CN113683649A

CN113683649A - Separation and purification method of pirarubicin

Info

Publication number: CN113683649A
Application number: CN202111044133.5A
Authority: CN
Inventors: 尹晓; 李绪振
Original assignee: Shandong Yinji Biotechnology Co ltd
Current assignee: Shandong Yinji Biotechnology Co ltd
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2021-11-23
Anticipated expiration: 2041-09-07
Also published as: CN113683649B

Abstract

The application discloses a method for separating and purifying pirarubicin, which at least comprises the following steps: step 1, obtaining a solution to be treated of a sample containing pirarubicin; step 2, separating and purifying the solution to be treated by adopting chromatography to obtain a pure pirarubicin product; the filler adopted in the chromatographic separation and purification is Diol-based bonded silica gel, namely Diol filler. According to the method, the Diol filler is used as the filler for chromatographic separation and purification, compared with the existing silica gel filler, the separation of the pirarubicin and the isomerized impurities thereof is more thorough, the purity of the pirarubicin can reach more than 99%, the content of the single impurity and the isomerized impurities is less than 0.1%, and the yield of the pirarubicin is more than 75%.

Description

Separation and purification method of pirarubicin

Technical Field

The application relates to a separation and purification method of pirarubicin, belonging to the technical field of drug separation and purification.

Background

Pirarubicin, also known as tetrahydropyraamycin, is an orange-red crystalline powder with a melting point of 188-192 ℃ (resolved), optical rotation of +195 ° -215 ° (C1 mg/mL, chloroform), maximum absorption of UV and visible light (methanol): 234nm, 252nm, 290nm, 498nm, 531.5nm, 580 nm. Is easily soluble in chloroform, dichloromethane and DMF, is slightly soluble in ethyl acetate, ethanol or methanol, and is hardly soluble in water, n-hexane or petroleum ether or diethyl ether. Pirarubicin is a semi-synthetic anthracycline antitumor antibiotic, synthesized from doxorubicin and dihydropyran, and rapidly enters cancer cells by inhibitingNucleic acid synthesis prevents cell division. After administration, the blood concentration is rapidly reduced and rapidly distributed in tissues, and the concentration in the lung and the spleen is higher than that of doxorubicin and is mainly excreted by bile. It can be used for relieving head and neck cancer, breast cancer, ovarian cancer, metrocarcinoma, and leukemia. And has less toxic and side effect on heart and marrow depression than doxorubicin. The molecular formula is as follows: c₃₂H₃₇NO₁₂The molecular weight is: 627.636, molecular structural formula:

pirarubicin has a chemical structure similar to that of doxorubicin and is a derivative of doxorubicin through addition reaction of 4' -OH of aminosugar moiety and pyranyl. In the pyranose reaction of doxorubicin, the reaction of 3, 4-dihydropyran with the hydroxyl group at the 4' -position produces pirarubicin and its isomerized product, which is the largest impurity in the pirarubicin synthesis reaction at a total product ratio of about 30%. Meanwhile, the isomerized product has very similar structure and physical and chemical properties with pirarubicin, and is difficult to remove by a conventional method.

Based on the characteristics, the research on the separation and purification technology of the pirarubicin has important value. There are relatively few patents and literature dealing with purification techniques for pirarubicin. Wherein patent CN101181280A provides a pirarubicin freeze-dried preparation and a preparation method thereof, patent CN101879144A provides a pirarubicin or pirarubicin hydrochloride liposome freeze-dried preparation and a preparation method thereof, and patent CN101352418A provides pirarubicin or pirarubicin hydrochloride lipid nanoparticles and a preparation method thereof, the methods related to the three patents are preparation methods of pirarubicin pharmaceutical preparations, and a purification method of pirarubicin is not mentioned. Patent CN111675738A discloses a method for purifying pirarubicin, which adopts crude pirarubicin product with mass content above 65%, and the crude product contains no isomer impurities, only impurities such as doxorubicin and doxorubicin ketone, and can be removed by extraction, without reference meaning. A method for purifying pirarubicin is disclosed in a document of Pirarubicin separation and purification published in the journal of northwest pharmacy by the university of Chinese pharmacy, namely Schweijian et al, and the purification process provided by the document is to purify a crude pirarubicin product by a silica gel chromatography column, purify the crude pirarubicin product with the content of 35.7 percent by taking methylene dichloride-methanol as eluent twice, and obtain a pure pirarubicin product with the purity of 99.6 percent. However, when silica gel is used for separation and purification, due to the fact that the hydrogen bond adsorption force of the silica gel to the pirarubicin is large, part of the pirarubicin cannot be eluted from the silica gel in the elution process, the yield of the pirarubicin is low, two times of silica gel column chromatography are needed, the solvent consumption is large, and meanwhile, due to the fact that the silica gel is serious in adsorption, the silica gel cannot be reused, and the environmental pollution is serious.

Therefore, how to further improve the purity of pirarubicin is a technical problem to be solved urgently by those skilled in the art.

Disclosure of Invention

According to the application, the method for separating and purifying the pirarubicin is provided, the purity of the pirarubicin is obviously improved by adopting the diol-based bonded silica gel as a separation and purification filler, and the content of isomer impurities is below 0.1%.

The separation and purification method of pirarubicin at least comprises the following steps:

step 1, obtaining a solution to be treated of a sample containing pirarubicin;

step 2, separating and purifying the solution to be treated by adopting chromatography to obtain a pure pirarubicin product;

the filler of the chromatographic column adopted in the chromatographic separation and purification is Doil filler.

Optionally, the concentration of the solution to be treated is 0.1mg/mL to 50 mg/mL.

Preferably, the concentration of the solution to be treated is 1mg/mL-20 mg/mL.

Specifically, the lower limit of the concentration of the solution to be treated can be independently selected from 0.1mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4 mg/mL; the upper concentration limit of the solution to be treated can be independently selected from 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50 mg/mL.

Optionally, the solution to be treated is prepared by a sample containing pirarubicin and a solvent I;

the solvent I is at least one selected from dichloromethane, trichloromethane, n-hexane and n-heptane.

Optionally, the sample containing pirarubicin has a content of pirarubicin of 30% to 98% by mass.

Specifically, in the sample containing the pirarubicin, the lower limit of the mass content of the pirarubicin can be independently selected from 30%, 38.1%, 42.8%, 45% and 53.1%; the upper limit of the mass content of the pirarubicin can be independently selected from 60%, 70%, 80%, 94.1% and 98%.

Optionally, the filler has a particle size of 5 μm to 100 μm.

Preferably, the particle size of the filler is between 25 μm and 45 μm.

Optionally, the sample containing pirarubicin is added in an amount of 0.1% to 15% of the mass of the filler.

Preferably, the sample containing pirarubicin is added in an amount of 1% -10% of the mass of the filler.

Specifically, the lower limit of the amount of the pirarubicin-containing sample can be independently selected from 0.1%, 1%, 5%, 6%, 7%, 8% of the mass of the filler; the upper limit of the amount of the pirarubicin-containing sample added can be independently selected from 10%, 11%, 12%, 13%, 14%, 15% of the mass of the filler.

Optionally, in the chromatographic separation and purification process, the eluent used is at least one selected from dichloromethane, chloroform, n-hexane, n-heptane, methanol, ethanol, acetone and ethyl acetate.

Preferably, the eluent is selected from a mixed solution of dichloromethane and methanol.

Optionally, during the chromatographic separation and purification, the elution speed is 5mL/min-500 mL/min.

Preferably, the elution rate is 150mL/min to 300 mL/min.

Specifically, the lower limit of the elution speed can be independently selected from 5mL/min, 10mL/min, 50mL/min, 100mL/min, 150 mL/min; the upper limit of the elution rate can be independently selected from 200mL/min, 250mL/min, 300mL/min, 400mL/min, 500 mL/min.

Optionally, in the step 2, the chromatographic separation and purification process is monitored by TLC point plate or HPLC.

Specifically, after loading to the chromatographic column, monitoring single-point elution of the pirarubicin by TLC spot plates or HPLC, and starting to collect eluent; and when the elution of impurities is monitored, the collection is stopped.

Optionally, the step 2 further includes: and concentrating and drying the eluent obtained by chromatographic separation and purification.

Specifically, the concentration is performed by reduced pressure rotary evaporation.

The application has no special requirements for the specific operation of concentration and drying, and the skilled person can select suitable parameters according to the operation requirements of the conventional pirarubicin. Specifically, the pressure of reduced pressure rotary evaporation concentration operation in the embodiment of the application is controlled to be below-0.01 MPa, and the temperature is controlled to be 25-35 ℃; the drying temperature is 25-35 ℃, and the drying time is 2-6 h.

Alternatively, the separation and purification method of pirarubicin comprises the following steps:

preparing a sample containing pirarubicin into a sample loading solution;

loading the sample loading liquid into a Diol chromatographic column for separation and purification;

monitoring the eluent by adopting a TLC (thin layer chromatography) spot plate or HPLC (high performance liquid chromatography), and starting to collect the eluent when the single spot elution of the pirarubicin exists; when monitoring that impurities are eluted, stopping collecting the eluent;

and concentrating the collected eluent, and drying (filtering) the solid obtained by concentration to obtain the pure product of the pirarubicin.

Specifically, the collected eluent is subjected to reduced pressure rotary evaporation concentration, and the operation is stopped when a large amount of solid appears; the product is concentrated by filtration and the resulting solid is dried to yield a pure product of the pirarubicin.

Optionally, the sample containing the pirarubicin is selected from at least one of commercially available pirarubicin or solution solid, a solution or solid containing the pirarubicin obtained by a synthetic reaction, and a solution or solid containing the pirarubicin obtained by extraction and purification after the synthetic reaction.

Diol fillers in this application refer to Diol-based bonded silica gels, also known as Diol-based compound bonded silica gels.

The beneficial effects that this application can produce include:

1) according to the separation and purification method of the pirarubicin, the filler contains the hydroxyl functional group, compared with a conventional silica gel column, the adsorption of the filler to the pirarubicin in the separation and purification process is greatly reduced, the filler is easy to elute, the yield of the pirarubicin is improved, and the yield of the pirarubicin is over 75%.

2) According to the separation and purification method of pirarubicin, the Diol filler is used as the filler for chromatographic separation and purification, the adsorption to a sample is small, the filler can be regenerated by using appropriate solvents such as methanol and the like, repeated reuse can be realized for many times, and the method is more beneficial to the environment compared with the existing silica gel column.

Drawings

FIG. 1 is a diagram of a TLC spot plate during collection of an eluate in example 1 of the present application;

FIG. 2 is a liquid chromatogram of a sample of pirarubicin according to example 1 of the present application before purification;

FIG. 3 is a liquid chromatogram of pure pirarubicin obtained in example 1 of the present application;

FIG. 4 is a liquid chromatogram of a sample of pirarubicin prior to purification according to example 2 of the present application;

FIG. 5 is a liquid chromatogram of pure pirarubicin obtained in example 2 of the present application;

FIG. 6 is a liquid chromatogram of a sample of pirarubicin according to example 3 of the present application before purification;

FIG. 7 is a liquid chromatogram of pure pirarubicin obtained in example 3 of the present application;

FIG. 8 is a diagram of a TLC spot plate during collection of an eluate in example 3 of the present application.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

Unless otherwise indicated, the materials in the examples of the present application are commercially available and the pirarubicin material is self-made by the unit of the applicant.

The method adopts Shimadzu high performance liquid chromatograph (model CBM-20A) for analysis and detection.

Example 1

1. Diol column packing: weighing 1kg of 25 μm Diol filler, soaking with 1000ml dichloromethane, stirring well, packing into chromatographic column by wet method, and compacting the filler by reducing the height of sieve plate to obtain Diol chromatographic column (phi 95mm × 250 mm).

2. Diol column equilibration: the column was equilibrated with 5L of solvent (dichloromethane) at an equilibrium flow rate of 200 ml/min.

3. Preparing a sample loading solution: weighing 50g of pirarubicin compared with a star sample (the mass content is 42.8%, and the isomeric impurities are 38.2%), dissolving the pirarubicin in 2L of solvent (dichloromethane) by stirring to obtain a pirarubicin loading solution.

4. Sample loading: the pirarubicin sample is pumped into a Diol chromatographic column at a flow rate of 200 ml/min.

5. Sample elution: the column was washed with 10L of an eluent (dichloromethane: methanol 900: 1) at an elution flow rate of 200 ml/min. As shown in fig. 1, the elution is started by single point elution of pirarubicin with monitoring by TLC plates; when the TLC spot plate was monitored for impurities, the collection was stopped.

6. And carrying out reduced pressure rotary evaporation concentration on the collected eluent, controlling the pressure to be-0.06 MPa and the temperature to be 35 ℃, and stopping operation when a large amount of solid appears. The concentrate was filtered and the resulting solid was dried under vacuum at 35 ℃ for 4h to yield pure pirarubicin. Liquid chromatograms before and after purification are shown in fig. 2 and 3, and liquid chromatogram data corresponding to the chromatograms are shown in tables 1a and 1 b.

TABLE 1a

Peak #	Retention time	Area of	Height	Area%	Theoretical plate #
						1	3.129	54373	8913	0.181	5196.225
2	4.319	4533716	383261	15.133	3329.298
						3	6.084	137723	12090	0.460	6403.438
4	6.685	325371	12426	1.086	165.713
						5	7.320	12827501	883407	42.817	6249.101
6	8.003	11442027	691244	38.192	5520.147
						7	10.355	199008	12820	0.664	10860.669
8	27.855	58833	1510	0.196	13256.312
						9	34.997	149307	1946	0.498	6056.490
10	40.038	231082	2569	0.771	5559.365
						Total of		29958939	2010184	100.000

TABLE 1b

Peak #	Retention time	Area of	Area%	Height	Theoretical plate #
						1	3.657	17284	0.089	2687	6094.857
2	4.047	9183	0.047	1510	9254.379
						3	4.603	7489	0.038	1123	9726.070
4	6.975	19403663	99.654	1221854	4083.169
						5	8.146	7360	0.038	781	15459.268
6	9.392	3645	0.019	459	22888.954
						7	12.695	6130	0.031	442	17971.357
8	15.655	16365	0.084	668	8600.446
						Total of		19471118	100.000	1229524

The pirarubicin recovery results are shown in table 1 c:

TABLE 1c

Name (R)	Quality (g)	Purity (%)	Isomerized impurity (%)	Recovery (%)
					Sample before purification	50.00	42.8	38.2	--
Purified sample	17.08	99.6	0.04	79.5

Example 2

The same Diol column and elution method as in example 1 was used, except that:

60g of pirarubicin solid with the mass content of 53.1 percent and the mass content of isomeric impurities of 39.8 percent is weighed and dissolved by stirring with 2L of solvent (dichloromethane). The Diol column was equilibrated with 5L of solvent (dichloromethane) at an equilibrium flow rate of 200mL/min, and then the column was loaded at a flow rate of 200 mL/min. After the sample loading is finished, the Diol chromatographic column is washed by an eluent (dichloromethane: ethanol volume ratio is 90: 1) with the elution flow rate of 200 mL/min. The HPLC assay collects a single-point component solution of pirarubicin (i.e., the eluate). Concentrating the eluate by rotary evaporation under reduced pressure at 32 deg.C under-0.07 MPa, and stopping operation when a large amount of solid appears. The concentrate was filtered and the resulting solid was dried under vacuum at 32 ℃ for 4h to yield pure pirarubicin. Liquid chromatograms before and after purification are shown in fig. 4 and 5, and liquid chromatogram data corresponding to the chromatograms are shown in tables 2a and 2 b.

TABLE 2a

Peak #	Retention time	Area of	Height	Area%	Theoretical plate #
						1	1.488	12739	318	0.023	25.426
2	4.050	24471	4848	0.045	1959.624
						3	4.166	92917	16225	0.170	3248.480
4	4.271	87687	17311	0.160	609.556
						5	4.397	224683	29314	0.410	869.706
6	4.646	1115837	90932	2.036	3463.489
						7	4.889	274290	34474	0.500	2065.515
8	5.086	1105934	110092	2.018	5967.582
						9	6.385	584035	54313	1.066	9509.756
10	6.995	29106116	1355335	53.103	2215.023
						11	8.114	21840914	1024361	39.848	2946.875
12	9.693	202720	8735	0.370	5214.153
						13	23.196	138758	2212	0.253	3503.536
Total of		54811100	2748470	100.000

TABLE 2b

Peak #	Retention time	Area of	Height	Area%	Theoretical plate #
						1	2.749	2759	164	0.047	478.655
2	3.347	84	18	0.001	10431.980
						3	4.029	2762	414	0.047	7959.182
4	4.551	5017	767	0.085	9677.229
						5	5.183	982	44	0.017	3065.848
6	5.611	1408	107	0.024	9032.471
						7	7.096	5906808	515735	99.641	8556.436
8	8.212	2360	260	0.040	17086.584
						9	12.933	1730	126	0.029	19270.243
10	15.542	811	26	0.014	0.000
						11	15.823	3383	186	0.057	15641.067
Total of		5928103	517846	100.000

The pirarubicin recovery results are shown in table 2 c:

TABLE 2c

Name (R)	Quality (g)	Purity (%)	Isomerized impurity (%)	Recovery (%)
					Sample before purification	60.00	53.1	39.8	--
Purified sample	24.9	99.6	0.04	77.8

Example 3

The same Diol column and elution method as in example 1 was used, with the difference that:

80g of doxorubicin is weighed, a reaction solution containing the pirarubicin is obtained through a pyranization reaction, and the content of the pirarubicin is detected by HPLC to be 38.1%, the content of isomerized impurities is 29.5%, and the content of the doxorubicin is 13.2%. After water was added to the reaction solution, the mixture was extracted with 3L of methylene chloride and dried over anhydrous sodium sulfate to obtain a sample solution having a pirarubicin concentration of 10 mg/mL. The Diol column was equilibrated with 5L of solvent (dichloromethane) at a flow rate of 200 mL/min; then, the Diol chromatographic column was loaded at a flow rate of 200 mL/min. After the sample loading is finished, the chromatographic column is washed by an eluent (dichloromethane and ethanol with the volume ratio of 90: 1) at the elution flow rate of 200 mL/min. The HPLC assay collects a single-point component solution of pirarubicin (i.e., the eluate). And (3) carrying out reduced pressure rotary evaporation and concentration on the eluent, controlling the pressure to be-0.06 MPa and the temperature to be 30 ℃, and stopping operation when a large amount of solid appears. And filtering the concentrate, and carrying out vacuum drying on the obtained solid at 30 ℃ for 4h to obtain the pure pirarubicin. Liquid chromatograms before and after purification are shown in fig. 6 and 7, and liquid chromatogram data corresponding to the chromatograms are shown in tables 3a and 3 b.

TABLE 3a

Peak #	Retention time	Area of	Area%	Height	Theoretical plate #
						1	3.723	383491	2.046	28414	1776.304
2	4.296	2482535	13.247	282993	5778.242
						3	4.876	207571	1.108	16246	3200.830
4	5.192	16923	0.090	2427	10898.122
						5	6.091	576631	3.077	59818	8880.219
6	6.322	54741	0.292	6662	64.926
						7	7.009	7141932	38.110	574085	6806.452
8	7.449	86842	0.463	10429	18835.039
						9	7.855	5531973	29.519	427352	7383.713
10	9.637	11323	0.060	573	4122.607
						11	10.232	278369	1.485	12590	7078.859
12	11.647	33721	0.180	2262	13014.077
						13	14.124	124641	0.665	5240	8451.529
14	25.322	795063	4.243	4756	787.225
						15	33.851	350310	1.869	4809	6193.349
16	39.471	520520	2.778	5984	4636.863
						17	47.430	143741	0.767	931	2105.427
Total of		18740326	100.000	1445570

TABLE 3b

Peak #	Retention time	Area of	Height	Area%	Theoretical plate #
						1	2.752	1871	137	0.011	1077.293
2	3.041	2177	483	0.012	8256.636
						3	3.701	1261	177	0.007	5754.732
，4	4.079	2666	377	0.015	7656.852
						5	4.623	14314	2162	0.081	9510.998
6	7.152	17638700	1077638	99.739	4122.318
						7	8.044	6030	648	0.034	16358.639
8	8.425	6066	630	0.034	15690.561
						9	8.962	1484	138	0.008	15519.459
10	9.743	4172	405	0.024	19345.878
						11	11.514	1876	129	0.011	14006.498
12	13.353	2200	135	0.012	16730.494
						13	16.369	2091	117	0.012	17313.398
Total of		17684909	1083177	100.000

The pirarubicin recovery results are shown in table 3 c:

TABLE 3c

Name (R)	Quality (g)	Purity (%)	Isomerized impurity (%)	Recovery (%)
					Sample before purification	80.00	38.1	29.5	--
Purified sample	23.41	99.7	0.03	76.5

Example 4

70g of pirarubicin solid with the content of 94.1 percent and the mass content of isomeric impurities of 2.3 percent are weighed and dissolved by stirring with 3L of solvent (dichloromethane). The Diol column was equilibrated with 5L of solvent (dichloromethane) at an equilibrium flow rate of 200mL/min, and then the Diol column was loaded at a flow rate of 200 mL/min. After the sample loading is finished, the chromatographic column is washed by an eluent (dichloromethane: ethanol volume ratio is 85: 1) with the elution flow rate of 200 mL/min. The TLC assay collects a single spot component solution of pirarubicin (i.e., the eluate). And (3) carrying out rotary evaporation and concentration on the elution liquid under reduced pressure, controlling the pressure to be-0.07 MPa and the temperature to be 28 ℃, and stopping operation when a large amount of solid appears. The concentrate was filtered and the resulting solid was dried under vacuum at 28 ℃ for 4h to give pure pirarubicin 61.17 g. The purity of pirarubicin is 99.6 percent by HPLC detection, the single impurity is less than 0.1 percent, and the isomerization impurity is 0.03 percent.

The pirarubicin recovery results are shown in table 4:

TABLE 4

Name (R)	Quality (g)	Purity (%)	Isomerized impurity (%)	Recovery (%)
					Sample before purification	70.00	94.1	2.3	--
Purified sample	61.17	99.6	0.03	92.5

Comparative example 1

The Pirarubicin purification method is mentioned in the Pirarubicin separation and purification literature published in the journal of northwest China pharmaceutical science, such as xueliang, the purification technology provided by the literature is to purify the crude Pirarubicin by a silica gel chromatographic column, and to purify the Pirarubicin twice by taking dichloromethane-methanol as eluent. The method disclosed by the method is used for purifying the pirarubicin, and the specific operation steps are as follows:

100g of 200-mesh 300-mesh silica gel is weighed, soaked in 100ml of dichloromethane and loaded into a glass column (phi 3.5cm multiplied by 20cm) for later use by a wet method. 5g of pirarubicin (42.8% by mass and 38.2% by mass of isomeric impurities) is weighed out and dissolved in 200ml of dichloromethane. The column was loaded at a flow rate of 10 ml/min. After the sample loading is finished, eluting with an eluent (dichloromethane: methanol volume ratio is 9: 1) at an elution flow rate of 10 ml/min. And collecting the pirarubicin component solution by TLC detection. And (3) carrying out rotary evaporation and concentration on the elution liquid under reduced pressure, controlling the pressure to be-0.06 MPa and the temperature to be 30 ℃, and stopping operation when a large amount of solid appears. The concentrate was filtered and the filter cake was dried under vacuum at 30 ℃ for 4h to give a solid 1.51g, 91.2% pure.

Further separation and purification of the solid: 40g of 200-mesh 300-mesh silica gel is weighed, soaked in 100ml of dichloromethane and loaded into a glass column (phi 2.5cm multiplied by 20cm) for later use by a wet method. The solid obtained in the above step (91.2% by mass) was taken and dissolved in 100ml of dichloromethane. The chromatographic column was loaded at a flow rate of 10 ml/min. After the sample loading, the mixture is eluted by an eluent (dichloromethane: methanol volume ratio is 9: 1) with the elution flow rate of 10 ml/min. And collecting the pirarubicin component solution by TLC detection. Concentrating the eluate by rotary evaporation under reduced pressure, and concentrating the eluate by rotary evaporation under reduced pressure at-0.06 MPa and 30 deg.C, and stopping operation when a large amount of solid appears. The concentrate was filtered and the filter cake was dried under vacuum at 30 ℃ for 4h to give a solid 1.03g, 98.5% purity and 0.23% isomerised impurities.

The pirarubicin recovery results are shown in table 5:

TABLE 5

Name (R)	Quality (g)	Purity (%)	Isomerized impurity (%)	Recovery (%)
					Sample before purification	5.00	42.8	38.2	--
Purified sample	1.03	98.5	0.234	47.4

Comparative example 2

Weighing 50g of 25-45 μm Diol filler, soaking in 100ml dichloromethane, stirring well, packing into chromatographic column by wet method, and compacting the filler by reducing the height of sieve plate to obtain Diol chromatographic column (phi 3.5cm × 10 cm).

The column was equilibrated with 100mL of solvent (dichloromethane) at an equilibrium flow rate of 10 mL/min.

5g (mass content 42.8%, isomeric impurity content 38.2%) of pirarubicin in a sample is weighed and dissolved in 200mL of a solvent (dichloromethane) with stirring.

The chromatographic column was loaded at a flow rate of 10 ml/min. The mixture was washed with 1L of an eluent (dichloromethane: methanol in a volume ratio of 900: 1) at an elution flow rate of 10 ml/min. And collecting single spots of the pirarubicin by TLC spot plate detection. Concentrating the eluate by rotary evaporation under reduced pressure, and concentrating the eluate by rotary evaporation under reduced pressure at-0.06 MPa and 30 deg.C, and stopping operation when a large amount of solid appears. And filtering the concentrate, and drying a filter cake for 4 hours under vacuum at 30 ℃ to obtain the pure pirarubicin.

The pirarubicin recovery results are shown in table 6:

TABLE 6

Name (R)	Quality (g)	Purity (%)	Isomerized impurity (%)	Recovery (%)
					Sample before purification	5.00	42.8	38.2	--
Purified sample	1.72	99.7	0.04	80.1

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

Claims

1. A method for separating and purifying pirarubicin, which is characterized by at least comprising the following steps:

step 1, obtaining a solution to be treated of a sample containing pirarubicin;

2. The method according to claim 1, wherein the concentration of the solution to be treated is 0.1mg/mL to 50 mg/mL.

3. The method according to claim 1, wherein the sample containing pirarubicin is present in an amount of 30% to 98% by weight.

4. A method according to claim 1, characterized in that the particle size of the filler is between 5 μm and 100 μm.

5. The method according to claim 1, wherein the sample containing pirarubicin is added in an amount of 0.1% to 15% by mass of the filler.

6. The method according to claim 1, wherein the eluent used in the chromatographic separation and purification process is at least one selected from dichloromethane, chloroform, n-hexane, n-heptane, methanol, ethanol, acetone and ethyl acetate.

7. The preparation method according to claim 1, wherein the elution rate during the chromatographic separation and purification is 5mL/min to 500 mL/min.

8. The method of claim 1, wherein in step 2, the chromatographic separation and purification process is monitored by TLC spot plate or HPLC.

9. The method of claim 1, wherein step 2 further comprises: and concentrating and drying the eluent obtained by chromatographic separation and purification.