Chiral chromatographic analysis method for synchronously separating phenoxyisopropanol and chloro-imidazole enantiomers

Abstract

The invention belongs to the technical field of chiral chromatographic separation, and discloses a chiral chromatographic analysis method for synchronously separating phenoxyisopropanol and chloro-imidazole enantiomers. The invention provides a chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and chloro-imidazole enantiomers for the first time. A mixed solution composed of n-hexane and lower alcohol (ethanol) is selected as a mobile phase, and synchronous chiral separation of enantiomers of the two compounds is successfully realized on a chiral stationary phase (bonded amylose-tri (3-chloro-5-methyl phenyl carbamate). The chiral chromatographic 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 analysis method for synchronously separating phenoxyisopropanol and climbazole enantiomers. Background Art

[0002] Phenoxyisopropanol (CAS No.: 770-35-4, structural formula as Formula I) and Climbazole (CAS No.: 38083-17-9, structural formula as Formula II) are commonly used preservatives in the cosmetics industry. They play a key role in preventing microbial contamination, ensuring product stability and protecting consumer health. However, with the in-depth study of the safety of these ingredients, their use has been strictly regulated and continued to be concerned. The China National Drug Administration stipulates that phenoxyisopropanol, as a restricted component, is only allowed to be used in rinse-off products with a maximum safe concentration of 2%; while the maximum safe concentration of Climbazole is 0.5%. In addition, in 2019, the European Union required that the maximum safe use concentration of Climbazole in products such as creams, hair lotions and foot care products be reduced from 0.5% to 0.2%.

[0003] J. Sumpter's research team at Brunel University in the UK found that some alkyl hydroxybenzoates exhibit estrogenic activity and recommended a re-evaluation of the safety of such substances. Although the European Cosmetic, Toiletries and Perfume Association emphasizes that parabens will hydrolyze after contact with the skin, limiting their ability to enter the blood circulation system, subsequent studies have shown that this hydrolysis process may not be complete. Given the similarity in chemical structure between phenoxyisopropanol and parabens, it is particularly important to explore their safety and reasonable application limits.

[0004]

[0005] The European Scientific Committee on Consumer Products reported that climbazole caused changes in blood biochemical indicators in rats and dogs in repeated dose toxicity tests and caused liver damage in rats. Researchers found that climbazole showed moderate toxicity in zebrafish embryos. After zebrafish were exposed to climbazole for 28 days, the fertility of all exposure groups decreased significantly, and it showed inhibitory ability on oocytes at different developmental stages. In view of the toxic effects of climbazole on organisms, it is necessary to conduct a more comprehensive safety assessment of climbazole.

[0006] Many basic biological molecules in organisms, such as amino acids, nucleotides and sugars, are chiral. When enantiomers interact with biomolecules, they may show different biological activities, metabolism and toxicity. As can be seen from Formula I and Formula II, both phenoxyisopropanol and climbazole contain a chiral center in their structures, and both have a pair of optical isomers, and their different enantiomers may have different interaction modes with biomolecules, thereby affecting their biological functions. At present, although the chiral separation of climbazole enantiomers in reverse phase chromatography has been reported, the chiral analysis of phenoxyisopropanol enantiomers is still in a blank state, especially the method for synchronously analyzing the enantiomer content of these two preservative ingredients in cosmetics has not been reported.

[0007] There are many factors that affect the separation of chiral compounds, such as the structure of the separation object compound, stationary phase, mobile phase and column temperature. Therefore, based on existing reports, it is not foreseeable what means can be used to effectively separate the four enantiomers of phenoxyisopropanol and climbazole. Therefore, a chiral chromatographic analysis method that can simultaneously separate the four enantiomers in a mixed sample of phenoxyisopropanol and climbazole is developed to provide key technical support for the cosmetics industry in preservative ingredient analysis, quality control and safety assessment. Summary of the invention

[0008] In order to solve the problem of lack of effective synchronous separation and analysis methods for phenoxyisopropanol and climbazole enantiomers in the prior art, the present invention aims to propose a chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and climbazole enantiomers. The present invention successfully achieves efficient synchronous separation of the enantiomers of the above two compounds by using bonded amylose-tris (3-chloro-5-methylphenylcarbamate) as a chiral stationary phase and matching a suitable mobile phase.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a chiral chromatographic analysis method for synchronously separating phenoxyisopropanol and climbazole enantiomers, comprising the following steps:

[0011] The chiral stationary phase was used to separate and analyze the mixed sample solution of phenoxyisopropanol and climbazole by liquid chromatography;

[0012] The mobile phase in the liquid chromatography comprises n-hexane and lower alcohol.

[0013] In some embodiments of the present invention, the volume ratio of n-hexane to lower alcohol in the mobile phase is 60-90:40-10.

[0014] In some embodiments of the present invention, the volume ratio of n-hexane to lower alcohol in the mobile phase is 60-80:40-20.

[0015] In some embodiments of the present invention, the volume ratio of n-hexane to lower alcohol in the mobile phase is 60-70:40-30.

[0016] As the content of lower alcohol in the mobile phase decreased, the separation between the four enantiomers in the mixed sample of phenoxyisopropanol and climbazole gradually increased, and the retention on the chiral stationary phase also gradually increased, and the retention time was significantly prolonged.

[0017] In some embodiments of the present invention, the lower alcohol is ethanol.

[0018] In some embodiments of the present invention, the chiral stationary phase is a polysaccharide derivative chiral stationary phase.

[0019] 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).

[0020] 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.

[0021] In some embodiments of the present invention, the solvent of the mixed sample solution is ethanol.

[0022] In some embodiments of the present invention, the concentration of phenoxyisopropanol or climbazole in the mixed sample solution is no more than 2.0 mg / mL.

[0023] In some embodiments of the present invention, the concentration of phenoxyisopropanol or climbazole in the mixed sample solution is 0.1-1.0 mg / mL.

[0024] In some embodiments of the present invention, the concentration of phenoxyisopropanol or climbazole in the mixed sample solution is 0.5 mg / mL.

[0025] 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.

[0026] In some embodiments of the present invention, the flow rate of the mobile phase in the liquid chromatography is 1 to 2 mL / min.

[0027] In some embodiments of the present invention, the injection volume in the liquid chromatography is 1 to 10 μL.

[0028] In some embodiments of the present invention, the injection volume in the liquid chromatography is 1 to 2 μL.

[0029] In some embodiments of the present invention, the column temperature in the liquid chromatography is 20-30°C.

[0030] In some embodiments of the present invention, the column temperature in the liquid chromatography is 25-30°C.

[0031] In some embodiments of the present invention, the detection wavelength in the liquid chromatography is 245-255 nm.

[0032] In some embodiments of the present invention, the detection wavelength in the liquid chromatography is 254 nm.

[0033] In some embodiments of the present invention, the chromatographic column is IG chiral chromatography column.

[0034] In some embodiments of the present invention, the detector in the liquid chromatography is a diode array detector.

[0035] The beneficial effects of the present invention are:

[0036] The present invention provides a chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and climbazole enantiomers for the first time. A mixed solution consisting of n-hexane and lower alcohol (ethanol) is selected as a mobile phase, and synchronous chiral separation of the enantiomers of the two compounds is successfully achieved on a chiral stationary phase (bonded amylose-tris (3-chloro-5-methylphenylcarbamate)). The chiral chromatographic analysis method has high separation, simple operation, easy control, low detection cost, and good stability.

[0037] Specifically, the chiral chromatography analysis method provided by the present invention has a separation degree between the enantiomers of phenoxyisopropanol and climbazole greater than 2.5, and can efficiently achieve the synchronous separation of the enantiomers of the above two compounds. Under preferred conditions, the selectivity factors are 1.26, 1.50 and 1.51, respectively; the analysis is completed within 9 minutes, the separation time is short, and it can be used to quickly analyze the composition of enantiomers in a mixed sample of phenoxyisopropanol and climbazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the HPLC spectrum of four enantiomers in the mixed sample of phenoxyisopropanol and climbazole obtained in Example 1 of the present invention.

[0039] Figure 2 This is the HPLC spectrum of the phenoxyisopropanol enantiomers obtained in Example 1 of the present invention.

[0040] Figure 3The HPLC spectrum of the climbazole enantiomers obtained in Example 1 of the present invention.

[0041] Figure 4 HPLC spectra of four enantiomers in the mixed samples of phenoxyisopropanol and climbazole obtained in Examples 2 to 4 of the present invention.

[0042] Figure 5 This is the HPLC spectrum of four enantiomers in the mixed sample of phenoxyisopropanol and climbazole obtained in Example 5 of the present invention.

[0043] Figure 6 This is the HPLC spectrum of four enantiomers in the mixed sample of phenoxyisopropanol and climbazole obtained in Example 6 of the present invention.

[0044] Figure 7 This is the HPLC spectrum of four enantiomers in the mixed sample of phenoxyisopropanol and climbazole obtained in Example 7 of the present invention. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below through specific examples.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0050] Example 1

[0051] A chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and climbazole enantiomers comprises the following steps:

[0052] (1) Preparation of test samples

[0053] Preparation of a mixed solution of phenoxyisopropanol (Shanghai Aladdin Biochemical Technology Co., Ltd., Catalog No. H2121122) and climbazole (Shanghai Anpu Laboratory Technology Co., Ltd., Catalog No. 2425188): Mix and dissolve the racemic samples of phenoxyisopropanol and climbazole in ethanol to form a sample solution 1 with a concentration of 0.5 mg / mL;

[0054] Preparation of phenoxyisopropanol racemate solution: dissolve phenoxyisopropanol racemate in ethanol to form a sample solution 2 with a concentration of 0.5 mg / mL;

[0055] Preparation of racemic solution of climbazole: the racemic climbazole was dissolved in ethanol to form a sample solution 3 with a concentration of 0.5 mg / mL;

[0056] (2) The sample solution is subjected to chiral analysis and separation by high performance liquid chromatography, wherein the specific parameters of the chromatography are as follows:

[0057] 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 (250 mm × 4.6 mm, 5 μm), the chiral stationary phase is silica gel surface bonded amylose-tris (3-chloro-5-methylphenylcarbamate), the mobile phase is n-hexane:ethanol with a volume ratio of 70:30; the flow rate is 1.0 mL / min, the injection volume is 2 μL, the column temperature is 25 ° C, the detection wavelength is 254 nm,

[0058] The test results are shown in Table 1 and Figures 1 to 3 .

[0059] Table 1 and Figure 1 The results showed that four completely baseline-separated chromatographic peaks were observed at retention times of 4.72, 5.21, 6.37, and 8.15 min, with resolutions of 2.79, 4.67, and 4.82, respectively, indicating that the chiral chromatographic analysis method can effectively separate the enantiomers of phenoxyisopropanol and climbazole simultaneously. Figure 2 and Figure 3 They are the comparison spectra of phenoxyisopropanol and climbazole racemate. By comparison, it can be found that in the synchronous separation process of phenoxyisopropanol and climbazole, the chromatographic peaks with shorter retention times (4.73 and 5.22min) are phenoxyisopropanol enantiomers, and the chromatographic peaks with longer retention times (6.43 and 8.21min) are climbazole enantiomers.

[0060] Example 2

[0061] A chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and climbazole enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:ethanol with a volume ratio of 90:10.

[0062] Example 3

[0063] A chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and climbazole enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:ethanol with a volume ratio of 80:20.

[0064] Example 4

[0065] A chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and climbazole enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:ethanol with a volume ratio of 60:40.

[0066] Example 5

[0067] A chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and climbazole enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:ethanol with a volume ratio of 50:50.

[0068] Example 6

[0069] A chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and climbazole enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:isopropanol with a volume ratio of 70:30.

[0070] Example 7

[0071] A chiral chromatographic analysis method for synchronous separation of phenoxyisopropanol and climbazole enantiomers, compared with Example 1, the only difference is that the mobile phase is n-hexane:n-butanol with a volume ratio of 70:30.

[0072] Effect Example

[0073] The chiral chromatography analysis methods of Examples 2 to 7 were used to perform chiral analysis and separation on the mixed solution of phenoxyisopropanol and climbazole racemate. Figures 4 to 7 And as shown in Table 1.

[0074] Under the chromatographic conditions of Examples 2 to 4, the four enantiomers in the mixed sample of phenoxyisopropanol and climbazole can be baseline separated. As the content of ethanol in the mobile phase decreases, the separation between the four enantiomers in the mixed sample of phenoxyisopropanol and climbazole gradually increases, and the retention on the chiral stationary phase also gradually increases, and the retention time is significantly prolonged ( Figure 4). When the volume ratio of n-hexane to ethanol in the mobile phase was 50:50 (ie, Example 5), the baseline separation of the phenoxyisopropanol enantiomers in the mixed sample of phenoxyisopropanol and climbazole was not achieved, but only the baseline separation of the climbazole enantiomers was achieved ( Figure 5 When isopropanol and n-butanol are selected as the alcohol components in the mobile phase (ie, Examples 6 to 7), the separation effect of the climbazole enantiomers in the synchronous separation target cannot be achieved ( Figure 6-7 ).

[0075] Table 1 Conditions and separation results of the chromatographic separation and analysis methods of Examples 1 to 7

[0076]

[0077] 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 analysis method for synchronously separating phenoxyisopropanol and climbazole enantiomers, comprising the following steps: The chiral stationary phase was used to separate and analyze the mixed sample solution of phenoxyisopropanol and climbazole by liquid chromatography; The mobile phase in the liquid chromatography comprises n-hexane and lower alcohol.

2. The chiral chromatography analysis method according to claim 1, characterized in that: The volume ratio of n-hexane to lower alcohol in the mobile phase is 60-90:40-10.

3. The chiral chromatography analysis method according to claim 2, characterized in that: The lower alcohol is ethanol.

4. The chiral chromatography analysis method according to claim 2, characterized in that: The chiral stationary phase is a polysaccharide derivative chiral stationary phase.

5. The chiral chromatography analysis method according to claim 4, 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-dimethylphenylcarbamate).

6. The chiral chromatography analysis method according to any one of claims 1 to 5, characterized in that: The solvent of the mixed sample solution is ethanol.

7. The chiral chromatography 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 analysis method according to claim 6, characterized in that: The column temperature in the liquid chromatography is 20-30° C.; and / or the detection wavelength in the liquid chromatography is 245-255 nm.

9. The chiral chromatography analysis method according to claim 6, characterized in that: The concentration of phenoxyisopropanol or climbazole in the mixed sample solution is no more than 2.0 mg / mL.

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

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