The express method of bisaprolol determination in substance and blood on hydrolysis products by gas chromatography has been developed. The optimal conditions of chromatography have been chosen.
Key words: beta-blockers,bisoprolol,gas-liquid chromatography,sodium nitrite.
Beta-blockers are widely used in the treatment of cardiovascular diseases, and the most cardioselective one is bisoprolol. The standard dose of the drug has little effect on blocking beta-2- adrenergic receptors and therefore devoid of many unwanted effects. Bisoprolol in therapeutic doses (2,510,0 mg/day) did not induce bronchoconstriction and does not violate the respiratory function in patients with chronic obstructive pulmonary disease (COPD). First pass effect through the liver is expressed very little, whereby the drug has a high bioavailability - about 90%. More than 95% of active substance excreted by the kidneys, 50% - unchanged.
Along with this drug in overdose shows bradycardia, conduction disturbances of cardiac rhythm, causing exacerbation of symptoms of chronic heart failure. Beta-blockers can be detected with a standard toxicological studies, but methods for measuring serum concentrations for most of them do not exist.
Therefore, the development of valid and reliable methods for the identification of bisoprolol from the blood is an urgent problem (Khodasevich A.P. et all., 1983; Anokhin V.T. et all., 1983; Belson et all., 2000; Kutyakov V.A., 2006; Semyanov et all., 2007; Knaub V.A., 2009; Lagutkina T.P. et all., 2011).
The aim of this research is to develop methods of identification of bisoprolol in a biological fluid by gasliquid chromatography.
Materials and methods. We used drug substance bisoprolol (British Pharmacopoeia, 2009.-V.I & II); solvents and reagents of the category —reagent grade" and —analytical grade". Used for the identification of a gas chromatograph —Crystal lux" -4000m. Temperature of the injector is 70-120 C, sample injection volume 0.5 cm3, type of detector - TCD, carrier gas - helium, carrier gas flow rate through the column - 2030 cm3/min. We used a packed column length of 2 m, diameter 3 mm, sorbent: Phase 7% PFMS-4 solid support Chromaton AW-DMCS fraction of 0.16 - 0.20 mm.
Results and discussion. The proposed method is based on the hydrolysis of bisoprolol with hydrochloric acid in the presence of sodium nitrite with propyl alcohol as the internal standard.
Gas chromatographic properties of the resulting hydrolysis product, as well as various experimental parameters affecting the stability and chromatographic separation of the obtained product were thoroughly studied and optimized. Trichloroacetic acid and hydrochloric acid were studied as derivatizing agents.The maximum chromatographic separation occurs by adding 10 ml of 2 M hydrochloric acid to the sample containing the substance to 1 mg/ml and adding 0.5 ml of sodium nitrite. Retention time of the obtained product is 1.11 min.
The developed technique used by us for the detection of bisoprolol in blood. For the preparation of mixtures used in the model whole blood samples obtained from healthy volunteers who were not taking drugs within a month prior to sampling. Modeling mixture of blood prepared by adding solutions containing substances at concentrations of 0.5; 1.0; 2,0; 5.0 ug/ml. The resulting solution was then transferred into vials and added to them in varying proportions of trichloroacetic acid, and propyl alcohol (internal standard) with sodium nitrite. In a medium TCA observed poor chromatographic separation of the peaks. Therefore, we used the hydrochloric acid, which showed highseparate ability. The retention time of the product obtained in the conditions we have developed methods for the substance and for the same biological fluid and is 1.11 m.
Conclusions. Wesuggest rapid procedure of bisoprolol detection in biological fluids using gas chromatography, allowing to identify the study drug with a minimum expenditure of time and in small quantities.
1. J. Bhatt, G. Subbaiah, S. Kambli, et al., —A high throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of bisoprolol in human plasma using multiplexing technique," Journal of Chromatography B, vol. 852, no. 1-2, pp. 374-381, 2007