A detection method for olafluanid and its related components

Abstract

The invention discloses a detection method for olaflu and related components thereof, which has high sensitivity and high accuracy. The invention newly discovers two impurity components existing in olaflu raw materials and toothpaste, and the chromatographic separation of adjacent peaks is greater than 1.5. The olaflu and four impurity components are successfully analyzed and detected by HPLC-ELSD. The contents of olaflu and related impurities can be calculated according to a peak area normalization method, or the contents of olaflu and related impurities in toothpaste can be calculated according to an external standard method. The operation is simple and fast, and the method is suitable for industrial production and quality control.

Description

Technical Field

[0001] The invention belongs to the technical field of compound detection, and particularly relates to a method for detecting olafluanid and related components thereof. Background Art

[0002] Olafur (English name: Olafur, CAS No: 6818-37-7) is an important organic fluoride and is widely used in the field of oral care. The applicant found through research that many related substances are produced during the preparation process of Olafur. The production of related substances not only affects the product purity and yield of Olafur, but also poses certain hidden dangers to the safety of the product. However, there are currently no reports on the analysis and detection methods of related substances of Olafur in Olafur raw materials or toothpaste. Therefore, in view of the defects existing in the prior art, it is particularly important to establish a comprehensive, effective and stable method for analyzing related substances in Olafur, to control the quality of Olafur raw materials or toothpaste products containing Olafur, to improve their product quality and reduce toxic side effects. Summary of the invention

[0003] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0004] As one aspect of the present invention, the present invention provides a method for detecting olafluanid and its related components, which adopts HPLC-ELSD method to detect toothpaste samples or olafluanid raw material samples, the chromatographic column adopts RP18 or C8 chromatographic column, the mobile phase A is 5-20mM NH4Ac, pH=3.5-4.5, and the mobile phase B is methanol; the eluent is: in volume ratio, mobile phase A: mobile phase B=13-17:83-87, isocratic elution; the ELSD evaporation tube temperature is 40-50°C, and the drift tube temperature is 40-50°C.

[0005] As a preferred embodiment of the method for detecting olaflu and its related components described in the present invention: the method for preparing the olaflu raw material sample comprises dissolving the olaflu raw material with methanol and mixing them evenly to obtain the olaflu raw material sample and perform detection.

[0006] As a preferred embodiment of the detection method of olafluanid and its related components described in the present invention: the preparation method of the toothpaste sample comprises: adding the toothpaste sample to a dichloromethane solution containing triethylamine, vortex mixing and extracting, centrifuging to remove the precipitate, adding water to the supernatant, vortex mixing and extracting, centrifuging to remove the lower organic phase, blowing nitrogen and concentrating to dryness, adding methanol to the obtained product, dissolving it and filtering it through a microporous filter membrane to obtain the toothpaste sample.

[0007] As a preferred embodiment of the method for detecting olafluanid and its related components described in the present invention, 0.5 to 10 parts of toothpaste sample are added to 20 to 60 parts of dichloromethane containing 0.05 to 0.2% triethylamine by weight.

[0008] As a preferred embodiment of the method for detecting olafluanid and its related components described in the present invention: the column temperature of the chromatographic column is 30-40°C.

[0009] As a preferred embodiment of the method for detecting olafluanid and its related components described in the present invention: the flow rate of the chromatographic column is 1.0-1.5 mL / min.

[0010] As a preferred embodiment of the detection method of olaflu and its related components described in the present invention: the olaflu and its related components are olaflu, impurity A, impurity B, impurity C and impurity D respectively;

[0011] The structural formula of the olaflurane is:

[0012]

[0013] The structural formula of the impurity A is:

[0014]

[0015] The structural formula of the impurity B is:

[0016]

[0017] The structural formula of the impurity C is:

[0018]

[0019] The structural formula of the impurity D is:

[0020] .

[0021] As a preferred embodiment of the method for detecting olafluanid and its related components described in the present invention: the gas flow rate of the ELSD is 1.0-1.6 SLM.

[0022] As a preferred embodiment of the method for detecting olafluanid and its related components described in the present invention, it also includes drawing a standard working curve, and the gradient concentration range of the quasi-working curve is 30-600 μg / mL.

[0023] As a preferred embodiment of the method for detecting olafluanid and its related components described in the present invention: the mobile phase A is 10 mM NH4Ac, pH=4.0, and the mobile phase B is methanol; the eluent is: in volume ratio, mobile phase A: mobile phase B=15:85.

[0024] Beneficial effects of the present invention: The present invention utilizes a high performance liquid phase to analyze the related substances of olaflu, which has high sensitivity and high accuracy. The present invention newly discovers two impurity components existing in olaflu raw materials and toothpaste, and the chromatographic separation of adjacent peaks is greater than 1.5. The olaflu and four impurity components are successfully analyzed and detected by HPLC-ELSD. The content of olaflu and related impurities can be calculated according to the peak area normalization method, or the content of olaflu and related impurities in toothpaste can be calculated according to the external standard method. The operation is simple and fast, and the present invention is suitable for industrial production and quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0026] Figure 1 This is the chromatogram of Example 1.

[0027] Figure 2 This is the LC-MS total ion current diagram of Example 2.

[0028] Figure 3 This is the chromatogram of Example 3.

[0029] Figure 4 This is the chromatogram of Control Example 1.

[0030] Figure 5 This is the chromatogram of Control Example 2.

[0031] Figure 6 This is the chromatogram of Control Example 3.

[0032] Figure 7 This is the chromatogram of Control Example 4. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0034] Embodiment 1:

[0035] Detection of related substances in olaflurane raw materials:

[0036] Take the raw material of olaflurane, dissolve it in methanol and quantitatively dilute it to make a solution with a raw material concentration of 1 mg / mL, and shake it well. Perform HPLC-ELSD detection, and the chromatographic conditions are as follows: detection instrument: Agilent 1260 InfinityⅡ liquid chromatograph equipped with ELSD detector; chromatographic column: Agilent Eclipse XDB-C8 (4.6*250mm, 5.0μm), column temperature: 30℃, mobile phase A: 10 mMNH4Ac (pH 4.0, pH adjusted with acetic acid) aqueous solution; mobile phase B: methanol; flow rate: 1.0 mL / min; eluent: mobile phase A: mobile phase = 15:85 (volume ratio), and use high performance liquid chromatography isocratic elution.

[0037] ELSD parameters: evaporation tube temperature: 50°C, drift tube temperature: 40°C, gas flow rate: 1.6SLM. The test results are as follows Figure 1 shown.

[0038] The retention time and peak area of ​​olafluanid and related impurities are shown in Table 1:

[0039] Table 1

[0040]

[0041] Embodiment 2:

[0042] Structural identification of olafluanid related substances in Example 1:

[0043] Using the same mobile phase and sample pretreatment conditions as in Example 1, LC-MS detection was performed on the olafluran raw material sample. The LC-MS results are shown in Table 2, and the LC-MS total ion current diagram is shown in Figure 2 .

[0044] Table 2

[0045]

[0046] The present invention discovers for the first time two new impurities mixed in the raw materials of Alaflu: impurity C and impurity D.

[0047] Embodiment 3:

[0048] Take an appropriate amount of olaflurane standard and impurity A, impurity B, impurity C and impurity D standards, add them to methanol and mix them evenly, as the system suitability test solution, inject them into the liquid chromatograph, and perform HPLC-ELSD detection. The chromatographic conditions are as follows: detection instrument: Agilent 1260 InfinityⅡ liquid chromatograph equipped with ELSD detector; chromatographic column: Agilent EclipseXDB-C8 (4.6*250mm, 5.0μm), column temperature: 30℃, mobile phase A: 10 mM NH4Ac (pH 4.0, pH adjusted by acetic acid) aqueous solution; mobile phase B: methanol; flow rate: 1.0 mL / min; eluent is mobile phase A: mobile phase = 15:85 (volume ratio), and elution isocratic by high performance liquid chromatography. ELSD parameters: evaporation tube temperature: 40℃, drift tube temperature: 40℃, gas flow rate: 1.6SLM. The peak order is impurity C, impurity D, impurity A, impurity B and olaflurane. The separation between the components is calculated as shown in Table 3. Figure 3 shown.

[0049] Table 3

[0050]

[0051] It can be seen from Table 3 that the separation between the components is greater than 1.5.

[0052] Comparative Example 1:

[0053] The difference between this comparative example and Example 3 is that the eluent is mobile phase A: mobile phase B = 10:90 (volume ratio), mobile phase A: 10 mM NH4Ac (pH 4.0, pH adjusted with acetic acid) aqueous solution; mobile phase B: methanol; flow rate: 1.0 mL / min, and the other conditions are the same as those in Example 3. See the chromatogram Figure 4 It can be seen that the chromatographic peaks of impurity C and impurity D overlap, baseline separation is not achieved, and the separation degree is less than 1.5.

[0054] Comparative Example 2:

[0055] The difference between this comparative example and Example 3 is that the eluent is mobile phase A: mobile phase = 20:80 (volume ratio), mobile phase A: 10 mM NH4Ac (pH 4.0, pH adjusted with acetic acid) aqueous solution; mobile phase B: methanol; flow rate: 1.0 mL / min, and the other conditions are the same as those in Example 3. See the chromatogram Figure 5 It can be seen that the chromatographic peaks of impurity C and impurity D overlap, baseline separation is not achieved, and the separation degree is less than 1.5.

[0056] Comparative Example 3:

[0057] The difference between this comparative example and Example 1 is: ELSD parameters: evaporation tube temperature: 30°C, drift tube temperature: 40°C, gas flow rate: 1.6 SLM. The other conditions are the same as those in Example 1. See the chromatogram Figure 6 , the baseline signal-to-noise ratio increased significantly, and the signal responses of impurities C and D disappeared.

[0058] Comparative Example 4:

[0059] The difference between this comparative example and Example 1 is: ELSD parameters: evaporation tube temperature: 70°C, drift tube temperature: 70°C, gas flow rate: 1.6 SLM. The other conditions are the same as those in Example 1. See the chromatogram Figure 7 , the chromatographic peak response decreased, and the signal responses of impurities C and impurity D disappeared.

[0060] Embodiment 4:

[0061] Detection of olaflurane-related substances in toothpaste:

[0062] Accurately weigh 1 g of toothpaste sample in a 50 mL centrifuge tube, add 30 mL of dichloromethane solution containing 0.1% triethylamine, vortex for 10 min for full extraction, centrifuge at 12000 rpm, remove the precipitate and transfer the supernatant, add 10 mL of deionized water to the supernatant, vortex for 3 minutes, centrifuge at 12000 rpm, transfer the lower organic phase to a clean nitrogen-blown bottle, concentrate to dryness with nitrogen, add 10 mL of methanol, and filter the solution through a 0.45 μm microporous filter membrane to obtain the test solution for use.

[0063] The test samples were subjected to HPLC-ELSD detection, and the chromatographic conditions were as follows: detection instrument: Agilent 1260 InfinityⅡ liquid chromatograph equipped with ELSD detector; chromatographic column: Agilent Eclipse XDB-C8 (4.6*250mm, 5.0μm), column temperature: 30℃, mobile phase A: 10 mM NH4Ac (pH 4.0, pH adjusted with acetic acid) aqueous solution; mobile phase B: methanol; flow rate: 1.0 mL / min; eluent: mobile phase A: mobile phase = 15:85 (volume ratio), and elution was performed by high performance liquid chromatography with isocratic elution.

[0064] ELSD parameters: evaporation tube temperature: 40°C, drift tube temperature: 40°C, gas flow rate: 1.6 SLM. The retention time and peak area of ​​olafluanid and related impurities are shown in Table 4:

[0065] Table 4

[0066]

[0067] Quantitative detection of olafluanid and its impurities by external standard method:

[0068] Impurities A, B, C, D and the olaflurane reference substance were respectively prepared into standard working solutions of 5 different concentrations (30, 100, 300, 600, 900ug / L), and analyzed by high performance liquid chromatography from low concentration to high concentration, and the standard working curve was drawn. The test results are shown in Table 5. Within a certain range, the concentrations of the four impurities showed a good linear relationship with the peak area.

[0069] Table 5 Linear regression equations and linear ranges of the methods for olafluanid and its impurities

[0070]

[0071] The present invention utilizes high performance liquid phase to analyze the related substances of olaflu, which has high sensitivity and high accuracy. The present invention newly discovers two impurity components existing in olaflu raw materials and toothpaste, and the chromatographic separation of adjacent peaks is greater than 1.5. The olaflu and four impurity components are successfully analyzed and detected by HPLC-ELSD. The content of olaflu and related impurities in the olaflu raw materials can be calculated according to the peak area normalization method, or the content of olaflu and related impurities in toothpaste or olaflu raw materials can be calculated according to the external standard method. The operation is simple and fast, and the present invention is suitable for industrial production and quality control.

[0072] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for detecting olafluanid and its related components, characterized in that: The toothpaste sample or the olafluran raw material sample is detected by HPLC-ELSD method, the chromatographic column is RP18 or C8 chromatographic column, the mobile phase A is 5-20mM NH4Ac, pH=3.5-4.5, the mobile phase B is methanol; the eluent is: in volume ratio, mobile phase A: mobile phase B=13-17:87-83, isocratic elution; the ELSD evaporation tube temperature is 40-50°C, and the drift tube temperature is 40-50°C; The preparation method of the olaflu raw material sample comprises dissolving the olaflu raw material with methanol and mixing them evenly to obtain the olaflu raw material sample and perform detection; The toothpaste sample preparation method comprises adding the toothpaste sample to a dichloromethane solution containing triethylamine, vortexing and mixing for extraction, centrifuging to remove precipitation, adding water to the supernatant, vortexing and mixing for extraction, centrifuging to remove the lower organic phase, blowing nitrogen to concentrate to dryness, adding methanol to the obtained product, dissolving it, and filtering it through a microporous filter membrane to obtain the toothpaste sample; The olafluanid and its related components are olafluanid, impurity A, impurity B, impurity C and impurity D; The structural formula of the olaflurane is: The structural formula of the impurity A is: The structural formula of the impurity B is: The structural formula of the impurity C is: The structural formula of the impurity D is:

2. The method for detecting olafluanid and its related components according to claim 1, characterized in that: The step of adding the toothpaste sample into the dichloromethane solution containing triethylamine is calculated by weight, that is, adding 0.5 to 10 parts of the toothpaste sample into 20 to 60 parts of dichloromethane containing 0.05 to 0.2% of triethylamine.

3. The method for detecting olafluanid and its related components according to claim 1 or 2, characterized in that: The column temperature of the chromatographic column is 30-40°C.

4. The method for detecting olafluanid and its related components according to claim 1 or 2, characterized in that: The flow rate of the chromatographic column is 1.0 to 1.5 mL / min.

5. The method for detecting olafluanid and its related components according to claim 1 or 2, characterized in that: The gas flow rate of the ELSD is 1.0-1.6 SLM.

6. The method for detecting olafluanid and its related components according to claim 1 or 2, characterized in that: The method also includes drawing a standard working curve, wherein the gradient concentration range of the quasi-working curve is 30 to 600 μg / mL.

7. The method for detecting olafluanid and its related components according to claim 1 or 2, characterized in that: The mobile phase A is 10 mM NH4Ac, pH=4.0, and the mobile phase B is methanol; the eluent is: in volume ratio, mobile phase A:mobile phase B=15:85.

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