol ysis). Plasma samples (100 mu l) were treated at pH 5.0 with 10 mg of the e nzyme (Limpet Acetone Powder type I) for 16 hr at 37 degrees C and extracte d at pH 10.4 with diisopropyl ether. Chiral mexiletine discrimination was o btained by reaction with o-phthalaldehyde/N-acetyl-L-cysteine, separation o f the resulting diastereomers on a C-18 reversed-phase column with a mobile phase of methanol-0.05 N acetate buffer, pH 5.5 (6.5:3.5, v/v), and fluore scence detection (lambda(ex) 350 nm, lambda(em) 455 nm). The performance ch aracteristics for the enantioselective analysis of mexiletine preceded by e nzymatic hydrolysis were recovery similar to 90%, quantification limit 1 ng /ml and linearity up to 1000 ng/ml plasma for both enantiomers. The coeffic ients of variation obtained in the study of intra- and inter-day precision were respectively 5% and 7% for both enantiomers. The assay was shown to be suitable for a pharmacokinetic study performed in a patient with the arrhy thmic form of chronic Chagas' heart disease treated with 200 mg t.i.d. of r acemic mexiletine hydrochloride. The high sensitivity of the method allows analysis of only 100 mu l plasma. (C) 1999 Wiley-Liss, Inc.