CN115219621A - Method for quantitatively analyzing p-acetamido-benzenesulfonyl chloride based on derivatization method - Google Patents
Method for quantitatively analyzing p-acetamido-benzenesulfonyl chloride based on derivatization method Download PDFInfo
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- 238000001212 derivatisation Methods 0.000 title claims abstract description 57
- GRDXCFKBQWDAJH-UHFFFAOYSA-N 4-acetamidobenzenesulfonyl chloride Chemical compound CC(=O)NC1=CC=C(S(Cl)(=O)=O)C=C1 GRDXCFKBQWDAJH-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 36
- 239000000243 solution Substances 0.000 claims abstract description 32
- 229960005404 sulfamethoxazole Drugs 0.000 claims abstract description 26
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 21
- 238000012360 testing method Methods 0.000 claims abstract description 19
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 14
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 8
- VKPQLZPZPYQFOK-UHFFFAOYSA-N 2-acetamidobenzenesulfonyl chloride Chemical compound CC(=O)NC1=CC=CC=C1S(Cl)(=O)=O VKPQLZPZPYQFOK-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000012490 blank solution Substances 0.000 claims abstract description 3
- 238000012545 processing Methods 0.000 claims abstract description 3
- 238000003860 storage Methods 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- 239000000523 sample Substances 0.000 claims description 26
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000012535 impurity Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 10
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 238000009210 therapy by ultrasound Methods 0.000 claims description 8
- 239000013068 control sample Substances 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 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 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 229960001413 acetanilide Drugs 0.000 claims description 3
- 239000012467 final product Substances 0.000 claims description 3
- OXHFLUZEVFZEEF-UHFFFAOYSA-N acetonitrile;aniline Chemical compound CC#N.NC1=CC=CC=C1 OXHFLUZEVFZEEF-UHFFFAOYSA-N 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 239000007921 spray Substances 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- 238000004885 tandem mass spectrometry Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 239000007788 liquid Substances 0.000 abstract description 6
- 238000007865 diluting Methods 0.000 description 7
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000004949 mass spectrometry Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000013558 reference substance Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229960005489 paracetamol Drugs 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- ZQPVMSLLKQTRMG-UHFFFAOYSA-N 4-acetamidobenzenesulfonic acid Chemical compound CC(=O)NC1=CC=C(S(O)(=O)=O)C=C1 ZQPVMSLLKQTRMG-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 1
- FKPXGNGUVSHWQQ-UHFFFAOYSA-N 5-methyl-1,2-oxazol-3-amine Chemical compound CC1=CC(N)=NO1 FKPXGNGUVSHWQQ-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 150000001263 acyl chlorides Chemical class 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 108010014404 dihydrofolate synthetase Proteins 0.000 description 1
- OZRNSSUDZOLUSN-LBPRGKRZSA-N dihydrofolic acid Chemical compound N=1C=2C(=O)NC(N)=NC=2NCC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OZRNSSUDZOLUSN-LBPRGKRZSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical compound ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
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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
-
- 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
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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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- G01N30/72—Mass spectrometers
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Abstract
The invention relates to a derivatization method based on quantitative p-acetamido-benzenesulfonyl chloride, and belongs to the field of analytical chemistry. The method of the invention comprises the following steps: (1) preparing a derivative reagent; (2) preparing a derivative blank solution; (3) preparing a control group storage solution and a sample; (4) processing of the test sample: performing derivatization treatment on the acetamidobenzenesulfonyl chloride in the test sample by using a derivatization reagent; (5) preparing a sample to be marked; (6) quantitative analysis of the sample: and (3) detecting the sample by triple quadrupole liquid chromatography-mass spectrometry. The method adopts a derivatization method combined with a triple quadrupole liquid chromatograph-mass spectrometer to determine the content of p-acetamido-benzene sulfonyl chloride in sulfamethoxazole, the prepared derivative product is stable and easy to detect, and finally lower detection limit and higher sensitivity in the determination of p-acetamido-benzene sulfonyl chloride are realized.
Description
Technical Field
The invention relates to a method for quantitatively analyzing p-acetamido-benzenesulfonyl chloride based on a derivatization method, belonging to the field of analytical chemistry.
Background
Sulfamethoxazole, also known as sulfamethoxazole, is a broad-spectrum antibacterial drug, has strong effects on staphylococcus and escherichia coli, and is used for treating urinary tract infection, fowl cholera and the like. The sulfamethoxazole belongs to a middle-effect sulfonamide medicine applied to the whole body, and can act on dihydrofolate synthetase in a bacterial body in a competitive way with PABA to prevent the synthesis of bacterial dihydrofolate, thereby inhibiting the growth and the reproduction of bacteria.
At present, the production process of sulfamethoxazole comprises the following steps: is prepared from p-acetamido-benzenesulfonyl chloride and 3-amino-5-methylisoxazole through condensation and hydrolysis. Among the residual impurities, the p-acetamidobenzenesulfonyl chloride is genotoxic impurity with warning structure, and is impurity mainly removed in the purification process of sulfamethoxazole, and in view of the requirements of removing impurity and detecting reaction proceeding degree, the accurate quantitative analysis of p-acetamidobenzenesulfonyl chloride is an essential link, and the content limit of p-acetamidobenzenesulfonyl chloride is only 1.25ppm, and meanwhile, the property of acyl chloride is active, so that the p-acetamidobenzenesulfonyl chloride has very high reactivity, and the direct analysis and detection usually can produce very large error.
The conventional analysis methods for detecting p-acetamido-benzenesulfonyl chloride comprise Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC), and the sensitivity of the conventional analysis method is difficult to achieve during GC or HPLC determination
With the rapid development of analysis technology, the LC-MS technology has become the mainstream technology due to its advantages of strong selectivity, high sensitivity, simple sample pretreatment, etc. The method has certain advantages by using a hydrolysis method and a liquid chromatograph-mass spectrometer for determination, but p-acetamido-benzenesulfonyl chloride and water are hydrolyzed to generate p-acetamido-benzenesulfonic acid which is a main process impurity of sulfamethoxazole, and the original p-acetamido-benzenesulfonic acid can interfere with a determination result.
Therefore, finding a method to combine the advantages of LC-MS technology for accurate quantitative analysis of acetamidobenzenesulfonyl chloride is a problem to be solved.
Disclosure of Invention
The invention aims to provide a method for quantitatively analyzing p-acetamido benzene sulfonyl chloride based on a derivatization method, which comprises the steps of selecting aniline as a derivatization reagent, fully reacting the aniline with p-acetamido benzene sulfonyl chloride in sulfamethoxazole, and substituting chlorine atoms by amino groups to form amide in the presence of amino groups to obtain 4- (N-acetamido benzene sulfonyl) aniline, wherein the product property is stable, taking the 4- (N-acetamido benzene sulfonyl) aniline as the content index of the p-acetamido benzene sulfonyl chloride, and then measuring the content of the p-acetamido benzene sulfonyl chloride in the sulfamethoxazole by using a triple quadrupole liquid mass spectrometer, so that the sensitivity of the measurement result is improved, and the detection limit is greatly reduced.
The invention provides a method for quantitatively analyzing p-acetamido-benzenesulfonyl chloride based on a derivatization method, which comprises the following steps:
(1) Preparing a derivatization reagent;
(2) Preparing a derivative blank solution;
(3) Preparing a control group storage solution and a sample;
(4) Processing of the test sample: derivatization treatment is carried out on the acetamido benzene sulfonyl chloride in the sample to be tested by a derivatization reagent;
(5) Preparing a marked sample;
(6) Quantitative analysis of the sample: and (3) detecting the sample by triple quadrupole liquid chromatography-mass spectrometry. Wherein, the triple quadrupole liquid chromatography-mass spectrometry detection is to adopt a high performance liquid chromatography system-triple quadrupole mass spectrometer to separate and detect a sample.
Preferably, in (1), the derivatization reagent is 0.3% (volume concentration) aniline acetonitrile solution; the preparation conditions of the derivatization reagent are as follows: weighing aniline solution, placing in a measuring flask, adding acetonitrile to dissolve and dilute to scale, and shaking up to obtain the final product.
(3) The control group sample is 4- (N-acetamido-benzenesulfonyl) aniline solution with known concentration, and is obtained by adding a derivatization reagent into p-acetamido-benzenesulfonyl chloride solution for derivatization.
(4) The test sample is sulfamethoxazole containing p-acetamidobenzenesulfonyl chloride impurities, and the treatment steps comprise:
s1, weighing sulfamethoxazole containing p-acetamido-benzenesulfonyl chloride impurities;
s2, dissolving the sulfamethoxazole in acetonitrile;
s3 adding the derivatizing reagent of any one of claims 1 or 2 to acetonitrile-dissolved sulfamethoxazole;
s4, after adding a derivatization reagent, standing at room temperature for 0-30min or performing ultrasonic treatment for 0-45min or performing water bath oscillation for 0-45min, so that the p-acetamidobenzenesulfonyl chloride reacts with the derivatization reagent to generate the 4- (N-acetamidobenzenesulfonyl) aniline.
Furthermore, the method for quantifying p-acetamidobenzene sulfonyl chloride based on the derivatization method realizes the application of quantitatively analyzing the impurity content of p-acetamidobenzene sulfonyl chloride in sulfamethoxazole.
The quantitative analysis of the sample comprises: specificity, linearity and range, accuracy, precision (repeatability, system precision).
Preferably, the conditions of the high performance liquid chromatography system-triple quadrupole mass spectrometer are as follows:
a chromatographic column: waters Xbridge C 18 A column, 250 mm. Times.4.6 mm,5 μm;
the mobile phase is as follows: phase A: formic acid aqueous solution; phase B: acetonitrile; preferably, the concentration of the aqueous formic acid solution is 0.1% (volume concentration);
column temperature: 40 ℃;
flow rate: 1mL/min;
sample injection amount: 20 mu L of the solution;
gradient elution conditions:
| time (min) | Phase A (%) | Phase B (%) |
| 0.00 | 60 | 40 |
| 2.00 | 60 | 40 |
| 6.00 | 10 | 90 |
| 10.00 | 10 | 90 |
| 10.10 | 60 | 40 |
| 15.00 | 60 | 40 |
Preferably, the high performance liquid chromatography system-triple quadrupole mass spectrometer of the invention has the mass spectrum conditions:
ESI source positive ion detection mode, i.e. ESI (+);
in ESI (+) ionization mode:
spray voltage: 4500V;
atomizing gas pressure: 60psi;
auxiliary gas pressure: 50psi;
air curtain air: 35psi;
source temperature: 500 ℃;
collision air pressure: 9psi.
Preferably, the mass spectrometric operating parameters for the acetaminophen sulfonyl chloride analysis in the ESI (+) ionization mode are shown in table 1 below:
table 1: mass spectrum working parameters of poly-p-acetamido benzene sulfonyl chloride in ESI (+) ionization mode
Further preferably, the steps of preparing the solution and reagents are as follows:
(a) Derivatizing reagent (0.3% aniline in acetonitrile): weighing 1.5mL of aniline solution, placing the aniline solution in a 500mL measuring flask, adding acetonitrile to dissolve and dilute the aniline solution to a scale, and shaking up to obtain the aniline solution;
(b) Reference stock solution: taking 25mg of p-acetamidobenzenesulfonyl chloride reference substance, precisely weighing, placing in a 200mL measuring flask, adding acetonitrile for dissolving and diluting to a scale, shaking up, precisely weighing 1mL, placing in a 50mL measuring flask, diluting with acetonitrile to a scale, shaking up, precisely weighing 1mL again, placing in a 50mL measuring flask, diluting with acetonitrile to a scale, and shaking up to obtain the p-acetamidobenzenesulfonyl chloride contrast substance;
(c) Control sample solution: precisely measuring 1mL of a reference substance stock solution, placing into a 20mL measuring flask, precisely adding 10mL of a derivatization reagent, shaking up, performing ultrasonic treatment for 30min, taking out, cooling to room temperature, diluting with water to a scale, and shaking up to obtain the reagent;
(d) Sample solution to be tested: taking 40mg of sulfamethoxazole, precisely weighing, placing in a 20mL measuring flask, adding 1mL of acetonitrile for dissolving, adding 10mL of derivatization reagent, shaking up, carrying out ultrasonic treatment for 30min, taking out, cooling to room temperature, diluting with water to a scale, and shaking up to obtain the sulfamethoxazole;
(e) Deriving a blank reagent: measuring acetonitrile 1mL, placing in a 20mL measuring flask, precisely adding derivatization reagent 10mL, shaking up, performing ultrasonic treatment for 30min, taking out, cooling to room temperature, diluting with water to a scale, and shaking up to obtain the final product;
(f) Adding a standard test sample solution: taking 40mg of sulfamethoxazole, precisely weighing, placing in a 20mL measuring flask, precisely adding 1.5mL of a reference substance stock solution for dissolution, adding 10mL of a derivatization reagent, shaking up, carrying out ultrasonic treatment for 30min, taking out, cooling to room temperature, diluting with water to a scale, and shaking up to obtain the sulfamethoxazole.
It should be understood that the above mentioned reagent and solvent are used for clarity and understanding, and are used in conventional dosage and common preparation container, and do not limit the total dosage of the solution of the present invention, and those skilled in the art can prepare other dosage according to the above mentioned ratio.
Further, the invention provides a method for calculating the content of p-acetamido-benzenesulfonyl chloride, which comprises the following steps:
precisely measuring the solutions with 20 μ L each, respectively injecting into a liquid chromatography-mass spectrometer, and calculating impurity content according to an external standard method.
The invention has the beneficial effects that:
(1) The derivatization method is combined with a triple quadrupole liquid chromatograph to determine the content of p-acetamido-benzenesulfonyl chloride in sulfamethoxazole, the derivatization product is stable, and the high sensitivity completely meets the determination requirement;
(2) The technical effect is excellent, the limit of the quantification of the p-acetamidobenzenesulfonyl chloride is 0.0049ng, the minimum detection limit is 0.0015ng, the p-acetamidobenzenesulfonyl chloride is in the range of 0.2449 ng/mL-4.899 ng/mL, which is equivalent to the range of 10% -200%, and the peak area and the concentration present a good linear relationship.
It will be understood by those skilled in the art that, unless otherwise specifically indicated, the numerical values set forth in the present specification may vary from 0 to ± 5% due to the presence of systematic errors.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a derivative blank map;
FIG. 2 is a graph of a p-acetamido-benzenesulfonyl chloride derivative product;
FIG. 3 is a test solution spectrum;
FIG. 4 is a graph of a solution of a labeled test article;
FIG. 5 is a graph comparing the amount of derivatizing agent added;
FIG. 6 is a comparison map of different derivative times;
FIG. 7 is a peak area comparison map for different ultrasonic conditions;
FIG. 8 is a comparison graph of peak areas under different water bath oscillation conditions;
FIG. 9 is a linear plot of the concentration of p-acetamidobenzenesulfonyl chloride versus the peak area.
Detailed Description
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1
When a liquid chromatograph-mass spectrometer is used for measuring a sample, an accurate mass-to-charge ratio (m/z) is required to achieve the effects of high sensitivity and low detection limit, so that a control sample solution (subjected to derivatization reaction) is used for mass spectrometry scanning, and the optimal mass spectrometry parameters, sample configuration and mass spectrometry scanning results are determined as shown in the following table:
table 2: selection of Mass Spectrometry parameters
Table 3: optimization of mass spectrometry parameters
When the detection ion (m/z) is 291.2/198.1, the sensitivity of the 4- (N-acetamidobenzenesulfonyl) aniline is high, the peak type is better (shown as a graph of an acetamidobenzenesulfonyl chloride derivative product in figure 2), and the mass spectrum parameter of the detection ion (m/z) is 291.2/198.1 is selected for determination.
Example 3
Discussing the optimal dosage of the derivatization reagent, so that the p-acetamido-benzenesulfonyl chloride is completely derivatized, the detection result is more accurate, and the test steps are as follows:
table 4: selection of the amount of derivatizing agent added
Table 5: results of measurements of different amounts of derivatizing reagent added
| Name (R) | Volume of derivatizing reagent (mL) | Peak area |
| Control sample solution (1) | 5 | 34000 |
| Control sample solution (2) | 10 | 48400 |
| Control sample solution (3) | 15 | 48000 |
As shown in FIG. 2, under the condition of standing at room temperature for 10 minutes, the peak areas of the control samples at 10mL and 15mL of the added amount of the derivatization reagent are substantially consistent (as shown in the comparison graph of the added amount of the derivatization reagent in FIG. 5), the control samples are completely derivatized at 10mL of the added amount of the derivatization reagent, and the added amount of the selected derivatization reagent is 10mL.
Example 4
The optimal derivatization reaction conditions are discussed, and the test conditions and the steps are as follows:
table 6: selection of derivatization reaction time
The results of the different derivatization reaction times are shown in the table below:
table 7: results of different derivatization reaction times
| Name (R) | Peak area | Name (R) | Peak area | Name(s) | Peak area |
| Room temperature for 0min | 33900 | Ultrasonic for 0min | 33900 | Shaking in water bath for 0min | 55900 |
| Room temperature for 10min | 49200 | Ultrasonic treatment for 10min | 54700 | Shaking in water bath for 10min | 56790 |
| Room temperature for 30min | 54700 | Ultrasonic treatment for 30min | 58000 | Shaking in water bath for 30min | 57550 |
| / | / | Ultrasonic for 45min | 56300 | Shaking in water bath for 45min | 58490 |
Under the ultrasonic condition, the derivatization reaction of the p-acetamidobenzenesulfonyl chloride is more complete, and the peak areas of the reference substance are basically consistent when the chromatogram is 30min and 45min through triple quadrupole liquid chromatography-mass spectrometry (as shown in a comparison graph under the room temperature condition of fig. 6, a comparison graph under the ultrasonic condition of fig. 7 and a comparison graph under the water bath oscillation condition of fig. 8), which indicates that the reference substance is completely derivatized when the chromatogram is 30min, and the derivatization condition is selected as ultrasonic 30min.
Example 5
The detection method comprises the following steps: the final detection method and the preparation method of each solution are determined by optimizing mass spectrum parameters, selecting the addition amount of the derivatization reagent and selecting the derivatization time, and are shown in the table below.
Table 8: determination of detection method
The results of the above table methods and parameter measurements are as follows:
table 9: test result of detection limit of p-acetamidobenzenesulfonyl chloride
Table 10: quantitative limit test result of p-acetamidobenzenesulfonyl chloride
The limit of quantification of acetaminophen is 0.0049ng, which corresponds to 0.0000122% (0.122 ppm) of the concentration of the test sample; the minimum detection limit is 0.0015ng, which is equivalent to 0.0000037% (0.037 ppm) of the concentration of the test sample, and the RSD of 6 repeated determinations is less than 10% at the quantification limit, wherein the quantification limit and the detection limit meet the determination requirements of p-acetamidobenzenesulfonyl chloride in sulfamethoxazole.
Table 11: stability results for p-acetamidobenzenesulfonyl chloride solution
| Time | Control solution |
| 0h | 8932 |
| 3h | 8915 |
| 5h | 8658 |
| 7h | 8517 |
| Mean value of | 8755.5 |
| RSD％ | 2.32 |
The reference solution is stable after being placed at room temperature for 7 hours, and the p-acetamido-benzenesulfonyl chloride derivative product has good stability and is easy to detect. In addition, the test samples were also tested and plotted as a linear relationship between peak areas corresponding to acetaminophen sulfonyl at different concentrations (FIG. 9), and the data results are shown in the following table:
table 12: linear relationship test of p-acetamidobenzenesulfonyl chloride
The p-acetamido-benzenesulfonic acid chloride is in the range of 0.2449 ng/mL-4.899 ng/mL, which is equivalent to the range of 10% -200%, and the peak area and the concentration present a good linear relationship. Through triple four-stage rod liquid-mass combined detection, a spectrum is shown in figure 4, a blank solvent and a test sample solution do not interfere with impurity detection, the sensitivity of the method meets the detection requirement, and the labeling recovery is better.
Claims (10)
1. A method for quantitatively analyzing p-acetamidobenzenesulfonyl chloride based on derivatization, comprising:
(1) Preparing a derivatization reagent;
(2) Preparing a derivative blank solution;
(3) Preparing a control group storage solution and a sample;
(4) Processing of the test sample: derivatization treatment is carried out on the acetamido benzene sulfonyl chloride in the sample to be tested by a derivatization reagent;
(5) Preparing a labeled test sample;
(6) Quantitative analysis of the sample: and detecting the sample by triple quadrupole liquid chromatography-mass spectrometry.
2. The method for quantitative analysis of p-acetamidobenzenesulfonyl chloride based on derivatization method as claimed in claim 1, wherein in (1), the derivatization reagent is 0.3% (volume concentration) aniline acetonitrile solution; the preparation conditions of the derivatization reagent are as follows: weighing aniline solution, placing in a measuring flask, adding acetonitrile to dissolve and dilute to scale, and shaking up to obtain the final product.
3. The method according to claim 1, wherein the control sample in (3) is 4- (N-acetamidobenzenesulfonyl) aniline solution with known concentration, and the control sample is obtained by adding a derivatization reagent into the p-acetamidobenzenesulfonyl chloride solution for derivatization.
4. The method for the quantitative analysis of p-acetamidobenzenesulfonyl chloride based on derivatization method as claimed in claim 1, wherein in (4), the sample is sulfamethoxazole containing p-acetamidobenzenesulfonyl chloride impurity, and the treatment step comprises:
s1, weighing sulfamethoxazole containing p-acetamido-benzenesulfonyl chloride impurities;
s2, dissolving the sulfamethoxazole in acetonitrile;
s3, adding sulfamethoxazole dissolved in acetonitrile into the derivatization reagent in the step (1);
s4, after adding a derivatization reagent, carrying out any operation of placing for 0-30min at room temperature or carrying out ultrasonic treatment for 0-45min or carrying out water bath oscillation for 0-45min, and enabling the p-acetamido benzene sulfonyl chloride to react with the derivatization reagent to generate the 4- (N-acetamido benzene sulfonyl) aniline.
5. The method for the quantitative analysis of p-acetamidobenzenesulfonyl chloride based on derivatization method as claimed in claim 1, wherein the triple quadrupole LC-MS test is performed by separating and testing the sample using HPLC-triple quadrupole MS-MS.
6. The method for the quantitative analysis of p-acetamidobenzenesulfonyl chloride based on derivatization according to claim 5, wherein the HPLC system conditions are as follows:
a chromatographic column: waters Xbridge C 18 Column, 250mm × 4.6mm,5 μm;
the mobile phase is as follows: phase A: formic acid aqueous solution; phase B: acetonitrile;
column temperature: 40 ℃;
flow rate: 1mL/min;
sample introduction amount: 20 mu L of the solution;
gradient elution conditions:
0 to 2min and 40 percent of B phase, wherein the A phase is eluted by 60 percent of B phase, 2 to 6min of A phase is uniformly reduced to 10 percent from 60 percent, the B phase is uniformly increased to 90 percent from 40 percent, 6 to 10min is kept to be 10 percent, the A phase is eluted by 90 percent of B phase, 10 to 10.10minA phase is uniformly increased to 60 percent from 10 percent, the B phase is uniformly reduced to 40 percent from 90 percent, and 10.10 to 15min is kept to be 60 percent, the B phase is eluted by 40 percent, and the column balance is carried out by 60 percent of A phase.
7. The method of claim 5, wherein the mass spectrometric conditions comprise:
ESI source positive ion detection mode, i.e. ESI (+);
while in ESI (+) ionization mode:
spray voltage: 4500V;
atomizing gas pressure: 60psi;
auxiliary gas pressure: 50psi;
air curtain air: 35psi;
source temperature: 500 ℃;
collision air pressure: 9psi.
8. The method for quantitatively analyzing p-acetamidobenzenesulfonyl chloride based on derivatization according to claim 6, wherein the concentration of the aqueous formic acid solution is 0.1% (v/v).
9. An application of a derivatization method based on quantitative analysis of p-acetamido-benzenesulfonyl chloride in quantitative analysis of p-acetamido-benzenesulfonyl chloride impurity content in sulfamethoxazole is provided.
10. The use of the method of claim 9 for the quantitative analysis of the impurity content of p-acetamidobenzenesulfonyl chloride in sulfamethoxazole, wherein the quantitative analysis of the sample comprises: specificity, linearity and range, quantitation limit, detection limit, accuracy, repeatability, system precision.
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