High-performance liquid chromatographic method for resolving the enantiomers of mexiletine and two major metabolites isolated from microbial fermentation media.
(+/-)-Mexiletine is a class Ib antiarrhythmic drug useful in the treatment of premature ventricular contractions. It is predominantly metabolized by the liver with less than 15% being excreted in urine as unchanged drug. p-Hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM) are the two major mammalian metabolites. The purpose of our study was to develop a stereospecific high-performance liquid chromatographic (HPLC) method to determine whether the fungus, Cunninghamella echinulata (UAMH 4145), was able to biosynthesize these same two metabolites from the substrate (+/-)-mexiletine. Furthermore, it was desirable to ascertain whether metabolism of mexiletine was stereoselective. The method requires pre-column derivatization of the drug and metabolites with S-(+)-1-(1-naphthyl)ethyl isocyanate (NEIC) followed by normal-phase HPLC. Mexiletine, PHM, HMM and (+/-)-1-(4-hydroxyphenoxy)-3-isopropylaminopropan-2-ol (internal standard) were extracted from microbial broth using two volumes of diethyl ether after basifying with sodium carbonate. The combined ether extracts were evaporated to dryness, using a gentle stream of nitrogen, and reconstituted in 0.3 ml of chloroform to which was added 0.075 ml of NEIC (0.1%, v/v, in chloroform). This solution was immediately evaporated to dryness under a nitrogen stream. The residue was reconstituted with 0.220 ml of chloroform and 0.030 ml of n-butylamine (0.33%, v/v, in chloroform) and injected into the HPLC system.
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
D G
DG
Freitag
R T
RT
Foster
R T
RT
Coutts
F M
FM
Pasutto
D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000032:analysis; D005285:Fermentation; D007202:Indicators and Reagents; D008801:Mexiletine / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D009090:Mucorales / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism
D003470:Culture Media; D007202:Indicators and Reagents; D008801:Mexiletine; C045807:6-hydroxymethylmexiletine; C045808:4-hydroxymexiletine