Analysis of Cardioactive Drugs

Immunoassays are commercially available for many drugs including digoxin, procainamide, lidocaine, quinidine, and disopyramide. The FPIA for disopyramide was used for determination of both total and free disopyramide concentration in serum. Chen et al. (27) observed good correlation between total and free disopyramide concentrations determined by FPIA assay and HPLC protocol. The EMIT assay of disopyramide can also be used for measuring both total and free concentrations of disopyramide (28). Several immunoassays are commercially available from different diagnostic companies for determination of serum digoxin concentrations. However, these immunoassays are subjected interferences. This aspect is discussed in detail in Chapter 6. HPLC/MS method, which is very specific for digoxin measurement in biological matrix, is useful to investigate fatal poisoning of digoxin in medical legal situation (29).

Verbesselt et al. described a rapid HPLC assay with solid phase extraction for analysis of 12 antiarrhythmic drugs in plasma: amiodarone, aprindine, disopyramide, flecainide, lidocaine, lorcainide, mexiletine, procainamide, propafenone, sotalol, tocainide, and verapamil. Because most of these drugs are basic compounds, alkalin-ization of column produced good absorption of these drugs in the extraction column. However, for amiodarone, an acidic pH (3.5) was maintained, and aprindine was eluted at neutral pH. After washing with water, the compounds were eluted with methanol except for amiodarone, which was eluted with acetonitrile and acetate buffer (8:5 by volume) at pH 5. Chromatographic separation was achieved by using a Spherisorb hexyl column (150 x 4.6 mm ID with particle size of 5 ^m) and mobile phase was composed of a mixture of acetonitrile or methanol with phosphate or acetate buffer at a different pH. Detection of peaks was achieved by either a UV detector or a fluorescence detector (30).

Concentrations of encainide and its metabolites can be determined in human plasma by using HPLC (31). A liquid chromatographic analysis of mexiletine in human serum with alternate application to determine concentrations of procainamide and its active metabolite N-acetyl procainamide has also been reported. The authors used N-propionyl procainamide as the internal standard (32). McErlance described stereoselective analysis of mexiletine enantiomers using HPLC. Resolution of mexiletine enantiomers as their 2-napthol derivatives was achieved by using a Prikle type 1A chiral phase column and fluorescence detection (33). However, concentration of mexiletine in serum can also be measured by GC couple with mass spectrometry with selected ion monitoring after derivatization of mexiletine and the internal standard. Minnigh et al. used p-chlorophenylalanine as the internal standard. The drug and the internal standard were extracted from plasma by a combination of ethyl acetate, hexane, and methanol (60:40:1 by volume) followed by evaporation of organic phase and derivatization to pentafluoropropyl derivatives. The mass spectrometer was operated in a selected ion-monitoring mode (34). Other derivatization techniques for determination of mexiletine concentration in serum have been described. Mexiletine can be extracted from alkaline serum with dichloromethane followed by derivatization with 2,2,2-trichloroethyl chloroformate. The reaction was completed in 30min at 70°C. N-propylamphetamine was used as the internal standard. The derivatized internal standard separated well from derivatized mexiletine. A representative total ion chromatogram showing analysis of mexiletine in serum is given in Fig. 1. The mass spectrometer was operated in selected ion-monitoring mode (m/z at 58, 102, 122, 232, 234 monitored for derivatized mexiletine and mz/z at 56, 91, 131, 260, and 262 for derivatized internal standard). The total ion chromatogram (EI) of mexiletine is given in Fig. 2. The assay was linear for serum mexiletine concentrations between 0.2 to 2.5 mg/L (35). GC/MS analysis of mexiletine in human serum after extraction and derivatization with perfluorooctanoyl chloride has also been reported (36). Although an FPIA assay for

Fig. 1. Total ion chromatogram analysis of a patient's sample containing 0.6mg/L of mexiletine. Peak A was derivatized internal standard (N-propylamphetamine) whereas Peak B was derivatized mexiletine (2,2,2,-trichloroethyl carbamate).

Fig. 1. Total ion chromatogram analysis of a patient's sample containing 0.6mg/L of mexiletine. Peak A was derivatized internal standard (N-propylamphetamine) whereas Peak B was derivatized mexiletine (2,2,2,-trichloroethyl carbamate).

50 100 150 200 250 300 350

Mass/Charge

Fig. 2. Electron ionization full scan mass spectrum of 2,2,2-trichloroethyl carbamate derivative of mexiletine.

50 100 150 200 250 300 350

Mass/Charge

Fig. 2. Electron ionization full scan mass spectrum of 2,2,2-trichloroethyl carbamate derivative of mexiletine.

flecainide was commercially available from the Abbott Laboratories, Abbott Park, IL in the past, this assay is no longer available and HPLC is the preferred method of determination of flecainide concentration in serum. Determination of serum flecainide concentration along with its metabolites using HPLC can be achieved by using octade-cylsilyl silica (ODS) column and fluorescence detection. Flecainide and its metabolites were extracted from serum with ethyl acetate (37). Another report described GC/MS validation of HPLC analysis of flecainide enantiomers in serum (38).

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