CN114460203B - Method for simultaneously detecting methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in medicine - Google Patents
Method for simultaneously detecting methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in medicine Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 64
- SDQCGKJCBWXRMK-UHFFFAOYSA-N propan-2-yl 4-methylbenzenesulfonate Chemical compound CC(C)OS(=O)(=O)C1=CC=C(C)C=C1 SDQCGKJCBWXRMK-UHFFFAOYSA-N 0.000 title claims abstract description 21
- VRZVPALEJCLXPR-UHFFFAOYSA-N ethyl 4-methylbenzenesulfonate Chemical compound CCOS(=O)(=O)C1=CC=C(C)C=C1 VRZVPALEJCLXPR-UHFFFAOYSA-N 0.000 title claims abstract description 20
- VUQUOGPMUUJORT-UHFFFAOYSA-N methyl 4-methylbenzenesulfonate Chemical compound COS(=O)(=O)C1=CC=C(C)C=C1 VUQUOGPMUUJORT-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 239000003814 drug Substances 0.000 title claims abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 30
- 239000013558 reference substance Substances 0.000 claims abstract description 16
- HTSCTBSPMLCBLT-UHFFFAOYSA-N ethyl 4-methylbenzenesulfinate Chemical compound CCOS(=O)C1=CC=C(C)C=C1 HTSCTBSPMLCBLT-UHFFFAOYSA-N 0.000 claims abstract description 14
- MGPLBSPZSIFUQX-UHFFFAOYSA-N methyl 4-methylbenzenesulfinate Chemical compound COS(=O)C1=CC=C(C)C=C1 MGPLBSPZSIFUQX-UHFFFAOYSA-N 0.000 claims abstract description 14
- OYULADAATOHCOM-UHFFFAOYSA-N propan-2-yl 4-methylbenzenesulfinate Chemical compound CC(C)OS(=O)C1=CC=C(C)C=C1 OYULADAATOHCOM-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000012488 sample solution Substances 0.000 claims abstract description 11
- 238000001819 mass spectrum Methods 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 3
- 238000010812 external standard method Methods 0.000 claims abstract description 3
- 238000012544 monitoring process Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 33
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 239000003085 diluting agent Substances 0.000 claims description 20
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 18
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 9
- 239000000377 silicon dioxide Substances 0.000 claims description 9
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 8
- 150000002500 ions Chemical class 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 7
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 7
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 abstract description 37
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000001195 ultra high performance liquid chromatography Methods 0.000 abstract description 6
- 239000011159 matrix material Substances 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 abstract description 4
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 238000004949 mass spectrometry Methods 0.000 description 9
- 238000011084 recovery Methods 0.000 description 8
- 238000007865 diluting Methods 0.000 description 7
- 239000011550 stock solution Substances 0.000 description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000004744 fabric Substances 0.000 description 6
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- DOUHZFSGSXMPIE-UHFFFAOYSA-N hydroxidooxidosulfur(.) Chemical compound [O]SO DOUHZFSGSXMPIE-UHFFFAOYSA-N 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- DILXLMRYFWFBGR-UHFFFAOYSA-N 2-formylbenzene-1,4-disulfonic acid Chemical compound OS(=O)(=O)C1=CC=C(S(O)(=O)=O)C(C=O)=C1 DILXLMRYFWFBGR-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 231100000024 genotoxic Toxicity 0.000 description 3
- 230000001738 genotoxic effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- -1 para-toluene sulfinate methyl ester Chemical class 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000066 reactive distillation Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical class CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- 230000002110 toxicologic effect Effects 0.000 description 2
- 231100000027 toxicology Toxicity 0.000 description 2
- FXJVNINSOKCNJP-UHFFFAOYSA-N 4-methylbenzenesulfinic acid Chemical compound CC1=CC=C(S(O)=O)C=C1 FXJVNINSOKCNJP-UHFFFAOYSA-N 0.000 description 1
- 241000221931 Hypomyces rosellus Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
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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)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a method for simultaneously detecting methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a medicine, which comprises the steps of respectively detecting a reference substance solution and a sample solution by adopting an ultra-high performance liquid chromatography-mass spectrometry combination and using a multi-reaction monitoring technology, and calculating according to an external standard method to determine the contents of the methyl p-toluenesulfonate, the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate in a sample. The invention adopts ultra-high performance liquid chromatography for separation, and high selectivity and high sensitivity mass spectrum are used for simultaneously detecting methyl p-toluenesulfinate, ethyl p-toluenesulfinate and isopropyl p-toluenesulfinate, the sample pretreatment is simple, the sample matrix has no interference to the determination of a target peak, and the method has the advantages of good durability, high detection efficiency and the like.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to an analysis and detection method for methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the field of medicines.
Background
Para-toluene sulfinic acid is an important raw material in organic synthesis, and is easy to form genotoxic impurities such as methyl para-toluene sulfinate, ethyl para-toluene sulfinate and isopropyl para-toluene sulfinate with trace methanol, ethanol and isopropanol in the process, and the substances can react with DNA in an alkylation way, so that the substances possibly become causes for causing cancers, and therefore, the control of the toxicological attention threshold (TTC) level of the impurities in the medicine is very important. The European Medicine Evaluation Agency (EMEA), the United states Food and Drug Administration (FDA), and the International conference on drug registration (ICH) all make limit regulations on genotoxic impurities that must be controlled below the toxicological attention threshold (TTC) level. Wherein the structure of the methyl p-toluene sulfinate, the ethyl p-toluene sulfinate and the isopropyl p-toluene sulfinate is as follows:
maritimibe is disclosed in WO 201017907 and has the following structural formula:
the intermediate in the process of the maritima cloth is likely to generate by-product p-toluene sulfinic acid under alkaline conditions, and p-toluene sulfinic acid and trace methanol, ethanol and isopropanol in the process are likely to form genotoxic impurities such as methyl p-toluene sulfinate, ethyl p-toluene sulfinate and isopropyl p-toluene sulfinate. The method is developed to detect the stability of the Haibomai cloth process and ensure the quality of the product.
The sulfonate detection method mainly comprises Gas Chromatography (GC), liquid chromatography (HPLC) and a combination method thereof for analysis, and currently, the sulfonate detection method is mainly more, and the sulfinate detection method is not yet found. The method adopts ultra-high performance liquid chromatography for separation, and high selectivity and high sensitivity mass spectrum for detection of methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate, so that sample pretreatment is simple, and a sample matrix has no interference to target peak determination; the method has good durability, and can well detect the contents of the methyl p-toluenesulfonate, the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate in the sample so as to ensure the product quality.
Disclosure of Invention
The invention aims to provide a method for detecting methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a medicine, which adopts ultra-high performance liquid chromatography-mass spectrometry to detect methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate simultaneously, and has the advantages of high selectivity, high sensitivity, simple sample pretreatment, no interference of a sample matrix on target peak measurement, good method durability and the like, and the specific scheme is as follows:
a method for simultaneously detecting methyl p-toluenesulfinate, ethyl p-toluenesulfinate and isopropyl p-toluenesulfinate in a medicament, the method comprising:
step 1: adding a diluent into a p-toluene sulfinate methyl ester, p-toluene sulfinate ethyl ester and p-toluene sulfinate isopropyl ester reference substance to prepare a reference substance solution;
step 2: adding a diluent into a sample to be tested to prepare a sample solution to be tested;
step 3: detecting the reference substance solution in the step 1 and the sample solution in the step 2 by adopting an ultra-high performance liquid chromatography-mass spectrometer, and determining the contents of methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the sample according to an external standard method;
wherein, the chromatographic conditions of the ultra performance liquid chromatography in the step 3 are as follows:
chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
mobile phase: mobile phase A is 0.002-0.05 moL/L ammonium formate aqueous solution, mobile phase B is methanol; the elution mode of the mobile phase is gradient elution, and the gradient elution is carried out according to the following procedures:
| time (min) | Mobile phase A% | Mobile phase B% |
| 0.0 | 50 | 50 |
| 4.0 | 50 | 50 |
| 5.0 | 10 | 90 |
| 7.0 | 10 | 90 |
| 7.1 | 50 | 50 |
| 9.0 | 50 | 50 |
The medicine is selected from p-toluene sulfinic acid, and trace amount of methanol, ethanol and isopropanol, preferably, the medicine is cobweb.
The diluent is 10-90% acetonitrile water solution (V/V), preferably 30-70% acetonitrile water solution (V/V), more preferably 40-60% acetonitrile water solution (V/V), and most preferably 50% acetonitrile water solution (V/V);
the reference substance solution is prepared into linear solutions containing methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate which are 1.25ng/mL,2.50ng/mL,5.00ng/mL,7.50ng/mL and 10.00ng/mL by adopting a progressive dilution method.
The column diameter of the octadecylsilane chemically bonded silica chromatographic column is 2.1 mm-4.6 mm, the column length is 50 mm-150 mm, and the particle size is 1.6 mu m-3.5 mu m; preferably, the column diameter of the octadecylsilane chemically bonded silica chromatographic column is 2.1 mm-3.5 mm, the column length is 50 mm-100 mm, and the particle diameter is 1.6 mu m-2.5 mu m; more preferably, the octadecylsilane chemically bonded silica chromatographic column has a column diameter of 2.1 to 3.0mm, a column length of 50 to 100mm and a particle diameter of 1.7 to 2.5. Mu.m; most preferably Agilent Eclipse Plus C RRHD 50 x 2.1mm 1.8 μm.
The column temperature of the chromatographic column is 25℃to 45℃and preferably 30℃to 45℃and more preferably 30℃to 40℃and most preferably 35 ℃.
The flow rate of the mobile phase is 0.2mL/min to 0.6mL/min, preferably 0.3mL/min to 0.5mL/min, more preferably 0.3mL/min to 0.4mL/min, and most preferably 0.4mL/min.
The mobile phase A is preferably 0.005 to 0.02moL/L of an aqueous ammonium formate solution, more preferably 0.01moL/L of an aqueous ammonium formate solution.
The mass spectrum conditions are as follows:
scanning mode: monitoring mass spectrum multiple reactions; ion source: an electrospray ion source; ion source mode: a positive mode; capillary voltage: 0.5-3.0 KV, preferably 1.0-2.0 KV, more preferably 1.0KV; drying gas temperature: 300 to 650 ℃, preferably 350 to 600 ℃, more preferably 400 to 550 ℃, most preferably 500 ℃; drying gas flow rate: 1000L/Hr; taper hole voltage: 15 to 40V, preferably 20 to 30V, more preferably 25V; source temperature: 150 ℃.
The ultra-high performance liquid chromatography-mass spectrometer is Waters ACQUITY I UPLC Class-TQ-S micro.
The method for detecting the methyl p-toluenesulfonate, the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate by using the ultra-high performance liquid-mass spectrometry has the following advantages: the contents of methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the sample can be detected simultaneously; the pretreatment of the sample is simple, the sample adopts a direct dissolution mode, and the derivatization treatment is not needed; the separation degree is good, and the methyl paratoluenesulfinate, the ethyl paratoluenesulfinate and the isopropyl paratoluenesulfinate are completely and effectively separated from the sample within 9 minutes; the linear correlation coefficient is good, and the linear correlation coefficients r of methyl paratoluenesulfinate, ethyl paratoluenesulfinate and isopropyl paratoluenesulfinate are all more than or equal to 0.999 in the respective linear ranges; the sensitivity is high, the detection quantitative limit of the instrument is 1.3ng/mL, and the detection limit is 0.4ng/mL; the recovery rate is high, and the standard adding accuracy recovery rates of the methyl paratoluenesulfinate, the ethyl paratoluenesulfinate and the isopropyl paratoluenesulfinate are all 90-110%. According to the method, three types of p-toluenesulfonic acid esters are detected simultaneously, so that the detection efficiency is improved, and meanwhile, the method adopts high-selectivity and high-sensitivity mass spectrum to detect the methyl p-toluenesulfonate, the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate, so that the sample pretreatment is simple, and the sample matrix has no interference to the target peak measurement; the method has the advantages of good durability, high detection efficiency and the like, and no report of detecting the p-toluenesulfonate is found at present.
Description of the drawings:
FIG. 1 is a diagram showing the ultra-high performance liquid-mass spectrum combination of a blank solution of methyl p-toluenesulfinate, ethyl p-toluenesulfinate and isopropyl p-toluenesulfinate in the method of the invention.
FIG. 2 is a solution chart of the ultra-high performance liquid-mass spectrometry reference substance of the method of the invention, namely, methyl p-toluenesulfinate, ethyl p-toluenesulfinate and isopropyl p-toluenesulfinate.
FIG. 3 is a linear plot of methyl p-toluenesulfinate in the process of the invention.
FIG. 4 is a linear plot of ethyl p-toluenesulfinate in the process of the invention.
FIG. 5 is a linear plot of isopropyl tosylate in the process of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail below by referring to the accompanying drawings and by referring to examples.
The apparatus and reagents used in the following examples:
methanol and acetonitrile are commercial chromatographic purity, ammonium formate is commercial mass spectrum purity, and water is self-made ultrapure water. The detection instrument is Waters ACQUITY I UPLC Class-TQ-S micro.
EXAMPLE 1 method for detecting methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate by ultra-high performance liquid-mass spectrometry
(1) Preparation of test solutions
The method comprises the steps of weighing a proper amount of the Haibomab sample, dissolving and diluting the sample into a solution containing 1mg/mL of the Haibomab by using a 50% acetonitrile water solution (V/V), and taking the solution as a sample solution.
(2) Preparation of control stock solutions
And respectively precisely weighing a proper amount of p-toluene sulfinate methyl ester, p-toluene sulfinate ethyl ester and p-toluene sulfinate isopropyl ester reference substances, and dissolving and diluting the reference substances by using a diluent to obtain reference substance stock solutions with the concentration of the p-toluene sulfinate methyl ester, the p-toluene sulfinate ethyl ester and the p-toluene sulfinate isopropyl ester of 25 ng/mL.
(3) Ultra-high performance liquid-mass spectrometry detection condition
Instrument: wo-superfine ultra-high performance liquid-triple quadrupole mass spectrometer (Waters ACQUITY I UPLC Class-TQ-S micro);
chromatographic conditions:
chromatographic column: agilent Eclipse Plus C18 RRHD 50 x 2.1mm 1.8 μm
Flow rate: 0.4mL/min
Column temperature: 35 DEG C
Sample injection amount: 10. mu L (mu L)
Mobile phase a:0.01moL/L ammonium formate aqueous solution
Mobile phase B: methanol
A diluent: 50% acetonitrile in water (V/V)
Blank solvent: same diluent
Gradient:
| time (min) | Mobile phase A% | Mobile phase B% |
| 0.0 | 50 | 50 |
| 4.0 | 50 | 50 |
| 5.0 | 10 | 90 |
| 7.0 | 10 | 90 |
| 7.1 | 50 | 50 |
| 9.0 | 50 | 50 |
Mass spectrometry conditions
(4) The specificity experiment is carried out on the ultra-high performance liquid-mass spectrum combined detection method, and the specificity experiment is specifically as follows:
a) Blank solvent: 50% acetonitrile aqueous solution (V/V), wherein the ion chart is shown in FIG. 1;
b) Control solution: transferring 4.0mL of the control stock solution into a 20mL volumetric flask, diluting to scale with a diluent, and mixing uniformly, wherein the characteristic ion diagram is shown in figure 2;
c) Adding a labeled test sample solution: weighing 20mg of the Haibomai sample in a 20mL volumetric flask, adding 4.0mL of a reference substance stock solution, dissolving with a proper amount of diluent, diluting to a scale with the diluent, and uniformly mixing.
According to the method, the specific solutions of the blank solvent, the reference substance solution and the labeled test sample solution are detected, the detection results are shown in table 1, and from the experimental results, the detection of the blank solution, the sample solution, the p-toluene sulfinic acid methyl ester, the p-toluene sulfinic acid ethyl ester and the p-toluene sulfinic acid isopropyl ester is not interfered, and the specificity of the ultra-high performance liquid-mass spectrometry detection method is good.
TABLE 1 determination of specific solutions
Example 2 ultra high Performance liquid chromatography Mass Spectrometry detection limit and quantitative limit
(1) Quantitative limiting solution: transferring 1.0mL of the reference substance stock solution into 6 20mL volumetric flasks, diluting to scale with a diluent, and uniformly mixing;
(2) Detection limit solution: transferring 3.0mL of the first quantitative limiting solution, diluting to a scale with a diluent in a 10mL volumetric flask, and uniformly mixing;
the quantitative limit and the detection limit solutions were tested according to the method of example 1, the signal to noise ratio (S/N) of the quantitative limit solution was not less than 10, the detection limit solution (S/N) was not less than 3, and the test results are shown in Table 2 and Table 3:
TABLE 2 quantitative limits for ultra high performance liquid chromatography-mass spectrometry
TABLE 3 ultra high Performance liquid chromatography mass spectrometry detection limits
Example 3 ultra high Performance liquid chromatography mass spectrometer detection method linearity
Linear solution: and (3) transferring 1.0mL, 2.0mL, 4.0mL, 6.0mL and 8.0mL of the stock solution of methyl paratoluenesulfinate, ethyl paratoluenesulfinate and isopropyl paratoluenesulfinate, respectively placing the stock solution into 5 volumetric flasks of 20mL, diluting to scale with a diluent, uniformly mixing, and preparing two parts of each linear concentration in parallel.
A linear equation and linear correlation coefficient were calculated for each linear solution sample by the method of example 1, and the results are shown in Table 4.
Table 4 linear regression equation, linear correlation coefficient r for ultra high performance liquid-mass spectrometer detection method
According to the experimental results, the high performance liquid-mass spectrometer detection method has good linear relation in the detection range, and the content of the methyl p-toluenesulfonate, the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate can be accurately quantified.
Example 4 accuracy and repeatability experiments of the ultra high Performance liquid chromatography Mass Spectrometry detection method
(1) Accuracy experiment
Preparing a sample solution of the Haibomai cloth, adding three para-toluene sulfinate methyl ester, para-toluene sulfinate ethyl ester and para-toluene sulfinate isopropyl ester reference substance solutions with different concentration levels, and preparing three parts of each concentration in parallel, wherein the preparation concentrations are shown in Table 5. Each aliquot of the labeled test sample solution was analyzed once by sample injection as in example 1, and the recovery per aliquot and the average recovery per concentration level were calculated. The results are shown in Table 5.
Table 5 accuracy of ultra-high performance liquid-mass spectrometer detection method in Haibomai cloth
(2) Repeatability experiments
According to the method, 6 parts of marked Haibo cloth solution with medium concentration level are prepared, each part of solution is injected and analyzed once according to the method of the example 1, and RSD (reactive distillation) of the recovery rate of the 6 parts of marked solution is calculated. The results are shown in Table 6.
Table 6 repeatability of ultra-high performance liquid-mass spectrometer detection method in Haibomai cloth
From the experimental results, the accuracy of the detection method and the recovery rate of the repeatability experiment of the high performance liquid-mass spectrometer are knownRSD is less than 3%, and accuracy and repeatability are good.
Example 5 detection of samples of methyl p-toluene sulfinate, ethyl p-toluene sulfinate, isopropyl p-toluene sulfinate in Haibumab
The maritima sample (20 mg) was weighed into a 20mL volumetric flask, dissolved with a proper amount of diluent, diluted to the scale with the diluent, and mixed uniformly, and the detection results are shown in Table 7.
TABLE 7 sample detection results
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From the above experimental results, no methyl p-toluenesulfinate, ethyl p-toluenesulfinate, isopropyl p-toluenesulfinate were detected in the Haibuji samples, and the detection limit was reported to be smaller.
The above embodiments can show that the detection method provided by the invention adopts a sample direct dissolution mode, and sample pretreatment is simple; the methyl paratoluenesulfinate, the ethyl paratoluenesulfinate and the isopropyl paratoluenesulfinate are completely and effectively separated from the sample within 9min, and the separation degree is good; the linear correlation coefficients r of methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate are all more than 0.999, and the linear correlation coefficients are good; the minimum quantitative limit of the instrument detection is 1.3ng/mL, the minimum detection limit is 0.4ng/mL, and the sensitivity is high; the standard adding accuracy recovery rate of the methyl p-toluenesulfonate, the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate is 90-110%, and the recovery rate is high. The method has the characteristics of rapidness, high efficiency, high sensitivity, no matrix interference in sample detection, good durability and the like, and can be suitable for detecting the methyl p-toluenesulfonate, the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate in the medicine.
Claims (28)
1. A method for simultaneously detecting methyl p-toluenesulfinate, ethyl p-toluenesulfinate and isopropyl p-toluenesulfinate in a medicament, the method comprising:
step 1: preparing a reference substance solution by adding a diluent into the reference substances of the methyl paratoluenesulfinate, the ethyl paratoluenesulfinate and the isopropyl paratoluenesulfinate;
step 2: adding a diluent into a sample to be tested to prepare a sample solution to be tested;
step 3: detecting the reference substance solution in the step 1 and the sample solution in the step 2 by adopting an ultra-high performance liquid chromatography-mass spectrometer, and determining the contents of methyl p-toluenesulfonate, ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the sample according to an external standard method;
wherein, the chromatographic conditions of the ultra performance liquid chromatography in the step 3 are as follows:
chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
mobile phase: mobile phase A is 0.002-0.05 moL/L ammonium formate aqueous solution, mobile phase B is methanol; the elution mode of the mobile phase is gradient elution, and the gradient elution is carried out according to the following procedures:
The medicine is Haibomab.
2. The method of claim 1, wherein the diluent in step 1 or step 2 is 10% to 90% acetonitrile in water (V/V).
3. The method of claim 1, wherein the diluent in step 1 or step 2 is 30% to 70% acetonitrile in water (V/V).
4. The method of claim 1, wherein the diluent in step 1 or step 2 is 40% to 60% acetonitrile in water (V/V).
5. The method of claim 1, wherein the diluent in step 1 or step 2 is 50% acetonitrile in water (V/V).
6. The method of any one of claims 1 to 5, wherein the octadecylsilane chemically bonded silica column in step 3 has a column diameter of 2.1mm to 4.6mm, a column length of 50mm to 150mm, and a particle diameter of 1.6 μm to 3.5 μm.
7. The method according to claim 6, wherein the octadecylsilane chemically bonded silica column in step 3 has a column diameter of 2.1mm to 3.5mm, a column length of 50mm to 100mm, and a particle diameter of 1.6 μm to 2.5. Mu.m.
8. The method according to claim 6, wherein the octadecylsilane chemically bonded silica column in step 3 has a column diameter of 2.1mm to 3.0mm, a column length of 50mm to 100mm, and a particle diameter of 1.7 μm to 2.5. Mu.m.
9. The method of claim 6, wherein the octadecylsilane chemically bonded silica column of step 3 is Agilent Eclipse Plus C RRHD 50 x 2.1mm 1.8 μm.
10. The method of any one of claims 1-5 or 7-9, wherein the column temperature of the chromatographic column in step 3 is 25 ℃ to 45 ℃.
11. The method of claim 10, wherein the column temperature of the chromatographic column in step 3 is from 30 ℃ to 45 ℃.
12. The method of claim 10, wherein the column temperature of the chromatographic column in step 3 is from 30 ℃ to 40 ℃.
13. The method of claim 10, wherein the column temperature of the chromatographic column in step 3 is 35 ℃.
14. The method of any one of claims 1-5, 7-9, or 11-13, wherein the mobile phase flow rate in step 3 is from 0.2mL/min to 0.6mL/min.
15. The method of claim 14, wherein the mobile phase in step 3 has a flow rate of 0.3mL/min to 0.5mL/min.
16. The method of claim 14, wherein the mobile phase in step 3 has a flow rate of 0.3mL/min to 0.4m L/min.
17. The method of claim 14, wherein the mobile phase in step 3 has a flow rate of 0.4mL/min.
18. The process of any one of claims 1-5, 7-9, 11-13 or 15-17, wherein mobile phase a in step 3 is 0.005 to 0.02moL/L ammonium formate in water.
19. The method of claim 18, wherein the mobile phase a in step 3 is 0.01moL/L ammonium formate aqueous solution.
20. The method of any one of claims 1-5, 7-9, 11-13, 15-17, or 19, wherein the parameters of the mass spectrum in step 3 are:
scanning mode: monitoring mass spectrum multiple reactions; ion source: an electrospray ion source; ion source mode: a positive mode; capillary voltage: 0.5 KV to 3.0KV; drying gas temperature: 300-650 ℃; drying gas flow rate: 1000L/Hr; taper hole voltage: 15-40V; source temperature: 150 ℃.
21. The method of claim 20, wherein the capillary voltage is 1.0KV to 2.0KV.
22. The method of claim 20, wherein the capillary voltage is 1.0KV.
23. The method of claim 20, wherein the drying gas temperature is 350-600 ℃.
24. The method of claim 20, wherein the drying gas temperature is 400 to 550 ℃.
25. The method of claim 20, wherein the drying gas temperature is 500 ℃.
26. The method of claim 20, wherein the cone voltage is 20-30V.
27. The method of claim 20, wherein the cone voltage is 25V.
28. The method of any one of claims 1-5, 7-9, 11-13, 15-17, 19, or 21-27, wherein the ultra performance liquid chromatography-mass spectrometer used in step 3 is Waters ACQUITYIUPLC Class-TQ-S micro.
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