Method for extracting and purifying tetrodotoxin

Abstract

A method for extracting TTX from a biological material containing TTX, wherein the method comprises the steps of extraction with a first solvent, concentration, washing with a second solvent, re-concentration and purification by chromatography.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under the Paris Convention to U.S. application No. 63 / 358,595 filed on July 6, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to a method for extracting and purifying tetrodotoxin from biological material. Background Art

[0003] Tetrodotoxin, also known as "TTX", is a naturally occurring toxin found in many terrestrial macroorganisms, marine macroorganisms, and microorganisms.

[0004] TTX was first isolated from the ovary of pufferfish in 1909. As a transmitter of voltage-gated sodium channels, TTX is a pharmaceutically relevant compound due to its potential application as an anesthetic, chemotherapeutic agent and in rehabilitation therapy for the treatment of heroin withdrawal reactions (Makarova M. et al., Angewandte Chemie, International Edition 2019, 58(51), 18338-18387). The structure of TTX and its analogs is shown in the following scheme:

[0005] Scheme 1 (TTX and analogs (hemiacetal type))

[0006]

[0007] Scheme 2 (TTX and analogs (lactone type))

[0008]

[0009] Scheme 3 (4,9-AnhydroTTX and analogs)

[0010]

[0011] Solution 4

[0012]

[0013] TTX is usually obtained by extraction from biological materials such as pufferfish, salamanders, frogs, toads and microbial fermentation broth (Pratheepa V. et al., Environmental Toxicology and Pharmacology 2016, 36, 1046-1054). Since the ovaries, livers and other internal organs of several pufferfish have high TTX content, these biological materials are usually used for industrial processing.

[0014] In CN112920192, TTX is extracted from pufferfish viscera by a method comprising ultrasonic extraction with an acidic ethanol solution, decolorization with activated carbon, purification on an ion exchange resin column, purification on an adsorption resin column and finally recrystallization of TTX.

[0015] In CN101891751, TTX is extracted from the ovaries of pufferfish by a method comprising extracting the biological material with an acidic alcohol solution, centrifuging / filtering the extract solution, purifying on a cation exchange resin column, concentrating the extract solution by vacuum distillation and a glucose gel column, and separating TTX by precipitation by adding an organic base. The yield of TTX obtained by this method is about 0.002% (calculated as grams of TTX / grams of fish viscera).

[0016] In CN102584843, TTX is extracted from roe or liver of puffer fish by a method comprising extracting the biological material with water, denaturing the protein in the extract by heat treatment, defatting with dichloromethane, purifying on an ion exchange resin column, ultrafiltration or nanofiltration to further purify / concentrate the eluate, purifying with preparative high performance liquid chromatography and crystallizing / precipitating TTX.

[0017] In WO2016061874, TTX is extracted from the liver or roe of pufferfish by a method comprising water extraction, separation of floccules obtained by adding alkali to the extraction solution, purification on an ion exchange resin column, purification on a silica gel column, and purification by preparative high performance liquid chromatography after precipitation / crystallization; the total yield of TTX obtained is about 0.001% (calculated as gram TTX / gram fish viscera).

[0018] Considering the potential pharmaceutical applications and, therefore, the market demand, alternative methods for TTX extraction and purification still need to be developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a flow chart showing the steps of a TTX extraction and purification method according to one aspect.

[0020] Figure 2 is a flow chart showing the steps of a TTX extraction and purification method according to one embodiment.

[0021] Figure 3 It is a graph showing the elution of the TTX peak (crude extract relative to RST (reference substance)) by the RP18 HPLC method using sodium 1-heptanesulfonate as the mobile phase (analysis scale ᴓ=4.6 mm).

[0022] Figure 4It is a graph showing the TTX purity (HPLC A%) of the intermediate after preparation. RP18-HPLC step (a) and CN-HPLC after purification (b). The corresponding data are shown in the table below.

[0023] Figure 5 a to Figure 5 c is a photograph of ovarian paste being stirred in the reactor. DETAILED DESCRIPTION

[0024] As described herein, provided herein are methods for extracting and purifying TTX from biological materials. definition

[0025] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of this application, the following terms are defined as follows.

[0026] As used herein, a term without a quantifier means one or more than one (ie, at least one). For example, "an element" means one element or more than one element.

[0027] As used herein, the term "about" is synonymous with "approximately" and is used to provide flexibility for the starting and ending points of a value or range by specifying that a given value can be "slightly above" or "slightly below" the value. For example, "about" can be expressed within three or more standard deviations. "About" can be expressed within a percentage range of a given value. For example, the range can be ±1%, ±5%, ±10%, ±20%, ±30%, ±40%, or ±50% of a given value. "About" can represent the order of magnitude of a given value, for example, within 2 times, within 3 times, within 4 times, or within 5 times of the value. However, it should be understood that even when a value is characterized by the term "about" in this article, clear support should be provided for the exact value at least, just as if the term "about" did not exist. In one aspect, the term "about" will be understood to include a range of ±10% of the corresponding value.

[0028] The term "adsorption chromatography" refers to the analytical separation of chemical mixtures based on the interaction of an adsorbate with an adsorbent. When a gaseous or liquid mixture passes through an adsorbent bed that adsorbs different compounds at different rates, the mixture is separated. Examples of suitable adsorbents are silica gel, modified silica gel, microcrystalline cellulose, and alumina.

[0029] The term "and / or" can mean "and" or "or".

[0030] The term "biological material" as used herein refers to any type of microorganism or macroorganism containing TTX. The biological material can include a whole organism or a part of an organism, such as a tissue obtained from these organisms. The tissue can include ovaries, livers, gonads, muscles, skin or other tissues of an organism containing TTX.

[0031] The terms "comprises" or "includes" may be used in this specification. As used herein (including the specification and / or claims), these terms should be interpreted as specifying the presence of the features, elements, steps or components, but do not exclude the presence of one or more other features, elements, steps, components or combinations thereof that are obvious to a person skilled in the relevant art. Therefore, the term "comprises / includes" as used in this specification means "consisting at least in part of..." When interpreting statements in this specification that contain this term, the features introduced by the term in each statement need to be present, but other features may also be present. Related terms, such as "comprises" and "comprised of", should be interpreted in the same manner.

[0032] The phrases "consisting essentially of" or "consisting essentially of" will be understood as generally closed terms, but allow for the inclusion of additional items, materials, components, steps, or elements that do not materially affect the basic and novel features or functions of the associated term. For example, if trace elements that are present in the composition but do not affect the properties or characteristics of the composition are present in the "consisting essentially of" language, they are allowed even if they are not explicitly listed in the list of items following the term. When open-ended terms such as "comprising" or "including" are used, it should be understood that the statement "consisting mainly of" and the statement "consisting of" are also directly supported, just as if these statements were explicitly written, and vice versa. In essence, the use of one of these terms in this specification can provide support for all other terms.

[0033] The term "dry residue" refers to the inorganic and / or organic solid material obtained after evaporation of the liquid phase or the wash phase. The amount of dry residue can be measured by weighing on a balance or using a thermogravimeter.

[0034] The term "extraction solid residue" refers to the residual biological material obtained after extraction with an organic solvent.

[0035] The term "liquid phase" defines any liquid, such as a solution, suspension, emulsion, slurry, foam, etc., which is collected and further processed in one or more steps of a process.

[0036] The term "organic solvent" as used herein refers to a carbon-based solvent that can dissolve or disperse one or more than one other substance. In one aspect, the organic solvent is selected from acetone, acetonitrile, benzene, n-butanol, n-butyl acetate, carbon tetrachloride, chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, ether, isopropyl ether, dimethylformamide, dimethyl sulfoxide, dioxane, ethanol, ethyl acetate, heptane, hexane, isooctane, isopropanol, methanol, methyl ethyl ketone, methyl tert-butyl ether, nitromethane, pentane, 1-propanol, tetrahydrofuran, toluene, trichloroethylene, xylene or a mixture thereof. The term "organic solvent" as used herein may also refer to a mixture of an organic solvent miscible with water and water.

[0037] The term "wash phase" defines any liquid, such as a solution, suspension, emulsion, slurry, foam, etc., that is collected and discharged during one or more steps of the process.

[0038] The term "recovery" or "percent recovery" refers to the amount of TTX obtained from an extraction or purification process relative to the total amount of TTX available in the starting material. The percent recovery can be determined for the entire process, where the amount of TTX in the final product is defined relative to the amount of TTX in the first starting material, or the percent recovery can be determined for a single step of a process, where the amount of TTX is defined relative to the amount of TTX available in the material obtained after the previous step of the process.

[0039] The term "yield" or "percent yield" refers to the amount of TTX obtained from the extraction process relative to the amount of total biomass in the starting material. Since the percentage yield of TTX is measured relative to the amount of total biomass in the starting material, this measurement is affected by the concentration of TTX available in the biomass and the effectiveness of the extraction and purification processes.

[0040] Applicants have developed a method for extracting and purifying TTX from biological material, wherein the biological material is extracted with an organic solvent. In one embodiment, the water content of the organic solvent is no higher than 40%.

[0041] In one aspect, the method comprises the steps of:

[0042] a) extracting a biological material with a first organic solvent (OS 1) to provide a first extracted solid residue ("ESR 1") and a first liquid phase (LP 1);

[0043] b) concentrating LP 1 to provide a concentrated liquid phase (C-LP 1);

[0044] c) washing C-LP 1 with a second organic solvent (OS 2) to provide a second liquid phase (LP 2) and a wash phase (WP 1);

[0045] d) concentrating LP 2 to provide pre-purified TTX (PP-TTX);

[0046] e) PP-TTX was purified by silica gel column chromatography to provide crude TTX (C-TTX).

[0047] In the above method, step (a) may be repeated (step (a1)).

[0048] In the embodiments of the above method, OS 1 and OS 2 may be different from each other.

[0049] In another aspect, the method comprises the steps of:

[0050] a) extracting the biological material with a first organic solvent (OS 1) to provide a first extracted solid residue (ESR 1) and a first liquid phase (LP 1);

[0051] a1) extracting the extracted solid residue (ESR 1) with a further organic solvent (OS 3) to provide a further extracted solid residue (ESR 2) and a further liquid phase (LP 3);

[0052] a2) combining LP 1 and LP 3 to provide a combined liquid phase (LP 4);

[0053] b) concentrating LP 4 to provide a concentrated liquid phase (C-LP 4);

[0054] c) washing the CLP 4 with a second organic solvent (OS 2) to provide a second liquid phase (LP 2) and a wash phase (WP 1);

[0055] d) concentrating LP 2 to provide pre-purified TTX (PP-TTX);

[0056] e) purifying PP-TTX by silica gel column chromatography to provide crude TTX (C-TTX); and, optionally

[0057] f) Purifying C-TTX by preparative HPLC to obtain purified TTX (P-TTX).

[0058] In an embodiment of the method, there is an additional step g) which comprises a further preparative HPLC purification step to reduce the content of residual ion-pairing agents in P-TTX.

[0059] In embodiments of the above methods, OS 3 is different from OS 1. In other embodiments, OS 2 is different from one or both of OS 3 and OS 1.

[0060] The advantages of the extraction and purification methods are discussed in the following sections.

[0061] The method can be performed using any tissue from an organism from which TTX has been extracted. This includes the whole body, skin, flesh, viscera or eggs of any biological material containing TTX. Examples of organisms from which TTX can be extracted include, but are not limited to, pufferfish (Takifugu spp., Tetradon spp., Lagocephalus spp., Spheroides spp., Arothon spp.), gastropods (Rapana spp., Nassarius spp., Oliva spp.), flatworms or hammerhead worms (Bipalium spp., Notoplana spp., Stylochus spp., Planocera spp.), octopuses (Octopus spp.), toads (Atelopus spp.), spp.), salamanders (Taricha spp., Notophthalmus spp., Cynops spp., Pachytriton spp., Paramesotriton spp., Laotriton spp.), crabs (Tachypleus spp., Atergatis spp.), mussels (Mytilus spp.), starfish (Astropecten spp.), and Proteobacteria (Vibrio spp., Pseudomonas spp., Serratia spp.), spp.), Roseobacter spp., Aeromonas spp., Shewanella spp., Marinomonas spp., Plesiomonas spp., Alteromonas spp., Actinobacter spp., Caulobacter spp.), Actinomycetes (Microbacterium spp., Micrococcus spp., Kytococcus spp., Cellulomonas spp.), Actinomycete spp.), Nocardiopsis spp., Streptomyces spp.), Firmicutes (Bacillus spp., Lysinibacillus spp.) and Bacteroidetes (Flavobacterium spp., Tenacibaculm spp.).

[0062] In one aspect, each organic solvent, OS 1 and OS 3, used in step a) and step a1) of the process is selected from the group consisting of acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, ethanol, isopropanol, methanol, 1-propanol and tetrahydrofuran and mixtures thereof with water. In a preferred aspect, the organic solvent is selected from the group consisting of acetone, methanol and ethanol and mixtures thereof with water. In a further preferred aspect, the organic solvent is methanol or a mixture of methanol and water.

[0063] In an embodiment, an organic acid is added to OS 1 and / or OS 3, wherein the organic acid is formic acid, acetic acid, trifluoroacetic acid, propionic acid, valeric acid, caproic acid, butyric acid, sorbic acid, lactic acid, citric acid, ascorbic acid, fumaric acid, malic acid, tartaric acid, oxalic acid, citric acid, gluconic acid, glutaric acid, glutamic acid, benzoic acid, benzyl acid, acetylsalicylic acid, cinnamic acid, and / or gallic acid; preferably acetic acid. The amount of organic acid added is about 0.01% to about 5% of the total volume of OS 1 and / or OS 3; preferably, about 0.1% to about 2.5%; more preferably, about 0.5% to about 1%.

[0064] The amount of OS 1 and / or OS 3 may independently be 1 V to 10 V, or 3 V to 6 V, wherein V is litres of solvent per kg of biological material. In a preferred aspect, the amount of solvent is 5 V.

[0065] Each step in step a) and step a1) can be independently repeated once or any number of times. In one aspect, these steps are implemented twice, up to 5 times or up to 10 times. It is understood that such multiple washings will help increase the extraction of the target product.

[0066] In another aspect, the water content of OS 1 and / or OS 3 is no more than 40 vol%, no more than 35 vol%, no more than 30 vol%, no more than 25 vol%, no more than 20 vol%, no more than 15 vol%, no more than 10 vol%, no more than 5 vol%; more preferably, no more than 20 vol%, no more than 15 vol%, no more than 10 vol%, no more than 5 vol%; even more preferably, no more than 10 vol%, no more than 5 vol%.

[0067] It is particularly advantageous to use an organic solvent having a water content not exceeding 40% of its total volume. In fact, the use of water disclosed in CN102584843 and WO2016061874 or the use of an organic solvent having a water content greater than 40% of its total volume disclosed in CN101891751 has several disadvantages for extracting biological materials. For example, due to extremely low filtration speeds and possible blockages, it is challenging to separate the extracted solid residue from the aqueous liquid phase by filtration or decantation. In addition, the more water used to extract biological materials, the greater the amount of protein extracted. When extracting larger amounts of protein, a denaturation step will be required, as described in CN102584843, which generally involves heat treatment. However, TTX is unstable at high temperatures and can be degraded by heat treatment. In addition, due to the extremely low content of TTX in biological materials, a large amount of solvent is required in order to effectively extract TTX. Furthermore, the use of an organic solvent having a water content of not more than 40% is advantageous, since the removal of the organic solvent is easier than the removal of water, for example by vacuum distillation, and the time and energy costs are lower.

[0068] Another unexpected discovery relates to the stability of TTX in organic solvents with a water content of less than 40%. As shown in Table 1, for example, when LP 1 is stored at room temperature (rt) or low temperature (It, 2°C to 8°C) for up to 15 days, 4-epi-TTX undergoes epimerization reaction to convert to TTX. Without being limited by theory, it is believed that the presence of methanol and acetic acid in the solution promotes the epimerization reaction.

[0069] Table 1

[0070]

[0071] In step b), LP 1 or LP 4 can be concentrated by distillation, vacuum distillation, ultrafiltration or nanofiltration; preferably by vacuum distillation. After step b), LP 1 or LP 4 is reduced by 80%, 85%, 90%, 95%, 98% or 99% of its initial volume; preferably, by 90% to 95%.

[0072] By step c), the amount of unwanted compounds contained in C-LP 1 or C-LP 4 is reduced, these compounds are for example, but not limited to, organic and inorganic salts, fatty acids, waxes, polysaccharides, amino acids. In the prior art, the first purification step is usually a chromatographic purification involving the use of ion exchange resins or adsorption resins. Even if a partially purified solution is obtained by these methods, the use of resins has several disadvantages; for example, large amounts of solvents are required to wash and activate the resins, then to elute the desired compounds and finally to regenerate the resins, thus requiring the concentration or discharge of large amounts of solvent. In contrast to the prior art, the method described herein does not comprise any step involving chromatographic purification using resins.

[0073] Furthermore, Applicants have observed that in attempts to purify, for example, LP 4 using ion exchange resins or adsorption resins, as shown in Table 2, TTX co-eluted with most impurities after 1 bed volume.

[0074] Table 2

[0075]

[0076] In one aspect, in step c), OS 2 is selected from: benzene, n-butanol, n-butyl acetate, carbon tetrachloride, chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, ethyl ether, diisopropyl ether, ethyl acetate, heptane, hexane, isooctane, methanol, methyl ethyl ketone, methyl tert-butyl ether, nitromethane, pentane, toluene, trichloroethylene, xylene; preferably selected from, methanol, dichloromethane, hexane and butanol and mixtures thereof.

[0077] Optionally, in step c), C-LP 1 or C-LP 4 is subjected to more washings, wherein OS 2 can be independently the same organic solvent or a different organic solvent or mixture of solvents in each separate washing. In one embodiment, step c) comprises up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 washings. It is understood that such multiple washings can be used to increase the extraction of TTX.

[0078] In step c), the amount of OS 2 used in each wash comprises 0.1 to 2 volumes per volume of C-LP 1 or C-LP 4, or comprises 0.1 to 2 volumes of the liquid phase obtained by the previous wash. In another aspect, a ratio of 0.5 to 1 volume may be used.

[0079] In an embodiment, washing is performed with hexane. This was observed to reduce the dry residue by up to 5% without loss of TTX.

[0080] Unexpectedly, when LP 2 was stored at room temperature (rt) or low temperature (It, 2°C to 8°C) for up to 15 days, not only did the epimerization of e-TTX to TTX occur, but also an increase in TTX concentration was observed, as shown in Table 3:

[0081] Table 3

[0082]

[0083] Typically C-LP 1 or C-LP 4 shows floating solid matter on the surface, which may be due to flocculation of some of the unwanted compounds. For example, washing with a mixture of dichloromethane and methanol can remove floating solids with a loss of TTX of less than 3%, preferably less than 2%. In one embodiment, the ratio of dichloromethane to methanol is from 99:1 to 70:30; preferably from 98:2 to 85:15; more preferably from 95:5 to 90:10. Repeated washing with another organic solvent selected from dichloromethane, ethyl acetate, hexane, diethyl ether, n-butanol, more preferably another organic solvent of n-butanol, can further reduce the dry residue content to 40%, preferably to 25%. The degree of reduction in the dry residue content depends on the number of washes, the pH and the concentration of C-LP 1 and / or C-LP 4. In one embodiment, in order to reduce the loss of TTX, C-LP 1 and / or C-LP 4 has a pH of 3 to 6; preferably, a pH of 3.5 to 5.5; more preferably, a pH of 4 to 4.5.

[0084] Table 4

[0085]

[0086] Preferably, in order to reduce the loss of TTX (i.e. improve the extraction efficiency), the solvent washing in step (c) can be repeated at least once, preferably at least four times. Examples are shown in Table 5, which provide data using n-butanol as the solvent, and washing was performed twice and four times.

[0087] Table 5

[0088]

[0089] LP2 obtained from step c) is concentrated in step d). In step d), LP2 can be concentrated by the same method as described in step b), including distillation, vacuum distillation, ultrafiltration or nanofiltration; preferably vacuum distillation. Preferably, LP2 is concentrated to dryness by vacuum distillation or freeze drying to provide PP-TTX.

[0090] Advantageously, in contrast to LP 1 or LP 4, the PP-TTX obtained in step d) can be purified by chromatography, for example silica gel chromatography, as described in the next section. Other column media, such as reverse phase RP18, cationic and anionic resins, molecular exclusion resins and activated carbon have also been tried, but have not provided satisfactory results.

[0091] In step e), the particle size of the silica gel used for chromatographic purification can be 10 to 500 μm; preferably 60 to 200 μm. In addition, the ratio between silica gel and PP-TTX is 10 to 200; preferably 50 to 150; more preferably 120.

[0092] In step e), one or more than one solvent selected from methanol, ethanol, propanol, isopropanol, acetone, acetonitrile, water and ethyl acetate can be used in the mobile phase. Preferably, the solvent comprises a combination of methanol, water and ethyl acetate. In other preferred aspects of this specification, the ratio of ethyl acetate, methanol and water is 10:10:3, 5:5:1 or 4:4:1.

[0093] In another aspect, an organic acid such as acetic acid, formic acid and / or trifluoroacetic acid is added to the mobile phase. Preferably, the organic acid is acetic acid. The amount of the acid added to the mobile phase can be 0.1% to 10% by volume of the mobile phase; and preferably 1% to 5% by volume.

[0094] Table 6 provides examples of solvent mixtures for chromatographic separation in step e).

[0095] Table 6

[0096]

[0097] Step e) provides C-TTX. In optional step f), the method further comprises a preparative HPLC step for purifying C-TTX. In this step, one or more than one solvent selected from methanol, ethanol, propanol, isopropanol, acetone, acetonitrile, water and ethyl acetate is used as a mobile phase. In a preferred embodiment, the mobile phase also comprises an organic acid selected from formic acid, acetic acid and trifluoroacetic acid.

[0098] In one aspect, the solvent used in step f) may further comprise an ion pairing agent selected from the group consisting of sodium 1-propanesulfonate, sodium 1-butanesulfonate, sodium 1-pentanesulfonate, sodium 1-hexanesulfonate, sodium 1-heptanesulfonate, sodium 1-octanesulfonate, sodium 1-nonanesulfonate, sodium 1-decanesulfonate, sodium 1-undecanesulfonate, sodium 1-dodecanesulfonate, sodium 1-tridecanesulfonate, sodium dodecyl sulfate, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium phosphate, tetrabutylammonium hydrogen sulfate, dodecyltrimethylammonium chloride and tetra(decyl)ammonium bromide.

[0099] In one aspect, the organic acid is trifluoroacetic acid and the ion pairing agent is sodium 1-heptane sulfonate.

[0100] In step f), the concentration of the organic acid may be 1 ppm to 100 ppm; preferably 15 ppm to 85 ppm; more preferably 40 ppm to 60 ppm. In addition, in step f), the concentration of the ion pair reagent is preferably 0.5 mmol to 12 mmol; more preferably 4 mmol to 10 mmol; and even more preferably 8 mmol.

[0101] Step f) allows for further extraction of TTX after step e). In step f), the percent recovery of TTX obtained from the C-TTX of step e) is greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99%.

[0102] Optionally, the above method comprises step g), which comprises a further preparative HPLC purification step to reduce the content of residual ion pair reagent in P-TTX 1. For this purpose, one or more than one solvent selected from methanol, ethanol, propanol, isopropanol, acetone, acetonitrile, water and ethyl acetate can be used as mobile phase. Preferably, the mobile phase also comprises an organic acid selected from formic acid, acetic acid and trifluoroacetic acid and an organic base selected from triethylamine, diethylamine and ammonia. Preferably, the organic acid is formic acid and the organic base is ammonia.

[0103] In one aspect, the concentration of the organic acid is 50 ppm to 200 ppm; preferably 80 ppm to 180 ppm; more preferably 140 ppm to 160 ppm; the concentration of the organic base is 100 ppm to 1500 ppm; preferably 300 ppm to 1000 ppm; more preferably 600 ppm.

[0104] Table 7 shows another step g) of this example.

[0105] Table 7

[0106]

[0107] For step g), after multiple HPLC runs, preferably no less than 3, 5, 8, 12, 17, 23, 25 or 30 runs, the content of the ion pair reagent can be reduced by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% relative to the initial amount. Example

[0108] Example 1 - Extraction with methanol + 1 v / v % acetic acid

[0109] 0.20 kg (TTX = 10 ppm) of puffer ovaries was thawed for about 45 minutes and cut into small pieces, then ground with a meat grinder to obtain 0.14 kg of ovarian paste. The ovarian paste was then transferred to a 1L reactor and extracted by adding 5 volumes (V) of solvent (methanol + 1 volume / volume % acetic acid) (where V is 1L / kg ovarian paste). The mixture was stirred for 30 minutes. Afterwards, the reactants were left in the reactor and complete phase separation was observed within 10 minutes. Despite this separation, the solution was discharged from the reactor and then filtered on a 240 mm Buchner funnel equipped with No. 42 filter paper. The clear solution was collected after filtration. The solid residue (about 100 grams) was subjected to a second extraction step (i.e., step a1). For the second extraction, 5V of solvent (methanol + 1% v / v acetic acid) was added and the mixture was stirred for 30 minutes. After mixing, phase separation was observed within 30 minutes, and the mixture was then discharged and filtered using a smaller Buchner funnel (110 mm) equipped with No. 42 filter paper. No obvious problems were observed in the filtration step, and 90 grams of wet residue were collected after filtration. After two extractions, the concentration of TTX in the combined extraction solution was about 9.3 ppm, and no TTX was detected in the wet residue. TTX recovery R% = 93% (calculated as: [ppm of TTX in the extraction solution] / [ppm of TTX in the ovary]).

[0110] Example 2 - Extraction with ethanol + 1 v / v % acetic acid

[0111] 0.61 kg (TTX 1 = 84 ppm) of puffer ovaries was thawed for about 45 minutes and cut into small pieces, then ground with a meat grinder to obtain 0.61 kg of ovarian paste. The ovarian paste was then transferred to a 1L reactor and extracted by adding 5 volumes (V) of solvent (ethanol + 1 volume / volume % acetic acid). The mixture was stirred at 200 rpm for 60 minutes and then discharged. After 90 minutes, phase separation was observed. Despite this separation, the solid was resuspended by stirring the mixture and the mixture was filtered on a 240 mm Buchner funnel equipped with No. 42 filter paper. A clear solution was collected after filtration. The solid residue (about 370 grams) was subjected to a second extraction step (i.e., step a1). For the second extraction, 5 volumes (V) of solvent (ethanol + 1 volume / volume % acetic acid) were added and the mixture was stirred at 200 rpm for 60 minutes. After mixing, phase separation was observed within 60 minutes, and the mixture was then filtered on a 240 mm Buchner funnel equipped with No. 42 filter paper. A clear solution was collected after filtration. No significant problems were observed in the filtration step, and 315 grams of wet residue were isolated. After two extractions, the concentration of TTX in the combined extraction solution was about 36.4 ppm and in the wet residue was 27 ppm. TTX recovery R% = 93% (calculated as: [ppm of TTX in the extraction solution] / [ppm of TTX in the ovary].

[0112] Example 3 - Extraction with acetone + 1 v / v % acetic acid

[0113] 0.61 kg of ovaries (TTX = 63 ppm) were thawed for about 45 minutes and cut into small pieces, then ground with a meat grinder to obtain 0.61 kg of ovarian paste. The ovarian paste was then transferred to a 1L reactor and extracted by adding 5 volumes (V) of solvent (acetone + 1 volume / volume % acetic acid). The mixture was stirred at 200 rpm for 60 minutes and then discharged. After 15 minutes, phase separation was observed. Despite this separation, the solid was suspended again by stirring the mixture and the mixture was filtered on a 240 mm Buchner funnel equipped with No. 42 filter paper. A clear solution was collected after filtration. The solid residue (about 353 grams) was subjected to a second extraction step ("step a1"). For the second extraction step, 5 volumes (V) of solvent (acetone + 1 volume / volume % acetic acid) were added and the mixture was stirred at 200 rpm for 60 minutes. After mixing, phase separation was observed within 120 minutes, and the mixture was then filtered on a 240 mm Buchner funnel equipped with No. 42 filter paper. A clear solution was collected after filtration. No obvious problems were observed during the filtration step and 315 g of wet residue were isolated. After two extractions, the recovery of TTX was R% = 5.2% (TTX = 3.27 ppm).

[0114] Example 4 - Extraction with methanol + 0.5 v / v % acetic acid

[0115] 0.51kg ovary (TTX=48ppm) was thawed for about 45 minutes and cut into small pieces, then ground with a meat grinder to obtain 0.51kg ovary paste. The ovary paste was then transferred to a 1L reactor and extracted by adding 5 volumes (V) of solvent (methanol + 0.5 volume / volume % acetic acid). The mixture was stirred at 200rpm for 60 minutes and then discharged. After 45 minutes, phase separation was observed. Despite this separation, the solid was suspended again by stirring the mixture and the mixture was filtered on a 240mm Buchner funnel equipped with No. 42 filter paper. A clear solution was collected after filtration. The solid residue (about 350 grams) was subjected to a second extraction step. For the second extraction, 5 volumes (V) of solvent (methanol + 0.5 volume / volume % acetic acid) were added and the mixture was stirred at 200rpm for 60 minutes. After mixing, phase separation was observed within 45 minutes, and the mixture was then filtered on a 240mm Buchner funnel equipped with No. 42 filter paper. A clear solution was collected after filtration. No obvious problems were observed during the filtration step and 307 g of wet residue were isolated. After two extractions, the recovery of TTX was R% = 70% (TTX 1 = 33.5 ppm).

[0116] Example 5 - Extraction with methanol / water / acetic acid (79:20:1 vol / vol %)

[0117] 0.51 kg (TTX = 51 ppm) of puffer ovaries were thawed for about 45 minutes and cut into small pieces, then ground with a meat grinder to obtain 0.51 kg of ovarian paste. The ovarian paste was then transferred to a 1L reactor and extracted by adding 5 volumes (V) of solvent (methanol / water / acetic acid, 79:20:1 volume / volume %). The mixture was stirred at 200 rpm for 60 minutes and then discharged. After 45 minutes, phase separation was observed. Despite this separation, the solid was suspended again by stirring and the mixture was filtered on a 240 mm Buchner funnel equipped with No. 42 filter paper. A clear solution was collected after filtration. The solid residue (about 465 grams) was subjected to a second extraction step. In this case, filtration seemed to be slower than other experiments and was completed in about 60 minutes. For the second extraction, 5 volumes (V) of solvent (methanol / water / acetic acid, 79:20:1 volume / volume %) were added and the mixture was stirred at 200 rpm for 60 minutes. After mixing, phase separation was observed within 45 minutes, and the mixture was then filtered on a 240 mm Buchner funnel equipped with filter paper No. 42. In this step, the filtration was very slow and an overnight stop was added: the mixture was recovered from the filter and stored overnight at 2°C to 8°C. After overnight, the mixture was filtered again, but even in this case, a slow filtration process was observed. Taking into account the overnight stop, it took about 7 hours and 30 minutes to filter the mixture. The resulting clear solution was collected and stored in a refrigerator at 2°C to 8°C; in addition, 606 g of wet residue was separated after filtration. After the first extraction, the recovery of TTX was R% = 70% (TTX = 33.5 ppm). After two extractions, the recovery of TTX was R% = 94% (TTX = 47.8 ppm).

[0118] Example 6 - Extraction with methanol / water / acetic acid (79.5:20:0.5 vol / vol %)

[0119] 0.51 kg (TTX = 39 ppm) of puffer ovaries were thawed for about 45 minutes and cut into small pieces, then ground with a meat grinder to obtain 0.51 kg of ovarian paste. The ovarian paste was then transferred to a 1L reactor and extracted by adding 5 volumes (V) of solvent (methanol / water / acetic acid, 79.5:20:0.5 volume / volume %). The mixture was stirred at 200 rpm for 60 minutes and then discharged. After 30 minutes, phase separation was observed. Despite this separation, the solid was suspended again by stirring and the mixture was filtered on a 240 mm Buchner funnel equipped with No. 42 filter paper. A clear solution was collected after filtration. The solid residue (about 405 grams) was subjected to a second extraction step. For the second extraction, 5 volumes (V) of solvent (methanol / water / acetic acid, 79.5:20:0.5 volume / volume %) were added and the mixture was stirred at 200 rpm for 60 minutes. After mixing, phase separation was observed within 45 minutes, and the mixture was then filtered on a 240 mm Buchner funnel equipped with filter paper No. 42. Even in this case, some problems were encountered during the second filtration process. When this step was performed, a slower filtration was initially observed. An overnight stop was again performed, so the mixture was recovered from the filter and stored overnight at 2°C to 8°C. After overnight, filtration was performed again, but no problems were observed in this case and the filtration was performed very efficiently. Taking into account the overnight stop, the filtration was completed in about 90 minutes. The resulting clear solution was collected and stored in a refrigerator at 2°C to 8°C; in addition, 404 g of wet residue were separated after filtration. After two extractions, the recovery of TTX was quantitative, R%>95%.

[0120] Example 7 - Extraction with: step a) methanol + 0.5 volume / volume % acetic acid, step a1) methanol / water / acetic acid (90:10:0.5 volume / volume %)

[0121] Before use, 3.78 kg (TTX = about 50 ppm) of pufferfish ovaries stored at 5°C to 10°C overnight were thawed. The material was ground with a meat grinder to obtain a dense ovary paste (3.70 kg). In order to improve the homogeneity of the solid sample for the assay sampling, the material was further thawed for about 45 minutes and manually mixed using a mechanical stirrer.

[0122] Step a)

[0123] Charge the glass jacketed reactor (in order of addition):

[0124] •Ovarian paste, 3.7 kg;

[0125] • Methanol, 18.5 L, equivalent to 5 volumes (V);

[0126] • Glacial acetic acid, 93 mL, equivalent to 0.5 v / v%.

[0127] The reactor was maintained at room temperature (20°C to 25°C) and a simple paddle was used to stir the material at the bottom of the reactor. A stirring rate of 150 rpm was applied to produce a vortex stirring. Once stirring began, the ovaries seemed to disperse rapidly. In Figures 6a to 6c, the appearance of the suspension is shown. The suspension was kept stirring for 60 minutes, and then the suspension was allowed to decant naturally, and the phase separation was monitored over time to qualitatively assess the relative volume of the precipitated solid. After overnight precipitation, it was observed that the volume ratio of the solution to the solid was about 3.2, and then the liquid phase was filtered on a 240 mm Buchner funnel equipped with No. 42 filter paper. A peristaltic pump was used to extract the liquid, while the solid residue (about 7 L) was left in the reactor for step a1). After filtration, a clear solution (12.1 kg, measured density = 0.839 g / mL, corresponding to 14.4 L, pH of about 4.3 to 4.6) was collected and transferred to a second jacketed reactor equipped with a distillation apparatus to concentrate the solution. The concentration step was carried out by heating the solution to 50°C (jacket temperature) and applying a vacuum of about 200 mbar; during the distillation, the stirring rate was set at 100 rpm. After about 26 hours, the resulting concentrated solution was discharged from the reactor (1.877 kg, measured density = 0.925 g / mL, corresponding to 2.03 L, pH of about 4.3 to 4.6). The solution was concentrated by 6.4 w / w (7.1 v / v) times.

[0128] Step a1

[0129] To the solid residue (20 L volume) obtained in step a) (about 7 L) was added the following extraction solvent (total volume 14.0 L):

[0130] •Methanol, 12.6 L, equivalent to 90 v / v%;

[0131] • Water, 1.4 L, equivalent to 10 vol / vol%;

[0132] • Glacial acetic acid, 69 mL, equivalent to 0.5 v / v%.

[0133] The temperature of the reactor was monitored (20°C to 25°C) and the material was stirred at the bottom of the reactor using a simple paddle. A stirring rate of 150 rpm was applied to produce a vortex stirring. The suspension was kept stirring for 60 minutes and then the suspension was naturally decanted overnight. After overnight precipitation, the volume ratio of solution / solid was observed to be about 3.2, and the liquid phase was then extracted with a peristaltic pump and filtered on a 240 mm Buchner funnel equipped with No. 42 filter paper. After filtration, a clear solution (13.92 kg, measured density = 0.833 g / mL, corresponding to 16.7 L, pH of about 4.3 to 4.6) was collected and transferred to the reactor for the concentration step. 2.5 kg of residual biomass was discharged from the reactor (filtered with qualitative filter paper) and stored in a refrigerator. The concentration step was carried out by heating the solution to 50°C (jacket temperature) and applying a vacuum of about 200 mbar; during the distillation, the stirring rate was set to 100 rpm. After about 19.5 hours, the concentrated solution obtained was discharged from the reactor (1.94 kg, measured density = 0.923 g / mL, corresponding to 2.1 L, pH of about 4.3 to 4.6). The solution was concentrated by 7.2 w / w (7.9 v / v) times. After recovering the concentrated solution, the formation of a gel-like solid was observed at the bottom of the solution. The obtained solid was separated by filtration (Whatman™ filter paper porosity 42-2.5 µm) (60 g). After step a) and step a1), the recovery of TTX was R% = 70% (TTX 1 = 34.4 ppm).

[0134] Example 8 - Extraction with: step a) methanol + 0.5 volume / volume % acetic acid, step a1) methanol / water / acetic acid (90:10:0.5 volume / volume %)

[0135] 2.15 kg (TTX = 42 ppm) of pufferfish ovaries were thawed, minced, loaded into a reactor, and extracted with 10.75 L (5 V) of methanol + 0.5 v / v % acetic acid; the mixture was stirred at room temperature for about 1 hour and then decanted for at least 4 hours; the solution was separated from the solid by vacuum suction, while the solid remained in the reactor and the solid was extracted again with 8 L (3.75 vol) of a 90 / 10 v / v methanol / water + 0.5 v / v % acetic acid mixture according to the same procedure, but the decanting was kept without stirring overnight. The liquid phase was separated, combined with the liquid phase of the first extraction, and filtered on a Buchner funnel equipped with filter paper No. 42. The filtered solution V = 17.5 L was concentrated to V = 1.7 L.

[0136] Example 9 - Extraction of the extract solution prepared according to Example 4 with n-hexane

[0137] A portion of the extract solution obtained after extraction prepared according to Example 4 (about 800 mL) was placed in a 1 L reactor and then concentrated under vacuum (about 100 mbar) at 50° C. (jacket temperature) until a final volume of about 100 mL was reached. At this point, the concentrate was washed with 1 volume (100 mL) of hexane in a separatory funnel, and the aqueous phase (76.2 g) was recovered and stored in a refrigerator. After phase separation, the dry residue in the organic phase was about DR% = 3.6% of the dry residue of the extract solution.

[0138] Example 10 - Extraction of the extract solution prepared according to Example 6 with n-hexane

[0139] A portion of the extract solution obtained after the extraction prepared according to Example 6 (about 800 mL) was placed in a 1 L reactor and then concentrated under vacuum (about 100 mbar) at 50° C. (jacket temperature) until a final volume of about 100 mL was reached. At this point, the concentrate was washed with 1 volume (100 mL) of hexane in a separatory funnel, and the aqueous phase (93 g) was recovered and stored in a refrigerator. After phase separation, the dry residue in the organic phase was about DR% = 3.1% of the dry residue of the extract solution.

[0140] Example 11 - Extraction of the extraction solution according to Example 8 with dichloromethane:methanol and n-butanol

[0141] The filtered concentrated solution (1.7 L) of Example 8 was extracted twice with a mixture of 90 / 10 volume / volume dichloromethane / methanol (0.9 L x 2), and the organic phase was removed. The aqueous phase of V = 1.55 L was concentrated to V = 0.85 L (maximum vacuum, external temperature = 50 ° C); 0.5 L of n-butanol was then added and the mixture was concentrated to V = 300 mL. The concentrated solution was extracted 4 times with n-butanol (first = 300 mL, second = 250 mL; third = 250 mL; fourth = 180 mL), and the aqueous phase was concentrated to dryness (weight = 60 g). The resulting material (60 g) was pre-purified TTX.

[0142] Example 12 - Purification of pre-purified TTX on silica gel 60

[0143] Pre-purified TTX (60 g) from Example 12 was suspended with methanol (480 mL = 8 V) and loaded onto a silica gel 60 glass column (900 g = 15 times the load weight; ᴓ = 8 cm, silica gel height = 48 cm) pre-filled with a 1 / 1 methanol / ethyl acetate mixture. TTX was eluted using the following gradient mixture (elution flow = 1.5 L / hour, linear velocity = 30 cm / hour): 1 / 1 methanol / ethyl acetate (5 L), 4 / 4 / 2 ethyl acetate / methanol / water (4.5 L), 4 / 4 / 2 ethyl acetate / methanol / water + 5% acetic acid (8 L), 8 / 2 methanol / water + 5% acetic acid (8 L). The fractions containing TTX were collected and concentrated to dryness to give a pasty brown oil = 50 g.

[0144] Example 13 - Preparative HPLC RP18 column

[0145] The paste oil from Example 13 was dissolved to V = 80 mL with water + 1% acetic acid and further diluted to V = 200 mL (4 V relative to the weight of the oil) with the same mixture. The diluted solution was loaded onto a RP-HPLC column (Agilent Zorbax™ SB300-C18 250×21.2, 7 µm) in 1 mL aliquots and purified according to the following purification HPLC parameters provided in Table 9.

[0146] Table 8: Mobile phase gradient:

[0147]

[0148] The main fractions collected from 200 preparative runs (collection time 12 min to 17.5 min) were pooled together and concentrated by freeze dryer, first to V = 1.2 L and finally to V = 100 mL.

[0149] Example 14 - Preparative CN-HPLC - for removal of ion-pairing agents.

[0150] An aliquot corresponding to the partial fraction (15 mL) obtained in Example 14 was loaded onto a CN-HPLC column (ADAMAS CN, 250 × 21.2 mm × ID 5µm) in 0.6 mL equal amounts and purified according to the following preparative HPLC purification parameters to remove the ion-pairing agent: Eluent: water + 600 ppm NH3 33% + 150 ppm HCOOH. The main fractions collected from 25 preparative runs (collected at 8.5 minutes to 15.5 minutes) were mixed together and concentrated by freeze drying, first to V = 200 mL and finally to V = 20 mL.

[0151] Although the above description mentions certain specific embodiments, it will be apparent to those skilled in the art. Any embodiments provided herein are included for illustrative purposes only and are not intended to be limiting in any way. Any drawings provided herein are only for illustrating various aspects of this specification and are not intended to be drawn to scale or limited in any way. The scope of the appended claims should not be limited to the preferred embodiments set forth in the above description, but should be given the broadest interpretation consistent with the entirety of this specification. The disclosures of all prior art cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for extracting TTX from a biological material containing TTX, the method comprising: a) extracting TTX from the biological material with a first organic solvent (OS 1) to provide a first extracted solid residue (ESR1) and a first liquid phase (LP 1); b) concentrating LP 1 to provide a concentrated liquid phase (C-LP 1); c) washing C-LP 1 with a second organic solvent (OS 2) to provide a second liquid phase (LP 2) and a wash phase (WP 1); d) concentrating LP 2 to provide pre-purified TTX (PP-TTX); and, e) PP-TTX was purified by silica gel column chromatography to provide crude TTX (C-TTX).

2. The method according to claim 1, wherein the first organic solvent or the second organic solvent is one or more of acetone, acetonitrile, benzene, n-butanol, n-butyl acetate, carbon tetrachloride, chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, ethyl ether, isopropyl ether, dimethylformamide, dimethyl sulfoxide, dioxane, ethanol, ethyl acetate, heptane, hexane, isooctane, isopropanol, methanol, methyl ethyl ketone, methyl tert-butyl ether, nitromethane, pentane, 1-propanol, tetrahydrofuran, toluene, trichloroethylene, and xylene, or a mixture thereof with water.

3. The method according to claim 1 or 2, wherein the first organic solvent and / or the second organic solvent further comprises an organic acid.

4. The method of claim 3, wherein the organic acid is present in a concentration of less than about 5% of the total volume of the first organic solvent or less than about 5% of the total volume of the second organic solvent, respectively.

5. The method according to any one of claims 1 to 3, wherein the organic acid is one or more than one of formic acid, acetic acid, trifluoroacetic acid, propionic acid, valeric acid, caproic acid, butyric acid, sorbic acid, lactic acid, citric acid, ascorbic acid, fumaric acid, malic acid, tartaric acid, oxalic acid, citric acid, gluconic acid, glutaric acid, glutamic acid, benzoic acid, benzyl acid, acetylsalicylic acid, cinnamic acid, gallic acid and acetic acid.

6. The method according to any one of claims 1 to 5, wherein the first organic solvent and the second organic solvent each have a water content of less than 40%.

7. The method according to any one of claims 1 to 6, wherein the first organic solvent is different from the second organic solvent.

8. The method according to any one of claims 1 to 7, wherein step (a) is repeated at least once.

9. A method for extracting TTX from a biological material containing TTX, the method comprising: a) extracting TTX from the biological material with a first organic solvent (OS 1) to provide a first extracted solid residue (ESR1) and a first liquid phase (LP 1); a1) extracting ESR 1 with a further organic solvent (OS 3) to provide a further extracted solid residue (ESR 2) and a further liquid phase (LP 3); a2) combining LP 1 and LP 3 to provide a combined liquid phase (LP 4); b) concentrating LP 4 to provide a concentrated liquid phase (C-LP 4); c) washing C-LP 4 with a second organic solvent (OS 2) to provide a second liquid phase (LP 2) and a wash phase (WP 1); d) concentrating LP 2 to provide pre-purified TTX (PP-TTX); and e) PP-TTX was purified by silica gel column chromatography to provide crude TTX (C-TTX).

10. The method according to claim 9, further comprising: f) purifying C-TTX by preparative HPLC to provide purified TTX (P-TTX).

11. The method according to claim 9 or 10, wherein at least one of step (a), step (a1) and step (c) is repeated at least once.

12. The method according to any one of claims 9 to 11, wherein OS 2 is one or more than one of benzene, n-butanol, n-butyl acetate, carbon tetrachloride, chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, ethyl ether, diisopropyl ether, ethyl acetate, heptane, hexane, isooctane, methanol, methyl ethyl ketone, methyl tert-butyl ether, nitromethane, pentane, toluene, trichloroethylene, and xylene.

13. The method according to any one of claims 9 to 11, wherein OS 2 is one or more than one of methanol, dichloromethane, hexane, and butanol.

14. The method according to any one of claims 9 to 13, wherein step (c) is repeated at least once.

15. The method according to claim 10, wherein step f) comprises the use of a solvent, and wherein the solvent comprises an ion pairing agent, wherein the ion pairing agent is one or more than one of sodium 1-propane sulfonate, sodium 1-butane sulfonate, sodium 1-pentane sulfonate, sodium 1-hexane sulfonate, sodium 1-heptane sulfonate, sodium 1-octane sulfonate, sodium 1-nonane sulfonate, sodium 1-decane sulfonate, sodium 1-undecane sulfonate, sodium 1-dodecane sulfonate, sodium 1-tridecane sulfonate, sodium dodecyl sulfate, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium phosphate, tetrabutylammonium hydrogen sulfate, dodecyltrimethylammonium chloride, and tetra(decyl)ammonium bromide.

16. The method according to claim 15, further comprising: g) P-TTX 1 was purified by HPLC to remove the ion-pairing agent.

17. The method of claim 16, wherein the purification of P-TTX comprises using one or more than one of methanol, ethanol, propanol, isopropanol, acetone, acetonitrile, water, and ethyl acetate as a mobile phase.

18. The method according to claim 17, wherein the mobile phase further comprises: - an organic acid, wherein the organic acid is one or more than one of formic acid, acetic acid and trifluoroacetic acid; and - an organic base, wherein the organic base is one or more than one of triethylamine, diethylamine and ammonia.

19. The method according to any one of claims 9 to 18, wherein the first solvent OS 1 and the third solvent OS 3 each independently comprise one or more of acetone, acetonitrile, benzene, n-butanol, n-butyl acetate, carbon tetrachloride, chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, ethyl ether, isopropyl ether, dimethylformamide, dimethyl sulfoxide, dioxane, ethanol, ethyl acetate, heptane, hexane, isooctane, isopropanol, methanol, methyl ethyl ketone, methyl tert-butyl ether, nitromethane, pentane, 1-propanol, tetrahydrofuran, toluene, trichloroethylene, and xylene, or a mixture thereof with water.

20. The method of claim 19, wherein OS1 and OS3 are different from each other.

21. The method of claim 19 or 20, wherein OS 2 is different from OS 1 and OS 3.

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