CN103645259B - Method for simultaneously determining 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione and related substances thereof - Google Patents
Method for simultaneously determining 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione and related substances thereof Download PDFInfo
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- CN103645259B CN103645259B CN201310683222.3A CN201310683222A CN103645259B CN 103645259 B CN103645259 B CN 103645259B CN 201310683222 A CN201310683222 A CN 201310683222A CN 103645259 B CN103645259 B CN 103645259B
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Abstract
The invention discloses a method for simultaneously determining 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione and related substances thereof. According to the method, the 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione and the related substances thereof are separated through high performance liquid chromatography, wherein a chromatographic column is bonded by octadecyl silica gel; a mobile phase is a methanol:acetic acid aqueous solution. The flow velocity in the high performance liquid chromatography is 0.5 to 1.2ml/min. The detection wavelength in the high performance liquid chromatography is 226nm. The method is simple, convenient and practical, can be used for quickly achieving the aims of simultaneously determining and separating the 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione and the related substances thereof, can be applied to determination of molecular weights of unknown impurities through liquid chromatography-mass spectrometry (LC-MS), provides a reliable basis for structure inference of the unknown impurities, and facilitates quality control of raw materials and preparations of the 4-amino-2-(2,6-dioxo-3-piperidyl)isoindoline-1,3-dione.
Description
Technical field
The present invention relates to a kind of separation, measure the method for pool horse degree amine and related substance thereof, especially relate to the method for a kind of Simultaneously test pool horse degree amine, synthesis initiation material, intermediate, accessory substance, degradation product and other unknown impurities.
Background technology
Pool horse degree amination formal name used at school is called 4-amino-2-(2,6-dioxo-piperidin-3-base)-isoindoline-1,3-diketone, being a kind of thalidomide analogs, is a kind of immunomodulator having antitumor activity.In vitro in cytologic experiments, can suppress hematopoietic tumor cell breed and apoptosis-induced.In addition, moor horse degree amine can suppress lenalidomide resistance multiple myeloma cell line propagation and at lenalidomide-sensitivity and lenalidomide-drug-resistant cell strain and dexamethasone co-induction apoptosis of tumor cells.Pool horse degree amine strengthens T cell-immunity cell-mediated with NK cell (NK) and suppresses the generation (e.g., TNF-α and IL-6) of monocyte pro-inflammatory cytokine.Mouse tumor model and and external umbilical cord model in moor horse degree amine display anti-angiogenesis activity, this medicine is at Clinical practice at present.
Structural formula is:
With 3-nitrophthalic acid and 3-amino-2,6 in this product synthesis technique ,-piperidine dione is as raw material, and intermediate is 3-aminophthalic acid, may produce the accessory substance of 4 nitros or 5 bit aminos in technological process, and the impurity of the unknown.The conventional high-efficient liquid phase method of current detection related substance is adopted to detect these impurity difficulty larger, be difficult to be separated above known impurities completely in existing known method in Same Way, so that there is certain difficulty, for safe medication brings relatively large hidden danger in the quality control of this product.
Summary of the invention
The object of this invention is to provide the method for a kind of Simultaneously test pool horse degree amine and related substance thereof.Through repeatedly optimizing, with efficient liquid-phase chromatography method, reaching and being separated object simultaneously, and the mobile phase of the high-efficient liquid phase method of Mass Spectrometer Method can be applicable to, the convenient structure being determined known and unknown related substance by mass spectrophotometry.
Technical scheme of the present invention is:
A method for Simultaneously test pool horse degree amine and related substance thereof, comprises the following steps:
(1) preparation system applicability solution:
Get pool horse degree amine and accessory substance 5-amino-2-(2,6-dioxo-piperidin-3-base)-isoindoline-1,3-diketone Working Control product, accurately weighed, add acetonitrile and make dissolving in right amount, add mobile phase A again: pH value be 2.6 ~ 3.0 acetic acid make the solution that every 1ml be 0.01mg containing pool horse degree amine, accessory substance, the final volume of described acetonitrile and mobile phase A acetic acid is than being 20:80;
(2) preparation pool horse degree amine need testing solution:
Get pool horse degree amine, accurately weighed, add acetonitrile and make dissolving in right amount, add mobile phase A: pH value be 2.6 ~ 3.0 acetic acid to make every 1ml containing pool horse degree amine be the solution of 0.125mg, the final volume of described acetonitrile and mobile phase A acetic acid is than being 20:80;
(3) determination of related substances is carried out by following high-efficient liquid phase chromatogram condition:
A. taking octadecylsilane chemically bonded silica as filling agent, take pH value as the acetum of 2.6 ~ 3.0 is mobile phase A, take methyl alcohol as Mobile phase B, gradient elution, and column temperature is 20 ~ 35 DEG C, and flow velocity is 0.5 ~ 1.2ml/min, and determined wavelength is 226nm;
Described condition of gradient elution is:
B. precision draws reference substance solution and need testing solution respectively, injection liquid chromatography, measures pool horse degree amine and related substance thereof.
Preferably, the pH of acetum described in step 3 is 2.6.
Preferably, described a. is with octadecylsilane chemically bonded silica for 150 × 4.0mm, and 5 μm of filling agents are mobile phase A with acetum, take first as Mobile phase B, gradient elution, and column temperature is 30 DEG C, and flow velocity is 1ml/min, and determined wavelength is 226nm.
Advantage of the present invention is by the high performance liquid chromatography set up, optimize, Simultaneously test pool horse degree amine, synthesis initiation material, intermediate, accessory substance, degradation product and other unknown impurities and related substance thereof, provide a kind of stability, reproducible assay method, the molecular weight that LC-MS (LC-MS) determines unknown impuritie can be applied to, for inferring that the structure of unknown impuritie provides reliable basis, for the quality control of pool horse degree amine raw material and preparation provides reliable basis, this new drug Quality Control can be convenient to.
By above analytical approach, can effectively pool horse degree amine and related substance thereof be separated, degree of separation meets the requirement of chromatographic resolution all far away, and not saliferous in mobile phase, noiseless to Mass Spectrometer Method, can be used for Mass Spectra, determine the molecular weight of each impurity peaks, for inferring that unknown impuritie is for reliable foundation.
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, makes various replacement and change according to ordinary skill knowledge and customary means, include within the scope of the invention.
Accompanying drawing explanation
Fig. 1 is that horse degree amine on the berth adds related impurities chromatographic fractionation figure (embodiment 7).
In figure, the chromatographic peak retention time of known raw material, intermediate or accessory substance sees the following form:
Embodiment
Instrument and reagent in following examples, be common commercially available except specified otherwise.
Instrument: Agilent1260series high performance liquid chromatograph, chromatographic grade acetonitrile, methyl alcohol (Merck company), glacial acetic acid (analyzing pure).
Chromatographic column: Agilent ZORBAX SB-C185 μm of 4.6 × 150mm
Reagent: raw material 1:3-amino-2,6,-piperidine dione (market purchasing), intermediate: 3-aminophthalic acid (market purchasing), raw material 2:3-nitrophthalic acid (market purchasing), accessory substance 1:4 – nitro-2-(2,6-dioxo-piperidin-3-base)-isoindoline-1,3-diketone (self-control), accessory substance 2:5-amino-2-(2,6-dioxo-piperidin-3-base)-isoindoline-1,3-diketone (self-control), pool horse degree amine raw material (Shenzhen Haiwang Pharmaceutical Co., Ltd provides, lot number 20130401,20130402,20130403).
The preparation of embodiment 1 system suitability solution
Get pool horse degree amine and accessory substance 2:5-amino-2-(2,6-dioxo-piperidin-3-base)-isoindoline-1,3-diketone; In right amount, make dissolving in right amount with acetonitrile, add mobile phase A pH value be 2.6 ~ 3.0 acetic acid to be diluted to containing pool horse degree amine and accessory substance 2 be all the solution of 10 μ g/ml.Acetonitrile and mobile phase A pH value are that the final volume of 2.6 ~ 3.0 acetic acid is than being 20:80.Accessory substance 2 is the accessory substances closest to pool horse degree amine, has been separated with accessory substance 2, other impurity and separable.
The screening of embodiment 2 chromatographic condition and system suitability
The selection of 2.1 mobile phases
Measure related request according to mass detector, successively select the separating effect following (see table 1) under different mobile phase condition; Acetum is with vinegar acid for adjusting pH in water.
Table 1. mobile phase selection result
| Mobile phase | Separating effect |
| Methanol-acetic acid solution (pH2.2) (30:70) | Degree of separation is 4.3 |
| Methanol-acetic acid solution (pH2.6) (30:70) | Degree of separation is 8.5 |
| Methanol-acetic acid solution (pH3.0) (30:70) | Degree of separation is 6.1 |
| Methanol-acetic acid solution (pH2.6) (gradient 1) | Degree of separation is 11.2 |
| Methanol-acetic acid solution (pH2.6) (gradient 2) | Degree of separation is 9.2 |
The degree of separation result (see table 1) at horse degree amine peak and accessory substance 2 peak is moored in system suitability solution measured by different mobile phase, show the good separating effect of gradient elution than isocratic elution, the degree of separation adopting the gradient elution program of this law (shown in table 4) to obtain increases more obvious.
The selection of 2.2 chromatographic columns
The chromatographic column of different brands on probation, different model, finally determines to adopt Agilent ZORBAXSB-C18(150 × 4.6mm, 5 μm), chromatographic column selection result is in table 2.
Table 2. chromatographic column selection result
Adopt the gradient elution program shown in table 4, different brands C18 chromatographic column carries out comparison test, result shows (see table 2), use Agilent ZORBAX SB-C18(150 × 4.6mm, 5 μm) time, the post effect of pool horse degree amine main peak is higher, and in system suitability solution, the degree of separation of two-component is better.
Finally determining with acetum to be mobile phase A, take methyl alcohol as Mobile phase B, and the optimal pH of acetum is about 2.6, carries out gradient elution according to following table 4.
The selection of 2.3 column temperatures
Select column temperature to be 25 DEG C, 30 DEG C and 35 DEG C to investigate, finally determine that column temperature is 30 DEG C, column temperature selection result is in table 3.
Table 3. column temperature selection result
Adopt the gradient elution program shown in table 4, use Agilent ZORBAX SB-C18(150 × 4.6mm, 5 μm) chromatographic column, test under different column temperature, result shows (see table 3), and when column temperature is 30 DEG C, degree of separation is better, and main peak post effect is higher.
The determination of chromatographic condition: through above-mentioned Selection experiment, the chromatographic condition determined is: with octadecylsilane chemically bonded silica (150 × 4.0mm, 5 μm) be filling agent, take acetum as mobile phase A, take methyl alcohol as Mobile phase B, gradient elution, column temperature is 30 DEG C, flow velocity is 1ml/min, and determined wavelength is 226nm; Described condition of gradient elution is in table 4.
2.4 system suitabilities:
Chromatographic condition and system suitability: with octadecylsilane chemically bonded silica (Agilent ZORBAXSB-C18150 × 4.6mm, 5 μm) be filling agent, take acetum as mobile phase A, take methyl alcohol as Mobile phase B, gradient elution, column temperature is 30 DEG C, and flow velocity is 1ml/min, determined wavelength is 226nm, carries out gradient 1 wash-out according to following table 4.
Table 4. gradient 1 mobile phase elution program
The preparation of embodiment 3 detectability solution
Detectability solution: precision measures need testing solution 0.11ml, puts in 100ml measuring bottle, adds mobile phase A-Mobile phase B (80:20(v/v)) be diluted to scale, shake up, to obtain final product.
System suitability solution: get pool horse degree amine and accessory substance 2 appropriate, make dissolving in right amount with acetonitrile, adds Mobile phase B and is diluted to solution containing mooring horse degree amine and accessory substance 2 and be respectively 10 μ g/ml.
Embodiment 4 detectability is tested
Accurate detectability solution 20ul described in extraction embodiment 3, injection liquid chromatography, record chromatogram, calculates main peak signal to noise ratio (S/N ratio) (S/N), 3<(S/N) <5, shows that this law measures the detectability concentration of mooring horse degree amine and is about 0.12 μ g/ml.
The preparation of embodiment 5 specificity solution
Need testing solution: precision takes pool horse degree amine 12.5mg, puts in 100ml measuring bottle, adds acetonitrile 20ml and make dissolving, add Mobile phase B and be diluted to scale, shake up, to obtain final product.
Specificity solution: get pool horse degree amine need testing solution, add 5 known impurities in right amount: raw material 1:3-amino-2,6,-piperidine dione, intermediate: 3-aminophthalic acid, raw material 2:3-nitrophthalic acid, accessory substance 1:4 – nitro-2-(2,6-dioxo-piperidin-3-base)-isoindoline-1,3-diketone, accessory substance 2:5-amino-2-(2,6-dioxo-piperidin-3-base)-isoindoline-1,3-diketone.
Embodiment 6 specificity is tested
Accurate specificity solution 20ul described in extraction embodiment 5, injection liquid chromatography, record chromatogram, the degree of separation of pool horse degree amine main peak and 5 known impurities is all greater than 10, degree of separation between 5 known impurities is all greater than 5, shows that this law measures the specificity of mooring horse degree amine and 5 known impurities better.
Embodiment 7 determination of related substances is tested
Accurate need testing solution 20ul described in extraction embodiment 5, injection liquid chromatography, record chromatogram.All do not detect 5 known impurities in the pool horse degree amine of three lot numbers (Shenzhen Haiwang Pharmaceutical Co., Ltd provides, lot number 20130401,20130402,20130403), unknown impuritie total content is respectively 0.03%, 0.04% and 0.02%.
The contrast of embodiment 8 and existing known method
Existing known method chromatographic condition:
Chromatographic column: ACE C18 post 150 × 4.6mm, 3 μm
Mobile phase A: methyl alcohol
Mobile phase B: 25mM ammonium acetate (add formic acid and adjust pH=5.5)
Both press gradient elution, as following table by volume:
Table 5. existing known method mobile phase elution program
Flow velocity: 0.7ml/min,
Column temperature: 30 DEG C.
By above-mentioned existing method chromatographic condition, the specificity solution 20ul described in accurate extraction embodiment 5, injection liquid chromatography, record chromatogram, result compares in table 6 with this law
The existing known method of table 6. compares with this law
Claims (5)
1. measure a method for pool horse degree amine, comprise the following steps:
(1) preparation system applicability solution;
(2) preparation pool horse degree amine need testing solution;
(3) measure by following high-efficient liquid phase chromatogram condition: a. chromatographic column take octadecylsilane chemically bonded silica as filling agent, take pH value as the acetum of 2.6 ~ 3.0 be mobile phase A, take methyl alcohol as Mobile phase B, gradient elution, column temperature is 20 ~ 35 DEG C, flow velocity is 0.5 ~ 1.2ml/min, and determined wavelength is 226nm;
Described condition of gradient elution is:
B. accurate absorption need testing solution, injection liquid chromatography, measures pool horse degree amine.
2. the method measuring pool horse degree amine as claimed in claim 1, it is characterized in that: described step (1) preparation system applicability solution: get pool horse degree amine and accessory substance 5-amino-2-(2,6-dioxo-piperidin-3-base)-isoindoline-1,3-diketone Working Control product, accurately weighed, add acetonitrile and make dissolving in right amount, add mobile phase A again: pH value be 2.6 ~ 3.0 acetic acid make the solution that every 1ml be 0.01mg containing pool horse degree amine, accessory substance, the final volume of described acetonitrile and mobile phase A acetic acid is than being 20:80.
3. the method measuring pool horse degree amine as claimed in claim 1, it is characterized in that: described step (2) preparation pool horse degree amine need testing solution: get pool horse degree amine, accurately weighed, add acetonitrile and make dissolving in right amount, add mobile phase A: pH value be 2.6 ~ 3.0 acetic acid to make every 1ml containing pool horse degree amine be the solution of 0.125mg, the final volume of described acetonitrile and mobile phase A acetic acid is than being 20:80.
4. the method for the mensuration pool horse degree amine as described in as arbitrary in claims 1 to 3, is characterized in that: the pH of step (3) described acetum is 2.6.
5. the method for the mensuration pool horse degree amine as described in as arbitrary in claims 1 to 3, it is characterized in that: described a. chromatographic column take octadecylsilane chemically bonded silica as filling agent, 150 × 4.0mm, 5 μm, being mobile phase A with acetum, take methyl alcohol as Mobile phase B, gradient elution, column temperature is 30 DEG C, and flow velocity is 1ml/min, and determined wavelength is 226nm.
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