A method for the quantitative determination of olaflur in raw materials and oral care products

Abstract

The application discloses a method for quantitatively detecting olaflur in raw materials and oral care products, and belongs to the technical field of high performance liquid chromatography-evaporative light scattering detection, and comprises the following steps: detecting olaflur raw material samples or toothpaste samples by using a high performance liquid chromatography-evaporative light scattering detection method; wherein the conditions of the high performance liquid chromatography comprise the following steps: a C18 chromatographic column with octadecylsilane-bonded silica gel as filler; a mobile phase, wherein phase A is a water solution containing 0.03-0.08 vol% trifluoroacetic acid, phase B is an acetonitrile solution containing 0.03-0.08 vol% trifluoroacetic acid; and gradient elution. The sample pre-treatment process is convenient, does not need solid phase extraction, is suitable for rapid detection, the mobile phase and elution gradient are optimized, the peak broadening and residual problems are effectively inhibited, the baseline stability is improved under the gradient condition, the sensitivity and accuracy are improved, the method can be used for detecting high and low olaflur content samples, and is suitable for industrial production and quality control.

Description

Technical Field

[0001] This invention belongs to the field of high performance liquid chromatography-evaporative light scattering detection technology, specifically relating to a quantitative detection method for olaflur in raw materials and oral care products. Background Technology

[0002] Olafur (CAS: 6818-37-7, C) 27 H 60 F2N2O3 is an organic fluoride source in toothpaste, possessing a triple mechanism of "film formation + remineralization + long-lasting antibacterial effect." Current detection methods primarily employ ion-selective electrodes or gas chromatography, which suffer from drawbacks such as cumbersome pretreatment, low sensitivity, and significant matrix interference. Because olafur (fluoride) lacks a chromophore in its structure and exhibits weak absorption in the UV region, conventional HPLC-UV detection suffers from low sensitivity and severe peak tailing. Therefore, developing a simple, accurate, and applicable analytical method for production and quality control is crucial. Due to olafur's high boiling point, low volatility, lack of a chromophore, and absence of derivatizable sites, conventional gas chromatography and HPLC-UV methods are not well-suited for its application. Ion chromatography methods suffer from significant matrix interference and can only measure the total fluoride ion content, not the concentration of olafur itself. Furthermore, this method cannot distinguish between olafur and other fluoride sources such as sodium fluoride and sodium monofluorophosphate in toothpaste.

[0003] The existing solid-phase extraction-ultra-high performance liquid chromatography-electrospray detector method can achieve qualitative and quantitative analysis of olafrine in products. However, this method still has many problems: ① Sample pretreatment is complex, requiring multiple vortex centrifugation extractions. After the supernatants are combined and concentrated, they still need to be further enriched by solid-phase extraction, followed by nitrogen blowing concentration and filtration to obtain the analyte; ② The mobile phase is complex, requiring gradient elution of three components: acetonitrile, ultrapure water, and ammonium acetate-acetic acid aqueous solution; ③ According to the actual detection spectrum, there are obvious matrix peaks between the three components. When different products have different matrices, it can easily affect the quantification of the three components.

[0004] Therefore, developing a method for detecting olafrondole with short pretreatment time, high efficiency, low cost, high separation, high sensitivity, and wide detection linear range is a technical problem that needs to be solved. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a method for quantitative detection of olaflu in raw materials and oral care products, comprising detecting olaflu raw material samples or toothpaste samples by high performance liquid chromatography-evaporative light scattering detection method.

[0007] The conditions for the high-performance liquid chromatography include:

[0008] Chromatographic column: C18 chromatographic column packed with octadecylsilane-bonded silica gel;

[0009] Mobile phase: Phase A is an aqueous solution containing 0.03-0.08 vol% trifluoroacetic acid, and Phase B is an acetonitrile solution containing 0.03-0.08 vol% trifluoroacetic acid;

[0010] Gradient elution: 0–11 min 45–55% phase A; 11.1–16 min 0% phase A; 16.1–19 min 45–55% phase A;

[0011] The temperature of the evaporation tube for evaporative light scattering is 75-85℃; the temperature of the drift tube is 75-85℃.

[0012] As a preferred embodiment of the quantitative detection method for olaflur in the raw materials and oral care products described in this invention, the column temperature of the chromatographic column is 25-35℃.

[0013] As a preferred embodiment of the quantitative detection method for olaflur in the raw materials and oral care products described in this invention, the flow rate of the chromatographic column is 0.8-1.2 mL / min.

[0014] As a preferred embodiment of the quantitative detection method for olaflur in the raw materials and oral care products described in this invention, the carrier gas flow rate of the evaporative light scattering is 1.4-1.8 SLM.

[0015] As a preferred embodiment of the quantitative detection method for olaflu in the raw materials and oral care products described in this invention: the preparation method of the olaflu raw material sample or toothpaste sample includes dissolving the olaflu raw material or toothpaste in methanol.

[0016] As a preferred embodiment of the quantitative detection method for olaflur in the raw materials and oral care products described in this invention, the toothpaste sample preparation method further includes adding quartz sand to assist dispersion.

[0017] As a preferred embodiment of the quantitative detection method for olaflur in the raw materials and oral care products described in this invention: the mobile phase is: phase A is an aqueous solution containing 0.05 vol% trifluoroacetic acid, and phase B is an acetonitrile solution containing 0.05 vol% trifluoroacetic acid.

[0018] As a preferred embodiment of the quantitative detection method for olaflur in the raw materials and oral care products described in this invention: the evaporation tube temperature is 80 ℃; the drift tube temperature is 80 ℃.

[0019] As a preferred embodiment of the quantitative detection method for olaflur in the raw materials and oral care products described in this invention, the structural formula of olaflur is:

[0020] .

[0021] As a preferred embodiment of the quantitative detection method of olaflu in the raw materials and oral care products described in this invention, it further includes detecting related derivatives of olaflu.

[0022] The beneficial effects of this invention are as follows: The sample pretreatment process of this invention is convenient, requiring no solid-phase extraction, and is suitable for rapid detection; the optimized mobile phase and elution gradient effectively suppress peak broadening and residue problems, and the baseline stability is improved under gradient conditions, thus improving both sensitivity and accuracy. It can be used to detect samples with high and low olafron content, and is suitable for industrial production and quality control. In addition, this invention unexpectedly discovered that the method of this invention can separate and detect 5 olafron-related impurities (olafron-related derivatives). Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0024] Figure 1 This is the HPLC-ELSD chromatogram of Example 1.

[0025] Figure 2 The HPLC-ELSD chromatogram is shown for Comparative Example 2.

[0026] Figure 3 The HPLC-ELSD chromatogram is shown for Comparative Example 3.

[0027] Figure 4 The HPLC-ELSD chromatogram is shown for Comparative Example 4.

[0028] Figure 5 The HPLC-ELSD chromatogram is shown for Comparative Example 5.

[0029] Figure 6 The HPLC-ELSD chromatogram is shown for Comparative Example 6.

[0030] Figure 7 This is the HPLC-ELSD chromatogram of Example 2. Detailed Implementation

[0031] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0032] Example 1:

[0033] The olaflurane sample was dissolved in methanol to prepare a 1 mg / mL test solution. The test solution was then analyzed by HPLC-ELSD. The chromatographic conditions were as follows: Instrument: Agilent 1260 Infinity liquid chromatograph equipped with an ELSD detector; Column: CAPCELL PAK C18 (4.6*250 mm, 5.0 μm); Column temperature: 30℃; Mobile phase A: 0.05 vol% trifluoroacetic acid aqueous solution; Mobile phase B: 0.05 vol% trifluoroacetic acid acetonitrile solution; Flow rate: 1.0 mL / min; Eluent: 0–11 min 45% A; 11.1–16 min 0% A; 16.1–19 min 45% A (v / v).

[0034] ELSD parameters: Evaporator temperature 80 ℃, drift tube temperature 80 ℃, carrier gas flow rate 1.6 SLM.

[0035] HPLC-ELSD detection chromatogram is shown below Figure 1 .

[0036] Comparative Example 1:

[0037] The same olaflue raw material sample as in Example 1 was subjected to HPLC-ELSD detection. The injection volume was adjusted according to the specific detection conditions. The chromatographic conditions were as follows: Detection instrument: Agilent 1260 Infinity liquid chromatograph equipped with an ELSD detector; Column: Agilent Eclipse XDB-C8 (4.6*250mm, 5.0μm), column temperature: 30℃, mobile phase A: 10 mM ammonium acetate (pH adjusted to 4.0 by acetic acid) aqueous solution; mobile phase B: methanol; flow rate: 1.0 mL / min; eluent: mobile phase A: mobile phase = 15:85 (volume ratio), isogradient elution.

[0038] ELSD parameters: Evaporator temperature: 40℃, Drift tube temperature: 40℃, Gas flow rate: 1.6SLM.

[0039] Table 1 shows the comparison of the proportion of free alkali and impurities (Araflu-related derivatives) in the raw material samples of Example 1 and Comparative Example 1, as well as the tailing factor and resolution.

[0040] Table 1

[0041]

[0042] As can be seen from Comparative Example 1 and Example 1, under the detection conditions of Example 1, the peak shape is narrower and the separation is higher. Impurity E can be separated under the conditions of Example 1. Based on the peak elution time, it is speculated that the peak may be the cis-trans isomer of impurity B. Under the conditions of Comparative Example 1, impurity E may be wrapped by the peak of impurity B.

[0043] The structural formulas of impurities A, B, C, D, and olaflurium have been identified in patent CN 119335103 A, and their structural formulas are as follows:

[0044] The structural formula of olaraflu is:

[0045]

[0046] The structural formula of impurity A is:

[0047]

[0048] The structural formula of impurity B is:

[0049]

[0050] The structural formula of impurity C is:

[0051]

[0052] The structural formula of impurity D is:

[0053] .

[0054] Using the same mobile phase and sample pretreatment conditions as in Example 1, LC-MS was performed on the olaflue feedstock sample. The LC-MS results of impurity E are shown in Table 2, and its structural formula is deduced as follows:

[0055] .

[0056] Table 2. Molecular ion peak and molecular formula of impurity E

[0057]

[0058] Table 3 shows the linear regression equations, linear ranges, limits of detection (LOD), and limits of quantitation (LOQ) for the free base of olaflurane and the two main impurities, impurity A and impurity B, in Example 1.

[0059] Table 3. Linear regression equation, linear range, detection limit, and quantitation limit for Example 1

[0060]

[0061] The linear range, limit of detection (LOD), and limit of quantitation (LOQ) of olafron and the two main impurities, impurity A and impurity B, in Comparative Example 1 are shown in Table 4.

[0062] Table 4. Linear range, limit of detection, and limit of quantitation for Comparative Example 1

[0063]

[0064] Comparative Example 2:

[0065] In Example 1, the chromatographic column was selected as a ZORBAX Eclipse XDB-C8 column, and the injection volume was adjusted according to the specific detection conditions. All other conditions were the same as in Example 1.

[0066] HPLC-ELSD detection chromatogram is shown below Figure 2 At 6.3 min, the resolution of impurity E was 1.3, which was insufficient to achieve baseline separation.

[0067] Table 5. Comparison of tailing factors between the methods of Example 1 and Comparative Example 2

[0068]

[0069] Comparative Example 3:

[0070] In Example 1, mobile phase B was a pure methanol solution, and the injection volume was adjusted according to the specific detection conditions. All other conditions were the same as in Example 1.

[0071] HPLC-ELSD detection chromatogram is shown below Figure 3 .from Figure 3 It can be seen that the peak emergence time is delayed and baseline separation cannot be achieved.

[0072] Comparative Example 4:

[0073] In Example 1, mobile phase B was a pure acetonitrile solution, and the injection volume was adjusted according to the specific detection conditions. All other conditions were the same as in Example 1.

[0074] HPLC-ELSD detection chromatogram is shown below Figure 4 .from Figure 4 It can be seen that the peak emergence time is delayed and baseline separation cannot be achieved.

[0075] Comparative Example 5:

[0076] In Example 1, the elution gradient conditions were: 0–5 min 95% A; 20–24 min 10% A; 25–30 min 95% A (volume ratio). The injection volume was adjusted according to the specific detection situation, and the other conditions were the same as in Example 1.

[0077] HPLC-ELSD detection chromatogram is shown below Figure 5 .from Figure 5 It can be seen that, compared with Example 1, the separation degree of impurity E is significantly reduced. The linear regression equations, linear ranges, limits of detection (LOD), and limits of quantitation (LOQ) for the free base of olaflurane and the two main impurities, impurity A and impurity B, in Comparative Example 5 are shown in Table 6.

[0078] Table 6. Linear regression equations, linear range, detection limit, and quantitation limit for Comparative Example 5.

[0079]

[0080] As shown in Table 6, the linearity of the standard curve is poor, with the linear correlation coefficient r only reaching >0.99 (Table 6), which is less than >0.999, failing to meet the requirements for quantitative detection, and significantly extending the detection time.

[0081] Comparative Example 6:

[0082] In Example 1, the elution gradient conditions were: 0–1 min 80% A; 5 min 40% A; 6–8 min 32% A; 10 min 20% A; 11.5–12 min 10% A; 13–17 min 80% A (volume ratio). The injection volume was adjusted according to the specific detection situation, and the other conditions were the same as in Example 1.

[0083] HPLC-ELSD detection chromatogram is shown below Figure 6 .from Figure 6 It can be seen that, compared with Example 1, the separation degree of impurity E is significantly reduced. The linear regression equations and linear ranges of the free olaflurane base and the two main impurities, impurity A and impurity B, in Comparative Example 6 are shown in Table 7.

[0084] Table 7. Linear Regression Equations and Linear Ranges for Comparative Example 6

[0085]

[0086] Example 2:

[0087] External standard method for quantitative determination of olafol content in toothpaste:

[0088] 1) Preparation of standard curve: Accurately weigh 25 mg of standard into a 25 mL volumetric flask, dissolve in HPLC-grade methanol and dilute to 25 mL to prepare a standard stock solution with a concentration of 1 mg / mL; serially dilute the standard stock solution to prepare standard working solutions of 3 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL and 300 μg / mL respectively, and elute by high performance liquid chromatography. Analyze the solutions sequentially from low concentration to high concentration using high performance liquid chromatography. Fit the concentration and peak area using a double logarithmic method, requiring a linear correlation coefficient r>0.999.

[0089] 2) Toothpaste sample pretreatment: Accurately weigh 1.0 g of toothpaste sample, add an appropriate amount of quartz sand to assist dispersion, add 5 mL of methanol, vortex mix for 5 min, sonicate for 10 min, centrifuge at 5000 rpm for 10 min, repeat the above operation twice, combine the supernatant, make up to 10 mL with methanol, mix well and filter through a 0.45 μm microporous membrane to obtain the test solution.

[0090] 3) High-performance liquid chromatography (HPLC) detection: The test solution was subjected to HPLC gradient elution, and the chromatogram was recorded. The HPLC conditions were as follows: a CAPCELL PAK C18 column (4.6*250 mm, 5.0 μm), column temperature: 30 ℃, mobile phase A was 0.05% trifluoroacetic acid aqueous solution, mobile phase B was 0.05% trifluoroacetic acid acetonitrile solution (v / v), and the eluent was: 0–11 min 45% A; 11.1–16 min 0% A; 16.1–19 min 45% A (v / v), using HPLC gradient elution. The detector was ELSD, the evaporation tube temperature was 80 ℃, the drift tube temperature was 80 ℃, and the carrier gas flow rate was 1.6 SLM.

[0091] 4) Based on the standard working curve, qualitative and quantitative analysis of olaflurane in the test sample was performed.

[0092] Under the above conditions, the olafral content was determined in three different batches of toothpaste samples, with RSD < 5% and spiked recoveries of 90–105%. The HPLC-ELSD chromatograms are shown below. Figure 7 .

[0093] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for quantitative detection of olaflurium in raw materials and oral care products, characterized in that: High performance liquid chromatography-evaporative light scattering detection method was used to detect olaflue raw material samples or toothpaste samples; The conditions for the high-performance liquid chromatography include: Chromatographic column: C18 chromatographic column packed with octadecylsilane-bonded silica gel; Mobile phase: Phase A is an aqueous solution containing 0.03-0.08 vol% trifluoroacetic acid, and Phase B is an acetonitrile solution containing 0.03-0.08 vol% trifluoroacetic acid; Gradient elution: 0–11 min 45% phase A; 11.1–16 min 0% phase A; 16.1–19 min 45% phase A; The temperature of the evaporation tube for evaporative light scattering is 75-85℃; the temperature of the drift tube is 75-85℃. The structural formula of the olafur is as follows: ； It also includes detecting related derivatives of the olaflu; the related derivatives of the olaflu are as follows: ， ， ， ,as well as 。 2. The quantitative detection method for olaflur in raw materials and oral care products according to claim 1, characterized in that: The column temperature of the chromatographic column is 25-35℃.

3. The quantitative detection method for olaflur in raw materials and oral care products according to claim 1 or 2, characterized in that: The flow rate of the chromatographic column is 0.8-1.2 mL / min.

4. The quantitative detection method for olaflur in raw materials and oral care products according to claim 1 or 2, characterized in that: The carrier gas flow rate for the evaporative light scattering is 1.4-1.8 SLM.

5. The quantitative detection method for olaflur in raw materials and oral care products according to claim 1 or 2, characterized in that: The preparation method of the olaflue raw material sample or toothpaste sample includes dissolving the olaflue raw material or toothpaste in methanol.

6. The quantitative detection method for olaflur in raw materials and oral care products according to claim 5, characterized in that: The method for preparing the toothpaste sample also includes adding quartz sand to assist dispersion.

7. The quantitative detection method for olaflur in raw materials and oral care products according to claim 1 or 2, characterized in that: The mobile phases are: Phase A is an aqueous solution containing 0.05 vol% trifluoroacetic acid, and Phase B is an acetonitrile solution containing 0.05 vol% trifluoroacetic acid.

8. The method for quantitative detection of olaflur in raw materials and oral care products according to claim 1 or 2, characterized in that: The temperature of the evaporator tube is 80 ℃; the temperature of the drift tube is 80 ℃.

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