Large Volume Injection of Biological Samples Dissolved in a Non-Eluting Solvent: A Way to Increase Sensitivity and a Means of Automating Drug Determination Using Hplc

Abstract

The injected volume of a sample dissolved in the mobile phase of an HPLC system must be maintained as small as possible so as to minimize the loss in efficiency. Generally this requirement limits the sensitivity of HPLC methods devoted to trace quantity determinations of drugs in biological fluids. In order to avoid this limitation and to increase the effective sensitivity of HPLC methods for determination of drugs such as antrafenine, nifuroxazide and cipropride, the samples were dissolved in a non-eluting solvent and a large volume (> 100 μl) was injected on to the chromatographic column.

The above-mentioned compounds and their internal standards were dissolved in a series of eluting and non-eluting solvents and increasing volumes (5 to 1000 μl) were injected. Peaks corresponding to injections made in an eluting solvent showed retention times independent of the injection volume but their variances increased with the volume injected. In contrast, peaks corresponding to injections made in a non-eluting solvent, similar to the mobile phase, had a variance independent of the injection volume but their retention times increased linearly with the injection volume. The repeated injection of such non-eluting solvents had no influence on chromatographic behaviour. Peaks corresponding to compounds injected in a non-eluting solvent made with components different from those of the mobile phase had a variance independent of the injection volume but their retention times varied both with the injection volume and with the interval between injection.

The application of non-eluting solvents has been defined theoretically and it has been demonstrated that solutions composed of 25% of the mobile phase diluted with the least eluting of its components act as non-eluting solvents and can be injected in large volume without loss in efficiency. This feature could be used to inject all the samples volume or only part of it, manually or automatically, since any automatic injector can be used with large volumes.

Thus, using the relatively simple procedure of making injections with a non-eluting solvent it is possible to increase both sensitivity and the rate of sample analysis.