Method of determining opium alkaloids

FIELD: chemistry.

SUBSTANCE: disclosed method of determining opium alkaloids involves extraction sample preparation carried out using a water-acetonitrile mixture with ratio of water to acetonitrile ranging from 38:12 to 42:8, as well as analysis of the obtained extract through high-performance liquid chromatography, carried out at wavelength 210 and 220 nm. The rate of extraction of alkaloids can be increased by adding mineral acid, for example orthophosphoric acid, to the extracting water-acetonitrile mixture.

EFFECT: shorter analysis time and fewer operations while preserving the degree of extraction of alkaloids.

3 cl, 9 dwg, 1 ex

 

The invention relates to analytical chemistry, and in particular to methods of determination of opium alkaloids, which can be used when assessing compliance with poppy nutritional requirements of GOST R 52533-2006 "Mack the food and forensics for the quantification of drug components.

The known method of determination of thebaine and oripavine in poppy seeds, including sample preparation and analysis by high performance liquid chromatography (HPLC). Sample preparation in the analysis was the fact that the extraction of alkaloids was carried out buffer solution with pH 4.5, containing 0.2 M ammonium phosphate, 20%ethanol, followed by separation of the mother liquor from the solid residue by centrifugation. Analysis was performed by HPLC with spectrophotometric detector at the analytical wavelength of 254 nm. The mobile phase to include 30% methanol and buffer with pH 4.5. This method does not allow to determine the content of morphine and codeine, which are the main components of opium.

The above HPLC method is only suitable for the determination of thebaine and oripavine and does not ensure complete extraction of other opium alkaloids. It should also be noted that this technique does not allow to determine trace amounts of the analyzed substances, because their most intensiveeconomy absorption lie in the shortwave region of the spectrum (patent No. 6723894 US, A01B 79/00. Production of thebaine and oripavine. April 20, 2004, Appl. No. 047206, January 15, 2002, Tasmanian Alkaloids PTY. Ltd. (Westbury, AU), Fist; Anthony John (Norwood, AU); Byrne; Christopher James (Westbury, AU); Gerlach; Wayne Lyie (Killara, AU)).

The known method of extraction of opium alkaloids from opium, including sample preparation, including extraction of different acids at different temperatures, and the analysis of the obtained extract by HPLC. In the process of analysis of the resulting extract may determine almost all of the opium alkaloids. This method with all its virtues labor-intensive and material-intensive, requires a strict control of conditions and is not Express. In addition, this method does not allow to extract opium alkaloids from poppy seeds (WO 2005/123743, October 16, 2006, C07D 489/02 (2006.01.01), PCT/US 2005/016512, December 29, 2005, Tomazi; Keith G. (Florissant, MO), Mallinckrodt Inc, Hazelwood, MO)).

Known extraction method determination of opium alkaloids in narcotic drugs derived from the poppy plant (V.I. Sorokin, Savenko TUE, The Semkin, H.E. Determining the type of drugs derived from cannabis and poppy guidelines / Ed. Dr. med. Sciences, Professor Aaabaa. - M: RF Ministry of internal Affairs of Russia, RTCSA United Russia, 1995). As extractants it is recommended to use chloroform or a mixture of chloroform-ethanol-25% aqueous ammonia at a ratio of 1:2:0.1, respectively, or slobodkina water. This must be strictly observed is the odds ratio of the sample (g) - the extractant (ml) 1:10. The determination is performed using thin layer and gas chromatography.

In the analysis by thin layer chromatography (TLC) on chromatographic plate put standard samples representing the solutions of the active narcotic alkaloids of opium and 3-4 ál of the extract of the test object. The obtained chromatogram examine in ultraviolet light, noting areas of absorption fluorescence. The identified areas are reagent Grade. Zones morphine and codeine are colored in purple, and the area of thebaine - yellow. Chromatogram can be shown by the reagent Dragendorff, which stains all identified areas in brown color. The division performed in the following solvent system: toluene (benzene)-ethanol-triethylamine - 9:1:1; toluene-acetone-ethanol-25% aqueous ammonia - 45:45:7:3; ethanol-diethyl ether-25% aqueous ammonia - 3:6:1.

In this method for identification of opium and aqueous extracts used high quality color reaction on the content macanawai acid is a natural satellite of opium. Prepare a 3%solution of ferric chloride, which is made of the extract of the test object. This reaction allows to distinguish extract of poppy straw from opium and its aqueous extracts. While a portion of opium dissolved in water in a ratio of 1:100 and 10 ml of water and the desire add one drop of an aqueous solution of iron chloride (III). If there macanawai acid appears in red color.

For the quantitative determination of morphine and codeine used method of gas chromatography. The analyzed object, dried at 110-115°C until constant weight, ground; then a sample of 0.5 g was placed in a conical flask with a glass stopper, ml 1.25 ml 5% aqueous potassium carbonate solution, mix thoroughly until a uniform moisture and leave for 15 minutes to swell. Then poured 25 ml of chloroform, stoppered flask and stirred for 30-40 minutes Stuck to the walls of the flask particles periodically immersed in a solution with a glass rod. The solution is filtered through a cotton filter, take 20 ml of the filtrate, take it to a round bottom flask, add 1 ml of a standard solution of methyltaurine in methanol, ethanol or chloroform (concentration methylesterase - 1 mg/ml) and the solvent is distilled off to dryness. After removal of the solvent extract process similitude mixture in the flask with the extract, add 1 ml of pyridine and gently wash away the film of the extract from the walls of the flask. Then to the pyridine solution was successively added 0.6 ml hexamethyldisilazane and 0.2 ml of trimethyl-CHLOROSILANES, stirring the mixture after the addition of each reagent. The mixture was incubated 20 min at a temperature of 40-50°C. Determination hold to nab the main column with a length of 2-3 m with applied phase OV-101 or SE-30 (3 or 5%) or quartz capillary column with a length of 10-25 m and a diameter of 0.2 mm with an applied phase OV-101. The carrier gas is helium or nitrogen. Chromatograph conditions were as follows: detector - flame ionization; evaporator temperature 275°C, detector 290°C; isothermal mode (optimum temperature is selected in the range of 230-250°C) or with the programming of the column temperature from 200 to 280°C at a rate of 10°C/min; the division of the flow of carrier gas for capillary column - 1:55.

To quantify the content of morphine and codeine can use a relative mass ratio. The relative mass ratio seligpreisung morphine and codeine to methyltaurine equal to 0.94 and 0.96, respectively.

Although GOST R 52533-2006 "Mack the food has a reference to this technique, but as a way of determining the narcotic opium alkaloids in poppy seeds food above method does not consider poppy seeds food as a source of active narcotic alkaloids of opium and does not contain guidance on the extraction and analysis of opium alkaloids from poppy seeds. Moreover, the procedure indicates that poppy seeds are narcotic alkaloids of opium does not contain drugs do not apply.

The closest analogue is the method of determination of opium alkaloids in poppy seeds (Kolychev I.A. Forensic study of drugs derived from plants of the poppy /Iagolai, Vshadow The / forensics. - 2007. No. 1. - Pp. 102-109).

In the process of sample preparation take an average sample of poppy seed mass on a 25.00 g and placed in a flask, to which is added 50 ml of a mixture of toluene: acetone: ethanol: ammonia solution in the ratio 25:25:2:1 and the extraction is carried out with shaking for one hour. The obtained extracts are merged in a separate flask, and the flask hitch again pour 50 ml of the same extraction mixture, the procedure is repeated twice more. The combined extracts evaporated until no odor of organic solvent. To the resulting viscous oily substances dark brown color add 25 ml of a 0.3%aqueous solution of hydrochloric acid and boiled for 15-20 minutes, cooled and carefully selected water fraction, for example, a syringe with a needle. This procedure was repeated twice. To the remainder after the second addition of hydrochloric acid oily residue add 15-20 ml of acetone, and to the resulting solution of 0.3%solution of hydrochloric acid with a volume of 20-25 ml thus formed a stable emulsion, which was first incubated for 10-15 minutes in normal conditions, and then boil until no odor of acetone, periodically adding water. The extract is filtered and evaporated at a temperature of 110°C. before the formation of dry residues. The solids dissolved in 10 ml of eluent (acetonitrile - phosphate is Ufer (3:7), phosphate buffer: 0.04 M K2HPO4, pH 3.5 by addition of ion-pair reagent (dodecyl-sulfate sodium)). The obtained solutions defend and filtered, and then analyzed by HPLC method.

Extracts of drugs derived from the poppy plant are dark brown, so the holding known drip chemical reactions in their study impractical. For conducting chemical studies of opium using quality color reaction on the content macanawai acid is necessary a portion of a substance to dissolve in water in the ratio 1:100, and 10 ml of water extraction to add one drop of 0.3%aqueous solution of ferric chloride (III). If there macanawai acid appears red staining. This reaction allows to distinguish the extraction of opium from opium, which contains macanova acid. It should be noted that macanova acid is a natural satellite of opium.

This method of sample preparation is very time consuming, but it provides a high degree of extraction of the alkaloids of opium. In the analysis of samples with the introduction of morphine was determined that his losses during extraction is not more than 5%. This method is able to analyze six samples within two working days.

The technical objective of the claimed invention is the simplification of the method definition wide-angle the opium alkaloids from poppy seeds food, reducing the complexity and reducing the time of determination of opium alkaloids.

To solve the technical problem is proposed extraction sample preparation for the determination of opium alkaloids to implement the extractive agent, representing water and the combined acetonitrile mixture. The original components are taken in the range of ratios of water: acetonitrile 38:12 42:8, respectively. The resulting extract was analyzed by high-performance liquid chromatography with wavelengths 210, 220 nm. When translating alkaloids in the salt form increases the rate of extraction by adding a mineral acid. It should also be noted that the determination of opium alkaloids can be carried out without the addition of mineral acid. Additive in a mixture of mineral acids is due to the fact that it makes no interfering influence in their denition and can be easily removed by thermal treatment.

The most efficient sample preparation to carry out water and the combined acetonitrile mixture, in which the original components are taken in the ratio of 40:10.

Figure 1 shows a graph of the studies according to the degree of extraction of alkaloids from the ratio of water: acetonitrile; figure 2 - graph of the degree of extraction of alkaloids from the volume of added mineral acid; figa - chromatogram of the extract of poppy seeds with a ratio of water: clonicel 35:15, respectively; on 3b is a chromatogram of the extract of poppy seeds with a ratio of water: acetonitrile 40:10, respectively; 3V - chromatogram of the extract of poppy seeds with a ratio of water: acetonitrile 45:5, respectively; 3G - chromatogram of the extract of poppy seeds with a ratio of water: acetonitrile 40:10, respectively, with the addition of phosphoric acid in a volume of 0.01 ml; 3D chromatogram of the extract of poppy seeds with a ratio of water: acetonitrile 40:10, respectively, with the addition of phosphoric acid in a volume of 0.03 ml; 3rd - chromatogram of the extract of poppy seeds oil at a ratio of water: acetonitrile 40:10, respectively, with the addition of phosphoric acid in a volume of 0.05 ml; g - chromatogram of the extract of poppy seeds with a ratio of water: acetonitrile 40:10, respectively, with the addition of orthophosphoric acid in the amount of 0.07 ml

Selection of optimal conditions for solving technical tasks performed by taking a sample of poppy seeds placed in a flask, pouring an aqueous solution of acetonitrile as a solvent. Studies to determine the optimal proportions of water: acetonitrile, in which the extraction of opium alkaloids as presented in figure 1.

As can be seen from the graph in figure 1, the optimum ratio of water: acetonitrile, equal 38:12-42:8 with the NGOs, the best - 40:10. The efficiency of extraction of alkaloids increased by adding a mineral acid due to the formation of their salt forms. With regard to the level defined concentrations of the alkaloids concentration of mineral acid, for example, 85% o-phosphoric acid minimally from 0,045 ml (figure 2). As can be seen from the chromatograms shown in figa - figv the extraction of alkaloids useful to the ratio of water: acetonitrile 38:12-42:8, respectively. From the chromatograms on high - z we see that the addition of mineral acid increases the degree of extraction of alkaloids. The best analytical conditions for determination of alkaloids are the wavelengths of 210, 220 nm.

The degree of extraction of alkaloids in the process of extraction increases with shaking due to the intensification of contact of poppy seeds with the extractant.

Example: 20 ml of extractant consisting of water, acetonitrile and phosphoric acid, taken in the ratio of 40:10:0.05 respectively (the ratio can vary depending on the reagents and conditions of extraction). Bulb mounted on the shaker, the extraction is carried out for 10-15 minutes, after which the extract obtained from each flask is poured into a separate flask, the remaining sample re-fill the same mixture and repeat the extraction. The third time it is sufficient to rinse with 10 ml of extractant. Joint the United extracts filtered and adjusted to the mark with water in the flask 50 ml The extract obtained from the seeds of poppy analyzed by high performance liquid chromatography under the following conditions:

1. Liquid chromatograph with a photometric detector in the UV region of the spectrum.

2. Column reversed-phase sorbent.

3. Perhaps both isocratic and gradient elution, the eluent composition is selected depending on the column. When gradient elution is achieved highest separation efficiency components. The most versatile part of eluent when working with reversed-phase chromatographic columns may be given by the following variant: the first stage: acetonitrile - phosphate buffer (3:7); the second stage - acetonitrile - phosphate buffer (6:4).

4. Phosphate buffer: 0.04 M K2HPO4, pH 3.5 by addition of ion-pair reagent (sodium dodecyl sulphate).

5. Analytical wavelength was 210, 220 nm.

The results presented in figb.

The content of morphine in the seeds, mg/g seeds of 4.1±0.5 in.

The codeine in the seeds, mg/g seeds 0,61±0,08.

On the resulting chromatogram (figb) there are peaks corresponding macanawai acid, morphine, codeine, thebaine. The possibility of defining macanawai acid eliminates the additional step of its analysis method drip reactions. Analysis of opium alkaloids in seeds m the ka food in this scenario, it takes less than one hour. The degree of extraction of alkaloids is 93-95%.

Unlike the prototype in the claimed method is used extractant another composition. In addition, this method is cheaper and more rapid in comparison with the prototype.

1. The method of determination of opium alkaloids, including extraction sample preparation and analysis by high performance liquid chromatography, characterized in that the extractant use of water and the combined acetonitrile mixture, the components are taken in the ratio of 38:12-42:8, respectively, and analysis by high performance liquid chromatography is carried out at wavelengths of 210, 220 nm.

2. The method according to claim 1, characterized in that water and the combined acetonitrile mixture of the initial components are taken in the ratio of 40:10, respectively.

3. The method according to claim 1 or 2, characterized in that the determination is performed with the addition of mineral acid, for example phosphoric acid.



 

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