CN105334270A - Liquid chromatography for separating and determining pomalidomide intermediate related substance - Google Patents
Liquid chromatography for separating and determining pomalidomide intermediate related substance Download PDFInfo
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- CN105334270A CN105334270A CN201410305848.5A CN201410305848A CN105334270A CN 105334270 A CN105334270 A CN 105334270A CN 201410305848 A CN201410305848 A CN 201410305848A CN 105334270 A CN105334270 A CN 105334270A
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Abstract
The invention discloses a liquid chromatography for separating and determining a pomalidomide intermediate related substance. The method comprises the following steps: 1) a tested object solution and a reference solution are prepared; 2) the tested object solution and the reference solution are detected through high-performance liquid chromatographic determination; wherein chromatogram condition is characterized in that octadecylsilane chemically bonded silica (250*4.0 mm, 5 [mu]m) is taken as a filler, an acetonitrile/phosphoric acid buffer (a pH value is 2.6) with a ratio of 10:90 is taken as a mobile phase A, the acetonitrile/phosphoric acid buffer (the pH value is 2.6) with the ratio of 90:10 is taken as the mobile phase B, gradient elution is carried out, a column temperature is 25-40 DEG C, a flow velocity is 0.8-1.2 ml/min, and a detection wavelength is 254 nm.
Description
Technical field
The invention belongs to analytical chemistry field, a kind of liquid phase chromatography of separated island form pool horse degree amine intermediate related substance.
Background technology
Pool horse degree amine (pomalidomide) is the new immunomodulator of Celgene drugmaker of U.S. exploitation, and commodity are called Pomalyst.On February 8th, 2013, U.S. food and Drug Administration (FDA) ratify this medicine due to treatment recurrent and Refractory Multiple Myeloma.
Pool horse degree amine intermediated chemistry is called 3-aminophthalic acid, and molecular formula is C
8h
7nO
4, its structural formula is:
In the process of this compound of synthesis, there is the important impurity of a few step may affect purity and the quality of medicine owing to removing not exclusively, related substance for the synthesis major control of pool horse degree amine intermediate has 3, related substance 1:3-aminophthalicanhydride respectively, related substance 2:4-amino-2-(1, 3-dioxo-1, 3-dihydroisobenzofuran-4-yl) isoindoline-1, 3-dione, related substance 3:3-(4-amino-1, 3-dioxoisoindolin-2-yl) phthalicacid, structure respectively as figure below from left to right shown in:
Related substance 1 (Impurity1)
Related substance 2 (Impurity2)
Related substance 3 (Impurity3)
The impurity introduced for synthesis pool horse degree amine intermediate needs to carry out quality control in the feed, and therefore, realize the separation of pool horse degree amine intermediate and related substance thereof, synthesis and the quality control aspect thereof of horse degree amine on the berth have important practical significance.
Summary of the invention
The object of the present invention is to provide and a kind ofly analyze pool horse degree amine intermediated chemistry purity and be separated the method for its related substance, thus realize the separated island form of pool horse degree amine intermediate and its related substance, ensure pool horse degree amine and the quality control containing pool horse degree amine preparation.
Technical scheme of the present invention is:
A method for liquid chromatography for separating and determining pool horse degree amine intermediate related substance, comprises the following steps:
(1) preparation of need testing solution and reference substance solution;
(2) described need testing solution and reference substance solution is detected by high-performance liquid chromatogram determination;
Wherein chromatographic condition is: with octadecylsilane chemically bonded silica (250 × 4.0mm, 5 μm) be filling agent, with the acetonitrile of 10:90 (v/v)/phosphate buffer pH=2.6 for mobile phase A, with the acetonitrile of 90:10 (v/v)/phosphate buffer pH=2.6 for Mobile phase B, gradient elution, column temperature is 25 ~ 40 DEG C, preferably 30 DEG C, flow velocity is 0.8 ~ 1.2ml/min, preferred 1.0mL/min, and determined wavelength is 254nm.
Further, the preparation of described need testing solution and reference substance solution comprises: get pool horse degree amine intermediate testing sample, solubilizer makes the solution that concentration is 1mg/ml, as need testing solution; Measure described need testing solution, solubilizer makes the solution that concentration is 0.01mg/ml, solution in contrast.
Further, described mobile phase A and the Mobile phase B of described solvent to be volume ratio be 50:50.
Further, the condition of described gradient elution is: with volume basis, and when 0 minute, mobile phase A is 90%, and Mobile phase B is 10%; When 5 minutes, mobile phase A is 90%, and Mobile phase B is 10%; When 35 minutes, mobile phase A is 20%, and Mobile phase B is 80%; When 36 minutes, mobile phase A is 90%, and Mobile phase B is 10%; When 40 minutes, mobile phase A is 90%, and Mobile phase B is 10%.
Further, described chromatographic column is Kormasil chromatographic column.
The present invention still further provides described method at monitoring pool horse degree amine or containing the purposes in the quality of pool horse degree amine preparation.
Accompanying drawing explanation
Fig. 1 is the chromatogram of pool horse degree amine intermediate sample.
Wherein, retention time is the chromatographic peak of 2.319 is related substance 1 (Impurity1), retention time to be 15.873 related substances 2 (Impurity2) and retention time be 16.358 chromatographic peak be related substance 3 (Impurity3).
Embodiment
Advantage and disadvantage of the present invention can be understood further by following examples, should not be construed and limit the scope of the present invention.
Instrument and reagent in following examples, be common commercially available except specified otherwise.
Instrument and reagent
Instrument: Agilent1260series high performance liquid chromatograph.
Chromatographic column: C18 (Kromasil250*4.6mm5 μm).
Test sample: pool horse degree amine intermediate that Shenzhen Haiwang Pharmaceutical Co., Ltd produces (synthetic route: 3-nitrophthalic acid obtains 3-aminophthalic acid through palladium carbon hydrogenating reduction, then obtain mooring horse degree amine with amino-2, the 6-piperidine dione condensations of 3-).
Reagent: chromatographic grade acetonitrile (Merck company), phosphoric acid (analyzing pure).
Solvent: volume is mobile phase A and the Mobile phase B of (50:50v/v).
The screening of [embodiment 1] chromatographic condition and system suitability
The selection of 1.1 mobile phase pH value
Selective flow phase pH value is 2.0,2.6 and 3.0 to investigate, and finally determine that mobile phase pH value is 2.6, mobile phase pH value selection result is in table 1.
Table 1. mobile phase pH value selection result
The selection of 1.2 mobile phases
With reference to pertinent literature binding tests concrete condition, successively select acetonitrile-phosphate buffer (be 2.6 with phosphoric acid adjust pH), acetonitrile-acetate buffer solution (be 2.6 with phosphoric acid adjust pH), acetonitrile-formic acid buffer (be 2.6 with formic acid adjust pH), acetonitrile-TFA damping fluid
(be 2.6 with TFA adjust pH), the separating effect under each mobile phase condition is following (see table 2
Table 2. mobile phase selection result
The selection of 1.3 determined wavelength
Select determined wavelength value to be that 240nm, 247nm, 250nm and 254nm investigate, finally determine that determined wavelength is 254nm, determined wavelength selects survey to the results are shown in Table 3
Table 3. determined wavelength selection result
In order to detect more impurity, better to control the quality of mooring horse degree amine intermediate, therefore select 254nm for pool horse degree amine intermediate related substance determined wavelength.
The selection of 1.4 solvents
1.4.1 the study on the stability of horse degree amine intermediate in methyl alcohol is moored
Investigated pool horse degree amine intermediate stability in methanol solution, institute's obtain solution was respectively at 0,1,2,4,8 hour injection liquid chromatography, and measurement result is in table 4
Selection result selected by table 4. solvent
Pool horse degree amine intermediate is unstable in methyl alcohol, and main peak is degraded into RRT:4.64 impurity gradually, and methyl alcohol therefore can not be used as the solvent dissolving pool horse degree amine intermediate.
1.4.2 the study on the stability of horse degree amine intermediate in acetonitrile is moored
Investigated pool horse degree amine intermediate stability in acetonitrile solution, institute's obtain solution was respectively at 0,1,2,8,12 hour injection liquid chromatography, and measurement result is in table 5
Selection result selected by table 5. solvent
Pool horse degree amine intermediate (3-aminophthalic acid) is more stable in acetonitrile, and relative retention time is that 4.64 mass peak areas increase gradually.Therefore after wiring solution-forming in 1 hour sample introduction.
The determination of chromatographic condition: through above-mentioned Selection experiment, the chromatographic condition determined is: with octadecylsilane chemically bonded silica (250 × 4.0mm, 5 μm) be filling agent, with acetonitrile/phosphate buffer pH=2.6 (10:90) for mobile phase A, with acetonitrile/phosphate buffer pH=2.6 (90:10) for Mobile phase B, gradient elution, column temperature is 25 DEG C, flow velocity is 1ml/min, and determined wavelength is 254nm; Described condition of gradient elution is: with volume basis, and when 0 minute, mobile phase A is 90%, and Mobile phase B is 10%; When 5 minutes, mobile phase A is 90%, and Mobile phase B is 10%; When 35 minutes, mobile phase A is 20%, and Mobile phase B is 80%; When 36 minutes, mobile phase A is 90%, and Mobile phase B is 10%; When 40 minutes, mobile phase A is 90%, and Mobile phase B is 10%.
The preparation of [embodiment 2] need testing solution
Take pool horse degree amine intermediate 10mg, put in 10ml measuring bottle, solubilizer dissolves and is diluted to scale, shakes up, and filters, gets subsequent filtrate, to obtain final product.
[embodiment 3] pool horse degree amine intermediate sample determination of related substances
Adopt method described in embodiment 2 to prepare need testing solution, take pool horse degree amine intermediate 10mg, put in 10ml measuring bottle, solubilizer dissolves and is diluted to scale, shakes up, and filters, gets subsequent filtrate, as need testing solution; Precision measures need testing solution 1ml and puts in 100ml measuring bottle, and solubilizer, to scale, shakes up, in contrast solution.Under following selected chromatographic condition: with octadecylsilane chemically bonded silica (250 × 4.0mm, 5 μm) be filling agent, with acetonitrile/phosphate buffer pH=2.6 (10:90) for mobile phase A, with acetonitrile/phosphate buffer pH=2.6 (90:10) for Mobile phase B, gradient elution, column temperature is 25 DEG C, and flow velocity is 1ml/min, and determined wavelength is 254nm; Number of theoretical plate calculates should be not less than 2000 by pool horse degree amine intermediate 3-aminophthalic acid peak, and the degree of separation at main peak and other impurities peak should be greater than 1.5.Precision measures contrast solution 10 μ l injection liquid chromatography, regulate detection sensitivity, major component chromatogram peak height is made to be about 10 ~ 20% of full scale, precision measures need testing solution 10 μ l injection liquid chromatography again, record chromatogram is to 10 times of pool horse degree amine intermediate 3-aminophthalic acid peak retention time, if any impurity peaks in need testing solution chromatogram, measure each impurity peak area and, must not be greater than 6 times (6%) of contrast solution main peak area.HPLC result shows, and the retention time of pool horse degree amine intermediate 3-aminophthalic acid is 3.4min, as shown in Figure 1.Measure the pool horse degree amine intermediate of three batches (20140301,20140401,20140501) respectively, wherein, the measurement result of pool horse degree amine intermediate related substance is as shown in table 6 below.
Horse degree amine intermediate determination of related substances result moored by table 6.
| Lot number | 20140301 | 20140401 | 20140501 |
| RRT2.34 impurity (%) | 0.20 | 0.22 | 0.21 |
| RRT4.64 impurity (%) | 3.99 | 4.06 | 3.98 |
| RRT4.78 impurity (%) | 0.62 | 0.62 | 0.62 |
| Other impurity (%) | 0.59 | 0.50 | 0.59 |
| Total impurities (%) | 5.40 | 5.40 | 5.40 |
Claims (7)
1. a method for liquid chromatography for separating and determining pool horse degree amine intermediate related substance, comprises the following steps:
(1) preparation of need testing solution and reference substance solution;
(2) described need testing solution and reference substance solution is detected by high-performance liquid chromatogram determination;
Wherein chromatographic condition is: with octadecylsilane chemically bonded silica (250 × 4.0mm, 5 μm) be filling agent, with the acetonitrile of 10:90/phosphate buffer pH=2.6 for mobile phase A, with the acetonitrile of 90:10/phosphate buffer pH=2.6 for Mobile phase B, gradient elution, column temperature is 25 ~ 40 DEG C, and flow velocity is 0.8 ~ 1.2ml/min, and determined wavelength is 254nm.
2. the method for claim 1, is characterized in that, the preparation of described need testing solution and reference substance solution comprises: get pool horse degree amine intermediate testing sample, solubilizer makes the solution that concentration is 1mg/ml, as need testing solution; Measure described need testing solution, solubilizer makes the solution that concentration is 0.01mg/ml, solution in contrast.
3. method as claimed in claim 2, is characterized in that, described mobile phase A and the Mobile phase B of described solvent to be volume ratio be 50:50.
4. the method for claim 1, is characterized in that, described column temperature is 30 DEG C, and flow velocity is 1.0mL/min.
5. the method for claim 1, is characterized in that, the condition of described gradient elution is: with volume basis, and when 0 minute, mobile phase A is 90%, and Mobile phase B is 10%; When 5 minutes, mobile phase A is 90%, and Mobile phase B is 10%; When 35 minutes, mobile phase A is 20%, and Mobile phase B is 80%; When 36 minutes, mobile phase A is 90%, and Mobile phase B is 10%; When 40 minutes, mobile phase A is 90%, and Mobile phase B is 10%.
6. point method as claimed in claim 1, it is characterized in that, described chromatographic column is Kormasil chromatographic column.
7. the method for claim 1 is at monitoring pool horse degree amine or containing the application in the quality of pool horse degree amine preparation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105866297A (en) * | 2016-06-24 | 2016-08-17 | 合肥久诺医药科技有限公司 | High performance liquid chromatography method of pomalidomide related substance |
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| WO2011050962A1 (en) * | 2009-10-29 | 2011-05-05 | Ratiopharm Gmbh | Acid addition salts of lenalidomide |
| CN103645259A (en) * | 2013-12-12 | 2014-03-19 | 深圳海王药业有限公司 | Method for simultaneously determining 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione and related substances thereof |
| CN103787956A (en) * | 2014-01-20 | 2014-05-14 | 上海医药工业研究院 | Preparation method of midbody for preparing pomalidomide |
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2014
- 2014-06-30 CN CN201410305848.5A patent/CN105334270A/en active Pending
Patent Citations (3)
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|---|---|---|---|---|
| WO2011050962A1 (en) * | 2009-10-29 | 2011-05-05 | Ratiopharm Gmbh | Acid addition salts of lenalidomide |
| CN103645259A (en) * | 2013-12-12 | 2014-03-19 | 深圳海王药业有限公司 | Method for simultaneously determining 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione and related substances thereof |
| CN103787956A (en) * | 2014-01-20 | 2014-05-14 | 上海医药工业研究院 | Preparation method of midbody for preparing pomalidomide |
Non-Patent Citations (3)
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| MATTHEW HOFFMANN等: "Absorption, metabolism and excretion of [14C]pomalidomide in humans following oral administration", 《CANCER CHEMOTHER PHARMACOL》 * |
| PADIGELA SWETHA等: "Estimation of pomalidomide in capsule dosage form by RP-HPLC", 《DER PHARMACIA LETTRE》 * |
| 邱娟等: "HPLC法测定沙利度胺片中有关物质的含量", 《中国药房》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105866297A (en) * | 2016-06-24 | 2016-08-17 | 合肥久诺医药科技有限公司 | High performance liquid chromatography method of pomalidomide related substance |
| CN105866297B (en) * | 2016-06-24 | 2019-01-04 | 合肥久诺医药科技有限公司 | A kind of HPLC analytical method of the pomalidomide in relation to substance |
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