CN117849217A - Method for detecting genotoxic impurities in propranolol hydrochloride tablet - Google Patents
Method for detecting genotoxic impurities in propranolol hydrochloride tablet Download PDFInfo
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- CN117849217A CN117849217A CN202311785636.7A CN202311785636A CN117849217A CN 117849217 A CN117849217 A CN 117849217A CN 202311785636 A CN202311785636 A CN 202311785636A CN 117849217 A CN117849217 A CN 117849217A
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- MEAPRSDUXBHXGD-UHFFFAOYSA-N 3-chloro-n-(4-propan-2-ylphenyl)propanamide Chemical compound CC(C)C1=CC=C(NC(=O)CCCl)C=C1 MEAPRSDUXBHXGD-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 229960004604 propranolol hydrochloride Drugs 0.000 title claims abstract description 32
- AQHHHDLHHXJYJD-UHFFFAOYSA-N propranolol hydrochloride Natural products C1=CC=C2C(OCC(O)CNC(C)C)=CC=CC2=C1 AQHHHDLHHXJYJD-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 239000012535 impurity Substances 0.000 title claims abstract description 28
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 23
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims description 22
- 239000013558 reference substance Substances 0.000 claims abstract description 25
- 238000012360 testing method Methods 0.000 claims abstract description 23
- TXYRHXNLIUKPMM-UHFFFAOYSA-N n-(2-hydroxy-3-naphthalen-1-yloxypropyl)-n-propan-2-ylnitrous amide Chemical compound C1=CC=C2C(OCC(O)CN(C(C)C)N=O)=CC=CC2=C1 TXYRHXNLIUKPMM-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 239000000243 solution Substances 0.000 claims description 88
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 56
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 28
- 235000019253 formic acid Nutrition 0.000 claims description 28
- 150000002500 ions Chemical class 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 238000001819 mass spectrum Methods 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 13
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000002552 multiple reaction monitoring Methods 0.000 description 15
- 239000003153 chemical reaction reagent Substances 0.000 description 13
- 239000000523 sample Substances 0.000 description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- 239000012488 sample solution Substances 0.000 description 10
- 239000000843 powder Substances 0.000 description 9
- 238000005303 weighing Methods 0.000 description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 8
- 229910021642 ultra pure water Inorganic materials 0.000 description 8
- 239000012498 ultrapure water Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000012085 test solution Substances 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 5
- 239000003651 drinking water Substances 0.000 description 5
- 235000020188 drinking water Nutrition 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- -1 2-hydroxy-3- (naphthalene-1-oxy) propyl Chemical group 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000004885 tandem mass spectrometry Methods 0.000 description 3
- 239000012490 blank solution Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000007872 degassing Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 2
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002876 beta blocker Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 150000003943 catecholamines Chemical class 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 238000002540 product ion scan Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012954 risk control Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000009863 secondary prevention Effects 0.000 description 1
- 230000002889 sympathetic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8641—Baseline
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a detection method of genotoxic impurities in propranolol hydrochloride tablets, which comprises the following steps: (1) Taking N-nitrosopropranolol solutions with different concentrations as reference substances, adopting a high performance liquid chromatography-mass spectrometer to record corresponding chromatograms, and testing the peak area of each reference substance; (2) Drawing a standard curve by taking the concentration of the N-nitrosopropranolol solution as an abscissa and the corresponding peak area as an ordinate; (3) And (3) testing the peak area of the propranolol hydrochloride tablet sample, carrying the peak area into the standard curve in the step (2), and calculating to obtain the content of genotoxic impurities in the propranolol hydrochloride tablet. The detection method provided by the invention has the advantages of strong specificity, high accuracy and sensitivity, simple and convenient operation, and is suitable for daily detection of genotoxic impurities in propranolol hydrochloride tablets.
Description
Technical Field
The invention belongs to the field of medicine analysis, and particularly relates to a method for detecting genotoxic impurities in propranolol hydrochloride tablets.
Background
The propranolol hydrochloride tablet is a beta receptor blocker which can antagonize the effects of sympathetic excitation and catecholamine, has the adaptation diseases of hypertension and labor angina, is also used for secondary prevention, and reduces the death rate of myocardial infarction. Nitrosamine impurities belong to the "queue of interest" substance mentioned in the guide [ 1 ] of ICH M7 (R1) (assessment and control of DNA-reactive (mutagenic) impurities in drugs to limit potential carcinogenic risk). In order to ensure the safety and quality control of the pharmaceutical product, effective risk control must be achieved. According to the related literature, N-nitrosopropranolol is known to be a genotoxic impurity which is easily generated in the preparation of propranolol hydrochloride tablets.
N-nitrosopropranolol (N-Nitroso Propranolol) with a chemical name of N- (2-hydroxy-3- (naphthalene-1-oxy) propyl) -N-isopropyl nitrosamine and a chemical structural formula of N- (2-hydroxy-3- (naphthalene-1-oxy) propyl) -N-isopropyl nitrosamine:
at present, few documents relate to detection of N-nitrosopropranolol in a propranolol hydrochloride tablet, so that a detection method with high sensitivity and strong specificity is provided, effective control of genotoxic impurities in the propranolol hydrochloride tablet is realized, drug administration safety is ensured, and great research significance is achieved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for detecting genotoxic impurities in propranolol hydrochloride tablets.
The aim of the invention is achieved by the following technical scheme:
a method for detecting genotoxic impurities in propranolol hydrochloride tablets comprises the following steps:
(1) Taking N-nitrosopropranolol solutions with different concentrations as reference substances, adopting a high performance liquid chromatography-mass spectrometer to record corresponding mass spectrograms, and testing the peak area of each reference substance;
(2) Drawing a standard curve by taking the concentration of the N-nitrosopropranolol solution as an abscissa and the corresponding peak area as an ordinate;
(3) And (3) testing the peak area of the propranolol hydrochloride tablet sample, carrying the peak area into the standard curve in the step (2), and calculating to obtain the content of genotoxic impurities in the propranolol hydrochloride tablet.
Preferably, in the step (1), the concentration of the N-nitrosopropranolol solution is 0.5 ng/mL-20.0 ng/mL.
Preferably, in the step (1), the N-nitrosopropranolol solution is prepared according to the following method: n-nitrosopropranolol is added into a methanol solution with the concentration of 80 percent to be dissolved and prepared into a solution with the corresponding concentration.
Preferably, in the step (1), the high performance liquid chromatography mass spectrometer uses octadecylsilane chemically bonded silica as a filler, the length of a chromatographic column is 150mm, the diameter is 3.0mm, and the particle size of the filler is 1.8 mu m.
Preferably, in step (1), the column temperature of the high performance liquid chromatography mass spectrometer is 45-55 ℃, more preferably 50 ℃.
Preferably, in the step (1), the mobile phase of the chromatograph of the high performance liquid chromatograph mass spectrometer is composed of phase A and phase B according to the following ratio of 30-45: 70-55 volume ratio; the A phase is an aqueous solution containing ammonium formate with a concentration of 0.009-0.011 mol/L and formic acid with a concentration of 0.09-0.11%, and the B phase is a methanol solution containing formic acid with a concentration of 0.09-0.11%.
More preferably, the A phase is an aqueous solution containing ammonium formate at a concentration of 0.01mol/L and formic acid at a concentration of 0.1%, and the B phase is a methanol solution containing formic acid at a concentration of 0.1%.
Preferably, in step (1), the flow rate of the mobile phase of the chromatograph-mass spectrometer is 0.3 to 0.5mL/min, more preferably 0.4mL/min.
Preferably, in step (1), the sample injection amount of the chromatograph-mass spectrometer for high performance liquid chromatography is 1 to 20uL, more preferably 10uL.
Preferably, in the step (1), the elution mode of the high performance liquid chromatography-mass spectrometer is gradient elution.
Preferably, in step (1), the mass spectrum parameters of the high performance liquid chromatography mass spectrometer are as follows: the capillary voltage is 3500-4500V, the temperature of the drying gas is 280-350 ℃, the flow rate of the drying gas is 10-15L/min, and the quantitative ion pair is parent ion m/z 289.00 and child ion m/z 72.00.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS method) for detecting the content of N-nitrosopropranolol in a propranolol hydrochloride tablet, which can rapidly and accurately detect genotoxic impurities in the propranolol hydrochloride tablet and raw materials thereof, effectively ensure the quality of products and reduce the medication risk. The method has the advantages of strong specificity, high accuracy and sensitivity, simple and convenient operation and suitability for daily detection.
Drawings
FIG. 1 is a MRM chart obtained with a 0.1% formic acid acetonitrile solution as mobile phase B.
FIG. 2 is a MRM chart of a methanol solution of 0.1% formic acid as mobile phase B.
FIG. 3 is a MRM chart obtained for mobile phase A formulated with ultrapure water.
Fig. 4 is an MRM plot of mobile phase a of a drohent drinking water formulation.
FIG. 5 is a MRM obtained by selecting Agilent ZORBAX SB-C18 3.0X105 mm,1.8 μm as a column.
FIG. 6 is a MRM obtained by selecting Waters ACQUITY UPLC C18 to be 3.0X105 mm and 1.7 μm as a column.
Fig. 7 is a linear diagram corresponding to the section of example [ 3.2 linearity ].
FIG. 8 is a graph comparing test results corresponding to the example [ 3.5.2 assay and acceptance criteria ].
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Quality standard:
the method is measured according to high performance liquid chromatography (the rule 0512 of the fourth edition of the Chinese pharmacopoeia 2020 edition) and mass spectrometry (the rule 0431 of the fourth edition of the Chinese pharmacopoeia 2020 edition).
Control line solutions: accurately weighing a proper amount of N-nitrosopropranolol reference substance, adding a methanol solution with the concentration of 80% for dissolution, and respectively quantitatively diluting to prepare solutions with the concentration of 0.5, 1, 2, 5, 10 and 20ng of N-nitrosopropranolol in each 1 mL.
Test solution: taking 10 pieces of a sample to be measured, precisely weighing, grinding, uniformly mixing, precisely weighing about 440mg of fine powder, placing in a 5mL volumetric flask, adding 80% methanol solution to dilute to scale, swirling for 5 minutes, filtering, and taking the subsequent filtrate as a sample solution.
Chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler (such as Agilent ZORBAX SB-C18 3.0X105 mm,1.8 μm or a chromatographic column with equivalent performance), an aqueous solution of 0.01mol/L ammonium formate-0.1% formic acid is used as a mobile phase A, a methanol solution of 0.1% formic acid is used as a mobile phase B, gradient elution is carried out according to Table 1, the flow rate is 0.40mL/min, the column temperature is 50 ℃, and the sample injection volume is 10. Mu.L.
TABLE 1 gradient elution schedule
| Time (min) | A％ | B％ |
| 0.0 | 37 | 63 |
| 3.0 | 37 | 63 |
| 5.0 | 60 | 40 |
| 8.0 | 60 | 40 |
| 10.0 | 45 | 55 |
| 16.0 | 45 | 55 |
| 17.0 | 37 | 63 |
| 22.0 | 37 | 63 |
Mass spectrometry conditions: detection was performed with a triple quadrupole tandem mass spectrometer using electrospray ionization source (ESI), using positive ion scan mode, capillary voltage 4000V, and dryer temperature 300 ℃. The detection mode was Multiple Reaction Monitoring (MRM), and the monitored ion pairs of the compounds are shown in table 2.
Table 2 monitoring ion pair checklist
System applicability requirements: taking a reference substance solution with the concentration of 1ng/mL, repeating the sample injection for 6 times, wherein the relative standard deviation of the main peak area is not more than 10.0%, and the concentration is 0.5ng/mL of reference substance solution, the signal to noise ratio of the obtained main peak is not lower than 10, the reference line column solutions are respectively taken for sample injection, a standard curve is drawn according to the peak area corresponding to the concentration, a linear regression equation is calculated, and R is calculated 2 Should not be lower than 0.99.
Assay: and respectively taking a reference substance solution and a sample solution, injecting the reference substance solution and the sample solution into a high performance liquid chromatography-mass spectrometer, recording a mass spectrum, measuring the peak area, and calculating according to a standard curve method to obtain the propranolol hydrochloride marked amount of 0.23ppm.
The development process of the analysis method comprises the following steps:
in terms of mass spectrometry conditions, QQQ parameters were established through a whole logical sequence of Scan full Scan (determining compound mass number) -SIM selection Ion monitoring (optimizing fragmentation voltage and collision cell voltage) -Product Ion Scan sub-Ion Scan (optimizing collision energy) -MRM multi-reaction monitoring (confirming quantitative ions).
In terms of chromatographic conditions, common LC/MS solvents are generally high-quality methanol, acetonitrile, water and volatile solvents, so that an aqueous solution of 0.01mol/L ammonium formate-0.1% formic acid is selected as a mobile phase A, a methanol solution of 0.1% formic acid or an acetonitrile solution of 0.1% formic acid is selected as a mobile phase B, and proper flow rate and gradient elution are set.
FIG. 1 is a MRM diagram obtained by taking an acetonitrile solution of 0.1% formic acid as a mobile phase B, taking an aqueous solution (ultrapure water) of 0.01mol/L ammonium formate-0.1% formic acid as a mobile phase A, taking a reference substance solution (5 ng/ml), injecting into a high performance liquid chromatography mass spectrometer, and recording a mass spectrum.
FIG. 2 is a MRM chart obtained by taking a methanol solution of 0.1% formic acid as a mobile phase B, taking an aqueous solution (ultrapure water) of 0.01mol/L ammonium formate-0.1% formic acid as a mobile phase A, taking a reference substance solution (5 ng/ml), injecting into a high performance liquid chromatography mass spectrometer, and recording a mass spectrum.
Referring to fig. 1 to 2, it can be seen that mobile phase B prepared with acetonitrile has a sharp peak front, and mobile phase B prepared with methanol has a symmetrical peak shape, so that a methanol solution of 1% formic acid is selected as mobile phase B.
The mass spectrum has higher requirements on water quality, and ultrapure water and chen drinking water prepared by a laboratory water purifier are respectively selected to prepare a mobile phase A.
FIG. 3 is a MRM chart obtained from a mobile phase A prepared from ultrapure water, wherein a methanol solution of 0.1% formic acid is taken as a mobile phase B, the ultrapure water is taken, and the ultrapure water is injected into a high performance liquid chromatography mass spectrometer to record a mass spectrum.
Fig. 4 is an MRM diagram obtained by a mobile phase a prepared from the drinking water of the minister, wherein a methanol solution of 0.1% formic acid is taken as a mobile phase B, the drinking water of the minister is taken, and the drinking water is injected into a high performance liquid chromatography-mass spectrometer for recording a mass spectrum.
Referring to fig. 3 to 4, it can be seen that the baseline noise is similar to that of the two, so that the ultrapure water with more convenience and low detection cost is selected.
Agilent and Waters columns were selected for comparison.
FIG. 5 is a MRM chart obtained by selecting Agilent ZORBAX SB-C18 3.0X105 mm,1.8 μm as a chromatographic column, 0.01mol/L ammonium formate-0.1% formic acid aqueous solution as mobile phase A,0.1% formic acid methanol solution as mobile phase B, taking reference substance solution (10 ng/ml), injecting into a high performance liquid chromatography mass spectrometer, and recording mass spectrograms.
FIG. 6 is a MRM chart obtained by selecting Waters ACQUITY UPLC C18 to be 3.0X105 mm and 1.7 μm as a chromatographic column, wherein 0.01mol/L ammonium formate-0.1% formic acid aqueous solution is mobile phase A,0.1% formic acid methanol solution is mobile phase B, taking a reference substance solution (10 ng/ml), injecting into a high performance liquid chromatography-mass spectrometer, and recording a mass spectrum.
Referring to FIGS. 5-6, it can be seen that the peak response of the Agilent column is higher and that under the same conditions the pressure at which the Agilent column is run is about 200Bar lower than that of the waters column, so that an Agilent column is selected for use.
Analytical method validation
1. Instrument, chemical reagent and sample information
1.1. Instrument information
Table 3 is an instrument information statistics table.
Table 3 instrument information list
| Instrument name | Grade (model) |
| Electronic balance | ME204 |
| Electronic balance | XPR36 |
| Chromatographic column | Agilent ZORBAX SB-C18 |
| High performance liquid mass spectrometer | Agilent 1290-6460 |
1.2. Reagent information
Table 4 is a statistical table of reagent information.
TABLE 4 list of reagent information
| Reagent name | Lot number | Source |
| Formic acid | 223682 | Siemens fly |
| Ammonium formate | 63C1503NG | ACS |
| Methanol | 11243235 239 | Merck (Merck) |
1.3. Sample information
Table 5 is a sample information statistics table.
Table 5 sample information list
2. Solution preparation
On the premise that the target concentration and the weighing accuracy meet the requirements, the volume of all the solutions can be enlarged or reduced to adapt to the actual needs.
2.1 mobile phase/Diluent/blank solution/blank adjuvant solution
2.1.1 mobile phase A:0.01mol/L ammonium formate-0.1% formic acid aqueous solution
Precisely weighing 0.63g of ammonium formate in a reagent bottle, adding 1000ml of water to dissolve, adding 1.0ml of formic acid into the reagent bottle, uniformly mixing, and carrying out ultrasonic degassing to obtain the product.
2.1.2 mobile phase B: methanol solution of 0.1% formic acid
1000ml of methanol is measured in a reagent bottle, 1.0ml of formic acid is added into the reagent bottle, and the reagent bottle is evenly mixed and subjected to ultrasonic degassing to obtain the reagent.
2.1.3 diluent/blank solution: 80% methanol
Weighing 800ml of methanol and 200ml of water in the same reagent bottle, and uniformly mixing to obtain the reagent.
2.1.4 blank adjuvant solution: and weighing about 400mg of propranolol hydrochloride tablet auxiliary material, placing the tablet auxiliary material into a 5mL volumetric flask, adding a diluent to dilute to a scale, swirling for 5 minutes, filtering, and taking a subsequent filtrate to obtain the propranolol hydrochloride tablet auxiliary material.
Table 6 shows the statistics of the ingredients of the blank auxiliary materials.
TABLE 6 list of blank adjuvant ingredients
2.2 control series of solutions
Table 7 shows statistics of compositions of the control series solutions.
TABLE 7 list of reference series solution compositions
3. Verification project
3.1 repeatability
3.1.1 preparation of solutions
And (2) preparing the reference substance solution 2 under the item of solution preparation.
3.1.2 measurement and acceptance criteria
Taking a reference substance solution 2, repeating sample injection for 6 times, wherein the main peak area RSD is less than or equal to 10.0%, and the retention time RSD is less than or equal to 2.0%.
High performance liquid chromatography tandem mass spectrometry
The chromatographic conditions are as follows:
chromatographic column: agilent ZORBAX SB-C18 3.0X105 mm,1.8 μm
Mobile phase a:0.01mol/L ammonium formate-0.1% formic acid aqueous solution
Mobile phase B: methanol solution of 0.1% formic acid
Elution mode: gradient elution (see Table 1 for details)
Flow rate: 0.40mL/min
Column temperature: 50 DEG C
The mass spectrum conditions are as follows:
ion source and ionization mode: ESI source, positive ion mode
Monitoring mode: MRM mode
Capillary voltage: 4000V
Drying gas temperature: 300 DEG C
Dry air flow: 13L/min
The test records are shown in Table 8.
Table 8 list of repeatability test results
3.2 linearity
3.2.1 preparation of solutions
Control series of solutions under the item "2. Solution formulation".
3.2.2 measurement and acceptance criteria
Sampling the solution of the control series, drawing a standard curve according to the peak area corresponding to the concentration, calculating a linear regression equation, R 2 And more than or equal to 0.99. The high performance liquid chromatography tandem mass spectrometry is adopted, and the test parameters are the same as the conditions under the item of '3.1 repeatability'.
The test records are shown in Table 9, and the corresponding linear graphs are shown in FIG. 7:
table 9 list of linear test results
3.3 quantitative limit and detection limit
3.3.1 preparation of solutions
Taking a proper amount of stock solution 2 under the item of solution preparation, and gradually diluting the stock solution with a diluent until the S/N of the target impurity is approximately equal to 10, wherein the stock solution is taken as a quantitative limiting solution; S/N is approximately equal to 3, and the solution is used as a detection limit solution.
3.3.2 measurement and acceptance criteria
The LOQ should not exceed 50% of its limit.
Test records are shown in Table 10:
table 10 quantitative limit and detection limit test results list
3.4 accuracy
3.4.1 preparation of solutions
Test solution: taking 10 pieces of the product (see 1.3. Sample information), precisely weighing, grinding, uniformly mixing, precisely weighing about 440mg of fine powder, placing into a 5ml volumetric flask, adding a diluent to dilute to scale, swirling for 5 minutes, filtering, and taking subsequent filtrate to obtain the product.
Control solution: and the same as the reference substance solution 2, the reference substance solution 4 and the reference substance solution 5 under the item of solution preparation.
Adding a labeled test sample solution:
low concentration: taking about 440mg of fine powder of the product, placing the fine powder into a 5ml volumetric flask, adding the reference substance solution 2 to dilute to a scale, swirling for 5 minutes, filtering, and taking the subsequent filtrate to obtain the product (three parts are prepared in parallel).
Medium concentration: taking about 440mg of fine powder of the product, placing the fine powder into a 5ml volumetric flask, adding the reference substance solution 4 to dilute to a scale, swirling for 5 minutes, filtering, and taking the subsequent filtrate to obtain the product (three parts are prepared in parallel).
High concentration: taking about 440mg of fine powder of the product, placing the fine powder into a 5ml volumetric flask, adding the reference substance solution 5 to dilute to a scale, swirling for 5 minutes, filtering, and taking the subsequent filtrate to obtain the product (three parts are prepared in parallel).
3.4.2 assay and acceptance criteria
The recovery rate of nine parts of marked sample solution is 70% -130%, and RSD is less than or equal to 10%.
Test records are shown in Table 11:
table 11 accuracy test results list
Note that: PNH concentration is the API concentration calculated from the powder sample weight, average tablet weight and index.
3.5 specificity
3.5.1 preparation of solutions
Blank auxiliary material solution: preparing blank auxiliary material solution under the item of '2' solution;
control solution: preparing a reference substance solution 5 under the item "2" by the solution;
test solution: test solution under the same "3.4 accuracy" term;
adding a labeled test sample solution: high concentration under the term "3.4 accuracy".
3.5.2 measurement and acceptance criteria
The blank auxiliary material solution has no interference to the determination of the target impurity, and adjacent peaks near the target impurity in the sample solution and the labeled sample solution are not generated.
The test results are shown in fig. 8, and from fig. 8, we can see that the test method of the invention has better specificity.
3.6 solution stability
3.6.1 preparation of solutions
Control solution: preparing a control solution 2 under the item "2" by the solution;
test solution: test solution under the same "3.4 accuracy" term;
3.6.2 measurement and acceptance criteria
Under the detection condition, selecting at least 3 time points for testing, wherein the ratio of the measured concentration at each time point to the measured concentration at 0h is 70% -130%.
Test records are shown in tables 12 and 13:
table 12 list of test results for stability of control solutions
TABLE 13 test results list of stability of sample solutions
Referring to tables 12 to 13, it can be derived that: the reference substance solution and the sample solution adopted by the invention have good stability, are used for detecting the solution, and ensure the accuracy of measurement.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any of various other corresponding changes and modifications made according to the technical idea of the present invention should be included in the scope of the claims of the present invention.
Claims (10)
1. The method for detecting the genotoxic impurities in the propranolol hydrochloride tablet is characterized by comprising the following steps of:
(1) Taking N-nitrosopropranolol solutions with different concentrations as reference substances, adopting a high performance liquid chromatography-mass spectrometer to record corresponding chromatograms, and testing the peak area of each reference substance;
(2) Drawing a standard curve by taking the concentration of the N-nitrosopropranolol solution as an abscissa and the corresponding peak area as an ordinate;
(3) And (3) testing the peak area of the propranolol hydrochloride tablet sample, carrying the peak area into the standard curve in the step (2), and calculating to obtain the content of genotoxic impurities in the propranolol hydrochloride tablet.
2. The method for detecting genotoxic impurities in propranolol hydrochloride tablets according to claim 1, wherein the concentration of the N-nitrosopropranolol solution in the step (1) is 0.5 ng/mL-20.0 ng/mL.
3. The method for detecting genotoxic impurities in propranolol hydrochloride tablets according to claim 2, wherein the N-nitrosopropranolol solution in the step (1) is prepared according to the following method: n-nitrosopropranolol is added into a methanol solution with the concentration of 80 percent to be dissolved and prepared into a solution with the corresponding concentration.
4. The method for detecting genotoxic impurities in propranolol hydrochloride tablets according to any one of claims 1 to 3, wherein the high performance liquid chromatography-mass spectrometry combined instrument in step (1) uses octadecylsilane chemically bonded silica as a filler, the length of a chromatographic column is 150mm, the diameter is 3.0mm, and the particle size of the filler is 1.8 μm.
5. The method for detecting genotoxic impurities in propranolol hydrochloride tablets according to claim 4, wherein the chromatographic column temperature of the high performance liquid chromatography-mass spectrometer in the step (1) is 45-55 ℃.
6. The method for detecting genotoxic impurities in propranolol hydrochloride tablets according to claim 5, wherein the mobile phase of the chromatograph of the high performance liquid chromatograph-mass spectrometer in the step (1) consists of a phase A and a phase B according to 30-45: 70-55 volume ratio; the A phase is an aqueous solution containing ammonium formate with a concentration of 0.009-0.011 mol/L and formic acid with a concentration of 0.09-0.11%, and the B phase is a methanol solution containing formic acid with a concentration of 0.09-0.11%.
7. The method for detecting genotoxic impurities in propranolol hydrochloride tablets according to claim 6, wherein the flow rate of the mobile phase of the chromatograph-mass spectrometer of the high performance liquid chromatograph in the step (1) is 0.3-0.5 mL/min;
and (3) the sample injection amount of the chromatograph of the high performance liquid chromatograph-mass spectrometer in the step (1) is 1-20 uL.
8. The method for detecting genotoxic impurities in propranolol hydrochloride tablets according to claim 7, wherein the flow rate of the mobile phase of the chromatograph-mass spectrometer of the high performance liquid chromatograph in the step (1) is 0.4mL/min;
and (3) the sample injection amount of the chromatograph of the high performance liquid chromatograph-mass spectrometer in the step (1) is 10uL.
9. The method for detecting genotoxic impurities in propranolol hydrochloride tablets according to any one of claims 1 to 3, wherein the elution mode of the high performance liquid chromatography-mass spectrometer in the step (1) is gradient elution.
10. The method for detecting genotoxic impurities in propranolol hydrochloride tablets according to claim 9, wherein the mass spectrum parameters of the high performance liquid chromatography-mass spectrometer in the step (1) are as follows: the capillary voltage is 3500-4500V, the temperature of the drying gas is 280-350 ℃, the flow rate of the drying gas is 10-15L/min, and the quantitative ion pair is parent ion m/z 289.00 and child ion m/z 72.00.
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