Chiral chromatographic separation and analysis method of homosalate enantiomer

Abstract

The invention belongs to the technical field of chiral chromatographic separation, discloses a chiral chromatographic separation and analysis method for a rimosalate enantiomer, and provides the chiral chromatographic separation and analysis method for the rimosalate enantiomer for the first time. According to the chiral chromatographic separation analysis method, a mixed solution composed of n-hexane and MTBE (methyl tert-butyl ether) is selected as a mobile phase, and chiral separation of four enantiomers of homosalate is successfully achieved on a polysaccharide derivative chiral stationary phase (bonded amylose-tri (3-chloro-5-methyl phenyl carbamate). The chiral chromatographic separation analysis method is high in separation degree, simple and convenient to operate, easy to control, low in detection cost and good in stability.

Description

Technical Field

[0001] The invention belongs to the technical field of chiral chromatographic separation, and particularly relates to a chiral chromatographic separation and analysis method for homosalate enantiomers. Background Art

[0002] Homosalate (there are four stereoisomers, as shown in formula I), the molecular formula is C 16 H 22 O3, as a salicylic acid derivative, is one of the most common ingredients in sunscreen products. Thanks to the small solvent discoloration displacement unique to salicylate compounds, the protective efficacy of homosalate is almost unaffected by changes in solvents in the formula, and is therefore widely used in a variety of sunscreen products. Research reports show that from 2018 to 2021, the use rate of homosalate in imported sunscreen products increased from 5.2% to 21.2%. In addition, survey data from the Portuguese market from 2015 to 2021 also found that the frequency of use of homosalate increased from 9% to about 17%. Given the widespread use of homosalate in sunscreen products, its potential impact on human health needs to be given more attention and research.

[0003]

[0004] Homosalate has been identified as an emerging endocrine disruptor (EDCs) that may affect the development and reproductive system of organisms. The European Scientific Committee on Consumer Safety (SCCS) has included homosalate in the list of 14 potential EDCs cosmetic raw materials, and some researchers have shown that it has anti-estrogenic and anti-androgenic activities. Not only that, homosalate has also been found to significantly increase the malformation rate of zebrafish F1 embryos and have a negative impact on the survival and proliferation of human trophoblast cells. In addition, homosalate can also enter the human circulatory system through skin absorption. Using the human physiological pharmacokinetics dermal model, researchers found that the cumulative range of homosalate entering the blood within 24 hours was 2.07 to 5.46 μg. Through systemic absorption experiments on healthy volunteers, it was found that the maximum concentration of homosalate in plasma was 23.1 ng / mL, which is 46 times the TTC threshold (0.5 ng / mL). According to the study, SCCS calculated that the skin absorption rate of homosalate was 5.3%. Taken together, these data highlight the continued concern regarding the safety of homosalate and the need for further research to ensure its safe use in cosmetics.

[0005] The enantiomers of chiral substances are very similar in physical and chemical properties, but may show completely different effects on biological activity or toxic effects. The molecular structure of homosalate contains two chiral centers and exists in two pairs of enantiomeric forms. Studies have shown that racemic homosalate has diastereoselectivity in drug metabolism. Existing data show that the oral bioavailability of cis-homosalate (6R, 8R + 6S, 8S) is one order of magnitude lower than that of trans-homosalate (6R, 8S + 6S, 8R), and the Cmax of cis-homosalate is 2.2 times that of trans-homosalate. However, since the prior art has not yet developed a chiral separation analysis method for the four enantiomers of homosalate, this has led to a lack of research reports on the enantiomer level of homosalate in terms of stereoselective safety assessment. In view of this, the development of a chiral chromatographic method capable of separating homosalate enantiomers is of vital importance for a deep understanding and evaluation of the safety of homosalate. Summary of the invention

[0006] In order to solve the problem of lack of effective separation methods for homosalate enantiomers in the prior art, the present invention aims to develop a chiral chromatographic separation and analysis method for homosalate enantiomers. The present invention successfully achieves chiral separation of the four enantiomers of homosalate by using bonded amylose-tris (3-chloro-5-methylphenylcarbamate) as a chiral stationary phase and matching a suitable mobile phase.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a chiral chromatographic separation and analysis method for homosalate enantiomers, comprising the following steps:

[0009] The chiral stationary phase was used to perform liquid chromatography separation and analysis on the homosalate sample solution;

[0010] The mobile phase in the liquid chromatography comprises n-hexane and methyl tert-butyl ether (MTBE).

[0011] In some embodiments of the present invention, the volume ratio of n-hexane to MTBE in the mobile phase is 75-95:25-5.

[0012] In some embodiments of the present invention, the volume ratio of n-hexane to MTBE in the mobile phase is 80-90:20-10.

[0013] In some embodiments of the present invention, the volume ratio of n-hexane to MTBE in the mobile phase is 85-90:15-10.

[0014] In some embodiments of the present invention, the chiral stationary phase is a polysaccharide derivative chiral stationary phase.

[0015] In some embodiments of the present invention, the polysaccharide derivative chiral stationary phase is selected from any one of bonded amylose-tris(3-chloro-5-methylphenylcarbamate), bonded p-chloro-phenylcarbamylated β-cyclodextrin, coated amylose tris((S)-(ɑ)-phenylethylcarbamylated), coated cellulose tris(3,5-dimethylphenylcarbamylated), coated cellulose tris(4-methylphenylcarbamylated) or coated amylose tris(3,5-dimethylphenylcarbamate).

[0016] In some embodiments of the present invention, the chiral stationary phase of the polysaccharide derivative is a bonded amylose-tris(3-chloro-5-methylphenylcarbamate) chiral stationary phase.

[0017] In some embodiments of the present invention, the column temperature in the liquid chromatography is 10-40°C.

[0018] In some embodiments of the present invention, the column temperature in the liquid chromatography is 15-35°C.

[0019] In some embodiments of the present invention, the column temperature in the liquid chromatography is 20-35°C.

[0020] In some embodiments of the present invention, the solvent of the mixed sample solution is a mixed solution of n-hexane and ethanol.

[0021] In some embodiments of the present invention, the solvent of the mixed sample solution is a mixture of n-hexane and ethanol in a volume ratio of 1:1.

[0022] In some embodiments of the present invention, the flow rate of the mobile phase in the liquid chromatography is 0.5 to 2 mL / min.

[0023] In some embodiments of the present invention, the flow rate of the mobile phase in the liquid chromatography is 1 to 2 mL / min.

[0024] In some embodiments of the present invention, the injection volume in the liquid chromatography is 1 to 10 μL.

[0025] In some embodiments of the present invention, the injection volume in the liquid chromatography is 1 to 2 μL.

[0026] In some embodiments of the present invention, the detection wavelength in the liquid chromatography is 245-255 nm.

[0027] In some embodiments of the present invention, the detection wavelength in the liquid chromatography is 254 nm.

[0028] In some embodiments of the present invention, the chromatographic column is IG chiral chromatography column.

[0029] In some embodiments of the present invention, the detector in the liquid chromatography is a diode array detector.

[0030] In some embodiments of the present invention, the concentration of homosalate in the sample solution is no greater than 2.0 mg / mL.

[0031] In some embodiments of the present invention, the concentration of homosalate in the sample solution is 0.5-1.0 mg / mL.

[0032] The beneficial effects of the present invention are:

[0033] The present invention provides a chiral chromatographic separation and analysis method for homosalate enantiomers for the first time. The chiral chromatographic separation and analysis method uses a mixed solution consisting of n-hexane and MTBE as a mobile phase, and successfully achieves chiral separation of four enantiomers of homosalate on a chiral stationary phase of a polysaccharide derivative (bonded amylose-tris(3-chloro-5-methylphenylcarbamate)). The chiral chromatographic separation and analysis method has high separation degree, simple operation, easy control, low detection cost, and good stability.

[0034] Specifically, the chiral chromatographic separation and analysis method of the present invention has a separation degree of greater than 1.5 between the enantiomers of homosalate, and can efficiently achieve the chiral separation of the four enantiomers of homosalate. Under preferred conditions, the selectivity factors are 1.28, 1.54 and 1.68, respectively; the analysis is completed within 9 minutes, the separation time is short, and it can be used to quickly analyze the composition of homosalate enantiomers in actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 HPLC spectra of four enantiomers of homosalate obtained in Example 1 of the present invention.

[0036] Figure 2 HPLC spectra of four enantiomers of homosalate obtained in Examples 1 to 3 and Examples 8 to 9 of the present invention.

[0037] Figure 3 HPLC spectra of four enantiomers of homosalate obtained in Examples 1 and 4 to 7 of the present invention.

[0038] Figure 4 HPLC spectra of four enantiomers of homosalate obtained in Comparative Examples 1 to 3 of the present invention. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below through specific examples.

[0040] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0041] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0042] In the present invention, the chiral stationary phase used is silica gel surface bonded amylose-tris(3-chloro-5-methylphenylcarbamate), which is produced by Daicel Pharmaceutical Chiral Technology (Shanghai) Co., Ltd., and products produced by other companies can also be used.

[0043] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0044] Example 1

[0045] A chiral chromatographic separation and analysis method for homosalate enantiomers comprises the following steps:

[0046] Preparation of the test sample: Racemic homosalate (Shanghai Aladdin Biochemical Technology Co., Ltd., Catalog No. L2216409) was dissolved in a mixed solution of n-hexane and ethanol (50:50, v / v) to form a sample solution with a concentration of 1.0 mg / mL.

[0047] High performance liquid chromatography separation: High performance liquid chromatography is used for chiral analysis and separation, wherein the specific parameters of the chromatography are as follows:

[0048] The high performance liquid chromatograph used was an Agilent 1200 series liquid chromatograph, and the detector was a diode array detector; the chromatographic column was produced by Daicel Pharmaceutical Chiral Technology (Shanghai) Co., Ltd. IG chiral column (250mm×4.6mm, 5μm), the chiral stationary phase was straight-chain starch-tris(3-chloro-5-methylphenylcarbamate) bonded to the silica surface, the mobile phase was n-hexane:MTBE in a volume ratio of 85:15, the flow rate was 1.0mL / min, the injection volume was 2μL, the column temperature was 35℃, and the detection wavelength was 254nm.

[0049] The test results are shown in Table 1 and Figure 1 As shown, at retention times of 4.54, 4.93, 5.92 and 7.82 min, four completely baseline-separated chromatographic peaks were observed, the three resolutions were 2.01, 2.56 and 3.25, and the three selectivity factors were 1.28, 1.54 and 1.68, respectively, indicating that the chiral chromatographic separation and analysis method of this embodiment can effectively perform chiral separation on the four enantiomers of homosalate.

[0050] Example 2

[0051] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:MTBE in a volume ratio of 90:10.

[0052] Example 3

[0053] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:MTBE in a volume ratio of 80:20.

[0054] Example 4

[0055] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, the only difference is that the column temperature of the chromatographic column is 30°C.

[0056] Example 5

[0057] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, the only difference is that the column temperature of the chromatographic column is 25°C.

[0058] Example 6

[0059] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, the only difference is that the column temperature of the chromatographic column is 20°C.

[0060] Example 7

[0061] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, the only difference is that the column temperature of the chromatographic column is 15°C.

[0062] Example 8

[0063] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:MTBE in a volume ratio of 75:25.

[0064] Example 9

[0065] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:MTBE in a volume ratio of 95:5.

[0066] Comparative Example 1

[0067] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:ethanol in a volume ratio of 85:15.

[0068] Comparative Example 2

[0069] A chiral chromatographic separation and analysis method for homosalate enantiomers, compared with Example 1, wherein the mobile phase is n-hexane:isopropanol in a volume ratio of 85:15.

[0070] Effect Example

[0071] The chiral chromatography separation and analysis methods of Examples 2 to 3 and Examples 8 to 9 were used to perform chiral analysis and separation of the racemic homosalate sample. The results are shown in Tables 1 and Figure 2 As shown, under the chromatographic conditions of Examples 2 to 3, the four enantiomers of homosalate can be baseline separated. However, under the conditions of Example 8 (n-hexane: MTBE = 75:25) and Example 9 (n-hexane: MTBE = 95:5), the four enantiomers failed to achieve complete baseline separation. Among them, the separation degree Rs 12 and Rs. 23 As the MTBE content in the mobile phase increases or decreases, the resolution Rs decreases. 34 It becomes smaller as the MTBE content decreases.

[0072] The chiral chromatography separation and analysis method of Examples 4 to 7 was used to perform chiral analysis and separation of the racemic homosalate sample. The results are shown in Tables 1 and Figure 3 As shown in the figure, the four enantiomers of homosalate can be baseline separated, but as the column temperature decreases, the retention time of the four enantiomers on the chiral stationary phase is prolonged, and the three resolutions gradually decrease. As the column temperature decreases, the selectivity factor gradually increases, which is opposite to the resolution trend.

[0073] The chiral chromatography separation and analysis method of Comparative Examples 1 to 2 was used to perform chiral analysis and separation of the racemic homosalate sample. The results are shown in Tables 1 and Figure 4 As shown, the chiral separation of the four enantiomers of homosalate could not be achieved using ethanol or isopropanol as the mobile phase component.

[0074] Table 1 Conditions and separation results of the chromatographic separation and analysis methods of Examples 1 to 9 and Comparative Examples 1 to 2

[0075]

[0076] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A chiral chromatographic separation and analysis method for homosalate enantiomers, comprising the following steps: The chiral stationary phase was used to perform liquid chromatography separation and analysis on the homosalate sample solution; The mobile phase in the liquid chromatography comprises n-hexane and methyl tert-butyl ether.

2. The chiral chromatography separation and analysis method according to claim 1, characterized in that: The volume ratio of n-hexane to methyl tert-butyl ether in the mobile phase is 75-95:25-5.

3. The chiral chromatographic separation and analysis method according to claim 2, characterized in that: The chiral stationary phase is a polysaccharide derivative chiral stationary phase.

4. The chiral chromatographic separation and analysis method according to claim 3, characterized in that: The polysaccharide derivative chiral stationary phase is selected from any one of bonded amylose-tris(3-chloro-5-methylphenylcarbamate), bonded p-chloro-phenylcarbamylated β-cyclodextrin, coated amylose tris((S)-(ɑ)-phenylethylcarbamylated), coated cellulose tris(3,5-dimethylphenylcarbamylated), coated cellulose tris(4-methylphenylcarbamylated) or coated amylose tris(3,5-dimethylphenylcarbamylated).

5. The chiral chromatography separation and analysis method according to any one of claims 1 to 4, characterized in that: The column temperature in the liquid chromatography is 10-40°C.

6. The chiral chromatography separation and analysis method according to any one of claims 1 to 4, characterized in that: The solvent of the mixed sample solution is a mixed solution of n-hexane and ethanol.

7. The chiral chromatography separation and analysis method according to claim 6, characterized in that: The flow rate of the mobile phase in the liquid chromatography is 0.5 to 2 mL / min.

8. The chiral chromatography separation and analysis method according to claim 6, characterized in that: The detection wavelength in the liquid chromatography is 245-255 nm.

9. The chiral chromatography separation and analysis method according to claim 7, characterized in that: The injection volume in the liquid chromatography is 1 to 10 μL.

10. The chiral chromatography separation and analysis method according to claim 6, characterized in that: The concentration of homosalate in the sample solution is no more than 2.0 mg / mL.

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