SEMI-SYNTHETIC COMPOUNDS

The object of the invention is a novelty mode of extraction of sulphonamides, secondary metabolites, pesticides or semi-synthetic compounds for analytical and preparative purposes, by using the mixture of tetrahydrofurans with water and the addition of non-organic salts as solvents with the possibility of simultaneous hydrolysis of compounds in the glycoside form.

Both science and industry search for new efficient methods of extraction for diagnostic purposes and to obtain compounds for more technical scale.

There has been a requirement lately for the increase of environmentally friendly methods in the field of modern extraction methods. Tetrahydrofura, by virtue of its similarity to the diethylic ether, proves to be an equally good agent for most organic compounds, being also safer in use due to the high boiling temperature and not revealing narcotic effects. Tetrahydrofuran is mainly used as a substratum for the production of a poliglycol (tetrahydromethyloethyl) used in elastomers manufacturing process. The dose LD ₅₀ for tetrahydrofuran is 1650 mg/kg (for rats) which allows to compare its toxicity with acetone so commonly used at households. A relatively low boiling temperature equal to 66°C allows for its easy vaporization as well as its recirculation in industrial installations, which limits its negative impact on the environment, lowers costs as well as enables easier processing of the extracted compounds.

Glycosides are a class of organic compounds made of sugar and aglicon part. A glycoside bond appears usually between a hemiacetal hydroxylate group and the micromocular compound. In nature, and especially among plants, the glycoside form is used to deactivate secondary metabolites to enable their storage in cells. In order to activate them, a hydrolysis of glycoside bond is necessary. Marking numerous compounds, including sulphonamides and secondary metabolites in the glycoside, is rather an arduous process. Therefore, the possibility of conducting hydrolysis is so significant. In the case of living organisms, the enzyme hydrolysis is used, which in diagnostic sampling, and at the preparative stage in particular, is a very expensive process. The less expensive acid hydrolysis creates the effect of sample neutralization and thus hinders the extraction. In the mode used in the invention described, a caching solution was used to neutralize the sample in order to ease its extraction; the sample has been further processed by selecting the most efficient and optimum pH level.

Sugars are the basis spare materials among plants. Extraction and marking compounds not being sugars from fruits and honey causes a lot of problems. This is mainly due to a huge predominance of saccharides in a sample. Through their structure, the soluble solutions of sugars in water are characterized by a high level of viscosity, which hinders a direct analysis of samples or makes it impossible to conduct it due to neglecting the de-sugaring process. The solubility of saccharides in tetrahydrofuran is disregardingly limited, thanks to which the extraction of compounds by means of this solution allows to remove sugars from the analyzed sample.

The psychoactive substances as chemical compounds, through their activity on the human organism, cause psycho changes and influence the perception of the surrounding reality.

The mode applied in the invention allows to achieve a higher level of organic compounds recovery, especially sulphonamides, secondary metabolites and semi-synthetic compounds.

The process of divisional "liquid-liquid" extraction has been long known in the literature and is commonly used even today. It assumes the application of two non-mixable liquids. One of them is usually water and the other a non-polar organic solvent. This process relies on the phenomenon that organic compounds are most often soluble in organic solvents, which allows their extraction and concentration. The applied solvents often have lower boiling temperature, as compared with water, which enables an easy evaporation process and obtaining clear form of the extracted compounds. The weakness of this method is the sue of solvents which do not mix with water, namely the ones of lower polarity level. Many organic compounds, including sulphonamides, secondary metabolites and semi-synthetic compounds, due to their chemical properties, are extracted to the lower polarity solvents and thus classic divisional extraction of the "liquid-liquid" type becomes a useless part of both analytical and industrial process.

It is known that by adding non-organic salts to the mixture of water with another organic water mixable solvent causes the appearance of two phases - organic and aqueous. Depending on the used organic solvent, salt and their concentration in the mixture, different effects are achieved. Currently the most frequent pair of solvents for analytical processes in the salting treatment is the combination of acetonitrile with water. The most popular procedure with the use of acetonitrile is based on "clearing" the sample by means of extraction to the solid state which increases the cost of a single analysis and makes this method impossible to be applied on the industrial scale. Acetonitrile solvent used in this method, due to the presence of nitrile group, (- CN) is metabolized in living organisms to (HCN), being a strong poison even in low concentrations.

The application of trahydrofuran in the invention, as well as eliminating the stage of the sample "thorough cleaning", have allowed for the lowering of costs and have enabled for the industrial use of the process.

The choice of the tetrahydrofuran and water mixture was dictated by the fact that tetrahydrofuran is a good solvent for many organic compounds, including sulphonamides. During the process of salting treatment it reveals better dissociation from the water chase making the solvent recovery an easier process and enabling the application of acetonitrile in a smaller volume.

Any non-organic salt may be used in the process as the salting treatment substance in this method, although the preferred substance is sodium chloride due to its low price and low level of toxicity.

The object of the invention is the mode of extraction of sulphonamides, secondary metabolites and semi-synthetic compounds including the following stages: a. The mixture of extracted material with the tetrahydrofuran and water mixture; b. Simultaneous acid and neutralizing hydrolysis of a mixture by means of caching solution;

c. Salting treatment of water and tetrahydrofuran mixture.

The mode according to the invention may be applied with the omission of the acid hydrolysis stage.

The mode according to the invention, where the ratio of tetrahydrofuran to water is between 1:10 and 2:1.

The mode according to the invention, where the hydrolysis is conducted by means of hydrochloric acid of the concentration between 0.5 and 4 mol.

The mode according to the invention, where the salting treatment is conducted by means of non-organic salt.

The mode according to the invention, where the caching is the citrate buffer of pH between 2-5, preferably 3.5. The mode according to the invention, where the secondary metabolites are extracted in the form of glycosides with active aglycones.

The mode according to the invention is used for the extraction of psychoactive agents from the food samples.

The mode according to the invention is used for the extraction of organic compounds from environmental samples.

The mode according to the invention is used for the extraction of active pharmacologic agents in order to mark their concentration in various formulas.

The mode according to the invention, where gas or liquid chromatographic technique is used for the sample analysis.

The mode according to the invention, where the process is conducted in a larger scale than the analytical one.

Fifiures description:

Fig.l - presents a chromatogram of sulphonamides marking in honey samples.

Fig.2 - presents a chromatogram of naphthoquinone extraction from the plants of Drosera and Dionea family.

Fig.3 - presents a chromatogram of the caffeine contents in coffee analysis.

Fig.4 - presents a chromatogram of the analysis of e-cigarette fluid for the presence of nicotine

Fig.5 - presents a chromatogram of the pesticides contents sample analysis Fig.6 - presents the extraction process according to the invention The invention is illustrated by the following examples of performances, which should not be treated as limitations.

The method relies on the combination of sample acid hydrolysis, neutralization and adding non-organic salt to the extracting mixture until its saturation (preferred salts: sodium chloride, magnesium sulphite). Such a procedure causes the separation of organic layer from aquatic.

1. A known amount of prepared earlier homogenous sample is measured;

2. In case of solid samples of dense liquids water is to be added to the extracted mixture;

3. In case of the lack of glycoside compounds in the sample proceed to the step 6;

4. Instead of water, acid is to be added to the extracting mixture - the stage of acid hydrolysis (preferably 0.5 to 4 mol concentration of hydrochloric acid);

5. The sample is neutralized by means of caching solution (preferable neutralizing agents: amon acetate, sodium acetate, sodium citrate, sodium hydrocarbonate, sodium hydroxide);

6. After mixing the sample, tetrahydrofuran in the volume from 0.2 up to 1.2 of the mixture is added and mixed accurately (preferably 0.5 of the volume);

7. After accurate mixing a solid form of salt is added (preferred salts: sodium chloride, magnesium sulphate or their mixtures) until the sample saturation;

8. Some amount of time should be given to observe the stages separation in case the separation is not clearly visible (or is not present). A centrifuge for speeding the process is recommended in cases of not clear a separation or when the lack of separation is observed;

9. The top layer containing the extracted compound should be collected; the top layer contains an organic solvent.

Example 1

Marking sulphonamides in honey samples:

1. 5g of honey was collected and placed in a centrifuge test tube (15 ml);

2. 4ml of 1 mol concentrated hydrochloric acid was added and treated for 45 min to hydrolize the glycoside bonds between sugars and sulphonamides;

3. 3ml of 6 mol amone acetate was added to neutralize the sample;

4. 4ml of tetrahydrofuran was added and shaken energetically for 1 minute, then 1.5g of sodium chloride was added and shaken again; 5. The sample was placed in a centrifuge to accelerate the process of phases separation;

6. 20pL of organic phase was collected, then thinned with 70pL of citrate buffer;

7. The sulphonamides in the mixture were marked by fluorescamine and then analyzed by means of a highly efficient liquid chromatography associated with fluorometric detector.

The obtained results were presented in a chromatogram - Fig.l

Example 2

Naphthoquinone extraction from the plants of Drosera and Dionea

1. lg of plant material was collected and placed in a centrifuge test tube (15 ml);

2. 12ml of tetrahydrofuran : water in proportion 1:1 proportion mixture was added;

3. The sample was soniced in an ultrasound sonic bath for 30 min;

4. 1.5g of sodium chloride was added and shaken for 1 min and then placed in a centrifuge;

5. 1ml of organic phase was collected for the analysis with highly efficient liquid chromatography connected to a spectrophotometric detector.

The obtained results were presented in a chromatogram - Fig.2 Example 3

The analysis of an e-cigarette fluid for the presence of nicotine

1. 0. 5g of finely grinded coffee beans were collected and placed in a test tube (15 ml);

2. 10ml of tetrahydrofuran and water mixture was added (1:1 proportion);

3. The sample was soniced in a ultrasonic bath for 30 min;

4. 1.5g of sodium chloride was added and shaken for 1 min, then placed in a centrifuge;

5. 1ml of organic phase was added which was later thinned with water at 1: 200 proportion for the analysis with a highly efficient liquid chromatography associated with a spectrophotometric detector.

The obtained results were presented in a chromatogram - Fig.3 Example 4

The analysis of the caffeine contents in coffee

1. 5g fluid containing nicotine was placed in a test tube;

2. 2ml of water and 3ml of tetrahydrofuran were added at the 1:1 proportion; the sample was then shaken for about 5 min;

3. 1.5g of sodium chloride was added and shaken for 1 minute;

4. 1ml of organic phase was collected and next thinned at the 1:12 proportion for the

analysis with a highly efficient liquid chromatography associated with spectrometric detector.

The obtained results were presented in a chromatogram - Fig.4

Example 5

Pesticides contents sample analysis

1. 6ml of water containing pesticides was placed in a 15 ml centrifuge test tube;

2. 3ml of tetrahydrofuran was added and next the sample was shaken for about 5 min;

3. 1.5g of sodium chloride was addend and nest shaken for 1 min;

4. 1ml of organic chase was collected, next evaporated and suspended in the volume of ΙΟΟμί. for the analysis with a highly efficient liquid chromatography associated with spectrophotometric detector.

The obtained results were presented in a chromatogram - Fig.5

The extraction method presented above is characterised by its simplicity and the relatively short time required for obtaining the extract.

Moreover, the solution, according to the invention, as compared to other commonly used extraction of plant tissues methods, does not require the initial phase of tissue maceration which in the case of standard solvents including methanol and chloroform, provides a higher recovery level of extracted substances. For plant tissues, including dionaea muscipula, it has been observed that the extraction process brings about from 30% to 50% higher recovery level as compared to the remaining contemporary recovery methods, with the 5 times lower use of organic solvent. Thanks to such a significant level of decrease in the use of solvents with the simultaneous increase of the extraction method efficiency, this method is a good candidate to replace other, more expensive and more complex methods currently in use.