Method of determining platelet resistance to acetylsalicylic acid
SUBSTANCE: invention relates to method of determining platelet resistance to acetylsalicylic acid (ASA) by impedance analysis of aggregation function of platelets in vivo, in which aggregation activity is analyses after incubation of biological material sample with ASA with application of aggregation inducer, and aggregation of platelets is induced by collagen in optimal concentration 2 mg/ml, and simultaneously with measurement of impedance determination of dynamics of platelet granules release is performed by luminescent method, where before carrying out aggregation samples are calibrated by means of adenosine triphosphate (ATP) standard, value of aggregation amplitude are determined in Ohms by obtained aggregatograms and obtained values are given points: values ≤6 correspond to 0 points, values 7-9 correspond to 1 point, values 10-12 correspond to 2 points, values >12 correspond to 3 points; after that, intensity of ATP release from platelet granules is determined in nmoles and obtained values are given points: values <0.5 correspond to 0 points, values 0.5-1.0 correspond to 1 point, values 1.0-1.5 correspond to 2 points, values 1.5 correspond to 3 points, and then resistance index (RI) is calculated by formula, value of IR parameter points to presence of aspirin-resistance of platelets.
EFFECT: carrying out fast complex diagnostics of platelet resistance to ASA with estimation of functional state of platelet granules before beginning treatment and possibility of preventing development of undesirable thrombotic or hemorrhagic complications.
2 dwg, 1 tbl, 2 ex
The invention relates to the problem of clinical and preventive personalized medicine - definition individual characteristics reaction platelet drugs, namely preventive methods in vitro diagnostics resistance of platelets to acetylsalicylic acid (ASA).
In connection with certain preventive effectiveness for cardiovascular diseases and affordability ASC is the most commonly prescribed antiplatelet drug [Volkov, C. I., rabuka centuries, Zapravlena O. E., fine A. I. Diagnosis of resistance to aspirin in patients with ischemic heart disease. RBM. Cardiol. zhurn., January 6, 2006 [online]. Found in the Internet: <URL: http://rq1.net.ua/cardio_j/2006/3/IMAG306/volkkov.gif>]. However, it is known that to achieve adequate reduction of platelet aggregation and prevent re-thrombosis and the occurrence of cardiovascular events not all patients taking ASA [Ashikhmin, J. I. the Phenomenon of resistance to the antithrombotic action of aspirin and ways to overcome it / I. I. Ashikhmin O. M. Drapkin // Russian medical news, 2008. - So 13. No. 2]. According to some authors, this phenomenon is called "resistance" to aspirin, distributed among the population with a frequency of from 5 to 48% [Hankey, G. Aspirin resistance / G. Hankey, J. Eikelboom // Lancet, 2006 - Vol.367]. About the Noah of the most important issues occurs when you attempt in any way to measure (evaluate) the degree of inhibition of platelet function after exposure to pharmacological agents is that none of the existing laboratory tests does not include the evaluation of all the versatility of platelet function [Bugaenko, Century Century Modern principles of antiplatelet therapy in patients with ischemic heart disease. Part 2. Tolerability and possible side effects / centuries Bugaenko // heart journal, 2009. No. 5]. This in vitro diagnostic efficiency ASC does not evaluate mechanisms to reduce the function of platelets, which may be due to defects such an important structural and functional component of platelets as granules [Vorobyev A. I. Manual of Hematology / A. I. Vorobiev. - M.: Medicine, 1985]. Impaired functioning of these specific organelles of platelets is the pathogenetic basis of a number of diseases in which the receiving ASA is contraindicated. It is known that in certain pathological conditions of blood there is inadequate response of platelets to acetylsalicylic acid, expressed in occurrence of hemorrhage [Avram, S., Lupu, A. Abnormalities of platelet aggregation in chronic myeloproliferative disorders / S. Avram, A. Lupu // J. Cell. Mol. Med., 2001 - Vol 5]. Use ASC causes an increased risk of gastrointestinal bleeding and hemorrhage what is a mini stroke [J. He, Whelton, P. K. Aspirin and risk of haemorrhagic stroke: a meta-analysis of randomized controlled trials // JAMA. 1998. Vol.280. P. 1930-1935]. In this regard, it is highly important to develop diagnostic tests conducted before administration of the drug by the patient to forecast individual reactivity of platelets to the action of the ASC, including the assessment of secretory activity.
There is a method of definition of aspirin resistance by measurement of light transmission in induced platelet aggregation in blood plasma [RF Patent №2413953C1, G01N 33/86 (2006.01). Publ. 10.03.2011. Bull. No. 7]. The method allows to quantify the amplitude of the aggregation process in vitro. To study the antiaggregatory properties in the test tube with platelet-rich plasma was pre-made solution of ASC and incubated. Then made an inductor - ADP and recorded the amplitude of the aggregation. The results were compared with the parameters of the aggregation caused by the inductor without prior exposure of the ASC with the calculation of the coefficient of inhibition of aggregation (KIA).
Diagnostic value of this method is limited to the obvious drawbacks: 1) the duration, complexity and lack of standardization of the process of preparing platelet-rich plasma; 2) during centrifugation of the blood platelets are exposed to significant mechanical stress to the e significantly alters their functional viability; 3) at the stage of sample preparation disturb the natural environment of platelets, which does not take into account the effects of other blood cells, modulation of platelet function; 4) increases the likelihood of a significant reduction in the concentration of labile mediators to the time of measurement. All of this allows us to create optimal standard conditions necessary to ensure the full realization of the functions of platelets after stimulation of the inductor. A significant drawback of optical aggregatometry is the inability to examine the blood of patients with severe hyperlipidemia, common in cardiology practice. In addition, the above measurement method skips 67% of patients with a defect in the accumulation of granules and the threat of hemorrhagic complications during therapy [Research society Clinical hemostaseology". Diagnostics [online]. Found in the Internet: <URL:http://www.hemostas.EN/society/diagnostics/ecomeds/index.shtml>].
Known another method for diagnosis of aspirin resistance, which is the study of the functional activity of platelets before and after incubation of the specimen with acetylsalicylic acid, while aggregation activity of platelets is determined in whole blood impedance method to calculate the inhibitory capacity of ASC in percent [atent of the Russian Federation No. 2379684 C2, G01N 33/48 (2006.01). Publ. 20.01.2010. (prototype)]. This method allows to predict the resistance to the ASC before therapy, but does not provide information about the state of the granular unit of platelets, which are important for their functioning in the hemostatic system.
The technical result of the invention consists in carrying out a quick, comprehensive diagnosis of resistance of platelets to aspirin with the assessment of the functional status of granules of platelets prior to treatment and possible prevention of adverse thrombotic or hemorrhagic complications.
The technical result is achieved in that in the method for determining the resistance of platelets to acetylsalicylic acid (ASA) by impedance studies of the aggregation of platelets in vitro, which investigate aggregation activity after incubation of the specimen with an ASC using aggregation inductor new is that the aggregation of platelets induce collagen in optimal concentration of 2 mg/ml and simultaneously with the measurement of impedance spend the determination of release granules of platelets by the luminescence method, where prior to the aggregation of the samples are calibrated using standard adenosine triphosphate (ATP), on the obtained agregatogramme determine the values of the amplitude and is regali in ohms and assign the obtained values of the points: values < 6 correspond to 0 points, the values of 7-9 correspond to 1 point, the values of 10-12 correspond to 2 points, the values of >12 correspond to 3 points, then determines the rate of ATP release from granules of platelets in nolah and assign the obtained values of the points: values <0,5 0 correspond to points, values of 0.5-1.0 correspond to 1 point, the values of 1.0 to 1.5 corresponds to 2 points, the values of >1.5 to correspond to 3 points, and then calculate the index resistivity (ER) by the formula:
where Bandand Bl- the degree of sensitivity to aspirin points in the impedance and fluorescence tests, respectively, with the value of the index IL more than 4 indicates the presence of userinitiated platelets.
The proposed method differs from the prototype in that the definition of aspirin resistance is carried out in vitro by studying the functional activity of platelets only after incubation of the specimen with an ASC, while aggregation activity of platelets and their granules is determined in whole blood complex impedance-fluorescent method that allows to obtain additional diagnostic parameter is the degree of influence of ASA on the release of the contents of platelet granules and allows for a more objective approach to the assessment of individual sensitivity is lnasty patient to the drug ASC.
Thus, the above distinctive features of the prototype characteristics allow to draw a conclusion on the conformity of the proposed technical solution the criterion of "novelty". The features distinguishing the claimed technical solution to the prototype, not identified in other technical solutions and, therefore, provide the claimed solution according to the criterion of "inventive step".
The invention is illustrated using graphic materials:
In Fig. 1 shows curves of collagen-induced aggregation and release curves granules of platelets in whole blood up to (1-2) and after (3-4) incubation of samples with ASA for individuals who are sensitive to aspirin.
In Fig. 2 presents curves of collagen-induced aggregation and release curves granules of platelets in whole blood up to (1-2) and after (3-4) incubation of samples with ASA for the individual, resistant to aspirin (1 - the original curve aggregation, 2 - the original curve of the intensity of the release pellets, 3 - curve aggregation after incubation with ASA, 4 - curve intensity release pellets after incubation of blood samples with ASA).
The method is as follows.
Blood samples obtained from the cubital vein and stabilize a 3.8% solution of sodium citrate in a volume ratio of 9:1. During the study blood remain in closed plastic probescope room temperature in accordance with accepted safety precautions when working with blood cells.
Analysis of the functional activity of platelets is carried out not earlier than 15 minutes and no later than 3 hours after receipt of the blood using a Lumi-aggregometer at 37°C and stirring the contents of a ditch with the mixer rotation speed of 1000 rpm, the Ratio of the volume of blood samples and the inductor is 100:1.
Prior to the aggregation of fluorescent method samples are calibrated using standard ATP. When this is set individually for the patient gain (in %) response of luminescence. Further registers the intensity of the release of ADP from granules of platelets (in nmol) with simultaneous recording of impedance parameters induced platelet aggregation in the same cell.
To induce aggregation, use a solution of collagen at a final concentration of 2.0 mg/ml, shipped ready to use.
Study of the functional activity of platelets each patient in the sample after pre-incubation of blood with ASA.
The incubation is conducted for 15 minutes at 37°C. in a pilot sample of whole blood add 40 ál 3,36 mm solution of ASC.
Processing agregators is generally characteristic points of the curves of aggregation to determine the maximum amplitude of rise of impedance in ohms to 5 minutes from the bearing into the measuring cell of the inductor - collagen and determination of the luminescence intensity when ATP release from granules of platelets in nolah. Identifying the source of low release granules may indicate pathology of platelets and contraindications to the appointment of the ASC. In addition to routine evaluation settings agregatogramme calculated the resistance index (IR). This parameter represents the amount of points characterizing the degree of sensitivity of the patient impedance test and fluorescent test for the maximum amplitudes of aggregation and release of granules, and is calculated by the formula: IL=Band+Blwhere Bandand Bl- the degree of sensitivity to aspirin points in the impedance and fluorescence test, respectively. This parameter determines the final degree of individual sensitivity to aspirin and includes a comprehensive assessment of the inhibitory effect of ASA on the accumulation of ATP, the intensity of the release granules, the dynamics of the membrane potential of platelets and agregation-adhesive. IL allows you to determine the individual sensitivity of the patient to ask in absolute units of the scale that can serve as a criterion to identify individuals with high and low response to the drug. This will allow you to assign therapy with aspirin, a reasonable timely is th predictive laboratory evaluation, and to avoid undesirable consequences of inadequate treatment.
Examples of the obtained agregators with individual assessment of aspirin resistance is shown in Fig. 1-2.
The degree of resistance is determined in accordance with the proposed scale:
Table 1 presents the results of a survey of 13 clinically healthy people, conducted on the basis of the Krasnoyarsk branch of the fgbi Hematological scientific center of the Russian Ministry of health. The proposed method was determined by the magnitude of the IL. In samples of ten patients, the value of this indicator was below 4. Three people according to the test results IL was equal to or more than 4.
Because healthy people who didn't ask, is the IR source without conducting incubation with the ACU were equal to or greater than 4, the level values of IL was adopted as the criterion of resistance. After the patient was admitted per os single dose ASA in the amount of 125 mg/day, the next day the newly defined metrics aggregation in a sample of fresh blood without pre-incubations. Three patients revealed by the results of preliminary testing with incubation in vitro IL was again more than 4, as before taking the drug. This confirms that the proposed solution allows to identify patients with resistance to ant is the platelet effect of aspirin. In contrast, among individuals with indicator IL for less than 4, a single receiving 125 mg ASA caused a significant inhibition of indicators aggregation, indicating their sensitivity to this drug.
The result shows a high diagnostic capabilities of the proposed method and the clinical usefulness of preventive determine antithrombotic actions ASC in vitro complex impedance-luminescent technique.
|The results of the research degree of resistance to aspirin|
|№ p/p||In the test, IN VITRO||In the test, IN VIVO||Forecast|
|The amplitude, Ω||Release granules, nmol||Band||Bl||IL||IL|
|12||13||0,73||3||1||4td align="center"> 6||resistant|
The method for determining the resistance of platelets to acetylsalicylic acid (ASA) by impedance studies of the aggregation of platelets in vitro, which investigate aggregation activity after incubation of the specimen with an ASC using aggregation inductor, characterized in that the aggregation of platelets induce collagen in optimal concentration of 2 mg/ml and simultaneously with the measurement of impedance spend the determination of release granules of platelets by the luminescence method, where prior to the aggregation of the samples are calibrated using standard adenosine triphosphate (ATP), resulting agregatogramme determine the amplitude value aggregation in ohms and assign the obtained values of the points: values ≤6 correspond to 0 points, the values of 7-9 correspond to 1 point, the values of 10-12 correspond to 2 points, the values of >12 correspond to 3 points, then determines the rate of ATP release from granules of platelets in nolah, and assign the obtained values of the Alla: value <
0,5 0 correspond to points, values of 0.5-1.0 correspond to 1 point, the values of 1.0 to 1.5 corresponds to 2 points, the values of >1.5 to correspond to 3 points, and then calculate the index resistivity (ER) by the formula:
where Bandand Bl- the degree of sensitivity to aspirin points in the impedance and fluorescence tests, respectively, with the value of the index IL more than 4 indicates the presence of userinitiated platelets.
SUBSTANCE: invention represents a diagnostic technique for the disturbed thrombocyte aggregation accompanying mucoviscidosis in children involving a thrombocyte aggregation test using the Multiplate aggregometer inducers. Trays with a magnetic mixer and electrodes are added with NaCl 400 mcl at 37°C and immediately added with whole blood 400 mcl from a hirudin test tube, incubated in the chamber for two minutes; the tray is added with 30 mcl of an aggregation inducer specified in a group: soluble thrombin receptor - peptid-6, adenosine diphosphate, arachidonic acid. The thrombocyte aggregation rate is displayed on the screen in the form of a curve, and the sub-curve area U is automatically calculated; the sub-curve area U shows the thrombocyte aggregation state as compared to reference values in the group of healthy children; if the threshold area U has appeared to exceed the reference, the thrombocyte hyperaggregation, while the threshold area U being less than the reference, the thrombocyte hypoaggregation is stated.
EFFECT: invention provides the timely diagnosis of microcirculatory disorders accompanying mucoviscidosis.
2 ex, 1 dwg
SUBSTANCE: invention refers to medicine, particularly to clinical biochemistry, and aims at determining oxidative protein modification in a substance pool of an average molecular weight in a biological medium accompanying any pathological conditions by biochemical examination. The biological medium specified in the blood plasma, erythrocyte or urine is sampled; proteins are deposited by adding 10% trichloroacetic acid; if observing sedimentation, a centrifugation cycle follows at 1000 rpm for 15 minutes; thereafter, 2,4-dinitrophenylhydrazine 0.05 M in hydrochloric acid 2 M is added; the sample is centrifuged at 1000 rpm for 20 minutes; and if observing sedimentation, the sediment is washed twice in ethanol-ethylacetate (1:1), dried on a water bath for 10 minutes and dissolved in urea 8 M; the sample is kept in a boiling water bath for 10 minutes until dissolved completely; the prepared solution is analysed by spectrophotometry. The method is applicable both for single use, and for monitoring of the postoperative oxidation protein modification and average molecule levels.
EFFECT: method provides higher information value of biochemical tests, reducing consumption of the biological material.
9 tbl, 2 ex
SUBSTANCE: invention includes determination of content of soluble fibrin and D-dimers, formed in the process of fibrinolysis, activated in blood sample. In method, in accordance with the claimed invention, level of D-dimers, corresponding to destruction of soluble fibrin and level of D-dimers in sample with border values of the norm, are compared.
EFFECT: test in accordance with the claimed invention can be applied for determining whether resistance to blood coagulation in patient is sufficient.
4 tbl, 3 ex, 2 dwg
SUBSTANCE: method for thrombin activity test in an initially non-reacted mixture of thrombin and fibrinogen (versions) involving the stages: (a) reversible thrombin inhibition by adding an inhibitory solution having pH varying within the range of 8.5 to 11.5; (b) addition of the known amount of fibrinogen to the mixture (or the known amount of a chromogenic or fluorogenic thrombin substrate), (c) reversible thrombin activation by pH reduction to approximately 6.0 to less than 8.5, (d) enabling thrombin reacting with fibrinogen, (e) thrombin activity test initially found in the dry mixture. The method for fibrinogen functionality test in an initially non-reacted mixture of thrombin and fibrinogen (versions) involving the stages: (a) reversible thrombin inhibition by adding an inhibitory solution having pH varying within the range of 8.5 to 11.5; (b) addition of the known amount of thrombin to the mixture (or a thrombin-like enzyme), (c) reversible thrombin activation by pH reduction to approximately 6.0 to less than 8.5, (d) enabling thrombin reacting with fibrinogen, (e) fibrinogen functionality test initially found in the dry mixture.
EFFECT: group of inventions enables higher accuracy of thrombin and fibrinogen activity test.
32 cl, 1 dwg
SUBSTANCE: analyser has a revolving cuvette with a sample in which there is a control ferromagnetic ball, a magnet which can interact with said ball, a coagulation sensor which transmits signals from the ball and having a Hall sensor and a magnet, a signal processing device in form of a power supply unit, a Hall sensor, a microprocessor and a display device included in a common measuring circuit. The analyser is multi-channelled by fitting at least one additional revolving cuvette to form several channels. All cuvettes lie in a temperature-controlled unit included in the common measuring circuit. The longitudinal axis of each cuvette is inclined at an acute angle to the vertical. In the coagulation sensor, the magnet lies opposite the Hall sensor on the opposite side of the cuvette. The magnet is in form of a flat cylinder mounted with possibility of displacement along the cuvette. The analyser is also fitted with a unit for controlling rotation of the cuvettes and, included in the common measuring a circuit, a measurement parameter adjustment unit having rewritable read-only memory, and a timer configured to automatically switch on when a reactant is fed into the cuvette.
EFFECT: use of the invention increases reliability and broadens functional capabilities of the analyser owing to use of a multichannel measuring circuit, and simplifies the measuring procedure by automating the process.
4 cl, 2 dwg
SUBSTANCE: blood plasma is examined in 4 minutes after the beginning of spontaneous red blood cell aggregation for free red blood cell count and cell count in aggregates. A percentage of non-aggregated red blood cells (PNA RBC) by formula PNA RBC=FRBSC×100/(TRBCA+FRBSC) wherein FRBSC is the free red blood cell count, TRBCA are total red blood cells in aggregates. If the PNA RBC is 56 to 30%, I degree of severity is stated, 30% to 4% - II degree of severity, less than 4% - III degree of severity.
EFFECT: use of the invention enables objectifying and increasing precision of evaluation of red blood cell aggregation, evaluating an intensity of patient's microcirculation disorders in a relatively short time, and thereby ensuring well-timed adequate complex of therapeutic measures or corrected therapy.
SUBSTANCE: quantity of fibrin-monomers, dissolved in 0.5 N sodium hydroxide, is determined spectrophotometrically with application of ethanol test. Claimed method of quantitative determination of fibrin-monomers in blood makes it possible to reveal pathological process in organism with 95% reliability.
EFFECT: increase of determination accuracy.
2 tbl, 4 ex
SUBSTANCE: thrombosis monitor comprises: a thrombosis chamber, at least in a part of which there is a thrombogenic material; an inlet tube connected to the thrombosis chamber through which blood flows into the thrombosis chamber; a blood supply container connected to the inlet tube; a feed pump for the container; a pressure sensor for measuring pressure applied to the container. A method of thrombosis monitoring consists in the fact that after introduction of an anticoagulant, blood is supplied from the container to the thrombosis chamber by pressing on a fluid placed on a blood layer and having density less than that of the blood layer; it is combined with anticoagulation blood processing or blood coagulation stimulation, and measurement of pressure applied to the container; the thrombogenic material is placed at least in a part of the thrombosis chamber.
EFFECT: group of inventions provides overall assessment of blood coagulation and platelet-cell thrombosis in a medium equivalent to blood flow for evaluation of efficacy of an antithrombotic drug.
11 cl, 15 dwg, 23 ex
SUBSTANCE: for determination of functional state of hemostasis system record of blood coagulation process is performed, current amplitude of blood resistance in first time moment is registered and second resistance of blood at multiple time moment from initial time value is measured. Two resistances and time moments are used to determine maximum blood resistance and time constant, by which blood resistance at the beginning and end of coagulation process is calculated. Obtained parameters are used to determine indices of beginning and end of blood coagulation process. Obtained indices are compared with of the same name indices of blood coagulation process in norm and in case of differently directed deviations disturbances of functional state of hemostasis system are diagnosed.
EFFECT: invention makes it possible to increase measurement accuracy and reduce examination time.
1 tbl, 4 dwg
SUBSTANCE: method is based on a method of observing turbidimetric fibrin clot formation with optical transmission of an incubation medium recorded by ultraviolet radiation band 230 to 320 nm by means of UV-spectrophotometre as a fibrin-polymer detector.
EFFECT: invention enables higher accuracy and sensitivity of the method.
4 ex, 4 dwg
SUBSTANCE: claimed is application of fat emulsion for parenteral feeding as solvent for compounds which are poorly soluble in water. Fat emulsion contains in 1 l of solution: 30 g of refined soybean oil, 30 g of triglycerides with the average chain length, 25 g of olive refined oil, 15 g of purified fish oil.
EFFECT: obtaining solvent for compounds, poorly soluble in water, which makes it possible to determine parameters and spectrum of biological activity of novel compounds of chemical nature at the stages of pre-clinical and clinical tests, which does not change basic biological constants and possesses biological inertness.
2 tbl, 2 ex
SUBSTANCE: invention relates to biology, forensic and analytical chemistry and specifically to methods of determining procaine in blood plasma. The method comprises adding sodium fluoride to blood plasma containing procaine to achieve concentration of 10 mg/ml; treating the obtained mixture with acetone; separating the extract from the precipitate by filtering; evaporating acetone from the filtrate in an air current at room temperature; diluting the aqueous residue by adding water; saturating the obtained solution with ammonium sulphate; alkalising with an ammonium buffer solution to pH 9.0-9.5; extracting twice with portions of an organic extraction agent in the form of 30% camphor solution in methyl acetate, with ratio of the aqueous to the organic phase of 1:1 by volume; separating the organic extracts; combining; evaporating the solvent from the combined extract in an air current at room temperature; chromatographing the residue in a thin layer of silica gel STKH-1A on Sorbfil PTSKH-AF-A-UF plates, using a dichloromethane-ethanol mobile phase in ratio of 6:4 by volume; developing the chromatogram in UV light; eluting the analysed substance from the sorbent with a mixture of acetonitrile-methanol-0.025 M potassium dihydrogen phosphate solution with pH 3.0 in ratio of 10:10:90 by volume; chromatographing by HPLC method using a reversed-phase sorbent Nucleosil C18, a polar mobile phase acetonitrile-methanol-0.025 M potassium dihydrogen phosphate solution with pH 3.0 in ratio of 10:10:90 by volume and a UV detector; measuring optical density at wavelength 298 nm and calculating the amount of the analysed compound from the area of the chromatographic peak.
EFFECT: method improves sensitivity of determination.
3 tbl, 2 ex
SUBSTANCE: invention relates to analytical chemistry. The method is characterised by electrochemically concentrating benzoic acid on the surface of a graphite electrode for 90 s at electrolysis potential of (-0.500) V on a background of 0.1 mol/l sodium hydrogen phosphate, recording polarisation curves with linear potential sweep rate of 25 mV/s and determining concentration of benzoic acid from the peak height in potential range of 0.5-1.6 V relative to a silver chloride electrode.
EFFECT: method provides highly sensitive and rapid determination of benzoic acid in medicinal drugs.
3 ex, 6 tbl
SUBSTANCE: sample is applied on a paper filter, and standard calibration solutions of metronidazole in the concentration range of 10-100 mcl are radially applied on the same filter. The filter is processed with a developing agent containing 5% potassium hydroxide and acetone taken in ratio 2:1 and thermostated at 100°C until coloured spots are visible. A colour intensity of the sample spot is compared to the colour of the spot colour of the reference calibration solutions to determine the metronidazole concentration in the analysed sample.
EFFECT: method enables the fast and reliable monitoring of the metronidazole content and the timely correction of the therapeutic process.
SUBSTANCE: invention refers to medicine, namely to studying and analysing medical preparations, and can be used for standardising herbal raw materials. A method for identification and qualitative measurement of chlorophyll, carotinoids and hydroxycinnamic acids in a combination in great nettle leaves involves a 1-hour fractional extraction, 30 min each of the ground raw material having a particle size of 1.0 mm on a water bath at a temperature of 100°C with 70% ethanol in a ratio of the herbal raw material to the extractant of 1:100, the combination of the extracts and reduction to 100 ml with a solvent, dilution of the prepared solution in a ratio of 2:25 in 96% ethanol, measuring an optical density of the solution in relation to 96% ethanol at maximum absorption 328±1 nm, 442±1 nm and 667±1 nm, calculation of the total content of hydroxycinnamic acids equivalent to chlorogenic acid, carotinoids equivalent to violaxanthin and chlorophyll in the percentage equivalent to an absolute dry mass of the raw material by formulas.
EFFECT: method provides availability, simplicity, efficiency and low error of measurement.
3 dwg, 1 ex
SUBSTANCE: method of determining fat-soluble vitamins A, D2, E and β-carotene which are present at the same time includes separating the fat-soluble vitamins from a substance by extraction with 96% ethanol, separating the alcohol extract of vitamins using a separating funnel, successive chromatography using Sorbfil PTSKH-P-A silica gel plates on a polymer substrate using two eluents with a different range, time of saturating the chamber with eluent vapour of 20 minutes and elution time of 55 min; drying the plates at temperature not lower than 80°C in a temperature-controlled chamber for 3-5 min, treating the plates with a developer - 5% alcohol solution of phosphatomolybdic acid; according to the invention, the eluents used are hexane:chloroform (19:1) and hexane:chloroform (3:1), and detection of the chromatographic zone of β-carotene is carried out before treating the plates with a developer in day light.
EFFECT: simpler and faster process of determining fat-soluble vitamins.
10 dwg, 3 ex
SUBSTANCE: invention relates to medicinal drug quality control and provides a method of authenticating and determining the quantitative content of benzethonium chloride in medicinal drugs, which includes separating components of the drug via high-performance liquid chromatography, where the eluent used is a mixture of acetonitrile, tetrabutylammonium hydrophosphate, disubstituted potassium phosphate and water, with content of tetrabutylammonium hydrophosphate of 2.5 mM, content of disubstituted potassium phosphate of 5 mM, wherein separation of the components of the drug is carried out with column length of 125 mm.
EFFECT: high accuracy and faster analysis since there is no need for special sample preparation.
SUBSTANCE: invention relates to a method of screening a non-teratogenic substance comprising bringing a test substance into contact with cereblon or a fragment of cereblon, evaluating the capacity of the test substance to bind to cereblon or the fragment of cereblon, and selecting a test substance that does not bind to cereblon or the fragment of cereblon or a test substance exhibiting lower capacity to bind to cereblon or the fragment of cereblon compared with thalidomide.
EFFECT: improved method.
8 cl, 19 dwg, 8 ex
SUBSTANCE: method involves taking blood at 1 ml per a sample into the Vakutainer evacuated test tube with sprayed ethylene diaminetetraacetic acid 2.2 mg; a reference sample 1.0 ml and a sample 1.0 ml with weighted xenobiotics are taken from the total volume into the Eppendorf tubes; 30-minute incubation is followed by using a uniform procedure to prepare blood smears by applying on a dry slide to prepare a smear to be stained according to Leishman; blood corpuscles are analysed by the Zeiss light microscope at a magnification 1500. A microscope field showing echinocytes with styloid processes of the membrane not found in the reference sample is a sign of the xenobiotic membrane toxicity.
EFFECT: invention enables an instant assessment of the membrane toxicity of the substance.
SUBSTANCE: lactic acid is transferred from a sample into a solution and voltammetric accumulation of lactic acid in the stirred solution is performed while bubbling with an inert gas for 30 s and with electroaccumulation potential of 1.2-1.4 V relative to a saturated silver chloride electrode on a background electrolyte of 0.1 M Na2HPO4, followed by detection of cathode peaks in differential mode of recording voltamperograms with potential sweep rate of 30-40 mV/s; concentration of lactic acid is determined from peak height in the potential range of 0.25-0.40 V by a standard addition method.
EFFECT: method is simple, does not require a large amount of reactants and labour costs.
2 ex, 1 tbl
FIELD: microbiology, pharmaceutical agents.
SUBSTANCE: invention relates to method for determination of genome response of specific cells to vegetable extract action. Method for drug screening includes 1) treatment of specific cells with vegetable extract; 2) protein or RNA isolation from said treated cells; 3) identification of isolated protein or RNA; 4) determination of compound(s) in said vegetable extract; 5) treatment of said cells with determined compound(s); 6) protein or RNA isolation from said treated cells; and 7) determination of compounds providing expression or suppression of said protein or RNA which have another concentration than in untreated said specific cells.
EFFECT: identification of individual compound(s) for screening of new pharmaceutical agents or new pharmaceutical application of existing drugs.