Different assays for hingamine in the study of biological fluids

The authors present a method of hingamine identification in non-biological substances (tablets, powder, syringes) and biological fluids (blood, urine). Isolation was made with chloroform in pH 10. Identification was conducted with thin-layer chromatography, gas chromatography/mass-spectrometry, high-performance liquid chromatography, spectrophotometry in UV region. The quantity was estimated with spectrophotometry in UV region, high-performance liquid chromatography and high-performance thin-layer chromatography. The results of the three methods are comparable.     

The use of enzymatic hydrolysis for isolation of barbituric acid derivatives from blood (as exemplified by phenobarbital and barbamyl)

Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented.     

Assessment for efficiency of isolation of toxic substances from blood

The authors gave assessment for efficiency of existing methods of isolation of toxic substances from blood with model substance. Some validation characteristics of the method of direct extraction from blood with organic solvent were determined.     

Isolation of blood caffeine as a model substance by enzymatic hydrolysis

Organic solvent extraction of toxic compounds from blood either directly or following precipitation of serum proteins yields only free fractions. It is proposed to dissociate toxin-protein complexes in blood samples by enzymatic hydrolysis. Model caffeine complexes in an albumin solution were treated by tripsin, chemotripsin, and papain. The best results were obtained using chemotripsin and papain with which 70.4 +/- 1.6% and 62.2 +/- 1.2% of the caffeine was isolated from blood compared with 45.6% by direct extraction. Validation characteristics of the proposed method are specified.