Patent EA-004663-B1 (EA)
Title: Method for chromatographic finger printing and standardization of single medicines and formulations
Inventor: DADALA VIJAYA KUMAR
IPC: G01N30/88
Filed: 2000-12-08
Abstract: 1. A method for detection and identification of constituents of extracts of plants or animals, natural or synthetic sources possessing medicinal value, using chromatographic finger printing techniques, the said method comprising steps of: i) extracting organic or organo-metallic compounds from plants or animal natural or synthetic sources using suitable solvent; ii) subjecting the extract obtained in step (i) to separation based on pH and polarity, using High Pressure Liquid Chromatography techniques; iii) generating contour and 3D chromatograms of the constituents eluted in step (ii); iv) converting the 3D and contour chromatogram obtained into a colored image, analyzing the colored image for its individual colons using the co-ordinates denoting all its 3-dimensional properties of the said image by using an in built software; v) denoting the concentrations of the various constituents eluted with time; vi) generating a chromatogram based on solar analyzed, having peaks at various retention times along with conjugative properties of the constituents; vii) identifying the compounds in the said elated constituents by the UV-VIS absorptive properties of the various compounds in the image, identifying, determining and classifying the compounds eluted as polar, medium polar and less or non-polar based on the polarity and conjugative properties; viii) generating a barcode for a selected peak using X axis as Retention Time, Y axis as Wavelength, R as number of Red Pixels, G as number of Green Pixels and B as number of Blue Pixels; and ix) generating a database of fingerprints and barcodes and identifying the respective compounds of the extract. 2. A method as claimed in claim 1 wherein, the solvents with different polarities are selected based on the hydrophilic and hydrophobic nature of the sample under study, ethyl alcohol is used for standardization of medicines. 3. A method as claimed in claim 1 wherein, the fingerprints are developed for the same medicine extracted under different pH ranges. 4. A method as claimed in claim 1 wherein, the HPLC apparatus used is selected from any commercially available HPLC apparatus with the Photo Diode Array detector, preferably with a gradient or ternary system of pumps. 5. A method as claimed in claim 1 wherein, the pH and polarity of the mobile phase is controlled by varying the ratio of the mixture 0 to 100% of an aqueous solvent, water or a buffer at a required pH by using a salt (like Potassium Di-Hydrogen orthophosphate or Di potassium hydrogen orthophosphate and phosphoric acid maintaining the required pH) with a non-aqueous solvent. 6. A method as claimed in claim 1 wherein, the non-aqueous, organic and aqueous, water or buffer at a known pH are the solvents used in step (iii) and are selected based on the range of polarity. 7. A method as claimed in claim 1 wherein, converting the contour chromatograms into a colored image comprising the conjugative and polarity properties of the constituents of the medicine under study. 8. A method as claimed in claim 1 wherein, the therapeutic efficacy of a medicine (single or formulated) is assessed using the quality of the constituents present in a particular polarity and W-Vis absorptive zone. 9. A method as claimed in claim 1 wherein, the software generates a barcode for a selected peak or peaks or image using the X axis as Retention Time, Y axis as Wavelength, R as number of Red Pixels, G as number of Green Pixels and B as number of Blue Pixels as the coordinates, provided by the software, which makes the product propriety for an industry. 10. A method as claimed in claim 1 wherein, the software used for detection and identification of constituents of extracts of plants or animals, natural or synthetic sources possessing medicinal value, is called Rainbow having the following features: (a) a software with a facility of opening chromatographic fingerprint images in different Formats (extensions) like. BMP, JPEG, TIF, GIF from the file folders and analyze it for different colors present in the image with single pixel sensitivity; (b) a software with a facility of display of the pixel information in the form of (l) a graph having a scale of X (0 min. time scale) and Y (200-800 nm) coordinates and (2) a Pie diagram with individual values of each peak (Automatic and Manual) in two separate columns beside the graph; (c) software with a facility of printing all the data generated after analysis using PRINT Icon; (d) a software with a facility of changing the page setup for printing using PAGE SETUP Icon, (e) a software with a facility of selecting a part of the image and analyze using RESIZE Icon; (f) a software with a facility of opening any number of image analysis windows for different images, and display of status in WINDOW icon; (g) a software with a facility of dividing the image in to three Zones at 20 min interval, using ZONE icon; (h) a software with a facility of inverting the selected image using INVERT icon; (i) a software with a facility of switching over to Notepad, Word pad and MS Word, using EDITOR icon; (j) a software with a facility of operational information about various features of the Software using, the HELP icon; and (k) software with a facility of saving the data generated using SAVE AS icon as JPEG file format. 1l. A software as claimed in claim 10, wherein extracts of plants or animals, natural or synthetic sources possessing medicinal value are used in data processing of 3 D chromatograms and color contour image of an ingredient, said processing comprising the steps of: a) generating a coloured contour image of extract, analyzing (extracting colors) the colored contour image based on the selection of various colors (with standards mentioned in release notes, life cycle, processing) denoting the concentrations of the various constituents eluted with time, and polarity based on retention time; b) analyzing the 3-D chromatograms of the medicine using all its 3 dimensional properties of the image; c) generating a chromatogram having peaks at various retention times along with conjugative properties of the molecules eluted with time in a specified order of polarity; d) identifying the compounds in the said molecules by the UV-Vis absorptive properties of the various constituents in the image; e) correlating the reported biological, therapeutic activity of the of various constituents present in the medicines understudy based on the polarity and the conjugative properties of the molecules by dividing the fingerprint into therapeutic zones on X and Y axis; f) generating a barcode for a selected peak (s) using the image coordinates viz., X for retention time, Y for wavelength, R for number of red pixels, G for number of green pixels and B for number of blue pixels, provided by the proposed software; g) generating a database of fingerprints and barcodes for the samples, facilitating all kinds of database utilities like Enterprise Resource Planning (ERP) and Customer Resource Management (CRM) applications; and h) generating a database of the display widows for all the samples to be used by the ENTERPRISE RESOURCE PLANNING (ERP) and CUSTOMER RELATIONSHIP MANAGEMENT (CRM) type of business applications. 12. A software as claimed in claim 11 wherein, the solvents used for extraction is selected based on the polarity, hydrophilic and hydrophobic nature of the constituents present in the plant, plant or animal, natural or synthetic in origin having medicinal value. 13. A software as claimed in claim 11 wherein, the HPLC apparatus used is selected from any commercially available HPLC apparatus with the Photo Diode Array detector, preferably with a gradient or ternary system of pumps. 14. A software as claimed in claim 11 wherein, the polarity of the mobile phase of a non-aqueous and an aqueous solvent of a specific pH is controlled by varying the ratio of the mobile phase from 0% to 100% of an aqueous solvents like water or a buffer of a known pH, along with a non-aqueous solvent. 15. A software as claimed in claim 11 wherein, on analysis of 3-D and contour chromatograms using new software called Rainbow, that gives a chromatogram with retention time and wavelength on its X and Y-axis. 16. A software as claimed in claim 11 wherein, on analysis of 3-D and contour chromatograms using new software which gives a data having indicated the vitiation of doses quantitatively in percentage ratio. 17. A software as claimed in claim 11 wherein, a single solvent ethanol is used for extraction of the constituents; same analytical conditions and instrumental parameters were used for all samples to bring the therapeutic generalizations and to achieve the therapeutic standardization. 18. A computational method of chromatographic finger printing, chemical and therapeutic standardization and bar coding of organic and organo-metallic molecules from a plant, animal or a naturally available or man made materials used as medicines, said method comprising: a) selecting plant, animal or a naturally available or man made materials which possess medicinal value, and extracting the constituents, b) separating the constituents into individual compounds, generating and converting the 3-D and contour chromatograms into fingerprints, c) analyzing the fingerprints using the software developed, and d) interpreting the data. 19. A method as claimed in claim 1 wherein, the software is used in step (iv) which provides chemical analysis of the constituents present in the extract under study and their conjugative and polarity properties indicating the therapeutic efficacy as per the traditional concepts of the medicine. 20. A method as claimed in claim 1 wherein, the software is used in step (iv) which provides a novel concept of chromatographic finger printing possessing medicinal value which is useful for the quick identification of the actual profile of the compounds present in the medicine under use along with their therapeutic efficacy of the constituents. 2