DEVELOPMENT OF THE G C -M S /M S METHOD FOR QUALITATIVE AND QUANTITATIVE DETERMINATION OF CLOZAPINE IN HUMAN BLOOD.

A simple, rapid, selective, sensitive gas chromatography-mass spectrometry (GC-MS/MS) method was developed and validated for the determination of clozapine in human blood. For isolation was used solid phase extraction. The calibration standards range was 5-1500ng/mL. Analysis was carried out on a gas chromatograph Agilent 7000A Quadrupole GC-MS/MS. GC run conditions - capillary column HP-5MS, 30m × 0.25mm, coated with a 0.25µm film, column temperature from 50°C to 305°C with an increase of 10°C/min; the injection port and the transfer line temperature were 310°C; carrier gas - helium at flow rate of 1.0 mL/min; Ionization was performed in the positive mode (EI+). Acquisition was made in TIC and MRM mode, electron impact 70Ev. Quantitative analysis was carried out by recording ions m/z 326 → 256, m/z 326 → 243 and m/z 326 → 192 for clozapine. The method was validated by investigating the following parameters: selectivity, linearity, accuracy, precision, identification of LOD and LLOQ and recovery. Method, given in this paper can be useful for determination of clozapine in forensic investigations and for TDM of schizophrenic patients to improve therapeutic efficacy during clozapine treatment.

DEVELOPMENT OF LC-MS/MS METHOD FOR DETERMINATION OF THE OLANZAPINE IN HUMAN PLASMA.

The study aim was to develop a validated selective, reproducible and sensitive Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) method for qualitative and quantitative determination of olanzapin (OLZ ) in human plasma. For isolation was used solid phase extraction with the Gilson GX-271 ASPEC Solid phase extraction system, cartridge Supelclean LC-18 SPE 500mg. Elution solvent contained 40mL of isopropanol, 4mL of concentrated ammonium hydroxide and 156mL of freshly prepared methylene chloride. Samples containing OLZ and IS were chromatographed on AGILENT TECHNOLOGIES 1290 Infinity AGILENT TECHNOLOGIES 6460 Triple quad LC-MS/MS system. Separation was performed by isocratic elution on Zorbax Eclipse plus C18 (100´4.5 mm, 3.8 µm) column, equipped with pre-column: UHPLC GUARD Zorbax Eclipse plus C18 (5×2.1 mm, 3.8 µm); column temperature was 35°C. The mobile phases consisted of 0.1 % water solution of formic acid HCOOH (H2O):0.1 % acetonitrile solution of formic acid HCOOH (CH3CN) in 70 :30 (v/v) proportion. The flow rate was 0.4 mL/min. Determination was performed on a triple-quadrupole mass spectrometer employing electrospray ionization technique (ESI+), operating in multiple reaction monitoring (MRM) and positive ion mode. Total chromatographic run time was 5 min. The optimized MRM transitions for OLZ were m/z 313 ® 256, m/z 313 →213 and m/z 313→198 as the quantitative and confirmative traces, respectively. Method was validated on following parameters: specificity, linearity, LLOQ, accuracy and precision, recovery, matrix effect and stability. The method was subsequently applied to in vitro plasma samples for determination of OLZ. The obtained data allows the opportunity for forensic investigation of the intoxication in postmortem cases.

Analysis of Perilla nankinensis decne essential oil using gas chromatography coupled with time-of-flight mass spectrometry.

Perilla is the genus of herbaceous plants of Lamiaceae family. The essential oil of Perilla is believed to be essential part of the herb. It was claimed to have anti-inflammatory, anti-hyperlipidemia, antioxidative and antimicrobial activities. Apart from pharmaceutical and nutrition purposes, It is an edible plant frequently used as a fresh vegetable. It was also applied to produce perfume and cosmetics. Due to the importance of the essential oil from the P. nanakinensis and the lack of detailed studies of them, our work aimed investigation of contents of essential compositions by GC- TOF/MS method. The materials of the study were the aerial parts of P. nankinesis collected at full flowering stage, Guria Region, Georgia. A qualitative analysis of the individual compounds in P. nankinensis essential oil performed by gas chromatography (GC) coupled with time-of-flight mass spectrometer (TOF/MS) for the identification of the resolved peaks. 28 components, which accounted for 77,7% of the oil, were identified. The main components of this essential oil were 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-, (Z,E)- (α-Farnesene) (34.3%), Caryophyllene oxide (10.2%), 1,6-Octadien-3-ol, 3,7-dimethyl- (linalool) (10.2%). Essential oil also contained humulene (3.9%),caryophyllene (1.9%), methyl salicilate (1.15%), 2-hexanoylfuran (1.0%), gernacrene (1.1%). Based on the results of our experimental data and literature reviews, Perilla essential oil can represent special interest and can be used as an important natural substance for obtaining medicinal and preventive products with the several biological activities.

The efficacy of topical drugs in treatment of noninflammatory acne vulgaris.

Treatment of Acne Vulgaris still remains as an actual problem. The purpose of the research was to study the curing process and side effects of topical drugs - Diacneal and Skinoren in patients with non-inflammatory acne vulgaris. The results of the research has confirmed the swift and stable treating effect of both preparations, that expressed in decrease in number and size of comedonal and papulo-comedonal formations, actually without side effects. Thus, in spite of divergence of either chemical composition or mechanism of action, both, Diacneal and Skinoren, may be recommended as a very good choice for monotherapy of non-inflammatory acne vulgaris.

Optical spectroscopy of parotid gland in case of adenopathy.

Reflectance and laser induced fluorescence spectroscopies were employed for the first time in a limited pilot study to probe freshly excised human parotid tissues (adenomatous and adjacent). In human parotid tissue, measurements were associated with endogenous fluorophores NADH and collagen, as well as tissue optical properties, with larger relative collagen content detected from normal to adenomatous tissues. The observed significant differences between the fluorescence and reflectance properties of normal, and adenomatous tissues present an opportunity for future statistical validation on a larger patient pool and indicate a potential application of multimodal optical spectroscopy to differentiate between pathological and normal parotid tissue states at different stages of disease.

[Methemoglobin and osmoresistance of erythrocytes in patients with different types of goiter].

The purpose of this study was to determine the level of methemoglobin in the blood and osmoresistance of erythrocytes in patients with different types of goiter and control subjects. Investigated parameters indicate to the antioxidant status of human organism. We examined 12 healthy controls and 49 hyperthyroid patients: 17 patients with nodular goiter (mild thyrotoxicosis), 22 patients with diffuse toxic goiter (high level of thyrotoxicosis) and 10--with euthyroid nodule goiter. Osmoresistance of erythrocytes was measured by differential photoelectrocolorymetric method and methemoglobin--by spectroscopic method. We had received significant correlation between the level of methemoglobin, osmoresistance of erythrocytes and the degree of thyrotoxicosis. The results once again prove sensitivity of antioxidant status in response to toxicity, in particular against thyrotoxicosis.

[Influence of 4-nitrophenol on some biochemical and morphological indices of rat blood].

Influence of different doses of 4-nitrophenol on some biochemical and morphological indices of rat blood was studied. Glucose and beta-lipoprotein concentration increased, but total protein concentration decreased in response to intraperitoneal injection of xenobiotic. As a result of xenobiotic action, the total amount of leukocytes and thrombocytes increased, while the amount of erythrocytes decreased. The morphological study of structural indices of blood formed elements has demonstrated, that intensity of the defensive response of blood formed elements was 4-nitrophenol dose-dependent.

[Vitamins C, B12 and folic acid in latent iron deficiency].

The aim of the work was to study the level of vitamins C, B12 and folic acid in latent iron deficiency of different etiology (hipo- and anacid gastritis and menorrhagia). 81 patients with latent iron deficiency were investigated. Vitamin C levels were measured by refractometry, folic acid and vitamin B12 by radioimmune assay. The obtained results showed significant decrease of ascorbic acid and less apparent decrease of folic acid in the blood plasma. The content of vitamin B12 was unchanged. Decrease of vitamin C level is related to the changes of initial stages of iron metabolism and its further absorption. Less apparent changes of folic acid and vitamin B12 indicates, that such important stages of erithropoesis as DNA replication and cell proliferation are intact. Our results indicate to the development of metabolic disorders prior to revelation of iron deficiency anaemia, which need timely correction.

Hysteretic interaction of NADH and Mg2+ with mammalian NADH:CoQ reductase from beef heart.

Preincubation of submitochondrial particles (SMP) from beef heart in a reaction mixture containing low concentrations of Mg2+ induces a time lag in the NADH:oxidase activity. Preconditioning of the SMP by NADH, but not by NAD+, prevents the Mg2+-related time lag. The data obtained show that there exists a tight binding site for Mg2+ regulating the rate of electron transfer from NADH to the natural acceptor. The ability of Mg2+ to form a catalytically inactive complex with the enzyme is regulated by NADH.

Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase.

A simple procedure for preparation of highly purified soluble succinate-ubiquinone reductase from bovine heart mitochondrial particles is described. The enzyme exhibits four major bands on sodium dodecyl sulfate gel electrophoresis and contains (nmol per mg protein): covalently bound flavin, 6; non-heme iron, 53; acid-labile sulfur, 50; cytochrome b-560 heme, 1.2. The enzyme catalyzes thenoyltrifluoroacetone, or carboxin-sensitive (pure non-competitive with Q2) reduction of Q2 by succinate with a turnover number close to that in parent submitochondrial particles. The succinate reduced enzyme exhibits ferredoxin-type iron-sulfur center EPR-signal (g = 1.94 species) and a semiquinone signal (g = 2.00). An oxidized preparation shows a symmetric signal centered around g = 2.01. An unusual dissociation of the enzyme in the absence of a detergent is described. When added to the assay mixture from a concentrated protein-detergent solution, the enzyme does not reduce Q2 being highly reactive towards ferricyanide ('low Km ferricyanide reactive site'; Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Commun. 65, 1264-1269). The ubiquinone reductase, not the ferricyanide reductase was observed when the enzyme was added to the assay mixture from the diluted protein-detergent solutions. Thus the dissociation of succinate dehydrogenase from the complex occurs in the absence of a detergent dependent on the concentration of the protein-detergent complex in the stock preparation where the samples for the assay are taken from. An active antimycin-sensitive succinate-cytochrome c reductase was reconstituted by admixing of the soluble succinate-ubiquinone reductase and the cytochrome b-c1 complex, i.e., from the complexes which both contain the ubiquinone reactivity conferring protein (QPs). Cytochrome c reductase was also reconstituted from the succinate-ubiquinone reductase and succinate-cytochrome c reductase containing inactivated succinate dehydrogenase. The reconstitution experiments suggest that there exists a specific protein-protein (or lipid) interaction between QPs and a certain component(s) of the b-c1 complex.

Pyridoxal phosphate-induced dissociation of the succinate: ubiquinone reductase.

Treatment of the soluble ubiquinone-deficient succinate: ubiquinone reductase with pyridoxal phosphate results in the inhibition of the carboxin-sensitive ubiquinone-reductase activity of the enzyme. The inactivation is prevented by the soluble homolog of ubiquinone (Q2) but is insensitive to the dicarboxylates interacting with the substrate binding site of succinate dehydrogenase. The reactivity of the pyridoxal phosphate-inhibited enzyme with different electron acceptors suggests that the observed inhibition is due to the dissociation of succinate dehydrogenase from the enzyme complex. The soluble succinate dehydrogenase was recovered in the supernatant after treatment of the insoluble succinate: ubiquinone reductase with pyridoxal phosphate. The data obtained strongly suggest the participation of amino groups in the interaction between succinate dehydrogenase and the ubiquinone reactivity conferring peptide within the complex.