Determination of ferrocene-a in blood serum by reversed-phase HPLC using an immobile phase of the C8 type

Abstract

Dihydroxyacetylene compounds of ferrocene are suecessfully used for the prophylaxis of malignant tumors [1]. The most pronounced activity was reported for ferrocene-A (l-ferrocenyl-l-phenyl-2-butin-l,4-diol, FA): the joint introduction of this compound with carcinogens into experimental animals prevented development of the malignant process. FA was also reported to produce an antibacterial effect [2]. In order to elucidate mechanisms of the pharmacological action of FA and study the FA pharmacokinetics, it is necessary to develop a method for determination of this compound in biological fluids.The best method for determining trace amounts of organic compounds in biological objects is high-performance liquid chromatography (HPLC), which is capable of distinguishing a given substance from its metabolites or endogenic compounds present in the blood serum. However, the retention of FA in the normal-phase regime (with a Silasorb-600 silica gel stationary phase) is hindered by a nonpolar character of the FA molecules. The hydrophobicity of FA is caused by the presence of eyelopentadiene nuclei, as well as phenyl and methine groups, in the molecule. For this reason, we have used HPLC in the reversed-phase mode with an immobile phase of the Cs type. This sorbent is characterized by the moderate polarity and binds hydrophobic compounds less tightly than does the Cl8 sorbent, ensuring the maximum retention [3].The experiments were performed on an automatically controlled Milikhrom-4 ("Nauchpfibor" plant, Orel, Russia) equipped with a UV detector. A metal column (62 x 2 mm) was filled with a Silasorb Cs immobile phase having a particle size of 5 pan (Lachema, Brno, Czech Republic). The "dead" volume of the column was determined by introducing a few microliters of sodium bromide. The mobile phase was selected by testing various ethanol-water, acetonitrilewater, and ethanol -0.05 M KaHPO4 mixtures with different component ratios; the best results were obtained for the I Dzhavakhishvili State University, Tbilisi, Republic of Georgia. 282 55 : 45 ethanol -water mixture. The mobile phase was supplied at a rate of 50 ~tl / rain. The sample detection was performed at a wavelength of 210 nm. The I-IPLC analysis was performed at room ternperamre. The internal standard was 5ethyl-5-(2-pentyl)barbiturate sodium (nembutal, NB).FA was extracted from a sample of blood serum by the method of liquid-liquid extraction using a hexane-isoamyl alcohol mixture (98 : 2). To 0.5 ml of the blood serum was added 0.1 ml of an NB solution, 0.5 rnl of 1 M NaOH, and 3 ml of the hexane-isoamyl alcohol extraetant. This mixture was shaken manually for I rain and then centrifuged for 10 min at 3000 rpm. The upper organic layer was transferred to another tube and dried in a weak air flow at 40~ on a water bath. The dry residue was dissolved in 50 lal of the mobile phase, of which 25-30 ~tl was applied onto the chromatographic column.Acceptable values of the capacity factor for the Ca immobile phase were obtained using the ethyl alcohol-water (55 : 45) and a...