Method for determining metronidazole in biosubstrate
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.
The present invention relates to medicine, namely to therapy, and can be used to control the content of metronidazole in liquid media (blood, saliva, lymph, cerebrospinal fluid, urine, pus) and the tissues of the body.
Metronidazole is used to treat clostridial anaerobic infections. Control over the level of this drug is necessary, since at lower concentrations below the minimum inhibitory growth of anaerobic bacteria in the septic focus, not suppressed. The excess concentration of the drug is associated with the risk of undesirable side effects, such as nausea, vomiting, diarrhea, urticaria, headache, weakness, drowsiness (Reference Vidal drugs in Russia: a Handbook. M: Estrofem Service, 1998, S. 148).
There is a method of determining the concentration of metronidazole in the blood, including extraction of sample (blood, tissue and other) ethanol exposure during the day at zero temperature, removing the spectrum of the resulting supernatant on a spectrophotometer at a wavelength of 320 nm and calculating the concentration of metronidazole using the calibration curve. (Baisogala D., Burdov B. A., Konoplyannikov A., and other Direct results of radiation and combined treatment of patients with malignant tumors with the use of metronidazole. J. Med. Radiola. - 1983, No. 2, S. 7-12).
The disadvantages of this method include the duration of the analysis (24-30 hours).
The closest to the technical nature of the claimed and adopted for the prototype is a method of determining the content of metronidazole in biosubstrate by conducting chromatographic analysis.
For the implementation of this method take 1 ml or 1 g of sediment sample (blood, saliva, cerebrospinal fluid, pus, tissue sampling), to which was added 1 ml of acetonitrile to precipitate high molecular weight fraction. The resulting mixture was centrifuged for 20 minutes at 3000 rpm and the supernatant evaporated to dryness in a stream of nitrogen at a temperature of 80-90°C. the Dry residue is completely dissolved in eluent acetonitrile-water, taken in the ratio 10:90, and then centrifuged for 20 minutes. The obtained supernatant injected with mobile phase: acetonitrile-water (10:90), a flow rate of 100 µl/min eluent Degassing with helium within 1-2 minutes quantification of metronidazole is carried out at a wavelength of 318 nm. The analysis, including sample preparation, an average of 2 hours (Kuznetsova, E. E., Gorokhova C. G., Andreeva T. A. and other high-performance liquid chromatography prolonged metronidazole - back. Chem-Pharm. J.-L. 2000, No. 8, S. 46-47).
The disadvantages of this method include the use of Dor is goteamsgo device, toxic solvents and multi-stage operations.
The objective of the proposed technical solution is the advanced diagnostic capabilities of the laboratory of control over concentration of metronidazole contained in the patient's blood.
The technical result of the proposed method is to create a simple, affordable rapid method of determining the concentration of metronidazole in fluids and tissues of the body, allowing the clock to be monitored.
The technical result is achieved in that the method of determining the content of metronidazole in biosubstrate carried out by chromatographic analysis of the sample.
Distinctive techniques of the proposed method lies in the fact that the sample assay applied on filter paper and on the same filter, radial, put the standard calibration solutions of metronidazole in the concentration range 10-100 μl. After that, the filter is treated with a reagent developer containing a 5% aqueous solution of caustic potash and acetone, taken in the ratio 2:1, and thermostatic at 100°C until the appearance of colored spots. The concentration of metronidazole in the analyzed sample set by matching the intensity of staining spot sampling spot one of the standard calibration solutions.
A comparative analysis was performed with a prototype of the m showed the proposed method differs from the known above-mentioned methods, and therefore meets the criterion of the invention of "novelty."
Comparison of the proposed technical solution is not only the prototype, but also with other technical solutions in medicine failed to reveal any sign of the claimed invention. In the available literature, the authors found no way of determining the content of metronidazole in biosubstrate using paper chromatography.
The authors of the proposed method experimentally established that the colour intensity of the analyzed sample is directly proportional with the concentration of metronidazole 10-100 ál.
Feature of the proposed method is that for the determination of metronidazole in samples no prior sample preparation and special laboratory equipment.
Clinical studies of the authors of the proposed method show that the proposed technical solution is available, simply and quickly to monitor drug levels in biosubstrate and make appropriate adjustments in the process protivoavariynogo treatment of severely ill patients.
The above can conclude that the technical solutions according to the criterion "and oprettelse level.
The method constituting the invention, intended for use in medicine, namely in clinical practice (therapy of purulent surgery, proctology, Oncology, and others). The possibility of its fulfillment is confirmed as described in the application techniques and, consequently, the proposed solution meets the criteria of the invention "industrial applicability".
The inventive method is carried out as follows.
On filter paper by using the pipettor put the sample assay (serum, saliva, urine, lymph, cerebrospinal fluid, pus, exudate). On the same radial filter is applied and 10 standard calibration solutions of metronidazole in the concentration range 10-100 μl. After that, the filter is carefully sprayed pre-prepared reagent developer containing a 5% aqueous solution of caustic potash and acetone, taken in the ratio of 2:1. Then the filter is placed in a thermostat with a temperature of 100°C. and dried until the appearance of colored spots. The intensity of yellow spots appeared depends on the concentration of metronidazole. The concentration of metronidazole in the analyzed sample set visually by matching the intensity of staining spot sampling spot one of the standard calibration solutions. The total analysis time is 20-30 minutes.
The proposed method is illustrated in the examples of the specific implementation.
Example 1. Patient Century, history No. 19140, was admitted to the intensive care unit of septic center Regional hospital with the diagnosis of widespread purulent peritonitis. To inhibit the growth of anaerobic clostridial infection, isolated from the patient, was appointed metronidazole in a dose of 500 mg intravenously. After 3 and 6 hours after administration of metronidazole carried out a determination of its content in the serum of this patient by the present method.
After 3 hours, the concentration of the administered drug was 4,34 µg/ml after 6 hours, the concentration of metronidazole in the serum of this patient was 3.24 µg/ml.
Identified a number of metronidazole was below the minimum bactericidal concentration that caused a subsequent correction dose.
Example 2. The patient Was, the case history No. 17390, enrolled in septic Department Bureau diagnosed with widespread purulent peritonitis, anaerobic clostridial infection. In complex drug therapy was included metronidazole endolymphatic at a dose of 500 mg
After 3, 6 and 9 hours after administration of metronidazole was definitely its content in the lymph offer this sick way: 3 hours - 35,3 µg/ml after 6 hours - 29,7 µg/ml and 9 hours - to 25.3 mg/ml
The obtained data testify that in sidemy period, the contents of metronidazole exceeded its minimum bactericidal concentration, that did not require changes in the tactics of antimicrobial therapy.
Example 3. Patient K., case history No. 21019, enrolled in septic Department Bureau diagnosed with cellulitis of thigh. From the wound was selected anaerobic infection (Bac. fragilis).
The patient was assigned metronidazole intravenous dose of 500 mg After 3 and 6 hours after administration conducted the determination of metronidazole in purulent discharge from the wound. Determined after 3 hours of 3.8 µg/ml after 6 hours and 2.9 µg/ml.
Installed the concentration of metronidazole was below the minimum bactericidal, which required an increase in dose.
Clinical studies of the proposed method was conducted at the clinic FBGU "NCRV" SB RAMS in the Regional clinical hospital. We examined 39 patients, including: widespread purulent peritonitis - 20, pancreonecrosis - 9, cellulitis of thigh - 5, ulcerative colitis - 5.
For comparison, the concentration of metronidazole in the serum of these patients was also determined and by spectrophotometry. When comparing results, the error definitions are not exceeded 3.2%. Thus the sensitivity of the proposed method is 89,8%, a specificity of 87.8%.
Thus, the proposed method is simple, fast and reliably determine the content of metronidazole in biosubstrate that is allows the opportunity for rapid analysis and thereby, allows timely adjustments to the treatment process. The method does not require the preliminary sample preparation and does not require special instrumentation.
The method of determining the content of metronidazole in biosubstrate by chromatographic analysis of a sample, wherein the sample assay applied on filter paper and on the same radial filter applied standard calibration solutions of metronidazole in the concentration range 10-100 μl, after which the filter is treated with a reagent developer containing a 5% aqueous solution of caustic potash and acetone, taken in the ratio 2:1, and thermostatic at 100°C until the appearance of colored spots, the color intensity of the spots of the sample is compared with the color spots of standard calibration solutions, which determine the concentration of metronidazole in the analyzed sample.
SUBSTANCE: invention relates to medicine, namely to method of estimating degree of community-acquired pneumonia severity. Essence of method consists in the following: in patient's blood determined are absolute quantity of leukocytes, relative quantity of eruthrocytes-macrocytes, absolute quantity of 3-class monocytes with segmented lobed nucleus, relative quantity of granular middle size lymphocytes, relative quantity of neutrophils with 7 segments in nucleus, quantitative index of C-reactive protein, as well as physiological index of patient's respiratory movements per 1 minute. Mathematical calculation of outhospital pneumonia severity index (SI) is carried out,and if value is lower than 0.795, pneumonia is estimated as being of medium severity, and if value is higher or equals 0.795, it is estimated as severe.
EFFECT: application of claimed method makes it possible to estimate degree of outhospital pneumonia severity in efficient way.
SUBSTANCE: invention relates to medicine, namely to cardiovascular diseases, and can be used for estimation of severity of endothelial dysfunction in patients with rheumatoid arthritis. Essence of method is based on determination of complex of parameters, ranges (R) of values of which are used for assigning diagnostic coefficients (DC). After that, DC values are summed up to obtain value of diagnostic index (DI). If its value is lower or equals minus 50, absence of ED is determined, if its value is higher than minus 50 or equals 0, indefinite state is determined, if its value is higher than 0 and lower than 40, initial manifestations of ED are determined, if its value is higher or equals 40, expressed endothelial dysfunction is determined.
EFFECT: application of invention makes it possible to differentiate severity of endothelial dysfunction, specify tactics of patient managing and achieve higher therapeutic effect.
1 tbl, 4 ex
SUBSTANCE: technique involves a blood serum examination for thyrotropic hormone, mcUnit/ml, free T4, pmole/l, prolactine, mIU/ml, follicle-stimulating hormone, mIU/ml, lutenizing hormone, mIU/ml, testosterone, nmole/l, dehydroepiandrosterone sulphate, mcg/ml, cortisol, nmole/l, oestradiol, pg/ml, 17-OH progesterone, nmole/l. Calculating an integral hormone state (HIS) by formula follows. If the HIS value is below 0.799, a habitual disorder is stated, which is accompanied by a minimum risk of the reproductive disorders. The HIS values falling from the range of 0.800 to 1.000 provides stating the tensed hormonal regulation reflecting a moderate risk of the reproductive disorders. The disturbed interhormonal cooperation corresponds to the HIS value falling within the range of 1.001 to ≤1.210 that represents a high risk of the reproductive disorders. The HIS value of more than 1.211 shows the lack of reserves that testifies to a very high risk of the reproductive disorders.
EFFECT: technique enables improving the diagnosis of the adolescent's reproductive disorders.
5 tbl, 3 ex
SUBSTANCE: at the first stage of a subtotal vitrectomy, the vitreous body is sampled with its biochemical parameters to be analysed. In an air-free environment, the vitreous body samples are analysed to determine the following values at 37°C by means of an automatic blood gas and acid-base balance analyser: pH, partial carbon dioxide pressure pCO2, actual bicarbonate ion
EFFECT: method enables assessing the severity and potential outcome of retinopathy in newborn children as shown by analysing the acid-base balance of the vitreous body.
SUBSTANCE: invention represents an instant diagnostic technique for acute intestinal infections (AIIs), involving detecting indication markers of the AII aetiology with the use of laboratory immunology tests, differing by the fact that the AII aetiology is stated in children of an early age category, preferentially in the newborn children; that is accompanied by measuring the concentration of cytokine, interleukin IL-10 in coprofiltrate and diagnosing chronic placental insufficiency (CPI); a probability (P) of the bacterial AII aetiology is calculated; the value P of more than 50% testifies to the bacterial AII aetiology, while the value P being less than 50% shows the absence of the bacterial AII aetiology, and enables considering the diagnostics second stage to be necessary, which implies measuring the concentration of cytokine, interleukin IL-4 in coprofiltrate; the time of latching the newborn child to the breast is established with considering the type of feeding; that is combined with calculating a probability (P) of the viral or viral-bacterial AII aetiology, with the value P of more than 50% testifying to the viral AII aetiology, while the value being less than 50% makes it possible to state the viral-bacterial AII aetiology.
EFFECT: more accurate diagnosing of the aetiology of acute intestinal infection and simplifying the diagnostic procedure.
SUBSTANCE: assessing the inducing action of cytomegalovirus infection on a newborn's erythrocyte methaemoglobin and oxyhaemoglobin in peripheral blood in aggravated cytomegalovirus infection in a pregnant woman in the third trimester of gestation is ensured by determining erythrocyte count pre-stained with 0.1% methylene blue. The cytomegalovirus antibody titre being 1:1600 accompanied by an increase of the erythrocyte count 45 min after the staining to 62.5±1.9%, an increase of methaemoglobin to 1.8±0.08%, a decrease of oxihaemoglobin to 96.5±2.9% is stated as a disturbed methaemoglobin recovery and reduced haemoglobin oxygenation that leads to developing haemic anaemia in a newborn.
EFFECT: method enables studying the haemoglobin oxygenation in peripheral blood erythrocytes of newborns of the mothers suffering from aggravated cytomegalovirus infection in the third trimester of gestation.
SUBSTANCE: invention refers to medicine, namely to neurology, can be used to specify a stage of aggravation or remission for the patients suffering multiple sclerosis (MS). The method is based on using the biochemical status values normally measured in the patient. A regression equation is used to determine the stage of multiple sclerosis: the stage of the clinical course of MS=2.5235 - 0.914006*sex+0.00355359*age+0.0702341*glucose+0.0131744*aspartate transaminase - 0.00412952*alanine transaminase - 0.0484807*bilirubin+0,153119*thymol test+0.196675*carbamide+0.00223117*cholesterol - 0.207918*amylase -0.00755805*total protein, wherein females are indicated as 0, females - as 1; the age is expressed in completed years at the moment of examination; glucose, alanine transaminase, bilirubin, thymol test, carbamide, cholesterol are absolute values measured in venous blood. If the derived value falls within the range of 2.89-3.48, the patient is stated to have remitted MS, while the value exceeding 3.48 enables diagnosing aggravated MS.
EFFECT: improved prediction procedure.
SUBSTANCE: invention represents a method for identifying the factors of genetic predisposition to breast cancer involving genetic marker recording by a massive parallel sequencing followed by sampling and analyzing in depth injurious mutations only among the patients suffering breast cancer; a differential characteristic of the method is that identifying the recessive factors of the genetic predisposition to breast cancer enables involving the patients with a medical history having an early age of the onset, primary multiple cancer and no cases of breast cancer or ovarian cancer in the family; the analysis procedure covers only the mutations found in the homozygous state.
EFFECT: expanding the methods for identifying the genetic predisposition to breast cancer.
SUBSTANCE: for immature girls after day 3-5 of menstrual cycle a level of Anti-Mullerian Hormone is determined in blood serum by method of enzyme multiplied immunoassay (ELISA), and if its value exceeds 5.2 ng/ml the menstrual dysfunction is diagnosed against the background of polycystic ovarian syndrome.
EFFECT: use of specified method increases accuracy of diagnostics and execution of the appropriate remedial measures relating polycystic ovarian syndrome.
SUBSTANCE: lipoaspirate is collected into a container; a liquid portion of the lipoaspirate is sampled from the bottom of the container and diluted in normal saline; that is followed by red blood count in the Goryaev's chamber in five large squares, each of which consists of 16 small squares by formula:
EFFECT: invention is easy to implement, objective, provides the high measurement accuracy.
3 tbl, 3 ex
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
SUBSTANCE: method consists in the application of glutationreductase and catalase enzymes as test-objects for determination of the anti-oxidant activity by the ratio of the rate of enzymatic reaction on the test-object after the substance addition and the rate of enzymatic reaction before the substance addition, which must be larger than 1. Preliminarily, before addition to an incubation medium, the samples of essential oil of Siberian fir are diluted with dimethylsulphoxide in a ratio of 1:1.
EFFECT: increased accuracy of determination.
2 ex, 1 tbl
SUBSTANCE: invention relates to a method of early detection of muscular degenerative diseases and to a method of prediction and/or determination of a therapeutic efficiency of a therapeutic preparation and/or a method of disease therapy by measurements of tetranor-PGDM (11,15-dioxo-9α-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic acid) in a subject's urine sample and comparison of its content relative to a sample, separated from a healthy individual. A muscular degenerative disease is identified in a patient if the concentration or content of tetranor-PGDM in a sample, separated in the patient is higher than the concentration or content of tetranor-PGDM in a sample, separated from a healthy individual. An efficiency of the therapeutic preparation and/or the method of therapy of the muscular degenerative disease is determined by comparison of the content of tetranor-PGDM in the sample, separated from the patient with the muscular degenerative disease before and after introduction of the therapeutic preparation. If the measured content of tetranor-PGDM in the sample considerably or inconsiderably decreases after the introduction of the therapeutic preparation, the method of therapy is efficient. The invention also relates to a set for diagnostics of muscular degenerative diseases, which includes an antibody to tetranor-PGDM, labelled tetranor-PGDM and optionally, at least, one compound, selected from a group, consisting of an antibody to immunoglobulin, a diluting solution for the sample, a diluting solution for the antibody and labelled tetranor-PGDM, tetranor-PGDM standard of the known concentration, a substrate for an enzyme immunoassay and a stopping solution for the enzyme immunoassay.
EFFECT: invention is efficient and simple in implementation.
7 cl, 1 tbl, 1 ex, 2 dwg
SUBSTANCE: method of codeine identification includes separation of mixture components by a method of thin-layer chromatography with separation of a chromatographic zone of codeine by a reagent of Dragendorff, and quantitative determination of codeine is carried out in an area of its manifested zone directly in a solid phase by measuring diffusion reflection coefficient at a wave length of 520 nm.
EFFECT: reduction of time of identification and quantitative determination of codeine, reduction of the process labour consumption, reduction of a probability of the target component loss.
2 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents a preparation for involving a mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow, which is introduced into the blood vessel or muscle and which contains any of components: (a) protein HMGB1; (b) HMGB1 protein-secreting cell; (c) a vector, into which HMGB1 protein-coding DNA is inserted; (d) protein HMGB2; (e) HMGB2 protein-secreting cell; (f) a vector, into which HMGB2 protein-coding DNA is inserted; (g) protein HMGB3; (h) HMGB3 protein-secreting cell; and (i) a vector, into which HMGB3 protein-coding DNA is inserted.
EFFECT: elaboration of the preparation for involving the mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow.
3 cl, 6 ex, 1 tbl, 14 dwg
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.