Method for analyzing labeled organic compound sample with high effectiveness liquid chromatography approach being applied

FIELD: chemical engineering; medical engineering.

SUBSTANCE: method involves plotting two chromatograms one of which is based on radioactivity (No 1) and the other one on ultraviolet absorption (No 2) or on radioactivity (No 1) and on fluorescence (No 2) and chromatogram specific relative to ultraviolet absorption (No 3) or relative to fluorescence (No 3). Material quality is estimated to be the more high the more close studied labeled compound peak shape is to trapezoid shape on the third chromatogram.

EFFECT: high accuracy of the method.

8 dwg

 

The invention relates to analytical organic chemistry and can find application in biological chemistry and medicine.

There is a method of analysis of a sample of labeled organic compounds by high performance liquid chromatography (HPLC) by plotting pairs of chromatograms using two detectors: radioactivity and UV absorption or by radioactivity and fluorescence, and then defining the qualities of the labeled sample by results of the visual assessment of the nature of the peaks of the analyzed compounds in the chromatograms (Patent RU №2183123, CL G 01 N 33/74, publ. 10.06.2001,)

However, the accuracy of determination of the purity of the analyzed sample labeled organic compounds when using this known method is low.

The technical result achieved by the present invention is to improve the accuracy of determining the purity of the sample labeled compounds.

This technical result is achieved in that in the method of analysis of a sample of labeled organic compounds by high performance liquid chromatography by plotting pairs of chromatograms using two detectors: radioactivity and UV absorption or by radioactivity and fluorescence and subsequent determination of the quality of the analyzed sample R. the results of the visual assessment of the nature of the peaks labeled compounds in the chromatograms distinctive feature is further draw the chromatogram specific radioactivity relative to the UV-absorption or the chromatogram specific radioactivity relative to the fluorescence, and the quality of the analyzed sample labelled compound appreciate the higher, the closer the shape of the peak labeled connection on an additional chromatogram to a trapezoid shape.

When analyzing samples labeled organic compounds by HPLC their purity can be estimated higher than “careful” peak, they formed on the chromatogram. However, subsequent application of quantitative methods of analysis of the sample, the degree of purity of which is estimated by HPLC as high, shows the fallacy of such a conclusion.

Our own research has shown that the results of analysis of samples labeled organic compounds can be made more informative and reliable, if in the process HPLC trace not only the chromatogram of their radioactivity and the chromatogram their UV absorption or chromatogram radioactivity in combination with fluorescence chromatogram, but additionally to draw a third chromatogram the chromatogram specific radioactivity relative to the UV absorption of the compounds or with respect to its fluorescence.

When high purity of the sample in the process of release of labeled compounds, analyses what has been created by HPLC, the ratio of its radioactivity to its UV absorption or fluorescence remains constant and therefore the curve in the area of the peak takes shape close to a trapezoid. If the specific radioactivity varies as the analyzed compounds, it indicates a lack of purity of the sample.

1 shows a General view of the setup used for analyzing a sample labeled connection method according to the invention.

Figure 2 gives the schematic of the setup is depicted in figure 1.

3 shows the chromatogram of the reaction mixture during the synthesis of labeled arachidonic acid. Column: Kromasil 100C18(4×150 mm) 7 microns. Eluent: 20% methanol - 50 mm ammonium phosphate buffer (pH 2,8).

1 - chromatogram radioactivity;

2 is a chromatogram of UV absorption;

3 is a chromatogram radioactivity relative to the UV-absorption.

4 shows the chromatogram of a sample of labeled arachidonic acid after isolation and purification (after preparative HPLC). Column: Kroinasil 100S18(4×150 mm) 7 microns. Eluent: 15% methanol - 50 mm ammoniumphosphate buffer (pH 2,8).

1 - chromatogram radioactivity;

2 is a chromatogram of UV absorption;

3 is a chromatogram of the specific radioactivity relative to the UV-absorption.

Figure 5 presents the chromatogram of the sample labeled olanzapine, after E. what about the cleaning (after preparative HPLC). Column: Kromasil 100C18(4×150 mm) 7 microns. Eluent: acetonitrile-water(1:5)+0.1% of triperoxonane acid.

1 - chromatogram radioactivity;

2 is a chromatogram of UV absorption;

3 is a chromatogram of the specific radioactivity relative to the UV-absorption.

Figure 6 presents the chromatogram of the sample labeled olanzapine, after re-treatment (after re-preparative HPLC). Column: Kromasil 100C18(4×150 mm) 7 microns. Eluent: acetonitrile-water(1,5)+0,1% triperoxonane acid.

1 - chromatogram radioactivity;

2 is a chromatogram of UV absorption;

3 is a chromatogram of the specific radioactivity relative to the UV absorption;

Figure 7 presents the chromatogram of the sample danilinho derived, tritium-labeled Proline. Column: Kromasil 100S18(4×150 mm) 7 microns. Eluent: methanol - 50 mm ammoniumphosphate buffer (pH 2.8) (65:35).

1 - chromatogram radioactivity;

2 - fluorescence chromatogram;

3 is a chromatogram of the specific radioactivity relative fluorescence.

On Fig presents the chromatogram of the sample danilinho derived, tritium-labeled Proline after the analytical treatment (analytical HPLC). Column: Kromasil 100S18(4×150 mm) 7 microns. Eluent: methanol - 50 mm ammoniumphosphate buffer (pH 2.8) (65:35),

1 - chromatogram radioactivity;

2 - fluorescence chromatogram;

3 - hromatography specific radioactivity relative fluorescence.

For implementing the method according to the invention is convenient to use the installation, General view and its schema are presented respectively in figures 1 and 2. The installation consists of two vessels (1, 2) with suenami, steering pump Gilson 305 (3), the slave pump Gilson 303 (4), manometric module (pressure gauge) Gilson 802C (5), active mixer Gilson 811 (6), 7125 injector (7) with hinge (8), predalone (9), column (10), UV detector (Jasco UVIDEC-100-1V, consisting of the measuring unit (11) and the cell (12), radioactivity detector, consisting of the cell (13) and the measuring unit (unit of account) (14), scintillator (vessel with scintillation fluid) (15), pump scintillation fluid (16), mixer (17), a vessel for collecting waste eluent (18), analog-to-digital Converter (19) and the computer (20). Instead of the UV detector can be used detector for fluorescence.

For the analysis in such a setup eluent of the vessels 1 and 2 are fed by pumps 3 and 4 under the control of the pump 3 and for a given program while monitoring the pressure in the system using the pressure gauge 5 are mixed in the active mixer 6. From it the mixture of eluents is served in the injector 7 loop 8, where in the eluents flow is injected samples. Then the mixture passes through predalone 9 and column 10 and enters the measuring unit 11 UV detector is a (or detector fluorescence) in his cell 12. Next, the sample is introduced into a cuvette 13 detector radioactivity measuring unit 14. Pre-sample is mixed with scintillation fluid, which is supplied from the scintillator 15 by pump 16 to the passive mixer 17. Exhaust eluent discharged into the vessel 18. Information from the detectors for UV-absorption (or fluorescence) and radioactivity processed in the analog-to-digital Converter 19 to the computer 20 using Multirom for Windows. The computer display will receive two pairs of chromatograms: radioactivity and UV absorption or by radioactivity and fluorescence. In addition, the display of the computer watching the chromatogram specific radioactivity relative to the UV-absorption or the chromatogram specific radioactivity relative to the fluorescence, which is judged on the quality of the analyzed sample labeled organic compounds.

Below are examples illustrating the invention.

Example 1. Determination of the purity of the tritium-labeled arachidonic acid. The reaction mixture was analyzed by HPLC, column Kromasil 100S18(4×150 mm) 7 μm, eluent: 20% methanol - 50 mm ammoniumphosphate buffer (pH of 2.8). Presented on Fig.3 chromatogram indicate low purity of the analyzed sample. The peak of arachidonic acid in the chromatogram 3 specific radioactives and UV absorption has the shape of a triangle (time out of 9.4 and 10.8). The selected sample of tritium-labeled arachidonic acid was subjected to preparative purification (preparative HPLC). The results of the analysis of a purified sample is presented in figure 4. Column Kromasil 100C18(4×150 mm) 7 microns. Eluent: 15% methanol - 50 mm ammoniumphosphate buffer (pH of 2.8). The peak tritium-labeled arachidonic acid in the chromatogram of the specific radioactivity relative to the UV absorption coming to a trapezoidal shape, this indicates a high degree of purification of the analyzed sample.

Example 2. Determination of the purity of the tritium-labeled olanzapine. Sample tritium-labeled olanzapine were analyzed by the method according to the invention after preparative HPLC. Column: Kromasil 100S18(4×150 mm) 7 microns. Eluent: acetonitrile-water (5:1)+0.1% of triperoxonane acid. The analysis of the curves of figure 5 shows the approximation of the shape of the peak on the chromatogram specific radioactivity relative to the UV-absorption to a trapezoidal form.

After re-cleaning shape of the peak (smpeg) indicates the high purity of the analyzed sample.

Example 3. Determination of purity danilinho derived tritium-labeled Proline. The reaction mixture was analyzed by HPLC, column Kromasil 100C18(4×150 mm) 7 microns. Eluent: methanol - 50 mm ammoniumphosphate buffer (rn) (65:35). Presented on Fig.7 chromatogram the s indicate low purity of the analyzed sample. Peak danilinho derivative labeled with tritium Proline on the chromatogram specific radioactivity relative fluorescence has the shape of a triangle (time ~7 min). The selected sample danilinho derivative labeled with tritium Proline was subjected to analytical treatment (analytical HPLC). The results of the analysis of a purified sample presented on Fig. Column: Kromasil 100C18(4×150 mm) 7 microns. Eluent: methanol - 50 mm ammoniumphosphate buffer (pH 2.8) (65:35). The ratio of peaks of radioactivity and fluorescence becomes constant, this indicates a high degree of purification of the analyzed sample.

Thus, the method according to the invention improves the accuracy of estimation of quality of the sample labeled compounds analyzed by HPLC method.

The method of analysis of a sample of labeled organic compounds by high performance liquid chromatography by plotting pairs of chromatograms using two detectors: radioactivity and UV absorption or by radioactivity and fluorescence and subsequent determination of the quality of the analyzed sample by results of the visual assessment of the nature of the peaks labeled compounds in the chromatograms, characterized in that it further draw the chromatogram specific radioactivity relative to the UV poglasheniye the chromatogram specific radioactivity relative fluorescence, and the quality of the analyzed sample appreciate the higher, the closer the shape of the peak labeled connection on an additional chromatogram to a trapezoid shape.



 

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