A method for chiral chromatographic separation and analysis of homosalate enantiomers

Abstract

The application belongs to the technical field of chiral chromatographic separation, and discloses a chiral chromatographic separation and analysis method for homosalate enantiomers. The chiral chromatographic separation and analysis method is used for the first time for homosalate enantiomers, and the chiral chromatographic separation and analysis method selects a mixed solution composed of n-hexane and MTBE (methyl tert-butyl ether) as a mobile phase, and successfully realizes chiral separation of four enantiomers of homosalate on a polysaccharide derivative chiral stationary phase (bonded type straight-chain amylose-tris (3-chloro-5-methyl phenyl urea)). The chiral chromatographic separation and analysis method has high resolution, is simple and easy to operate and control, has low detection cost, and has good stability.

Description

Technical Field

[0001] This invention belongs to the field of chiral chromatography separation technology, specifically relating to a chiral chromatographic separation and analysis method for enantiomers of humosalidinyl. Background Technology

[0002] Homosalate (exists in four stereoisomers, as shown in Formula I), with the molecular formula C 16 H 22 O3, a salicylic acid derivative, is one of the most common ingredients in sunscreen products. Thanks to the characteristic of salicylates having minimal solvent discoloration shift, homosalate's protective efficacy is almost unaffected by changes in the solvent in the formula, thus leading to its widespread use in various sunscreen products. Research reports show that from 2018 to 2021, the usage rate of homosalate in imported sunscreen products increased from 5.2% to 21.2%. Furthermore, survey data from the Portuguese market from 2015 to 2021 also found that the frequency of homosalate use increased from 9% to approximately 17%. Given the widespread use of homosalate in sunscreen products, its potential impact on human health requires greater attention and research.

[0003]

[0004] Homosalt has been identified as an emerging endocrine disruptor (EDC) that may affect the development and reproductive systems of organisms. The European Union Scientific Committee on Consumer Safety (SCCS) has included homosalt in its list of 14 potential EDC cosmetic ingredients, and researchers have shown that it possesses anti-estrogenic and anti-androgenic activities. Furthermore, homosalt has been found to significantly increase the malformation rate of zebrafish F1 embryos and negatively impact the survival and proliferation of human trophoblast cells. In addition, homosalt can be absorbed into the human circulatory system through the skin. Using a human physiological pharmacokinetic dermal model, researchers found that the cumulative amount of homosalt entering the bloodstream within 24 hours ranged from 2.07 to 5.46 μg. Systemic absorption experiments on healthy volunteers revealed a maximum plasma concentration of 23.1 ng / mL, which is 46 times the TTC threshold (0.5 ng / mL). According to the study, the SCCS calculated the skin absorption rate of homosalt to be 5.3%. In conclusion, these data underscore the ongoing concern about the safety of homosalate and the necessity for further research to ensure its safe use in cosmetics.

[0005] Enantiomers of chiral substances are extremely similar in physical and chemical properties, but can exhibit drastically different effects on biological activity or toxicity. Homosalidine contains two chiral centers and exists in two pairs of enantiomers. Previous studies have shown that the racemic homosalidine exhibits diastereoselectivity in drug metabolism. Existing data indicate that the oral bioavailability of cis-homosaidine (6R, 8R+6S, 8S) is an order of magnitude lower than that of trans-homosaidine (6R, 8S+6S, 8R), and the Cmax of cis-homosaidine is 2.2 times that of trans-homosaidine. However, due to the lack of chiral separation and analytical methods for the four enantiomers of homosalidine, there is a lack of reports on enantiomer-level studies in stereoselective safety assessments of homosalidine. Therefore, developing a chiral chromatographic method capable of separating homosalidine enantiomers is crucial for a deeper understanding and assessment of the safety of homosalidine. Summary of the Invention

[0006] To address the lack of effective separation methods for homosalate enantiomers in existing technologies, this invention aims to develop a chiral chromatographic separation method for homosalate enantiomers. This invention successfully achieves chiral separation of four enantiomers of homosalate by using bonded linear starch-tris(3-chloro-5-methylphenylcarbamate) as the chiral stationary phase and combining it with a suitable mobile phase.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

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

[0009] A chiral stationary phase was used for liquid chromatography separation and analysis of the homosalate sample solution;

[0010] The mobile phase in the liquid chromatography includes 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 chiral stationary phase of the polysaccharide derivative is selected from any one of the following: bonded amylose-tris(3-chloro-5-methylphenylcarbamate), bonded p-chloro-phenylcarbamylated β-cyclodextrin, coated amylose-tris((S)-(α)-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 polysaccharide derivative is chirally immobilized as a bonded linear starch-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 mixture of n-hexane and ethanol.

[0021] In some embodiments of the present invention, the solvent of the mixed sample solution is obtained by mixing 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 chromatograph 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 chromatograph is 1 to 2 mL / min.

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

[0025] In some embodiments of the present invention, the injection volume in the liquid chromatograph 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 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 not 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 this invention are:

[0033] This invention provides, for the first time, a chiral chromatographic separation method for the enantiomers of homosalide. This method uses a mixed solution of n-hexane and MTBE as the mobile phase and successfully achieves chiral separation of the four enantiomers of homosalide on a chiral stationary phase of polysaccharide derivatives (bonded amylose-tris(3-chloro-5-methylphenylcarbamate)). This chiral chromatographic separation method offers high resolution, is simple and easy to control, has low detection cost, and exhibits good stability.

[0034] Specifically, the chiral chromatographic separation and analysis method of the present invention achieves a resolution greater than 1.5 between the enantiomers of homosalidine, enabling efficient chiral separation of the four enantiomers of homosalidine. Under preferred conditions, the selectivity factors are 1.28, 1.54, and 1.68, respectively; the analysis is completed within 9 minutes, with a short separation time, making it suitable for rapid analysis of the composition of homosalidine enantiomers in real samples. Attached Figure Description

[0035] Figure 1 The images show the HPLC spectra of the four enantiomers of humosafil obtained in Example 1 of this invention.

[0036] Figure 2 The images show the HPLC spectra of the four enantiomers of humosafil obtained in Examples 1-3 and Examples 8-9 of this invention.

[0037] Figure 3 The images show the HPLC spectra of the four enantiomers of humosafil obtained in Examples 1 and 4-7 of this invention.

[0038] Figure 4 The images show the HPLC spectra of the four enantiomers of humosafil obtained in Comparative Examples 1-3 of this invention. Detailed Implementation

[0039] The present invention will be further described in detail below through specific embodiments.

[0040] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

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

[0043] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0044] Example 1

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

[0046] Preparation of the test sample: Racemic homosalate (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number 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 (HPLC) separation: Chiral analysis and separation were performed using HPLC, with the specific chromatographic parameters as follows:

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

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

[0050] Example 2

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

[0052] Example 3

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

[0054] Example 4

[0055] A chiral chromatographic separation and analysis method for enantiomers of humosafil is provided, which differs from Example 1 only in that the column temperature is 30°C.

[0056] Example 5

[0057] A chiral chromatographic separation and analysis method for enantiomers of humosafil is provided, which differs from Example 1 only in that the column temperature is 25°C.

[0058] Example 6

[0059] A chiral chromatographic separation and analysis method for enantiomers of humosafil is provided, which differs from Example 1 only in that the column temperature is 20°C.

[0060] Example 7

[0061] A chiral chromatographic separation and analysis method for enantiomers of humosafil is provided, which differs from Example 1 only in that the column temperature is 15°C.

[0062] Example 8

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

[0064] Example 9

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

[0066] Comparative Example 1

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

[0068] Comparative Example 2

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

[0070] Effect Example

[0071] Chiral separation of racemic homosalate samples was performed using the chiral chromatographic separation methods of Examples 2-3 and 8-9. The results are shown in Table 1 and 2. Figure 2 As shown, under the chromatographic conditions of Examples 2 and 3, baseline separation of the four enantiomers of homosalide was achieved. However, under the conditions of Example 8 (n-hexane:MTBE = 75:25) and Example 9 (n-hexane:MTBE = 95:5), complete baseline separation of the four enantiomers was not achieved. The resolution Rs... 12 and Rs 23 The resolution Rs decreases as the MTBE content in the mobile phase increases or decreases. 34 The value decreases as the MTBE content decreases.

[0072] Chiral separation and analysis of racemic homosalyl ester samples were performed using the chiral chromatographic separation and analysis methods described in Examples 4-7. The results are shown in Table 1 and... Figure 3 As shown, baseline separation of the four enantiomers of homosalide was achieved, but as the column temperature decreased, the retention time of the four enantiomers on the chiral stationary phase increased, and the resolution of the three enantiomers gradually decreased. Conversely, as the column temperature decreased, the selectivity gradually increased, showing the opposite trend to the resolution.

[0073] Chiral chromatographic separation and analysis methods were used in Comparative Examples 1 and 2 to separate racemic homosalyl ester samples. The results are shown in Table 1 and 2. Figure 4 As shown, using ethanol or isopropanol as the mobile phase component cannot achieve chiral separation of the four enantiomers of homosalide.

[0074] Table 1. Condition settings and separation results of the chromatographic separation analysis methods in Examples 1-9 and Comparative Examples 1-2.

[0075]

[0076] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A chiral chromatographic separation and analysis method for four enantiomers of homosalyl ester, comprising the following steps: A chiral stationary phase was used for liquid chromatography separation and analysis of the homosalate sample solution; The mobile phase in the liquid chromatography includes n-hexane and methyl tert-butyl ether; The homosalate sample solution is a racemic homosalate solution dissolved in a mixture of n-hexane and ethanol. The chiral stationary phase is silica gel surface-bonded linear starch-tris(3-chloro-5-methylphenylcarbamate). The volume ratio of n-hexane to methyl tert-butyl ether in the mobile phase is 80–90: 20–10; The detection wavelength in the liquid chromatography is 245–255 nm.

2. The chiral chromatographic separation and analysis method according to claim 1, characterized in that, The column temperature in the liquid chromatography is 10–40 °C.

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

4. The chiral chromatographic separation and analysis method according to claim 1, characterized in that, The injection volume in the liquid chromatography is 1–10 μL.

5. The chiral chromatographic separation and analysis method according to claim 4, characterized in that, The concentration of homosalate in the sample solution is no greater than 2.0 mg / mL.

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