A method for preparing dopamine hydrochloride by separating and purifying from a fermentation broth

Abstract

The application discloses a preparation method for separating and purifying dopamine hydrochloride from a fermentation liquor. The method comprises the following steps: (1) filtering dopamine fermentation liquor through a ceramic membrane to obtain a filtrate, decolorizing the filtrate through a macroporous resin, exchanging and adsorbing the filtrate through an ion exchange resin, and desorbing the filtrate into a salt by using an ethanol and dilute hydrochloric acid solution; (2) concentrating and cooling desorption liquid to obtain crystallization liquid; and (3) filtering the crystallization liquid, washing wet powder sufficiently, and drying the wet powder to obtain dopamine hydrochloride finished products. The dopamine hydrochloride prepared by the method meets the quality standard of the Chinese Pharmacopoeia, the total yield is greater than 70%, the whole purification process uses less organic solvent, is safe and environment-friendly, the process is simple and easy to implement, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of industrial microbial technology, specifically relating to a method for separating and purifying dopamine hydrochloride from fermentation broth. Background Technology

[0002] Dopamine hydrochloride, chemically named 4-(2-aminoethyl)-1,2-benzenediol hydrochloride, belongs to the catecholamine class of drugs and has a wide range of clinical applications. It stimulates adrenaline and dopamine receptors and is used as an emergency treatment for various types of shock.

[0003] The structural formula of dopamine hydrochloride is shown below.

[0004]

[0005] Currently, the reported methods for preparing dopamine hydrochloride are mainly synthetic. Patent CN111925293 B discloses several chemical synthesis methods: Synthesis Route 1: Using piperoethylamine as the starting material, a ring-opening reaction is carried out in the presence of hydrochloric acid and phenol to obtain dopamine hydrochloride. This method involves expensive starting materials, high reaction temperatures, and difficulties in impurity control. Synthesis Route 2: Using vanillin as the starting material, dopamine hydrochloride is obtained through a four-step reaction involving condensation, reduction, demethoxylation, and salt formation. In this method, vanillin is an aldehyde, which is unstable, and nitromethane is a Group 2B carcinogen, prone to explosion. Furthermore, the process is lengthy and has a low yield. Synthesis Route 3: Using catechol as the starting material, it reacts with pyruvate and ammonia to obtain 3,4-dihydroxy-L-phenylalanine, which is then decarboxylated by decarboxylase to obtain dopamine. This method requires harsh reaction conditions and is costly. Synthetic route 4: Starting with veratral, it first condenses with nitromethane to produce 3,4-dimethoxy-β-nitrostyrene, then undergoes catalytic reduction to obtain 3,4-dimethoxyphenylethylamine, which reacts with hydrobromic acid to remove the methoxy group, and finally reacts with hydrochloric acid to form a salt to give dopamine hydrochloride. This method involves more steps and uses palladium as a catalyst.

[0006] Synthetic methods have many drawbacks, such as high starting material prices, complicated synthesis steps, low recovery rate, and non-green and environmentally friendly processes.

[0007] With the development of synthetic biology technology, literature reports and patents (CN112941002B) disclose the construction and application of recombinant Escherichia coli strains for dopamine production, using glucose as a carbon source and inorganic salt culture medium for fermentation to produce dopamine. Biosynthetic production of dopamine has the advantages of simple process, high product conversion rate, and green sustainability. Currently, no literature reports have been found regarding the separation and purification methods for dopamine production via biosynthesis.

[0008] As a raw material for pharmaceuticals, the quality of dopamine hydrochloride is directly related to the safety of clinical use. Breaking through traditional chemical synthesis processes and developing an extraction process to separate and purify dopamine hydrochloride from fermentation broth has become an urgent need. Summary of the Invention

[0009] This invention provides a novel process for preparing high-purity, low-cost, and industrially suitable dopamine hydrochloride. The prepared dopamine hydrochloride has a purity higher than 99.90%, meets the relevant standards of the Chinese Pharmacopoeia, has a total yield greater than 70%, and features low production cost, safety, and environmental friendliness, making it suitable for large-scale industrial production.

[0010] The present invention will now be described in detail:

[0011] The method of the present invention includes the following steps: the dopamine fermentation broth is filtered through a ceramic membrane to obtain a clear filtrate, the filtrate is decolorized by a macroporous resin and then enriched by an ion exchange resin, desorbed by an ethanol-hydrochloric acid solution and formed into a salt, the salt-forming desorbed solution is concentrated and crystallized to obtain dopamine hydrochloride crystallization solution, the crystallization solution is filtered, thoroughly washed with a cold ethanol solution and dried to obtain dopamine hydrochloride powder.

[0012] Specifically, the present invention relates to a method for separating and purifying dopamine hydrochloride from fermentation broth, comprising the following steps:

[0013] 1) The dopamine fermentation broth was filtered through a ceramic membrane to obtain a clear dopamine filtrate;

[0014] 2) The dopamine filtrate was decolorized using a macroporous resin to obtain a decolorized solution;

[0015] 3) The decolorized solution is adsorbed by ion exchange resin, the saturated resin is washed with deionized water, and desorbed by ethanol-hydrochloric acid aqueous solution. The desorbed solution is collected by HPLC.

[0016] 4) Concentrate the desorption solution under reduced pressure and cool it to 2-8℃ to crystallize, obtaining a crystalline solution;

[0017] 5) Filter the crystallization liquid, wash the wet powder 2-3 times with washing liquid, and dry at 40-50℃ to obtain dopamine hydrochloride.

[0018] The ceramic membrane used in step 1) has a pore size of 50 nm or 100 nm, preferably 50 nm. Using ceramic membrane filtration can significantly improve the quality of the filtrate.

[0019] In step 2), the pH of the filtrate is 6.0-6.5.

[0020] The macroporous resin used in step 2) is one of XR205, XRK209, and XRTS2, with XR205 being preferred. The amount of resin used is 0.06 to 0.10 liters per liter of dopamine filtrate.

[0021] The ion exchange resin used in step 3) is any one of HD-2, HZD-2, and D152 resins, preferably HZD-2. The resin dosage is 0.10-0.15 liters per liter of dopamine filtrate. The exchange adsorption can be performed using an ion exchange resin column for sample loading. The flow rate for loading the ion exchange resin onto the column (sample) is 0.8-1.2 BV / h, and the desorption flow rate is 0.4-0.6 BV / h. Preferably, the flow rate for loading the column (sample) is 1.0 BV / h, and the desorption flow rate is 0.5 BV / h.

[0022] In step 3), after the ion exchange resin has finished adsorbing, the column can be washed with 10 column volumes of deionized water to remove a large amount of pigment impurities.

[0023] In step 3), the volume content of ethanol in the ethanol-hydrochloric acid aqueous solution is 30-50%, and the concentration of hydrochloric acid is 0.40-0.60 mol / L.

[0024] In step 4), the concentration of the desorption solution after vacuum concentration is 200–300 mg / mL.

[0025] In step 5), the crystallization liquid is filtered, and the washing solution used for wet powder washing is 90-95% cold ethanol (2-8℃), and the washing volume is 0.3-0.5 times the volume of the crystallization liquid.

[0026] The dopamine hydrochloride obtained by this invention can be used as a raw material for pharmaceuticals, or as an intermediate in the synthesis of high-value-added products such as hydroxytyrosol and Rhodiola rosea. It can also be used as an intermediate in the formation of polydopamine for use in the materials field. The dopamine hydrochloride concentrate obtained by this invention has a purity of over 99.90%, and the overall extraction yield is greater than 70%.

[0027] The preparation method of dopamine hydrochloride of this invention has the following advantages: 1. The fermentation broth is filtered using a ceramic membrane, which can effectively remove various insoluble culture media and impurities from the bacteria and fermentation broth, improving filtration efficiency and obtaining a high-quality clear filtrate. 2. The use of decolorizing resin can remove a large amount of pigment impurities from the filtrate, which is beneficial to improving the quality of the eluent. 3. Dopamine is enriched using cation exchange resin and desorbed using dilute hydrochloric acid solution. During the desorption process, pharmaceutical-grade dopamine hydrochloride is generated simultaneously. Adding a certain proportion of ethanol to the eluent improves the desorption rate, making the eluent more concentrated and also facilitating eluent concentration. 4. During the filtration of the crystallization liquid, the wet powder is thoroughly washed with 90-95% cold ethanol to remove some pigments and inorganic salts, significantly improving the quality of the finished product and ensuring the yield. 5. The various steps of the process are closely coordinated and connected. The process is simple, environmentally friendly, easy to operate, highly efficient, and produces a high-purity product. All inspection indicators meet the standards of the Chinese Pharmacopoeia, and the overall yield reaches over 70%, making it suitable for industrial production. Attached Figure Description

[0028] Appendix Figure 1HPLC chromatogram of dopamine hydrochloride concentrate Detailed Implementation

[0029] The following examples are for illustrative purposes only and should not be construed as limiting the invention. Unless otherwise specified, all percentages mentioned in the examples are volume percentages. Dopamine fermentation broth can be prepared using synthetic biotechnology (e.g., Wang Qinhong, Song Fuqiang, et al., CN112941002B "Recombinant Escherichia coli strains for producing dopamine and their construction methods and applications"). The dopamine fermentation broth used in this invention was provided by North China Pharmaceutical Group New Drug Research and Development Co., Ltd., and all equipment and reagents used are commercially available products.

[0030] Macroporous resins: XR205, XRK209, XRTS2, Shanghai Xuner Technology Co., Ltd.;

[0031] Ion exchange resins: HD-2, HZD-2, D152, Shanghai Huazhen Technology Co., Ltd.

[0032] Detection method: High performance liquid chromatography

[0033] The HPLC detection conditions were as follows: column: octadecylsilane-bonded silica column (length: 250 mm, inner diameter: 4.6 mm, packing particle size: 5 μm); mobile phase: 0.1% phosphoric acid aqueous solution: methanol = 90:10; detector: UV detector; detection wavelength: 280 nm; column temperature: 30 ℃; flow rate: 1.0 mL / min; injection volume: 10 μL.

[0034] Example 1

[0035] 30 L of dopamine fermentation broth, with a fermentation unit of 42 g / L, was filtered through a 50 nm ceramic membrane, yielding a 40 L filtrate. The filtrate, with a pH of 6.3, was decolorized using an XR205 resin column (1:5 diameter-to-height ratio, 4 L resin charge, 6 L / h flow rate). The decolorized solution was then adsorbed onto a D152 ion exchange resin column (1:6 diameter-to-height ratio, 6 L resin charge, 6 L / h flow rate). The saturated resin was washed with 10 column volumes of deionized water, followed by desorption with 50% ethanol + 0.5 mol / L hydrochloric acid solution at a flow rate of 3 L / h. HPLC analysis was performed, and the eluent was collected. The eluent was concentrated under reduced pressure at 50–60 °C to 240 mg / mL, then slowly cooled to 2–8 °C and crystallized for 8 h, yielding a 4.5 L crystallization solution. The crystallization liquid was filtered, and the wet powder was washed twice with 1.8 L of 95% cold ethanol and rinsed once. The wet powder was then vacuum dried at 50 °C for 6 h to obtain 910 g of dopamine hydrochloride concentrate with a content of 100.00% and a purity of 100%. The largest single impurity was not detected, and the total yield was 72.2%.

[0036] Example 2

[0037] 30 L of dopamine fermentation broth (45 g / L fermentation unit) was filtered through a 50 nm ceramic membrane, yielding a 40 L filtrate. The filtrate, with a pH of 6.4, was decolorized using an XRK209 resin column (1:5 diameter-to-height ratio, 3.5 L resin charge, 6 L / h flow rate). The decolorized solution was then adsorbed onto an HZD-2 ion exchange resin column (1:6 diameter-to-height ratio, 6 L resin charge, 6 L / h flow rate). The saturated resin was washed with 10 column volumes of deionized water, followed by desorption with 50% ethanol + 0.4 mol / L hydrochloric acid solution at a flow rate of 3 L / h. HPLC analysis was performed, and the eluent was collected. The eluent was concentrated under reduced pressure at 50–60 °C to 250 mg / mL, then slowly cooled to 2–8 °C and crystallized for 8 h, yielding a 4.6 L crystallization solution. The crystallization liquid was filtered, and the wet powder was washed twice with 2L of 90% cold ethanol and rinsed once. The wet powder was then vacuum dried at 50℃ for 6 hours to obtain 950g of dopamine hydrochloride concentrate with a content of 99.98%, a purity of 99.95%, a maximum single impurity of <0.1%, and a total yield of 70.4%.

[0038] Example 3

[0039] 30 L of dopamine fermentation broth, with a fermentation unit of 44 g / L, was filtered through a 50 nm ceramic membrane, yielding a 40 L filtrate. The filtrate, with a pH of 6.4, was decolorized using an XRTS2 resin column (1:5 diameter-to-height ratio, 4 L resin charge, 6 L / h flow rate). The decolorized solution was then adsorbed onto an HD-2 ion exchange resin column (1:6 diameter-to-height ratio, 6 L resin charge, 6 L / h flow rate). The saturated resin was washed with 10 column volumes of deionized water, followed by desorption with 30% ethanol + 0.6 mol / L hydrochloric acid solution at a flow rate of 3 L / h. HPLC analysis was performed, and the eluent was collected. The eluent was concentrated under reduced pressure at 50–60 °C to 270 mg / mL, then slowly cooled to 2–8 °C and crystallized for 8 h, yielding a 4.2 L crystallization solution. The crystallization liquid was filtered, and the wet powder was washed twice with 1.5L of 95% cold ethanol and rinsed once. The wet powder was then vacuum dried at 50℃ for 6 hours to obtain 942g of dopamine hydrochloride concentrate with a content of 99.96%, a purity of 99.94%, a maximum single impurity of <0.1%, and a total yield of 71.34%.

[0040] Example 4

[0041] 320 L of dopamine fermentation broth, with a fermentation unit of 45 g / L, was filtered through a 50 nm ceramic membrane, yielding a filtrate volume of 410 L. The filtrate, with a pH of 6.3, was decolorized using an XR205 resin column (1:5 diameter-to-height ratio, 40 L resin charge, 60 L / h flow rate). The decolorized solution was then adsorbed onto an HZD-2 ion exchange resin column (1:6 diameter-to-height ratio, 60 L resin charge, 60 L / h flow rate). The saturated resin was washed with 10 column volumes of deionized water, followed by desorption with 50% ethanol + 0.5 mol / L hydrochloric acid solution at a flow rate of 30 L / h. HPLC analysis was performed, and the eluent was collected. The eluent was concentrated under reduced pressure at 50–60 °C to 260 mg / mL, then slowly cooled to 2–8 °C for 8 h of crystallization, yielding a crystallization liquid volume of 47.2 L. The crystallization solution was filtered, and the wet powder was washed twice with 20 L of 95% cold ethanol, followed by rinsing once. The wet powder was then vacuum dried at 50°C for 6 hours to obtain 10.50 kg of dopamine hydrochloride concentrate with a content of 100.01% and a purity of 100%. No maximum single impurity was detected. The HPLC chromatogram is attached. Figure 1 The overall yield was 72.9%.

[0042] Comparative Example 1

[0043] 30 L of dopamine fermentation broth (45 g / L fermentation unit) was filtered through a 50 nm ceramic membrane, yielding a 40 L filtrate. The filtrate, with a pH of 6.9, was decolorized using an XRTS2 resin column (1:5 diameter-to-height ratio, 4 L resin charge, 6 L / h flow rate). The decolorized solution was then adsorbed onto an HD-2 ion exchange resin column (1:6 diameter-to-height ratio, 6 L resin charge, 6 L / h flow rate). The saturated resin was washed with 10 column volumes of deionized water, followed by desorption with 30% ethanol + 0.6 mol / L hydrochloric acid solution at a flow rate of 3 L / h. HPLC analysis was performed, and the eluent, which was dark in color, was collected. The eluent was concentrated under reduced pressure at 50–60 °C to 250 mg / mL, then slowly cooled to 2–8 °C and crystallized for 8 h, yielding a 4.2 L crystallization solution. The crystallization liquid was filtered, and the wet powder was washed twice with 1.5L of 95% cold ethanol and rinsed once. The wet powder was then vacuum dried at 50℃ for 6 hours to obtain 910g of dopamine hydrochloride concentrate with a content of 98.10%, a purity of 99.30%, a maximum single impurity of <0.3%, and a total yield of 66.13%.

[0044] Comparative Example 2

[0045] 30 L of dopamine fermentation broth, with a fermentation unit of 44 g / L, was filtered through a 50 nm ceramic membrane, yielding a 40 L filtrate. The filtrate, with a pH of 5.8, was decolorized using an XRTS2 resin column (1:5 diameter-to-height ratio, 4 L resin charge, 6 L / h flow rate). The decolorized solution was then adsorbed onto an HD-2 ion exchange resin column (1:6 diameter-to-height ratio, 6 L resin charge, 6 L / h flow rate). The saturated resin was washed with 10 column volumes of deionized water, followed by desorption with 30% ethanol + 0.6 mol / L hydrochloric acid solution at a flow rate of 3 L / h. HPLC analysis was performed, and the eluent was collected. The eluent was concentrated under reduced pressure at 50–60 °C to 240 mg / mL, then slowly cooled to 2–8 °C and crystallized for 8 h, yielding a 4.0 L crystallization solution. The crystallization liquid was filtered, and the wet powder was washed twice with 1.3L of 95% cold ethanol and rinsed once. The wet powder was then vacuum dried at 50℃ for 6 hours to obtain 820g of dopamine hydrochloride concentrate with a content of 98.10%, a purity of 99.60%, a maximum single impurity of <0.1%, and a total yield of 60.94%.

[0046] Comparative Example 3

[0047] 30 L of dopamine fermentation broth, with a fermentation unit of 42 g / L, was filtered through a 50 nm ceramic membrane, yielding a 40 L filtrate. The filtrate, with a pH of 6.3, was decolorized using an XRTS2 resin column (1:5 diameter-to-height ratio, 4 L resin charge, 6 L / h flow rate). The decolorized solution was then adsorbed onto an HD-2 ion exchange resin column (1:6 diameter-to-height ratio, 6 L resin charge, 6 L / h flow rate). The saturated resin was washed with 10 column volumes of deionized water, followed by desorption with 20% ethanol + 0.3 mol / L hydrochloric acid solution at a flow rate of 5 L / h. HPLC analysis was performed, and the eluent was collected. The eluent showed tailing and a relatively large volume. It was concentrated under reduced pressure at 50–60 °C to 250 mg / ml, then slowly cooled to 2–8 °C and crystallized for 8 hours, yielding a 4.0 L crystallization solution. The crystallization liquid was filtered, and the wet powder was washed twice with 1.5L of 95% cold ethanol and rinsed once. The wet powder was then vacuum dried at 50℃ for 6 hours to obtain 865g of dopamine hydrochloride concentrate with a content of 98.70%, a purity of 99.65%, a maximum single impurity of <0.1%, and an overall yield of 67.8%.

[0048] Comparative Example 4

[0049] 30 L of dopamine fermentation broth (45 g / L fermentation unit) was filtered through a 50 nm ceramic membrane, yielding a 40 L filtrate. The filtrate, with a pH of 6.4, was decolorized using an XRK209 resin column (1:5 diameter-to-height ratio, 3.5 L resin charge, 6 L / h flow rate). The decolorized solution was then adsorbed onto an HZD-2 ion exchange resin column (1:6 diameter-to-height ratio, 6 L resin charge, 6 L / h flow rate). The saturated resin was washed with 10 column volumes of deionized water, followed by desorption with 60% ethanol + 0.6 mol / L hydrochloric acid solution at a flow rate of 1.8 L / h. HPLC analysis was performed, and the eluent was collected. The eluent was dark in color and concentrated under reduced pressure at 50–60 °C to 250 mg / ml. The solution was then slowly cooled to 2–8 °C and crystallized for 8 hours, yielding a 4.1 L crystallization solution. The crystallization liquid was filtered, and the wet powder was washed twice with 2L of 90% cold ethanol and rinsed once. The wet powder was then vacuum dried at 50℃ for 6 hours to obtain 869g of dopamine hydrochloride concentrate with a content of 98.38%, a purity of 99.45%, a maximum single impurity of <0.1%, and a total yield of 63.3%.

Claims

1. A method for separating and purifying dopamine hydrochloride from fermentation broth, characterized in that... The method includes the following steps: 1) The dopamine fermentation broth is filtered through a ceramic membrane to obtain dopamine filtrate. The ceramic membrane used has a specification of 50nm or 100nm. 2) The dopamine filtrate is decolorized by passing it through a macroporous resin to obtain a decolorized solution, wherein the macroporous resin is XR205, XRK209, or XRTS2; 3) The decolorized solution is adsorbed by ion exchange resin. After the resin is saturated, it is washed with deionized water. After washing, it is desorbed by a desorbent containing ethanol and hydrochloric acid. The desorbed solution is collected by HPLC. 4) Concentrate the desorption solution under reduced pressure and cool it to 2-8℃ to crystallize, obtaining a crystalline solution; 5) Filter the crystallization liquid, wash the wet powder thoroughly with washing liquid, and dry at 40-50℃ to obtain dopamine hydrochloride concentrate powder; In step 2), the pH of the filtrate is 6.0-6.5; in step 3), the ion exchange resin used is any one of HD-2, HZD-2, or D152 resin, and the desorbent used is a solution containing 30-50% ethanol and 0.40-0.60 mol / L hydrochloric acid.

2. The method according to claim 1, wherein the amount of ion exchange resin used in step 3) is 0.10 to 0.15 liters per liter of dopamine filtrate, and the exchange adsorption is performed by loading the sample onto an ion exchange resin column at a flow rate of 0.8 to 1.2 BV / h and a desorption flow rate of 0.4 to 0.6 BV / h.

3. The method according to claim 1, wherein the deionized water washing in step 3) is a 10-column volume deionized water washing.

4. The method according to claim 1, wherein in step 4), the desorption solution is concentrated to a concentration of 200-300 mg / ml.

5. The method according to claim 1, wherein the washing solution in step 5) is 90-95% cold ethanol, and the amount used is 0.3-0.5 times the volume of the crystallization liquid.

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