CN111007184A - Method for detecting content of 4-methylpiperazine-1-formyl chloride hydrochloride - Google Patents
Method for detecting content of 4-methylpiperazine-1-formyl chloride hydrochloride Download PDFInfo
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Abstract
The invention relates to a method for detecting the content of 4-methylpiperazine-1-formyl chloride hydrochloride, which comprises the following steps: 1) preparation of a standard solution: weighing 10-15mg of a 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing the standard substance in a 25ml brown volumetric flask, dissolving the standard substance in methanol, diluting the solution to a scale, and shaking up; 2) preparing a test solution: weighing 10-15mg of a 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 25ml brown volumetric flask, dissolving the sample with methanol, diluting the sample to a scale, and shaking up; 3) content determination: respectively and precisely measuring 10 mul of standard sample and sample solution, injecting into a liquid chromatograph, wherein the sample injection time is at least 2 times of the retention time of the main peak, and calculating according to an area normalization method.
Description
Technical Field
The invention relates to the field of analytical chemistry, and particularly relates to a method for detecting the content of 4-methylpiperazine-1-formyl chloride hydrochloride.
Background
1-chloroformyl-4-methylpiperazine hydrochloride, also known as: 4-methylpiperazine-1-formyl chloride hydrochloride having the following structural formula:
CAS:55112-42-0。
1-chloroformyl-4-methylpiperazine hydrochloride was used in the prior art for the synthesis of zopiclone or eszopiclone, such as: chinese patent No. 200710115636.0 (publication No. CN101195624A, entitled method for preparing zopiclone) discloses a method for preparing zopiclone by reacting 6- (5-chloro-2-pyridyl) -5-hydroxy-7-oxo-5, 6-dihydropyrrolo [3, 4-b ] pyrazine with 1-chloroformyl-4-methylpiperazine hydrochloride as raw materials. Chinese patent 201110057089.1 (publication No. CN102675318A, entitled chiral synthesis of eszopiclone) and chinese patent 201110057090.4(CN102675319A) disclose a preparation method of zopiclone using 1-chloroformyl-4-methylpiperazine hydrochloride as a raw material.
4-methylpiperazine-1-carbonyl chloride hydrochloride as a synthetic material for zopiclone had a significant effect on the purity of the synthetic zopiclone.
4-methylpiperazine-1-formyl chloride hydrochloride is mainly prepared from N-methylpiperazine and phosgene (carbonyl chloride, CoCl2) or diphosgene, and Chinese patent No. 03129559.2(CN1566105A) discloses a method for preparing the compound by reacting N-methylpiperazine and bis (trichloromethyl) carbonate in an organic solvent, wherein the main factors influencing the purity of the compound comprise intermediates generated in the reaction process, reaction raw materials, solvents, degradation products in the storage process and the like.
In order to enable the detection method of the 4-methylpiperazine-1-formyl chloride hydrochloride to be faster, more accurate and more efficient, the inventor obtains a novel high-efficiency liquid-phase content determination method of the 4-methylpiperazine-1-formyl chloride hydrochloride through research and screening.
Disclosure of Invention
The invention aims to provide a novel method for detecting the content of 4-methylpiperazine-1-formyl chloride hydrochloride.
The method of the invention also adopts high performance liquid chromatography, and is different from the prior art, the method adopts methanol to dissolve 4-methyl piperazine-1-formyl chloride hydrochloride to prepare solution, and then standard solution and test solution are obtained to be injected into a high performance liquid chromatography instrument, the invention also improves the chromatographic conditions, and the detection method of the invention is specifically described as follows:
a method for detecting the content of 4-methylpiperazine-1-formyl chloride hydrochloride comprises the following steps:
1) preparation of a standard solution:
weighing 10-15mg of a 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing the standard substance in a 25ml brown volumetric flask, dissolving the standard substance in methanol, diluting the solution to a scale, and shaking up;
2) preparing a test solution:
weighing 10-15mg of a 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 25ml brown volumetric flask, dissolving the sample with methanol, diluting the sample to a scale, and shaking up;
3) the determination method comprises the following steps:
the chromatographic conditions are as follows: the chromatographic column is an amino chromatographic column; mobile phase: acetonitrile-acidic water (58-62: 42-38); detection wavelength: 198-; flow rate: 0.8-1.2 ml/min; column temperature: normal temperature;
4) and (3) determination: respectively and precisely measuring 10 mul of standard sample and sample solution, injecting into a liquid chromatograph, wherein the sample injection time is at least 2 times of the retention time of the main peak, and calculating according to an area normalization method.
The method comprises the following steps:
the amino chromatographic column in the step 3) is Waters Xbridge Amide;
the preparation method of the acidic water comprises the following steps: adding phosphoric acid to the purified water to adjust pH to 2.85-2.75, preferably pH2.8, filtering with filter membrane, and degassing.
Preferably, the method comprises the steps of:
1) preparation of a standard solution:
weighing 12.5mg of a 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing the standard substance in a 25ml brown volumetric flask, dissolving the standard substance in methanol, diluting the solution to a scale, and shaking up;
2) preparing a test solution:
weighing 12.5mg of a 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 25ml brown volumetric flask, dissolving the sample with methanol, diluting the sample to a scale, and shaking up;
3) the chromatographic conditions are
A chromatographic column: waters Xbridge Amide, 3.5 μm, 4.6 х 250 mm; mobile phase: acetonitrile-acidic water at pH2.8 (60: 40); detection wavelength: 199 nm; flow rate: 1.0 ml/min; column temperature: normal temperature;
4) and (3) determination: respectively and precisely measuring 10 mul of standard sample and sample solution, injecting into a liquid chromatograph, wherein the sample injection time is at least 2 times of the retention time of the main peak, and calculating according to an area normalization method.
The method of the invention is obtained by screening, and the screening process is as follows:
first, screening of solvent species
The chromatographic conditions were set as follows: a chromatographic column: c18, 250mm × 4.6mm, 5 μm, mobile phase: acetonitrile + buffer (potassium dihydrogen phosphate 0.8 g in 1000ml water) + phosphoric acid ═ 10+90+0.1 (V/V), filtered through a filter membrane and degassed; the detection wavelength is 220nm, the flow rate is 1.0ml/min, the column temperature is normal temperature.
And (3) respectively selecting different solvents to perform dissolution test on the sample under the chromatographic conditions:
1. solvent 1: acetonitrile: potassium dihydrogen phosphate buffer: phosphoric acid (10:90:0.1)
The method specifically comprises the following steps: acetonitrile + buffer (potassium dihydrogen phosphate 0.8 g in 1000ml of water) + phosphoric acid ═ 10+90+ 0.1.
Preparing a test solution: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 50ml volumetric flask, adding a mixed solution of acetonitrile, potassium dihydrogen phosphate buffer solution and phosphoric acid (the volume ratio is 10:90:0.1), carrying out ultrasonic dissolution, cooling to room temperature, adding a mobile phase to dilute to a scale, shaking up, and carrying out continuous sampling for 5 needles.
2. Solvent 2, 3: respectively methanol and acetonitrile
The solution 1 in the preparation of the above sample solution was replaced with methanol, acetonitrile, respectively, namely: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 50ml volumetric flask, respectively adding methanol and acetonitrile, ultrasonically dissolving, cooling to room temperature, adding methanol or acetonitrile to dilute to a scale, shaking up, and continuously injecting samples for 5 needles.
3. As a result: compared with chromatograms (fig. 1, fig. 2 and fig. 3, wherein fig. 1 is solvent 1, fig. 2 is methanol and fig. 3 is acetonitrile), the sample peak patterns of methanol and solvent 1 are better.
4. To further compare the effects of methanol and solvent 1, the inventors repeatedly injected 5 samples of methanol neutralized solvent 1 and examined the reproducibility of the sample peaks in methanol and solvent 1, and the results are shown in table 1.
Table 1: peak area comparison of samples from different solvents
| Kind of solvent | 1 | 2 | 3 | 4 | 5 | RSD％ |
| Solvent 1 | 512285 | 442936 | 383511 | 328595 | 286171 | 23.0％ |
| Methanol | 5247657 | 5143062 | 5110128 | 5097919 | 5077793 | 1.3％ |
The following conclusions are drawn from table 1: compared with data, the sample is more stable in methanol, and the system repeatability is good. When 4-methylpiperazine-1-formyl chloride hydrochloride is dissolved in the solvent 1, the solution is unstable and is easily degraded. Therefore, the invention selects methanol as solvent.
Two, chromatographic column combined screening
The chromatographic conditions are the same as the previous solvent screening method, and screening is carried out on the basis of the conditions:
1. condition 1: mobile phase acetonitrile, potassium dihydrogen phosphate buffer and phosphoric acid (10:90:0.1 by volume, supra) adjusted ph4.25 prior to use with a C18 chromatography column. Sample solution: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 50ml volumetric flask, adding methanol, ultrasonically dissolving, cooling to room temperature, shaking up, and injecting samples.
2. Condition 2: mobile phase acetonitrile, potassium dihydrogen phosphate buffer and phosphoric acid (volume ratio 10:90:0.1) were adjusted to ph5.5 and then combined with a C18 column. Sample solution: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 50ml volumetric flask, adding methanol, ultrasonically dissolving, cooling to room temperature, shaking up, and injecting samples.
3. Condition 3: mobile phase acetonitrile, potassium dihydrogen phosphate buffer and phosphoric acid (volume ratio 10:90:0.1) were adjusted to ph5.0 and then combined with a C18 column. Sample solution: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 50ml volumetric flask, adding methanol, ultrasonically dissolving, cooling to room temperature, shaking up, and injecting samples.
4. Condition 4: mobile phase acetonitrile, potassium dihydrogen phosphate buffer and phosphoric acid (volume ratio 10:90:0.1) were adjusted to ph6.4 and then combined with a C18 column. Sample solution: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 50ml volumetric flask, adding methanol, ultrasonically dissolving, cooling to room temperature, shaking up, and injecting samples.
Summarizing the chromatographic conditions, wherein a C18 column is generally applied to RP-HPLC, separating less polar or non-polar substances; for substances with larger polarity, the retention time is close to the dead time, and the quantitative analysis is not easy to realize due to the influence of a solvent peak.
The present invention contemplates the use of an amino column that provides suitable retention of strongly polar and strongly hydrophilic compounds, the mechanism of which mainly includes the following three aspects: (1) partitioning mechanism (2) ion exchange (3) dipole-dipole interaction. There are also phenomena indicating that the mechanism of HILIC retention involves multiple secondary effects such as hydrogen bonding, dipolar interactions and electrostatic interactions, which are difficult to distinguish. Regardless of the mechanism, only the separating ability is of interest. Acetonitrile-water system is generally adopted as a mobile phase, wherein the proportion of the water phase is 20-60% to ensure the obvious hydrophilic effect of the mobile phase and realize the separation of strong polar substances. Quantitative analysis is realized by adjusting the proportion of the water phase. So the test was continued using condition 5.
5. The conditions 5 acetonitrile-acidic water (60:40), pH2.8+ amino column was adjusted by adding phosphoric acid to the acidic water. Sample solution: weighing 12.53mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 25ml volumetric flask, adding methanol, ultrasonically dissolving, cooling to room temperature, shaking up and injecting. (this sample solution, unlike the sample solutions of conditions 1 to 4, increases in the concentration of the sample solution, can improve the sensitivity of the method, but the increase in the concentration has no effect on the peak shape and the degree of separation, and this condition improves the peak shape, the degree of separation, and the stability of the solution mainly by changing the column and the mobile phase)
And (4) conclusion: from the chromatograms (fig. 4, 5, 6, 7, 8), it was found that the peak patterns of the mobile phase ph2.8 and the amino column were more effective in terms of the degree of separation, and therefore the mobile phase acetonitrile-acidic water (60:40) ph2.8 and the amino column were selected.
Thirdly, screening sample injection amount
1、20μ1：
1.1 sample solution: weighing 12.5mg of a 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 25ml volumetric flask, adding methanol, ultrasonically dissolving, cooling to room temperature, shaking up, and continuously injecting a sample of 6 needles.
The 1.2 chromatogram is shown in FIG. 9, and FIG. 9 is a representative chromatogram.
Data results are shown in table 2:
table 2: data with a sample size of 20 μ 1
| Numbering | Peak area | Number of theoretical plate | Degree of separation |
| 1 | 17559270 | 6101 | 2.0 |
| 2 | 17345890 | 6335 | 2.0 |
| 3 | 17531653 | 6191 | 1.9 |
| 4 | 17329319 | 6324 | 2.0 |
| 5 | 17510893 | 6225 | 1.9 |
| 6 | 17505049 | 6206 | 1.9 |
Table 2 data the results illustrate: the system has good applicability.
2、10μ1：
2.1 sample solution: weighing 12.5mg of a 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 25ml volumetric flask, adding methanol, carrying out ultrasonic dissolution, cooling to room temperature, shaking up, and injecting samples.
2.2 chromatogram is shown in FIG. 10, and FIG. 10 is a representative chromatogram.
Data results are shown in table 3:
table 3: data with a sample size of 10 μ 1
| Numbering | Peak area | Number of theoretical plate | Degree of separation |
| 1 | 8665236 | 11208 | 2.5 |
| 2 | 8648206 | 11152 | 2.6 |
| 3 | 8508247 | 11515 | 2.6 |
| 4 | 8689819 | 11126 | 2.2 |
| 5 | 8690018 | 11093 | 2.5 |
| 6 | 8707492 | 10944 | 2.5 |
Comparison of sample size by table 2 and table 3: 10. mu.l is better than 20. mu.l theoretical plate number and degree of separation, so a 10. mu.l sample size was chosen.
Third, methodology verification
The following chromatographic conditions were used: the chromatographic column is Waters Xbridge Amide, 3.5 μm, 4.6 × 250 mm; mobile phase: acetonitrile-acidic water (58: 42-62: 38); detection wavelength: 198-; flow rate: 0.8-1.2 ml/min; column temperature: and (5) normal temperature.
1. Specificity test
1.1 preparation of the solution:
blank solvent: methanol
Preparing a reference substance solution: accurately weighing a 12.54mg 4-methylpiperazine-1-formyl chloride hydrochloride reference substance to a 25ml brown volumetric flask, dissolving methanol, fixing the volume to a scale, and shaking up.
Preparing a sample solution: accurately weighing a 12.48mg sample of 4-methylpiperazine-1-formyl chloride hydrochloride into a 25ml brown volumetric flask, dissolving the sample with methanol to a constant volume to a scale, and shaking up.
And taking blank solvent, reference substance solution and sample solution for respective injection.
1.2 Experimental results: see fig. 11, 12, 13.
The results show that: the blank solvent was not perturbed at the control peak position and the target peak retention time was 6.164. The degree of separation of the target peak and impurity peak in the sample solution was 2.3.
The results show that: the method has good specificity.
2. Linear test
2.1 sample solution preparation:
preparation of a reference stock solution: the control sample was weighed precisely at 50.22mg, placed in a 50ml volumetric flask, dissolved in methanol, diluted to the mark and shaken well. Taking the corresponding volume of the stock solution according to the following table 4, adding the stock solution into a corresponding volumetric flask, adding methanol to dilute the stock solution to the scale, shaking the stock solution uniformly, taking 10 mu l of the solution respectively, and recording the chromatogram. Three injections were injected per concentration. Taking the peak area (A) of each concentration as the ordinate and the mass concentration (C, mg/ml) as the abscissa, linear regression was performed to draw a standard curve.
2.2 the results of the experiment are shown in Table 4:
table 4: linear assay dilution method:
| numbering | Volume ml of stock solution | Volumetric flask volume ml | Diluted concentration mg/ml |
| 1 | 5 | 20 | 0.2483 |
| 2 | 4 | 10 | 0.3973 |
| 3 | 5 | 10 | 0.4967 |
| 4 | 6 | 10 | 0.5960 |
| 5 | 8 | 10 | 0.7947 |
Table 4 the results show: Y18246482.0X + 241783.8; r is 0.9999
3. Accuracy test
3.1 sample solution preparation:
preparation of a reference stock solution: a control (purity: 98.9%) was weighed precisely at 50.22mg, placed in a 50ml volumetric flask, dissolved in methanol, diluted to the mark and shaken well. The diluted stock solution is test solution with different known concentrations, 10 mu l of the solution is respectively taken, chromatogram is recorded, and the sample recovery rate is calculated.
3.2 the results are shown in Table 5:
table 5: and (3) an accuracy test result:
| numbering | Added in an amount of mg | Measured value mg | The recovery rate is high |
| 1 | 4.0176 | 4.0055 | 99.7 |
| 2 | 4.0176 | 4.0015 | 99.6 |
| 3 | 4.0176 | 4.0015 | 99.6 |
| 4 | 5.0220 | 4.9969 | 99.5 |
| 5 | 5.0220 | 4.9969 | 99.5 |
| 6 | 5.0220 | 4.9919 | 99.4 |
| 7 | 6.0264 | 5.9481 | 98.7 |
| 8 | 6.0264 | 6.0143 | 99.8 |
| 9 | 6.0264 | 6.0143 | 99.8 |
4. Precision test
4.1 Experimental methods: 6 parts of sample solution were prepared on different days by two analysts using different analysis instruments, purity was tested, and the relative standard deviation of the purity results and the relative deviation of the average of the two results were calculated for each analyst.
4.2 results are shown in Table 6
Table 6: results of precision measurement
Through the above experiments, it was finally confirmed that: the testing method provided by the invention has linearity and precision within the concentration range of 0.02483 mg/ml-0.7947 mg/ml, and has good accuracy.
The invention has the beneficial effects that:
the 4-methylpiperazine-1-formyl chloride hydrochloride is a small molecular compound and is extremely unstable in an aqueous solution, and the invention develops an HPLC method which has the advantages of good repeatability, high precision, good stability and simplicity and is used for testing the purity of the 4-methylpiperazine-1-formyl chloride hydrochloride.
Drawings
FIG. 1 is a high performance liquid chromatogram of 4-methylpiperazine-1-formyl chloride hydrochloride in solvent 1;
FIG. 2 is a high performance liquid chromatogram of 4-methylpiperazine-1-carbonyl chloride hydrochloride in methanol;
FIG. 3 is a high performance liquid chromatogram of 4-methylpiperazine-1-formyl chloride hydrochloride in acetonitrile;
FIG. 4 is a high performance liquid chromatogram of a mobile phase pH4.25+ C18 column;
FIG. 5 is a high performance liquid chromatogram of a mobile phase pH5.0+ C18 column;
FIG. 6 is a high performance liquid chromatogram of a mobile phase Ph5.5+ C18 column;
FIG. 7 is a high performance liquid chromatogram of a mobile phase pH6.44+ C18 column;
FIG. 8 is a high performance liquid chromatogram of a mobile phase Ph2.8+ amino chromatography column;
FIGS. 9 and 10 are high performance liquid chromatograms with sample volumes of 20. mu.l and 10. mu.l, respectively;
FIG. 11, FIG. 12, FIG. 13 are high performance liquid chromatograms of samples of methanol, 4-methylpiperazine-1-carbonyl chloride hydrochloride control and 4-methylpiperazine-1-carbonyl chloride hydrochloride, respectively, for a specificity test;
FIGS. 14-1, 14-2, and 14-3 are high performance liquid chromatograms of three batches of 4-methylpiperazine-1-carbonyl chloride hydrochloride samples.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The experiment involves instrument, reagent, sample and chromatographic column information:
1) high performance liquid chromatography, model e2695, brand Waters;
2) analytical balance, XS105, Mettler;
3) acetonitrile, grade chromatographically pure, lot number JA064320, source Merck;
4) phosphoric acid, grade analytical grade, lot number 20151121, source Tianjin Koimeu;
5) methanol, grade chromatographically pure, batch No. I0901407732, source Merck;
6) 4-methylpiperazine-1-formyl chloride hydrochloride standard, lot No. 1627151, source Aladin;
7) 4-methylpiperazine-1-carbonyl chloride hydrochloride sample, batch No.: 20170721, Y1803027, 17000318, from suppliers dongyo pharmaceuticals, inc.
Example 1: detection method of 4-methylpiperazine-1-formyl chloride hydrochloride
1. Preparation of a standard solution: 12.5mg of 4-methylpiperazine-1-formyl chloride hydrochloride standard substance is weighed, placed in a 25ml brown volumetric flask, dissolved by methanol, diluted to the scale and shaken up.
2. Preparing a test solution:
12.5mg of 4-methylpiperazine-1-carbonyl chloride hydrochloride sample is weighed out, placed in a 25ml brown volumetric flask, dissolved in methanol and diluted to the mark and shaken up.
3. The chromatographic conditions of the liquid chromatograph are
A chromatographic column: waters Xbridge Amide 3.5 μm 4.6 x 250 mm;
mobile phase: acetonitrile-acidic water (60:40) (also 58: 42-62: 38) was prepared as follows: adding phosphoric acid into purified water to adjust pH to 2.8, filtering with filter membrane, and degassing;
detection wavelength: 199 nm; flow rate: 1.0 ml/min; column temperature: and (5) normal temperature.
4. And (3) determination: respectively and precisely measuring 10 mul of standard sample and sample solution, injecting into a liquid chromatograph, wherein the sample injection time is at least 2 times of the retention time of a main peak, and the sample purity is not less than 95.0% calculated according to an area normalization method.
Experimental example 1: practical application of the method provided by the invention
1. See example 1 for the detection method.
2. The samples tested were three commercially available samples of 4-methylpiperazine-1-carbonyl chloride hydrochloride.
3. And (3) detection results: see FIGS. 14-1 through 14-3 and Table 7
Table 7: content detection of three batches of 4-methylpiperazine-1-formyl chloride hydrochloride
| Batch number | Content (wt.) |
| 20170721 | 98.0％ |
| Y1803027 | 99.7％ |
| 17000318 | 98.6％ |
Claims (5)
1. A method for detecting the content of 4-methylpiperazine-1-formyl chloride hydrochloride comprises the following steps:
1) preparation of a standard solution:
weighing 10-15mg of a 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing the standard substance in a 25ml brown volumetric flask, dissolving the standard substance in methanol, diluting the solution to a scale, and shaking up;
2) preparing a test solution:
weighing 10-15mg of a 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 25ml brown volumetric flask, dissolving the sample with methanol, diluting the sample to a scale, and shaking up;
3) the determination method comprises the following steps:
the chromatographic conditions are as follows: the chromatographic column is an amino chromatographic column; mobile phase: acetonitrile-acidic water (58-62: 42-38); detection wavelength: 198-; flow rate: 0.8-1.2 ml/min; column temperature: and (5) normal temperature.
4) And (3) determination: respectively and precisely measuring 10 mul of standard sample and sample solution, injecting into a liquid chromatograph, wherein the sample injection time is at least 2 times of the retention time of the main peak, and calculating according to an area normalization method.
2. The content detecting method according to claim 1, wherein the amino chromatographic column in the step 3) is Waters XBridgeamide.
3. The detection method according to claim 1, wherein the preparation method of the acidic water comprises: adding phosphoric acid into purified water to adjust pH to 2.85-2.75, filtering with filter membrane, and degassing.
4. The detection method according to claim 3, wherein the pH is 2.8.
5. The detection method according to claim 1, the method comprising the steps of:
1) preparation of a standard solution:
weighing 12.5mg of a 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing the standard substance in a 25ml brown volumetric flask, dissolving the standard substance in methanol, diluting the solution to a scale, and shaking up;
2) preparing a test solution:
weighing 12.5mg of a 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing the sample in a 25ml brown volumetric flask, dissolving the sample with methanol, diluting the sample to a scale, and shaking up;
3) the chromatographic conditions are
A chromatographic column: waters Xbridge Amide, 3.5 μm, 4.6 х 250 mm; mobile phase: acetonitrile-acidic water at pH2.8 (60: 40); detection wavelength: 199 nm; flow rate: 1.0 ml/min; column temperature: normal temperature;
4) and (3) determination: respectively and precisely measuring 10 mul of standard sample and sample solution, injecting into a liquid chromatograph, wherein the sample injection time is at least 2 times of the retention time of the main peak, and calculating according to an area normalization method.
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| CN114839308A (en) * | 2021-01-30 | 2022-08-02 | 江苏天士力帝益药业有限公司 | Method for detecting L-tryptophan methyl ester hydrochloride in D-tryptophan methyl ester hydrochloride |
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