HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY IONSPRAY MASS-SPECTROMETRY FORTHE SPECIFIC DETERMINATION OF DIGOXIN AND SOME RELATED CARDIAC-GLYCOSIDES IN HUMAN PLASMA

An original method based upon high-performance liquid chromatography c oupled to ionspray mass spectrometry (HPLC-ISP-MS) has been developed for the identification and quantification in plasma of several cardiac glycosides, namely digoxin, digitoxin, lanatoside C and acetyldigitox in. After single-step liquid-liquid extraction by chloroform-2 propano l (95:5, v/v) at pH 9.5 using oleandrin as an internal standard, solut es are separated on a 4 mu m NovaPak C-18 (Waters) column (150x2.0 mm, I.D.), using a gradient of acetonitrile-2 mM NH4COOH, pH 3 buffer (fl ow-rate 200 mu 1/min, post-column split 1:3). Detection is done by a P erkin-EImer Sciex API-100 mass analyzer equipped with an ISP interface . In most instances the major ion observed is not [M+H](+) as expected , but [M+NH4](+). The mean retention times (min) an: lanatoside C, 5.7 4; digoxin, 6.00; digitoxin, 8.08, oleandrin, 8.30, acetyldigitoxin, 8 .66 and 9.01 (isomers alpha and beta, respectively). The lower limits of detection in single ion monitoring mode range from 0.15 ng/ml (alph a- and beta-acetyldigitoxin) to 0.60 ng/ml (lanatoside C), making the method less sensitive than radioimmunoassay, whereas it is much mon sp ecific.