Automated liquid chromatographic analysis of the anti-tumorigenic drugs etoposide (VP 16-213) and teniposide (VM 26).

A method is described for the fully automated analysis of large numbers of 1--2 ml serum and plasma or urine samples containing the anti-tumorigenic drugs etoposide and teniposide and their aglycone. The blood samples are hydrolysed by a proteolytic enzyme, subtilisin A, prior to preconcentration on a small precolumn. The hydrolysis step serves both to release the strongly protein-bound drugs and to prevent clogging of the chromatographic system. On-line preconcentration is carried out with precolumns packed with PRP1, a micro-particulate divinylbenzene-styrene copolymeric sorbent. Chromatography takes place, after column switching, in a C18/methanol--water system. After a post-column clean-up step using continuous extraction with dichloroethane in an autoanalyzer system, native fluorescence of these analytes is used for detection of the drugs. Recovery of etoposide and teniposide from spiked serum and plasma samples was 100%. Calibration curves of etoposide and teniposide typically show correlation coefficients of 0.9994 over a two-to-three order linear range. The detection limit of etoposide is approx. 8 ng per sample. Repeatability was found to be excellent. Unattended overnight routine analysis is possible without any problems. This method, considering optimal sample throughput, reliability and selectivity, competes favourably with existing techniques for the analysis of etoposide and teniposide.

Automated determination of drugs in blood samples after enzymatic hydrolysis using precolumn switching and post-column reaction detection.

Enzymatic hydrolysis of blood samples with subtilisin-A releases protein-bound drugs and permits the repeated (10-50 times) injection of up to 1-ml volumes on short (2-30 mm) precolumns without appreciable build-up of pressure or loss of performance of the precolumn. The principle of fully automated serum and plasma analysis is demonstrated with the drug secoverine as a model compound. After enzymatic hydrolysis of the sample with an equal volume of a 1 mg/ml solution of subtilisin-A for 15 min at 55 degrees C, the model compound is preconcentrated using a microprocessor-controlled column switching unit. Separation occurs in a reversed-phase liquid chromatographic system using a CN-type stationary phase and a buffered aqueous dioxane solution as mobile phase. Detection is done by UV spectrophotometry or fluorometrically after post-column ion-pairing reaction with dimethoxyanthracenesulphonate. The relative standard deviation of the procedure is less than +/- 6% (n = 10).

Liquid chromatographic detector for organosulphur compounds based on a ligand-exchange reaction.

A method is described for the post-column reaction detection of organosulphur compounds in liquid chromatography, which is based on a ligand-exchange reaction between the palladium(II)-calcein complex and the sulphur-containing compounds. The release of free calcein provides an indirect measure of the amount of organosulphur compounds via fluorescence detection. The kinetics of the reaction and the signal intensity have been studied as a function of the structure of selected organosulphur compounds and of parameters such as carrier- and reagent-stream composition and temperature. Optimal conditions are as follows: an aqueous mobile phase of pH 5-7, to which may be added up to 20% of methanol; an elevated reaction temperature of around 60 degrees C and a palladium(II)-calcein reagent solution of ca. 10(-5) M, which should contain some zinc(II) to enhance the fluorescence intensity. Detection limits, which are dependent on the structure of the compounds, typically are between 0.5 and 1.0 ng. Good linearity is observed over a two- to three-order concentration range. The method has been applied to the determination of ethylene thiourea in e.g. wash-water of apples and tomatoes, and to the detection of penicillamine in spiked serum and urine.