Liquid Chromatographic Determination of Six Antiepileptic Drugs and Two Metabolites in Microsamples of Human Plasma

Abstract

A simple, rapid, sensitive, and reproducible high-performance liquid chromatographic (HPLC) method for simultaneous determination of the antiepileptic drugs (ethosuximide, primidone, lamotrigine, phenobarbital, phenytoin, and carbamazepine) and two metabolites (carbamazepine-diol and carbamazepine-epoxide) in human plasma is described. The procedure involves extraction of the drugs from human plasma (100 microL) with ether using 9-hydroxymethyl-10-carbamyl acridan as an internal standard. The extract was evaporated and reconstituted with mobile phase and then injected onto the chromatograph. The drugs and the internal standard were eluted from a Supelcosil LC-18 stainless steel column at ambient temperature with a mobile phase consisting of a 0.01M phosphate buffer/methanol/acetonitrile (65/18/17, v/v/v) adjusted to a pH of 7.5 with phosphoric acid and a flow rate of 1 mL/min. The effluent was monitored at 220 nm. Quantitation was achieved by using peak area ratio of each drug to the internal standard. The intraassay and interassay coefficients of variation (CV) ranged from 2.43% to 6.25% and from 3.02% to 5.85%, respectively. The absolute (extraction) and relative (analytical) recoveries for the drugs ranged from 70.7% to 104.4% and from 88.3% to 106.1%, respectively. Stability tests showed that the drugs were stable in plasma for at least 4 weeks when stored at -20 degrees C. The method was applied clinically for monitoring the AEDs in epileptic patients.

Dried blood spot assay for the quantification of phenytoin using Liquid Chromatography-Mass Spectrometry

“…Over the last years, a number of validated methods for PHT measurement have been reported, usually requiring a large amount of plasma sample [6,[10][11][12][13]. In pediatric patients the available amount of sample …”

Determination of phenobarbital in human plasma by a specific liquid chromatography method: application to a bioequivalence study

“…The peak areas were integrated automatically by computer using a Shimadzu Class VP V 6.14 software program. The experiments were carried out on a reversed phase Phenomenex (Torrance, USA) Synergi MAX-RP C 12 analytical column (150 x 4.6mm I.D., with a particle size of 4 µm and pore size of 80 Å) maintained at 35 ºC. The LC system was operated isocratically using a mobile phase of water:acetonitrile:methanol (58.8:15.2:26, v/v/v).…”

“…[6][7][8][9][11][12][13][14][15][16][17][18][19][20][21][22] A chiral LC method was developed and validated for the determination of phenobarbital and methylphenobarbital enantiomers in human plasma using a C 18 precolumn and a chiral column for the enantiomeric separation. 23 The RP-LC methods were developed with UV detection and using stir bar-sorptive extraction, 11 liquid-liquid extraction (LLE), 12,13,21 direct injection of plasma with manual column-switching technique, 17 SPE, 22 or protein precipitation. [18][19][20] However, some of these methods show disadvantages such as low sensitivity, complicated and extensive sample preparation, and most of them did not demonstrate any applicability for pharmacokinetic or bioequivalence studies.…”

Analysis of antiepileptic drugs in biological fluids by means of electrokinetic chromatography

“…Several HPLC methods have been developed for the determination of LTG in biological fluids [118][119][120][121][122]. Shihabi et al [123] reported a CZE method for the determination of LTG in human serum.…”