Method for identification and classification of 3-oxosteroids and their metabolites in doping test of sportsmen |
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IPC classes for russian patent Method for identification and classification of 3-oxosteroids and their metabolites in doping test of sportsmen (RU 2452967):
    
Method of antenatal prediction of consequences of perinatal lesions of nervous system in children / 2449287
Clinical examination of pregnant woman is carried out, additionally performed are Stange's and Hench's functional respiratory tests in early period at terms of 11-19, 21-29, 31-39 weeks of gestation period, also in dynamics in I and III trimesters determined are indices of blood hormonal spectrum: T4, TSH, T3, cortisole, vitamin E, insulin, indices of lipid peroxidation and antioxidative blood activity, blood indices - hemoglobin, platelets, total protein, fibrinogen. Analysis of risk factors is carried out, their gradations and numerical values are determined and prognostic coefficients S1 and S2 are calculated by formulas. If S1>S2, presence of moderate or severe CNS lesion in children at the age of 4 is predicted. If S1<S2, presence of light degree of severity, or absence of consequences of CNS lesion in children at the age of 4 is predicted.
Method for prediction of dysthyroidism / 2446401
Method involves woman's pre-delivery blood serum examination for nitrogen oxide and relaxin that is followed by calculation of the NO/relaxin relation. If the relation is 3.6 and less, dysthyroidism is predicted.
Method of predicting development of cerebral metastases in case of lung cancer in women / 2439579
In women in tissue of lung malignant tumour after radical surgery in pulmonectomy volume and in intact lung tissue, obtained from ablated from lung section of the same lung, level of progesterone is determined. If it reduces in tumour in 5 times and more relative the level of progesterone in intact lung tissue, development of cerebral lung cancer metastases in women within the term from 3 to 8 months is predicted.
Method of diagnosing androgenic deficiency / 2439578
In blood serum bioavailable testosterone fraction bound to albumin is determined. If its value is lower than 185.7 pg/ml, disease is diagnosed.
 Method of predicting survival potential of patients with malignant gliomas / 2439577
In patient's urine content of 6-sulfatoximelatonin is determined. If determined content of 6-sulfatoximelatonin after operation before complex treatment equals 292.7 nmol/ml survival to 2.3±0.5 months is predicted, if 22.4 nmol/ml is determined, survival for more than 12 months is predicted. If after chemical therapy determined content of 6-sulfatoximelatonin equals 268.2 nmll/ml, predicted survival time equals to 2.1±0.6 months, if content is 32.8 nmol/ml - more than 12 months. If after a month after treatment determined content of 6-sulfatoximelatonin equals 479.3 nmol/ml predicted is survival for 1.2±0.5 months, in case of 37.4 nmol/ml - survival for more than 12 months.
Method for prediction of cervical cancer metastases / 2436102
In 10-14 days following a surgical removal of a tumour in a patient, daily urine is analysed for sex hormones: oestradiol and pregnandiol. Then, they are related to each other. If the value exceeds 0.8, the onset of metastases or a recurrent disease for the following one and a half or two years is predicted. If the relation is less than 0.8, a metastases-free and recurrence-free period for more than 8-10 years is predicted.
Method of predicting state of fertility in women of reproductive age with uterus myoma / 2433411
At lutein phase of menstrual cycle concentrations of 2-hydroxyestrone 16α - hydroxyestrone in urine, concentration of progesterone in blood serum are determined. Then, canonical value is calculated by formula: K=4.23-0.04×A+0.0016×B-0.18×C, where A is concentration of progesterone in blood serum on 20-21 days of menstrual cycle (lutein phase) (nmol/l), B - concentration of 2-hydroxyestrone in urine (nmol/l), C - concentration of 16α - hydroxyestrone in urine (nmol/l). If K is higher than -0.175, infertility is predicted, if K is lower than -0.175 - safe fertility.
Method of predicting pregnancy in programme of extracorporal fertilisation and transfer of embryos in standard long protocol of superovulation stimulation / 2430379
Before programme of ECF and TE in standard long protocol of superovulation stimulation in patient analysis of hormonal status is performed - content of progesterone receptors in mononuclear fraction of peripheral blood cells is determined. Content of progesterone receptors in mononuclear fraction of peripheral blood cells is determined in the middle of luteal phase of patient's menstrual cycle, before administration of gonadotropin-releasing hormone agonist when carrying out treatment in accordance with standard long protocol of superovulation stimulation. If value is more than 700 progesterone receptors per cell, pregnancy as a result of programme of ECF and TE in standard long protocol of superovulation stimulation is predicted.
Method for prediction of clinical effectiveness of metformin and/or weight-loss therapy in patients with polycystic ovarian syndrome / 2427842
Patient is examined for an anti-Mueller hormone level. If its value is increased by 1/3 and less from an upper limit of normal, a positive menstrual response to treatment is predicted.
Method of predicting risk of thyroid gland diseases in women of perimenopausal age / 2421127
Invention relates to field of medicine, namely to endocrinology. The following data are determined: cause of menopause, application of estrogen preparations, value of index of body weight and thyroid gland volume, concentration of antibodies to thyroid peroxidase and thyrotropic hormone. On the basis of obtained data prognostic coefficient which makes it possible to predict presence or absence of risk of developing thyroid gland diseases in the nearest five years is calculated.
 Method of temperature profile presentation of biological object / 2452925
Method involves input in a database of an image of an examined body area of a biological object which is displayed on a monitor with temperature measuring points on the examined area of the object displayed on the image of the object on the monitor, and after measurements and processing of measuring results in the computer, a computer program forms a temperature profile image on the image of the examined area of the object.
 Method of treating children suffering neurogenic bladder dysfunction with decreased urinate activity by biological feedback / 2452531
Method involves a standard paediatric session of biological feedback - the therapy with using the Urosteam apparatus. The standard session involves training of rectal and urethral sphincters and pelvic floor muscles by voluntary contractions in bladder filling to 1/3-1/2 of the volume. It is combined with recording of bioelectric activity of said muscles in the form of visual graphic objects and providing feedback for achievement of the specified effect. Perianal and abdominal muscle work is differentiated. Duration of a procedure is 28 minutes. The therapy involves 10 procedures. It is followed by a course of the mixed stimulating biological feedback - the Stim Bio therapy. Each procedure is preceded by natural ultrasound-aided bladder filling to threshold sensitivity. It is followed by stimulation at current intensity 5 mA and frequency 75 Hz, with throughput to 500 ms. The 8-second relaxation phases are alternated with 12-second stimulation phases. Duration of a procedure is 28 minutes. The procedure efficacy is controlled by urofluometry followed by determination of residual volume. Each session is followed by controlled urination for the purpose of encoding of urinate skills. The therapeutic course consists of 10 sessions.
Method of treating psychogenic erectile dysfunction / 2452380
Invention relates to field of medicine, namely to sexopathology, and deals with treatment of psychogenic erectile dysfunction (impotence). Preliminary examination of patient aimed at identifying said disorder is performed by registration of nocturnal tumenescence, preferably in home conditions. Signal, corresponding to nocturnal tumenescence norm is selected. In accordance with patient's wishes a relaxing melody is selected. The selected signal is converted into a sound signal, the converted signal is superposed onto the relaxing melody in form of soling sound with patient listening to the obtained audio file for 10 minutes an hour before the sexual intercourse.
 Method of estimating motivational orientation of personality / 2452379
Invention relates to medicine, psychology, and is intended for determination of motivational orientation (MO) of individual. Tested person selects 10 cards with the most important for them value orientations (VO) from 36 cards with given in them VO and arranges them in successive order in accordance with rank importance. Selected VO are related to one of 8 basic types of human activity: perfectional (PF), status (ST), self-realisation (SR), communicative (CM), hedonistic (HD), cognitive CG), esthetic (ES) and altruistic (AL). On the basis of frequency of selection of VO which refer to certain MO types and their rank estimation, final estimation of each MO type is performed in points from 1 to 5 as the arithmetic mean of its all constituents by formula:
Method of quantitative assessment of parameters of life quality of mentally ill people / 2452378
Invention relates to psychiatry and medical psychology and can be used in psychodiagnostics of mentally ill people's attitude to their life. Assessment of patients' life quality is carried out with further processing of obtained answers. Patient answers questions of the questionnaire "Life quality is a two-format instrument". After that, on the basis of obtained results, value of index of psychotics-dependent life quality (PKJ) is calculated: low, when patient requires medical interventions, aimed at improvement of life quality; medium life quality, when patient requires differential observation; high life quality, when patient does not require medical interventions, aimed at improvement of life quality. Assessment is performed in dynamics and targets for further work with patient are selected with setting new therapeutic tasks aimed at correction of life aims.
Method of predicting development of acute endometriosis after medical abortion / 2452377
Invention relates to field of medicine, namely to gynecology. In order to predict development of acute endometriosis after medical abortion before performing abortion performed is analysis of enzymes of lymphocytes in peripheral blood of pregnant women: glucose-6-phosphatedehydrogenase (G6PDG), NADP-dependent reaction of glutamate dehydrogenase (NADPGDG), malic-enzyme (NADPMDG), NADPH-dependent reaction of glutamate dehydrogenase (NADPHGDG) and glutathione reductase (GR). Coefficient of NADP+ (CR NADP), which represents the ratio of the product of activities of G6PDG, NADPMDG and NADPGDG to the product of activities of GR and NADPH-GDG, that is: CR NADP=(G6PDG-NADPMDGNADPGDG)/( GR-NADPH-GDG), is calculated. If the value of CR NADP equals or is higher than 0.09 development of acute endometriosis is predicted, and if the value of CR NADP is lower than 0.09, absence of development of acute endometriosis after medical abortion is predicted.
 Instrument for introduction / 2452376
Invention relates to medical equipment, namely to instrument for introduction of transcutaneous sensor, which contains sensitive part, for instance, for registration of glucose in blood. Instrument for introduction of transcutaneous sensor contains case, sensor case, needle bush, spring element and bearing site. Case is provided with guiding means on its internal surface for guided travel of bearing site. Sensor case contains sensitive part, introduced under the skin. Needle bush contains needle for introduction for puncturing skin. Sensor case and needle bush are connected to each other in a separable way and in their connected state needle for introduction contacts with sensitive part. Bearing site on its internal surface is provided with guiding means which ensures guided travel relative to case between retracted and extended positions. Spring element is connected with trigger means. When trigger means is set into operation sensor case, needle bush and bearing site under the impact of spring element are put into extended position, in which needle and sensitive part can be introduced under the skin. Needle bush and bearing site are provided with inseparable means of fixation. Needle for introduction, at least partially, surrounds sensitive part, introduced under the skin. Introduced sensor of the said instrument for introduction can register glucose in patient's blood. In the second version of implementation of instrument for transcutaneous sensor, needle bush and bearing site are provided with inseparable means of fixation. Lower base of case is made with protruding part. Protruding part forms an angle with needle for introduction in longitudinal direction, setting for the person who introduces the needle the required angle in introduction.
Method of determining optimal volume of infusion therapy for patients with thermal burns who are in state of alcoholic intoxication / 2452375
Invention relates to medicine, namely to combustiology, traumatology, resuscitation science and addictology, and can be used in treatment of patients with thermal burns, who are in state of alcohol intoxication. For this purpose determined are severity of burn disease, degree of alcohol intoxication, area of burn surface, physiological needs and pathologic loss during a day. After that volume of infusion therapy is calculated by formula: V=(Co × ABS)+(Ca x SVIT)+PN+PL, where V is volume of infusion therapy, in ml; Co is coefficient of burn disease severity: 0.5 in case of light burn shock; 0.75 in case of medium burn shock; 1.0 - in case of severe burn shock; 1.5 - in case of extremely severe burn shock. ABS is the area of burn surface, in cm2; Ca is coefficient of alcoholic intoxication degree: 0.5 - in case of light degree; 0.75 - in case of medium degree; 1.0 - in case of severe degree; 1.5 - in case of alcoholic coma. SVIT is standard volume of infusion therapy; PN stands for physiological needs of organism during a day, in ml; PL stands for pathologic loss during a day, in ml.
Method of determining optimal volume of infusion therapy for patients with thermal burns who are in state of alcoholic intoxication / 2452375
Invention relates to medicine, namely to combustiology, traumatology, resuscitation science and addictology, and can be used in treatment of patients with thermal burns, who are in state of alcohol intoxication. For this purpose determined are severity of burn disease, degree of alcohol intoxication, area of burn surface, physiological needs and pathologic loss during a day. After that volume of infusion therapy is calculated by formula: V=(Co × ABS)+(Ca x SVIT)+PN+PL, where V is volume of infusion therapy, in ml; Co is coefficient of burn disease severity: 0.5 in case of light burn shock; 0.75 in case of medium burn shock; 1.0 - in case of severe burn shock; 1.5 - in case of extremely severe burn shock. ABS is the area of burn surface, in cm2; Ca is coefficient of alcoholic intoxication degree: 0.5 - in case of light degree; 0.75 - in case of medium degree; 1.0 - in case of severe degree; 1.5 - in case of alcoholic coma. SVIT is standard volume of infusion therapy; PN stands for physiological needs of organism during a day, in ml; PL stands for pathologic loss during a day, in ml.
Method of determining volume of detoxification-infusion therapy for patients in state of alcoholic intoxication / 2452374
Invention relates to medicine, namely to resuscitation science and addictology, and can be used in carrying out detoxification-infusion therapy in patients in state of alcoholic intoxication. For this purpose determined are degree of alcoholic intoxication, body weight, physiological needs of organism and pathologic loss during day. Volume of detoxification-infusion therapy is calculated by formula: V=Ca × PW+PN+PL, where: V is volume of detoxification-infusion therapy, in ml; Ca is coefficient of alcoholic intoxication degree: 0.25 in case of light degree, 0.5 - in case of medium degree, 0.75 in case of severe degree, 1.0 - in case of alcoholic coma; PW is patient's body weight in grams; PN - physiological needs of organism during a day, in ml; PL - pathologic loss during a day, in ml.
Method for diagnosing the cases of superior thorax aperture syndrome / 2243717
Method involves carrying out ultrasonic scanning examination of subclavian artery over its whole extent in physiological arm position with arterial blood pressure being measured in the middle one third of the arm. Next, when applying compression tests, blood circulation parameters variations are recorded in distal segment of the subclavian artery with arterial blood pressure being concurrently measured. Three degrees of superior thorax aperture syndrome severity are diagnosed depending on reduction of linear blood circulation velocity and arterial blood pressure compared to their initial values. Mild one takes place when linear blood circulation velocity reduction reaches 40% and arterial blood pressure 20% of initial level, moderate one when linear blood circulation velocity reduction reaches 70% and arterial blood pressure 50% and heavy one when linear blood circulation velocity reduction is greater than 70% of initial level and arterial blood pressure is greater than 50% to the extent of no blood circulation manifestation being observed in the subclavian artery.
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FIELD: medicine. SUBSTANCE: blood plasma is examined for the presence and the position of double bonds and the related differences in structural characteristics. Common and examined steroids are chemically modified by a carboxyl group (oximes); their mass spectra are read out and recorded; characteristic ions (lc) and neutral loss (Do) are specified in the mass spectra. The specified lc and Do values are related to those specific for common 3-oxosteroid oximes by the absolute value. Herewith it is only the similar lc and Do values from both sides differing by 0.3% and less that are taken into consideration; the comparison results are used to identify 3-oxosteroid oximes. EFFECT: high accuracy of identification and classification of 3-oxosteroids and their metabolites in doping test of sportsmen, lower detection threshold, reduced amount of blood plasma required for the analysis combined with cutting analysis time. 4 tbl, 1 ex, 5 dwg
The invention relates to medicine, more specifically to methods of identification and determination of steroids and their metabolites, and can be used for doping control. There is a method of recognition and identification of steroids by gas chromatography in combination with mass spectrometric detector [1]. The main disadvantage of this method is the low productivity of the process (at the same time you can analyze 2-3 substances), and the reliability of the results of the analysis do not exceed 0.85, which affects the interpretation of the results. There are also known methods of recognition and identification of anabolic androgenic steroids by high-performance liquid chromatography coupled with tandem mass spectrometry [2-5]. The main disadvantage of this technical solution is the low productivity of the process (at the same time you can analyze 2-3 substances) when the validity of the analysis results is not more than 0.95, and which also affects the interpretation of the results, and besides, it is necessary to detect a lower limit to the concentration of the detected substances are quite high and range from 10 to 2 ng/ml. The technical result, which directed the establishment of this invention is to provide a high precision and s is arnosti recognition and classification of steroids while reducing the number of the analyzed biological fluid (blood plasma), reducing the lower limit of detection and reduce the time of analysis. The technical result is achieved in that the method of identification of 3-oxosteroid in the study of blood plasma, which consists in determining the presence and position of double bonds and the related differences in the structural characteristics known and researched steroids are chemically modified at the carboxyl group (oximes), shoot and record their mass spectra, which are selected from the mass spectra characteristic ions (Iwith) and neutral loss (Do), compare the selected Iwithand Do with those of the known Akimov 3-oxosteroid in absolute values, while taking into account only the values are the same Iwithand Do both sides, varying not more than 0.3%, and on the basis of the comparison results identify oximes 3-oxosteroid. Anabolic androgenic steroids (AAS) is the most common class of compounds abused by athletes to achieve high results [6], and in the prohibited list world anti-Doping Agency (WADA) [7] they are in first place in group S1. According to the authors, getting those or other derivatives of anabolic steroids, namely the introduction of steroid structure of fragments constant is th charge or easily ionizable, in terms of the dissociation reactions induced by collisions allows to identify not only the known steroid, but also to explore ways of mass spectrometric collapse of new (previously unknown) steroid structures and their intended metabolites. 3-Oxosteroid is steroids containing as a substituent the oxygen at the carbon atom at position 3 in the molecule gonna (cyclopentanoperhydrophenanthrene), which is the basis of all steroids. 3-Oxosteroid are extremely important hormones and depending on the nature and location of the substituents, as well as the presence and location of double bonds in the molecule are different regulatory functions in the body. Currently synthesized a large number of different steroids (exogenous), including 3-of oxosteroid, the functional properties not only repeat, but significantly superior to natural steroids produced by the body (endogenous). Such exogenous steroids, particularly testosterone, Androstenedione, etc. can be used and are used as doping. So for doping control athletes definition 3-oxosteroid and products of their metabolism is extremely important, but at the same time, and extremely difficult. There are several reasons, but the most important one is by chemical inertness of steroids. So, for example, 3-oxosteroid can be obtained by dehydrogenation of hydrocortisone, and to restore in pretty tough conditions in the presence of a catalyst of Genesta. Steroids weakly ionized in the process of conducting HPLC-MS/MS analysis with electrospray ionization. This circumstance leads to the fact that to obtain reliable results of the analysis necessary to examine large quantities of analyte. In addition, the limits of detection of steroids when conducting the above analysis range from 10 ng/ml to 2.0 ng/ml, which is obviously insufficient for reliable analysis for the content of steroids and products of their metabolism <10,0 and up to 2.0 PG/ml It should be noted that in our case oxosteroid modify ketogroup. Standard solutions of analytes can be prepared by dissolving the exact portions at the exact volume of the solvent, for example methanol. Working solutions can be obtained by dilution to the appropriate concentration. As chemical modifiers can be used hydroxylamine. As the elution solvent can be used, for example, a mixture of methanol, ammonium acetate and formic acid. As extractant for IGE can be used methyl tertiary butyl ether (MTBE). As pH regulators can be used is sodium bicarbonate and potassium carbonate. As HPLC-MS/MS can be used gas chromatography-mass spectrometer triple quadrupole analyzer TSQ Quantum of Thermo Finnigan (USA), coupled with high performance liquid chromatograph model Surveyor, equipped with an autosampler, a high-pressure pump and degasser company Thermo Finnigan (USA). As accessories can be used: - automatic shaker firm Glas-Col®, USA - for JJA; - centrifuge brand Rotina 46R company Hettich (Germany) to obtain the contrast of the boundary surface between the organic and aqueous phases; - privately company Barnstead Inc. (CUJA), combined with a nitrogen generator Mistral-4 firms Schmidlin-DBS AG (Czech Republic) for evaporation of the organic extract; - column Eclipse XDB-C18, 150×2.1 mm, particle size 5 µm, pore size 100 Á, firms Agilent (USA) for chromatographic separation. As a mobile phase can be used system: 0.05% formic acid, 20 mm solution of ammonium acetate (pH 3.0) and methanol (B). As internal standards (ISTD) can be used, for example, D3-testosterone and methyltestosterone. As a 3-oxosteroid can be selected, for example, testosterone, methyl-1-testosterone, Boldin, androstendion and other similar 3-oxosteroid. Neutral loss is the loss of the investigated molecule with nocturnia fragments in the process of conducting mass spectrometric analysis. It should be noted that these losses of structural fragments in the process of electrospray ionization can be quite a large number, and structural fragments can be the same (for example, the simultaneous loss of two water molecules) or, what often happens in practice, the different nature and the different molecular mass. The contribution of neutral losses total information about the studied object is large enough and therefore the category "neutral loss" as a material object, collateral analysis, and was selected by the authors as the criteria that make a substantial contribution to the information about the investigated substance and, in particular, in establishing its structure. The invention can be implemented as follows. Dissolve accurately weighed substances standards in organic solvents, each aliquot modify hydroxylamine in an acidic environment, the organic components of each modified aliquots quantitatively separated, evaporated in a stream of nitrogen, pererastayut dry residues in the mobile phase and then each aliquot is injected into the system HPLC-MS/MS with electrospray ionization in the registration mode, the positive ions. Remove and register chromatographic and mass spectrometric characteristics of substances-standards (detects full the th MS/MS spectrum, determine the retention time, molecular mass precursor ions, characteristic ions, the lower the detection limit and define the neutral loss). At the same time preparing samples studied biological fluids on a similar scheme and also removed and register their chromatographic and mass spectrometric characteristics; then select from the mass spectra of the investigated biological fluids Iwithand Do, which carry information about the presence and/or position of double bonds in the molecule, which compare the selected Iwithand Do with those of the known Akimov 3-oxosteroid in absolute values, while taking into account only the values are the same Iwithand Do both sides, varying not more than 0.3%, and on the basis of results of comparison of crash data Iwithand Do on the group's compliance with the structural characteristics of known Akimov 3-oxosteroid, which constitute the correspondence table for the selected Iwithand Do the structural characteristics of known Akimov 3-oxosteroid and based on the location of the selected Iwithand Do group compliance classify the nature and structure recognized Akimov 3-oxosteroid. For a better understanding of the invention can be illustrated by, but is not exhausted by the following specific examples of its implementation. Example 1. A. Obtaining mass spectra, determination of characteristic ions, retention time and the limits of detection of known steroids. Prepare standard solutions of analytes (1 mg/ml): testosterone, methyltestosterone, Androstenedione, 1-testosterone, methyl-1-testosterone, 1-Androstenedione, boldenone, metandienon, Boldin, dihydrotestosterone, mestanolone, 5α-androstane-3,17-dione. Working solutions are prepared by dissolving accurately weighed steroids - standards in methanol. Then get working solutions of steroids dilution of the standard solutions to the content of 10 μg/ml Of each working solution selected aliquots of 100 µl injected internal standard (D3-testosterone and methyltestosterone) and chemically modify them. Parameters modification: hydroxylamine under acidic (HCl) environment - 30-35 min, 60-70°C. the Organic layer is modified aliquot quantitatively separated, evaporated in a stream of nitrogen, pererastayut dry residue in mobile phase composition: 0.05% formic acid, 20 mm solution of ammonium acetate (pH 3.0). Thus prepared working solutions is introduced into the system HPLC-MS/MS (gas chromatography-mass spectrometer triple quadrupole analyzer TSQ Quantum of Thermo Finnigan (USA), coupled with high performance liquid chromatograph model Surveyor, equipped with an autosampler, a pump is Alenia and degasser company Thermo Finnigan (USA)). The analysis is carried out at flow rate of the mobile phase of 0.2 ml/min analyte share gradient elution under conditions: 0 min - 40%; 8-9 min 90% B; 12-18 min - 40%; the total time of analysis, taking into account the stabilization of the system before writing the next sample is 18 minutes Ionization at atmospheric pressure is carried elektrorazpredelenie in the registration mode, the positive ions. The voltage on the capillary - 3.8 kV; capillary temperature 245°C; flow rate of drying gas (nitrogen) - 0.45 l/min; gas flow rate (argon) in the camera collision - 0.075 l/min; the temperature in the chamber ionization 200°C; pressure sprayer - 2 ATM. Detection of the detected substances is carried out in the registration mode selective reactions (SRM). The width of the peak for the precursor ions and the corresponding characteristic ions in the first quadrupole (Q1) and the third quadrupole (Q3) is 0.5 Amu (at half height), the delay time is 5 MS. Processing the received data (mass spectra, the characteristic ions, retention times and limits of detection of the investigated steroids) is carried out with application software Xcalibur version 1.3 of Thermo Finnigan, USA. The results of the analysis of steroids-standards presented in the table.
Mapping the selected characteristic ions, which carry information about the presence and/or position of double bonds in the molecule (Table 1, bold), and neutral losses in the analysis of the mass spectra of the 12 investigated Akimov 3-oxosteroid allows you to select four groups of conformity selected characteristic ions and neutral losses structural characteristics of the studied 3-oxosteroid (Table 1 in the column "remarks" is marked in Roman numerals from I to IV) B. sample Preparation and analysis of samples of blood plasma. Three athletes from risk groups (male, 22, 23 and 27 years old) receive blood samples by sampling from the finger (150,0 μl), OTDELA the t plasma and divide it into two aliquots (volume and 20,0 30,0 ml) and next to each sample of blood plasma of the first aliquot volume of 20.0 μl add 50 μl of solution vnutrennego standard containing D3-testosterone (10 ng/ál) and methyltestosterone (10 ng/μl), and chemically modify them. Parameters modification: hydroxylamine under acidic (HCl) environment - 30-35 min, 60-70°C. the Modified aliquots alkalinized to pH 9.5-10.0 with a mixture of sodium bicarbonate and potassium carbonate (1:2), add 5 g of ammonium sulfate and then extracted with 5 ml of MTBE for 10 min on automatic extractor. Centrifuged at 2000 rpm for 5 min, the organic extract is evaporated to dryness in a stream of nitrogen. The dry residue is dissolved in 100 μl of methanol, and then 15 μl of the solution is injected into the system HPLC-MS/MS with electrospray ionization at atmospheric pressure in the registration mode, the positive ions. The analysis is conducted at the following parameters. The flow rate of the mobile phase of 0.2 ml/min analyte share gradient elution under conditions: 0 min - 40%; 8-9 min 90% B; 12-18 min - 40%, the total time of analysis, taking into account the stabilization of the system before writing the next sample is 18 minutes Ionization at atmospheric pressure is carried elektrorazpredelenie in the registration mode, the positive ions. The voltage on the capillary - 3.8 kV; capillary temperature is 245°C; flow rate of drying gas (nitrogen) - 0.45 l/min; gas flow rate (argon) in the camera collision - 0.075 l/min; the temperature in the chamber ionization - 200°C; pressure spray on the e - 2 ATM. Detection of the detected substances is carried out in the registration mode selective reactions (SRM). The width of the peak for the precursor ions and the corresponding characteristic ions in the first quadrupole (Q1) and the third quadrupole (Q3) is 0.5 Amu (at half height), the delay time is 5 MS. The results of the analysis of the first aliquot blood plasma samples are presented in Tables 2, 3 and 4 (athletes 22, 23 and 27 years, respectively). Note: the table presents the data of mass spectra of steroids that match the characteristics of the considered four groups of steroid standards. Needless to say, that if you expand the list of steroid-standards (i.e. known steroids), then certainly consider 4 groups of steroids will be replenished with new steroids and their metabolites, and the number of groups will be more than four.
Tables 2-4 (column "PR is a comment") set (detected) belonging analyzed steroids to the corresponding groups, and further themselves steroids classified (compared selected characteristic ions). Thus, athletes from the risk group established a presence in plasma: 22 year - endogenous steroid methyl-1-testosterone and exogenous steroid Androstenedione, 23, exogenous steroid testosterone and endogenous steroid Boldin, 27 years - endogenous steroids mestanolone and 1-Androstenedione. C. Comparative analysis of samples of blood plasma without derivatization. a) sample preparation and analysis of blood plasma (30,0 second ál aliquots of each sample from part B of Example 1) is conducted as in Example 1, except that the samples do not modify hydroxylamine. The results of the analysis of negative - no steroid was not found. b) athlete (male, 22 years) receive a blood sample by a fence from Vienna (6.5 ml), separate the plasma (volume 1.5 ml), add 50 ál of solution vnutrennego standard containing D3-testosterone (10 ng/ál) and methyltestosterone (10 ng/μl), alkalinized to pH 9.5-10.0 with a mixture of sodium bicarbonate and potassium carbonate (1:2), add 5 g of ammonium sulfate and then extracted with 5 ml of MTBE for 10 min on automatic extractor. Centrifuged at 2000 rpm for 5 min, the organic extract is evaporated to dryness in a stream of nitrogen. The dry residue is dissolved in 100 μl of methanol, and then 15 μl of the solution is injected into the system HPLC-MS/MS. The analysis is conducted at the same settings as the part B of Example 1. The results of the analysis are presented in the form of MS-MS spectra, on page 16 description. Lower limit of detection of steroids is 8 ng/ml (8000 PG/ml) As can be seen from the description, the example of the method and comparative analysis, the claimed invention provides a high accuracy of recognition and classification of 3-oxosteroid and their metabolites in doping control athletes, reduces the detection threshold, reduces the amount of plasma required for analysis, while reducing the time of analysis. Mass spectra Akimov 3-oxosteroid (presented mass spectra of the three steroids-standards of 12). Arrows indicate neutral loss and presents their values.
1-oxime testosterone
Methyl-1-testosterone oxime
1-androstene the oxime **Actually neutral loss of oxime 1-Androsterone are 117 {99+18(-N20)} MS/MS spectra of blood plasma from part b of Example 1 (sopostavitelnaya)
It should be noted that the values of the characteristic ions of the MS-spectra are not the same and not corallium with any of the installed 4 groups of 3-oxosteroid-standards.
A method for the identification of 3-oxosteroid in the study of blood plasma, which consists in determining the presence and position of double bonds and the related differences in structural characteristics, with known and researched steroids are chemically modified at the carboxyl group (oximes), shoot and record their mass spectra, which are selected from the mass spectra characteristic ions (Iwith) and neutral loss (Do), compare the selected Iwithand Do with those of the known Akimov 3-oxosteroid in absolute values, while taking into account only the values are the same Iwithand Do both sides, varying not more than 0.3%, and on the basis of the comparison results identify oximes 3-oxosteroid.
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