Determination of mizolastine, a new antihistaminic drug, in human plasma by liquid-liquid extraction, solid-phase extraction and column-switching techniques in combination with high-performance liquid chromatography.

For the determination of mizolastine (2-[[[1-[(4-fluorophenyl)methyl]-1H-benzimidazol-2-yl]-4- piperidinyl]methylamino]-4(3H)-pyrimidinone, SL 85.0324), a new antihistaminic drug, in human plasma, three methods were developed based on liquid-liquid extraction, solid-phase extraction and column-switching in combination with high-performance liquid chromatography with ultraviolet detection. The liquid-liquid extraction method included a back-extraction step that preconcentrates the drug into a small aqueous volume, resulting in very high sensitivity (0.5 ng/ml of plasma); it can be used in conventional bioanalytical laboratories that do not have sophisticated automatic devices. The solid-phase extraction method is performed by using a robotic system (Benchmate). It is completely automated from the initial sampling to the final injection into the chromatograph. It has a good sensitivity (1 ng/ml of plasma), but requires an expensive apparatus and skilled analysts. The column-switching method is based on a solid-phase extraction performed on-line with chromatographic analysis; it is not completely automatic, because some operations are performed manually. The device required for valve switching is not expensive and can be managed by a simple integrator or a personal computer; it is very easy to use and affords a sensitivity (2.5 ng/ml of plasma) that generally satisfies the needs of pharmacokinetic investigations of mizolastine. The conditions were similar for all the three methods: a C8 type column, an eluent of phosphate buffer and acetonitrile, and a spectrophotometric ultraviolet detector operated at 285 nm.

Determination of zolpidem, a new sleep-inducing agent, and its metabolites in biological fluids: pharmacokinetics, drug metabolism and overdosing investigations in humans.

For the determination of zolpidem, a new sleep inducer, and its metabolites in human plasma and urine, three methods were developed that are suitable for pharmacokinetics, drug metabolism and overdosing investigations. The methods used for pharmacokinetic and drug metabolism studies are based on column-switching high-performance liquid chromatography; they do not require any sample manipulation because the plasma or diluted urine is injected into a pre-column where clean-up and preconcentration take place. The analytes are transferred by valve-switching to the C18 analytical column for chromatography. To investigate overdose cases, urine samples only are used: the method is simple, because the diluted urine can be injected directly into the analytical column (phenyl type). This allows the identification and quantification of the principal urinary metabolite of zolpidem, the unchanged drug being practically undetectable. All the methods use fluorescence detection, which affords high sensitivity and selectivity. It is necessary to use a method capable of the determination of metabolites even if these are apparently pharmacologically inactive, because in different physiopathological populations the qualitative and quantitative metabolic profiles of zolpidem could be different. The method designed for the investigation of (accidental or deliberate) overdose cases is, as required on such occasions, simple and rapid, with good selectivity with respect to commonly prescribed psychotropic drugs.

Plasma determination of the enantiomers of SL 84.0418, a new antihyperglycaemic drug, by HPLC on a chiral alpha 1-AGP column.

SL 84.0418 is a new antihyperglycaemic drug. It is an orally active and selective alpha 2-adrenoceptor antagonist. This molecule, a pyrroloindole derivative, contains an asymmetric center yielding two enantiomers. In order to evaluate the pharmacokinetic profile of both enantiomers following oral administration of the racemate we have developed an HPLC method for their separation and quantification. The liquid-liquid extraction involved three steps with two salting-out procedures at pH 11.5 before and after a back-extraction with 0.005 M H2SO4. The enantiomers were separated by HPLC on a stainless-steel column (100 x 4.0 mm) packed with a chiral alpha 1-acid. The UV response was linear from 1 to 250 ng/ml for both enantiomers. The relative standard deviation (RSD) for reproducibility was below 10.7%. Using quality control samples, precision was found below 7.8% and accuracy was 108%. Extraction recoveries were ca. 60% for both enantiomers and 94% for the internal standard. Column life was brought up to 2 months, which corresponds to about 1,000 injections of biological extract. No guard column was used, but a daily back-flush was carried out. This method is suitable for routine analysis and 30 to 40 plasma samples a day can be processed. This method allows the definition of the pharmacokinetic profile of both enantiomers of SL 84.0418 in human plasma after single oral administration of doses as low as 20 mg of the racemic drug.

Determination of mephenesin in plasma by high-performance liquid chromatography with fluorimetric detection.

A procedure for the determination of the CNS-active muscle-relaxant mephenesin in plasma samples is described. The method involved a single-step extraction with diethyl ether followed by high-performance liquid chromatography separation and fluorimetric detection. Linearity of the detection response was observed between 1 ng/ml, which is the limit of determination, and 500 ng/ml. Inter-day assays, including quality-control samples, performed over a 4-month period, showed the method to be reproducible and precise, and suitable for routine analysis in pharmacokinetic studies.

High-performance liquid and gas chromatographic methods for the determination of cicloprolol.

Two methods, one based on high-performance liquid chromatography (HPLC) and the other on gas chromatography (GC), were developed for the quantification of the partial adrenergic receptor antagonist cicloprolol. In the GC method, samples are cleaned up by back-extraction, then derivatized with heptafluorobutyric anhydride and separated on a capillary cross-linked methylsilicone column. This GC method is time-consuming but, with electron-capture detection, cicloprolol can be quantified at levels down to 1 ng/ml. The HPLC method, using a reversed ODS stationary phase and fluorimetric detection, is less sensitive (5 ng/ml) but, with a single-step extraction, is faster and simpler. The determination of cicloprolol in human blood samples by the two methods gave comparable results. Routine monitoring of cicloprolol can be done easily with the HPLC method, whereas the time-consuming GC method may be reserved for pharmacokinetic studies where late-sampled tubes, with low concentrations, must be analysed.

Determination of the GABAergic antidepressant drug fengabine and some of its metabolites in plasma by capillary gas chromatography with electron-capture detection.

A sensitive capillary gas chromatographic method was developed for the determination of fengabine (a GABAergic antidepressant drug) and some of its metabolites in plasma samples. The method involves a single and rapid liquid-liquid extraction of the parent drug and metabolites from plasma buffered at pH 5, evaporation of the organic phase under nitrogen, derivatization to tert.-butyldimethylsilyl ethers and esters and automatic gas chromatography on a fused-silica, silicone-bonded capillary column coupled to an electron-capture detector. The detection limit for fengabine and other compounds is lower than 1 ng/ml in plasma; the method was successfully applied to pharmacokinetic and drug monitoring clinical studies and tested on more than 2000 biological samples and was found not to suffer from endogenous or exogenous interferences.

A preliminary metabolic study of alpidem in rat and man.

The metabolism of alpidem, a new anxiolytic agent in the chemical series of imidazopyridines, was investigated in rat and man. In both species the compound is extensively metabolised by three main routes of biotransformation: aromatic oxidation of the imidazopyridine ring; n-dealkylation; and/or aliphatic oxidation of the substituted amide side chains.

Determination of alpidem and its metabolites in human plasma by high-performance liquid chromatography and fluorimetric detection.

A high-performance liquid chromatographic method has been developed for the simultaneous determination of alpidem and its metabolites in human plasma. The method involved a single extraction of the parent drug and metabolites into diethyl ether from alkalinized plasma, evaporation of the organic solution and chromatography of the extracts on a C18 column coupled to a fluorimetric detector. An internal standard was used for the quantitative determination of the compounds. The method was selective for alpidem and three of its metabolites and has a limit of detection of less than 1 ng ml-1 for all the compounds. Since the chromatographic run took more than 20 min, the chromatographic process was fully automated and performed overnight.

High-performance liquid chromatographic determination of alfuzosin in biological fluids with fluorimetric detection and large-volume injection.

A high-performance liquid chromatographic method has been developed for the determination of alfuzosin, a new antagonist of alpha 1 post-synaptic adrenergic receptors, in blood, plasma or urine. With fluorimetric detection and the large volume injection technique, the limit of detection in plasma is 0.5-1 ng ml-1, which is sensitive enough for pharmacokinetic studies in man. The calibration graph is linear between 1 and 200 ng ml-1 in blood plasma, with coefficients of variation of 6.2 and 1%, respectively. In urine, the linearity range is 0.05-10 micrograms ml-1; at the lowest concentration, the coefficient of variation is about 10%. A constant plasma/blood concentration ratio (1.25 +/- 0.05) allows the measurement of drug in either fluid. In blood or plasma, alfuzosin is stable at 37 degrees C for 24 h and at -20 degrees C for 6 months. As expected for reversed-phase chromatography, the retention times of alfuzosin and a few of its analogues decrease inversely with the concentration of acetonitrile in the mobile phase. However, as this concentration reaches about 75%, the retention times increase sharply. This U-shaped curve may be explained by interactions of the amino groups with silanol groups of the stationary phase.

Determination of antrafenine and its main acid metabolite, 2-{[7-(trifluoromethyl)-4-quinolinyl]amino}-benzoic acid, in biological fluids using high-performance liquid chromatography with large volume automatic injection and gas-liquid chromatography with derivative formation.

Specific and sensitive analytical methods have been developed for the measurement of antrafenine and its main acid metabolite, 2-([17-(trifluoromethyl)-4-quinolinyl] amino) benzoic acid (FQB), at therapeutic concentrations in plasma and urine. Following the addition of internal standards (the methyl ester of FQB and 2-([8-(trifluoromethyl)-4-quinolinyl] amino) benzoic acid) the parent drug and the metabolite were extracted from biological material with diethyl ether at a weakly acid pH. Drug extracts were evaporated to dryness prior to chromatographic analysis. Antrafenine was measured by high-performance liquid chromatography using a Spherisorb 5-micrometer ODS column with acetonitrile-0.1 M sodium acetate as the mobile phase. Samples were injected automatically using a 500-microliter injection loop. The detector wavelength was 353 nm corresponding to the maximum UV absorption of both drug and internal standard. The coefficient of variation (C.V.) for the determination of antrafenine concentrations between 5 and 250 ng/ml ranged between 24 and 3%, respectively. The acid metabolite of antrafenine was measured by gas-liquid chromatography with electron-capture detection using a 1 m column packed with 3% OV-225 on Gas-Chrom Q (100-120 mesh) at 240 degrees C and on-column methylation with trimethylphenyl ammonium hydroxide. The C. V. of the method for the analysis of metabolite concentrations between 10 and 500 ng/ml ranged between 3 and 9%, respectively.

Determination of nifuroxazide in biological fluids by automated high-performance liquid chromatography with large-volume injection.

A high-performance liquid chromatographic method for the measurement of nifuroxazide in plasma is described. The technique is based on the single extraction of the drug from buffered plasma with chloroform, using nifuratel as internal standard. The chromatographic system consisted of a 15 cm x 4.6 mm I.D. stainless-steel column packed with Spherisorb ODS, 5 micrometer, and the mobile phase was acetonitrile-orthophosphoric acid (pH 2.5) (30:70). The method was able to measure accurately plasma nifuroxazide concentrations down to 2 ng . ml-1 using 2 ml of sample with no interference from endogenous compounds. The coefficients of variation of the method at 200 and 2 ng . ml-1 were 3% and 15%, respectively, and the calibration graph was linear in this range. The use of automatic injection makes the method suitable for the routine analysis of large numbers of samples.