High-performance liquid chromatography of the anti-tumour agent triethylenethiophosphoramide and its metabolite triethylenephosphoramide with sodium sulphide, taurine and o-phthalaldehyde as pre-column fluorescent derivatization reagents.

The method described is based on the reaction of triethylenethiophosphoramide (ThioTEPA) and triethylenephosphoramide (TEPA), through their ethyleneimine groups, with sodium sulphide, taurine and o-phthalaldehyde to give fluorescent products, and separation of the derivatives by reversed-phase high-performance liquid chromatography. The method was successfully applied to the determination of ThioTEPA and TEPA in rabbit plasma samples after clean-up with an Extrelut 3 column. The recoveries of ThioTEPA and TEPA from plasma were 66.1-80.3% and the limits of determination in plasma were ca. 10 and 20 ng/ml, respectively.

Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase.

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.

Gas chromatographic analysis of triethylenethiophosphoramide and triethylenephosphoramide in biological specimens.

Comprehensive pharmacokinetic studies could realise a greater potential for the antitumour agent triethylenethiophosphoramide (ThioTEPA), and these would be aided by the development of a selective and sensitive assay. After extraction of ThioTEPA and its metabolite, triethylenephosphoramide (TEPA), from plasma using Sep-Pak C18 cartridges, the compounds were separated by capillary chromatography, detected using a nitrogen detector and quantified by reference to an internal standard, hexaethylphosphoramide. The limits of sensitivity were 1-5 ng/ml. Analytical recoveries were 74 and 95%, for TEPA and ThioTEPA, respectively, in the therapeutic range. At similar concentrations, extents of protein binding, determined by ultrafiltration, were not significant. Preliminary investigations of the elimination of ThioTEPA show that drug loss occurs more quickly in mice than in humans and in both species the metabolite is extensively recycled.

[Gas-liquid chromatographic assay of a new antineoplastic agent, 1,3,3,5,5-pentakis-(azaridino)lambda 6-2,4,6,3 lambda 5, 5 lambda 5-thiatriazadiphosphorine-1-oxide. Application to pharmacokinetic studies]. / Dosage par chromatographie gaz-liquide d'un nouvel antitumoral, 1,3,3,5,5-pentakis-(azaridino)-lambda 6,2,4,6,3 lambda 5,5 lambda 5-thiatriazadiphosphorine-1-oxyde. Applications aux études pharmacocinétiques.

A method for the assay of 1,3,3,5,5-pentakis-(azaridino)-lambda 6,2,4,6,3 lambda 5,5 lambda 5-thiatriazadiphosphorine-1-oxide (SOAz), a new anticancer drug of which the clinical trials are in progress, is described. This method is based on capillary gas chromatography using a thermionic detector. The lower detection limit was 100 pg per injection and a coefficient of variation smaller than 5% could be obtained when parathion was used as external standard. The method is suitable for biological samples and therefore has been proposed for clinical pharmacokinetic studies as well as for the determination of patterns of SOAz distribution in several organs of the mouse. A preliminary clinical study showed that the serum decay curves of SOAz could be fitted to an open two-compartment model for drug disappearance.