CN104297361A - Detection method for tetraacetylribose in azacitidine raw material - Google Patents
Detection method for tetraacetylribose in azacitidine raw material Download PDFInfo
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- CN104297361A CN104297361A CN201410443487.0A CN201410443487A CN104297361A CN 104297361 A CN104297361 A CN 104297361A CN 201410443487 A CN201410443487 A CN 201410443487A CN 104297361 A CN104297361 A CN 104297361A
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Abstract
The invention discloses an impurity detection method, in particular to a detection method for tetraacetylribose in azacitidine. The method adopts high performance liquid chromatography, a octadecyl silane bonded silica gel chromatographic column is selected, and water-methanol is employed as the mobile phase to conduct isocratic elution, the flow rate is 0.8-1.2mL/min, the column temperature is 25-35DEG C, and the detection wavelength of the sample is 205nm-215nm. The mobile phase is employed to perform isocratic elution, wherein the volume percentage of methanol is 30-45%. The detection method has the advantages of high sensitivity, high accuracy, good impurity peak shape symmetry, and high column efficiency, etc.
Description
Technical field
The present invention relates to a kind of detection method of impurity, particularly the detection method of 1,2,3,5-Tetra-O-Acetyl-D-Ribose in a kind of azacitidine.
Background technology
Azacitidine, English name Azacitidine, chemistry 1-(β-D-RIBOSE base)-4-amino-1,3,5-triazines-2 (1H)-one by name, have another name called 5-azacitidine, its structural formula is as follows:
Azacitidine is cytosine nucleoside medicine.Can directly mix in DNA, suppress DNA and RNA synthesis, the cell being in the S phase can be killed and wounded.U.S. FDA to go on the market this medicine early than ratifying Celgene corp in May, 2004, and commodity are called: Vidaza, are mainly used in treating myelodysplastic syndrome, acute nonlymphocytic leukemia.
1,2,3,5-Tetra-O-Acetyl-D-Ribose is one of raw material that synthesis azacitidine is conventional, may there is 1,2,3,5-Tetra-O-Acetyl-D-Ribose impurity in the azacitidine of synthesis.Defects inspecting is one of important indicator controlling drug quality.The impact of pharmaceutical purity on patient is very large.Injectable drug is impure is the main cause causing adverse drug reaction (as allergy, drug fever etc.).Oral drugs are impure, may increase the incidence of bad reaction, as Nausea and vomiting, stomachache, diarrhoea etc.Meanwhile, what may affect is the absorption of medicine, thus affects curative effect of medication.Therefore, what the detection method of impurity of the drug just showed is particularly important.
At present, the detection method of impurity in azacitidine raw material described in American Pharmacopeia 36 editions (USP36), adopt high performance liquid chromatography, mobile phase is adopted to be 1.54g/L ammonium acetate solution and acetonitrile, carry out gradient elution, column temperature is 10 DEG C, and elution time is 82min, and determined wavelength is 242nm.The method can not be used for detecting 1,2,3,5-Tetra-O-Acetyl-D-Ribose in azacitidine, even if select the maximum absorption wavelength 210nm of 1,2,3,5-Tetra-O-Acetyl-D-Ribose to be determined wavelength, other condition does not change, and can not detect the 1,2,3,5-Tetra-O-Acetyl-D-Ribose in azacitidine.
Summary of the invention
The object of the invention is to overcome above-mentioned deficiency existing in prior art, provide the detection method of 1,2,3,5-Tetra-O-Acetyl-D-Ribose in a kind of azacitidine raw material, the method can detect 1,2,3,5-Tetra-O-Acetyl-D-Ribose in azacitidine, have highly sensitive, accuracy is high, and impurity peak shape symmetry is good, and post imitates high advantage.
In order to achieve the above object, concrete technical scheme is as follows:
The detection method of 1,2,3,5-Tetra-O-Acetyl-D-Ribose in a kind of azacitidine raw material, adopt high performance liquid chromatography, select octadecylsilane chemically bonded silica chromatographic column, be that mobile phase carries out isocratic elution with water-methanol, flow velocity is 0.8-1.2mL/min, column temperature 25-35 DEG C, the determined wavelength of sample is 205nm to 215nm;
Select column temperature to be 10 DEG C in USP36, temperature is on the low side, causes post to be pressed higher, can reduce the serviceable life of chromatographic column, simultaneously wayward.The column temperature that the present invention selects is 25 DEG C-35 DEG C, temperature near room temperature, and post forces down, and easily controls.1,2,3,5-Tetra-O-Acetyl-D-Ribose has good absorption at 205nm-215nm.
Described mobile phase carries out isocratic elution, and wherein, the percent by volume of methyl alcohol is 30-45%.The mobile phase condition that the present invention selects, elution time is short, and impurity peaks is fully separated with main peak.If only select water as mobile phase, detection time can be very long; If only select methyl alcohol as mobile phase, impurity peaks cannot be separated with main peak.
Preferably, the detection method of 1,2,3,5-Tetra-O-Acetyl-D-Ribose in azacitidine raw material, adopts high performance liquid chromatography, select octadecylsilane chemically bonded silica chromatographic column, be that mobile phase carries out isocratic elution with water-methanol, flow velocity is 1.0mL/min, column temperature 30 DEG C, the determined wavelength of sample is 210nm; The maximum absorption wavelength of 1,2,3,5-Tetra-O-Acetyl-D-Ribose is 210nm, selects maximum absorption wavelength to detect, and is conducive to the accuracy improving testing result.
Preferably, described mobile phase carries out isocratic elution, and wherein, the percent by volume of methyl alcohol is 35%.Wash-out under this condition, 10min just can go out impurity peaks, and impurity peaks and main peak good separating effect.
The granularity of described octadecylsilane chemically bonded silica chromatographic column filler is 5 μm, and specification is 150mm × 4.6mm.
The detection method of 1,2,3,5-Tetra-O-Acetyl-D-Ribose in described azacitidine raw material, elution time is 20min.Method for detecting impurities in USP36, elution time is 82min.Elution time in the present invention only needs 20min.The while of time saving, there is good impurity 1,2,3,5-Tetra-O-Acetyl-D-Ribose Detection results.
Described sample is the azacitidine aqueous solution of 0.5-10mg/mL.
Measure described azacitidine aqueous solution 50 μ L, measure.
Compared with prior art, beneficial effect of the present invention:
(1) detection method in the present invention may be used for accurately detecting impurity 1,2,3,5-Tetra-O-Acetyl-D-Ribose in azacitidine.
(2) mobile phase in the present invention is common solvent, adopts isocratic elution simultaneously, and method is simple to operation.Elution time only needs 20min, and just can obtain good testing result, detection efficiency is high.
(3) adopt the method in the present invention to detect, degree of separation reaches more than 4, and usual degree of separation is greater than 2, and impurity peaks and main peak just can be separated very well, and degree of separation is larger, and effect is better.Tailing factor is close to 1, and peak shape symmetry is good.Theoretical cam curve is greater than 5000, and post effect is high.
Accompanying drawing illustrates:
Fig. 1 is embodiment 1 high-efficient liquid phase chromatogram.
Fig. 2 is embodiment 3 high-efficient liquid phase chromatogram.
Fig. 3 is comparative example 1 high-efficient liquid phase chromatogram.
Embodiment
Below in conjunction with test example and embodiment, the present invention is described in further detail.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment, all technology realized based on content of the present invention all belong to scope of the present invention.
Embodiment 1
A detection method for 1,2,3,5-Tetra-O-Acetyl-D-Ribose in azacitidine raw material, adopt high performance liquid chromatography, select Agilent zorbax SB-C18 chromatographic column, the granularity of filler is 5 μm, and specification is 150mm × 4.6mm.With the percent by volume 70%:30% of water and methyl alcohol for mobile phase carries out isocratic elution, flow velocity is 1.0mL/min, column temperature 25 DEG C, and measure 50 μ L, 5mg/mL azacitidine aqueous solution and detect, determined wavelength is 210nm, and elution time is 20min.Testing result as shown in Figure 1.1,2,3,5-Tetra-O-Acetyl-D-Ribose impurity peaks retention time (R=10.510min), adjacent peak obtains good separation (degree of separation R=4.54) simultaneously, peak theoretical cam curve higher (N=9662), peak type symmetry good (Symm=0.96).It shows that the method accuracy is high, highly sensitive, accurately can detect the content of 1,2,3,5-Tetra-O-Acetyl-D-Ribose impurity in azacitidine raw material.
Embodiment 2
A detection method for 1,2,3,5-Tetra-O-Acetyl-D-Ribose in azacitidine raw material, adopts high performance liquid chromatography, selects Agilent XDB-C
18chromatographic column, the granularity of filler is 5 μm, and specification is 150mm × 4.6mm.With the percent by volume 65%:35% of water and methyl alcohol for mobile phase carries out isocratic elution, flow velocity is 1.2mL/min, column temperature 30 DEG C, and measure 50 μ L, 0.5mg/mL azacitidine aqueous solution and detect, determined wavelength is 210nm, and elution time is 20min.Testing result: 1,2,3,5-Tetra-O-Acetyl-D-Ribose impurity peaks retention time (R=8.286min), adjacent peak obtains good separation, peak theoretical cam curve higher (N=9671), peak type symmetry good (Symm=0.98) simultaneously.It shows that the method accuracy is high, highly sensitive, accurately can detect the content of 1,2,3,5-Tetra-O-Acetyl-D-Ribose impurity in azacitidine raw material.
Embodiment 3
A detection method for 1,2,3,5-Tetra-O-Acetyl-D-Ribose in azacitidine raw material, adopt high performance liquid chromatography, select Agilent zorbax SB-C18 chromatographic column, the granularity of filler is 5 μm, and specification is 150mm × 4.6mm.With the percent by volume 55%:45% of water and methyl alcohol for mobile phase carries out isocratic elution, flow velocity is 0.8mL/min, column temperature 35 DEG C, and measure 50 μ L, 10mg/mL azacitidine aqueous solution and detect, determined wavelength is 215nm, and elution time is 20min.Testing result as shown in Figure 2.1,2,3,5-Tetra-O-Acetyl-D-Ribose impurity peaks retention time (R=4.540min), adjacent peak obtains good separation, peak theoretical cam curve higher (N=6417), peak type symmetry good (Symm=0.96) simultaneously.It shows that the method accuracy is high, highly sensitive, accurately can detect the content of 1,2,3,5-Tetra-O-Acetyl-D-Ribose impurity in azacitidine raw material.
Comparative example 1
The detection method of impurity in azacitidine raw material described in American Pharmacopeia 36 editions, adopt high performance liquid chromatography, select Zorbax Bonus RP brand of L60 chromatographic column, the granularity of filler is 5 μm, and specification is 250mm × 4.6mm.With the ammonium acetate of 1.54g/L water-soluble with acetonitrile for mobile phase carries out gradient elution, the gradient elution volume time is shown in Table 1.Flow velocity is 1.0mL/min, column temperature 10 DEG C, and measure 5 μ L, 0.8mg/mL azacitidine aqueous solution and detect, determined wavelength is 242nm, and elution time is 82min.We change determined wavelength into 210nm, and testing result as shown in Figure 3.As can be seen here, even if select 1,2,3,5-Tetra-O-Acetyl-D-Ribose absorption maximum as determined wavelength, the method also cannot detect 1,2,3,5-Tetra-O-Acetyl-D-Ribose.
Table 1 eluent gradient elution volume timetable
| Time (min) | The ammonium acetate solution (%) of 1.54g/L | Acetonitrile (%) |
| 0 | 100 | 0 |
| 10 | 100 | 0 |
| 26 | 93 | 7 |
| 37 | 85 | 15 |
| 51 | 73 | 27 |
| 61 | 20 | 80 |
| 71 | 20 | 80 |
| 72 | 100 | 0 |
| 82 | 100 | 0 |
Claims (7)
1. the detection method of 1,2,3,5-Tetra-O-Acetyl-D-Ribose in an azacitidine raw material, adopt high performance liquid chromatography, it is characterized in that, select octadecylsilane chemically bonded silica chromatographic column, be that mobile phase carries out isocratic elution with water-methanol, flow velocity is 0.8-1.2mL/min, column temperature 25-35 DEG C, and the determined wavelength of sample is 205nm to 215nm;
Described mobile phase carries out isocratic elution, and wherein, the percent by volume of methyl alcohol is 30-45%.
2. detection method according to claim 1, is characterized in that, selects octadecylsilane chemically bonded silica chromatographic column, and be that mobile phase carries out isocratic elution with water-methanol, flow velocity is 1.0mL/min, column temperature 30 DEG C, and the determined wavelength of sample is 210nm;
Described mobile phase carries out isocratic elution, and wherein, the percent by volume of methyl alcohol is 35%.
3. detection method according to claim 1 and 2, is characterized in that, the granularity of described octadecylsilane chemically bonded silica chromatographic column filler is 5 μm, and specification is 150mm × 4.6mm.
4. detection method according to claim 1 and 2, is characterized in that, elution time is 20 min.
5. detection method according to claim 1 and 2, is characterized in that, described sample is the azacitidine aqueous solution of 0.5-100mg/mL.
6. detection method according to claim 1 and 2, is characterized in that, described sample is the azacitidine aqueous solution of 5mg/mL.
7. detection method according to claim 5, is characterized in that, measures described azacitidine aqueous solution 50 μ L, measures.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111650301A (en) * | 2020-06-17 | 2020-09-11 | 普研（上海）标准技术服务股份有限公司 | Method for determining and analyzing azacitidine content and application |
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| WO2010017374A1 (en) * | 2008-08-06 | 2010-02-11 | Sicor Inc. | Process for preparing azacytidine intermediate |
| CN101974051A (en) * | 2010-10-08 | 2011-02-16 | 重庆泰濠制药有限公司 | Method for synthesizing azacitidine |
| CN103524584A (en) * | 2012-07-05 | 2014-01-22 | 天津九海医药科技有限公司 | Synthetic method of azacitidine |
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Patent Citations (3)
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| WO2010017374A1 (en) * | 2008-08-06 | 2010-02-11 | Sicor Inc. | Process for preparing azacytidine intermediate |
| CN101974051A (en) * | 2010-10-08 | 2011-02-16 | 重庆泰濠制药有限公司 | Method for synthesizing azacitidine |
| CN103524584A (en) * | 2012-07-05 | 2014-01-22 | 天津九海医药科技有限公司 | Synthetic method of azacitidine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111650301A (en) * | 2020-06-17 | 2020-09-11 | 普研（上海）标准技术服务股份有限公司 | Method for determining and analyzing azacitidine content and application |
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