Method for detecting tetrahydrocurcumin

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for detecting tetrahydrocurcumin by high performance liquid chromatography. Comprising the following steps: dissolving and diluting a sample with an organic solvent, and detecting a sample solution with high performance liquid chromatography; the high performance liquid chromatography conditions are as follows: a chromatographic column is a cyano column, and detection is performed under the wavelength of 220 nm or 280 nm; the mobile phase is composed of an organic phase and a water phase, and the volume ratio of the organic phase to the water phase is 1: (0.5-2.5); the water phase is a buffer solution with the pH value of 6.5-7.0. The method disclosed by the invention is high in detection speed, high in sensitivity and accuracy and good in quantitative detection effect; the method can be carried out under a nearly neutral mobile phase condition, a chromatographic column resistant to a high pH value does not need to be selected, an ion pair reagent does not need to be used, damage to the chromatographic column is small, and the service life of the chromatographic column is prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting tetrahydrocurcumin by utilizing high performance liquid chromatography. Background Art

[0002] Curcumin, a natural plant ingredient, has been widely used in cosmetics and medical fields since ancient times. However, its poor stability and bioavailability limit its application in cosmetics. Compared with curcumin, tetrahydrocurcumin exhibits higher chemical stability and water solubility.

[0003] Tetrahydrocurcumin, one of the active ingredients in turmeric and a metabolite of curcumin in the body, is known as (1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione). It exhibits anti-inflammatory, antioxidant, whitening, anti-tumor, and diabetic effects, and is used in pharmaceuticals and cosmetics. Its whitening mechanism primarily involves potent inhibition of tyrosinase activity and scavenging of oxygen free radicals. Therefore, in cosmetics, tetrahydrocurcumin is widely used in whitening, freckle removal, and anti-aging skincare products.

[0004] Tetrahydrocurcumin has a pentanedione structure in the molecule, with two isomers of enol and keto, and there is tautomerism of keto-enol. Therefore, using high performance liquid chromatography, eluting with C18 columns in combination with acidic to neutral flow, the mobile phase is an organic solvent-acid water system. When tetrahydrocurcumin is detected, both isomers exist simultaneously in the chromatographic system, and two isomers can be separated. Its chromatogram presents bimodal peaks, causing tetrahydrocurcumin to be unable to be accurately quantified, and the accuracy and precision of detection are disturbed. The ratio of tetrahydrocurcumin keto and enol has a great relationship with the type of chromatographic system, particularly chromatographic column, resulting in poor repeatability and precision.

[0005] Some literature has proposed improving ion-pair chromatography by adding ion-pairing reagents, such as alkyl quaternary ammonium salts such as dodecyltrimethylammonium chloride, to the mobile phase to overcome the double peak problem. However, the mobile phase system used has a pH of 9.5 to 11, which is highly alkaline and requires the use of specialized chromatographic columns resistant to high pH. Furthermore, the ion-pairing reagents affect the life of the column, making the reproducibility of the analytical method challenging. Therefore, it is necessary to improve existing technologies and achieve quantitative analysis of tetrahydrocurcumin using high-performance liquid chromatography under neutral or slightly acidic conditions. Summary of the Invention

[0006] The present invention aims to provide a method for detecting tetrahydrocurcumin, which can quickly detect tetrahydrocurcumin under neutral to acidic mobile phase conditions and improve the precision and sensitivity of detection.

[0007] The technical solution of the present invention is: a method for detecting tetrahydrocurcumin, characterized in that it comprises the following steps: The sample is dissolved and diluted with an organic solvent, and the sample solution is detected by high performance liquid chromatography.

[0008] The HPLC conditions were as follows: a cyano column and detection at a wavelength of 220 or 280 nm; The mobile phase consists of an organic phase and an aqueous phase, with a volume ratio of the organic phase to the aqueous phase of 1:0.5-2.5; the aqueous phase is a buffer solution with a pH of 6.5-7.0.

[0009] According to the test results and the standard curve, tetrahydrocurcumin can be quantitatively detected.

[0010] Preferably, the aqueous phase is a sodium acetate buffer, wherein the concentration of acetate is 10-120 mM, more preferably 10-100 mM; in a preferred embodiment of the present invention, it is 15-100 mM.

[0011] The concentration of acetate in the mobile phase is 4-60 mM; preferably, the concentration of acetate in the mobile phase is 10-50 mM.

[0012] The organic solvent and the organic phase are alcohols, including methanol, ethanol, propanol, isopropanol, butanol, and isobutanol.

[0013] Preferably, the organic solvent is methanol; and the organic phase is methanol.

[0014] Preferably, the volume ratio of the organic phase to the aqueous phase is 1:0.5-2; in a preferred embodiment of the present invention, the volume ratio of the organic phase to the aqueous phase is 1:0.6-1.5.

[0015] In a preferred embodiment of the present invention, the high performance liquid chromatography conditions are as follows: a cyano column is used, the mobile phase is methanol and sodium acetate buffer; the pH value of the sodium acetate buffer is 6.5-7.0, and the acetate concentration is 15-100 mM; the volume ratio of methanol to sodium acetate buffer is 1:0.6-1.5, and the acetate concentration in the mobile phase is 10-50 mM.

[0016] Preferably, the sample solution is filtered and then detected by high performance liquid chromatography.

[0017] This method can realize the quantitative or qualitative detection of tetrahydrocurcumin.

[0018] This method was ultimately established using a cyano column as the mobile phase, using a simple neutral to slightly acidic buffer, such as sodium acetate, to quantify and analyze tetrahydrocurcumin. This method can be applied to both the quality control of cosmetic raw materials and the analysis and testing of finished products.

[0019] The quantitative limit of this method for tetrahydrocurcumin is 20 mg / L, the detection limit is 6 mg / L, the linear range is 40-800 mg / L, the retention time is 3-5 minutes, the detection speed is fast, the sensitivity and accuracy are high, and the quantitative detection effect is good.

[0020] Furthermore, the method of the present invention can be used for detection under near-neutral mobile phase conditions, does not require the selection of a chromatographic column resistant to high pH values, and does not require the use of ion-pair reagents, thereby causing little damage to the chromatographic column and extending the service life of the chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The chromatogram of the tetrahydrocurcumin standard solution was obtained using a ZORBAX Eclipse Plus C18 column and a mobile phase of methanol and pure water at a ratio of 60:40. Figure 2 The chromatogram of the tetrahydrocurcumin standard solution was obtained by using a ZORBAX Eclipse Plus C18 column with a mobile phase of methanol and 0.1% phosphoric acid aqueous solution (pH 2.2) at a ratio of 60:40. Figure 3 The column is ZORBAX Eclipse Plus C18, and the mobile phase is methanol: 100mM sodium acetate aqueous solution (pH=6.8) = 60:40. The chromatogram of tetrahydrocurcumin standard solution is shown in Figure 2. Figure 4 The column is ZORBAX Eclipse Plus C18, and the mobile phase is methanol: 100mM sodium acetate aqueous solution (pH=6.8) = 60:40. The UV spectrum of the main peak of tetrahydrocurcumin Figure 5 Hypersil GOLD CN cyano column, mobile phase - methanol: pure water = 40:60, chromatogram of tetrahydrocurcumin standard solution Figure 6 Hypersil GOLD CN cyano column, mobile phase - methanol: 0.1% phosphoric acid aqueous solution (pH = 2.2) = 40:60, chromatogram of tetrahydrocurcumin standard solution Figure 7 Hypersil GOLD CN cyano column, mobile phase - methanol: 0.1% phosphoric acid aqueous solution (pH = 2.2) = 40:60, UV spectrum of the main peak of tetrahydrocurcumin Figure 8 Hypersil GOLD CN cyano column, mobile phase - methanol: 100mM sodium acetate aqueous solution (pH=6.8): pure water = 40:20:40, chromatogram of tetrahydrocurcumin standard solution Figure 9Hypersil GOLD CN cyano column, mobile phase - methanol: 100mM sodium acetate aqueous solution (pH=6.8) = 60:40, chromatogram of tetrahydrocurcumin standard solution Figure 10 Hypersil GOLD CN cyano column, mobile phase - methanol: 100mM sodium acetate aqueous solution (pH=6.8): pure water = 40:20:40, UV spectrum of the main peak of tetrahydrocurcumin Figure 11 Chromatogram of 1# raw material sample solution Figure 12 Chromatogram of 3# cosmetic product sample solution DETAILED DESCRIPTION Chromatographic columns: Hypersil GOLD CN cyano column, 4.6*250mm, 5μm; ZORBAX Eclipse Plus C18 column, 4.6*250mm, 5μm; Reagents: Accurately weigh approximately 80 mg of tetrahydrocurcumin standard into a 100 mL volumetric flask, dissolve with methanol, dilute to volume, and shake well to prepare the standard stock solution. Pure water; 100 mM sodium acetate buffer (pH = 6.5-6.8); 0.1% phosphoric acid aqueous solution (pH = 2.2); methanol.

[0022] Example 1 Selection of chromatographic column, mobile phase and detection wavelength A cyano column (CN column) is a versatile chromatographic column that has both normal-phase and reverse-phase separation capabilities. It is suitable for separating non-polar to moderately polar compounds, especially in normal-phase mode, and has unique selectivity for polar compounds. It is commonly used in pharmaceuticals, complex mixtures, and sample analysis containing polar groups such as carbonyls or hydroxyls.

[0023] Since the ratio of tetrahydrocurcumin keto form and enol form is closely related to the chromatographic system, especially the type of chromatographic column, we selected the commonly used C18 column and the cyano column with a large difference in bonded phase for detection in the mobile phase system.

[0024] (1) The mobile phase is a non-buffered salt system and an acidic system Accurately transfer 2.5 mL of the standard stock solution to a 10 mL volumetric flask, dilute to the mark with methanol, and shake well.

[0025] Injection volume: 5 μL; column temperature: 30°C; detector: DAD; detection wavelength: 280 nm; flow rate: 1.0 mL / min.

[0026] Methanol: pure water = 60:40 (volume ratio) and methanol: 0.1% phosphoric acid aqueous solution (pH = 2.2) = 60:40 (volume ratio) were used as mobile phases for elution. The chromatograms of tetrahydrocurcumin standard solution under different mobile phase conditions on the C18 column are shown in the figure. Figure 1 and Figure 2 shown.

[0027] The chromatograms of tetrahydrocurcumin standard solution under different mobile phase conditions on cyano column are as follows: Figure 5 and Figure 6 As shown, when the mobile phase is methanol: 0.1% phosphoric acid aqueous solution (pH = 2.2) = 60:40 (volume ratio), the UV spectrum of the main peak of tetrahydrocurcumin is as follows Figure 7 shown.

[0028] The results showed that on a C18 column, when the aqueous mobile phase was pure water or an acidic solution, the main peak of tetrahydrocurcumin split into two peaks, making quantification impossible. On a cyano column, when the aqueous mobile phase was pure water or an acidic solution, the main peak of tetrahydrocurcumin was broad and tailing, making integration difficult. Furthermore, the UV spectrum showed that the compound structure was not fixed, making it not recommended for quantitative analysis.

[0029] (2) Mobile phase is a buffer system Accurately transfer 2.5 mL of the standard stock solution to a 10 mL volumetric flask, dilute to the mark with methanol, and shake well.

[0030] Injection volume: 5 μL; column temperature: 30°C; detector: DAD; detection wavelength: 280 nm; flow rate: 1.0 mL / min.

[0031] 1. ZORBAX Eclipse Plus C18 Column When using a ZORBAX Eclipse Plus C18 column, the mobile phase is methanol: 100mM sodium acetate buffer (pH=6.8) = 60:40, and the chromatogram of the tetrahydrocurcumin standard solution is as follows Figure 3 As shown, the main peak UV spectrum of tetrahydrocurcumin is as follows Figure 4 shown.

[0032] The results showed that when sodium acetate buffer solution was added to the mobile phase on the C18 column, the main peak was symmetrical but the broad peak was not conducive to integration, and the UV spectrum showed that the compound structure was not fixed, so it was not recommended for use in quantitative analysis.

[0033] 2. Hypersil GOLD CN cyano column (1) When using Hypersil GOLD CN cyano column, the mobile phase is methanol: 100mM sodium acetate buffer (pH=6.8): pure water = 40:20:40 (volume ratio). The chromatogram of tetrahydrocurcumin standard solution is as follows: Figure 8 After 100 mM sodium acetate buffer was diluted twice with pure water, the pH was about 6.9.

[0034] (2) The mobile phase is methanol: 100mM sodium acetate aqueous solution (pH=6.8) = 60:40 (volume ratio). The chromatogram of tetrahydrocurcumin standard solution is as follows: Figure 9 shown.

[0035] Depend on Figure 8 、 9 The results show that using a cyano column, the chromatographic performance is similar under the two mobile phase conditions mentioned above. Retention times vary depending on the ratio of methanol in the organic phase. A higher methanol content results in shorter retention times.

[0036] The chromatogram results showed that the main peak had a sharp and symmetrical shape, and the peak area had good reproducibility, allowing for accurate quantification. Therefore, these conditions were selected for parameter validation of the analytical method, and the results showed that the method had good precision and reproducibility.

[0037] Furthermore, when using a cyano column, the salt content (ion concentration) in the two mobile phase conditions did not affect the retention time, peak shape, and response value of the chromatogram.

[0038] Combined with the chromatographic results of the mobile phases of methanol and acidic system (0.1% phosphoric acid aqueous solution, pH=2.2), it can be judged that when using a cyano column, the key condition affecting the detection results is pH.

[0039] When the volume ratio of sodium acetate buffer to pure water is adjusted and the pH of the buffer is controlled between 6.5 and 7.0, the acetate ion concentration in the range of 10-100 mM has no effect on the retention time, peak shape and response value of high performance liquid chromatography.

[0040] When the following mobile phase is used, the chromatogram results are similar to those of (1) and (2). The main peak is sharp and symmetrical, and the peak area has good reproducibility, which can be used for quantitative analysis.

[0041] (3) The mobile phase was methanol: 100 mM sodium acetate aqueous solution (pH = 6.6) = 60:40 (volume ratio); (4) The mobile phase was methanol: 100 mM sodium acetate aqueous solution (pH = 6.6): pure water = 40:10:50 (volume ratio).

[0042] (3) Selection of detection wavelength Chromatographic column: Hypersil GOLD CN cyano column, 4.6*250mm, 5μm; Mobile phase: methanol: 100 mM sodium acetate buffer (pH = 6.8): pure water = 40:20:40; Injection volume: 5 μL; column temperature: 30°C; detector: DAD; detection wavelength: 280 nm; flow rate: 1.0 mL / min.

[0043] Under the above optimized chromatographic column and mobile phase system, the UV spectrum of tetrahydrocurcumin was investigated. The results showed that tetrahydrocurcumin compound had characteristic absorption at 220nm and 280nm, such as Figure 10 shown.

[0044] Taking both selectivity and sensitivity into consideration, the detection wavelength of 280 nm was finally selected for the quantitative analysis of tetrahydrocurcumin.

[0045] Example 2 Standard curve, linear range, limit of quantification and limit of detection Accurately weigh about 80 mg of tetrahydrocurcumin standard into a 100 mL volumetric flask, dissolve it with methanol, dilute to the mark and shake well to prepare the standard stock solution; accurately pipette 0.5, 2.5 and 5.0 mL of the standard stock solution into a 10 mL volumetric flask, dilute to the mark with methanol and shake well to prepare a series of standard solutions. The concentrations of the series of standard solutions are shown in the table below: A series of standard solutions were injected and tested according to the following chromatographic conditions.

[0046] Chromatographic column: Hypersil GOLD CN cyano column, 4.6*250mm, 5μm; Mobile phase: methanol: 100 mM sodium acetate buffer (pH = 6.8): pure water = 40:20:40; Injection volume: 5 μL; column temperature: 30°C; detector: DAD; detection wavelength: 280 nm; flow rate: 1.0 mL / min.

[0047] With 10 times the signal-to-noise ratio as the instrument's limit of quantification and 3 times the signal-to-noise ratio as the limit of detection, the linear range, limit of quantification, and limit of detection of the method are shown in the table below.

[0048] Example 3 Method Precision The standard solution STD2 was injected and tested according to the chromatographic conditions described in Example 2. The precision of the method was RSD = 0.9% (n = 6). The data are shown in the table below.

[0049] Example 4 Actual sample detection Take one cosmetic raw material tetrahydrocurcumin sample (1#), accurately weigh 20 mg and put it into a 100 mL volumetric flask, dissolve it in methanol and dilute it to the mark and shake it well to serve as the raw material sample solution. Take three cosmetic products (2#, 3#, 4#), accurately weigh 1.0 g of each sample and put it into a 20 mL volumetric flask, dissolve it in methanol and dilute it to the mark and shake it well to serve as the test sample solution. Filter an appropriate amount through a 0.45 μm hydrophobic organic phase filter, and inject and test according to the chromatographic conditions described in Example 2. The test results of the samples are shown in the table below. The HPLC chromatograms of the 1# cosmetic raw material sample and the 3# cosmetic product are shown in Figures 11 and 12. Figure 12 shown.

[0050]

Claims

1. A method for detecting tetrahydrocurcumin, characterized in that, The steps include: The sample is dissolved and diluted with an organic solvent, and the sample solution is tested by high performance liquid chromatography; The HPLC conditions were as follows: a cyano column and detection at a wavelength of 220 nm or 280 nm; The mobile phase consists of an organic phase and an aqueous phase, with a volume ratio of the organic phase to the aqueous phase of 1:0.5-2.5; the aqueous phase is a buffer solution with a pH of 6.5-7.

0.

2. The method for detecting tetrahydrocurcumin according to claim 1, wherein Also includes: Tetrahydrocurcumin was quantitatively detected based on the test results and the standard curve.

3. The method for detecting tetrahydrocurcumin according to claim 1, wherein Detection was at 280 nm wavelength.

4. The method for detecting tetrahydrocurcumin according to claim 1, wherein The organic solvent and the organic phase are alcohol.

5. The method for detecting tetrahydrocurcumin according to claim 4, wherein The organic solvent and the organic phase are methanol.

6. The method for detecting tetrahydrocurcumin according to claim 1, wherein The buffer solution is sodium acetate buffer.

7. The method for detecting tetrahydrocurcumin according to claim 6, wherein The acetate concentration in sodium acetate buffer is 10-120 mM.

8. The method for detecting tetrahydrocurcumin according to claim 1, wherein In the mobile phase, the volume ratio of the organic phase to the aqueous phase is 1:0.6-2.

9. The method for detecting tetrahydrocurcumin according to claim 1, wherein The acetate concentration in the mobile phase was 4–60 mM.

10. The method for detecting tetrahydrocurcumin according to claim 1, wherein The sample solution was filtered and then detected by high performance liquid chromatography.

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