A method for the analysis of trace amounts of tert-butylamine residues with high sensitivity and low limits

Abstract

The present invention belongs to the field of drug analysis, and specifically relates to a method for analyzing residual traces of tert-butylamine with high sensitivity and low limit. The present invention creatively develops a highly sensitive derivatization reverse detection method, which can significantly reduce the detection limit and quantitative limit of tert-butylamine content, and can avoid the risk of salting out and improve the stability of the derivative product, and has extremely high commercial application value.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and in particular relates to a tert-butylamine residual trace analysis method with high sensitivity and low limit. Background Art

[0002] Tert-butylamine is a colorless, transparent liquid that is soluble in water, anhydrous ethanol, benzene, chloroform, ether, etc. It is often used as a rubber accelerator, synthetic medicine, dye, pesticide, and other chemical preparations. Tert-butylamine is toxic and may be fatal if inhaled, taken orally, or absorbed through the skin. Moreover, tert-butylamine has a strong irritating effect on the eyes, skin, mucous membranes, and respiratory tract. Therefore, the residual amount of tert-butylamine in the raw materials must be strictly tested and controlled.

[0003] Tert-butylamine is a volatile alkaline organic compound with a molecular weight of 73.14 and a boiling point of 46°C. In the field of drug analysis, gas chromatography (GC) and flame ionization detector (FID) are usually preferred for method development of drug molecules. Tert-butylamine has a strong alkalinity, is easy to remain on the chromatographic column, and has a poor peak shape, so it can be detected using a basic compound chromatographic column. However, the detection limit of tert-butylamine using conventional gas chromatography can only reach 100ppm at present, which is difficult to achieve the demand for lower and more accurate detection limits. In addition, the molecular weight of tert-butylamine is very close to the molecular weight of the column bleed peak of the gas chromatography-mass spectrometer (GCMS), and is easily interfered by the column bleed peak, so it is not suitable for detection by gas chromatography-mass spectrometry. In addition, tert-butylamine is an organic amine that exists in the form of tert-butylamine cation in aqueous solution. However, due to the poor solubility of the test chemical raw materials in large proportions of aqueous phase, when the method was developed in ion chromatography-cation mode, the detection limit of tert-butylamine was only 100 ppm, which also failed to meet the lower detection requirements. Summary of the invention

[0004] The existing technology has the problem of low sensitivity or poor popularity when detecting residual amine compounds in chemical raw materials. In view of this problem, the present invention creatively develops a highly sensitive derivatization reverse phase detection method, which can significantly reduce the detection limit and quantitative limit of tert-butylamine content, avoid salting out risks and improve the stability of derivative products, and has extremely high commercial application value.

[0005] The method for analyzing the residual trace amount of tert-butylamine with high sensitivity and low limit of the present invention comprises the following steps:

[0006] The first step: dissolving the chemical raw material pharmaceutical cosolvent and acetonitrile, adding an acid binding agent and a derivatizing agent for derivatization reaction, and after the reaction is completed, quenching the reaction with water to obtain a test solution;

[0007] Step 2: After dissolving the tert-butylamine reference substance with a co-solvent and acetonitrile, an acid-binding agent and a derivatizing agent are added to carry out a derivatization reaction, and after the reaction is completed, the reaction is quenched with water to obtain a reference substance solution;

[0008] Step 3: Take appropriate amounts of the test solution and the reference solution, inject them into the high performance liquid chromatograph, and record the peak area of ​​the tert-butylamine derivative in the chromatogram;

[0009] Step 4: Calculate the residual amount of tert-butylamine in the chemical raw material drug by external standard method; the calculation formula is:

[0010] ;

[0011] in:

[0012] A S : Peak area of ​​tert-butylamine derivatives in the test solution;

[0013] W S : The weight of the test sample (mg);

[0014] W STD : The weight of the reference substance (mg);

[0015] V STD : dilution volume of reference solution (ml);

[0016] V S : dilution volume of the test solution (ml);

[0017] A STD : Peak area of ​​tert-butylamine derivative in reference solution;

[0018] Pw: content of tert-butylamine reference substance.

[0019] Preferably, the chemical raw material is an acidic salt; preferably, the chemical raw material is clenbuterol hydrochloride.

[0020] Preferably, the co-solvent is N-methylpyrrolidone and / or N,N-dimethylformamide, wherein the volume ratio of the co-solvent to acetonitrile is 1:1 to 1:20.

[0021] Preferably, the acid binding agent is selected from a combination of one or more of triethylamine, diethanolamine and N,N-diisopropylethylamine.

[0022] Preferably, the molar ratio of the acid binding agent to the tert-butylamine reference substance is 10000:1 to 200000:1.

[0023] Preferably, the derivatizing agent is benzoyl chloride.

[0024] Preferably, the molar ratio of the derivatizing agent to the tert-butylamine reference substance is 100:1 to 1000:1.

[0025] Preferably, the tert-butylamine derivative is an amide compound.

[0026] Preferably, the reaction temperature is room temperature to 60°C.

[0027] Preferably, the concentration of the chemical raw material in the test solution is 36±2 mg / ml; the concentration of the tert-butylamine reference substance in the reference solution is 0.18±0.01 μg / ml.

[0028] Preferably, octadecyl bonded silica gel is used as filler, the column temperature is 35±3° C., mobile phase A is 0.1±0.01% phosphoric acid aqueous solution, mobile phase B is acetonitrile, and the flow rate is 1.0±0.2 ml / min.

[0029] Preferably, the detection wavelength of the high performance liquid chromatograph is 224±5 nm or 205±5 nm. Preferably, the detection wavelength of the high performance liquid chromatograph is 224 nm.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. In view of the problem of low sensitivity in detecting tert-butylamine residues, the present invention develops a derivatization reversed-phase liquid chromatography method to analyze the tert-butylamine content in raw materials, which can quantitatively and accurately detect tert-butylamine residues above 1.5 ppm, significantly enhancing the sensitivity of the detection method.

[0032] 2. The present invention uses benzoyl chloride as a derivatization reagent, which achieves rapid derivatization at room temperature, increases derivatization stability, and saves detection time.

[0033] 3. The present invention optimizes and verifies the detection method, which has the advantages of simplicity, rapidity, high accuracy, etc., and reduces the risk of salting out. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a representative spectrum for characterizing the specificity of blank solution, positioning solution, reference solution and test solution by HPLC overlay; wherein, 1-reference solution, 2-blank solution, 3-positioning solution, 4-test solution;

[0035] Figure 2It is a representative spectrum of the sensitivity of blank solution, detection limit solution, quantitative limit solution and reference solution by HPLC overlay; wherein, 1-reference solution, 2-quantification limit solution, 3-detection limit solution, 4-blank solution;

[0036] Figure 3 A linear graph showing a linear regression of the peak area of ​​the tert-butylamine derivative against the tert-butylamine concentration;

[0037] Figure 4 The representative graphs of the accuracy of blank solution, reference solution, test solution, and AC2-100% solution (accuracy limit spiked solution) are characterized by HPLC overlay; wherein, 1-AC2-100% solution, 2-test solution, 3-reference solution, 4-blank solution. DETAILED DESCRIPTION

[0038] The present invention is further described by the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention. Specific experimental methods not mentioned in the following examples are usually carried out according to conventional experimental methods.

[0039] How to obtain a detection method with good specificity, effectively separate the tert-butylamine derivatives and the impurities of the raw material drug, and improve the derivatization process to improve the sensitivity and accuracy of the detection method, is the technical problem to be solved by the present invention. The following is an exemplary description of the trace analysis method of the residual tert-butylamine in the chemical raw material drug of the present invention.

[0040] After dissolving the chemical raw material pharmaceutical cosolvent and the solvent acetonitrile, an acid binding agent and a derivatizing agent are added to carry out a derivatization reaction. After the reaction is completed, water is used to quench the reaction to obtain a test sample solution.

[0041] The chemical raw material is an acidic salt. Preferably, the chemical raw material is clenbuterol hydrochloride. The chemical raw material contains impurities. The impurities are fatty amines with strong alkalinity, including but not limited to tert-butylamine, n-butylamine, diethylamine, isopropylamine, etc.

[0042] Selection of derivatization agent. Tert-butylamine itself has no ultraviolet absorption. The selection of a suitable derivatization agent with ultraviolet absorption can make the tert-butylamine derivative have ultraviolet absorption. Preferably, the derivatization agent is benzoyl chloride. The derivative formed by the reaction of benzoyl chloride and tert-butylamine is an amide compound, which has ultraviolet absorption and is a neutral compound with stable properties. The derivatization reaction speed is fast and can shorten the derivatization time. If a salicylaldehyde-methanol derivatization system is used, the derivative formed is an imine compound, which has a potential decomposition risk. Therefore, a strong base such as sodium hydroxide needs to be added to form an alkaline environment to increase the stability of the derivative product imine compound.

[0043] The chemical raw material clenbuterol hydrochloride has limited solubility in acetonitrile, and it is difficult to obtain an acetonitrile solution of the desired concentration. Water and methanol react with the derivatization reagent benzoyl chloride, so they are not suitable as cosolvents. For example, the cosolvent is selected from N-methylpyrrolidone (NMP) and / or N,N-dimethylformamide (DMF). Preferably, NMP is selected as the cosolvent.

[0044] The volume ratio of the co-solvent to acetonitrile may be 1:1 to 1:20. As an example, the volume ratio of the co-solvent to acetonitrile is 1:10.

[0045] Selection of acid-binding agent. The acid-binding agent is selected from triethylamine (TEA), diethanolamine (DEA), and N,N-diisopropylethylamine (DIEA). Preferably, the acid-binding agent is DIEA. The acid-binding agent can adsorb hydrochloric acid contained in the API and the byproduct of the derivatization reaction, thereby prompting the derivatization reaction to reach the end point. Strong bases such as sodium hydroxide react with the derivatization reagent acyl chloride and are not suitable for the derivatization system described in the present invention.

[0046] The derivatization temperature is room temperature to 60°C. For example, the reaction temperature is room temperature, 40°C and 60°C. A water bath can be heated to maintain the desired derivatization reaction temperature. If not specified, room temperature refers to 15 to 25°C.

[0047] The derivatization time can be adjusted as needed. The derivatization time can be within 2 hours. For example, the derivatization time is 0.5 minutes to 2 hours. Preferably, the derivatization time is 1 minute to 1 hour. For example, the derivatization time is shaking (about 1 minute), 10 minutes and 1 hour.

[0048] The molar ratio of the acid binding agent to the tert-butylamine reference substance is 10000: 1 to 200000: 1. For example, the molar ratio of the acid binding agent to the tert-butylamine reference substance is 10000: 1, 120000: 1 and 200000: 1. Preferably, the molar ratio of the acid binding agent to the tert-butylamine reference substance is 120000: 1.

[0049] The molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine reference substance is 100: 1 to 1000: 1. For example, the molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine reference substance is 100: 1, 250: 1, 500: 1 and 1000: 1. Preferably, the molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine reference substance is 250: 1.

[0050] In order to eliminate the influence of excess derivatization reagent benzoyl chloride on the chromatographic system, purified water was used to quench benzoyl chloride and terminate the reaction.

[0051] The preparation process of the reference solution is basically the same as the preparation process of the test solution. For example, after dissolving the tert-butylamine reference substance with a cosolvent and a solvent acetonitrile, an acid binding agent and a derivatizing agent are added to carry out a derivatization reaction, and after the reaction is completed, water is used to quench the reaction to obtain a reference solution.

[0052] Accurately measure appropriate amounts of the test solution and reference solution, respectively, inject them into a high performance liquid chromatograph, and record the peak area of ​​the tert-butylamine derivative in the chromatogram. The detection wavelength of the high performance liquid chromatograph is 224±5nm or 205±5nm. For example, the detection wavelength of the high performance liquid phase condition is 224nm or 205nm. Preferably, the detection wavelength of the high performance liquid phase condition is 224nm.

[0053] Optimization of chromatographic parameters. The mobile phase, chromatographic column, and elution procedure were optimized, and finally a conventional C18 chromatographic column and phosphoric acid aqueous solution-acetonitrile were used as the mobile phase system. Specifically, octadecyl bonded silica gel was used as the filler, the column temperature was 35±3°C, the mobile phase A was a 0.1±0.01% phosphoric acid aqueous solution, the mobile phase B was acetonitrile, and the flow rate was 1.0±0.2ml / min. For example, octadecyl bonded silica gel was used as the filler, and the octadecyl bonded silica gel was Waters XBridge ® C18, 4.6*150mm*3.5μm, column temperature of 35℃, detection wavelength of 224nm, 0.1% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution, flow rate of 1.0 ml / min. If potassium dihydrogen phosphate solution is used as aqueous mobile phase, buffered salt solution has potential risk of salting out.

[0054] The residual tert-butylamine in the test sample was calculated according to the external standard method. The calculation formula is:

[0055] .

[0056] Among them: A S W: The peak area of ​​tert-butylamine derivatives in the test solution; S W: the weight of the test sample (mg); STD : The weight of the reference substance (mg); V STD V: volume of reference solution (ml); S : Volume of test solution (ml); A STD : Peak area of ​​tert-butylamine derivatives in reference solution; Pw: content of tert-butylamine reference.

[0057] In some embodiments, the present invention provides a method for analyzing the residual amount of tert-butylamine in the clenbuterol hydrochloride raw material, comprising the following steps:

[0058] Step 1: Take an appropriate amount of clenbuterol hydrochloride bulk drug sample, add NMP to dissolve by ultrasonication, add acetonitrile to dilute, add DIEA and shake well, add benzoyl chloride solution, shake well, add purified water to the scale as the test solution.

[0059] Step 2: Take an appropriate amount of NMP, add acetonitrile to dilute, add an appropriate amount of tert-butylamine reference solution, add DIEA, shake well, add benzoyl chloride, shake well, add purified water to the scale as the reference solution.

[0060] Step 3: Accurately measure appropriate amounts of the two solutions obtained in the first and second steps above, inject them into a high performance liquid chromatograph, and record the peak area of ​​the tert-butylamine derivative in the chromatogram. Among them, octadecyl bonded silica gel is used as filler, the column temperature is 35°C, the detection wavelength is 224nm, 0.1% phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is performed at a flow rate of 1.0 ml / min.

[0061] Step 4: Calculate the residue of tert-butylamine derivatives in the test sample according to the external standard method.

[0062] In summary, the derivatization reverse phase detection method of the present invention can accurately determine the residual amount of tert-butylamine in chemical raw materials by optimizing the cosolvent, acid binding agent and derivatization agent, and achieve a significant reduction in the detection limit and detection limit of the tert-butylamine content, especially being able to quantitatively detect tert-butylamine residues above 1.5ppm in chemical raw materials, with the advantages of high sensitivity and good reproducibility. Moreover, the derivatization process of the method of the present invention does not require heating, is convenient and fast, can avoid the risk of salting out, improve the stability of the derivative product, and is conducive to ensuring the quality of chemical raw materials, and has extremely high commercial application value.

[0063] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0064] The following are the instruments, reference substances, reagents and chromatographic conditions involved in the embodiments and comparative examples.

[0065] Table 1

[0066]

[0067] Table 2

[0068]

[0069] Example 1

[0070] Step 1: Derivatization of the test sample. Place 720 mg of clenbuterol hydrochloride raw material sample in a volumetric flask, add co-solvent NMP and ultrasonically dissolve it, then add 12.8 ml of solvent acetonitrile to dilute it, add acid-binding agent DIEA and shake well, add benzoyl chloride solution (1.7 mg / ml, acetonitrile) and continue to shake well, and add purified water to the mark to serve as the test sample solution.

[0071] Step 2: Reference substance derivatization. It is basically the same as the first step, except that the clenbuterol hydrochloride bulk drug sample is replaced with tert-butylamine reference substance solution. Take the co-solvent NMP and place it in a volumetric flask, add an appropriate amount of tert-butylamine reference substance solution (1.825μg / ml, acetonitrile) 2.0ml, add acetonitrile 10.8ml for dilution, add the acid binding agent DIEA, shake well, add benzoyl chloride (1.7mg / ml, acetonitrile) and continue to shake well, add purified water to the scale, and use it as the reference substance solution.

[0072] Step 3: Injection procedure and method parameters. Take 10 μl of the two solutions obtained in the first and second steps above, respectively, and inject them into the high performance liquid chromatograph to record the peak area of ​​the tert-butylamine derivative in the chromatogram. Octadecyl bonded silica gel is used as the filler. The octadecyl bonded silica gel is Waters XBridge ® C18, 4.6*150mm*3.5μm, column temperature of 35℃, detection wavelength of 224nm, 0.1% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution, flow rate of 1.0 ml / min.

[0073] Table 3

[0074]

[0075] Step 4: Calculate the residual amount of tert-butylamine derivatives in the test sample according to the external standard method. The calculation formula is:

[0076] .

[0077] Among them: A S W: The peak area of ​​tert-butylamine derivatives in the test solution; S W: the weight of the test sample (mg); STD : The weight of the reference substance (mg); V STD V: dilution volume of reference solution (ml); S : dilution volume of the test solution (ml); A STD : Peak area of ​​tert-butylamine derivatives in reference solution; Pw: content of tert-butylamine reference.

[0078] In the first and second steps of this embodiment, the cosolvent is NMP; the amount of the acid-binding agent DIEA is: the molar ratio of the acid-binding agent to the tert-butylamine reference substance is 200,000:1; the amount of the derivatizing agent benzoyl chloride is: the molar ratio of the derivatizing agent to the tert-butylamine reference substance is 250:1. The quantitative test results using different amounts of cosolvent NMP are shown in Table 4.

[0079] Table 4

[0080]

[0081] As can be seen from Table 4, when the volume ratio of co-solvent NMP: solvent acetonitrile is 1:10, the tert-butylamine derivative has a large peak area and the test sample can be completely dissolved. This shows that the larger the volume of the co-solvent, the greater the inhibition of the derivatization reaction, but if the volume of the co-solvent is too low, the test sample will precipitate. Therefore, the volume ratio of co-solvent NMP: solvent acetonitrile was selected to be 1:10 for subsequent experiments.

[0082] Example 2

[0083] It is basically the same as Example 1, except that in the first and second steps: the volume ratio of the cosolvent NMP: the solvent acetonitrile is 1:10; the molar ratio of the acid-binding agent (TEA or DIEA) to the tert-butylamine reference substance is 40000:1; the molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine reference substance is 100:1. The quantitative detection results using different types of acid-binding agents are shown in Table 5.

[0084] Table 5

[0085]

[0086] It can be seen from Table 5 that there is basically no interference when the acid binding agent is DIEA. Therefore, DIEA was selected as the acid binding agent for subsequent experiments.

[0087] Example 3

[0088] The method is basically the same as Example 1, except that in the first and second steps, the volume ratio of the cosolvent NMP to the solvent acetonitrile is 1:10; the acid-binding agent is DIEA; and the molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine reference substance is 100:1. The quantitative detection results using different amounts of the acid-binding agent are shown in Table 6.

[0089] Table 6

[0090]

[0091] Recovery rate = (tert-butylamine content of the sample after spike-tert-butylamine content of the sample itself) / amount of tert-butylamine added

[0092] As can be seen from Table 6, the best effect is achieved when the molar ratio of the acid-binding agent DIEA to the tert-butylamine reference substance is 120000:1, with a high recovery rate and no interference. If the amount of DIEA added is small, the recovery rate is low; if the amount of DIEA added is high, the interference becomes greater.

[0093] Example 4

[0094] The method is basically the same as Example 1, except that in the first and second steps, the volume ratio of the cosolvent NMP to the solvent acetonitrile is 1:10; the acid-binding agent is DIEA; and the molar ratio of the acid-binding agent to the tert-butylamine reference substance is 120000:1. The quantitative detection results of the molar ratio of the derivatizing agent to the tert-butylamine reference substance are shown in Table 7.

[0095] Table 7

[0096]

[0097] As can be seen from Table 7, the best effect is achieved when the molar ratio of benzoyl chloride to tert-butylamine reference substance is 250:1. If the amount of benzoyl chloride added is small, the recovery rate is low; if the amount of benzoyl chloride added is high, the interference is large and the detection cannot be performed.

[0098] Conclusion: The final proposed conditions are the volume ratio of cosolvent NMP: solvent acetonitrile is 1:10; the acid binder is DIEA; the molar ratio of the acid binder to the tert-butylamine reference substance is 120000:1; the molar ratio of benzoyl chloride to the tert-butylamine reference substance is 250:1. The specific derivatization conditions are:

[0099] Derivatization of test sample: About 720 mg of clenbuterol hydrochloride raw material sample was placed in a 20 ml volumetric flask, 1.2 ml of co-solvent NMP was added and dissolved by ultrasound, then 12.8 ml of solvent acetonitrile was added for dilution, 1 ml of DIEA was added and shaken, 1.0 ml of benzoyl chloride solution (1.7 mg / ml, acetonitrile) was added and continued to shake, and purified water was added to the volume to make up to the mark as the test sample solution;

[0100] Derivatization of reference substance: Take 1.2 ml of NMP and place it in a 20 ml volumetric flask, add 2.0 ml of tert-butylamine reference solution (1.825 μg / ml, acetonitrile), add 10.8 ml of acetonitrile to dilute, add 1 ml of DIEA, shake well, add 1.0 ml of benzoyl chloride (1.7 mg / ml, acetonitrile) and continue to shake well, add purified water to make up to the mark as the reference solution;

[0101] Preparation of blank solution: Take 1.2 ml of NMP and place it in a 20 ml volumetric flask, add 12.8 ml of acetonitrile to dilute, add 0.5 ml of DIEA, shake well, add 1.0 ml of benzoyl chloride (1.7 mg / ml, acetonitrile) and continue to shake well, add purified water to make up to the mark as the blank solution.

[0102] Example 5

[0103] 5.1 Specificity test

[0104] Take blank solution, positioning solution, reference solution, and test solution, and perform detection after derivatization according to the proposed chromatographic conditions, and record the chromatogram. The preparation of positioning solution is similar to that of reference solution, except that the amount of tert-butylamine added to positioning solution is 5 times that of reference solution.

[0105] from Figure 1 It can be seen that the retention time of the chromatographic peak of the tert-butylamine derivative is about 18.5 min, and there is no interference from other components in the blank solution and the test sample at the tert-butylamine peak position, indicating good specificity.

[0106] 5.2 Limit of quantitation test

[0107] Place NMP in a volumetric flask, add tert-butylamine to prepare a solution of the required concentration, perform derivatization according to the proposed method, record the chromatogram, and take the concentration corresponding to the solution with a signal-to-noise ratio greater than 10:1 as the quantitative limit. The preparation of the quantitative limit solution is similar to that of the reference solution, except that the amount of tert-butylamine added is 0.3 times that of the reference solution. The results of the quantitative limit test are shown in Table 8.

[0108] Table 8

[0109]

[0110] From the above test results, it can be seen that when the concentration of the raw material sample solution is 36 mg / mL and the injection volume is 10 μL, the quantification limit of tert-butylamine under the proposed method is equivalent to 30% (1.5 ppm) of the limit (5 ppm), and the peak area RSD of the quantification limit solution after three consecutive injections is less than 15%.

[0111] 5.3 Detection limit test

[0112] Place NMP in a volumetric flask, add tert-butylamine to prepare a solution of the required concentration, perform the derivatization according to the proposed method, record the chromatogram, and take the concentration corresponding to the solution with a signal-to-noise ratio greater than 3:1 as the detection limit. The preparation of the detection limit solution is similar to that of the reference solution, except that the amount of tert-butylamine added is 0.1 times that of the reference solution. The detection limit test results are shown in Table 9.

[0113] Table 9

[0114]

[0115] From the above test results, it can be seen that when the concentration of the raw material sample solution is 36 mg / mL and the injection volume is 10 μL, the detection limit under the proposed method is equivalent to 10% (0.5 ppm) of the limit (5 ppm).

[0116] from Figure 2 It can be seen that the blank solution has basically no interference, and the signal-to-noise ratio of the detection limit solution and the quantification limit solution can meet the requirements.

[0117] 5.4 Linearity

[0118] According to the proposed method, 30%, 50%, 100%, 150%, 250%, and 500% of the limit concentration level of tert-butylamine (5ppm) were prepared as linear solutions. The preparation of each linear solution was similar to that of the reference solution, except that only the amount of tert-butylamine added was adjusted. The linear test results are shown in Table 10.

[0119] Table 10

[0120]

[0121] Figure 3 This is a linear graph of the linear regression of peak area against concentration when tert-butylamine is between 30% and 500% of the limit concentration in the linear test.

[0122] from Figure 3 It can be seen that the linear test shows that the peak area of ​​the derivative is linearly regressed to the concentration between the limit concentration of 30% (1.5ppm) and the limit concentration of 500% (25ppm) of tert-butylamine, and the linear regression coefficient R is 0.9999, and the absolute value of the Y-axis intercept / 100% peak area of ​​the concentration is 6.10%. This shows that under the proposed method, the concentration of tert-butylamine and the peak area have a good linear relationship.

[0123] 5.5 Accuracy test

[0124] Prepare the test solution and reference solution according to the proposed method, prepare spiked test solution with limit concentration of 50%, 100% and 150%, prepare 2 copies of each concentration in parallel, measure according to the proposed chromatographic conditions, record the chromatogram, calculate the content of tert-butylamine by peak area according to the external standard method, and calculate the recovery rate of tert-butylamine in each spiked test solution. The preparation of each accuracy test solution is similar to that of the test solution. After the solution is dissolved, different amounts of tert-butylamine are added as required. The accuracy test results are shown in Table 11.

[0125] Table 11

[0126]

[0127] The test results show that the recovery of tert-butylamine in 6 samples of spiked API at 3 levels should be in the range of 96.30% to 100.50%, with an average value of 98.5% and an RSD of 1.6%. The experimental results prove that the accuracy and precision of this method are good.

[0128] from Figure 4It can be seen that the recovery at the 100% spike level was good.

[0129] Comparative Example 1

[0130] Step A, test sample derivatization: take 50 mg of arotinolol hydrochloride bulk drug sample into a 25 ml volumetric flask, add 10 ml of methanol to dissolve, add 0.5 ml of 0.1 mol / L sodium hydroxide and 1 ml of a derivatizing agent (10% salicylaldehyde-methanol), derivatize in a 60°C water bath for 30 min, take out and cool, and dilute to the mark with methanol;

[0131] Step B, reference substance derivatization: Take an appropriate amount of tert-butylamine reference substance, add methanol to dilute to make a 50g / ml solution; take 1ml of the above solution into a 25ml volumetric flask, add 10mL of methanol, 0.5ml of 0.1mol / L sodium hydroxide and 1ml of derivatization agent (10% salicylaldehyde-methanol), derivatize in a 60℃ water bath for 30min, take out and cool, and dilute to the mark with methanol;

[0132] Step C: Take the above two solutions, accurately measure 20 μl of each, and inject them into a liquid chromatograph, using octadecyl bonded silica gel (WELCH Ultimate AQ-C18, 5 μm, 4.6×250 mm) as the filler, the column temperature is 30°C, the detection wavelength is 348 nm, 0.02 mol / L KH2PO4 (phosphoric acid adjusted to pH 3.0±0.1) as the mobile phase A, acetonitrile as the mobile phase B, and the gradient elution is shown in Table 12, with a flow rate of 1.0 ml / min.

[0133] Table 12

[0134]

[0135] Step D: Record the peak area of ​​the tert-butylamine derivative in the chromatogram and calculate the amount of tert-butylamine in the test sample by the external standard method:

[0136] Calculation formula

[0137] .

[0138] Where:

[0139] A 样 - Obtain the residual peak area in the chromatogram of the test solution;

[0140] A 对 - Obtain the residual peak area in the chromatogram of the reference solution;

[0141] M 对 -Weighing amount of reference substance (mg);

[0142] M 样 -Weighing amount of test sample (mg).

[0143] The test results show that the detection limit of tert-butylamine in Comparative Example 1 is 1000 ppm, and the detection limit of quantification is 10 ppm.

Claims

1. A method for analyzing residual trace amounts of tert-butylamine with high sensitivity and low limit, characterized in that: The tert-butylamine residual trace analysis method comprises the following steps: Step 1: dissolve the chemical raw material clenbuterol hydrochloride in acetonitrile and a co-solvent, add an acid-binding agent and a derivatizing agent to carry out a derivatization reaction, and after the reaction is completed, quench the reaction with water to obtain a test solution; Step 2: dissolving the tert-butylamine reference substance in acetonitrile and a co-solvent, adding an acid-binding agent and a derivatizing agent to carry out a derivatization reaction, and after the reaction is completed, quenching the reaction with water to obtain a reference substance solution; Step 3: Take appropriate amounts of the test solution and the reference solution, inject them into the high performance liquid chromatograph, and record the peak area of ​​the tert-butylamine derivative in the chromatogram; Step 4: Calculate the residual amount of tert-butylamine in the chemical raw material clenbuterol hydrochloride by external standard method; the calculation formula is: ； in: A S : Peak area of ​​tert-butylamine derivatives in the test solution; W S : The weight of the test sample, mg; W STD : The weight of the reference substance, mg; V STD : Volume of reference solution, ml; V S : Volume of test solution, ml; A STD : Peak area of ​​tert-butylamine derivative in reference solution; Pw: content of tert-butylamine reference substance; in: The cosolvent is N-methylpyrrolidone; the volume ratio of the cosolvent to acetonitrile is 1:1 to 1:10; The acid binding agent is N,N-diisopropylethylamine; the molar ratio of the acid binding agent to the tert-butylamine reference substance is 120000:1; The derivatizing agent is benzoyl chloride; the molar ratio of the derivatizing agent benzoyl chloride to the tert-butylamine reference substance is 100:1 to 250:1; Octadecyl bonded silica was used as the filler, the column temperature was 35 ± 3 °C, the mobile phase A was 0.1 ± 0.01% phosphoric acid aqueous solution, the mobile phase B was acetonitrile, and the flow rate was 1.0 ± 0.2 ml / min; The detection wavelength of the high performance liquid chromatograph is 224±5nm or 205±5nm; The gradient elution parameters of the high performance liquid chromatograph are: 。 2. The method for analyzing residual trace amounts of tert-butylamine according to claim 1, wherein: The reaction temperature is room temperature ~ 60 °C.

3. The method for analyzing residual trace amounts of tert-butylamine according to claim 1, characterized in that: The tert-butylamine derivative is an amide compound.

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