Transfer of Mexiletine into Breast Milk: A Case Report.

Background: Mexiletine is a class 1B antiarrhythmic agent, used to treat ventricular arrhythmias, and noncardiac-related problems such as myotonia. Limited safety data are available on the transfer of this drug into breast milk. Case Report: We report the case of a woman diagnosed with myotonia congenita who breastfed two children after two consecutive pregnancies. During the breastfeeding of the first and the second infant, she collected, respectively, five and seven samples at 0, 2, 4, 6, and 8 hours and 0, 1, 2, 3, 4, 6, and 8 hours after taking 200 mg of mexiletine thrice daily for seven doses. One week after the collection, samples were analyzed with a validated liquid chromatography tandem mass spectrometry method. No side effect was observed in either child according to the mother. Results: Using the mean milk concentrations, it is estimated that an exclusively breastfed infant would receive a maximum of 5.14% of the initial pediatric mexiletine dosage. We calculated a maximum of 2.67% for the first infant in our case, considering a nonexclusive breastfeeding. Maximal concentrations were observed 1-2 hours after the dose of mexiletine. Results seem to be in accordance with the two cases previously published. Conclusions: Mexiletine transfers into breast milk in low levels. However, results are obtained from only one woman. Therefore, caution should be used when mexiletine is prescribed to breastfeeding women. More cases are needed to evaluate the interindividual variability and to guide women regarding breastfeeding with mexiletine.

Highly sensitive determination and validation of gabapentin in pharmaceutical preparations by HPLC with 4-fluoro-7-nitrobenzofurazan derivatization and fluorescence detection.

A sensitive HPLC method with pre-column fluorescence derivatization using 4-Fluoro-7-Nitrobenzofurazan (NBD-F) has been developed for the determination of gabapentin in pharmaceutical preparations. The method is based on the derivatization of gabapentin with (NBD-F) in borate buffer of pH 9.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Inertsil C(18) column (250 mm × 4.6 mm) using a mobile phase of methanol water (80:20, v/v) solvent system at 1.2 mL/min flow rate. Mexiletine was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 521 nm (emission). The assay was linear over the concentration range of 5 50 ng/mL. The method was validated for specificity, linearity, limit of detection, limit of quantification, precision, accuracy, robustness. Moreover, the method was found to be sensitive with a low limit of detection (0.85 ng/mL) and limit of quantitation (2.55 ng/mL). The results of the developed procedure for gabapentin content in capsules were compared with those by the official method (USP 32). Statistical analysis by t- and F-tests, showed no significant difference at 95 confidence level between the two proposed methods.

High performance liquid chromatography enantioseparation of the novel designed mexiletine derivatives and its analogs.

A series of novel designed mexiletine derivatives and its analogs were prepared, the structures were confirmed by Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared Spectroscopy (FTIR), and Electrospray Ionization-Mass Spectrometry (ESI-MS), and the enantioseparations were performed on polysaccharide-based chiral stationary phase (CSP), Chiralcel OD-H, and Chiralcel OJ-H, under normal-phase mode. The effects of the concentration of isopropanol in the mobile phase were studied, seven of the eight enantiomers got baseline separation on Chiralcel OD-H, and five of the eight enantiomers got successfully separation on Chiralcel OJ-H. The effects of structural features were also discussed.

Column-switching HPLC determination of mexiletine using tris(bipyridine)ruthenium(III) electrogenerated chemiluminescence and precolumn derivatization with divinylsulfone.

A sensitive HPLC method combined with a column-switching system and tris(bipyridine)ruthenium(III) electrogenerated chemiluminescence (ECL) detection has been developed for the quantitative determination of mexiletine (MEX). MEX was derivatized by divinylsulfone (DVS) and then measured. The optimum conditions for the derivatization reaction were 10 µL of sample solutions, 40 mM DVS (pH 8.0), a reaction temperature of 50°C, and a reaction time of 15 min. The derivatized samples were cleaned up by an on-line pretreatment column. Also, after column-switching to the analytical column, the derivatized MEX was separated and detected. The calibration curves of MEX in human control serum showed good linear regression (r = 0.9996) from 0.008 to 6.56 µg ml(-1). The detection limit of MEX was 0.008 µg ml(-1) (S/N = 3). At a concentration of 2.0 µg ml(-1) MEX, the relative standard deviation (n = 5) was 0.98%. In this method the concentration of MEX in human control serum was readily measured, and this method was successfully applied to the time courses of the concentration of MEX in rabbit plasma after intravenous administration. The proposed method involved a simple and minimum sample-preparation procedure and a short run time (<20 min). Therefore it can be applied to routine therapeutic monitoring and pharmacokinetic studies of MEX.

Validated HPTLC methods for quantification of mexiletine hydrochloride in a pharmaceutical formulation.

Two simple, accurate, and precise HPTLC methods have been established for the determination of mexiletine hydrochloride, an antiarrhythmic agent, in Mexicord capsules. Analyses were performed in horizontal chambers on RP C18F254s and normal-phase amino (NH2) HPTLC precoated plates with the mobile phases tetrahydrofuran-citrate buffer, pH 4.45 (3 + 7, v/v) and chloroform-tetrahydrofuran-hexane-ethylamine (3 + 2 + 5 + 0.1, v/v/v/v), respectively. The plates were developed for a distance of 40 mm in both cases. Densitometric measurements were achieved in the UV mode at 217 nm based on peak areas with semilinear calibration curves (R2 > or = 0.97) in the concentration range 0.5-8.0 microg/spot for the NH2 and C18 HPTLC methods. The elaborated chromatographic methods were validated in accordance with International Conference on Harmonization guidelines in terms of linearity, accuracy (99.64% for NH2 and 99.53% for C18), precision (intraday RSD 1.16 and 2.71%, respectively), sensitivity (LOD 0.1 microg/spot for both systems), and specificity.

Enantioselective determination of mexiletine and its metabolites p-hydroxymexiletine and hydroxymethylmexiletine in rat plasma by normal-phase liquid chromatography-tandem mass spectrometry: application to pharmacokinetics.

Mexiletine (MEX), hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM) were analyzed in rat plasma by LC-MS/MS. The plasma samples were prepared by liquid-liquid extraction using methyl-tert-butyl ether as extracting solvent. MEX, HMM, and PHM enantiomers were resolved on a Chiralpak(R) AD column. Validation of the method showed a relative standard deviation (precision) and relative errors (accuracy) of less than 15% for all analytes studied. Quantification limits were 0.5 ng ml(-1) for the MEX and 0.2 ng ml(-1) for the HMM and PHM enantiomers. The validated method was successfully applied to quantify the enantiomers of MEX and its metabolites in plasma samples of rats (n = 6) treated with a single oral dose of racemic MEX.

Novel automated assay for the quality control of mexiletine hydrochloride formulations using sequential injection and on-line dilution.

The first automated method for the determination of mexiletine hydrochloride - an antiarrhythmic agent - is reported. The method is based on the reaction of the analyte with o-phthalaldehyde (OPA) in the presence of sulfite in basic medium using a sequential injection (SI) manifold. The reaction product was monitored spectrofluorimetrically (lambda(ex)=350 nm/lambda(em)=446 nm). A simple and effective on-line dilution approach was adopted in order to expand the linearity and apply the method to assay, dosage uniformity and dissolution tests with minimum sample preparation. Chemical (pH, amount concentrations of OPA and sulfite) and instrumental variables (temperature, flow rate, injection volumes, etc.) that affected the determination were studied. The developed assay was validated in terms of linearity, range, limits of detection (LOD=3.4 mg L(-1)) and quantitation (LOQ=10 mg L(-1)), accuracy, precision (R.S.D.<3.4%) and selectivity. The method was applied successfully to the quality control of a mexiletine-containing formulation. The results were in good agreement with the US pharmacopoeia HPLC method.

Selective and stability-indicating methods for the simultaneous determination of mexiletine hydrochloride and/or its related substance: 2,6-dimethylphenol.

Four simple, rapid, sensitive, and selective analytical procedures were developed for determination of mexiletine hydrochloride (MX) and/or its related substance: 2,6-dimethylphenol (DMP). The latter is a synthetic impurity for which a maximum pharmacopeial limit is defined. The first method depends on derivative-ratio spectrophotometry, for which the first-derivative signals of the ratio spectra at 259 nm (Deltalambda = 3 nm) are selected for the determination of MX. The second method is based on the spectrofluorometric measurement of MX in alkaline solution in the presence of 15 mM sodium dodecyl sulfate micellar medium at 292 nm (lambdaEx 260 nm). The third method is based on liquid chromatographic (LC) separation of MX and DMP on an RP-C8 column with a mobile phase consisting of 50 mM Na2HPO4-acetonitrile (60 + 40, adjusted to pH 2.4), and quantification of the analytes is achieved with UV detection at 212 nm based on peak area. The fourth method uses the coupling reaction of DMP with 2,6-dibromo-quinone-4-chlorimide (DBQC) in borate buffer to form an intensely colored product that was spectrophotometrically measured using first-derivative amplitudes at 670 nm (Deltalambda = 6 nm) for the determination of DMP. Different variables affecting each method were carefully investigated and optimized. The reliability and analytical performance of the proposed methods, including linearity, range, precision, accuracy, and detection and quantitation limits, were statistically validated. The first 3 methods were successfully applied for the stability-indicating determination of MX in laboratory-prepared mixtures with DMP, as well as for the determination of MX in capsules. Also, the LC and the DBQC spectrophotometric methods permitted the selective determination of DMP in the presence of a large excess of the parent drug at or near the pharmacopeial limit (0.1-1%).

Simultaneous separation of 14 antiarrhythmic drugs and determination of mexiletine and flecainide by capillary zone electrophoresis.

A method using capillary zone electrophoresis was developed for the simultaneous separation of 14 antiarrhythmic drugs belonging to various classes. The drugs are separated on a fused-silica capillary, 90 cm x 75 microm (72 cm effective length), with phosphate and acetate buffers as background electrolytes and UV detection at 217 nm. The effects of buffer pH, temperature, and applied voltage on the migration of the drugs were studied. The pH was found to be the most significant factor determining effective separation. The antiarrhythmic compounds are completely separated within a relatively short time (< 7 min) by using 70 mM phosphate buffer at pH 7.91, an applied voltage of 28 kV, and a temperature of 32 degrees C. Mexiletine (MEX) and flecainide (FLE) were quantified under conditions of the optimum separation. The calibration graphs were constructed over the concentration range of 4.0-14.0 microg/mL for both drugs with good correlation (r > or = 0.9999). Detection and quantitation limits were found to be 0.5 and 1.5 microg/mL for FLE and 0.7 and 2.1 microg/mL for MEX, respectively. The proposed method was used for the determination of both drugs in their commercial forms with satisfactory precision (relative standard deviations of 0.36-1.21% for FLE and 0.78-1.66% for MEX) and accuracy (relative standard errors of 0.13-1.17% for FLE and 0.35-1.18% for MEX).

The effect of stationary phase on lipophilicity determination of beta-blockers using reverse-phase chromatographic systems.

Evaluation of lipophilicity parameters for basic compounds using different chromatographic stationary phases is presented. An HPLC method for determination of lipophilic molecule-stationary phase interactions was based on gradient analysis. Differences in correlation between the lipophilicity of compounds and experimental chromatographic results obtained in pseudo-membrane systems showed a strong influence of stationary phase structure and physico-chemical properties. beta-Blocker drugs with varying lipophilicity and bio-activity were chosen as test compounds. The stationary phases used for the study were monolithic rod-structure C18 and silica gel octadecyl phase SG-C18 as reference material. The second group was silica gel-based polar-embedded alkylamide and cholesterolic phases. The mobile phase was composed of acetonitrile or methanol with ammonium acetate, and a linear gradient of methanol and acetonitrile in mobile phase was performed. A linear correlation of plots of log k(g) = f(log P) was observed, especially for polar-embedded phases, and this allowed log P(HPLC) to be calculated. The behavior of stationary phases in methanol and acetonitrile buffer showed differences between obtained log P(HPLC) values.

Comparison of classical and derivative UV-spectrophotometric methods for the quantification of diltiazem and mexiletine.

A first order derivative UV-spectrophotometric method for the determination of diltiazem hydrochloride and mexiletine hydrochloride has been developed and validated. In the assay, the first- and second-order measurements with the use of the "peak-zero" and "peak-peak" techniques were applied. The linear correlation (r < 0.9999) between the amplitude of the peak and the concentration of the examined drugs in the range of 3.0-8.0 microg mL(-1) for diltiazem and 50-100 microg mL(-1) for mexiletine was obtained. The proposed method was successfully applied for accurate (mean recovery about 100%), precise (RSD about 1%) and selective determination of the studied drug in the pure and dosage forms.

[Enantioseparation of twelve pharmaceutical racemates with high performance capillary electrophoresis using L-leucine as chiral selector].

A rapid enantiomeric separation method using L-leucine as chiral selector was established. Capillary zone electrophoresis (CZE) has been used for the enantiomeric separation of twelve pharmaceutical racemates with bare fused silica capillary and employing L-leucine as chiral selector. The enantiomeric resolution was influenced by L-leucine concentration and pH of background electrolyte (BGE). The effects of the BGE types and concentrations on the enantiomeric separation were also investigated. The results showed that in the solution containing 50 mmol/L borax and 70 mmol/L L-leucine (pH 9.0), all the twelve drugs were on baseline separated in less than 11 minutes.

Mexiletine-sensitive membrane electrode for medical application.

Response characteristics of mexiletine-sensitive membrane electrodes based on crown ether and ion-exchanger were examined in a physiological saline in order to find an electrode suitable for determining concentrations of this drug under physiological conditions. Among various crown ethers screened, 4',4"(5")-di-tert-butyldicyclohexano-18-crown6 showed the highest sensitivity to mexiletine in physiological saline containing 0.15 M NaCl and 5 mM 4-(2-hydroxyethyl)-2-piperazineethanesulfonic acid (Hepes) NaOH (pH 7.4). However, the detection limit of 30 microM was 10 times higher than that of 3 microM observed with the electrode based on an ion-exchanger, sodium tetrakis[3,5-bis(2-methoxyhexafluoro-2-propyl)phenyl]borate. Having high selectivity against inorganic cations such as Na+ or K+, the electrode using the ion-exchanger enabled us to determine the level of mexiletine in saliva, the monitoring of which is quite effective for controlling the dose of this drug noninvasively. The mexiletine concentrations determined with the mexiletine electrode compared favourably with those determined by high-performance liquid chromatography which requires an additional procedure to extract mexiletine from saliva.

Stereoselective metabolism of rac-mexiletine by the fungus Cunninghamella echinulata yields the major human metabolites hydroxymethylmexiletine and p-hydroxymexiletine.

rac-Mexiletine is an orally effective class 1b antiarrhythmic agent used to treat ventricular arrhythmias. In vivo experiments have demonstrated. It is predominantly metabolized by the liver with < 10% excreted as unchanged drug. The major mammalian metabolites have been identified as p-hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM). The purpose of our study was to determine whether the fungus Cunninghamella echinulata, which possesses a cytochrome P450 system analogous to that found in humans, could be used as a suitable in vitro model for studying the oxidative metabolism of rac-mexiletine. To accomplish this, a high performance liquid chromatographic assay was used that was capable of simultaneously quantifying the enantiomers of mexiletine, HMM, and PHM. Utilizing this procedure, it was demonstrated that C. echinulata stereoselectively converted rac-mexiletine into HMM (4% of added drug) and PHM (32% of added drug) after an incubation period of 50 hr. In addition, metabolite biosynthesis could be optimized by altering fermentation media components. Seven media values and seven pH values were evaluated. It was determined that a medium at pH 7.0 containing yeast extract and sucrose yielded optimal amounts of metabolites.

Direct analysis of the enantiomers of mexiletine and its metabolites in plasma and urine using an HPLC-CSP.

A high-performance liquid chromatographic assay has been developed for the quantification of the enantiomers of mexiletine and its four major metabolites, in plasma and in urine. Mexiletine and all metabolites were determined, after derivatization of mexiletine and its hydroxymetabolites, p-hydroxymexiletine and hydroxymethylmexiletine, using a Chiralpak AD chiral stationary phase, based on a carbamoyl derivative of amylose. o-phthalaldehyde was chosen as derivatization reagent to increase the sensitivity of detection, to achieve separation of all compounds in one chromatographic system, and to avoid interferences.

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.

Primary amine drug selective electrodes with special crown ethers as neutral carriers.

Ionophores selectively sensitive to primary amines have been synthesized which display low potentiometric selectivity coefficients for K+, Na+ and NH4+ ions, secondary and tertiary amines as well as quaternary ammonium ions. These ionophores include macrocyclic polyethers with dinaphthyl subunits and azocrown ether with nitrogen donor atoms. The feasibility of these ionophores for preparing primary amine drug selective electrodes was investigated in detail. Practically usable PVC membrane electrodes sensitive to primary amine drugs, such as mexiletine, dopamine, metaraminol and tryptamine, and aliphatic primary amines have been prepared with these ionophores as neutral carriers. Direct potentiometric methods for assaying these drugs have been proposed by using the prepared electrodes. The proposed primary amine drug selective electrodes are remarkably superior to those based on ion-associates. Compared with the electrodes based on common ethers, the interference by K+, Na+ and NH4+ ions is substantially reduced. A digital simulation of the electrochemical process concerning the membrane transport was performed and some interesting conclusions have been drawn.

High-performance liquid chromatographic assay for mexiletine hydroxylation in microsomes of human liver.

A simple high-performance liquid chromatographic assay, using fluorescence detection, is described for determining simultaneously the production of the two major hydroxylated metabolites of mexiletine in human liver microsomes. The detection limits of hydroxymethylmexiletine and p-hydroxymexiletine are 0.35 and 0.08 nmol/ml, respectively. The assay is specific, reproducible and allows the simultaneous kinetic characterization of the reactions in small amounts of liver tissue. The assay may be used to acquire a better knowledge of the kinetic behaviour of mexiletine and of its metabolites, and to investigate if the large inter-individual variations of the mexiletine pharmacokinetics are of metabolic origin, due to variations of its hydroxylation processes.

Stereoselective analysis of the enantiomers of mexiletine by high-performance liquid chromatography using fluorescence detection and study of their stereoselective disposition in man.

A sensitive, stereoselective high-performance liquid chromatographic assay was developed for the resolution of the enantiomers of mexiletine as their 2-naphthoyl derivatives on a Pirkle type 1A chiral phase column. Detection of the derivatives was accomplished with a fluorescent detector. Maximum recovery of the enantiomers from plasma was 83% and was observed when plasma proteins were precipitated with a mixture of barium hydroxide-zinc sulphate. The calibration curve in plasma was linear over the concentration range 5-750 ng/ml for each enantiomer (r2 = 0.999) and in urine the linear range was 0.25-7.5 micrograms/ml (r2 = 0.999) for each enantiomer. The minimum detectable quantity of each enantiomer in plasma was 5 ng/ml at a signal-to-noise ratio of 5:1, representing 100 pg injected. A preliminary pharmacokinetic study was undertaken in one healthy male volunteer following an oral dose of 300 mg of racemic mexiletine hydrochloride. The apparent elimination half-lives determined from the plasma data were 12.1 and 14.1 h for the R(-) and S(+) enantiomers, respectively. The cumulative urinary excretion amounts of R(-)- and S(+)-mexiletine were found to be 8.01 and 10.46 mg, respectively. The plasma data indicated that a cross-over of the enantiomer ratios occurred at approximately 8 h. The urinary excretion of the enantiomers was consistent with the pattern found in plasma.