Method of blood plasma oligosaccharide analysis

FIELD: medicine.

SUBSTANCE: invention refers to a method of analysing oligosaccharides being components of low molecular weight heparins and very low molecular weight heparins in blood plasma. A method of analysing β-unsaturated oligosaccharides in blood plasma. Application of the method for quantitative analysis of enoxaparin, octaparin, bemiparin or tinsaparin. Application of the method for quantitative analysis of octasaccharides (versions).

EFFECT: more adequate oligosaccharide analysis.

15 cl, 2 dwg, 2 tbl, 2 ex

 

The present invention relates to a method of analysis of oligosaccharides, comprising the heparins low molecular weight heparins with very low molecular weight, in the blood plasma.

Heparins are biologically active compounds of the group of glycosaminoglycans, which have particularly useful properties of anticoagulants. The heparins of low molecular weight (HBPM) and heparins with very low molecular weight (HTBPM) is obtained by slicing a long polysaccharide chains of heparin into shorter chains with low molecular weight.

Oligosaccharides comprising HBPM and HTBPM usually characterized by macroscopic parameters such as the average molecular weight or biological activity, such as, for example, the activity of anti-Xa (IU/mg). Mentioned activity, which is determined by the interaction with antithrombin III, is the most commonly used.

However, measurement of such macroscopic parameters or biological activity does not allow to answer many important questions. In particular, on the basis of such criteria it is impossible to analyze the pharmacokinetic profiles of different oligosaccharides comprising HBPM and HTBPM. For example, when using the activity of anti-Xa only species having affinity for AT III, i.e. priblizitelen is only 20% of the mixture; therefore, the 80% of the oligosaccharides cannot be measured. Moreover, the contribution of each species, constituting an affine mixture, also cannot be determined.

Thus, it is impossible to determine the plasma concentration of each oligosaccharide and more good reasons, its pharmacokinetic profile by a simple measurement of the activity of anti-Xa. However, such profiles could provide very valuable information to improve posologie medicines based on HBPM and HTBPM.

In order to be able to analyze the content of various oligosaccharides comprising HBPM or HTBPM, in plasma, it is necessary to be able to separate completely and reproducibly such oligosaccharides from other plasma components, particularly proteins. In the methods described in the prior art, have resorted to continuous processing by proteases (Volpietal.,1995, Biochim. Biophys.Acta., 1243 (1):49-58).

Ways chromatography or electrophoresis to analyze the various components of HBPM. For example, capillary electrophoresis (EC) is used to separate oligosaccharides of heparin (Guerrini et al., Glycobiology, 2002,12: 713-719; Linhardt et al., BioMethods, 1997,9: 183-197). However, the selectivity of EC is much smaller in comparison with liquid chromatography. It is also possible, in some with whom ucah, for the analysis of heparin oligosaccharides to use mass spectrophotometry MALDI-TOF, but it cannot be used to analyze complex mixtures, not to mention the high cost of it.

As a consequence, the analysis of sulfated oligosaccharides, such as oligosaccharides, comprising HBPM and HTBPM, produced mainly by the method of high performance liquid chromatography (HPLC). Was recently described method of analysis HBPM, consisting of enzymatic depolymerization, combined, if necessary, with subsequent restoration, and quantitative analysis by HPLC method (WO 2004/027087). In a recent European application (EP 04290789.9) described a method for quantitative analysis of oligosaccharides comprising HBPM and HTBPM way anion-exchange chromatography CTA-SAX. In the above-mentioned method is used anion-exchange chromatography is carried out with a Quaternary ammonium salt, in particular of camillejimene adsorbed in dynamic conditions on a column of silica as inverted phases and keeping a clear and permanent positive charge at a pH in the range from 2 to 12. In this method may require pre-processing, depolymerization or separation exclusion chromatography prior to analysis HBPM and HTBPM method HPLC on a column of CTA-SAX.

The object of the present invention is a method of analysis oligos is haridev in plasma, characterized in that it is carried out in two stages:

processing samples of blood plasma;

- quantitative analysis by HPLC method.

In this method does not require any enzymatic depolymerization or fractionation exclusion chromatography of oligosaccharides present in the sample of blood plasma, prior to quantitative analysis by HPLC method. On the contrary, according to this method, a sample of blood plasma is treated by filtration thus, to separate oligosaccharides, passing into the filtrate, from polluting the plasma, in particular proteins, which remain in concentrate.

Thus, a more preferred object of the present invention is a method such as defined previously, in which a sample of blood plasma is treated by filtration. More preferably, the object of the present invention is a method such as defined previously, characterized in that molecules of molecular weight greater than 30 kDa, remain in concentrate.

To ensure the possibility to quantify this method of processing samples of blood plasma and to check the reproducibility of such processing with different samples, the sample is injected, if necessary, the internal standard. This standard should be the oligosaccharide, which is how b is more close structurally to analyze quantitatively the connection and which is added to the plasma immediately before filtering.

Thus, an object of the present invention is also a method such as defined previously, characterized in that the oligosaccharide witness added to the plasma sample immediately before filtering.

Although mostly oligosaccharides and are separated from the proteins by filtration, it is also possible that some part of them is captured concentrate due to the electrostatic interaction with him. In this case, you must implement one or more leaching of the concentrate brine.

Thus, an object of the present invention is also a method such as defined previously, characterized in that the concentrate is washed at least once with brine.

According to the present invention the salt solution is preferably a solution of potassium chloride.

More preferably, the object of the present invention is a method such as defined previously, characterized in that the concentration of the salt solution after adding the concentrate is greater than or equal to 1 M, in particular, the way in which the salt concentration is greater than or equal to 2 m.

After one or more washings with saline concentrate is washed, if necessary, water. Thus, an object of the present invention is a method such as defined previously, in which the Le at least one wash concentrate salt solution provides for a stage of washing with water.

The primary filtrate is combined with the filtrate produced during leaching, and the totality of the filtrate was diluted 5 times with water. This dilution allows to concentrate the totality of oligosaccharides after injection in the chromatographic column, avoiding peak broadening caused by the very high salt concentration. Just before the introduction of the pH of the solution was adjusted to 3 by adding 1 N HCl solution.

In the method according to the present invention, the total volume of the filtrate and, if necessary, the filtrate after washing directly injected and analyzed by HPLC method.

Anion-exchange HPLC is a method of separation, better adapted to such a complex mixture. In particular, for HPLC is used anion-exchange column with grafting of Quaternary ammonium due to the covalent bond. This type of column has the advantage of being able to use as a mobile phase solution of perchlorate, which is impossible with column CTA-SAX due to the formation of a strong complex perchlorate and CTA.

Thus, an object of the present invention is also a method such as defined previously, in which the oligosaccharide quantitatively analyze the way anion-exchange HPLC. More preferably, the object of the present invention is a method such as defined previously, in which oterom oligosaccharides quantitatively analyzed by HPLC method for anion-exchange column with an injection of Quaternary ammonium compounds through covalent bonds.

Can be used column type IonPAc AS11 (Dionex) with a grain size 9-13 μm and a length of 25 cm Can be used by speakers of any common diameter in the range from 1 to 4.6 mm, but preferably use a column with a diameter of 2 mm. In the more General case can be used a column of smaller diameter, for example 1 mm, the use of which is required due to the small amount of plasma and may also increase the sensitivity of analysis.

Used by the device can be chromatograph for creating gradient elution and equipped with a UV detector. Preferably use the detector, equipped with a diode matrix sensor element, which allows to obtain UV spectra of the components and to register complex signals, which is the difference of the absorption at 2 different wavelengths and detecting acetylated oligosaccharides among the compounds of plasmatic origin. The use of mobile phase, transparent to 200 nm, allows easy differentiation components. Used in the present invention the mobile phase is preferably a solution of sodium perchlorate, but can also be used methansulfonate or phosphates.

Thus, an object of the present invention is a method, such as measuring the Leno earlier, characterized in that the used mobile phase, transparent to 200 nm. In particular, an object of the present invention is a method such as defined previously, characterized in that the mobile phase is a solution of sodium perchlorate, methanesulfonate or phosphate. More preferably, the object of the present invention is a method such as defined previously, characterized in that the mobile phase is a solution of sodium perchlorate.

The pH value is recommended when the separation is in the range from 2 to 6.5. Preferably the pH is about 3. This pH value can be obtained by adding salts, such as phosphate, can form a buffer solution with pH=3 is better than perchlorate.

As an example, the following standard conditions chromatographic separation:

solvent A: NH2PO4, 2.5mm, brought to pH=2,9 addition of H3PO4;

solvent B: NaClO4, 1 N - NaH2PO4, 2.5mm, brought to pH=3,0 addition of H3PO4.

Gradient elution can be the following:

T=0 min: B=3%; T=40 min: B=60%, T=60 min: B=80%.

For columns with a diameter of 2 mm can be selected, for example, the feed rate is 0.22 ml/min and column temperature of 40°C.

The oligosaccharides present in samples of filtered plasma, detektyw the t in the UV spectrum. As deatsetilirovanie polysaccharides possess, at a given value of pH, almost identical UV spectrum, it is possible to selectively detect acetylated sugar, taking as a signal difference of the absorption at two wavelengths selected so that the absorption deacetylating saccharides is deducted.

The following case is chosen wavelengths 202 and 230 nm as a reference and register the signals at 202-230 nm. To a person skilled in the art it is obvious that the choice of wavelength will depend on the pH of the mobile phase, with adjustment for several nm may be necessary to ensure that the optimum of the above-mentioned conditions. Most appropriate for this technique the detector is a diode matrix detector DAD Agilent Technologies. In this case, carry out a dual detection for each sample at 234 nm, on the one hand, and 202-230 nm, on the other hand. You can also triple detection, measuring, in addition, the absorption of each sample at 280 nm, which allows you to verify that the signal is not distorted protein residues.

Thus, an object of the present invention is a method such as defined previously, characterized in that the method of detection allows you to selectively detect acetylated sugar.

The object of the present invention is also the method such as defined previously, characterized in that the method is selective detection of acetylated sugars is carried out, taking as a signal difference of the absorption at two wavelengths selected so that the absorption deacetylating saccharides is deducted.

Thus, an object of the present invention preferably is a method such as defined previously, characterized in that the method is selective detection of acetylated sugars carried out by measuring the absorption at 202-230 and 234 nm.

Thus, an object of the present invention preferably is a method such as defined previously, characterized in that the method is selective detection of acetylated sugars carried out by measuring the absorption at 202-230, 234 and 280 nm.

The way you defined earlier, allows more preferably to analyze any β-unsaturated oligosaccharides. More preferably the method is used for analysis of oligosaccharides comprising enoxaparin, octoberin, bemiparin, Tinzaparin.

Experimental example

Method

The plasma processing

Take samples of plasma volume in the range from 100 to 1000 μl and, if necessary, add the internal standard.

After homogenizing the resulting solution is injected into the cartridge ultrafiltration Ultrafree5, 30 kDa (Millipore), pre-washed 250 ál water by centrifugation for 5 minutes at 10000 rpm, the Plasma is injected into the upper part of the cartridge for 1 or 2 doses if the quantity to be filtered, in excess of 500 ál.

Chuck centrifuged at 10,000 rpm for 1 to 2 hours, then select the filtrate. If necessary, the second part of the plasma to be filtered, introduced into the upper part of the cartridge and centrifuged again. The exact amount of injected plasma is determined by weighing. Chuck centrifuged up until protein balance in the upper part of the cartridge will not be approximately no more than 10% of the original amount. Select the filtrate and concentrate add 210 ál of 3 M KCl solution per ml of the filtered plasma.

After homogenization cartridge centrifuged at 10,000 rpm for 1 to 4 hours. Chuck centrifuged up until protein balance in the upper part of the cartridge will not be approximately no more than 10% of the original amount.

Select the second filtrate and concentrate the second time add 210 ál of 3 M KCl solution per ml of the filtered plasma. After homogenization cartridge centrifuged at 10,000 rpm for 1 to 4 hours. Chuck centrifuged up until protein balance in the upper part of the cartridge will not be approximately no more than 10% from the original the number.

Select the third filtrate and add 250 ál of water per ml of the filtered plasma.

After homogenization cartridge centrifuged at 10,000 rpm for 1 to 4 hours. Chuck centrifuged up until protein balance in the upper part of the cartridge will not be approximately no more than 10% of the original amount.

The filtrates are combined diluted 5 times with water and the entire amount directly injected with anion-exchange HPLC.

Just before the introduction of the pH of the injected solution was adjusted to 3 by adding 1 N HCl solution.

Method HPLC

Filtered plasma analyzed by the method of ion-exchange HPLC. Used columns are columns IonPAc As11 (Dionex) with a length of 25 cm and an inner diameter of 2 mm is Used, the detector is a UV-spectrophotometer, equipped with a diode matrix sensor element.

Enter the total volume carried out after adjusting the pH to 3 by adding 1 N HCl solution. Choose the feed rate is 0.22 ml/min and a column temperature of 40°C.

Elution is carried out in gradient mode. Use two solutions:

solvent A: NH2PO4, 2.5mm, brought to pH=2,9 addition of H3PO4;

solvent B: NaClO4, 1 N, NaH2PO4, 2.5mm, brought to pH=3,0 addition of H3PO4.

Gradient elution assetsassets:

T=0 min: B=3%; T=40 min: B=60%, T=60 min: B=80%.

Example 1

Below is a changing mixture of three octasaccharide in the blood of dogs-males breed Beagle. Mentioned 3 octasaccharide denoted ΔIIaIIsIsIs, ΔIsIIaIIsIs and ΔIIaIIsIsIs1,6-anhydroare the main octasaccharide, affine with fractions octasaccharide Lovenox®. They have a strong affinity for AT III and, therefore, antithrombotic activity.

The mixture is injected subcutaneously with 3 dogs-males breed Beagle with a target dose for each of 3 these components 0.5 mg/kg of the Injected solution is a solution of a mixture of 3 octasaccharide with a target concentration of 0.5 mg/ml in 0.9%NaCl solution.

Sampling of blood (5 ml) is carried out in a moments time 0; 0,25; 0,5; 1; 2; 4; 6; 8; 24; 30 and 48 h after injection. Blood samples collected in tubes containing 400 μl of a mixture of CTAD (citrate, theophylline, adenosine, dipyridamole). After homogenization the solution is centrifuged at 3000×g for 15 min at 15°C. the Plasma supernatant is isolated and stored at -20°C until use.

For processing plasma before HPLC select approximately 1 ml of plasma and weighed, then add 50 ál of the internal standard solution with a concentration of approximately 0.02 g/l

The internal standard is chosen so that it was as close as possible to structural connections, subject to analysis. Its structure is presented below.

Used tetrasaccharide allows you to adjust the potential impact of changes in the degree of extraction. The plasma process and analyze by HPLC method.

The results obtained are presented in table 1. The corresponding chromatogram is presented in figure 1.

Example 2

HTBPM with an average molecular weight of 2500 Yes injected with a target dose of 1.4 mg/kg volunteers. Sampling of blood is carried out in a moments time 0; 0,25; 0,5; 0,75; 1; 1,25; 1,5; 2; 2,5; 3; 3,5; 4; 4,5; 5; 6; 7; 8; 9; 10; 12; 14 and 24 h after injection.

As used HTBPM is a complex mixture of oligosaccharides, the study was limited to only some of the elements selected from the above-mentioned mixture.

On the other hand, as it was not found internal standard, which does not interact under chromatography was carried out with any of the components of the mixture, a quantitative analysis was carried out, taking as a ratio of the molar response factor obtained in example 1 with affine octasaccharide.

The results reveal very large differences in the content of oligosaccharides, are what they studied heparin with low molecular weight. Oligosaccharides, which has active anti-Xa, show the kinetics of excretion is significantly slower than other sulfated oligosaccharides that cannot be detected on the basis of the activity monitoring anti-Xa.

The results obtained are presented in table 2. The corresponding chromatogram is presented in figure 2.

Table 2
The change in plasma concentration (μm) of the main oligosaccharides, depending on the time after injection
Time clockΔIsΔIsIIsΔIsIsΔIIaIIsIsΔIsIsIsΔIIaIIsIsIs
00,000,000,000,000,000,00
0,250,240,200,160,050,020,01
0,50,30 0,300,240,100,040,03
0,750,260,260,210,100,030,02
10,230,260,200,100,040,03
1,250,200,230,190,120,040,04
1,50,190,220,190,110,030,04
20,160,180,150,110,030,04
2,50,110,130,10 0,090,020,03
30,100,100,090,080,020,03
3,50,080,100,070,090,020,04
40,070,070,070,100,010,02
4,50,060,060,060,100,010,04
50,040,050,050,090,010,03
60,030,040,040,080,01 0,02
70,030,030,030,100,010,04
80,020,020,020,070,000,02
90,010,010,010,070,000,02
100,010,010,010,070,000,02
120,000,000,010,080,000,03
140,000,000,000,070,000,02
24 0,050,01

1

Chromatographic monitoring of pharmacokinetics 3 octasaccharide the dog 1.

2

Chromatographic control the major oligosaccharides comprising heparin, very low molecular weight, depending on the time after injection.

1. The method of analysis of β-unsaturated oligosaccharides in plasma, characterized in that it is carried out in two stages:
a) processing of plasma samples by filtration, and to the plasma before filtering add oligosaccharide witness and molecules of molecular weight greater than 30 kDa, remain in the concentrate, and the concentrate is washed at least once with a solution of potassium chloride concentration after addition of the concentrate is greater than or equal to 1 M;
(b) quantitative analysis method of high performance liquid chromatography (HPLC) of the total volume of filtrate and, if necessary, wash.

2. The method according to claim 1, characterized in that the concentration of the solution of potassium chloride after addition of the concentrate is equal to or exceeds 2 M

3. The method according to claim 1, characterized in that the concentrate is washed at least once with a solution of potassium chloride, and then washed with water the th.

4. The method according to claim 1, characterized in that the method chromatography is an anion-exchange chromatography.

5. The method according to claim 1, characterized in that the method chromatography is an anion-exchange chromatography on anion-exchange column with an injection of Quaternary ammonium compounds through covalent bonds.

6. The method according to claim 4, characterized in that the used mobile phase, transparent to 200 nm.

7. The method according to claim 6, characterized in that the mobile phase is a solution of sodium perchlorate, methanesulfonate or phosphate.

8. The method according to claim 6, characterized in that the mobile phase is a solution of sodium perchlorate.

9. The method according to claim 4, characterized in that it includes a method of detection that allows you to selectively detect acetylated sugar.

10. The method according to claim 9, characterized in that the method is selective detection of acetylated sugars is carried out, taking as a signal difference of the absorption at two wavelengths selected so that the absorption deacetylating saccharides is deducted.

11. The method according to claim 10, characterized in that the method is selective detection of acetylated sugars carried out by measuring the absorption at 202-230 and 234 nm.

12. The method according to claim 11, characterized in that the method is selective detection of acetylated sugars carried out by measuring the absorption at 202-230, 234 and 280 nm.

13. Application of the method according to claim 1 for the quantitative analysis enoksaparina, octopamine, bemiparin or Tinzaparin.

14. Application of the method according to claim 1 for the quantitative analysis of one or more of the following octasaccharide:


15. Application of the method according to claim 1 for the quantitative analysis of one or more of the following oligosaccharides:







 

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FIELD: medicine.

SUBSTANCE: solution of substance to be defined and a sample solution for comparison are prepared. As a diluent for the preparation of the test solutions 0.1 M solution of hydrochloric acid is used. As a sample for comparison yellow dimethyl is used. Optical density of solution of substance to be defined and of yellow dimethyl sample for comparison are measured by spectrophotometer at a wavelength of 318 nm. The results are calculated according to the formula with a conversion coefficient.

EFFECT: improving of results reproducibility; reducing of cost, complexity and analysis errors.

3 ex

FIELD: physics.

SUBSTANCE: method of determining quality of Iceland spar crystals for making polarisers involves measurement of the optical density spectrum of the crystal in the wavelength range from 220 to 400 nm in plane-polarised light. The electric vector of the plane-polarised light is parallel to the optical axis of the crystal. At least two opposite faces of the crystal, parallel to its optical axis, are polished, and the optical axis of the crystal is perpendicular to the incident light. The positions of maximums of the absorption band are determined, as well as the corresponding optical density values, and the quality class of the crystalline material is determined as follows: 1st class - not above 0.31 cm-1, 2nd class - not above 0.35 cm-1, 3rd category - not above 0.45 cm-1.

EFFECT: more accurate control of light absorption of crystalline material for making polarisers.

5 dwg, 1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: present invention refers to analytical chemistry and can be used for quantative determination of methionine in water solutions by spectrophotmetric method. The method includes the preparation of standard methionine solutions, determination of optical density at characteristic wavelength, plotting of the calibration function for standard methionine solutions (optical density against concentrations at characteristic wavelength), determination in the analysed methionine solution of the optical density and methionine concentration according to optical density at characteristic wavelength using the calibration function. The determination of the characteristic wavelength is implemented in ultraviolet spectral region according to the differential absorption in coordinates λ=f(ΔA) whereat ΔA=Afollowing-Aprevious.

EFFECT: enhancing of determination reliability, accuracy and selectivity as well as simplifying and time decrease of the determination.

3 dwg

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine and can be used in control and analytic laboratories for standardisation and control of medication quality. Solutions of substance to be determined and comparison sample are prepared. As solvent for preparation of tested solutions 0.1 M solution of hydrochloric acid is used. As comparison sample phenolphthalein or benzoic acid is used. Optical density of solution of substance to be determined and comparison sample of phenolphthalein or benzoic acid are measured by spectrophotometer. Calculation of results is carried out by formula by introducing to it scaling factor. The method allows for increasing sensitivity of analysis, reproducibility of results of determination, reducing toxicity, labour-intensiveness and analysis error, unifying analysis technique.

EFFECT: increase in analysis sensitivity and reproducibility of results of determination, unification of analysis technique.

4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used in analytical laboratories for standardisation and quality assurance of medical products. The method involves preparing solutions of defined substance and the standard reference solution. Clear water and 0.1 M sodium hydroxide solution are used as a dissolvent for preparation of a defined solution, nickel chloride being used as the standard reference solution. The method further includes measuring optical density of a defined solution and reference nickel chloride solution using spectrophotometer; calculating results from the formula and entering recalculation factor therein.

EFFECT: increased sensitivity of the analysis and reproducibility of results; reduced toxicity, labour input, and analytical error.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used in analytical laboratories for standardisation and quality assurance of medical products. Solutions of defined substance and the standard reference solution are prepared. The 0.1 M sodium hydroxide solution is used as a dissolvent for preparation of a defined solution, and as the standard reference solution - potassium chromate. Measure optical density of a defined solution and reference potassium chromate solution using spectrophotometer. Calculation of results is performed under the formula entering recalculation factor in it.

EFFECT: allows increasing reproducibility of results of definition, sensitivity of the analysis, reducing labour input, an analysis error, excluding use of toxic reactants and unifying analysis techniques.

5 ex

FIELD: measuring engineering.

SUBSTANCE: device comprises the source of ultraviolet radiation, pickups of ultraviolet radiation arranged in the liquid at various distances from the source, and appliance for cleaning.

EFFECT: enhanced precision and simplified design.

2 dwg

FIELD: analytical chemistry.

SUBSTANCE: invention relates to method for quantitative determination of thiotriazoline and pyracetam in complex drugs by high performance chromatography, wherein silicagel with grafted 3-(chlorodimethyl)-propyl-N-dodecylcarbamate having particle size of 5 mum is used as sorbent; and degassed 0.05 M aqueous solution of potassium dihydrophosphate is used as mobile phase. Mobile phase velocity is 1 ml/min, and column temperature is 30°C. Method of present invention makes it possible to determine content of two abovementioned active ingredients simultaneously.

EFFECT: simplified process of sample preparation.

3 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: conjunctival cell sample is collected by pressing a bulbar conjunctiva of an examined eyeball at 2-3 mm above a limb in the meridian 12 hours with a soft contact lens (SCL) placed with its concave side on a tonometre. Then, the SCL is removed from the tonometre and turned out so that the collected sample is at the bottom of the SCL which is fixed in 95% alcohol by single dip. Then, the SCL is washed with distilled water, then coloured with hematoxylin for 10-15 seconds, wash with distilled water and air-dried. Thereafter, the SCL is placed on a slide surface for cytological analysis.

EFFECT: invention allows simplifying the method for conjunctival cell preparation for cytological analysis, reduced analysis time and cost.

1 ex

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