Quantitative determination of diazoline in blood serum

Abstract

Diazoline, a 1,2,3,4-tetrahydrocarboline derivative, is capable of blocking Hwhistamine receptors and is used in medicine for the treatment of various allergic diseases [1].Specialists of the Pyatigorsk State Pharmaceutical Academy have developed a solid medicinal form ofdiazoline (tablets) based on the diazoline -13-cyclodextrin interaction.In order to study the pharmacokinetic characteristics of the new medicinal form, it was necessary to develop a method for monitoring the concentration of diazoline in the blood. The method based on chromatodensitometric analysis suggested in [2] is very cumbersome and labor-consuming. Moreover, in our opinion, the proposed technique does not ensure sufficiently complete isolation of diazoline from the biological liquid. For this reason, we have developed a new rapid technique for the quantitative determination of diazoline in the blood. We have selected HPLC as an analytical tool, since this method is now most widely used in drug analysis. EXPERIMENTAL PARTThe HPLC analyses were carried out on a Milikhrom-2 chromatograph (Russia) equipped with an UV detector and a KAKh 4-64-3 column filled with Separon C18.In order to select the optimum chromatographic conditions, we have preliminarily studied the retention time of diazoline and the coefficient of asymmetry of the corresponding peak as functions of the mobile phase composition.The chromatography was performed in the following regime: detection wavelength, 286 nm (maximum UV absorption of diazoline); scale factor, 1.6; eluent supply rate, 50 pl/min; chart velocity, 0.6 cm/min.As the mobile phase, we have studied aqueous ethyl alcohol solutions of various concentrations (60-90%). The samples for analysis were 2/al aliquots of a diazoline solution in DMSO with a concentration of 0.5 -5 mg/ml. I Pyatigorsk State Pharmaceutical Academy, Pyatigorsk, Russia.It was found that the HPLC chromatogram of diazoline has a single peak with the retention time and the asymmetry coefficient directly depending on the ethanol concentration in the mobile phase. The area under the peak is proportional to the diazoline solution concentration.The results of preliminary measurements showed that the optimum concentration of ethanol in the mobile phase is 80-90%, which corresponds to a retention time of 5.8 -6.5 min. Unfortunately, the peak of diazoline obtained for this eluent overlapped with peaks of the blood serum components and we had to reduce the ethanol concentration to 65-75%. However, this change led to asymmetry of the HPLC peak of diazoline. In order to improve the peak shape we have employed modifiers-glacial acetic acid (providing the regime of ionization suppression) and diethylamine (converting the drug into an ionized form). The amount of modifiers added to the eluent (0.5%) agrees with the commonly accepted level [2].Suppression of diazoline ionization in the presence of glacial acetic acid neither improved the peak shape nor 11 I1