Method for extraction and analysis of contents made from organic material

Abstract

The present invention relates to a process for isolating constituents from organic material, comprising the following process steps: a) freeze-drying of the organic material; b) extracting the constituents with a polar solvent or solvent mixture (A) and an organic solvent or solvent mixture (B), it being possible for the extracts of the extraction with (A) and (B) to form one phase; c) combining the extracts (A) and (B) to give one phase; and d) carrying out an esterification/transesterification in the unpolar phase with an alcohol, the esterification/transesterification being carried out in the presence of a volatile acid; the process being a high-throughput process.

1. A high-throughout high-throughput process for isolating constituents from an organic material, comprising:
 a) freeze-drying the organic material; 
 b) extracting the freeze-dried organic material with
 1) a polar solvent or solvent mixture to form an extract A, and 
 2) an organic solvent or solvent mixture to form an extract B; 
 
 c) combining the extract A and the extract B to form one phase; 
 d) treating the combined extracts of step c) with an alcohol in the presence of a volatile acid to carry out an esterfication/transesterfication esterification/transesterification; and 
 e) isolating the constituents. 
 
     
     
       2. The process of  claim 1 , wherein the polar solvent or solvent mixture comprises a monophasic mixture of 50 to 90% by volume of C 1 -C 6 -alkyl alcohol, 10 to 50% by volume of water, and 40 to 0% by volume of a further solvent or solvent mixture. 
     
     
       3. The process of  claim 1 , wherein the organic solvent or solvent mixture comprises a monophasic mixture of 30% by volume to 60% by volume of C 1 - to C 6 -alkyl alcohol, 40% by volume to 70% by volume of chloroform or dichloromethane, and 0% by volume to 30% by volume of a further solvent or solvent mixture. 
     
     
       4. The process of  claim 1 , wherein the the extraction is carried out by means of accelerated solvent extraction, pressurized liquid extraction, pressurized fluid extraction, ultrasonic extraction, shock-wave extraction, microwave extraction, or by means of a vibrating mill or an Ultraturrax. 
     
     
       5. The process of  claim 1 , wherein the extraction is carried out at a temperature of from 0° C. to 200° C., at a pressure of from 40 bar to 200 bar, or at both said temperature and said pressure. 
     
     
       6. The process of  claim 1 , wherein the volatile acid is HCl, HBr, BF 3 , BCl 3 , trifluoroacetic acid, trichloroacetic acid, or HI. 
     
     
       7. The process of  claim 1 , wherein the alcohol is saturated, unsaturated, straight-chain, branched-chain, or cyclic alkyl alcohol with 1 to 8 carbon atoms. 
     
     
       8. The process of  claim 1 , wherein the constituents are derivatized, chromatographed, and/or analyzed in one or more further steps. 
     
     
       9. The process of  claim 1 , further comprising:
 i) freezing the organic material; 
 ii) homogenizing and/or dispersing the organic material; 
 iii) following phase separation or esterification/transesterification, evaporating the extract to dryness; 
 iv) carrying out an oxime formation in the unpolar phase; 
 v) carrying out an oxime formation in the polar phase; 
 vi) carrying out a trialkylsilylation in the unpolar phase; and/or 
 vii) carrying out a trialkylsilylation in the polar phase. 
 
     
     
       10. The process of  claim 9 , wherein the trialkylsilylation is a trimethylsilylation. 
     
     
       11. The process of  claim 1 , wherein the extract A, the extract B or the combined extracts are treated with one or more internal standards. 
     
     
       12. The process of  claim 1 , wherein the extracted constituents are analyzed by LC, MS, GC, LC/MS, GC/MS and/or LC/MS/MS analysis. 
     
     
       13. The process of  claim 1 , wherein the solvent or solvent mixture further comprises up to 5% by weight of buffer salts, acids, and/or bases. 
     
     
       14. The process of  claim 1 , wherein the organic material consists of plant material. 
     
     
       15. A high-throughout high-throughput process for establishing a metabolic profile, comprising the process steps of  claim 1  and further comprising analyzing the isolated constituents by automatic peak recognition or peak integration. 
     
     
       16. A high-throughout process for the separation of constituents from an organic material, comprising:
 a) freeze-drying the organic material;   b) extracting the freeze-dried organic material with
 1) a polar solvent or solvent mixture to form an extract A, and 
 2) an organic solvent or solvent mixture to form an extract B; 
   c) combining the extract A and the extract B to form one phase;   d) to obtain an unpolar phase and a polar phase, treating the combined extracts of step c) with
 1) an unpolar solvent or solvent mixture, 
 2) a polar solvent or solvent mixture, or 
 3) an unpolar solvent or solvent mixture and a polar solvent or solvent mixture; and 
   e) performing an esterification/transesterification of the constituents contained in the unpolar phase with an alcohol in the presence of a volatile acid.   
     
     
       17. The process of claim 16, further comprising evaporating the solvent of the unpolar phase. 
     
     
       18. The process of claim 16, wherein the polar solvent or solvent mixture comprises a monophasic mixture of 50 to 90% by volume of C 1 -C 6 -alkyl alcohol, 10 to 50% by volume of water, and 40 to 0% by volume of a further solvent or solvent mixture. 
     
     
       19. The process of claim 16, wherein the organic solvent or solvent mixture comprises a monophasic mixture of 30% by volume to 60% by volume of C 1 - to C 6 -alkyl alcohol, 40% by volume to 70% by volume of chloroform or dichloromethane, and 0% by volume to 30% by volume of a further solvent or solvent mixture. 
     
     
       20. The process of claim 16, wherein the extraction is carried out by means of accelerated solvent extraction, pressurized liquid extraction, pressurized fluid extraction, ultrasonic extraction, shock-wave extraction, microwave extraction, or by means of a vibrating mill or an Ultraturrax. 
     
     
       21. The process of claim 16, wherein the extraction is carried out at a temperature of from 0° C. to 200° C., at a pressure of from 40 bar to 200 bar, or at both said temperature and said pressure. 
     
     
       22. The process of claim 16, wherein the volatile acid is HCl, HBr, BF 3 , BCl 3 , trifluoroacetic acid, trichloroacetic acid, or HI. 
     
     
       23. The process of claim 16, wherein the alcohol is saturated, unsaturated, straight-chain, branched-chain, or cyclic alkyl alcohol with 1 to 8 carbon atoms. 
     
     
       24. The process of claim 16, wherein the constituents are derivatized, chromatographed, and/or analyzed in one or more further steps. 
     
     
       25. The process of claim 16, further comprising one or more of:
 i) freezing the organic material;   ii) homogenizing and/or dispersing the organic material;   iii) following phase separation or esterification/transesterification, evaporating the extract to dryness;   iv) carrying out an oxime formation in the unpolar phase;   v) carrying out an oxime formation in the polar phase;   vi) carrying out a trialkylsilylation in the unpolar phase; or   vii) carrying out a trialkylsilylation in the polar phase.   
     
     
       26. The process of claim 25, wherein the trialkylsilylation is a trimethylsilylation. 
     
     
       27. The process of claim 16, wherein the extract A, the extract B, or the combined extracts are treated with one or more internal standards. 
     
     
       28. The process of claim 16, wherein the extracted constituents are analyzed by LC, MS, GC, LC/MS, GC/MS and/or LC/MS/MS analysis. 
     
     
       29. The process of claim 16, wherein the solvent or solvent mixture further comprises up to 5% by weight of buffer salts, acids, and/or bases. 
     
     
       30. The process of claim 16, wherein the organic material consists of plant material. 
     
     
       31. A high-throughout process for establishing a metabolic profile, comprising the process steps of claim 16 and further comprising analyzing the constituents by automatic peak recognition or peak integration. 
     
     
       32. The process of claim 16, further comprising adding water to the combined extracts of step c). 
     
     
       33. The process of claim 16, wherein the extraction of step (b)(1) and the extraction of step (b)(2) are carried out in succession, wherein the extraction of step (b)(1) is carried out followed by the extraction of step (b)(2), or the extraction of step (b)(2) is carried out followed by the extraction of step (b)(1). 
     
     
       34. The process of claim 16, wherein the extract A, the extract B, or the combined extracts are treated with one or more analytical standards, wherein the analytical standard is an isotope-labeled, radiolabeled, or fluorescence-labeled substance.