CN108375644B - Analysis method of neuromuscular blocker intermediate - Google Patents
Analysis method of neuromuscular blocker intermediate Download PDFInfo
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- CN108375644B CN108375644B CN201711268853.3A CN201711268853A CN108375644B CN 108375644 B CN108375644 B CN 108375644B CN 201711268853 A CN201711268853 A CN 201711268853A CN 108375644 B CN108375644 B CN 108375644B
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Abstract
A method for analyzing a neuromuscular blocker intermediate, the method comprising the step of isolating a micraconium chloride intermediate using high performance liquid chromatography. The method can effectively separate the intermediate 1-5 of the mikul ammonium chloride and impurities, simultaneously measures the contents of the intermediate 1-5 and the impurities, has high sensitivity and accuracy, has better separation degree and impurity detection limit, can be used for process control of the mikul ammonium chloride synthesis process, is beneficial to quality control of the finished mikul ammonium chloride products, can complete purity analysis and detection of the mikul ammonium chloride intermediate 1-5 at one time, is simple to operate, greatly simplifies the analysis process and the analysis time, improves the analysis efficiency, and saves the process period, material resources and labor cost.
Description
Technical Field
The invention relates to an analysis method of a neuromuscular blocker intermediate.
Background
Neuromuscular blockers provide skeletal muscle relaxation for surgery and tracheal intubation. Mickoamine is the shortest effective non-depolarizing neuromuscular blocker at present, and can be used as an auxiliary drug for general anesthesia to relax skeletal muscles so as to be beneficial to trachea intubation and mechanical ventilation. The accumulation is not obvious under clinical dosage, the histamine release promoting effect is small, no adverse effect is caused on intracranial pressure and intraocular pressure, the muscle relaxation concentration and range are easy to control, and the postoperative recovery is fast. The micstore ammonium chloride can be flexibly applied to various operations, and can be applied to patients with neuromuscular diseases and hyperkalemia besides general patients; especially, the cardiovascular disease of children is rarely influenced in pediatric operations, and the medicine is regarded as a substitute medicine of the succinylcholine. The good clinical appearance of mickoamine makes it more and more interesting in anesthesia surgery.
Mikamurammonium chloride, having a chemical name of Mivacurium chloride, (1R,1'R,) -2,2' - ((((E) -4-alkenylsuberoyl) bis (oxy)) bis (n-propyl-3, 1-disubstituted)) bis (6, 7-dimethoxy-2-methyl-1- (3,4, 5-trimethoxybenzyl) -1,2,3, 4-tetrahydroisoquinoline-2-quaternary ammonium) dichloride having a molecular formula of C58H80Cl2N2O14Molecular weight 1100.17, structural formula:
abbott, formerly available in the United states for the first time in 1992, is a white or off-white powder; has hygroscopicity, is very soluble in water or dichloromethane, and is almost insoluble in n-hexane.
1-5 intermediates are used in the synthesis of the mikul ammonium chloride raw material. Through research, when the intermediate of mickey ammonium chloride contains impurities, the impurities and the derived impurities thereof are introduced into the finally synthesized raw mickey ammonium chloride material through a series of reactions such as condensation, cyclization, reduction, methylation and the like, so that the quality of the product of the preparation related to mickey ammonium chloride is influenced.
If a correlation analysis method is only adopted in the quality standard of the mikul ammonium chloride bulk drug for detecting the impurities in the finished mikul ammonium chloride bulk drug product, the content of the impurities cannot be controlled from the source.
The literature (European Journal of Pharmaceutical Sciences,5(1997) 253-266) reports the synthetic procedure for the synthesis of intermediate 5 from intermediate 4, and the HPLC method which can only be used to monitor the reaction at this step and determine the purity of intermediate 5. The chromatographic column is 5mm silica column (Lichrosorb, E.Merck), the wavelength is 282nm, the mobile phase is acetonitrile-water-phosphoric acid (91:8:1), and the flow rate is 1.3-1.5 ml/min. The method has the defects that the method is only suitable for controlling the intermediate 5, but not suitable for other four intermediates, and the aim of complete process control on the whole synthesis of the mickojim cannot be achieved.
The content determination method of the intermediate 4 is reported in the literature (chemical reagent, 2013, 35(1), 94-96). The chromatographic column was a C18 column (Agilent 250 mm. times.4.6 mm, 5 μm), the mobile phase was acetonitrile-water (60:40), the detection wavelength was 281nm, the flow rate was 1.0mL/min, the column temperature was controlled at 25 ℃ and the calculation was performed by external standard method. The method is a content determination method of the intermediate 4, and a reference substance of the intermediate 4 is required to be taken to calculate the result. Firstly, the method is not beneficial to the impurity determination of the intermediate 4, and secondly, the purposes of efficiently and quickly obtaining results and providing data for process control cannot be achieved, so that the method cannot be applied to the process control of the synthesis process.
In view of the above published literature, the reported methods only allow analytical detection of one of the above-mentioned intermediates or process control of one of the reaction steps. No detection method is available for simultaneously carrying out quality analysis and detection on the 5 intermediates of the mikul ammonium chloride, and no detection method is available for carrying out process monitoring on the series of synthesis processes.
The structures of the 5 intermediates are very similar, and the separation of the 5 intermediates in the analysis method is a very challenging matter to perform process control on the synthetic route. Moreover, each intermediate is required to be subjected to quality control so as to meet the standard, and a corresponding analysis method is also urgently required to be established. In order to better achieve the process control of the mikul ammonium chloride synthesis process and the quality control of the mikul ammonium chloride intermediate, a reasonable and efficient analysis and detection method is needed to be established to control the steps of the synthesis process and detect and analyze the purity of the intermediate.
Disclosure of Invention
The invention creatively develops an analysis method which can detect the purity of five intermediates respectively or simultaneously and can realize the process control of a series of synthesis steps from the intermediate 1 to the intermediate 5.
The invention provides a separation method of a mickey ammonium chloride intermediate, which is characterized by comprising the step of separating the mickey ammonium chloride intermediate by adopting a high performance liquid chromatography, wherein the detection wavelength is 250-300 nm, and gradient elution is carried out by using a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution, the organic solvent is 100: 0-50: 50(v/v), the mobile phase B is an aqueous solution, the organic solvent is 0: 100-50: 50(v/v), and the gradient elution conditions are as follows:
a. the mobile phase B is changed from 0-30% to 45-100%, and the running time of the phase is 10-30 min;
b. on the basis of the stage a, the proportion of the mobile phase B is kept unchanged at 45-100%, and the running time of the stage is 10-30 min;
wherein, in each stage, the percentage of the mobile phase B changes or keeps unchanged along with the time, and the sum of the percentage of the mobile phase A and the percentage of the mobile phase B is 100 percent.
Optionally, the above separation method: in the high performance liquid chromatography, octadecyl bonded silica gel or octyl silane bonded silica gel is used as a stationary phase.
Optionally, the above separation method: the mobile phase A is an aqueous solution and an organic solvent is 95: 5-55: 45 (v/v).
Optionally, the above separation method: the mobile phase B is an aqueous solution and an organic solvent, wherein the ratio of the organic solvent to the mobile phase B is 5: 95-45: 55 (v/v).
Optionally, the aqueous solution in the mobile phase a and the mobile phase B is independently selected from at least one of pure water, acid-containing buffer, alkali-containing buffer, salt-containing buffer, and ion-pair-containing reagent aqueous solution.
Optionally, the aqueous solution in the mobile phase a and the mobile phase B is independently selected from at least one of pure water, acid-containing buffer, salt-containing buffer, and ion pair reagent-containing aqueous solution.
Optionally, the organic solvent in the mobile phase a and the mobile phase B is independently selected from at least one of acetonitrile and alcohols.
Optionally, the acid in the acid-containing buffer is selected from at least one of phosphoric acid, sulfuric acid, hydrochloric acid, formic acid, acetic acid, and trifluoroacetic acid.
Optionally, the acid in the acid-containing buffer is selected from at least one of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid.
Optionally, the concentration of the acid-containing aqueous solution is 0.005-2% (v/v), optionally 0.01-1% (v/v).
Optionally, the base in the base-containing buffer is at least one selected from ammonia, ammonia water, sodium hydroxide, potassium hydroxide, diethylamine and triethylamine.
Optionally, the salt in the salt-containing buffer is selected from at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium acetate, ammonium formate, and sodium acetate.
Optionally, the salt in the salt-containing buffer is selected from at least one of sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium acetate, ammonium formate, and sodium acetate.
Optionally, the concentration of the saline solution is 1.0mol/L to 0.005mol/L, optionally 0.5mol/L to 0.01 mol/L.
Optionally, the ion pair in the ion pair-containing reagent aqueous solution is selected from at least one of sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate and sodium decane sulfonate.
Optionally, the concentration of the ion pair-containing reagent aqueous solution is 0.001mol/L to 0.05mol/L, optionally 0.002mol/L to 0.01 mol/L.
Optionally, the pH of the aqueous solution is 2.0 to 10.0, optionally 2.0 to 7.0.
Optionally, the pH of the aqueous solution is adjusted by one or more of ammonia, phosphoric acid, sodium hydroxide, formic acid, acetic acid, potassium hydroxide, triethylamine, diethylamine, and trifluoroacetic acid.
Optionally, the alcohol is selected from at least one of methanol, ethanol, isopropanol.
Optionally, the above separation method, wherein the gradient elution conditions are:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100～70 | 0～30 |
| 5 | 80～60 | 20～40 |
| 10 | 70～40 | 30～60 |
| 15 | 65～20 | 35～80 |
| 20 | 55～0 | 45～100 |
| 40 | 55～0 | 45～100 |
Or
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100～80 | 0～20 |
| 5 | 80～60 | 20～40 |
| 15 | 70～40 | 30～60 |
| 20 | 50～20 | 50～80 |
| 30 | 30～0 | 70～100 |
| 50 | 30～0 | 70～100 |
Or
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100～75 | 0～25 |
| 10 | 80～60 | 20～40 |
| 15 | 65～40 | 35～60 |
| 25 | 50～15 | 50～85 |
| 30 | 35～0 | 65～100 |
| 45 | 35～0 | 65～100 |
Wherein the percentage of the mobile phase B changes or keeps unchanged along with the time, and the sum of the percentage of the mobile phase A and the percentage of the mobile phase B is 100 percent.
Optionally, the above separation method is characterized by:
the flow rate of the mobile phase is 0.5-2.0mL/min, optionally 0.5-1.5mL/min, or optionally 0.7-1.3 mL/min;
the detection wavelength is 260-290 nm, and can be 270-290 nm;
the sample amount is 1-100 μ L, optionally 5-50 μ L, or optionally 20 μ L.
Optionally, the separation method is characterized in that the micakammonium chloride intermediate comprises intermediate 1, intermediate 2, intermediate 3, intermediate 4, intermediate 5 selected from micakammonium chloride
The invention also provides a detection method of the mikuyammonium chloride intermediate, which is characterized by comprising the step of detecting the mikuyammonium chloride intermediate by adopting the separation method.
Optionally, the detection method comprises the following steps: taking a sample to be detected, preparing a sample solution, sucking the sample solution, injecting the sample solution into a high performance liquid chromatograph, and measuring by adopting the separation method;
optionally, the preparation of the test solution comprises the following steps: taking a sample to be tested, and preparing a test solution with the concentration of 0.1 mg/mL-5.0 mg/mL by using a diluent;
optionally, the diluent is selected from at least one of an aqueous solution and an organic solvent;
optionally, the aqueous solution is selected from at least one of pure water, acid-containing buffer, alkali-containing buffer, salt-containing buffer, and ion-pair reagent-containing aqueous solution;
optionally, the acid in the acid-containing buffer is selected from at least one of phosphoric acid, sulfuric acid, hydrochloric acid, formic acid, acetic acid and trifluoroacetic acid; optionally, the acid in the acid-containing buffer is selected from at least one of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid; optionally, the concentration of the acid-containing aqueous solution is 0.005-2% (v/v), optionally 0.01-1% (v/v);
optionally, the base in the base-containing buffer is selected from at least one of ammonia, ammonia water, sodium hydroxide, potassium hydroxide, diethylamine and triethylamine;
optionally, the salt in the salt-containing buffer is selected from at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium acetate, ammonium formate, sodium acetate; optionally, the concentration of the buffer solution containing salt is 1.0-0.001 mol/L, and preferably 0.5-0.005 mol/L;
optionally, the ion pair in the ion pair-containing reagent aqueous solution is selected from at least one of sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate and sodium decane sulfonate; optionally, the concentration of the ion pair-containing reagent aqueous solution is 0.001 mol/L-0.05 mol/L, optionally 0.002mol/L-0.01 mol/L;
optionally, the pH of the aqueous solution is 2.0-10.0, optionally 2.0-7.0;
optionally, the pH of the aqueous solution is adjusted by one or more of ammonia, phosphoric acid, sodium hydroxide, formic acid, acetic acid, potassium hydroxide, triethylamine, diethylamine, and trifluoroacetic acid.
Optionally, the detection method as described above, wherein the micakammonium chloride intermediate comprises intermediate 1, intermediate 2, intermediate 3, intermediate 4, intermediate 5 selected from micakammonium chloride
The invention also provides a purity determination method of the mikul ammonium chloride intermediate, which is characterized by comprising the step of determining the purity of the mikul ammonium chloride intermediate by adopting the detection method; optionally, the purity determination method is calculated by an area normalization method.
Optionally, the purity determination method as described above, wherein the micakammonium chloride intermediate comprises intermediate 1, intermediate 2, intermediate 3, intermediate 4, intermediate 5 selected from micakammonium chloride
The invention also provides a quality analysis method of mikul ammonium chloride, which is characterized by comprising a separation step, a detection step or a determination step of impurities, wherein the separation step comprises separation according to the separation method, the detection step comprises detection according to the detection method, and the determination step comprises determination according to the purity determination method.
The quality analysis method of mikuonium chloride can be applied to the analysis of impurities in mikuonium chloride bulk drugs or mikuonium chloride preparations.
The method for separating, detecting and measuring the content of the midbody of the mikamine has the following advantages: the method can effectively separate the intermediate 1-5 and impurities of the mikul ammonium chloride, can measure the respective contents of the intermediate 1-5 and the impurities, has high sensitivity, high accuracy and better separation degree and impurity detection limit, can realize the process control of a series of synthesis steps from the intermediate 1 to the intermediate 5, can be used for the process control of the mikul ammonium chloride synthesis process, is beneficial to the quality control of the finished mikul ammonium chloride product, can complete the purity analysis and detection of the mikul ammonium chloride intermediate 1-5 at one time, is simple to operate, greatly simplifies the analysis process and analysis time, improves the analysis efficiency, and saves the process period, material resources and labor cost.
Drawings
FIG. 1 is a high performance liquid chromatogram of a test solution of intermediate 1 of example 1;
FIG. 2 is a high performance liquid chromatogram of a test solution of intermediate 2 of example 1;
FIG. 3 is a high performance liquid chromatogram of a test solution of intermediate 3 of example 1;
FIG. 4 is a high performance liquid chromatogram of a test solution of intermediate 4 of example 1;
FIG. 5 is a high performance liquid chromatogram of a test solution of intermediate 5 of example 1;
FIG. 6 is a high performance liquid chromatogram of the mixed localization solution of intermediates 1-5 of example 1.
In the above figures, the ordinate is the response value in AU, and the abscissa is the retention time in min.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
Example 1
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: pure water-acetonitrile (95:5)
Mobile phase B: pure water-acetonitrile (25:75)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phaseB(％) |
| 0 | 100 | 0 |
| 5 | 80 | 20 |
| 10 | 70 | 30 |
| 15 | 65 | 35 |
| 20 | 55 | 45 |
| 40 | 55 | 45 |
Wavelength: 275nm
Flow rate: 0.5mL/min
Diluent agent: 0.45% aqueous acetic acid (adjusted to pH 4.0 with ammonia test solution) -acetonitrile (40:60)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 2.5mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring each sample solution by 5 μ L, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 2
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: 1% phosphoric acid in water (pH adjusted to 2.0 with phosphoric acid) -methanol (75:25)
Mobile phase B: 1% phosphoric acid in water (pH adjusted to 2.0 with phosphoric acid) -methanol (10:90)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 70 | 30 |
| 5 | 60 | 40 |
| 10 | 40 | 60 |
| 15 | 20 | 80 |
| 20 | 0 | 100 |
| 40 | 0 | 100 |
Wavelength: 260nm
Flow rate: 1.3mL/min
Diluent agent: 1% phosphoric acid in water (pH adjusted to 2.0 with phosphoric acid) -methanol (75:25)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 0.5mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring 10 μ L of each sample solution, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 3
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: 0.5% aqueous formic acid (pH adjusted to 4.6 with ammonia test solution) -methanol (90:10)
Mobile phase B: 0.5% aqueous formic acid (pH adjusted to 4.6 with ammonia test solution) -methanol (30:70)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 85 | 15 |
| 5 | 65 | 35 |
| 10 | 60 | 40 |
| 15 | 40 | 60 |
| 20 | 35 | 65 |
| 40 | 35 | 65 |
Wavelength: 285nm
Flow rate: 0.7mL/min
Diluent agent: 0.5% aqueous formic acid (pH adjusted to 4.6 with ammonia test solution) -methanol (90:10)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting by using a diluent to prepare a single intermediate solution containing 3.0mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: respectively and precisely measuring 10 μ L of test solution, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 4
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: 0.25% aqueous acetic acid (adjusted to pH 5.0 with Ammonia test solution) -methanol (95:5)
Mobile phase B: 0.25% aqueous acetic acid (adjusted to pH 5.0 with Ammonia test solution) -methanol (10:90)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 80 | 20 |
| 5 | 65 | 35 |
| 15 | 45 | 55 |
| 20 | 20 | 80 |
| 30 | 5 | 95 |
| 50 | 5 | 95 |
Wavelength: 280nm
Flow rate: 1.0mL/min
Diluent agent: pure water
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 1.0mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring 20 μ L of each sample solution, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 5
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: 0.01% aqueous trifluoroacetic acid (pH 3.0 adjusted with sodium hydroxide) -methanol-acetonitrile (60:30:10)
Mobile phase B: 0.01% trifluoroacetic acid in water (pH 3.0 adjusted with sodium hydroxide) -methanol-acetonitrile (20:60:20)
Gradient elution:
wavelength: 269nm
Flow rate: 1.5mL/min
Diluent agent: 0.01% aqueous trifluoroacetic acid (pH 3.0 adjusted with sodium hydroxide) -methanol-acetonitrile (60:30:10)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 0.1mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring each sample solution 40 μ L, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 6
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: 0.005mol/L aqueous sodium dihydrogen phosphate (adjusted to pH 7.0 with sodium hydroxide) -acetonitrile (85:15)
Mobile phase B: 0.005mol/L aqueous sodium dihydrogen phosphate (adjusted to pH 7.0 with sodium hydroxide) -acetonitrile (5:95)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 5 | 80 | 20 |
| 15 | 70 | 30 |
| 20 | 50 | 50 |
| 30 | 30 | 70 |
| 50 | 30 | 70 |
Wavelength: 265nm
Flow rate: 0.6mL/min
Diluent agent: 0.005mol/L aqueous sodium dihydrogen phosphate (adjusted to pH 7.0 with sodium hydroxide) -acetonitrile (85:15)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 2.5mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring each sample solution 25 μ L, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 7
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: 0.01mol/L aqueous potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) -methanol (90:10)
Mobile phase B: 0.01mol/L potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile (30:70)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 10 | 80 | 20 |
| 15 | 65 | 35 |
| 25 | 50 | 50 |
| 30 | 35 | 65 |
| 45 | 35 | 65 |
Wavelength: 280nm
Flow rate: 1.2mL/min
Diluent agent: 0.01mol/L potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) -acetonitrile (30:70)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 5.0mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring each sample solution by 15 μ L, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 8
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: 0.1mol/L ammonium formate solution in water (pH adjusted to 5.0 with formic acid or ammonia solution) -acetonitrile (55:45)
Mobile phase B: 0.1mol/L aqueous ammonium formate solution (pH adjusted to 5.0 with formic acid or ammonia solution) -methanol (40:60)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 75 | 25 |
| 10 | 60 | 40 |
| 15 | 40 | 60 |
| 25 | 15 | 85 |
| 30 | 0 | 100 |
| 45 | 0 | 100 |
Wavelength: 285nm
Flow rate: 0.7mL/min
Diluent agent: 0.1mol/L aqueous ammonium formate solution (pH adjusted to 5.0 with formic acid or ammonia solution) -methanol (40:60)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting by using a diluent to prepare a single intermediate solution containing 3.0mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring each sample solution by 30 μ L, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 9
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: 1.0mol/L aqueous ammonium acetate (adjusted to pH 3.0 with acetic acid) -methanol-acetonitrile (75:20:5)
Mobile phase B: 1.0mol/L aqueous ammonium acetate (adjusted to pH 3.0 with acetic acid) -methanol-acetonitrile (20:40:40)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 97 | 3 |
| 10 | 80 | 20 |
| 15 | 45 | 55 |
| 25 | 20 | 80 |
| 30 | 5 | 95 |
| 45 | 5 | 95 |
Wavelength: 290nm of
Flow rate: 1.0mL/min
Diluent agent: 1.0mol/L aqueous ammonium acetate solution (adjusted to pH 3.0 with acetic acid)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 2.0mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring 10 μ L of each sample solution, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 10
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: 0.5mol/L aqueous sodium acetate (adjusted to pH 4.5 with Ammonia test solution) -methanol-acetonitrile (60:30:10)
Mobile phase B: 0.5mol/L aqueous sodium acetate (adjusted to pH 4.5 with Ammonia test solution) -methanol-acetonitrile (45:50:5)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 10 | 70 | 30 |
| 15 | 52 | 48 |
| 25 | 40 | 60 |
| 30 | 25 | 75 |
| 45 | 25 | 75 |
Wavelength: 270nm
Flow rate: 0.9mL/min
Diluent agent: 0.5mol/L aqueous sodium acetate solution (adjusted to pH 4.5 with ammonia test solution)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 0.1mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring each sample solution 50 μ L, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 11
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: 0.001mol/L aqueous solution of sodium pentanesulfonate-acetonitrile (80:20)
Mobile phase B: 0.001mol/L aqueous solution of sodium pentanesulfonate-acetonitrile (45:55)
Gradient elution:
wavelength: 280nm
Flow rate: 1.0mL/min
Diluent agent: 0.001mol/L sodium pentane sulfonate water solution
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 0.5mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring 20 μ L of each sample solution, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 12
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: 0.05mol/L Pentanesulfonic acid sodium salt aqueous solution-methanol (90:10)
Mobile phase B: 0.05mol/L sodium pentane sulfonate aqueous solution-methanol (20:80)
Gradient elution:
wavelength: 282nm
Flow rate: 0.9mL/min
Diluent agent: 0.05mol/L Pentanesulfonic acid sodium salt aqueous solution-methanol (90:10)
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 1.0mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring each sample solution 25 μ L, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 13
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: 0.002mol/L sodium hexane sulfonate aqueous solution-methanol (95:5)
Mobile phase B: 0.002mol/L sodium hexanesulfonate aqueous solution-methanol (25:75)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 5 | 70 | 30 |
| 10 | 45 | 55 |
| 15 | 30 | 70 |
| 20 | 0 | 100 |
| 40 | 0 | 100 |
Wavelength: 275nm
Flow rate: 1.1mL/min
Diluent agent: 0.002mol/L sodium hexanesulfonate aqueous solution
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 0.8mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring 20 μ L of each sample solution, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
example 14
Main experimental instruments and chromatographic conditions:
an Agilent 1260-type high performance liquid chromatograph is adopted, a G1312X binary pump, a G1329B automatic temperature control autosampler, a G1316A column incubator and a G1315CD diode array detector are arranged, and an Agilent Chemstation chromatographic workstation is adopted as a chromatographic analysis processing system.
Chromatographic conditions are as follows:
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: 0.01mol/L aqueous solution of sodium decane sulfonate acetonitrile (65:35)
Mobile phase B: 0.01mol/L aqueous solution of sodium decane sulfonate acetonitrile (10:90)
Gradient elution:
| gradient (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 10 | 75 | 25 |
| 15 | 60 | 40 |
| 25 | 35 | 65 |
| 30 | 10 | 90 |
| 45 | 10 | 90 |
Wavelength: 285nm
Flow rate: 1.0mL/min
Diluent agent: 0.01mol/L aqueous solution of sodium decane sulfonate
Test solution: respectively weighing samples of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5, precisely weighing, and respectively dissolving and diluting with a diluent to prepare a single intermediate solution containing 1.0mg in each 1 mL; and taking a proper amount of the mixed samples of the intermediates 1-5, precisely weighing, and dissolving and diluting by using a diluent to prepare a mixed positioning solution containing 0.01mg of the mixed samples in every 1 mL.
The determination method comprises the following steps: precisely measuring each sample solution by 15 μ L, injecting into liquid chromatograph, and recording chromatogram. Calculated according to an area normalization method.
And (3) measuring results:
as can be seen from the results of the analytical tests on the single intermediate solution and the mixed localization solution in examples 1 to 14, the separation and detection method of the present invention can separate and detect the intermediates 1 to 5 well, respectively or simultaneously, and has good separation degree and impurity detection limit. According to the test result of the mixed positioning solution, the purity analysis and detection of the mikul ammonium chloride intermediates 1-5 can be completed at one time, the analysis process and the analysis time are greatly simplified, the analysis efficiency is improved, and the process period, the physical cost and the labor cost are saved. According to the test result of a single intermediate solution, the method also detects various impurities, and quantitatively analyzes the intermediate 1-5, the content of the impurity with the largest area in the impurity peak and the total impurity content, so that the separation, detection and content determination method can carry out more comprehensive and accurate analysis and detection on the mikul ammonium chloride, can be used for process control of the mikul ammonium chloride synthesis process, and is beneficial to quality control of the finished mikul ammonium chloride product.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
Claims (27)
1. A separation method of mickey ammonium chloride intermediate is characterized by comprising the step of separating the mickey ammonium chloride intermediate by adopting a high performance liquid chromatography, wherein the detection wavelength in the high performance liquid chromatography is 260-290 nm,
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: the volume ratio of pure water to acetonitrile is 95:5
Mobile phase B: the volume ratio of pure water to acetonitrile is 25:75
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: the volume ratio of 1% phosphoric acid aqueous solution to methanol adjusted to pH 2.0 with phosphoric acid was 75:25
Mobile phase B: the volume ratio of 1% phosphoric acid aqueous solution to methanol adjusted to pH 2.0 with phosphoric acid was 10:90
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: adjusting pH to 4.6 with ammonia test solution, and adjusting the volume ratio of 0.5% formic acid aqueous solution to methanol to be 90:10
Mobile phase B: adjusting pH to 4.6 with ammonia test solution, and adjusting the volume ratio of 0.5% formic acid aqueous solution-methanol to be 30:70
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: adjusting pH to 5.0 with ammonia test solution, and adjusting volume ratio of 0.25% acetic acid water solution-methanol to 95:5
Mobile phase B: adjusting pH to 5.0 with ammonia test solution, and adjusting volume ratio of 0.25% acetic acid water solution-methanol to be 10:90
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: adjusting pH to 3.0 with sodium hydroxide, and the volume ratio of 0.01% trifluoroacetic acid aqueous solution-methanol-acetonitrile is 60:30:10
Mobile phase B: adjusting pH to 3.0 with sodium hydroxide, and the volume ratio of 0.01% trifluoroacetic acid aqueous solution-methanol-acetonitrile is 20:60:20
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: adjusting pH to 7.0 with sodium hydroxide, wherein the volume ratio of 0.005mol/L sodium dihydrogen phosphate aqueous solution to acetonitrile is 85:15
Mobile phase B: adjusting the volume ratio of 0.005mol/L sodium dihydrogen phosphate aqueous solution to acetonitrile with sodium hydroxide to pH 7.0 to be 5:95
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: the volume ratio of 0.01mol/L potassium dihydrogen phosphate aqueous solution to methanol is adjusted to 2.5 by phosphoric acid, and the volume ratio is 90:10
Mobile phase B: adjusting the pH value to 2.5 by using phosphoric acid, wherein the volume ratio of 0.01mol/L potassium dihydrogen phosphate aqueous solution to acetonitrile is 30:70
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: adjusting the volume ratio of 0.1mol/L ammonium formate aqueous solution to acetonitrile with pH of 5.0 by using formic acid or ammonia test solution to be 55:45
Mobile phase B: adjusting the volume ratio of 0.1mol/L ammonium formate aqueous solution to methanol with pH of 5.0 by using formic acid or ammonia test solution to be 40:60
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: adjusting pH to 3.0 with acetic acid and the volume ratio of 1.0mol/L ammonium acetate aqueous solution-methanol-acetonitrile to be 75:20:5
Mobile phase B: adjusting pH to 3.0 with acetic acid, wherein the volume ratio of 1.0mol/L ammonium acetate aqueous solution-methanol-acetonitrile is 20:40
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: adjusting the volume ratio of 0.5mol/L sodium acetate aqueous solution-methanol-acetonitrile with ammonia test solution to pH 4.5 to be 60:30:10
Mobile phase B: adjusting the volume ratio of 0.5mol/L sodium acetate aqueous solution-methanol-acetonitrile with ammonia test solution to pH 4.5 to be 45:50:5
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: the volume ratio of 0.001mol/L sodium pentane sulfonate aqueous solution to acetonitrile is 80:20
Mobile phase B: the volume ratio of 0.001mol/L sodium pentane sulfonate aqueous solution to acetonitrile is 45:55
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: the volume ratio of 0.05mol/L sodium pentane sulfonate aqueous solution to methanol is 90:10
Mobile phase B: the volume ratio of 0.05mol/L sodium pentane sulfonate aqueous solution to methanol is 20:80
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octadecylsilane chemically bonded silica gel column
Mobile phase: mobile phase A: 0.002mol/L sodium hexanesulfonate aqueous solution-methanol volume is 95:5
Mobile phase B: the volume ratio of 0.002mol/L sodium hexanesulfonate aqueous solution to methanol is 25:75
Gradient elution:
alternatively, the first and second electrodes may be,
a chromatographic column: octane silane bonding silica gel column
Mobile phase: mobile phase A: the volume ratio of 0.01mol/L sodium decane sulfonate aqueous solution to acetonitrile is 65:35
Mobile phase B: the volume ratio of 0.01mol/L sodium decane sulfonate aqueous solution to acetonitrile is 10:90
Gradient elution:
wherein, in each stage, the percentage of the mobile phase B changes or keeps unchanged along with the time, and the sum of the percentage of the mobile phase A and the percentage of the mobile phase B is 100 percent;
the micakammonium chloride intermediate comprises an intermediate 5 and one or more of an intermediate 1, an intermediate 2, an intermediate 3 and an intermediate 4;
2. the separation method according to claim 1, characterized in that: the flow rate of the mobile phase is 0.5-2.0 mL/min.
3. The separation method according to claim 2, characterized in that: the flow rate of the mobile phase is 0.5-1.5 mL/min.
4. The separation method according to claim 3, characterized in that: the flow rate of the mobile phase is 0.7-1.3 mL/min.
5. The separation method according to claim 1, characterized in that: the detection wavelength is 270-290 nm.
6. The separation method according to claim 1, characterized in that: the sample amount is 1-100 μ L.
7. The separation method according to claim 6, characterized in that: the sample injection amount is 5-50 mu L.
8. The separation method according to claim 7, characterized in that: the sample size was 20. mu.L.
9. A method for detecting a micraconium chloride intermediate, comprising the step of detecting the micraconium chloride intermediate by the separation method according to any one of claims 1 to 8.
10. The detection method of micakammonium chloride intermediate examination as claimed in claim 9, wherein the detection method comprises the following steps: taking a sample to be tested, preparing a test solution, sucking the test solution, injecting the test solution into a high performance liquid chromatograph, and measuring by using the separation method of any one of claims 1 to 8.
11. The detection method of micstore ammonium chloride intermediate examination as claimed in claim 10, wherein the preparation of the test solution comprises the following steps: taking a sample to be tested, and preparing a test solution with the concentration of 0.1 mg/mL-5.0 mg/mL by using the diluent.
12. The detection method of micaceous ammonium chloride intermediate examination as claimed in claim 11, wherein the diluent is at least one selected from the group consisting of an aqueous solution and an organic solvent.
13. The detection method of micstore ammonium chloride intermediate physical examination as claimed in claim 12, wherein the aqueous solution used by the diluent is selected from at least one of pure water, acid-containing buffer, alkali-containing buffer, salt-containing buffer, and ion-pair reagent-containing aqueous solution;
the organic solvent used in the diluent is at least one selected from acetonitrile and alcohols.
14. The detection method of micstore ammonium chloride intermediate physical examination as claimed in claim 13, wherein the acid in the acid-containing buffer solution used in the dilution is selected from at least one of phosphoric acid, sulfuric acid, hydrochloric acid, formic acid, acetic acid and trifluoroacetic acid;
the alkali in the alkali-containing buffer solution used by the diluent is selected from at least one of ammonia, ammonia water, sodium hydroxide, potassium hydroxide, diethylamine and triethylamine;
the salt in the salt-containing buffer solution used by the diluent is selected from at least one of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium acetate, ammonium formate and sodium acetate;
the ion pair in the ion pair-containing reagent water solution used by the diluent is selected from at least one of sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate and sodium decane sulfonate;
the alcohol used in the diluent is at least one selected from methanol, ethanol and isopropanol.
15. The detection method of micstore ammonium chloride intermediate physical examination as claimed in claim 14, wherein the acid in the acid-containing buffer used in the dilution is at least one selected from phosphoric acid, formic acid, acetic acid and trifluoroacetic acid.
16. The detection method for mikahi ammonium chloride intermediate physical examination as claimed in any one of claims 13-15, wherein the dilution solution uses an acid-containing buffer solution with a volume concentration of 0.005% -2%.
17. The detection method for mikail ammonium chloride intermediate physical examination as claimed in claim 16, wherein the volume concentration of the acid-containing buffer solution used in the diluent is 0.01% -1%.
18. The detection method for mikail ammonium chloride intermediate physical examination as claimed in claim 13 or 14, wherein the concentration of the saline buffer used by the diluent is 0.001mol/L-1.0 mol/L.
19. The detection method for mikail ammonium chloride intermediate examination as claimed in claim 18, wherein the concentration of the saline buffer used in the dilution solution is 0.005 mol/L-0.5 mol/L.
20. The detection method for mikahi ammonium chloride intermediate physical examination as claimed in claim 13 or 14, wherein the concentration of the ion pair reagent-containing aqueous solution used in the diluent is 0.001mol/L to 0.05 mol/L.
21. The method for detecting micaceous ammonium chloride intermediate as claimed in claim 20, wherein the concentration of the aqueous solution containing the ion-pair reagent used in the diluent is 0.002mol/L to 0.01 mol/L.
22. The detection method of micaceous ammonium chloride intermediate examination as claimed in claim 12 or 13, wherein the pH of the aqueous solution used for the dilution is 2.0-10.0.
23. The detection method of micakammonium chloride intermediate examination as claimed in claim 22, wherein the pH of the aqueous solution used for the dilution is 2.0-7.0.
24. The detection method of micstore ammonium chloride intermediate examination as claimed in claim 22, wherein the pH value of the aqueous solution used in the dilution is adjusted by one or more of ammonia, phosphoric acid, sodium hydroxide, formic acid, acetic acid, potassium hydroxide, triethylamine, diethylamine and trifluoroacetic acid.
25. A method for determining the purity of a micraconium chloride intermediate, comprising the step of determining the purity of the micraconium chloride intermediate by the detection method according to any one of claims 9 to 24.
26. The method for determining the purity of the micstore ammonium chloride intermediate as claimed in claim 25, wherein the method for determining the purity is calculated by area normalization.
27. A method for mass analysis of mikuhi ammonium chloride comprising a separation step, a detection step or a measurement step of impurities, wherein the separation step comprises separation according to the separation method of any one of claims 1 to 8, the detection step comprises detection according to the detection method of any one of claims 9 to 24, and the measurement step comprises measurement according to the purity measurement method of claim 25 or 26.
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