Determination of retigabine and its acetyl metabolite in biological matrices by on-line solid-phase extraction (column switching) liquid chromatography with tandem mass spectrometry.
A HPLC assay with tandem mass spectrometric detection in the positive-ion atmospheric pressure chemical ionisation (APCI) mode for the sensitive determination of retigabine [(I), D-23129] and its acetyl metabolite [(II), ADW 21-360] in plasma was developed, utilising the structural analogue (D-10328), (III), as internal standard. Automated on-line solid-phase extraction of diluted plasma samples, based on 200-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of retigabine and the acetyl metabolite down to 1 ng/ml. Injection of 500 microl of diluted plasma onto a C2 stationary phase-based column switching system in combination with a 75 mm x 4 mm reversed-phase analytical column at a flow-rate of 0.5 ml/min provided cycle times of 4 min per sample. The standard curves were linear from 1 to 1000 ng/ml using weighted linear regression analysis (1/x2). The method is accurate (mean accuracy < or = +/- 10%), precise (RSD < +/- 15%) and sensitive, providing lower limits of quantification in plasma of 1 ng/ml for retigabine (I), and 2.5 ng/ml for the metabolite (II) with limits of detection of 0.5 ng/ml for both analytes. Up to 200 unknowns may be analysed each 24 h per analyst.
Knebel
N G
NG
Department of Biological Research Biochemistry, ASTA Medica AG, Frankfurt, Germany. norbert.knebel@astamedica.de
Grieb
S
S
Leisenheimer
S
S
Locher
M
M
D000927:Anticonvulsants / Q000097:blood* / Q000493:pharmacokinetics; D002138:Calibration; D002219:Carbamates / Q000097:blood* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010655:Phenylenediamines / Q000097:blood* / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000927:Anticonvulsants; D002219:Carbamates; D010655:Phenylenediamines; C101866:ezogabine