Development of a novel 96-well format for liquid-liquid microextraction and its application in the HPLC analysis of biological samples.

A novel 96-well liquid-liquid microextraction system combined with modern HPLC was developed and used for the simultaneous analysis of 96 biological samples. The system made use of hollow fibers, a 96-well plate, and a plastic base with a center hole and a side hole. One end of the hollow fiber was sealed, while the other end was attached to one of the holes positioned at the center for the plastic base. The needle was inserted into the liquid from inside or outside of the hollow fiber through the center or the side holes, respectively. The system was tested with plasma samples containing three compounds, acidic indomethacin, neutral dexamethasone, and basic propafenone. Some parameters, such as the kind and dimension of hollow fiber, pH and salt concentration of the donor phase, the selection of organic solvent for the acceptor phase, and the extraction time were investigated. Under the optimization conditions, the Log D and drug concentration of indomethacin, dexamethasone, and propafenone in plasma and urine samples were analyzed. Then, the methodology was validated. The results demonstrated that ng/mL levels could be exactly and rapidly analyzed by our system, which was equipped with an auto-injection sampler, making sample analysis more convenient.

Simultaneous analysis of six cardiovascular drugs by capillary electrophoresis coupled with electrochemical and electrochemiluminescence detection, using a chemometrical optimization approach.

CE coupled with dual electrochemical (EC) and electrochemiluminescence (ECL) detection was optimized for simultaneous analysis of six cardiovascular drugs (alprenolol, propafenone, acebutolol, verapamil, atenolol and metoprolol) via central composite design. Following this study, three critical electrophoretic factors governing the CE separation were investigated: Tris-H(3)PO(4) buffer concentration, buffer pH value and separation voltage. A modified chromatographic response was adopted for evaluating CE separation quality. Optimum conditions were achieved using Tris-H(3)PO(4) buffer 35.6 mM (pH 2.3) separated at 13.9 kV, which was employed experimentally and led to the successful simultaneous separation of the above six drugs. The good agreement of the chromatographic response was observed between predicted data and actual experimental results using these optimized conditions (RSD=3.75%). The proposed method was validated for linearity, repeatability and sensitivity, and subsequently successfully applied to determine six basic drugs in urine samples.

Enantioselective assay of S+ - and R- -propafenone in human urine by using RP-HPLC with pre-column chiral derivatization.

The enantioselective assay for S(+)- and R(-)-propafenone (PPF) in human urine that developed in this work involves extraction of propafenone from human urine and using S(+)-propafenone as internal standard, chiral derivatization with 2,3,4,6-tetra-O-beta-D-glucopranosyl isothiocyanate, and quantitation by an RP-HPLC system with UV detection (lambda=220 nm). A baseline separation of propafenone enantiomers was achieved on a 5-microm reverse phase ODS column, with a mixture of acetic acid (25:12:0.02,v/v) as mobile phase. There was good linear relationship from 24.9 ng/ml to 1875.0 ng/ml for both of enantiomers. The regression equations of the standard curves based on C(S-PPF) (or C(R-PPF)) versus ratio of A(S-PPF)/A(S) (or A(R-PPF)/A(S)) were y=0.0032x-0.081, (r=0.999) for S-PPF and y=0.0033x+0.0039, (r=0.998) for R-PPF, respectively. The method's limit of detection was 12.5 ng/ml for both enantiomers, and the method's limit of quantitation was 28.2+/-0.52 ng/ml for S-PPF, 30.4+/-methanol:water:glacial 0.53 ng/ml for R-PPF (RSD<8%, n=5). The analytical method yielded average recovery of 98.9% and 100.4% for S-PPF and R-PPF, respectively. The relative standard deviation was no more than 6.11% and 6.22% for S-PPF and R-PPF, respectively. The method enabled study of metabolism of S(+)- and R(-)-propafenone in human urine. The results from 7 volunteers administered 150 mg racemic propafenone indicated that propafenone enantiomers undergo stereoselective metabolism and that in the human body, S(+)-propafenone is metabolized more extensively than R(-)-propafenone.

Fatal propafenone overdoses: case reports and a review of the literature.

First synthesized in 1970, propafenone is a frequently used 1C antiarrhythmic drug metabolized into two major metabolites, 5-hydroxypropafenone and norpropafenone. Paradoxically, fatal intoxication is rarely described, and only six cases have been reported in the literature. We report our experience with two patients found dead of self-inflicted poisoning where the propafenone blood concentration was very high (one concentration to our knowledge is one of the highest reported in the literature). At autopsy, no evidence of significant pathological disease were found. Propafenone was detected in blood by gas chromatography-mass spectrometry and by high-performance liquid chromatography using a diode-array detector, respectively, as propafenone artifact and propafenone. Blood propafenone concentrations were 4180 ng/mL and 9123 ng/mL. The literature regarding propafenone pharmacokinetic and intoxication is reviewed, and we discuss the low death rate attributed to this drug in contrast to its frequent use.

Determination of propafenone and its phase I and phase II metabolites in plasma and urine by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A sensitive method was developed to determine propafenone, 5-hydroxypropafenone, N-despropylpropafenone and propafenone glucuronides in human plasma and urine by HPLC-electrospray ionization mass spectrometry with the respective deuterated analogues as internal standards. The analytes were extracted by a single solid-phase extraction, collecting two fractions, one containing the glucuronides and the other propafenone and the phase I metabolites 5-hydroxypropafenone and N-despropylpropafenone. The mobile phases used for HPLC were: (A) 5 mM ammonium acetate in water and (B) 5 mM ammonium acetate in methanol-tetrahydrofuran (50:50, v/v). Separation of the diastereoisomeric propafenone glucuronides was achieved on a Spherisorb ODS 2 column (150 x 2.0 mm I.D., particle size 5 microm) at a flow-rate of 0.3 ml/min using a linear gradient from 20% B to 50% B in 15 min. For separation of propafenone, 5-hydroxypropafenone and N-desalkylpropafenone a linear gradient from 50% B to 80% B in 10 min was employed. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions for quantification. The limits of quantification achieved with this method were 10 pmol/ml for propafenone, 5-hydroxypropafenone, R- and S-propafenone glucuronide and 20 pmol/ml for N-desalkylpropafenone using 0.5 ml of plasma. Reproducibility and accuracy was below 12% for each analyte over the whole concentration range measured. The method was applied to a pharmacokinetic study assessing the influence of rifampicin on propafenone disposition.

Pharmacokinetic and pharmacodynamic interaction between mexiletine and propafenone in human beings.

BACKGROUND AND OBJECTIVE: Mexiletine and propafenone are often used concomitantly and are metabolized by the same cytochrome P450 isozymes, namely CYP2D6, CYP1A2, and probably CYP3A4. Our objective was to study the potential pharmacokinetic and electrophysiological interactions between mexiletine and propafenone. METHODS: Fifteen healthy volunteers, 8 extensive metabolizers and 7 poor metabolizers of CYP2D6, received oral doses of mexiletine 100 mg two times daily from day 1 to day 8 and oral doses of propafenone 150 mg two times daily from day 5 to day 12. Interdose studies were performed at steady-state on mexiletine alone (day 4), mexiletine plus propafenone (day 8), and propafenone alone (day 12). RESULTS: In subjects in the extensive metabolizer group, coadministration of propafenone decreased oral clearances of R-(-)-mexiletine (from 41+/-11 L/h to 28+/-7 L/h) and S-(+)-mexiletine (from 43+/-15 L/h to 29+/-11 L/h) to an extent such that these values were no longer different between the extensive and the poor metabolizer groups. Propafenone coadministration also decreased partial metabolic clearances of mexiletine to hydroxymethylmexiletine, p-hydroxymexiletine, and m-hydroxymexiletine in extensive metabolizers by 71%, 67%, and 73%, respectively. In contrast, propafenone did not alter the kinetics of mexiletine enantiomers in subjects in the poor metabolizer group except for a slight decrease in the formation of hydroxymethylmexiletine. Pharmacokinetic parameters of propafenone were not changed during concomitant administration of mexiletine in subjects of either phenotype. Finally, electrocardiographic parameters (QRS duration, QTc, RR, and PR intervals) were not modified during the combined administration of the drugs. CONCLUSION: Propafenone is a potent CYP2D6 inhibitor that may cause an increase in plasma concentrations of coadministered CYP2D6 substrates.

[Characterization of some glucuronide conjugates by electrospray ion trap mass spectrometry].

To investigate the characteristics of mass spectra of some drug metabolites, solutions of glucuronide conjugates of N-(4-ethoxyphenyl)-2-hydroxyl-benzamide (etofesalamide), 4-(3H-1, 2-dihydro-1-pyrrolizinone-2-methylamino)-benzoic acid, 5-hydroxypropafenone and propafenone were analyzed using electrospray ion trap mass spectrometry in a multi-stage full scan mode performed on a Finnigan LCQ instrument. Sample solutions were directly introduced into the ESI source at a flow rate of 15-30 microliters.min-1 by a syringe pump. The mass spectrometer was operated in the negative ion mode. A full scan mass spectra of each analyte provided quasimolecular ion m/z M-1, and full scan MS2 and MS3 spectra gave characteristic fragment ions m/z 175 and m/z 113, respectively, which were derived from the glucuronate moiety of each analyte. The proposed interpretation of m/z 175 is the negative ion of glucuronic acid with a loss of 18 u (H2O), which produces m/z 113 after further losing 18 u (H2O) and 44 u (CO2). Fragmentation pathway has been established for each analyte. The results show that quasimolecular ion m/z M-1 and the characteristic fragment ions m/z 175 and m/z 113 in the multi-stage full scan mass analysis of each analyte can be predicted for future structure elucidation or quantitative determination of glucuronide conjugates by LC/MS.

Identification of propafenone metaboliser phenotype from plasma and urine excretion data.

The aim of the study was to validate a test based on analyses of urine to identify the propafenone metaboliser phenotype during routine chronic therapy. Twenty seven patients chronically treated with propafenone were studied. A debrisoquine test was performed in 10. Propafenone and its metabolites in plasma and urine were measured by HPLC. Propafenone, 5-hydroxypropafenone and N-depropylpropafenone concentrations in plasma were 1.09, 0.182 and 0.101 ng.ml-1, respectively. Total recovery of the administered dose in urine was 30.7%. Two patients were identified as PM, based on the result of the debrisoquine test (log D/4OHD of 1.26 and 1.36). This finding was confirmed by the propafenone metabolic ratio in urine, but the plasma data did not permit clearcut separation of the phenotypes. Propafenone/5-hydroxypropafenone in plasma was not a good predictor of metabolizer phenotype. Although the number of patients who completed all three tests was limited, it is concluded that analysis of propafenone/5-hydroxypropafenone in urine collected between two consecutive doses at steady-state is more practical than the debrisoquine test and more specific than determining the propafenone/5-hydroxypropafenone ratio in plasma, for identification of the propafenone metaboliser phenotypes.

Identification and determination of propafenone and its principal metabolites in human urine using capillary gas chromatography/mass spectrometry.

The capillary gas chromatography/mass spectrometry of trimethylsilyl-trifluoroacetyl, trifluoroacetyl and pentafluoropropionyl (PFP) derivatives of the antiarrhythmic agent propafenone (Rytmonorm), as well as its main metabolites N-despropyl-propafenone and 5-hydroxy-propafenone, have been investigated. Both electron impact and positive isobutane chemical ionization mass spectrometry using the Ion Trap Detector have been evaluated. The presence of propafenone and its co-extracted metabolites in human urine at time intervals after the oral administration of 150 mg Rytmonorm to healthy volunteers was established, and the urinary excretion of propafenone and 5-hydroxy-propafenone was calculated using selective chemical ionization mass spectrometric detection. Only a few per cent of the dose was excreted unchanged in the urine. Large intersubject variabilities had been observed also. The large dynamic range of the Ion Trap Detector and the high correlation coefficients (0.92-0.99) of the calibration curves were striking.

Formation of formaldehyde adducts from various drugs by use of methanol in a toxicological screening procedure with gas chromatography-mass spectrometry.

Use of methanol as a solvent in a toxicological screening procedure with gas chromatography-mass spectrometry may be associated with artifact formation. Artifacts with a molecular ion of [M + 12]+ are formed from various drugs, such as amphetamine, propafenone, flecainide, beta-blockers and prilocaine. The mechanism of artifact formation was studied by mass spectral techniques, labelling and nuclear magnetic resonance spectroscopy. It was shown that the artifacts were generated by the addition of formaldehyde and subsequent loss of water. Formaldehyde is probably formed by thermal dehydrogenation of methanol in the injection port of the gas chromatograph.

Reversed-phase LC resolutions of chiral antiarrhythmic agents via derivatization with homochiral isothiocyanates.

The search for new antiarrhythmic agents has been intense, because the established drugs for the treatment of cardiac arrhythmias are neither uniformly effective nor well-tolerated. Among the recently introduced new antiarrhythmic agents are tocainide (TOC), mexiletine (MEX), flecainide (FLE), and propafenone (PRO). Each of these drugs is a chiral amine used clinically as the racemic mixture. We have examined the high-performance liquid chromatographic chiral resolution of the above four drugs via derivatization with homochiral derivatizing agents (HDAs). The amino functionality of the drugs was reacted with four homochiral isothiocyanates, 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (TAGIT), (R)-alpha-methylbenzyl isothiocyanate (RAMBI), (S)-1-(1-naphthyl)ethyl isothiocyanate (SNEIT), and (R)-1-(2-naphthyl)ethyl isothiocyanate (RBEIT). Complete separation of the two peaks (resolution factor R = 1.5) was achieved with all four HDAs for TOC, with TAGIT, RBEIT, and RAMBI for MEX, with TAGIT and SNEIT for PRO, and only with TAGIT for FLE. SNEIT was used to develop analytical procedures for the determination of the enantiomeric composition of TOC in human urine and blood serum. The four HDAs offer several advantages over many other HDAs and should be useful in studies of enantioselective drug action and disposition.

Genetically-determined interaction between propafenone and low dose quinidine: role of active metabolites in modulating net drug effect.

1. Quinidine is a potent inhibitor of the genetically-determined debrisoquine 4-hydroxylation. Oxidation reactions of several other drugs, including the 5-hydroxylation of the new antiarrhythmic drug propafenone, depend on the isozyme responsible for debrisoquine 4-hydroxylation. 2. The effect of quinidine on the debrisoquine phenotype-dependent 5-hydroxylation and the pharmacological activity of propafenone was studied in seven 'extensive' metabolizers and two 'poor' metabolizers of the drug receiving propafenone for the treatment of ventricular arrhythmias. 3. In patients with the extensive metabolizer phenotype, quinidine increased mean steady-state plasma propafenone concentrations more than two fold, from 408 +/- 351 (mean +/- s.d.) to 1096 +/- 644 ng ml-1 (P less than 0.001), decreased 5-hydroxypropafenone concentrations from 242 +/- 196 to 125 +/- 97 ng ml-1 (P less than 0.02) and reduced propafenone oral clearance by 58 +/- 23%. 4. Despite these changes in plasma concentrations, electrocardiographic intervals and arrhythmia frequency were unaltered by quinidine coadministration, indicating that 5-hydroxypropafenone contributes to the pharmacologic effects of propafenone therapy in extensive metabolizers. 5. In contrasts, plasma concentrations of propafenone and 5-hydroxypropafenone remained unchanged in the two patients with the poor metabolizer phenotype. 6. Biotransformation of substrates for the debrisoquine pathway can be markedly perturbed by even low doses of quinidine; interindividual variability in drug interactions may have a genetic component.

Determination of propafenone in biological fluids by fused-silica capillary gas chromatography using electron-capture detection.

A gas-liquid chromatographic method with electron-capture detection using a capillary column with the inlet in the splitless injection mode is reported for the assay of propafenone. A 25 m X 0.31 mm cross-linked, 5% phenylmethylsilicone-coated fused-silica capillary column was employed for all analyses. The present method provides improved selectivity and sensitivity over other existing gas chromatographic and high-performance liquid chromatographic (HPLC) methods. Linearity was observed in the ranges 2.5-50 and 10-100 ng/ml. The coefficient of variation was found to be less than 10% over the concentration ranges studied. Application of the developed method is demonstrated by measuring serum propafenone concentrations over 24 h in a normal healthy volunteer after a single oral dose of propafenone and by measuring trough plasma propafenone concentrations at steady state in patients receiving this new antiarrhythmic drug. Validity of the present method is further demonstrated by comparison of analytical results obtained from measurement of patient samples using a modified published HPLC method.