CN103728393A - Method for separating and measuring deferasirox raw material related substances by liquid chromatography - Google Patents

Method for separating and measuring deferasirox raw material related substances by liquid chromatography Download PDF

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Publication number
CN103728393A
CN103728393A CN201410011654.4A CN201410011654A CN103728393A CN 103728393 A CN103728393 A CN 103728393A CN 201410011654 A CN201410011654 A CN 201410011654A CN 103728393 A CN103728393 A CN 103728393A
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China
Prior art keywords
deferasirox
separating
inorganic salts
assaying
buffer solution
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CN201410011654.4A
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Chinese (zh)
Inventor
罗祖良
方灿良
刘秋叶
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WUXI WANQUAN MEDICAL TECHNOLOGY Co Ltd
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WUXI WANQUAN MEDICAL TECHNOLOGY Co Ltd
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Abstract

The invention belongs to the field of analytical chemistry, and discloses a method for separating and measuring deferasirox related substances by liquid chromatography. By using a chromatographic column in which octadecyl silane bonded silica gel is used as a filler and using inorganic acid salt buffer solution-organic phase in a certain ratio as a mobile phase, the method can be used for measuring the content of deferasirox and related substances thereof, thereby effectively controlling the quality of the deferasirox. The method has the advantages of high specificity and high accuracy, and is simple to operate.

Description

A kind of method with liquid chromatography for separating and determining DEFERASIROX raw material related substance
Technical field
The invention belongs to analytical chemistry field, be specifically related to the method for liquid chromatography for separating and determining DEFERASIROX and related substance thereof.
Background technology
DEFERASIROX is used for the treatment of the age and is greater than the β-patients with thalassemia of 6 years old because of chronic iron overload due to frequent blood transfusion (the monthly administered dose >=7mL/kg of Red Blood Cells Concentrate).DEFERASIROX chemistry 4-[3 by name, 5-Bia (2-hydroxyphenyl)-1H-1,2.4-triazollyl]-benzoic acid molecular formula is C 21h 15n 3o 4.DEFERASIROX structural formula is:
Figure 2014100116544100002DEST_PATH_IMAGE002
In the process of synthetic this compound, there is the important intermediate of several steps not exclusively to affect purity and the quality of medicine due to removal, these intermediates are the related substance in Control of drug quality, related substance for the synthetic major control of DEFERASIROX has 4, respectively intermediate 1 2-Hydroxy-benzoic acid, intermediate 2 2-Hydroxy-benzamide, intermediate 3 2-(2-Hydroxy-phenyl)-benzo[e] [1,3] oxazin-4-one, intermediate 4 4-Hydrazino-benzoic acid structural formulas are respectively:
Figure 2014100116544100002DEST_PATH_IMAGE004
Figure 2014100116544100002DEST_PATH_IMAGE006
Figure 2014100116544100002DEST_PATH_IMAGE008
Figure 2014100116544100002DEST_PATH_IMAGE010
Intermediate 1 intermediate 2 intermediate 3 intermediates 4
For the related substance of introducing in synthetic DEFERASIROX, no matter be all, need to carry out quality control in bulk drug or preparation, therefore, realize the separation of DEFERASIROX and related substance thereof, aspect the quality control of the synthetic and preparation process of DEFERASIROX, having important practical significance.
Summary of the invention
The object of the present invention is to provide a kind of method of purity of analyzing DEFERASIROX and separated its related substance, thereby realize DEFERASIROX and the separated of its related substance and measure, guarantee DEFERASIROX and containing the quality control of DEFERASIROX preparation.
The method of the purity with liquid chromatography analysis DEFERASIROX of the present invention and separated its related substance, is to adopt the chromatographic column that octadecylsilane chemically bonded silica is filler, and a certain proportion of inorganic salts buffer solution-organic phase of take is mobile phase.
Above-mentioned said chromatographic column be take octadecylsilane chemically bonded silica as filler, is selected from Kromasil-C 18or Ultimate – C 18.
Above-mentioned said organic phase is selected from following compound: methyl alcohol, acetonitrile, propyl alcohol, isopropyl alcohol, tetrahydrofuran etc., be preferably acetonitrile.
Above-mentioned said method, its mobile phase inorganic salts buffer solution-organic phase adopts gradient elution.
In above-mentioned said method, the inorganic salts that comprise in inorganic salts buffer solution are selected from phosphate, carbonate, citrate, are preferably phosphate.
The concentration of the inorganic salts that wherein comprise in inorganic salts buffer solution is 0.01～0.1mol/L, and preferred concentration is 0.02mol/L.
Method of separating and assaying of the present invention, can realize in accordance with the following methods:
1) get DEFERASIROX or appropriate containing the formulation samples of DEFERASIROX, by methyl alcohol or mobile phase sample dissolution, be mixed with every 1mL containing the sample solution of DEFERASIROX 0.1～1.5mg.
2) flow rate of mobile phase being set is 0.5～1.5mL/min, and flow rate of mobile phase is preferably 1.0mL/min, and detection wavelength is 200～250nm, and optimum detection wavelength is 210nm, and column oven temperature is 10～40 ℃, and column oven temperature the best is 30 ℃.
3) get 1) sample solution 10～50 μ L, injection liquid chromatography, completes the separation determination of DEFERASIROX and related substance.Wherein:
The model of high performance liquid chromatograph, has no special requirements, and the chromatograph that the present invention adopts is Shimadzu: LC-20AT pump, SPD-M20A detecting device, SIL-20AC automatic sampler, CBM-20A controller, CTO-10AS column oven, LC solution workstation
Chromatographic column: C 18(Kromasil, 250 * 4.6 mm, 5 μ m)
Mobile phase: A: phosphate sodium dihydrogen buffer solution (takes 3.12gKH 2pO 4, add water 1000ml and make to dissolve, dilute phosphoric acid solution regulates pH to 3.0); B: acetonitrile; Adopt gradient elution
Flow velocity: 1.0mL/min
Detect wavelength: 210nm
Sampling volume: 10 μ L
The present invention adopts C 18(Kromasil, 250 * 4.6 mm, 5 μ m), can effective separated DEFERASIROX and related substance thereof.The invention solves the separation determination problem of DEFERASIROX and related substance thereof, thereby guaranteed the quality controllable of DEFERASIROX and preparation thereof.
Accompanying drawing explanation
DEFERASIROX when Fig. 1 is embodiment 1 and related substance HPLC figure thereof;
DEFERASIROX HPLC figure when Fig. 2 is embodiment 1;
DEFERASIROX when Fig. 3 is embodiment 2 and related substance HPLC figure thereof;
The HPLC figure of DEFERASIROX when Fig. 4 is embodiment 2;
Solvent HPLC figure when Fig. 5 is embodiment 3;
DEFERASIROX when Fig. 6 is embodiment 3 and related substance HPLC figure thereof;
The HPLC figure of DEFERASIROX when Fig. 7 is embodiment 3.
embodiment:
Following examples are used for further understanding the present invention, but are not limited to the scope of this enforcement.
embodiment 1
Instrument and condition
High performance liquid chromatograph: Shimadzu: LC-20AT, CBM-20A, SIL-20AC, SPD-M20A, CTO-10ASvp;
Chromatographic column: C 18(Kromasil, 250 * 4.6 mm, 5 μ m);
Mobile phase: A: phosphate sodium dihydrogen buffer solution (takes 3.12gKH 2pO 4, add water 1000ml and make to dissolve, sodium hydroxide solution regulates pH to 6.0); B: acetonitrile; By carrying out wash-out with Gradient
T(min) 0 5 10 12 30 35 40
B% 15 15 40 50 50 15 15
Flow velocity: 1.0mL/min
Detect wavelength: 210nm
Sampling volume: 10 μ L
Experimental procedure
Get DEFERASIROX and intermediate is appropriate, use respectively acetonitrile sample dissolution, be mixed with the sample solution containing DEFERASIROX and the about 0.25mg/mL of intermediate thereof.By above-mentioned condition, carry out efficient liquid phase chromatographic analysis, record chromatogram.The results are shown in accompanying drawing 1 ~ 2, the chromatographic peak that in Fig. 1, retention time is 16.784min is DEFERASIROX, and all the other chromatographic peaks are the chromatographic peak of each related substance of DEFERASIROX; The chromatographic peak that in Fig. 2, retention time is 16.763min is DEFERASIROX.
embodiment 2
Instrument and condition
High performance liquid chromatograph: Shimadzu: LC-20AT, CBM-20A, SIL-20AC, SPD-M20A, CTO-10ASvp;
Chromatographic column: C 18(Kromasil, 250 * 4.6 mm, 5 μ m);
Mobile phase: A: phosphate sodium dihydrogen buffer solution (takes 3.12gKH 2pO 4, add water 1000ml and make to dissolve, sodium hydroxide solution regulates pH to 6.0); B: acetonitrile; By carrying out wash-out with Gradient
T(min) 0 5 10 12 30 35 40
B% 25 25 40 50 50 25 25
Flow velocity: 1.0mL/min
Detect wavelength: 210nm
Sampling volume: 10 μ L
Experimental procedure
Get DEFERASIROX and intermediate is appropriate, use respectively acetonitrile sample dissolution, be mixed with the sample solution containing DEFERASIROX and the about 0.25mg/mL of intermediate thereof.By above-mentioned condition, carry out efficient liquid phase chromatographic analysis, record chromatogram.The results are shown in accompanying drawing 3 ~ 4, the chromatographic peak that in Fig. 3, retention time is 17.110min is DEFERASIROX, and all the other chromatographic peaks are the chromatographic peak of each related substance of DEFERASIROX; The chromatographic peak that in Fig. 4, retention time is 17.102min is DEFERASIROX.
embodiment 3
Instrument and condition
High performance liquid chromatograph: Shimadzu: LC-20AT, CBM-20A, SIL-20AC, SPD-M20A, CTO-10ASvp;
Chromatographic column: C 18(Kromasil, 250 * 4.6 mm, 5 μ m);
Mobile phase A: phosphate sodium dihydrogen buffer solution (takes 3.12gKH 2pO 4, add water 1000ml and make to dissolve, dilute phosphoric acid solution regulates pH to 3.0); B: acetonitrile; By carrying out wash-out with Gradient
T(min) 0 5 10 12 30 35 40
B% 25 25 40 50 50 25 25
Flow velocity: 1.0mL/min
Detect wavelength: 210nm
Sampling volume: 10 μ L
Experimental procedure
Get DEFERASIROX and intermediate is appropriate, use respectively acetonitrile sample dissolution, be mixed with the sample solution containing DEFERASIROX and the about 0.25mg/mL of intermediate thereof; Separately get acetonitrile in right amount as blank solvent.By above-mentioned condition, carry out efficient liquid phase chromatographic analysis, record chromatogram.The results are shown in accompanying drawing 5 ~ 7, Fig. 5 is solvent chromatogram; The chromatographic peak that in Fig. 6, retention time is 29.725min is DEFERASIROX, and all the other chromatographic peaks are the chromatographic peak of each related substance of DEFERASIROX, and as seen from the figure, DEFERASIROX and its related substance can reach baseline separation, meet the requirement of Chinese Pharmacopoeia; The chromatographic peak that in Fig. 7, retention time is 29.689min is DEFERASIROX, can find out that DEFERASIROX can be completely separated with its related substance under this condition.
From Fig. 1-Fig. 7, show: method of the present invention, can be clearly that DEFERASIROX is separated with its intermediate, and can accurately detect quantitatively, to calculate the content of DEFERASIROX, thereby effectively control the product quality of DEFERASIROX.

Claims (10)

1. a method for liquid chromatography for separating and determining DEFERASIROX related substance, is characterized in that: the chromatographic column that octadecylsilane chemically bonded silica is filler, a certain proportion of inorganic salts buffer solution-organic phase of take is mobile phase.
2. method of separating and assaying according to claim 1, chromatographic column is selected from the chromatographic column that brand is Ultimate and Kromasil.
3. according to the method for separating and assaying described in claim 1, said organic phase is selected from a kind of in following compound: methyl alcohol, acetonitrile, propyl alcohol, isopropyl alcohol, tetrahydrofuran.
4. according to the method for separating and assaying described in claim 3, said organic phase is acetonitrile.
5. method of separating and assaying according to claim 1, in said inorganic salts buffer solution, inorganic salts are selected from following inorganic salts: phosphate, carbonate, citrate.
6. method of separating and assaying according to claim 1, in said inorganic salts buffer solution, the concentration of contained inorganic salts optimum is 0.02mol/L, the pH value of inorganic salts buffer solution is 3.0.
7. according to the method for separating and assaying described in claim 5, inorganic salts preferably phosphate in said inorganic salts buffer solution.
8. according to the method for separating and assaying described in claim 1, it is characterized in that, comprise following step:
1) get DEFERASIROX or appropriate containing the formulation samples of DEFERASIROX, respectively by methyl alcohol or mobile phase sample dissolution, be mixed with every 1mL containing the sample solution of DEFERASIROX and intermediate 0.1～1.5mg thereof;
2) flow rate of mobile phase being set is 0.5～1.5mL/min, and detection wavelength is 200～250nm, and column oven temperature is 10～40 ℃;
3) get 1) sample solution 10～50 μ L, injection liquid chromatography, completes the separation determination of DEFERASIROX and related substance thereof.
9. Analyze & separate method according to claim 7, inorganic salts preferably phosphoric acid sodium dihydrogen.
10. Analyze & separate method according to claim 8, step 2) said flow rate of mobile phase is preferably 1.0mL/min, and said detection wavelength is 210 nm.
CN201410011654.4A 2014-01-10 2014-01-10 Method for separating and measuring deferasirox raw material related substances by liquid chromatography Pending CN103728393A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0017164A1 (en) * 1979-03-30 1980-10-15 Atto Corporation Method for determining catecholic compounds and their related compounds
WO2009147529A1 (en) * 2008-06-02 2009-12-10 Actavis Group Ptc Ehf Substantially pure deferasirox and processes for the preparation thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0017164A1 (en) * 1979-03-30 1980-10-15 Atto Corporation Method for determining catecholic compounds and their related compounds
WO2009147529A1 (en) * 2008-06-02 2009-12-10 Actavis Group Ptc Ehf Substantially pure deferasirox and processes for the preparation thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
N.PADMAJA ET AL.: "METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE DETREMINATION OF DEFERASIROX IN TABLETS", 《INTERNATIONAL JOURNAL OF PHARMACY AND BIOLOGICAL SCIENCES》, vol. 2, no. 4, 31 December 2012 (2012-12-31), pages 338 - 343 *
PRAVISH KUMAR TIWARI ET AL.: "Low Level Determination of Genotoxic Impurity in Deferasirox Formulation", 《JOURNAL OF ANALYTICAL SCIENCES, METHODS AND INSTRUMENTATION》, vol. 3, 30 September 2013 (2013-09-30), pages 179 - 183 *
RAVI KIRAN KAJA ET AL.: "A Stability Indicating LC Method for Deferasirox in Bulk Drugs and Pharmaceutical Dosage Forms", 《CHROMATOGRAPHIA》, vol. 72, 30 September 2010 (2010-09-30) *
SAJI THOMAS ET AL.: "Identification, characterization and quantification of a new impurity in deferasirox active pharmaceutical ingredient by LC–ESI–QT/MS/MS", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》, vol. 63, 28 January 2012 (2012-01-28), pages 112 - 119, XP028466844, DOI: 10.1016/j.jpba.2012.01.024 *

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Application publication date: 20140416