An HPLC method for the detection of substances related to adrenaline intermediates

Abstract

The invention belongs to the field of drug analysis technology, and is specifically related to a HPLC detection method for adrenaline intermediate related substances. The detection method is: using octadecyl bonded silica as a reverse phase chromatographic column of a filler, with a mixed solution of 50mmol / L sodium perchlorate and 5mmol / L potassium dihydrogen phosphate, pH=3.0 as a weak elution mobile phase, acetonitrile-methanol=3:7, V / V, the mixture is a strong elution mobile phase, with weak elution mobile phase: strong elution mobile phase=(95~20):(5~80)(v / v) Gradient elution is carried out, adrenaline intermediate related impurities are separated and measured, and chromatographic conditions are used: mobile phase flow rate is 0.8~1.2mL / min; chromatographic column temperature is 30℃~50℃; detection wavelength is 205nm~240nm. This detection method integrates multiple intermediate detections into one detection method. It is easy to operate, low-cost, and can effectively separate 20 impurities in epinephrine intermediates. The separation degree between all impurities is greater than 1.5, providing a simple, stable and reliable analytical detection method for product research and development and production.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a HPLC detection method for adrenaline intermediate-related substances. Background Art

[0002] As an α and β receptor agonist, epinephrine mainly constricts arterioles and precapillary sphincters, and secondarily constricts veins and aortas. Epinephrine is a hormone secreted by the human body and a catecholamine neurotransmitter produced in some neurons of the central nervous system and chromaffin cells of the adrenal medulla. Epinephrine has many functions in the body, including regulating heart rate, respiratory rate and metabolic conversion. Epinephrine increases the contractility of the heart, dilates the blood vessels of the heart, liver, and tendons and bones, and constricts the blood vessels of the skin and mucous membranes. It is essential for saving dying people or animals. Epinephrine was isolated and identified by Polish physiologist Napoleon Cybulski in 1895 and first synthesized artificially by Friedrich Stolz in 1904. Epinephrine injection was first approved for marketing in Canada in December 1951. Indications are to relieve respiratory distress caused by bronchospasm; to quickly relieve allergic reactions to drugs and other allergens and to prolong the effects of infiltration anesthetics; and to treat cardiac arrest caused by various reasons, but not for heart failure or hemorrhagic trauma or cardiogenic shock.

[0003] Artificial synthesis methods of medicinal adrenaline include chloroacetylation, ethylene oxide, hydrocyanic acid, etc. It is planned to use catechol as the raw material to synthesize adrenaline raw material, first through chloroacetylation to generate 2-chloro-3,4-dihydroxyacetophenone, then through amine nucleophilic substitution reaction to generate 3,4-dihydroxyphenyl N-benzyl-N-methylamine ketone, and then through palladium carbon catalytic hydrogenation debenzylation and carbonyl reduction, tartaric acid splitting and ammonia free tartrate to obtain adrenaline. Among them, 2-chloro-3,4-dihydroxyacetophenone (intermediate ADR-3) and 3,4-dihydroxyphenyl N-benzyl-N-methylamine ketone (intermediate ADR-4) are key intermediates, and due to the complex reaction system and many by-products, the pharmacopoeias of various countries currently do not include detection methods for controlling the quality of their intermediates. Develop detection methods with strong specificity and short analysis time to control the quality of key intermediates to ensure the quality of adrenaline and its preparations.

[0004] Based on the synthesis process, the impurities of the starting materials catechol (ADR-1), chloroacetyl chloride (ADR-2), N-methylbenzylamine and the finished product, the potential impurities were analyzed. The following Table-1 shows the names and structural formulas of each impurity.

[0005] Table-1 Potential impurities in the synthesis of adrenaline

[0006]

[0007] The applicant has consulted a large number of Chinese and foreign patents and non-patent literature and found that there is currently no literature reporting on good high-performance liquid chromatography methods for detecting substances related to adrenaline intermediates, which is not conducive to the company's control over product quality.

[0008] In order to solve the problems existing in the prior art, the inventors of the present application have provided a method for analyzing various impurities in adrenaline intermediates through a large amount of theoretical research and experimental exploration, so as to better control the product quality of adrenaline. Summary of the invention

[0009] In order to solve the problems existing in the prior art, the inventors of the present application have provided a liquid phase method for separating and analyzing adrenaline intermediate-related substances (20 impurities) through a large amount of theoretical research and experimental exploration, so as to better control the quality of adrenaline products.

[0010] In order to achieve the purpose of the present invention, the technical solution provided by the present invention is:

[0011] A HPLC detection method for adrenaline intermediate related substances: using octadecyl bonded silica gel (reverse phase) as a chromatographic column filler, using a mixture of 50mmol / L sodium perchlorate and 5mmol / L potassium dihydrogen phosphate (50mmol / L and 5mmol / L refer to the concentrations of sodium perchlorate and potassium dihydrogen phosphate in the mixture), pH=3.0 (adjusted with phosphoric acid) as a weak elution mobile phase, acetonitrile-methanol (3:7, V / V) mixture as a strong elution mobile phase, and gradient elution with weak elution mobile phase: strong elution mobile phase=(95-20):(5-80) (v / v) to separate and determine 20 impurities of adrenaline intermediates, the HPLC detection adopts the following chromatographic conditions:

[0012] The flow rate of the mobile phase is 0.8-1.2 mL / min;

[0013] The column temperature is 30℃~50℃;

[0014] The detector uses an ultraviolet detector with a detection wavelength of 205nm to 240nm;

[0015] Preferably, the chromatographic column is Waters Atlantis T3 (4.6 mm×150 mm), and the filler particle size is 3 μm;

[0016] Preferably, the column temperature of the chromatographic column is 35°C;

[0017] Preferably, the detection wavelength is 230 nm;

[0018] Preferably, the mobile phase flow rate is 1.0 mL / min;

[0019] Preferably, the injection volume is 10 μL.

[0020] Furthermore, the detection method comprises the following steps:

[0021] 1.1 Prepare the test solution with a diluent, wherein the diluent is: acetonitrile: water = 20:80, v / v;

[0022] 1.2 Inject the test solution obtained in step 1.1 into the chromatographic column of the high performance liquid chromatograph for detection.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] 1. Effective separation and detection of 20 impurities in adrenaline intermediates are achieved. Therefore, the detection method of the present invention can better achieve quality control of adrenaline intermediates, improve the quality of the final product, and ensure the safety and effectiveness of its clinical use.

[0025] 2. The detection method of the present invention integrates multiple intermediate detections into one detection method, which is easy to operate and low in cost. It can effectively separate 20 impurities in adrenaline intermediates, and the separation degree between all impurities is greater than 1.5, providing a simple, stable and reliable analytical detection method for product research and development and production.

[0026] It should be noted that, in the present invention, "%" refers to volume percentage unless otherwise specified. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the LogD graph of ADR-1 and related impurities;

[0028] Figure 2 It is the LogD graph of ADR-3 and related impurities;

[0029] Figure 3 It is the LogD graph of ADR-4 and related impurities;

[0030] Figure 4 The spectra were detected for ADR and related substances using mobile phases of pH 2.5, pH 5.0, pH 7.5, and pH 10.0, respectively. From top to bottom: pH 5.0, pH 10.0, pH 2.5, and pH 7.5;

[0031] Figure 5 To detect the spectra of ADR and related substances using different chromatographic columns;

[0032] Figure 6 To detect the spectra of ADR and related substances using different mobile phases A;

[0033] Figure 7 To detect the spectra of ADR and related substances using mobile phase A with different salt concentrations;

[0034] Figure 8 Create gradient-temperature-organic phase type 3D models for ADR intermediates and related substances ACD / LC-Simulator;

[0035] Fig. 9 This is the test diagram of the actual samples of ADR intermediates and related substances;

[0036] Fig.10 Create gradient-temperature-mobile phase pH 3D models for ADR intermediates and related substances ACD / LC-Simulator;

[0037] Fig.11 This is the test diagram of the actual samples of ADR intermediates and related substances;

[0038] Fig.12 Wavelength screening for ADR intermediates and related substances analysis methods (expander);

[0039] Fig.13 Wavelength screening for analysis of ADR intermediates and related substances (test samples and impurities). DETAILED DESCRIPTION

[0040] To facilitate those skilled in the art to understand the contents of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection requested by the claims of the present invention in any form.

[0041] In the following examples, the reagents used are as follows:

[0042] ADR-1, ADR-1-IMP-01, ADR-1-IMP-02, and ADR-1-IMP-04 were purchased from the China Food and Drug Administration;

[0043] ADR-3, ADR-3-IMP-01, ADR-3-IMP-02, ADR-3-IMP-03, ADR-3-IMP-04, ADR-3-IMP-06 and ADR-3-IMP-07 are manufactured by the applicant based on the prior art;

[0044] ADR-4-IMP-01 and ADR-4-IMP-02 were purchased from Aladdin, and ADR-4, ADR-4-IMP-03, ADR-4-IMP-04, ADR-4-IMP-06, ADR-4-IMP-07, ADR-4-IMP-09 and ADR-4-IMP-10 were manufactured by the applicant according to the prior art;

[0045] ADR-5 was manufactured by the applicant based on existing technology.

[0046] Example 1

[0047] Diluent screening

[0048] Through testing, it was found that all impurities had good solubility in 20 v / v% acetonitrile aqueous solution, so 20 v / v% acetonitrile aqueous solution was used as a diluent to prepare the impurity stock solution.

[0049] pH screening

[0050] Figure 1 The following is the LogD graph of ADR-1 and related impurities (drawn by ACD / Auto Chrom). It can be seen from the LogD graph of ADR-1 and related impurities that the LogD of all compounds is greater than 0, and they have a certain retention on the ordinary C18 chromatographic column. Under the mobile phase condition of pH less than 9, each compound shows a molecular form, and the peak order is not affected by pH. When the pH of the mobile phase is greater than 9, the retention of each compound is sharply weakened. When the pH of the mobile phase is greater than 12, the LogD of all compounds is less than 0. Therefore, the detection of ADR-1 and its related substances is suitable for acidic conditions.

[0051] Figure 2 The following is the LogD graph of ADR-3 and related impurities. From the LogD graph of ADR-3 and related impurities, we can see that compound 2 (1-(3,4-dihydroxyphenyl)-2-hydroxyethanone) has the smallest LogD, the worst hydrophobicity, and the weakest retention, but the LogD of all compounds is greater than 0, and they have a certain retention on the ordinary C18 chromatographic column. Compound 4 is in a molecular state in the pH range of 1-14, indicating that the compound is a neutral compound and is not affected by pH. Among ADR-3 and related substances, the six compounds except compound 4 show molecular ion forms under mobile phase conditions with a pH greater than 8, and the retention is sharply weakened. Under mobile phase conditions with a pH less than 6, each compound shows a molecular form, and the peak order is not affected by pH. Therefore, the detection of ADR-3 and its related substances is suitable for acidic conditions.

[0052] Figure 3 The following is the LogD graph of ADR-4 and related impurities. It can be seen from the LogD graph of ADR-4 and related impurities that the retention behaviors of each compound under different pH conditions are different. Compound 6 is in a molecular state in the pH range of 1-14, indicating that the compound is a neutral compound and is not affected by pH. Overall, the retention of each compound under acidic conditions is stronger than that under alkaline conditions. Therefore, the detection of ADR-4 and its related substances is suitable for acidic conditions.

[0053] In summary, intermediates ADR-3, ADR-4 and related impurities have different retention behaviors under different pH conditions. Under acidic conditions, most compounds exist in a single form; as the pH increases, the compounds appear in a molecular ion state, and the retention of most compounds decreases.

[0054] ADR and related substances were detected using mobile phases of pH 2.5, pH 5.0, pH 7.5, and pH 10.0, respectively. The specific chromatographic conditions are shown in Table 2.

[0055] Sample solution preparation (the samples used for subsequent parameter optimization tests are the same as those used for mobile phase pH screening, so they will not be repeated here):

[0056] ADR-1 mixed solution: Take 5 mg each of ADR-1, ADR-1-IMP-01, ADR-1-IMP-02 and ADR-1-IMP-04 in a 10 mL volumetric flask and dissolve and dilute with 20% acetonitrile to make a 0.5 mg / mL solution.

[0057] ADR-3 mixed solution: Take 2 mg each of ADR-3, ADR-3-IMP-01, ADR-3-IMP-02, ADR-3-IMP-03, ADR-3-IMP-04, ADR-3-IMP-06 and ADR-3-IMP-07 in a 10 mL volumetric flask and dissolve and dilute with 20% acetonitrile to make a 0.2 mg / mL solution.

[0058] ADR-4 mixed solution: Take 2 mg each of ADR-4, ADR-4-IMP-01, ADR-4-IMP-02, ADR-4-IMP-03, ADR-4-IMP-04, ADR-4-IMP-06, ADR-4-IMP-07, ADR-4-IMP-09 and ADR-4-IMP-10 in a 10 mL volumetric flask and dissolve and dilute with 20% acetonitrile to make a 0.2 mg / mL solution.

[0059] ADR mixed solution: Take 2 mL each of ADR-1 mixed solution, ADR-3 mixed solution and ADR-4 mixed solution, mix well, and obtain ADR mixed solution.

[0060] Table-2 Chromatographic conditions for pH screening

[0061]

[0062] Figure 4 The spectra were detected for ADR and related substances using mobile phases of pH 2.5, pH 5.0, pH 7.5, and pH 10.0, respectively.

[0063] Results analysis: The screening results of different pH values ​​show that under pH 2.5, the compound retention and peak shape are better; under pH 5.0, the baseline of the chromatogram fluctuates greatly, mainly because the mobile phase under pH 5.0 is ammonium acetate buffer solution, the UV absorption cutoff wavelength of which is 210nm, and there is terminal absorption, which affects the detection sensitivity; under pH 7.5 and pH 10.0, the compound retention and peak shape are poor. Therefore, the mobile phase system of pH 2.5 is more suitable for the detection of related substances of ADR intermediates.

[0064] Conclusion: The mobile phase system with pH 2.5 was selected for the detection of related substances of ADR intermediates.

[0065] Column Screening

[0066] according to Figure 1-3 The LogD graph shows that ADR and related substances are weakly hydrophobic, so the common C 18 The chromatographic column may cause some compounds to be unretained. Therefore, this study mainly selected the following chromatographic columns for screening based on their hydrophilicity to examine the peak shape and retention of each substance. The chromatographic columns and chromatographic conditions for column screening are shown in Table 3.

[0067] Table-3 Chromatographic conditions for column screening

[0068]

[0069]

[0070] Note: Column 1 was purchased from Waters Corporation, column 2 was purchased from Thermo Fisher Scientific, column 3 was purchased from Waters Corporation, column 4 was purchased from Agilent, column 5 was purchased from Shimadzu Corporation, column 6 was purchased from ACE, and column 7 was purchased from ACE.

[0071] Figure 5 The results of the chromatographic column screening are summarized in Table 4.

[0072] Table-4 Summary of test results of chromatographic column screening

[0073]

[0074]

[0075] Result analysis: The chromatograms and result summary tables obtained by chromatographic column screening were analyzed. Under the conditions of Waters Atlantis T3, the number of chromatographic peaks of ADR intermediates and related substances detected was the largest, the retention and peak shape symmetry of the chromatographic peaks were good, and the baseline was relatively stable. Therefore, Waters Atlantis T3 was selected as the chromatographic column for the detection of ADR intermediates and related substances.

[0076] Mobile phase screening

[0077] The chromatographic conditions for mobile phase screening are shown in Table 5.

[0078] Table-5 Chromatographic conditions for mobile phase screening

[0079]

[0080] Note: TFA is trifluoroacetic acid.

[0081] Figure 6 The spectra of ADR and related substances were detected using different mobile phases A. The test results of mobile phase A screening are summarized in Table 6.

[0082] Table-6 Summary of test results of mobile phase screening

[0083]

[0084]

[0085] Result analysis: The typical chromatograms of mobile phase screening show that under the three mobile phase conditions, ADR intermediates and related substances are retained, and some compounds have tailing phenomenon. Considering that under the conditions of mobile phase 3, the number of chromatographic peaks of ADR intermediates and related substances is the largest, mobile phase 3 is selected for the detection of ADR intermediates and related substances.

[0086] Salt concentration screening

[0087] The chromatographic conditions for screening the salt concentration in the mobile phase are shown in Table-7.

[0088] Table-7 Chromatographic conditions for screening salt concentration in mobile phase

[0089]

[0090]

[0091] Figure 7 The spectra of ADR and related substances were detected using mobile phase A with different salt concentrations.

[0092] Analysis of results: It can be seen from the typical chromatograms of salt concentration screening that under the three different salt concentration conditions of 10mmol / LNaClO4 solution, 50mmol / LNaClO4 solution and 100mmol / LNaClO4 solution, ADR intermediates and related substances are retained, and the separation effects of each compound under the conditions of 50mmol / LNaClO4 solution and 100mmol / LNaClO4 solution are better than those under the conditions of 10mmol / LNaClO4 solution, while there is no obvious difference in the separation effects of each compound under the conditions of 50mmol / LNaClO4 solution and 100mmol / LNaClO4 solution. Considering the high cost of chromatographic grade NaClO4, 50mmol / LNaClO4+5mmol / LKH2PO4 (H3PO4 adjusted to pH 3.0) / acetonitrile was initially selected for the detection of ADR intermediates and related substances.

[0093] Optimization of organic phase types by gradient temperature

[0094] A common goal of HPLC method development is to separate all target chromatographic peaks from their adjacent peaks (i.e., Rs ≥ 1.5). The formula for separation Rs is as follows:

[0095] Rs=(1 / 4)[k / (1+k)](α-1)N 0.5

[0096] Different separation conditions have different effects on the retention factor (k) and selectivity (α) of the chromatographic peak. The type of chromatographic column, the pH of the mobile phase, and the type of organic phase have a major impact on the selectivity (α), while temperature and gradient have a significant impact on the retention and selectivity of the chromatographic peak (especially for structural analogs). This study designed temperature (logk = logA + B / Tk), gradient The specific chromatographic conditions are shown in Table 8:

[0097] Table-8 Chromatographic conditions optimized by gradient temperature and organic phase type

[0098]

[0099]

[0100] Use ACD / LC-Simulator to create a 3D model of gradient temperature organic phase types and optimize the best gradient-temperature-organic phase type model. Figure 8 Create gradient-temperature-organic phase type 3D models for ADR intermediates and related substances ACD / LC-Simulator.

[0101] The chromatogram predicted by ACD / LC-Simulator shows that the separation of each impurity is best when the column temperature is 35°C, the organic phase is acetonitrile-methanol (30:70), and the gradient is 0min (5% B)-33min (80% B). The minimum separation Rs of ADR intermediates and related substances is 1.9. The chromatographic conditions predicted by ACD / LC-Simulator are shown in Table-9.

[0102] Table-9 Chromatographic conditions predicted by ACD / LC-Simulator

[0103]

[0104]

[0105] The chromatographic conditions predicted by ACD / LC-Simulator were used for actual sample testing. Fig. 9 This is a test diagram of actual samples of ADR intermediates and related substances.

[0106] Result analysis: There is no difference between the actual sample test chart using the chromatographic conditions predicted by ACD / LC-Simulator and the chromatogram predicted by ACD software, and the separation degree of each impurity Rs≥1.5.

[0107] Optimization of pH value of gradient temperature mobile phase

[0108] The chromatographic conditions for gradient temperature mobile phase pH optimization are shown in Table-9.

[0109] Table-9 Chromatographic conditions optimized by gradient temperature mobile phase pH

[0110]

[0111]

[0112] ACD / LC-Simulator was used to create a gradient temperature mobile phase pH 3D model and optimize the best gradient-temperature-mobile phase pH model. Fig.10 ACD / LC-Simulator was used to create a gradient-temperature-mobile phase pH 3D model for ADR intermediates and related substances.

[0113] The chromatogram predicted by ACD / LC-Simulator shows that the separation of each impurity is best when the column temperature is 35°C, the mobile phase pH is 3.0, and the gradient is 0min (5% B)-28min (80% B), and the minimum separation Rs of ADR intermediates and related substances is 1.6. The chromatographic conditions predicted by ACD / LC-Simulator are shown in Table-10.

[0114] Table-10 Chromatographic conditions predicted by ACD / LC-Simulator

[0115]

[0116]

[0117] The chromatographic conditions predicted by ACD / LC-Simulator were used for actual sample testing. Fig.11 This is a test diagram of actual samples of ADR intermediates and related substances.

[0118] Result analysis: There is no difference between the actual sample test chart using the chromatographic conditions predicted by ACD / LC-Simulator and the chromatogram predicted by ACD software, and the separation degree of each impurity Rs≥1.64.

[0119] Detection wavelength selection

[0120] The chromatographic conditions for detection wavelength screening are shown in Table 10.

[0121] Table-10 Chromatographic conditions for detection wavelength screening

[0122]

[0123]

[0124] Fig.12 For wavelength screening of ADR intermediates and related substances analysis methods (diluent).

[0125] Fig.13 Wavelength screening for the analysis of ADR intermediates and related substances (test samples and impurities). The test results of ADR mixed solution at different wavelengths are shown in Table 11.

[0126] Table-11 Test results of the same sample at different wavelengths

[0127]

[0128]

[0129] Result analysis: By extracting different detection wavelengths for the blank (diluent), the detection wavelengths were extracted at 205nm, 210nm, 220nm, 230nm and 240nm. Fig.12 It can be seen that the fluctuation of the blank baseline decreases with the continuous increase of the detection wavelength. When the detection wavelength is 230nm and 240nm, the fluctuation of the blank baseline is relatively stable and has little effect on sample detection. By extracting different detection wavelengths of the ADR mixed solution, the detection wavelengths of 205nm, 210nm, 220nm, 230nm and 240nm were extracted respectively. Fig.13 As can be seen from Table 11, with the continuous increase of the detection wavelength, the response of ADR intermediates and impurities is constantly decreasing. By comparing the peak areas of each impurity peak under the detection wavelength of 230nm and the detection wavelength of 240nm, it is found that the peak area of ​​ADR-1-IMP-04 is 8 times smaller under the detection wavelength of 240nm than under the detection wavelength of 230nm, the peak area of ​​ADR-1 and ADR-1-IMP-01 is 6 times smaller under the detection wavelength of 240nm than under the detection wavelength of 230nm, and the peak area of ​​ADR-4-IMP-04 and ADR-1-IMP-02 is 5 times smaller under the detection wavelength of 240nm than under the detection wavelength of 230nm. Taking into account the detection of impurities, the sensitivity of impurities and the fluctuation of the baseline, 230nm was finally determined as the detection wavelength.

[0130] Based on the results of the optimization of the above conditions, the final analysis method was determined and presented in the form of a list. The chromatographic conditions for the adrenaline intermediate detection method are shown in Table 12.

[0131] Table-12 Chromatographic conditions for detection of adrenaline intermediates

[0132]

[0133]

Claims

1. An HPLC detection method for adrenaline intermediate related substances, wherein the adrenaline intermediate related substances include the following types: Features: A reverse phase chromatographic column with octadecyl bonded silica as a filler is used, a mixture of 50 mmol / L sodium perchlorate and 5 mmol / L potassium dihydrogen phosphate, pH = 3.0 is used as a weak elution mobile phase, a mixture of acetonitrile-methanol with a volume ratio of 3:7 is used as a strong elution mobile phase, a gradient elution program: 0 to 28 min, the weak elution mobile phase is from 95% to 20%, and the adrenaline intermediate related substances are separated and determined. The HPLC detection adopts the following chromatographic conditions: The flow rate of the mobile phase is 0.8-1.2 mL / min; The column temperature is 30℃~50℃; The detector adopts an ultraviolet detector with a detection wavelength of 205nm to 240nm.

2. The detection method according to claim 1, characterized in that: The chromatographic column is Waters Atlantis T3.

3. The detection method according to claim 2, characterized in that: The specification of the chromatographic column is: 4.6mm×150mm, and the particle size of the filler is 3μm.

4. The detection method according to claim 1 or 2, characterized in that: The column temperature of the chromatographic column is 35°C.

5. The detection method according to claim 1 or 2, characterized in that: The detection wavelength is 230 nm.

6. The detection method according to claim 1 or 2, characterized in that: The flow rate of the mobile phase is 1.0 mL / min.

7. The detection method according to claim 1 or 2, characterized in that: The injection volume was 10 μL.

8. The detection method according to claim 1 or 2, characterized in that: The detection method comprises the following steps: 1.1 Prepare the test solution with a diluent, wherein the diluent is: acetonitrile: water = 20:80, v / v; 1.2 Inject the test solution obtained in step 1.1 into the chromatographic column of the high performance liquid chromatograph for detection.

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