A method for detecting the purity of dimethylpyridinamine by high performance liquid chromatography

Abstract

The application relates to the field of analytical chemistry, and particularly discloses a method for detecting the purity of dimethylpyridine amine by high performance liquid chromatography. The method for detecting the purity of dimethylpyridine amine by high performance liquid chromatography comprises the following steps: S1, preparing a dimethylpyridine amine detection sample: diluting dimethylpyridine amine with a mobile phase to prepare a dimethylpyridine amine detection sample; S2, high performance liquid detection: sampling and sample injection for detection, wherein the chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as the filler; the mobile phase is a water phase containing trifluoroacetic acid and an organic phase containing trifluoroacetic acid; the water phase containing trifluoroacetic acid accounts for 60-80 v / v% of the mobile phase, and the organic phase containing trifluoroacetic acid accounts for 20-40 v / v% of the mobile phase. The detection method has the effects of good separation effect, symmetrical peak, shortened analysis time and high detection sensitivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, more particularly, it relates to a method for detecting the purity of dimethylpyridinamine by high performance liquid chromatography. BACKGROUND

[0002] Dimethylpyridinamine is an important chemical and pharmaceutical raw material intermediate, with a molecular formula of C 12 H 13 N3, a molecular weight of 199.25, a CAS number of 1539-42-0, a chemical name of 2,2-dipicolylamine, and a structural formula of dimethylpyridinamine as follows:

[0003]

[0004] The structural formulas of two impurities, 2-cyanopyridine and 2-(aminomethyl)pyridine, in the synthesis process of dimethylpyridinamine are as follows, the former is 2-cyanopyridine, and the latter is 2-(aminomethyl)pyridine;

[0005]

[0006] In related technologies, the Chinese application file with the application number 202110382208 discloses an application of zinc-dimethylpyridinamine in preparing a drug for diagnosing thrombotic diseases; the Chinese application file with the application number CN201810019857.6 discloses a dimethylpyridinamine-based ultraviolet probe and its preparation and application; and it is indicated that the application field of dimethylpyridinamine is relatively wide. Therefore, a method for detecting dimethylpyridinamine with high sensitivity, speed and stability is needed to separate dimethylpyridinamine and two impurities, 2-cyanopyridine and 2-(aminomethyl)pyridine, in the synthesis process of dimethylpyridinamine under the same chromatographic conditions.

[0007] In the prior art, the detection method of dimethylpyridinamine is gas chromatography analysis method, and the chromatographic conditions are as follows: chromatographic column, HP-5; injection port temperature, 250 ℃; FID detector temperature, 300 ℃; flow rate, 2 ml / min; injection mode, split injection; split ratio, 50:1; carrier gas, high-purity nitrogen; injection volume, 0.5 μL; and the programmed temperature method is shown in the following table; and the detection results are shown in the description accompanying drawings. Figure 1

[0008]

[0009]

[0010] ​In the related art, the peak symmetry of dimethylpyridine amine detected by gas chromatography is poor, the relative retention time of the main peak dimethylpyridine amine is long, and due to the high boiling point of unknown components during the synthesis of dimethylpyridine amine, there are incomplete gasification substances in the sample in the gas chromatography method, and after multiple injections, it is found that the glass liner is contaminated, and in the continuous sample test, the unknown peak often appears in the gas chromatogram, thereby causing deviation in the sample purity analysis. Therefore, there is an urgent need for a method for detecting dimethylpyridine amine with good detection stability, high sensitivity, good separation effect and short analysis time. SUMMARY

[0011] In order to provide a method for detecting dimethylpyridine amine with good detection stability, high sensitivity, good separation effect and short analysis time, the application provides a method for detecting the purity of dimethylpyridine amine by high performance liquid chromatography.

[0012] The application provides a method for detecting the purity of dimethylpyridine amine by high performance liquid chromatography, which adopts the following technical scheme:

[0013] A method for detecting the purity of dimethylpyridine amine by high performance liquid chromatography, comprising the following steps:

[0014] Step S1, dimethylpyridine amine detection sample preparation: dilute dimethylpyridine amine with mobile phase to prepare dimethylpyridine amine detection sample;

[0015] Step S2, high performance liquid detection: sampling and sample injection for detection, wherein the chromatographic column adopts a chromatographic column with octadecylsilane bonded silica gel as the filler; the mobile phase adopts a water phase containing trifluoroacetic acid and an organic phase containing trifluoroacetic acid; wherein the water phase containing trifluoroacetic acid accounts for 60-80v / v% of the mobile phase, and the organic phase containing trifluoroacetic acid accounts for 20-40v / v% of the mobile phase.

[0016] By adopting the above technical scheme, unlike the prior art, the application uses high performance liquid chromatography to detect the sample dimethylpyridine amine, and the chromatographic column with octadecylsilane bonded silica gel as the filler used in the application is a general high performance liquid chromatographic column, which is cheap. The mobile phase in the application is a water phase containing trifluoroacetic acid and an organic phase containing trifluoroacetic acid, and the ratio of the water phase containing trifluoroacetic acid to the organic phase containing trifluoroacetic acid is adjusted; compared with the detection method of dimethylpyridine amine by gas chromatography, this method uses a specific chromatographic column and a specific ratio of mobile phase, so that the separation effect of dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl)pyridine is good, the peak is symmetrical, and the analysis time is shortened; the detection sensitivity is also high.

[0017] Optionally, in the water phase containing trifluoroacetic acid, the concentration of trifluoroacetic acid is 0.08v / v%-0.12v / v%.

[0018] By adopting the technical scheme, in combination with the different structure polarity of the substances to be separated, the concentration of trifluoroacetic acid in the aqueous phase is adjusted in a specific range, so that the polar part and the non-polar part in the sample driven into the chromatographic column by the mobile phase are well separated, the peak time of dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine in the sample is close to the peak time of dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine in the standard, the reproducibility of multiple experiments is good, the solution stability is good, and the linear relationship of detecting dimethylpyridine amine standard is good.

[0019] Optionally, in the trifluoroacetic acid-containing organic phase, the concentration of trifluoroacetic acid is 0.08v / v%-0.12v / v%.

[0020] By adopting the technical scheme, in combination with the different structure polarity of the substances to be separated, the concentration of trifluoroacetic acid in the aqueous phase is adjusted in a specific range, so that the polar part and the non-polar part in the sample driven into the chromatographic column by the mobile phase are well separated, the peak time of dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine in the sample is close to the peak time of dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine in the standard, the reproducibility of multiple experiments is good, the solution stability is good, and the linear relationship of detecting dimethylpyridine amine standard is good.

[0021] Optionally, in the trifluoroacetic acid-containing organic phase, the solvent is methanol.

[0022] By adopting the technical scheme, since the polarity of methanol is larger, the solubility of dimethylpyridine amine in methanol is very strong, when methanol and ethanol are used to dissolve dimethylpyridine amine respectively, methanol as a solvent dissolves dimethylpyridine amine more thoroughly, therefore, methanol is selected as the organic phase, and the separation degree of dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine in the sample is better.

[0023] Optionally, in the step S2, the flow rate of the mobile phase is 0.9-1.1mL / min.

[0024] By adopting the technical scheme, the appropriate flow rate of the mobile phase is selected, so that the residence time of the non-polar substances in the chromatographic column can be effectively controlled, the separation degree of the three substances dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine in the sample is good, the peak symmetry is good, there is no tailing, and the analysis time is shortened.

[0025] Optionally, in the step S1, the concentration of dimethylpyridine amine diluted with the mobile phase is 0.0026-0.012g / mL.

[0026] By adopting the technical scheme, the sample with the appropriate concentration can be fully contacted with the mobile phase under the driving of the mobile phase, so that the mobile phase can better separate dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine, and the peak symmetry is good, there is no tailing, and the analysis time is short.

[0027] Optionally, in the step S2, the column temperature of the chromatographic column is 30-40℃.

[0028] By adopting the technical scheme, the column temperature of 30-40℃ is selected, so that a large number of substance volatilization phenomena do not occur in the mobile phase and the sample. Compared with the gas chromatography in the prior art, the method of the application does not pollute the liquid chromatograph, so that the appearance of impurity peaks is avoided, and the reproducibility of multiple detections is good.

[0029] Optionally, in the step S2, the detection wavelength is 254-265nm.

[0030] By adopting the technical scheme, the sample is detected at the optimal wavelength, so that the interference of other components on the detection result is reduced, and the accuracy of the detection result is improved.

[0031] Optionally, in the step S2, the injection volume is 5-15μL.

[0032] By adopting the technical scheme, the small injection amount ensures that any substance in the sample can be identified and detected, so that the accuracy of the detection result is high.

[0033] In summary, the application has the following beneficial effects:

[0034] 1. In the application, high performance liquid chromatography is used to detect dimethylpyridine amine, and the mobile phase adopts a specific proportion of water phase containing trifluoroacetic acid and organic phase containing trifluoroacetic acid; compared with the detection method of dimethylpyridine amine by gas chromatography, this method has good separation effect of dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine, good peak symmetry, short analysis time, and high detection sensitivity;

[0035] 2. In the mobile phase of the application, the concentration of trifluoroacetic acid in the water phase and the concentration of trifluoroacetic acid in the organic phase are in a specific range, so that the polar part and the non-polar part in the sample can be well separated, the peak time of dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine in the sample is close to that of the standard dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine, the reproducibility of multiple experiments is good, the solution stability is good, and the linear relationship of the detection of dimethylpyridine amine standard is good;

[0036] 3, The application selects a suitable flow rate of the mobile phase, so that the residence time of the non-polar substances in the sample on the chromatographic column can be effectively controlled, and the separation degree of the three substances dimethylpyridine amine, 2-cyanopyridine and 2-(aminomethyl) pyridine detected finally is good, the peak symmetry is good, there is no tailing, and the analysis time is shortened. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a gas chromatogram intended to show the gas chromatography detection of dimethylpyridine amine in the sample in the background art;

[0038] Figure 2 is a liquid chromatogram intended to show the liquid chromatography detection of dimethylpyridine amine in the sample in Example 1 of the application;

[0039] Figure 3 is a linear regression graph intended to show the liquid chromatography detection of dimethylpyridine amine standard in Example 1 of the application. DETAILED DESCRIPTION

[0040] The application will be further described in detail below in combination with examples and comparative examples.

[0041] The sources of raw materials for the following examples and comparative examples are provided: The raw materials for the examples and comparative examples can be commercially purchased, and all samples of the application are from Shaanxi Dabing Pharmaceutical Technology Co., Ltd.; The dimethylpyridine amine standard is (aladdin batch number C2011152); The 2-(aminomethyl) pyridine is (macklin batch number C11980176); The 2-cyanopyridine is (macklin batch number C11980176).

[0042] An example of a method for detecting the purity of dimethylpyridine amine by high performance liquid chromatography

[0043] Method validation example

[0044] Example 1

[0045] A method for detecting the purity of dimethylpyridine amine by high performance liquid chromatography, comprising the following steps:

[0046] Preparation of mobile phase: mix the water phase containing 0.1% trifluoroacetic acid by volume concentration and the organic phase containing 0.1% trifluoroacetic acid by volume concentration according to the volume ratio of 70:30, and the organic solvent is methanol, to prepare the mobile phase;

[0047] Step S1, preparation of dimethylpyridine amine detection sample: weigh 2021060301 sample into a 10 mL volumetric flask, dilute to the mark with mobile phase, shake well, and prepare dimethylpyridine amine detection sample;

[0048] Step S2, high performance liquid detection: the chromatographic column is Waters 4.6 x 5 μL column; column temperature 35 °C, detection wavelength 265 nm, flow rate of mobile phase 1.0 mL / min, injection volume 10 μL, detection.

[0049] Performance test:

[0050] a, specificity test

[0051] 1) Take sample 0.1021 g and standard dimethylpyridine amine 0.1053 g, using the detection method of Example 1, the retention time is shown in Table 1 as follows;

[0052] Table 1

[0053]

[0054] 2) Take sample and standard 2-(aminomethyl)pyridine 0.0513 g, using the detection method of Example 1, the retention time is shown in Table 2 as follows;

[0055] Table 2

[0056]

[0057] 3) Take sample 0.1039 g and standard 2-cyanopyridine 0.0533 g, using the detection method of Example 1, the retention time is shown in Table 3 as follows;

[0058] Table 3

[0059]

[0060] From the specificity test results, under the same chromatographic conditions, using the same sample, the retention time of dimethylpyridine amine and two impurities 2-cyanopyridine and 2-(aminomethyl)pyridine in the synthesis process of dimethylpyridine amine are consistent with the standard, which proves that the detection method of the application is excellent.

[0061] b, retention time, resolution and theoretical plate number test

[0062] Take sample 0.1016 g, using the detection method of Example 1, the retention time, resolution and theoretical plate number are obtained, as shown in Table 4 as follows, as shown in the description Figure 2 ;

[0063] Table 4

[0064]

[0065]

[0066] From the above retention time, resolution and theoretical plate number detection results, it can be seen that the detection method of the application can separate dimethylpyridine amine, 2-(aminomethyl)pyridine and 2-cyanopyridine in the sample, and the separation effect is good, and the peak shape is symmetrical.

[0067] c. Reproducibility detection

[0068] Take 0.1062 g of the sample, use the detection method of Example 1 to continuously sample 6 times, and record the peak area of dimethylpyridine amine, as shown in Table 5 below;

[0069] Table 5

[0070]

[0071] From the above reproducibility detection results, it can be seen that the RSD% is 0.79, indicating that the detection method of the application has good reproducibility and high sensitivity.

[0072] d. Solution stability detection

[0073] Take 0.1062 g of the sample, use the detection method of Example 1, sample every 0, 1, 2, 4, 6, 8 hours, and record the peak area of dimethylpyridine amine, as shown in Table 6 below;

[0074] Table 6

[0075]

[0076] From the above reproducibility detection results, it can be seen that the RSD% is 0.94, indicating that in the detection method of the application, the prepared dimethylpyridine amine detection sample solution has good stability and high detection sensitivity.

[0077] e. Linear relationship detection of standard dimethylpyridine amine

[0078] Replace the 2021060301 sample in the application with standard dimethylpyridine amine, use the detection method of Example 1, and the standard concentration and detection peak area are shown in Table 7 below; the linear regression graph is shown in the specification Figure 3 ;

[0079] Table 7

[0080]

[0081] From Table 7 and the specification Figure 3 , it can be seen that the R 2 is 0.9931, indicating that the detection method of the application has good linear relationship for detecting standard samples.

[0082] Sample detection example

[0083] Example 2

[0084] The difference from Example 1 is that a different batch of sample of different mass is used in step S1 : 0.0273 g of sample 2021060701 is used.

[0085] Example 3

[0086] The difference from Example 1 is that a different batch of sample of different mass is used in step S1 : 0.1057 g of sample 2021060901 is used.

[0087] Example 4

[0088] The difference from Example 1 is that a different batch of sample of different mass is used in step S1 : 0.0273 g of sample 2021060701 is used.

[0089] Example 5

[0090] The difference from Example 1 is that a different batch of sample of different mass is used in step S1 : 0.0733 g of sample 2021060701 is used.

[0091] Example 6

[0092] The difference from Example 1 is that in the mobile phase preparation, the water phase containing trifluoroacetic acid with a volume concentration of 0.08% is mixed with the organic phase containing trifluoroacetic acid with a volume concentration of 0.12% according to a volume ratio of 80:20, and the organic phase is methanol.

[0093] In step S2, the column temperature is 30°C, the detection wavelength is 254 nm, and the flow rate of the mobile phase is 0.9 mL / min.

[0094] Example 7

[0095] The difference from Example 1 is that in the mobile phase preparation, the water phase containing trifluoroacetic acid with a volume concentration of 0.12% is mixed with the organic phase containing trifluoroacetic acid with a volume concentration of 0.08% according to a volume ratio of 60:40.

[0096] In step S2, the column temperature is 40°C, the detection wavelength is 260 nm, and the flow rate of the mobile phase is 1.1 mL / min.

[0097] Comparative Example 1

[0098] The difference from Example 1 is that in the mobile phase preparation, the water phase containing trifluoroacetic acid with a volume concentration of 0.1% is mixed with the organic phase containing trifluoroacetic acid with a volume concentration of 0.1% according to a volume ratio of 30:70.

[0099] Performance detection test

[0100] The peak area of dimethylpyridine amine obtained by the method of examples 1 to 7 and comparative example 1 was calculated to obtain the sample purity, which was compared with the sample purity of the same sample obtained by gas chromatography, and the comparison results are shown in Table 9 below, wherein the sample purity obtained by gas chromatography is purity 1, and the sample purity obtained by the method of the application is purity 2.

[0101] Gas chromatography conditions: chromatographic column: HP-5; injection port temperature: ; FID detector temperature: ; flow rate: 2 ml / min; injection mode: split injection; split ratio: 50:1; carrier gas: high-purity nitrogen; injection volume: 0.5 μL; gas chromatography temperature program is shown in Table 8 below;

[0102] Table 8

[0103] Rate Temperature Retention time 0°C / min 80.0℃ 2.0 min 20°C / min 300.0℃ 5.0 min

[0104] The calculation method of peak area for calculating sample purity is normalization method.

[0105] Calculation formula: Xi% = [Ai / (A1+A2+…+An)]x100, Xi: percentage content of component i; A1, A2…An: peak area of components 1, 2…n.

[0106] Table 9

[0107] Purity 1 (%) Purity 2 (%) Difference absolute value (%) Example 1 95.63 95.38 0.25 Example 2 96.95 96.63 0.32 Example 3 97.39 97.26 0.13 Example 4 96.72 96.59 0.13 Example 5 96.72 96.88 0.16 Example 6 96.37 96.54 0.17 Example 7 97.25 97.13 0.12 Comparative Example 1 90.68 96.64 5.96

[0108] In combination with examples 1 to 5, it can be seen that the difference between purity 2 and purity 1 detected by examples 1 to 5 using different batches of samples with different concentrations is relatively small, and the absolute value is controlled within 0.33; it is proved that the method for detecting dimethylpyridine amine purity of the application has universality for the detection of dimethylpyridine amine.

[0109] In combination with examples 1, 6 and 7, it can be seen that the difference between purity 1 and purity 2 detected by examples 1, 6 and 7 is relatively small, and the absolute value is controlled within 0.26; and examples 1, 6 and 7 cover the protection scope of the claims of the application, which proves that the protection scope of the claims of the application is reasonable.

[0110] In combination with examples 1 and comparative example 1, it can be seen that in comparative example 1, the volume ratio of the mobile phase is not within the protection scope of the application, so the purity detected by comparative example 1 is quite different from the purity detected by gas chromatography; it is proved that the mobile phase of the application must be selected from the mobile phase within the protection scope of claim 1.

[0111] The embodiments are only illustrative of the present application, and are not intended to limit the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for detecting the purity of dimethylpyridinamine by high performance liquid chromatography, characterized in that, The method comprises the following steps: Step S1, preparation of a dimethylpyridine amine detection sample: dilute the dimethylpyridine amine with a mobile phase to prepare a dimethylpyridine amine detection sample; The concentration of the dimethylpyridine amine diluted with the mobile phase is 0.0026-0.012 g / mL; Step S2, high-performance liquid detection: take a sample and inject the sample for detection, wherein the chromatographic column is an octadecylsilane-bonded silica gel chromatographic column; the mobile phase is a water phase containing trifluoroacetic acid and an organic phase containing trifluoroacetic acid; the water phase containing trifluoroacetic acid accounts for 60-80 v / v% of the mobile phase, and the organic phase containing trifluoroacetic acid accounts for 20-40 v / v% of the mobile phase; In the organic phase containing trifluoroacetic acid, the solvent is methanol; In the step S2, the column temperature of the chromatographic column is 30-40℃, the detection wavelength is 254-265 nm, and the injection volume is 5-15 μL; The detection sample is a sample with a batch number of 2021060301 produced by Shaanxi Dasheng Pharmaceutical Technology Co., Ltd.

2. The method for detecting the purity of dimethylpyridinamine by high performance liquid chromatography according to claim 1, characterized in that, In the water phase containing trifluoroacetic acid, the concentration of trifluoroacetic acid is 0.08 v / v%-0.12 v / v%.

3. The method for detecting the purity of dimethylpyridinamine by high performance liquid chromatography according to claim 1, characterized in that, In the organic phase containing trifluoroacetic acid, the concentration of trifluoroacetic acid is 0.08 v / v%-0.12 v / v%.

4. The method for detecting the purity of dimethylpyridinamine by high performance liquid chromatography according to any one of claims 1-3, characterized in that, In the step S2, the flow rate of the mobile phase is 0.9-1.1 mL / min.

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