CN115267002A - Method for measuring free hydrazine in fuliginib by GC-MS/MS (gas chromatography-Mass Spectrometry/Mass Spectrometry) combination - Google Patents
Method for measuring free hydrazine in fuliginib by GC-MS/MS (gas chromatography-Mass Spectrometry/Mass Spectrometry) combination Download PDFInfo
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- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract description 18
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention provides a method for detecting free hydrazine in fulguranib by GC-MS/MS combination. By the method, the content of free hydrazine in the drug-containing fulminant can be effectively detected. The method provided by the invention is simple to operate, low in detection limit, high in accuracy and good in repeatability, and can be used for quality control of the Limletinib production. Specifically, free hydrazine is derived from acetone under the conditions of benzoic acid and disodium ethylene diamine tetraacetate to generate diacetone hydrazone, and the diacetone hydrazone is detected by using an EI source of a headspace gas-phase mass spectrometer, so that ions 97 and 112 are quantified.
Description
Technical Field
The invention provides a method for detecting free hydrazine in fulguranib by GC-MS/MS combination, belonging to the field of medical analysis.
Background
Exatritinib is a selective, ATP-noncompetitive MEK inhibitor. Through the combination with MEK, the phosphorylation of downstream ERK1/2 is blocked, the over-activation of an ERK signal path caused by RAS or RAF mutation is inhibited, the proliferation of corresponding tumor cells is inhibited, and the target specific anti-tumor effect is exerted.
Ciclopirox selectively inhibits MEK1 activity with an IC50 of 1.9nM and a weak inhibitory effect on MEK2, with an IC50 of 888nM, with no significant inhibitory activity against other 70 more kinases. Has strong in vitro inhibitory activity on A375 cell ERK phosphorylation (IC 50 is 1.16 nM). Has selective in vitro antiproliferative effect on human tumor cells, especially RAF or RAS mutated human tumor cells, wherein colo-205, A375, HL-60 cells are most sensitive with IC50<1nM. While there was no significant inhibitory activity (IC 50>1000 nM) against normal cells (MRC-5). The in vitro antitumor activity is similar to that of Trametinib and is stronger than that of AZD6244.
Exampanib shows remarkable in-vivo anti-tumor effect on a human tumor nude mouse transplantation model with RAF or RAS mutation such as A375, COLO-205, calu-6, HT29 and the like, and the drug effect is dose-dependent in the range of 1-9 mg/kg. Of these, A375 and COLO-205 were the most sensitive, and some tumor regression was seen at high doses. In A375 nude mouse transplantation tumor model, HL-085 effective dose is 1mg/kg, and corresponding tumor suppressor Tumor (TGI): 60-70%, the inhibition rate of ERK phosphorylation of tumor tissues can reach 73-80%, and the corresponding plasma exposure level is as follows: cmax:309.8ng/ml, AUC (0-t): 605.8ng/mL × h. The tumor inhibition rate of 3mg/kg exceeds 90%, which is equivalent to Trametinib 0.3mg/kg QD and AZD6244 mg/kg BID, and the corresponding tumor tissue ERK phosphorylation inhibition rate can reach the highest: 90-100%, and the single administration inhibition rate of more than 50% can last for more than 12h. The antitumor effect of the product is related to the inhibition of ERK phosphorylation, and p-ERK can be used as a biomarker of the drug effect. In addition, HL-085 showed some in vivo anti-tumor effect on non-RAF or RAS mutated human non-small cell lung cancer cell NCI-H1975, although it had no significant in vitro inhibitory activity (IC 50>1000 nM). In the Calu-6 model, the combination of the product and Docetaxel has certain synergistic effect.
There was some toxic response observed in fultinib at high doses (6 mg/kg, qd or above), including symptoms of skin and gut abnormalities and reduced weight gain in animals, with 9mg/kg approaching the maximum tolerated dose. In the medium and low dose (3 mg/kg, qd and below), no obvious body weight change and other toxic reactions are observed, and the selective antitumor effect is shown.
Ribolitinib is known by the chemical name 4-fluoro-5- (2-fluoro-4-iodoaniline) -N- (2-hydroxyethoxy) benzo [ d ] thiazole-6-carboxamide, having the molecular formula C16H12F2IN3O3S, molecular weight 491.25 and chemical structure as follows:
the anti-tumor drug of the fulguranib can inhibit the proliferation of corresponding tumor cells, thereby exerting the specific anti-tumor effect of targets.
In the technical synthesis of pellitinib, hydrazine compounds are used, which are obvious radical-toxic compounds and must be strictly controlled.
The prior art has the following methods for detecting hydrazine:
a fluorescence probe detection method, chinese patent CN108982447A, discloses a method for detecting hydrazine by a ratio type fluorescence probe, and the method is complex and tedious in operation, long in time consumption and mainly applied to the field of biology.
An ion chromatography detection method, chinese patent CN109682921A, discloses a method for detecting free hydrazine by ion chromatography, which uses a conductivity detector for detection, a chromatographic column Thermo Dionex IonPac CS12A, 250mm x 4mm, a cation inhibitor CERS 4mm, and a mobile phase: 5mmol/l methane sulfonic acid; flow rate: 0.8ml/min-1.3ml/min; column temperature: 28-35 ℃.
An ultraviolet spectrophotometer method, chinese patent CN102478521A, discloses a method for detecting yellow condensate generated by reaction of free hydrazine and p-dimethylaminobenzaldehyde, which has maximum absorption at the wavelength of 456nm and then is detected by the ultraviolet spectrophotometer. The method requires pre-detection derivatization, which increases the difficulty of operation, and requires that the product has insufficient specificity without absorption at 456 nm.
A gas chromatography detection method, and a Chinese patent CN103698459A discloses a method for detecting free hydrazine contained in a medicament by using a headspace gas chromatography (provided with a FID detector).
At present, a headspace gas mass spectrometer (EI source) detection method of free hydrazine in the aequila is not found.
Disclosure of Invention
The invention provides a detection method for measuring free hydrazine in the fuliginib by GC-MS/MS combination aiming at the defects in the prior art. Can be used for the free hydrazine's of radical poison impurity test in the container lulitinib, ensure that the free hydrazine of radical poison impurity accords with the limit requirement, guarantee the quality of container lulitinib.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a detection and analysis method for free hydrazine in fultinib is characterized by comprising the following steps:
1. the utility model provides a survey and appear free hydrazine in litinib with GC-MS/MS allies oneself with detection method, its characterized in that selects quartz capillary chromatographic column, uses inert gas as the helium gas source, and the column adopts program heating mode, reposition of redundant personnel mode, EI detector, between 30 m-60 m of quartz capillary chromatographic column, the column diameter is 0.25 mm-0.53 mm, and the coating thickness is between 0.25 um-3 um, and the condition of program heating is: the initial temperature is 40-60 ℃, the temperature is 5-20 ℃/min, the final temperature is 120-200 ℃, the maintaining time is 2-10 min, the temperature is increased to 180-260 ℃ at 20-40 ℃/min, the maintaining time is 3-10min, and the constant flow rate is 1.0-5.0 mL/min; the headspace temperature is 85-120 ℃, the shaking time is 10-50min, the quantitative ring equilibrium temperature is 80-130 ℃, and the transmission line equilibrium temperature is 105-120 ℃;
2. the mass spectrum conditions are as follows: the ion source is an EI source. The ion source temperature is 200-220 ℃, the ion source interface temperature is 210-230 ℃, the solvent removal time is 5.5min, the mass spectrum acquisition time is 5.5-15min, the acquisition mode SIM, the quantitative ions 97 and the qualitative ions 112. In some embodiments of the invention, the quartz capillary chromatography column has a length of between 30m and 60m, a column diameter of between 0.25mm and 0.53mm, and a coating thickness of between 0.25um and 3 um. Wherein the quartz capillary chromatography column preferably has a length of 30m, a column diameter of 0.32mm, and a coating thickness of 1.8um.
In some embodiments of the invention, the conditions of temperature programming are: the starting temperature is 40 ℃ to 60 ℃, preferably 50 ℃; the heating rate is 5-20 ℃, and the preferential temperature is 15 ℃; the final temperature is 120-200 ℃, preferably 150 ℃; the maintaining time is 2-10 min, preferably 5min; then 20-40 ℃/min, preferably 30 ℃/min, the final temperature is 180-260 ℃, preferably 240 ℃, and the holding time is 3-10min; the constant flow rate is 1.0 to 5.0mL/min, preferably 2.0mL/min. The flow dividing ratio is 5.
In some embodiments of the invention, the headspace temperature is from 85 to 120 ℃, preferably 110 ℃; shaking for 10-50min, preferably 30min; the equilibrium temperature of the quantitative ring is 80-130 ℃, preferably 120 ℃; the equilibrium temperature of the transmission line is 105-120 ℃, and preferably 110 ℃; in some embodiments of the invention, the mass spectrometry ion source is EI, the ion source temperature is 200-220 ℃, preferably 210 ℃, at a voltage of 3.8-45 kV, preferably 4.0kV; the ion source interface temperature is 210-230 ℃, preferably 220 ℃.
Furthermore, the invention provides a GC-MS/MS combined method for detecting free hydrazine in the fulguranib, which comprises the following steps:
and (3) adopting a derivatization method to perform derivatization on the free hydrazine.
Preparing a derivatization reagent: weighing a proper amount of benzoic acid, adding a certain amount of N-methyl pyrrolidone for dissolving, adding a certain volume of acetone, uniformly mixing, and quantifying to a certain volume to obtain the derivatization reagent. In the derivatization reagent, the concentration of the benzoic acid is between 0.05mg/mL and 0.5mg/mL, and preferably 0.1mg/mL; the concentration of acetone is between 0.05mL/mL and 0.5mg/mL, preferably 0.1mL/mL.
Reference substance solution
1) Preparation of a reference substance: preparing hydrazine hydrate or hydrazine sulfate into a certain concentration by using a diluent and 0.5-2.0 mg/mL EDTA disodium solution, transferring a proper amount of the EDTA disodium solution into a headspace bottle, adding a derivatization agent, sealing, performing derivatization under the condition set by the headspace, and injecting the mixture into a gas phase mass spectrometer for detection; the content of the free hydrazine in the reference substance is between 0.01mg/mL and 0.1mg/mL, preferably 0.04mg/mL.
2) Preparation of a test solution: weighing a proper amount of the fullinide into a headspace bottle, adding a diluent and a derivatizing agent, sealing, derivatizing under the headspace setting condition, and injecting into a gas phase mass spectrometer for detection; the quantity of the fulotinib sample can be between 20mg and 100mg, preferably 50mg.
The invention provides the advantages of the detection method: (1) the invention provides a novel instrument for detecting free hydrazine in the Vanilotinib by gas mass spectrometry, which has strong specificity; (2) the operation is simple, and the derivation is carried out in the process of headspace shaking; (3) the method has the advantages of strong repeatability, high sensitivity and high accuracy.
Description of the drawings:
FIG. 1 is a special chromatogram of the derivatization product diacetone hydrazone;
FIG. 2 is a quantitative mass spectrum of a control solution;
FIG. 3 is a quantitative mass spectrum of a test solution.
Detailed Description
The invention is further illustrated by the following detailed description in conjunction with the accompanying drawings.
Example 1: detection of free hydrazine in fultinib.
The instrument comprises the following steps: SHIMADZU GCMS-TQ8040 (ion source: EI source), SHIMADZU HS-20 headspace sampler
And (3) chromatographic column: agilent VF-624MS,60m 0.32mm,1.8 μm
The instrument parameters are as follows:
hydrazine hydrate: the content is as follows: 87.3%, free hydrazine content: 62.5 percent.
Can Lutinib sample: the content is more than 99 percent
Preparing each solution:
the above solution is introduced into a gas phase mass spectrometer, mass spectrograms are recorded, mass spectrograms of reference solution are shown in figure 2, and mass spectrograms of test solution are shown in figure 3.
Example 2: and (5) system applicability inspection.
The systematic applicability of the hydrazine control solution was examined and the specific results are shown in table 1.
Table 1: systematic applicability results for hydrazine
All detection results meet the receiving standard.
Example 3: and (6) sensitivity inspection.
Preparing a quantitative limiting solution: remove 0.3mL of stock solution 2 to 20mL volumetric flasks and bring the volume to the mark with diluent. 3 portions were prepared in parallel, 0.1mL was transferred to a headspace bottle, 1mL of derivatizing agent was added, and the cap was sealed.
Preparing a detection limiting solution: transferring 0.15mL of stock solution into a 2-20 mL volumetric flask, fixing the volume to the scale with a diluent, transferring 0.1mL into a headspace flask, adding 1mL of a derivative, and sealing by a gland.
The results of the sensitivity measurements are shown in Table 2.
Table 2: hydrazine sensitivity test results
Example 4: and (6) inspecting the accuracy.
The samples with the concentration of 30% and 100% are respectively prepared and added with the standard solution for detection, and the detection results are shown in table 3.
Table 3: accuracy detection result
Claims (10)
1. An analysis method of free hydrazine in the fulgurib is characterized by comprising the following steps:
A. derivatization
Putting a free hydrazine reference substance solution or an exocellobinib test substance solution into a headspace bottle, adding a derivatization reagent, uniformly mixing, and performing derivatization to generate diacetone hydrazone, wherein the reaction formula is shown as formula I:
(1) Derivatization reagent:
weighing a proper amount of benzoic acid solid, dissolving a proper amount of N-methyl pyrrolidone, adding a certain volume of acetone, and uniformly mixing to obtain a derivatization reagent; the concentration of the benzoic acid is between 0.05mg/mL and 0.5mg/mL, and the concentration of the acetone is between 0.05mL/mL and 0.5 mg/mL;
(2) Taking a proper amount of free hydrazine reference substance, adding 0.5-2.0 mg/mL EDTA disodium solution for dissolving, putting a certain volume of EDTA disodium solution containing the free hydrazine reference substance and a derivatization agent into a headspace bottle, and uniformly mixing to obtain a reference substance solution;
(3) Taking a proper amount of the fultinib sample, adding 0.5-2.0 mg/mL of EDTA disodium solution for dissolving, putting a certain volume of the fultinib sample EDTA disodium solution and a certain volume of derivatization agent into a headspace bottle, adding into the headspace bottle, and uniformly mixing to obtain a sample solution;
the GC-MS/MS conditions are as follows:
the method is carried out by using a GC-MS/MS combined detection method, and the chromatographic conditions are as follows:
the gas chromatographic column adopts a quartz capillary chromatographic column, inert gas is used as a gas source, the column adopts a temperature programming mode, a shunt mode and an EI detector, wherein helium is selected as the inert gas, the quartz capillary chromatographic column is 30-60 m in thickness, the column diameter is 0.25-0.53 mm, the coating thickness is 0.25-3 um, and when the carrier gas source is helium, the temperature programming condition is as follows: the initial temperature is 40-60 ℃, the temperature is 5-20 ℃/min, the final temperature is 120-200 ℃, the maintaining time is 2-10 min, the temperature is raised to 180-260 ℃ at 20-40 ℃/min, the maintaining time is 3-10min, and the linear speed is 15-50 cm/s; the headspace temperature is 85-120 ℃, the shaking time is 10-50min, the quantitative ring equilibrium temperature is 80-130 ℃, and the transmission line equilibrium temperature is 105-120 ℃;
C. the mass spectrum conditions are as follows:
the ion source is an EI source. Ion source temperature is 200-220 deg.C, ion source interface temperature is 210-230 deg.C, and solvent
Cutting time is 5.5min, mass spectrum acquisition time is 5.5-15min, acquisition mode SIM, quantitative ions 97,
the ions 112 are characterized.
2. The method of claim 1 (A), wherein the solvent for preparing the reference and test samples is disodium EDTA solution, and the concentration is 0.5 mg/mL-2.0 mg/mL, preferably 1.0mg/mL.
3. The process according to claim 1 (a), characterized in that in the derivatizing agent, the concentration of benzoic acid is between 0.05mg/mL and 0.5mg/mL, preferably 0.1mg/mL; the concentration of acetone is between 0.05mL/mL and 0.5mg/mL, preferably 0.1mL/mL.
4. The process according to claim 1 (a), characterized in that the temperature of derivatization is controlled between 30 and 40 ℃, preferably 35 ℃.
5. The method of claim 1 (B), wherein the quartz capillary chromatography column has a length of 30m, a column diameter of 0.32mm, and a coating thickness of 1.8um.
6. The method of claim 1 (B), wherein the temperature programming is: the initial temperature is 50 ℃, the heating rate is 15 ℃/min, the final temperature is 150 ℃, the temperature is maintained for 5 minutes, the heating rate is 30 ℃/min, the final temperature is 220 ℃, and the temperature is maintained for 5 minutes; the linear velocity was 30cm/s.
7. The method according to claim 1 (B), wherein the split mode has a split ratio of 5.
8. The method of claim 1 (B), wherein the headspace temperature is 85-120 ℃, the shaking time is 10-50min, the quantitative loop equilibrium temperature is 80-130 ℃, and the transmission line equilibrium temperature is 105-120 ℃.
9. The method of claim 1 (C), the ion source parameters being:
an ion source: EI source
Voltage: 3.8-4.5 kV
Ion source temperature: 200-220 deg.C
The interface temperature of the ion source is 210-230 ℃, the solvent cutting time is 5.5min, the mass spectrum acquisition time is 5.5-15min,
acquisition mode SIM, quantitative ions 97, qualitative ions 112.
10. A method for measuring free hydrazine in fulgurtinib by GC-MS/MS combination is characterized by comprising the following steps:
(1) Weighing a proper amount of benzoic acid solid, adding a proper amount of N-methyl pyrrolidone for dissolving, adding a certain volume of acetone, and uniformly mixing to obtain the derivatization reagent. In a derivatization reagent, the concentration of the benzoic acid is between 0.05mg/mL and 0.5 mg/mL; the concentration of the acetone is between 0.05mL/mL and 0.5 mg/mL.
(2) Taking a proper amount of free hydrazine reference substance, adding 0.5-2.0 mg/mL EDTA disodium solution for dissolving, transferring a proper amount of EDTA disodium solution containing the free hydrazine reference substance and a proper amount of derivatization reagent, putting the EDTA disodium solution and the derivatization reagent into a headspace bottle, and uniformly mixing to obtain a reference substance solution; the content of the free hydrazine is between 0.01mg/mL and 0.1 mg/mL.
(3) Taking a proper amount of the fultinib test sample, adding 0.5-2.0 mg/mL of EDTA disodium solution for dissolving, transferring a proper amount of the fultinib test sample EDTA disodium solution and a proper amount of derivatization reagent, putting the mixture into a headspace bottle, and uniformly mixing to obtain a test sample solution; the amount of the fultinib sample is between 20mg and 100 mg.
(4) And (3) injecting 1mL of hydrazine reference substance solution and fulgurib test substance solution into a GC-MS/MS (gas chromatography-mass spectrometry) spectrometer, detecting according to the chromatographic conditions and mass spectrometry conditions of B and C, and recording a chromatogram.
(5) And (4) calculating the content of free hydrazine in the bulk drug according to the chromatogram obtained in the step (4) by using an external standard method.
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| CN202210945615.6A CN115267002A (en) | 2022-08-08 | 2022-08-08 | Method for measuring free hydrazine in fuliginib by GC-MS/MS (gas chromatography-Mass Spectrometry/Mass Spectrometry) combination |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101910154A (en) * | 2007-10-26 | 2010-12-08 | 阿米拉制药公司 | 5-lipoxygenase activating protein (FLAP) inhibitor |
| CN103698459A (en) * | 2013-12-30 | 2014-04-02 | 中美华世通生物医药科技（武汉）有限公司 | Detecting method of free hydrazine in drug |
| CN113804781A (en) * | 2021-09-06 | 2021-12-17 | 丽珠医药集团股份有限公司 | Detection and analysis method for hydrazine hydrate in dantrolene sodium |
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- 2022-08-08 CN CN202210945615.6A patent/CN115267002A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101910154A (en) * | 2007-10-26 | 2010-12-08 | 阿米拉制药公司 | 5-lipoxygenase activating protein (FLAP) inhibitor |
| CN103698459A (en) * | 2013-12-30 | 2014-04-02 | 中美华世通生物医药科技（武汉）有限公司 | Detecting method of free hydrazine in drug |
| CN113804781A (en) * | 2021-09-06 | 2021-12-17 | 丽珠医药集团股份有限公司 | Detection and analysis method for hydrazine hydrate in dantrolene sodium |
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| 周延生等: "药物中水合肼残留量的气相色谱分析", 分析试验室, vol. 27, no. 3, pages 84 - 86 * |
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