Development and validation of a HPLC-MS/MS method for the analysis of fatty acids - in the form of FAME ammonium adducts - in human whole blood and erythrocytes to determine omega-3 index.

Recent scientific studies in the field of health and nutrition have unanimously affirmed the importance of consuming the omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), because of their cardioprotective properties. Fatty acid profiling in erythrocyte membranes allows the omega-3 index, which is a recognized indicator of the risk of developing cardiovascular disease, to be calculated. One consequence of the upward trend in healthy lifestyles and longevity is an increase in the number of studies into the omega-3 index, which requires a reliable method for the quantitative analysis of fatty acids. This article describes the development and validation of a sensitive and reproducible liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the quantitative analysis of 23 fatty acids (in the form of fatty acid methyl esters, FAMEs) in 40 µl of whole blood and erythrocytes. The list of acids includes saturated, omega-9 unsaturated, omega-6 unsaturated and omega-3 unsaturated fatty acids as well as their trans-isomers. The limit of quantitation was 250 ng ml-1 for C12:0, C16:0 and C18:0; and 62.5 ng ml-1 for other FAMEs, including EPA, DHA and trans-isomers of FAME C16:1, C18:1 and C18:2 n-6. Sample preparation for fatty acid (FA) esterification/methylation with boron trifluoride-methanol (BF3) has been optimized. Chromatographic separation has been carried out on a C8 column in gradient mode using a mixture of acetonitrile, isopropanol and water with the addition of 0.1% formic acid and 5 mM ammonium formate. As a result, the problem of separating the cis- and trans-isomers of FAME C16:1, C18:1 and C18:2 n-6 has been solved. The electrospray ionization mass spectrometry (ESI-MS) detection of FAMEs, in the form of ammonium adducts, has been optimized for the first time, which has made the method more sensitive that when the protonated species are used. This method has been applied to 12 samples from healthy subjects that consumed omega-3 supplements and has proven to be a reliable tool for determining the omega-3 index.

[The detection of the 25B-NBOMe derivative of phenylethylamine in the biological material]. / Obnaruzhenie 25B-NBOMe - proizvodnogo fenilétilamina v biologicheskom materiale.

This article is focused on the conditions for the detection and identification of 2-[4-bromo-2.5-dimethoxyl]-N-[(2-methoxyphenyl)methyl] ethamine (25B-NBOMe) and its major metabolites by the combination of the HPLC/MS/MS techniques. The high-resolution mass spectra obtained with the use of a linear ion trap are described. The results of the study give evidence of the possibility for the detection of the analytes within 24 hours after drug consumption and within 3 months after the storage of the biological material of interest in a refrigerator at a temperature of 3-5 °C. The data obtained confirmed high stability of 2-(4-bromo-2.5-dimethoxyl]-N-[(2-methoxyphenyl)methyl] ethamine and its metabolites in the biological tissues.

[The development and validation of the method for the identification of ethyl glucuronide and ethyl sulfate as the markers of the consumption of ethyl alcohol during one's lifetime]. / Razrabotka i validatsiia metodiki opredeleniia étilgliukuronida i étilsul'fata kak markerov prizhiznennogo upotrebleniia étilovogo spirta.

The objective of the present study was the development and validation of the rapid reproducible method for the identification of ethyl glucuronide and ethyl sulfate allowing to store and transport the study specimens without the loss of the substances of interest by placing the samples on the paper. We have developed the validated technique for the detection and quantitative determination of ethyl glucuronide and ethyl sulfate in the cadaveric blood and urine by means of low-resolution tandem mass-spectroscopy with the use of deuterated derivatives of these substances as the internal standards. The low threshold for quantitative determination of both above substances is 50 ng/ml for the blood and 100 ng/ml for the urine. The method is characterized by the accuracy and precision with the coefficient of variation below 15% and the influence of the matrix with the coefficient of variation below 15%. The evaluation of stability of the two analytes in blood when stored in the dry condition on the paper carrier during 2 weeks showed that the coefficient of variation did not exceed 6.4%. The comparative study of ethyl glucuronide and ethyl sulfate in the samples of cadaveric blood and urine containing from 0 to 5.2% of ethyl alcohol was carried out. The methods for the transportation of the biological fluids and for the extraction of ethyl glucuronide and ethyl sulfate placed on the paper carrier (Whatman 903) have been proposed. The possibility has been demonstrated to use ethyl glucuronide and ethyl sulfate as the markers of the consumption of ethyl alcohol during one's lifetime for the purpose of investigation of the putrifactive changes of the blood components.

[The detection and identification of alpha-pyrrolidino-valerophenone (α-PVP) and its metabolites in the objects of the forensic chemical examination]. / Obnaruzhenie α-pirrolidinovalerofenona (α-PVP) i ego metabolitov v ob''ektakh sudebno-khimicheskogo issledovaniia.

The combined application of the IK 200609 chemical toxicological analyzer and the diagnostic biosensor (reagent) for the determination of synthetic cations (with due regard for compliance with the instruction for analysis) made it possible to detect during 15 minutes the presence of cations in the urine samples at the concentration of 376.64 ng/ml. This result was further confirmed by HPLC-Ms/MS and GH-MS. The use of the analyzer allowed the screening of the urine samples to be performed within several minutes at the preliminary stage of the study. A simplified method of tissue sample preparation with the use of the kits for extraction and solid-phase purification is proposed together with a variant of blood sample preparation with the use of filtration. The proposed approach can be employed for the rapid detection and identification of alpha-pyrrolidino-valerophenone (α-PVP) and its metabolites in urine, blood and tissues of various organs for the purpose of practical toxicological investigations in the framework of forensic chemical expertise.