CN116159338A - Column chromatography method for purifying nonionic iodine contrast agent - Google Patents

Abstract

The invention discloses a column chromatography method for purifying a nonionic iodine contrast agent. Belongs to column chromatography for purifying nonionic iodine contrast agents; the operation steps are as follows: 1. sample pretreatment: dissolving the chemically synthesized non-ionic iodine contrast agent crude product in water, and then filtering by using a water-based filter membrane with the diameter of 0.22 mu m; 2. loading: after balancing the chromatographic column by using a balancing liquid, loading the treated sample into the chromatographic column; 3. eluting: eluting with eluent, and collecting target components in segments; 4. regeneration: and cleaning the impurities by using a cleaning solution, and regenerating the chromatographic column. The invention discloses a column chromatography method for purifying a nonionic iodine contrast agent, which can improve the purity of the nonionic iodine contrast agent to more than 99.5%, has single impurity less than 0.05% and has yield as high as more than 90%. The purification effect is good, the efficiency is high, the cost is low, and the method can be used for large-scale industrial production.

Description

Column chromatography method for purifying nonionic iodine contrast agent

Technical Field

The invention belongs to the field of downstream purification of contrast agents, and relates to a column chromatography method for purifying a nonionic iodine contrast agent.

Background

Contrast agents are chemicals that are administered or injected into human tissue or organs to enhance image viewing, and generally have a higher density than surrounding tissue, and contrast is displayed by imaging with medical devices such as CT, nuclear magnetism, and the like. The second generation nonionic iodine contrast agent has low side reaction incidence and good organism tolerance, and is widely used for various angiography radiography examinations, and the nonionic iodine contrast agent is also divided into monomers and dimers. The nonionic iodic contrast agents which have been marketed are iopamidol, iohexol, iopromide, iomeprol, ioversol, iobitol, iodixanol, iotrolan, etc.

At present, the purification method of the nonionic iodine contrast agent mainly comprises recrystallization, macroporous adsorption resin purification, reversed phase chromatography purification and the like. Among them, the recrystallization method has limited improvement of purity, and requires more than two times of recrystallization, resulting in low yield; the purification of macroporous adsorption resin can improve the purity to more than 99.0 percent, but has the defects of long purification period and lower yield; the reverse phase chromatography purification can generally improve the purity to more than 99.5%, the yield can reach 90%, the purification time is short, the efficiency is high, but the service life of the silica gel matrix reverse phase filler is short, the price is high, and the production cost is high.

Therefore, in order to solve the problems of the current purification of nonionic iodine contrast agents, it is necessary to develop a purification method which has a good purification effect, high efficiency and low cost.

Disclosure of Invention

The invention aims to: the invention aims to provide a column chromatography method for purifying a nonionic iodine contrast agent, which adopts polyethylene-divinylbenzene (PS-DVB) microspheres as a stationary phase, adopts the principle that the retention of a target object and impurities on the stationary phase is different, and can obtain a high-purity target object by collecting corresponding components along with the elution in the purification process.

The technical scheme is as follows: the invention relates to a column chromatography method for purifying a nonionic iodine contrast agent, which comprises the following specific operation steps:

step (1), sample pretreatment: dissolving the chemically synthesized non-ionic iodine contrast agent crude product in water, and then filtering by using a water-based filter membrane with the diameter of 0.22 mu m;

step (2), loading: after balancing the chromatographic column by using the balancing liquid, loading the sample processed in the step (1) into the chromatographic column;

step (3), elution: eluting with eluent, and collecting target components in segments;

step (4), regeneration: and cleaning the impurities by using a cleaning solution, and regenerating the chromatographic column.

Further, in step (1), the chemically synthesized crude non-ionic iodic contrast agent includes, but is not limited to, iopamidol, iohexol, iopromide, iomeprol, ioversol, iodixanol, and iotrolan.

Further, in the step (2), the balance liquid is pure water.

Further, in the step (3), the eluent is pure water or a mixed solution of water and an organic solvent;

wherein the organic solution is at least one of ethanol, methanol, isopropanol, n-butanol or acetonitrile;

the elution modes include linear elution and isocratic elution.

Further, in the step (4), the cleaning liquid is a mixed liquid of water and an organic solvent;

wherein the organic solution is at least one of ethanol, methanol, isopropanol, n-butanol or acetonitrile.

Further, in the present column chromatography, the stationary phase involved is polyethylene-divinylbenzene (PS-DVB) microspheres, commercially available as PolyRP, having a particle size of 10-100 μm and a pore size

In the column chromatography, the chromatographic column is a single chromatographic column or a plurality of chromatographic columns are used in series.

The beneficial effects are that: compared with the prior art, the invention has the characteristics that: the invention discloses a column chromatography method for purifying a nonionic iodine contrast agent, which can improve the purity of the nonionic iodine contrast agent to more than 99.5%, has single impurity less than 0.05% and has yield as high as more than 90%. The purification effect is good, the efficiency is high, the cost is low, and the method can be used for large-scale industrial production.

Drawings

FIG. 1 is a purification scheme of the present invention for the isolation of iopamidol using the purification method of example 1;

FIG. 2 is an analytical test pattern of the separation of iopamidol using the purification method of example 1 of the present invention;

FIG. 3 is a purification scheme of the present invention for the isolation of iohexol using the purification method of example 2;

FIG. 4 is an analytical test pattern of the isolation of iohexol according to the invention using the purification method of example 2;

FIG. 5 is a purification scheme of the present invention for the isolation of iopromide using the purification method of example 3;

FIG. 6 is an analytical test pattern of the separation of iopromide using the purification method of example 3 of the present invention;

FIG. 7 is a purification scheme of the present invention for the isolation of iohexol using the purification method of example 4;

FIG. 8 is an analytical test pattern of the isolation of iohexol according to the invention using the purification method of example 4;

fig. 9 is a flow chart of the operation of the present invention.

Detailed Description

In order to more clearly describe the technical scheme of the invention, the technical scheme of the invention is further described in detail below with reference to the accompanying drawings:

example 1

This example uses PolyRP 30-300 (particle size 30 μm, pore size)

) And (3) filling the stationary phase into a chromatographic column with the specification of 21.2mm (ID) multiplied by 250mm (L), purifying the chemically synthesized lopamidol crude product, collecting target components in sections, detecting the purity of each collected fraction by HPLC, and carrying out sample combination on qualified fractions.

Sample pretreatment: dissolving the chemically synthesized crude lopamidol with pure water, and filtering with a 0.22 mu m water-based filter membrane, wherein the purity of the crude lopamidol is 95.290%;

column chromatography parameters:

apparatus and method for controlling the operation of a device	Preparative HPLC
		Chromatographic column	PolyRP 30-300(21.2mm(ID)×250mm(L))
Sample concentration	300mg/mL-400mg/mL
		Flow rate	10mL/min
Mobile phase	A: pure water; b: ethanol
		Operating temperature	Room temperature
Detection of	UV240nm
		Sample of	Crude lopamidol
Sample loading amount	80mg/mL

Column chromatography process:

step (a)	Time (min)	Gradient of
			Balancing	20	100％A
Loading sample	N/A	Pump A samples until the sample is completely applied
			Elution	200	Gradient elution of 0% B-20% B
Cleaning	30	80％B

Analysis column parameters:

analyzing column gradient information:

purifying a sample: the sample solution is separated and purified by column chromatography, a column with the specification of 21.2mm (ID) multiplied by 250mm (L) is filled with PolyRP 30-300, gradient elution is carried out, the fraction starts to fall from the peak of the target peak to the peak of the target peak, collection is stopped, and the column is washed by high-concentration organic solvent.

The fractions received were analyzed by HPLC and the acceptable fractions were pooled.

Using this purification method, the recovery rate of iopamidol was 93.8% and the purity was 99.947%; specifically as shown in table 1;

table 1 purity analysis integration table of purification method for iopamidol separation

Sequence number	Time	Peak area	Peak height	Peak width	Symmetry factor	Peak surfaceProduct%
							1	10.436	5.7	4.5E-1	0.2103	0.913	0.011
2	11.258	5.6	3.8E-1	0.2473	0.884	0.010
							3	13.198	2.5	1.6E-1	0.2618	0.802	0.005
4	14.781	53502.6	2633.5	0.314	0.618	99.947
							5	16.152	10.8	6.2E-1	0.2881	0.754	0.020
6	21.308	4	1.6E-1	0.4105	0.628	0.008

Example 2

This example uses PolyRP 30-100 (particle size 30 μm, pore size)

) The stationary phase is packed into a chromatographic column with the specification of 4.6mm (ID) multiplied by 250mm (L), the crude product of the chemically synthesized iobitol is purified, target components are collected in sections, the purity of each collected fraction is detected by HPLC, and qualified fractions are subjected to sample matching.

Sample pretreatment: dissolving the chemically synthesized crude product of the iohexol with pure water, and filtering the solution with a 0.22 mu m water-based filter membrane, wherein the purity of the crude product is 95.333%;

column chromatography parameters:

column chromatography process:

step (a)	Time (min)	Gradient of
			Balancing	20	100％A
Loading sample	N/A	Pump A samples until the sample is completely applied
			Elution	300	Gradient elution of 0% B-30% B
Cleaning	30	80％B

Analysis column parameters:

analyzing column gradient information:

time (min)	A(％)	B(％)
			0	100	0
40	100	0
			80	85	15
95	25	75
			100	100	0
110	100	0

Purifying a sample: separating and purifying the sample solution by column chromatography, using PolyRP 30-100 filler, connecting three columns with the specification of 4.6mm (ID) x 250mm (L) in series, performing gradient elution, starting from the peak of the target peak to the beginning of the target peak, dropping, stopping collecting, and cleaning the column with high-concentration organic solvent;

the fractions received were analyzed by HPLC and the acceptable fractions were pooled.

Using this purification method, the recovery rate of iohexol was 97.2% and its purity was 99.811%; as shown in the detail of the table 2,

table 2 purification method integration table for purity analysis of iobitol separation

Sequence number	Time	Peak area	Peak height	Peak width	Symmetry factor	Peak area%
							1	4.959	91	9.8	0.1547	0.269	0.035
2	5.576	35.7	2.3	0.2618	0.489	0.014
							3	8.258	15.9	7.1E-1	0.3726	0	0.006
4	8.605	21.3	8.7E-1	0.406	0.473	0.008
							5	9.902	3.8	2.5E-1	0.2521	0	0.001
6	10.148	14.1	4.5E-1	0.5175	0.602	0.005
							7	16.473	256748.4	1752.9	2.4412	2.251	99.811
8	21.049	21.9	5.1E-1	0.7141	0.909	0.009
							9	22.872	12.2	2.4E-1	0.8393	0.553	0.005
10	24.038	6.2	1.3E-1	0.8055	0.741	0.002
							11	26.555	56.5	1	0.9344	0.989	0.022
12	27.958	24.3	4.8E-1	0.8456	0.787	0.009
							13	39.648	56.3	9.2E-1	1.0166	0.771	0.022
14	67.862	50.4	1.6	0.5242	0.804	0.020
							15	81.051	77.1	2.8	0.4579	0.867	0.030

Example 3

This example uses PolyRP 30-100 (particle size 30 μm, pore size)

) Packing into chromatographic column of 4.6mm (ID) x 250mm (L) specification as stationary phase, purifying chemically synthesized crude iopromide, collecting target components in stages, detecting purity of each collected fraction by HPLC, and collecting the purified productAnd (5) carrying out sample matching on the qualified fraction.

Sample pretreatment: dissolving the chemically synthesized crude product of iopromide with pure water, and filtering with a 0.22 mu m water-based filter membrane, wherein the purity of the crude product is 99.355%;

column chromatography parameters:

apparatus and method for controlling the operation of a device

Preparative HPLC

Column chromatography process:

step (a)	Time (min)	Gradient of
			Balancing	10	100％A
Loading sample	N/A	Pump A samples until the sample is completely applied
			Elution	300	Gradient elution of 0% B-30% B
Cleaning	30	80％B

Analysis column parameters:

/>

analyzing column gradient information:

time (min)	A(％)	B(％)
			0	98	2
40	80	20
			60	40	60
61	98	2
			68	98	2

Purifying a sample: the sample solution is separated and purified by column chromatography, a PolyRP 30-100 packing is used, two columns with the specification of 4.6mm (ID) multiplied by 250mm (L) are connected in series, gradient elution is carried out, the fraction starts to fall from the peak of the target peak to the peak of the target peak, collection is stopped, and the columns are washed by high-concentration organic solvent.

The fractions received were analyzed by HPLC and the acceptable fractions were pooled.

Using this purification method, the recovery rate of iopromide was 94.5% and the purity was 99.897%; specifically as shown in table 3;

table 3 purity analysis integration table of purification method for iopromide separation

Sequence number	Time	Peak area	Peak height	Peak width	Symmetry factor	Peak area%
							1	21.278	3.5	1.2E-1	0.464	1.222	0.012
2	23.438	2.6	1E-1	0.428	1.595	0.009
							3	25.129	5.7	2.2E-1	0.4333	0	0.020
4	30.108	28730.4	646.9	0.7402	3.511	99.897
							5	31.016	12.5	4.4E-1	0.4689	0.649	0.043
6	31.816	5.4	2.1E-1	0.4239	1.119	0.019

Example 4

This example uses PolyRP 30-300 (particle size 30 μm, pore size)

) The stationary phase is packed into a chromatographic column with the specification of 4.6mm (ID) multiplied by 250mm (L), the crude product of the chemically synthesized iobitol is purified, target components are collected in sections, the purity of each collected fraction is detected by HPLC, and qualified fractions are subjected to sample matching.

Sample pretreatment: dissolving the chemically synthesized crude product of the iohexol with pure water, and filtering the solution with a 0.22 mu m water-based filter membrane, wherein the purity of the crude product is 95.333%;

column chromatography parameters:

apparatus and method for controlling the operation of a device	Preparative HPLC
		Chromatographic column	PolyRP 30-300(4.6mm(ID)×250mm(L))
Sample concentration	300mg/mL-400mg/mL
		Flow rate	1mL/min
Mobile phase	A: pure water; b: methanol
		Operating temperature	Room temperature
Detection of	UV242nm
		Sample of	Crude iodic alcohol
Sample loading amount	70mg/mL

Column chromatography process:

step (a)	Time (min)	Gradient of
			Balancing	30	100％A
Loading sample	N/A	Pump A samples until the sample is completely applied
			Elution	220	Isocratic elution of 10% B
Cleaning	30	80％B

Analysis column parameters:

analyzing column gradient information:

time (min)	A(％)	B(％)
			0	100	0
40	100	0
			80	85	15
95	25	75
			100	100	0
110	100	0

Purifying a sample: the sample solution is separated and purified by column chromatography, a column with the specification of 4.6mm (ID) multiplied by 250mm (L) is filled with PolyRP 30-300, isocratic elution is carried out, the fraction starts to fall from the peak of the target peak to the peak of the target peak, collection is stopped, and the column is washed by high-concentration organic solvent.

The fractions received were analyzed by HPLC and the acceptable fractions were pooled.

Using this purification method, the recovery of iobiol was 97.1% and its purity was 99.834%, as shown in table 4 in detail;

table 4 purification method integration table for purity analysis of iobitol separation

Sequence number	Time	Peak area	Peak height	Peak width	Symmetry factor	Peak area%
							1	5.375	5.1	5.2E-1	0.1633	0.749	0.013
2	5.657	5.3	4.6E-1	0.1902	0.729	0.014
							3	6.221	4	2.8E-1	0.2327	0.715	0.010
4	9.827	4.5	1.2E-1	0.6551	1.275	0.012
							5	20.569	38286.8	236.7	2.6961	2.861	99.834
6	26.641	1.7	2.9E-2	0.9738	0.711	0.004
							7	28.964	3	3.3E-2	1.5153	0.406	0.008
8	36.616	8.5	9.9E-2	1.4274	1.298	0.022
							9	38.622	4	4.5E-2	1.4844	0.75	0.010
10	50.429	6.4	1.3E-1	0.8402	0.843	0.017
							11	53.987	5.6	1.2E-1	0.7694	0.856	0.015
12	72.321	8.2	3E-1	0.4548	0.724	0.021
							13	85.26	7.4	3.4E-1	0.3701	0.859	0.019

Example 5

This example uses PolyRP 75-300 (particle size 75 μm, pore size)

) The crude product of chemically synthesized iodixanol was purified by packing a 21.2mm (ID) x 250mm (L) specification column as a stationary phase, collecting the target components in stages by the same method as in example 3, detecting the purity of each collected fraction by HPLC, and fitting the qualified fraction.

Using this purification method, the recovery of iodixanol was 93.4% and its purity was 95.217%.

Example 6

PolyRP 15-300 (particle size 15 μm, pore size)

) The crude iohexol product of chemical synthesis was purified by packing a 21.2mm (ID). Times.250 mm (L) specification column as a stationary phase, collecting the target components in stages by the same method as in example 1, detecting the purity of each collected fraction by HPLC, and fitting the qualified fraction.

Using this purification method, iohexol recovery was 95.3% and purity was 99.586%.

The gradient elution method used in the examples 1, 2, 3, 5 and 6 and the isocratic elution method used in the example 4 have the advantages of shortening the preparation time, improving the efficiency, reducing the time cost, reducing the consumption of methanol, being beneficial to recycling the methanol and greatly reducing the solvent cost.

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the invention without departing from the principles thereof are intended to be within the scope of the invention as set forth in the following claims.

Claims (6)

1. A column chromatography method for purifying a nonionic iodine contrast agent, which is characterized by comprising the following specific operation steps:

step (1), sample pretreatment: dissolving the chemically synthesized non-ionic iodine contrast agent crude product in water, and then filtering by using a water-based filter membrane with the diameter of 0.22 mu m;

step (2), loading: after balancing the chromatographic column by using the balancing liquid, loading the sample processed in the step (1) into the chromatographic column;

step (3), elution: eluting with eluent, and collecting target components in segments;

step (4), regeneration: and cleaning the impurities by using a cleaning solution, and regenerating the chromatographic column.

2. A column chromatography for purifying a non-ionic iodinated contrast medium according to claim 1,

in step (1), the chemically synthesized crude non-ionic iodic contrast agent includes, but is not limited to, iopamidol, iohexol, iopromide, iomeprol, ioversol, iodixanol, and iotrolan.

3. A column chromatography method for purifying a non-ionic iodinated contrast agent according to claim 1, characterized in that:

in the step (2), the balance liquid is pure water.

4. A column chromatography for purifying a non-ionic iodinated contrast medium according to claim 1,

in the step (3), the eluent is pure water or a mixed solution of water and an organic solvent;

wherein the organic solution is at least one of ethanol, methanol, isopropanol, n-butanol or acetonitrile;

the elution modes include linear elution and isocratic elution.

5. A column chromatography for purifying a non-ionic iodinated contrast medium according to claim 1,

in the step (4), the cleaning liquid is a mixed liquid of water and an organic solvent;

wherein the organic solution is at least one of ethanol, methanol, isopropanol, n-butanol or acetonitrile.

6. A column chromatography for purifying a non-ionic iodinated contrast medium according to claim 1,

in the column chromatography, the stationary phase is polyethylene-divinylbenzene microsphere with particle size of 10-100 μm and pore diameter

In the column chromatography, the chromatographic column is a single chromatographic column or a plurality of chromatographic columns are used in series.

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