CN111007184B - 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) Standard substance solution preparation: weighing 10-15mg of 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking; 2) Sample solution preparation: weighing 10-15mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking; 3) And (3) content measurement: respectively precisely measuring 10 μl of standard sample and sample solution, and 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, in particular 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-carbonyl chloride hydrochloride has 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, as follows: chinese patent 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 material. Chinese patent 201110057089.1 (publication No. CN102675318a, the chiral synthesis of eszopiclone) and chinese patent 201110057090.4 (CN 102675319 a) disclose a process for preparing zopiclone using 1-chloroformyl-4-methylpiperazine hydrochloride as starting material.
The purity of the 4-methylpiperazine-1-formyl chloride hydrochloride serving as a synthetic material of zopiclone has a great influence on the synthesis of zopiclone.
The 4-methylpiperazine-1-formyl chloride hydrochloride is mainly prepared from N-methylpiperazine and phosgene (carbonyl chloride, coCl 2) or diphosgene, and Chinese patent 03129559.2 (CN 1566105A) discloses a method for preparing the compound by reacting N-methylpiperazine and bis (trichloromethyl) carbonate as raw materials in an organic solvent, wherein the main factors influencing the purity of the compound are intermediates generated in the reaction process, reaction raw materials, solvents, degradation products in the storage process and the like.
In order to make the detection method of the 4-methylpiperazine-1-formyl chloride hydrochloride faster, more accurate and more efficient, the inventor obtains a novel method for measuring the high-efficiency liquid phase content 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 in that the method adopts methanol to dissolve 4-methylpiperazine-1-formyl chloride hydrochloride to prepare a solution, and then a standard solution and a sample solution are obtained for being injected into a high performance liquid chromatography instrument, and 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, comprising the following steps:
1) Standard substance solution preparation:
weighing 10-15mg of 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking;
2) Sample solution preparation:
weighing 10-15mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking;
3) Assay:
the chromatographic conditions are as follows: the chromatographic column is an amino chromatographic column; mobile phase: acetonitrile-sour water (58-62:42-38); detection wavelength: 198-201nm; flow rate: 0.8-1.2ml/min; column temperature: normal temperature;
4) And (3) measuring: respectively precisely measuring 10 μl of standard sample and sample solution, and 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: the purified water is taken, added with phosphoric acid to adjust the pH to 2.85-2.75, preferably pH2.8, filtered by a filter membrane and degassed.
Preferably, the method comprises the steps of:
1) Standard substance solution preparation:
weighing 12.5mg of 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking;
2) Sample solution preparation:
weighing 12.5mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking;
3) The chromatographic conditions are
Chromatographic column: waters XBridge Amide,3.5 μm,4.6 х mm; mobile phase: acetonitrile-acidic water at pH2.8 (60:40); detection wavelength: 199nm; flow rate: 1.0ml/min; column temperature: normal temperature;
4) And (3) measuring: respectively precisely measuring 10 μl of standard sample and sample solution, and 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 is obtained through screening, and the screening process is as follows:
1. screening of solvent species
The chromatographic conditions were set as follows: chromatographic column: c18 250 mm. Times.4.6 mm,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 respectively selecting different solvents under the chromatographic conditions to carry out dissolution test on the sample:
1. solvent 1: acetonitrile: potassium dihydrogen phosphate buffer: phosphoric acid (10:90:0.1)
The method comprises the following steps: acetonitrile + buffer (potassium dihydrogen phosphate 0.8 g in 1000ml water) +phosphoric acid=10+90+0.1.
Sample solution preparation: 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 (volume ratio is 10:90:0.1), performing ultrasonic dissolution, cooling to room temperature, adding a mobile phase to dilute to a scale, shaking uniformly, and continuously injecting 5 needles.
2. Solvents 2, 3: methanol and acetonitrile respectively
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 in a 50ml volumetric flask, respectively adding methanol and acetonitrile, performing ultrasonic dissolution, cooling to room temperature, adding methanol or acetonitrile to dilute to a scale, shaking uniformly, and continuously injecting into 5 needles.
3. Results: the peak patterns of the samples of methanol and solvent 1 are better compared by chromatograms (fig. 1, 2, 3, wherein fig. 1 is solvent 1, fig. 2 is methanol, and fig. 3 is acetonitrile).
4. To further compare the effect of methanol and solvent 1, the inventors repeatedly sampled the samples in methanol and solvent 1 for 5 needles, examined the reproducibility of the sample peaks in methanol and solvent 1, and the results are shown in table 1.
Table 1: peak area contrast for different solvent samples
| Solvent species | 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: the data comparison sample is more stable in methanol, and the system repeatability is good. When the solvent 1 dissolves the 4-methylpiperazine-1-formyl chloride hydrochloride, the solution is unstable and is easy to degrade. Therefore, the invention selects methanol as solvent.
2. Chromatographic column combination screening
Chromatographic conditions were the same as the previous solvent screening methods, screening was performed on the basis of the conditions:
1. condition 1: the mobile phase acetonitrile, potassium dihydrogen phosphate buffer and phosphoric acid (volume ratio 10:90:0.1, supra) were adjusted to pH4.25 and then used with a C18 chromatographic column. Sample solution: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing in a 50ml volumetric flask, adding methanol, performing ultrasonic dissolution, cooling to room temperature, shaking uniformly, and injecting sample.
2. Condition 2: the mobile phase acetonitrile, potassium dihydrogen phosphate buffer and phosphoric acid (volume ratio 10:90:0.1) were adjusted to pH5.5 and then used with a C18 chromatographic column. Sample solution: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing in a 50ml volumetric flask, adding methanol, performing ultrasonic dissolution, cooling to room temperature, shaking uniformly, and injecting sample.
3. Condition 3: the mobile phase acetonitrile, potassium dihydrogen phosphate buffer and phosphoric acid (volume ratio 10:90:0.1) were adjusted to pH5.0 and then used with a C18 chromatographic column. Sample solution: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing in a 50ml volumetric flask, adding methanol, performing ultrasonic dissolution, cooling to room temperature, shaking uniformly, and injecting sample.
4. Condition 4: the mobile phase acetonitrile, potassium dihydrogen phosphate buffer and phosphoric acid (volume ratio 10:90:0.1) were adjusted to pH6.4 and then used with a C18 chromatographic column. Sample solution: weighing 0.03g of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing in a 50ml volumetric flask, adding methanol, performing ultrasonic dissolution, cooling to room temperature, shaking uniformly, and injecting sample.
Summarizing chromatographic conditions, wherein the C18 column is typically used for RP-HPLC, separating less polar or non-polar materials; for substances with larger polarity, the retention time is close to dead time, and is influenced by solvent peaks, so that quantitative analysis is not easy to realize.
The present invention contemplates the use of amino columns which provide suitable retention for highly polar and highly hydrophilic compounds, the mechanism of which consists essentially of the following three aspects: (1) partitioning mechanism (2) ion exchange (3) dipole-dipole interactions. There are also phenomena that indicate that the retention mechanism of HILIC involves various secondary effects such as hydrogen bonding, dipole effect and electrostatic effect, which are difficult to distinguish. Irrespective of its mechanism, only its separation capacity is of concern. Acetonitrile-water system is generally adopted as mobile phase, wherein the proportion of water phase is 20% -60% to ensure the remarkable hydrophilic effect, and separation of strong polar substances is realized. Quantitative analysis is realized by adjusting the proportion of the water phase. The test was continued using condition 5.
5. Condition 5 acetonitrile-acidic water (60:40), acidic water plus phosphoric acid to adjust ph2.8+ amino column. Sample solution: 12.53mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample is weighed, placed in a 25ml volumetric flask, added with methanol, ultrasonically dissolved, cooled to room temperature, shaken well and injected. (this sample solution is different from the sample concentration in conditions 1 to 4 in that the concentration of the sample solution is increased to improve the sensitivity of the method, but the increase in concentration has no influence on the peak type and the degree of separation, and the conditions improve the peak type, the degree of separation, and the stability of the solution by mainly changing the column and the mobile phase)
Conclusion: from the chromatograms (FIGS. 4, 5,6, 7, 8), it can be judged that the peak types of the mobile phase Ph2.8 and the amino column, the separation degree and the like are better, so that the mobile phase acetonitrile-acidic water (60:40) Ph2.8 and the amino column are selected.
3. Sample size screening
1、20μ1：
1.1 sample solution: 12.5mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample is weighed, placed in a 25ml volumetric flask, added with methanol, ultrasonically dissolved, cooled to room temperature, shaken uniformly and continuously injected into 6 needles.
1.2 chromatograms are shown in fig. 9, with fig. 9 being a representative map.
The data results are shown in Table 2:
table 2: data with sample injection amount of 20 mu 1
| Numbering device | Peak area | Theoretical plate number | 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 results illustrate: the system has good applicability.
2、10μ1：
2.1 sample solution: 12.5mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample is weighed, placed in a 25ml volumetric flask, added with methanol, ultrasonically dissolved, cooled to room temperature, shaken well and injected.
2.2 chromatograms are shown in fig. 10, with fig. 10 being a representative map.
The data results are shown in Table 3:
table 3: data with sample injection amount of 10 mu 1
| Numbering device | Peak area | Theoretical plate number | 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 |
Sample injection amount comparison by table 2 and table 3: 10. Mu.l was better than 20. Mu.l theoretical plate number and degree of separation, so 10. Mu.l of the sample was selected.
3. Methodological verification
The following chromatographic conditions were used: the column was Waters XBridge Amide,3.5 μm,4.6 x 250mm; mobile phase: acetonitrile-sour water (58:42-62:38); detection wavelength: 198-201nm; flow rate: 0.8-1.2ml/min; column temperature: normal temperature.
1. Specificity test
1.1 preparation of solution:
blank solvent: methanol
Preparing a reference substance solution: the reference substance of the 4-methylpiperazine-1-formyl chloride hydrochloride is precisely weighed to be dissolved in methanol in a 25ml brown volumetric flask to be fixed to a scale, and then the mixture is shaken uniformly.
Sample solution preparation: a sample of 12.48mg of 4-methylpiperazine-1-carbonyl chloride hydrochloride was precisely weighed into a 25ml brown volumetric flask, dissolved in methanol to a fixed volume to a scale, and shaken well.
Taking blank solvent, and respectively injecting a reference substance solution and a sample solution.
1.2 experimental results: see fig. 11, 12, 13.
The results show that: the blank solvent did not interfere with the control peak position, target peak retention time 6.164. The separation degree of the target peak and the impurity peak in the sample solution was 2.3.
The results show that: the method has good specificity.
2. Linearity test
2.1 sample solution preparation:
preparation of control stock solution: the reference substance 50.22mg is precisely weighed, placed in a 50ml volumetric flask, dissolved with methanol, diluted to a scale and shaken well. The volume of the stock solution was taken as shown in Table 4, diluted with methanol to the scale, shaken well, and the chromatograms were recorded by taking 10. Mu.l of the above solutions, respectively. Each concentration was sampled three times. And (3) carrying out linear regression by taking the area (A) of each concentration peak as an ordinate and the mass concentration (C, mg/ml) as an abscissa, and drawing a standard curve.
2.2 experimental results are shown in Table 4:
table 4: linear test dilution method:
| numbering device | Stock solution volume ml | 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 that: y=18246482.0x+241783.8; r=0.9999
3. Accuracy test
3.1 sample solution preparation:
preparation of control stock solution: the control (purity: 98.9%) was precisely weighed at 50.22mg, placed in a 50ml volumetric flask, dissolved in methanol, diluted to scale and shaken well. The diluted stock solutions are test solutions with different known concentrations, 10 μl of the above solutions are respectively taken, a chromatogram is recorded, and the sample recovery rate is calculated.
3.2 results are shown in Table 5:
table 5: accuracy test results:
| numbering device | Added in an amount of mg | Measurement value mg | Recovery% |
| 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 method: the two analysts used different analytical instruments to prepare 6 portions of sample solution at different dates, test the purity, and calculate the relative standard deviation of the purity results for each person and the relative deviation of the average of the two results.
4.2 results are shown in Table 6
Table 6: results of precision test
Through the above experiments, it was finally confirmed that: the testing method provided by the invention has good linearity and precision and accuracy within the concentration range of 0.02483 mg/ml-0.7947 mg/ml.
The invention has the beneficial effects that:
the 4-methylpiperazine-1-formyl chloride hydrochloride is a small molecular compound and is extremely unstable in aqueous solution, and the invention develops a simple HPLC method for testing the purity of the 4-methylpiperazine-1-formyl chloride hydrochloride, which has good repeatability, high precision and good stability.
Drawings
FIG. 1 is a high performance liquid chromatogram of 4-methylpiperazine-1-carbonyl 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-carbonyl chloride hydrochloride in acetonitrile;
FIG. 4 is a high performance liquid chromatogram of a mobile phase pH4.25+C18 chromatographic column;
FIG. 5 is a high performance liquid chromatogram of a mobile phase pH5.0+C18 chromatographic column;
FIG. 6 is a high performance liquid chromatogram of a mobile phase Ph5.5+C18 chromatographic column;
FIG. 7 is a high performance liquid chromatogram of a mobile phase pH6.44+C18 chromatographic column;
FIG. 8 is a high performance liquid chromatogram of a mobile phase Ph2.8+ amino chromatographic column;
FIGS. 9 and 10 are high performance liquid chromatograms with sample injection amounts of 20. Mu.l and 10. Mu.l, respectively;
FIGS. 11, 12 and 13 are high performance liquid chromatograms of methanol, 4-methylpiperazine-1-formyl chloride hydrochloride control and 4-methylpiperazine-1-formyl chloride hydrochloride samples, respectively, in the specific test;
FIGS. 14-1, 14-2, 14-3 are high performance liquid chromatograms of three batches of 4-methylpiperazine-1-carbonyl chloride hydrochloride samples.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The experiments involved instrument, reagent, sample and column information:
1) High performance liquid chromatograph, model e2695, brands Waters;
2) Analytical balance, XS105, mettler;
3) Acetonitrile, grade chromatographic purity, lot number JA064320, source Merck;
4) Phosphoric acid, grade analytically pure, lot number 20151121, source of the Tianjin dense Europe;
5) Methanol, grade chromatographic purity, lot I0901407732, source Merck;
6) 4-methylpiperazine-1-carbonyl chloride hydrochloride standard, lot number 1627151, source Aladin;
7) 4-methylpiperazine-1-carbonyl chloride hydrochloride sample, lot number: 20170721, Y1803027, 17000318, from Chi Zhoudong liters of the supplier of the pharmaceutical company Limited.
Example 1: method for detecting 4-methylpiperazine-1-formyl chloride hydrochloride
1. Standard substance solution preparation: 12.5mg of 4-methylpiperazine-1-formyl chloride hydrochloride standard is weighed, placed in a 25ml brown volumetric flask, dissolved with methanol, diluted to a scale and shaken well.
2. Sample solution preparation:
12.5mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample was weighed, placed in a 25ml brown volumetric flask, dissolved with methanol, diluted to scale and shaken well.
3. The chromatographic conditions of the liquid chromatograph are that
Chromatographic column: waters XBridge Amide 3.5.5 μm 4.6×250mm;
mobile phase: acetonitrile-sour water (60:40) (58:42-62:38 also applicable), the sour water was prepared as follows: adding phosphoric acid into the purified water to adjust the pH to 2.8, filtering by a filter membrane, and degassing;
detection wavelength: 199nm; flow rate: 1.0ml/min; column temperature: normal temperature.
4. And (3) measuring: respectively precisely measuring 10 μl of standard sample and sample solution, and injecting into a liquid chromatograph, wherein the sample injection time is at least 2 times of the retention time of main peak, and the sample purity is not less than 95.0% calculated by area normalization method.
Experimental example 1: the method provided by the invention is practically applied
1. The detection method is described in example 1.
2. The test samples were three commercially available batches of 4-methylpiperazine-1-carbonyl chloride hydrochloride samples.
3. Detection result: see FIGS. 14-1 to 14-3 and Table 7
Table 7: three batches of content detection of 4-methylpiperazine-1-formyl chloride hydrochloride
| Lot number | Content of |
| 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, comprising the following steps:
1) Standard substance solution preparation:
weighing 10-15mg of 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking;
2) Sample solution preparation:
weighing 10-15mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking;
3) Assay:
the chromatographic conditions are as follows: the chromatographic column is an amino chromatographic column; mobile phase: the dosage ratio of acetonitrile to acidic water is 58-62:42-38; detection wavelength: 198-201nm; flow rate: 0.8-1.2ml/min; column temperature: normal temperature;
4) And (3) measuring: respectively precisely measuring 10 μl of standard sample and sample solution, and 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 method according to claim 1, wherein the amino chromatographic column in step 3) is Waters XBridge Amide.
3. The detection method according to claim 1, wherein the acidic water is prepared by: adding phosphoric acid into purified water to adjust the pH to 2.85-2.75, filtering with a 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) Standard substance solution preparation:
weighing 12.5mg of 4-methylpiperazine-1-formyl chloride hydrochloride standard substance, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking;
2) Sample solution preparation:
weighing 12.5mg of 4-methylpiperazine-1-formyl chloride hydrochloride sample, placing in a 25ml brown volumetric flask, dissolving with methanol, diluting to scale, and shaking;
3) The chromatographic conditions are
Chromatographic column: waters XBridge Amide,3.5 μm,4.6 х mm; mobile phase: the dosage ratio of the acid water of acetonitrile-PH 2.8 is 60:40; detection wavelength: 199nm; flow rate: 1.0ml/min; column temperature: normal temperature;
4) And (3) measuring: respectively precisely measuring 10 μl of standard sample and sample solution, and 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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