[THE TECHNIQUE OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY FOR SIMULTANEOUS DIAGNOSTIC OF INHERENT HYPERPLASIA OF ADRENAL GLANDS TYPE I AND II].

The article considers the technique of high-performance liquid chromatography making it possible simultaneously detect cortisol, cortisone and secondary steroids in serum for consequent analysis of common reversed-phase high-performance liquid chromatography with ultraviolet under 240 nm. The liquid-liquid extraction from alkaline medium in diethyl ether The separation using column of 150x4.6 size ODS 3.5 mkm in isocratic mode. The eluent acetonitrile--0.02 M phosphate buffer pH 8.0--isopropanol (40:60:1). The application of proposed technique managed to separate cortisol, cortisone, dexamethasone, corticosterone, 11-desoxicortisol, testosterone, desoxicorticosterone, 17α-gidroxiprogesterone and androstendion in 20 minutes. The simplicity, reproducibility and sufficient selectivity and sensitivity of technique permit implement it in clinical practice for simultaneous diagnostic of inherent hyperplasia of adrenal glands type I and II.

[THE SIMULTANEOUS ANALYSIS OF FREE CATECHOLAMINES AND METANEPHRTNES IN URINE USING TECHNIQUE OF HIGHLY EFFECTIVE LIQUID CHROMATOGRAPHY WITH FLUORIMETRIC DETECTION AND SOLID PHASE EXTRACTION ON POLYMERIC SORBENT (PUROSEP-200)].

The article considers the technique of simultaneous detection of free catecholamines and free metanephrines in urine using inverse phase highly effective liquid chromatography with fluorimetric detection. The solid phase extraction was implemented on cartridges with 30 mg of hyper cross-linked polystyrene (Purosep-200). The simplicity, reproducibility and sufficient sensitivity of technique permit applying it in clinical practice to diagnose pheochromocytoma.

Lymphocyte Subpopulations and Cytokine Levels in Experimental Hyperhomocysteinemia.

Moderate exogenous hyperhomocysteinemia is associated with an increase in lymphocyte count (mainly at the expense of cytotoxic T cells), a 2-fold drop of the immunoregulatory index, and increase in TNF-α, IFN-γ, and IL-17α concentrations in the serum and in mononuclear cells. Homocysteine concentration in the mononuclears remains unchanged.

[HPLC analysis of dehydroepiandrosterone sulfate in serum with use of solid-phase extraction on hyper cross-linked polystyrene (Purosep-200)].

We have developed a simple HPLC method for analysis of the dehydroepiandrosterone sulfate (DHEA-sulfate) in serum with use a new procedure of solid-phase extraction (SPE) on hyper cross-linked polystyrene (Purosep-200) and fast chromatographic separation on the monolithic column under isocratic elution and UV detection at 200 nm. Complete SPE procedure lasts for about 7 min, chromatographic separation takes less than 6 min. Simplicity and high reproducibility of this method makes it attractive in routine clinical practice.

[Modified HPLC method of determination of the valproic acid in biological fluids].

Proposed modified HPLC method for determination of valproic acid in biological fluids. Created solid-phase extraction of valproic and heptanoic acids (internal standard, IS) on the cartridges packed hyper cross-linked polystyrene which maintain some tens extractions without losses of efficiency. Carboxylic acids are derivative with 1-(bromoacetyl)pyrene in acetone at presence of triethylamine. Chromatographic separation of derivatives is performed on Chromolith Perfonnance RP-18e columns, which packed unique monolithic sorbent. UV detection at 360 nm. Mobile phase acetonitrile - water (90:10, v/v) plus 1% isopropanol, speed flow 2000 microL/min, pressure 21 bar. Complete chromatographic cycle less than 3 minutes. Yield of IS and valproic acid (extraction plus derivatization) was 101-106%. Sensitivity (limit detection) was near 1 ng for valproic and near 0.6 ng for heptanoic acid during signal/noise ratio = 3.

[The HPLC analysis of vanillil-almond, 5-hydroxiindolacetic, homovanillic and homogentisic acids in biologic fluids in clinical laboratory].

The simple and fast HPLC technique of analysis of vanillil-almond, 5-hydroxiindolacetic, homovanillic and homogentisic acids in urine with solid-phase extraction using ultra cross-linked polystyrene (Purosep-200) is proposed. The separation on column Chromolith Performance RP-18e "Merck" 100x4.6 mm with monolithic phase-reversed silica gel in isocratic or gradient mode with ultraviolet detection under 285 nm. The isocratic mode is applied in case of ordinary analysis of vanillil-almond or homogentisic acids. The composition of eluent is isopropanol - water - TFA (1:99:0.025, v/v/v), flow speed is 1400 mkl/min; pressure is 37 bar, full separation less than in 4 min. The gradient low pressure mode is applied to analyze vanillil-almond, hydroxiindolacetic and homovanillic acids. Under this approach the switching to second eluent occurred from second minute. The composition of eluent is isopropanol-water-TFA (6:94:0.025, v/v/v), flow speed is 1400 mkl/min, pressure is 43 bar, full separation is less than in 7 min. The output (extraction share) consisted 78-113%. The simplicity reproducibility and sufficient sensitivity of technique in combination with the possibility of its application on standard chromatographic equipment (isocratic pump and ultraviolet detector) made it useful for routine clinical application.

[HPLC determination of plasma/serum homocysteine and cysteine with UV detection and solid-phase extraction on a polymeric sorbent].

Isocratic HPLC determination of plasma/serum homocysteine and cysteine with separation on reversed-phase column and UV detection at 330 nm is proposed. The mobile phase consist of acetonitrile - 0.05 M citrate-phosphate buffer with pH 2.4 - isopropanol (15:85:1, v/v/v). Full separation of cysteine, cysteamine (IS), glutathione and homocysteine was achieved within less than 10 minutes. Reduction of thiols from disulfides was performed by 1,4-dithioerithreitol, and derivatization by with Ellman's reagent [5'5-dithiobis-(2-nitrobenzoic acid)]. After that plasma/serum, containing derivatives of thiols, is cleared and concentrated on cartridge packed with 10 mg of hypercross-linked polystyrene (Purosep-200). Elution from cartridge is made with water-organic solvent (without evaporation and concentration, but without dilution), as well as waterless solvents (with evaporation and concentration). Simplicity, reproducibility in combination with high cleanliness of extracts and sufficient sensitivity (0.4 ng for homocysteine, 2 ng for glutathione and 0.2 ng for cysteine and cysteamine at a signal/noise ratio > 3), make this method suitable for routine clinical application.

[A simple and highly sensitive high-performance liquid chromatography assay of salivary putrescine and cadaverine].

A simple and highly sensitive high-performance liquid chromatography assay is proposed to test salivary diamines (putrescine and cadaverine). Derivation was carried out with the orthophthalic aldehyde 2-mercaptoethanol. A rapid purification procedure for derivatives on the cartridges packed with 10 mg of hypercrosslinked polystyrene (Purosep-200) was first developed. Separation was made on a Chromolith (Merck), 100 x 4.6 mm in size, with monolithic reverse-phase silica gel (RP-18e) in the isocratic mode with ultraviolet (UV) detection at 230 nm. The eluent contained 55% acetonitrile and 45% 0.01 M phosphate buffer pH 6.8, added by 1% of isopropanol; flow rate was 1400 pl/min; pressure was 28 bars. Complete separation of diamine derivatives lasted at least 5 min. The sensitivity of the assay with UV detection (230 nm) was about 0.1 ngfor diamines and about 0.5 ng for the internal standard (IS) at a signal/noise ratio of 3.0, which enabled diamines to be determined in I pl (0.001 ml) of saliva. The simplicity, reproducibility, and high sensitivity of the assay along with the feasibility of its application on standard chromatographic equipment (an isocratic pump and an UV detector) make it suitable for routine clinical application.

[HPLC for the diagnosis and monitoring of phenylketonuria and ketoaciduria].

A simple, rapid, and sensitive HPLC is proposed to test six amino acids in plasma/serum. Deproteinization was carried out with acetonitrile; derivation was made with the orthophthalic aldehyde 2-mercaptoethanol. Separation was accompanied on a Chromolith (Merck), 4.6 mm in size, with monolytic reverse-phase silica gel in the isocratic mode with ultraviolet detection at 230 nm. The eluent contained a 50% mixture of methanol-acetonitrile-isopropanol (90:5:5, v/v/v) and 50% 0.01 M phosphate buffer (pH 6.7); flow rate 1000 ul/min; pressure 42 bars. Complete separation lasted at least 10 min. The detection limit was about 1 ng for phenylalanine, leucine, and isoleucine and less than 0.5 ng for tryptophan, valine, and methionine at a signal/noise ratio of 3.0. The simplicity, reproducibility, and sufficient sensitivity of the technique along with the feasibility of its application on standard chromatographic equipment (an isocratic pump and a ultraviolet detector) make it suitable for routine clinical application.

[High-performance liquid chromatographic method for the determination of dihydroquercetin in extracts of medicinal plants].

Determination of dihydroquercetin in extracts of medicinal plants by a high-performance liquid chromatography is presented. The method is simple, quick, highly reproducible and can be widely applied in phytopharmacology.

[HPLC analysis of urinary metabolites of catecholamines and serotonin for routine laboratory practice].

A simple high performance liquid chromatographic technique has been developed to analyze urinary metabolites of catecholamines along with ultraviolet detection. The metabolites were extracted in ethyl acetate when a biospecimen was saturated with ammonium sulfate. Separation was effected on a column containing 5 microm of Diasphere C16 in the two-stage low-pressure gradient. The eluents were based on acetonitrile-isopropanol-0.02 M citric acid, added by potassium perchlorate, 5 g/l (3 and 10% of its organic phase). The complete chromatographic cycle was about 30 min. The extraction degree was 86-106%. The sensitivity (detection limit) of the technique at a signal/noise ratio of > 3 was 2 ng (vanilylmandelic acid), 3 ng (iso-vanilylmandelic acid) and 3,4-dioxyphenylacetic acid), 1 ng (5-hydroxyindoleacetic acid), and 5 ng (homovanillic acid). The linear dependence was in the range of 5 to 1,000 ng. The author presents his own findings and the data available in the literature on the content of metabolites of catecholamines and serotonin in healthy individuals and in patients with catecholamine-secreting tumors (pheochromocytoma and neuroblastoma).

[A simple method for determining antiepileptic preparations in biological fluids by using high-pressure liquid chromatography (HPLC)]. / Prostoi metod opredeleniia protivoépilepticheskikh preparatov v biologicheskikh zhidkostiakh s pomoshch'iu vysokoéffektivnoi zhidkostnoi khromatografii (VEZhKh).

The authors suggest a simple HPLC method with ultraviolet detection at 200 nm for finding barbiturates, diphenine, etosuccimide, and carbamazepin in plasma and saline using an original technology of liquid-liquid extraction. It allows clear chromatograms with a minimal amount of interfering compounds, a high extraction percentage (90-100%) and sufficient sensitivity to be obtained. A method has also been worked out for detecting clonazepam on the basis of solid-phase extraction (cartridge "Diapak-C16") precolumn concentration, and ultraviolet detection at 200 and 215 nm. The suggested extraction technologies also make possible successful monitoring of antiepileptics in the saline, for which the plasma/saline coefficients are calculated and the dependence between the degree of phenobarbital extraction into the saline and its pH is demonstrated.

[Analysis of functional and biochemical changes in feline cerebral hemispheres after subchronic haloperidol administration]. / Analiz funktsional'nykh i biokhimicheskikh izmenenii v polushariiakh mozga u koshek posle subkhronicheskogo vvedeniia galoperiodol.

Feline experiments have revealed that subchronic haloperidol causes asymmetric changes in the mesencephalic levels of dopamine and serotonin and impairs intrahemispheric balance between the dopaminergic and serotoninergic systems. At the same time the neuroleptic increases motor asymmetry, which appears in response to apomorphine, and changes the nature of interhemispheric relationships. The findings suggest that these changes may be one of the causes of extrapyramidal disorders associated with long-term use of the neuroleptic.

[Cavinton in the prevention of the convulsive syndrome in children after birth injury]. / Kavinton v profilaktike sudorozhnogo sindroma u detei, perenesshikh rodovuiu travmu.

The authors studied the efficacy of cavinton as an agent helpful in preventing neurologic disorders in the newborn with hypoxic ischemic encephalopathy due to intracranial birth trauma. The short-term results of the treatment were elucidated in 61 children. In group I including 20 persons given conventional therapy, the disappearance of seizures was recorded in 6 patients; out of 41 children (group II) given additionally cavinton, in 27. Twenty-nine children were followed up for a year. In group I, convulsive paroxysms recurred in 4 patients, whereas in the group II children, no convulsive syndrome was recorded on the follow-up. The group II children also showed a decrease of the phenomena of intracranial hypertension and normalization of the psychomotor development. The preventive effect of cavinton seen in children with a history of birth trauma may be accounted for by its capacity of normalizing cerebrovascular disorders and by its own anticonvulsive properties.

[Behavioral and EEG signs of the convulsive action of kynurenine and quinolinic acid in cats]. / Povedencheskie i EEG-priznaki sudorozhnogo deistviia kinurenina i khinolinovoi kisloty u koshek.

Administration of quinolinic acid but not L-kynurenine into the cat caudate nucleus was found to produce the appearance of epileptiform charges in the EEG with concomitant behavioral convulsions. A combination of quinolinic acid injections with systemic administration of penicillin resulted in the development of the status of myoclonic convulsions alternating with generalized tonicoclonic seizures. These states were eliminated by diazepam. The right caudate nucleus was revealed to be more sensitive as compared to the left one to the convulsant action of combined administration of quinolinic acid with penicillin.

[Pharmacological analysis of the anticonvulsant action of kavinton]. / Farmakologicheskii analiz protivosudorozhnogo deistviia kavintona.

In experiments on rats it was found that cavinton administered in doses of 1-30 mg/kg exerted a distinct dose-dependent anticonvulsant effect, according to the test of maximal electroshock, not less than that of phenobarbital, difenin and carbamazepine. Combined use of cavinton and these drugs potentiated its anticonvulsant action. The involvement of noradrenergic systems, in particular, alpha 1-adrenoceptors, in the anticonvulsant effect of cavinton is suggested.

[Changes in cat behavior as affected by intracerebral microinjections of kynurenine and quinolinic acid]. / Izmeneniia povedeniia koshek pod vliianiem vnutrimozgovykh mikroin"ektsii kinurenina i kholinovoi kisloty.

Behavioural changes induced by microinjections of neuroactive tryptophan metabolites: L-kynurenine sulfate (KYN, 50-1,000 micrograms) and quinolinic acid (QA, 20-1,000 micrograms) were studied in chronic experiments on cats with cannulas implanted into nuclei caudati. Unlike KYN, whose effects were scarce and nonspecific, QA (500 and 1,000 micrograms) produced marked motor and emotional shifts. The effects were manifested in contralateral rotatory movements, limb hyperkinesis, emotional strain, malice, fear, aggression with snarling and hissing, attack on provocation. It is suggested that kynurenines and QA can participate in the generation of emotional disorders peculiar for epilepsy.