J.P. Dubois

Chromatography of benzodiazepines

An overview of methods for the determination of benzodiazepines in biological media, based on the application of chromatographic techniques, is presented. A general discussion of the techniques in terms of stability, selectivity, validation, standardization, detection and sensitivity is given. No single technique can be claimed as the method of choice for benzodiazepines. Gas chromatography with electron-capture detection has some strong claims and shows generally good sensitivity and reproducibility. High-performance liquid chromatographic equipment is readily available in most laboratories. The ultimate choice of an assay method for benzodiazepines will be determined by the clinical application (routine monitoring, pharmacokinetics, overdose, forensic medicine) and by the characteristics of the benzodiazepine, the expertise of the analyst, the equipment available, the desired sensitivity and specificity and the time involved in method development or adaptation and validation.

Simultaneous determination of carbamazepine, oxcarbazepine and their main metabolites in plasma by liquid chromatography

Carbamazepine (WI-dibenz [ b,f] azepine-5-carboxamide) (IV, Fig. 1) , the active ingredient of Tegretol, is an anticonvulsant that is widely used in the treatment of both partial and generalized tonic-clonic seizures [ 11. Many methods have been published for the analysis of carbamazepine and its epoxide metabolite (V) . However, only one liquid chromatographic (LC) method has been described that allows the simultaneous determination of carbamazepine, its epoxide metabolite and its truns-diol metabolite (III) [ 21. Under steady-state conditions, the truns-diol metabolite can reach plasma concentrations even higher than those of the epoxide metabolite. Oxcarbazepine (lO,ll-dihydro-lo-oxo-5H-dibenz[ b,f]azepined-carboxamide) (I), the ketohomologue of carbamazepine, is a new anticonvulsant in’development [ 3, 41. Three LC methods have been described for the simultaneous determination of oxcarbazepine and two of its metabolites [ 5-71. No analytical method was available to measure oxcarbazepine, carbamazepine and their metabolites simultaneously. The need to monitor the plasma concentrations of both oxcarbazepine, carbamazepine and their respective metabolites arose during clinical trials in which carbamazepine was to be replaced by oxcarbazepine and vice versa. Therefore, the specific and sensitive LC assay described was developed to allow the

Simultaneous determination of clomipramine and its N-desmethyl metabolite in human whole blood by capillary gas chromatography with mass-selective detection

A method for the simultaneous determination of clomipramine and its N-desmethyl metabolite at concentrations down to ca. 2 nmol/l in human whole blood is described. After addition of a known amount of deuterium-labelled internal standards, compounds are extracted into n-heptane-isoamyl alcohol (99:1, v/v) at basic pH, back-extracted into an acidic aqueous solution and re-extracted at basic pH into n-heptane. N-Desmethylclomipramine and the internal standard are derivatized with pentafluoropropionic anhydride. The compounds are determined by capillary gas chromatography with mass-selective detection. The technique was applied to determine the human blood concentrations of clomipramine and its N-desmethyl metabolite after oral administration of Anafranil; mean blood concentrations are reported.

Determination of oxiracetam in plasma and urine by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic method was developed for the determination of oxiracetam in plasma and urine. A sample of plasma (250 microliters) or urine (10 microliters) is mixed with the internal standard solution, 4.2 ml of acetonitrile-water (1000:4, v/v) and 0.8 ml of dichloromethane, and 1 ml of the clear solution is injected onto a first column filled with Li-Chrosorb NH2. The sample is eluted with acetonitrile-water (95:5, v/v). The portion of the eluate (heart-cutting) from this column containing the compounds of interest is selected and loaded on a Nucleosil NH2 column and eluted with acetonitrile-water (90:10, v/v). During this chromatography the first column (LiChrosorb NH2) is rinsed with acetonitrile-water (50:50, v/v). Ultraviolet detection at 200 nm is used for quantitation. The limit of quantitation of oxiracetam is ca. 1.5 microM (240 ng/ml) in plasma and 76 microM (12 micrograms/ml) in urine. Oxiracetam was stable in plasma and urine samples kept frozen at -20 degrees C for nine months and one year, respectively.

High-performance liquid chromatographic determination of the penem antibiotic (5R,6S)-2-aminomethyl-6-[(1R)-hydroxyethyl]-2-penem-3-carboxylic acid in human plasma and urine

High-performance liquid chromatographic methods for the determination of (5R,6S)-2-aminomethyl-6-[(1R)-hydroxyethyl]-2-penem-3-carboxylic acid in plasma and urine have been developed, validated and applied to clinical samples. After addition of another penem, (5R,6S)-2-aminoethyl-6-[(1R)-hydroxyethyl]-2-penem-3-carboxylic acid, as an internal standard, plasma proteins are precipitated with a saturated solution of ammonium sulphate. A portion of the supernatant is injected on to a reversed-phase column (RP 8-10 micron) that is eluted with pH 6 phosphate buffer. The urine assay entails a 25-fold dilution with pH 6 buffer and addition of the internal standard prior to injection. The detector response at 320 nm is a linear function of concentration over the ranges 1.6-410 mumol/l (0.4-100 micrograms/ml) and 41-1025 mumol/l (10-250 micrograms/ml) for the plasma and urine assays, respectively. These methods have proved to be suitable for pharmacokinetic investigations in man.

Determination of the two dinitate metabolites of nitroglycerin in human plasma by capillary gas chromatography with electron-capture detection

This paper describes a sensitive method for the specific determination of 1,2-glyceryl dinitrate and 1,3-glyceryl dinitrate as metabolites of nitroglycerin at concentrations down to 250 pg/ml plasma. After addition of a known amount of 2-isosorbide mononitrate as internal standard, plasma is introduced onto an Extrelut cartridge and the compounds of interest are eluted with dichloromethane. The glyceryl dinitrates are then quantitated by capillary gas chromatography with electron-capture detection.

Determination of xylometazoline in plasma and urine by gas chromatography using a fused-silica capillary column and an electron-capture detector.

A sensitive method is described for the determination of unchanged xylometazoline in plasma and urine at concentrations down to 35 nmol/l. After addition of naphazoline as an internal standard, both compounds are extracted with dichloromethane-diethyl ether (20:80) at pH 10, back-extracted with an acidic solution and re-extracted from a sodium hydroxide solution with dichloromethane-diethyl ether (20:80). The compounds are then derivatized with heptafluorobutyric anhydride in the presence of pyridine. The derivatives are determined by capillary gas chromatography using electron-capture detection.

High-performance liquid chromatographic determination of pirprofen and five of its metabolites in human plasma without hydrolysis and in human urine before and after chemical hydrolysis

Selective high-performance liquid chromatographic methods for the simultaneous determination of pirprofen and five of its metabolites either in plasma or in urine before and after chemical hydrolysis were developed. After addition of an internal standard and a buffer, the compounds were extracted from plasma using reversed-phase C18 Bond-Elut columns and from urine using pre-packed silica Extrelut 1 columns, back-extraction into sodium hydroxide and acidification of the alkaline phase before injection. Pirprofen, its five metabolites and the internal standard were separated using a linear elution gradient chromatographic system and wavelength programming. The analysis of spiked samples demonstrated the good accuracy and precision of the methods with limits of quantitation of 100 or 200 ng/ml for the different compounds in plasma, 200 or 360 ng/ml in urine without hydrolysis and 1 or 1.8 micrograms/ml in urine after chemical hydrolysis.

The Routine Use, and Column-Stability Implications, of Several Column-Switching HPLC Methods for Determining Drugs in Plasma and Urine

Several possible configurations can be used for column-switching systems: trace enrichment on a pre-column and automatic back-flush or forward-flush injection of the purified sample on an analytical column; injection of the sample onto one column and automatic selection of a portion of the eluate (heart-cutting) for chromatography on a second column; combination of these two systems: trace enrichment and heart-cutting. These approaches are briefly described for metoprolol, CGP 6140 and oxiracetam.