An ultra-high performance liquid chromatography method for simultaneously determining multiple kavain ester components in kava extract
By employing ultra-high performance liquid chromatography (UHPLC) with specific mobile phases and gradient elution conditions, the problem of incomplete component monitoring in kava extract was solved, enabling simultaneous quantification and efficient separation of nine components, thus improving the accuracy and stability of detection.
Patent Information
- Application Number
- CN202611000624.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-07
- Publication Date
- 2026-08-04
AI Technical Summary
Existing technologies cannot fully monitor the nine key components in kava extract, especially the chemically unstable yellow kava piperine C, and the separation effect is poor, affecting the accuracy and reliability of quantitative results.
Ultra-high performance liquid chromatography (UHPLC) was employed, using octadecylsilane-bonded silica gel as the packing material. The mobile phase was prepared as a ternary gradient elution of phosphoric acid aqueous solution, acetonitrile, and isopropanol. The optimized detection wavelength was 220 nm, and the flow rate was 0.3 mL/min, enabling gradient elution to be completed within 25-35 minutes and ensuring baseline separation of all components.
Simultaneous quantitative analysis of all nine components in kava extract was achieved, with significantly improved separation effect, shortened analysis time, enhanced detection sensitivity and stability, and greatly improved accuracy and reliability of the method.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical analysis and plant extract detection technology, specifically relating to an ultra-high performance liquid chromatography method for simultaneously determining the content of multiple calvalactones and chrysone active ingredients in calva extract. Background Technology
[0002] Kava extract, a natural plant preparation derived from the roots of Pipermethysticum, contains a series of kava lactone compounds (such as capsaicin and narcoticin) and chondroitin and other chondroitin compounds. These components are widely used in pharmaceuticals and health supplements due to their significant sedative and anti-anxiety physiological activities. However, the kava lactone compounds have similar chemical structures but vary considerably in content, and some components (such as chondroitin C) are chemically unstable. This presents a significant challenge to establishing a quality control method capable of simultaneously, rapidly, and accurately monitoring all key active ingredients.
[0003] Existing technologies have explored methods for detecting kava extracts. In particular, existing technology CN110658274A discloses a "Quantitative Detection Method for Active Components in Kava Pepper Herbs." This document aims to address the scarcity of kava pepper reference standards and the high cost of testing. Its core contribution lies in providing a "one-test-multiple-evaluations" strategy: by determining the percentage absorption coefficient of each reference standard to calculate the relative correction factor, subsequent detection only requires the use of one reference standard (such as dihydrocapsaicin) to simultaneously calculate the content of eight components (including capsanthin, dihydrocapsanthin, capsanthin, dihydrocapsanthin, capsanthin, demethoxycapsanthin, kava piperine A, and yellow kava piperine B). This method simplifies the operation and reduces costs to a certain extent.
[0004] However, in-depth analysis reveals significant technical limitations and application defects in the existing method: First, regarding detection coverage, the method fails to include "yellow kava piperine C" in the monitoring scope, resulting in blind spots in the characterization of the complete quality spectrum of kava extract and failing to comprehensively reflect product quality. Second, in terms of chromatographic separation capability, although it uses a 1.7 μm particle size column, the method is essentially based on the separation logic of high-performance liquid chromatography (HPLC), with a flow rate set at 0.2 mL / min and a total analysis cycle of 18 minutes. Practice shows that under these conditions, the separation effect between the target component and adjacent impurity peaks is still unsatisfactory, making it difficult to achieve baseline separation in all batches, directly affecting the accuracy and reliability of the quantitative results. Finally, regarding detection conditions, the method selects 232 nm as the detection wavelength and uses an aqueous acetic acid solution as the aqueous phase. These conditions are not optimal for all nine key components (especially the newly added yellow kava piperine C), potentially leading to insufficient detection sensitivity and stability for some components.
[0005] Therefore, there is an urgent need in the field for a novel detection method that can simultaneously cover all nine key components (including yellow kava piperine C) and achieve effective baseline separation of all target peaks and impurity peaks within a shorter analysis time. This method requires fundamentally improving chromatographic separation efficiency and analytical throughput, and optimizing detection conditions to ensure sensitive and stable responses for all components, thereby providing a solid technical guarantee for the high-quality control and standardization of kava extracts. Summary of the Invention
[0006] To address the aforementioned problems, this invention discloses an ultra-high performance liquid chromatography method for the simultaneous determination of multiple kavalactone components in kavalactin extract.
[0007] This invention includes the following technical solutions: An ultra-high performance liquid chromatography method for the simultaneous determination of multiple kavalactone components in kava extract includes the following steps: S1. Mobile phase preparation step: Prepare a mobile phase, which contains three components: an aqueous phosphoric acid solution, acetonitrile, and isopropanol; S2. Preparation steps of reference solution: Prepare a mixed reference solution containing narcotic capsanthin, demethoxynarcotic capsanthin, dihydronarcotic capsanthin, capsanthin, dihydronarcotic capsanthin, kavalpin A, kavalpin B and kavalpin C. S3. Preparation steps of test solution: Prepare test solution of kava extract; S4. Chromatographic determination procedure: Ultra-high performance liquid chromatography (UHPLC) was used with octadecylsilane-bonded silica gel as the packing material for gradient elution determination. The gradient elution is a ternary gradient elution, with phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and isopropanol as mobile phase C. During the total elution cycle of 25 to 35 minutes, the proportions of mobile phases A, B, and C are linearly adjusted in multiple stages, so that the proportion of mobile phase B gradually increases from the initial 5% to 15% to the final 65% to 75%, while the proportion of mobile phase A is correspondingly reduced, and the proportion of mobile phase C is maintained in the range of 10% to 30% throughout the elution process. The column temperature was 25–45℃, the flow rate was 0.1–0.5 mL / min, and the detection wavelength was 210–250 nm.
[0008] Furthermore, in the above-mentioned ultra-high performance liquid chromatography method, in step S1, the mass fraction of the phosphoric acid aqueous solution is 0.05% to 0.2%.
[0009] Furthermore, in the above-mentioned ultra-high performance liquid chromatography method, in step S2, the preparation of the reference solution, the concentrations of capsaicin, demethoxycapsaicin, dihydrocapsaicin, capsaicin, capsaicin, capsaicin, and dihydrocapsaicin in the mixed reference solution are each independently 0.2–0.3 mg / mL, and the concentrations of kavalpin A, kavalpin B, and kavalpin C are each independently 0.02–0.03 mg / mL.
[0010] Furthermore, in the above-mentioned ultra-high performance liquid chromatography method, in step S4, the gradient elution procedure specifically includes: initially, the volume ratio of mobile phases A, B, and C is (65-75):(5-15):(15-25); during the middle stage of elution, the proportion of mobile phase A is reduced to 10%-20%, the proportion of mobile phase B is increased to 65%-75%, and the proportion of mobile phase C is adjusted to 8%-15%; finally, the mobile phase ratio is restored to the initial state and maintained for a period of time to balance the system.
[0011] Furthermore, in the above-mentioned ultra-high performance liquid chromatography method, in step S4, the gradient elution program is specifically as follows: Within 0 to 0.5 minutes, maintain the volume ratio of mobile phases A, B, and C at 70:10:20; within 0.5 to 8 minutes, linearly change the proportion of mobile phase A from 70% to 58%, the proportion of mobile phase B from 10% to 22%, and maintain the proportion of mobile phase C at 20%; within 8 to 21 minutes, linearly change the proportion of mobile phase A from 58% to 15%, the proportion of mobile phase B from 22% to 72%, and the proportion of mobile phase C from 20% to 13%; within 21 to 24 minutes, maintain the volume ratio of mobile phases A, B, and C at 15:72:13; within 24 to 25 minutes, restore the mobile phase ratio to 70:10:20 and maintain this ratio for 30 minutes.
[0012] Furthermore, in the above-mentioned ultra-high performance liquid chromatography method, in step S4, the column temperature of the chromatographic column is 35℃, the flow rate is 0.3mL / min, and the detection wavelength is 220nm.
[0013] The present invention also discloses a quality control method for kava extract, wherein the content of the nine kava lactone components in the kava extract is simultaneously determined by any of the above methods, and the content of each component is compared with a preset standard to evaluate the product quality.
[0014] The present invention also discloses an ultra-high performance liquid chromatography detection system for implementing the above method, comprising: Ultra-high performance liquid chromatograph, equipped with an octadecylsilane-bonded silica column and an ultraviolet detector; The system control unit is configured to control the chromatograph to perform the gradient elution program as defined above.
[0015] This invention also discloses a mixed reference standard for the quality testing of kava extract, which consists of a solvent and nine kava lactone standards dissolved therein. The nine kava lactones are: capsanthin, demethoxycapsanthin, dihydrocapsanthin, capsanthin, capsanthin, dihydrocapsanthin, yellow capsanthin A, yellow capsanthin B, and yellow capsanthin C. The concentration ratio of the nine standards is such that the peak area response values of each component are on the same order of magnitude when performing ultra-high performance liquid chromatography analysis.
[0016] The present invention also discloses the application of the above method in the preparation of kava extract formulations for relieving anxiety, promoting relaxation or improving sleep.
[0017] Compared with the prior art, the present invention has the following outstanding advantages: 1. More comprehensive component monitoring is achieved. Existing technology 1 (CN110658274A) can only detect 8 components, while this invention is the first to achieve simultaneous quantitative analysis of all 9 key components in kava extract (including yellow kava piperine C, which is not covered by existing technology 1) under single chromatographic conditions, eliminating blind spots in quality monitoring and making quality control more comprehensive.
[0018] 2. Significantly improved chromatographic separation. By optimizing ultra-high performance liquid chromatography (UPLC) conditions and employing a specific ternary gradient elution system of "phosphoric acid aqueous solution-acetonitrile-isopropanol," this invention effectively solves the problem of insufficient separation in the prior art 1. Experimental results show that all nine target components can achieve baseline separation from adjacent impurity peaks, with stable chromatographic baselines and good peak shapes, thus ensuring the accuracy of quantitative results.
[0019] 3. Balancing analytical efficiency and methodological robustness. While ensuring good separation of all nine components, this invention can complete the analysis within 30 minutes, significantly reducing the analysis time compared to traditional HPLC methods. Method validation data show that this method outperforms the levels reported in the prior art in terms of key indicators (RSD%) such as precision, repeatability, and solution stability, demonstrating excellent methodological robustness.
[0020] 4. Improved detection conditions are provided. By optimizing the detection wavelength to 220 nm and using phosphoric acid instead of acetic acid to adjust the mobile phase, this invention provides higher detection sensitivity and more stable chromatographic response for all nine components, especially kavalpinx, thus enhancing the applicability of the method.
[0021] In summary, the analytical method provided by this invention is superior to existing technologies in terms of detection coverage, separation capability, analytical efficiency, and method robustness, providing a more reliable and efficient solution for the quality control of kava extract. Attached Figure Description
[0022] Figure 1 The chromatogram of the reference standard was obtained by high performance liquid chromatography, where peaks 1-7 are characteristic peaks. Figure 2 For high-performance liquid chromatography (HPLC) to detect the chromatogram of the test sample, peaks 1-7 are characteristic peaks; Figure 3 The chromatogram of the reference standard was obtained by ultra-high performance liquid chromatography, where peaks 1-9 are characteristic peaks. Figure 4 For the detection of the chromatogram of the test sample by ultra-high performance liquid chromatography, peaks 1-9 are characteristic peaks; Figure 5 This is the chromatogram of the mobile phase blank solution; Figure 6 The solvent blank is the solvent chromatogram; Figure 7 Linearity graph of anesthetic capsaicin reference standard; Figure 8 Linearity graph for demethoxycapsaicin reference standard; Figure 9 Linearity graph for dihydrocapsaicin reference standard; Figure 10 Linear relationship graph of capsaicin reference standard; Figure 11 Linearity graph for anesthetic capsaicin reference standard; Figure 12 Linearity graph for dihydrocapsaicin reference standard; Figure 13 Linearity graph for yellow kavapiperidine A reference standard; Figure 14 Linearity graph for yellow kavapiperine B reference standard; Figure 15 Linearity graph for yellow kavapiperine C reference standard. Detailed Implementation
[0023] Example: 1. Determination of detection method Through comparative analysis, it was found that ordinary liquid chromatography conditions could not achieve effective separation of the target peak. The comparison results are shown in Table 1 below:
[0024] Characteristic peak attribution and identification: Take the reference solution and the test solution, and inject them separately under the above chromatographic conditions. By comparing the retention time and ultraviolet absorption spectrum, the main chromatographic peaks in the characteristic chromatogram of the test sample are assigned. The results are as follows: Peak 1 is capsaicin, Peak 2 is demethoxycapsaicin, Peak 3 is dihydrocapsaicin, Peak 4 is capsaicin, Peak 5 is capsaicin, Peak 6 is dihydrocapsaicin, Peak 7 is capsaicin A, Peak 8 is capsaicin B, and Peak 9 is capsaicin C (see...). Figure 3 , Figure 4 ).in, Figure 3 This is the chromatogram of the reference solution, showing the positions of each peak compared to the test solution. Figure 4 The retention times of the corresponding peaks in the samples were consistent, indicating that all nine components could be accurately identified in the test sample.
[0025] Compared to the test conditions determined in the subsequent "Chromatographic Conditions and System Suitability Test", the only difference in this set of tests was the analytical chromatograph and flow rate; all other conditions and parameters were the same. The results were examined. In summary, through comparative studies of different detection instruments, flow rates, and chromatographic columns, it was found that under the same detection conditions, the target peak in the chromatogram determined by high-performance liquid chromatography (HPLC) could not be completely separated from surrounding impurities, even after adjusting other chromatographic conditions. Therefore, this method was invented, and the detection conditions were changed to ultra-high-performance liquid chromatography (UHPLC). The results are as follows: like Figure 3 , 4 As shown, it can be observed that under ultra-high performance liquid chromatography (UHPLC) conditions, the separation effect between the target peak and the subsequent impurity peak is significantly better than under other chromatographic conditions. Therefore, the chromatographic conditions of this invention are determined as follows: The determination was performed using ultra-high performance liquid chromatography.
[0026] Chromatographic conditions and system suitability test Octadecylsilane-bonded silica gel was used as the packing material; 0.1% phosphoric acid (0.1% aqueous phosphoric acid solution) was used as mobile phase A, acetonitrile as mobile phase B, and isopropanol as mobile phase C, and gradient elution was performed according to the specifications in Table 1; the column temperature was 35℃, the flow rate was 0.3 ml per minute, and the detection wavelength was 220 nm.
[0027] Preparation of reference solution Reference standards narcotic capsanthin, demethoxynarcotic capsanthin, dihydronarcotic capsanthin, capsanthin, narcotic capsanthin, dihydronarcotic capsanthin, kavalpin A, kavalpin B, and kavalpin C were added to the mobile phase to prepare solutions with concentrations of 0.26 mg / ml, 0.25 mg / ml, 0.24 mg / ml, 0.25 mg / ml, 0.25 mg / ml, 0.25 mg / ml, 0.026 mg / ml, 0.026 mg / ml, and 0.026 mg / ml, respectively.
[0028] Preparation of test solution Preparation of test solution: Take 50.0 mg ± 0.05 mg of the sample to be tested, put it in a 50 ml volumetric flask, add 30 ml of ethanol solution, sonicate for 30 minutes until completely dissolved, place at room temperature, dilute to the mark with ethanol, shake well, filter, and take the filtrate to obtain the test solution. Determination method Accurately pipette 2 μl of the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result.
[0029] 2. Method Validation 2.1) Specificity The specificity of this method was examined using sample, mobile phase blank, and solvent blank as controls. The results are shown in Table 2 below:
[0030] Figure 5 The curves in the chromatogram, from top to bottom, are: mobile phase blank, solvent blank, reference solution, and test solution; the solvent blank chromatogram (see...) Figure 6 The display shows no interference peaks.
[0031] Conclusion: The above results show that the present invention has no interference with the detection of kavalactone solvents and mobile phases, demonstrating the specificity of the present invention.
[0032] 2.2) Precision The test solution was prepared using the same test solution preparation method, and injected under chromatographic conditions. Six consecutive determinations were performed. The peak area RSDs (n=6) for narcotic capsanthin, demethoxycapsanthin, dihydrocapsanthin, capsanthin, narcotic capsanthin, dihydronarcotic capsanthin, kavalpin A, kavalpin B, and kavalpin C were 1.02%, 0.87%, 0.68%, 0.54%, 0.77%, 0.62%, 0.45%, 0.57%, and 0.61%, respectively, indicating good instrument precision.
[0033] 2.3) Repeatability The test solution was prepared using the same test solution preparation method, and injected under chromatographic conditions. The results were repeated six times. The peak area RSDs (n=6) for narcotic capsanthin, demethoxycapsanthin, dihydrocapsanthin, capsanthin, narcotic capsanthin, dihydronarcotic capsanthin, kavalpin A, kavalpin B, and kavalpin C were 0.89%, 0.78%, 0.62%, 0.49%, 0.39%, 1.04%, 1.25%, 0.99%, and 0.87%, respectively, indicating good repeatability of the method.
[0034] 2.4) Stability The test solution was prepared using the same preparation method. After being placed at room temperature for 0, 2, 4, 8, 12, and 24 hours, the sample was injected under chromatographic conditions and measured six times consecutively. The peak areas RSD (n=6) of narcotic capsanthin, demethoxycapsanthin, dihydrocapsanthin, capsanthin, narcotic capsanthin, dihydronarcotic capsanthin, kavalpin A, kavalpin B, and kavalpin C were 1.45%, 1.32%, 1.36%, 1.65%, 1.05%, 1.36%, 1.15%, 0.99%, and 1.27%, respectively, indicating that the test solution had good stability within 24 hours.
[0035] 2.5) Spiking and Recovery Six test solutions were prepared using the same test solution preparation method. An appropriate amount of reference stock solution was added to each solution, and the solutions were prepared according to the same method. The solutions were then injected and analyzed under chromatographic conditions. The average recovery rate and RSD were calculated. The results are shown in Tables 3 and 3(continued).
[0036]
[0037]
[0038] 2.6) Linearity and Range Accurately pipette 1, 2, 4, 6, 8, and 10 ml of the mixed reference solution into separate 10 ml volumetric flasks. Add the mobile phase to dilute to volume, mix well, and prepare mixed reference solutions of different concentrations. Inject and determine the solutions according to chromatographic conditions. Perform linear regression with the mass concentration of the reference solution as the abscissa (X) and the peak area as the ordinate (Y). The results show that capsaicin exhibits a good linear relationship with the peak area integral value within the range of 25.4–254 μg / ml (see [reference]). Figure 7 The equation is Y = 24017x + 7071.7, R 2 =0.9998, demethoxycapsaicin showed a good linear relationship with the peak area integral value within the range of 25.2–252 ug / ml (see [reference]). Figure 8 The equation is Y = 9215.7x + 3019.2, R 2 =0.9994, dihydrocapsaicin showed a good linear relationship with the peak area integral value within the range of 26.3–263 ug / ml (see [reference]). Figure 9 The equation is Y = 15704x - 16170, R 2 =0.9997, and capsaicin showed a good linear relationship with the peak area integral value within the range of 24.3–243 ug / ml (see [reference]). Figure 10 The equation is Y = 10746x + 12830, R 2 =0.9992, and the concentration of anesthetic capsaicin within the range of 25.8–258 ug / ml showed a good linear relationship with the peak area integral value (see [reference]). Figure 11 The equation is Y = 22239x - 32748, R 2 =0.9996, dihydrocapsaicin showed a good linear relationship with the peak area integral value within the range of 23.1–231 ug / ml (see [reference]). Figure 12 The equation is Y = 20942x - 29099, R 2 =0.9995, and the concentrations of 2.8–28 ug / ml of kavalpin A showed a good linear relationship with the peak area integral value (see [reference]). Figure 13 The equation is Y = 16721x - 5827.4, R 2 =0.9991, and the concentrations of kavalpin B 2.78–27.8 ug / ml showed a good linear relationship with the peak area integral value (see [reference]). Figure 14 The equation is Y = 16932x - 0.3233, R 2 =1. The concentrations of kavalpin C2.89–28.9 ug / ml showed a good linear relationship with the peak area integral value (see [reference]). Figure 15 The equation is Y = 20194x - 23510, R 2 =0.9992.
[0039] 3. Determination of the content of active ingredients Take the test samples (batch numbers 20250101, 20250102, and 20250103), prepare three test solutions in parallel, and measure each sample three times in parallel. The results are shown in Table 4.
[0040]
[0041] As can be seen from the above embodiments, the present invention is not a simple optimization of existing detection methods, but rather provides a completely new technical solution, which is reflected in the following aspects: 1. This invention solves a long-standing technical problem: Existing technologies (such as CN110658274A) lack sufficient separation capabilities, making it impossible to effectively monitor the chemically unstable kavalpin C. This invention, by establishing a specific ultra-high performance liquid chromatography (UPLC) method and a ternary gradient elution system, achieves for the first time the simultaneous and accurate quantification of all nine key active ingredients (including kavalpin C) in kava extract, overcoming the technical deficiency of incomplete component coverage in existing methods.
[0042] 2. Unexpected separation results achieved: Addressing the poor separation problem in existing technology 1, this invention unexpectedly achieved complete baseline separation of all nine target components from adjacent impurity peaks by optimizing the detection wavelength to 220 nm, replacing the mobile phase acid adjuster with phosphoric acid, and employing a lower flow rate (0.3 mL / min) and a matching gradient program. This separation effect is unattainable by conventional high-performance liquid chromatography (HPLC) or unoptimized UPLC methods, providing a prerequisite for accurate quantification.
[0043] 3. Verification of the superiority of the overall method performance: Systematic methodological verification shows that the data results of this method are significantly better than those disclosed in the prior art 1 in terms of key indicators such as linear range, precision (RSD%), repeatability and solution stability.
[0044] The above are merely a few preferred embodiments of the present invention, described in a relatively specific and detailed manner, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.
Claims
1. An ultra-high performance liquid chromatography method for simultaneously determining a plurality of kavain components in a kava extract, characterized by, Includes the following steps: S1. Mobile phase preparation step: Prepare a mobile phase, which contains three components: an aqueous phosphoric acid solution, acetonitrile, and isopropanol; S2. Preparation steps of reference solution: Prepare a mixed reference solution containing narcotic capsanthin, demethoxynarcotic capsanthin, dihydronarcotic capsanthin, capsanthin, dihydronarcotic capsanthin, kavalpin A, kavalpin B and kavalpin C. S3. Preparation steps of test solution: Prepare test solution of kava extract; S4. Chromatographic determination procedure: Ultra-high performance liquid chromatography (UHPLC) was used with octadecylsilane-bonded silica gel as the packing material for gradient elution determination. The gradient elution is a ternary gradient elution, with phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and isopropanol as mobile phase C. During the total elution cycle of 25 to 35 minutes, the proportions of mobile phases A, B, and C are linearly adjusted in multiple stages, so that the proportion of mobile phase B gradually increases from the initial 5% to 15% to the final 65% to 75%, while the proportion of mobile phase A is correspondingly reduced, and the proportion of mobile phase C is maintained in the range of 10% to 30% throughout the elution process. The column temperature was 25–45℃, the flow rate was 0.1–0.5 mL / min, and the detection wavelength was 210–250 nm.
2. The method of claim 1, wherein, In step S1, the mass fraction of the phosphoric acid aqueous solution is 0.05% to 0.2%.
3. The method of claim 1, wherein, In step S2, the preparation of the reference solution, the concentrations of narcotic capsanthin, demethoxycapsanthin, dihydrocapsanthin, capsanthin, narcotic capsanthin, and dihydronarcotic capsanthin in the mixed reference solution are each independently 0.2–0.3 mg / mL, and the concentrations of kavalpin A, kavalpin B, and kavalpin C are each independently 0.02–0.03 mg / mL.
4. The method of claim 1, wherein, In step S4, the gradient elution procedure specifically includes: initially, the volume ratio of mobile phases A, B, and C is (65-75):(5-15):(15-25); during the middle stage of elution, the proportion of mobile phase A is reduced to 10%-20%, the proportion of mobile phase B is increased to 65%-75%, and the proportion of mobile phase C is adjusted to 8%-15%; finally, the proportion of mobile phases is restored to the initial state and maintained for a period of time to balance the system.
5. The method of claim 4, wherein, In step S4, the gradient elution program is as follows: Within 0 to 0.5 minutes, maintain the volume ratio of mobile phases A, B, and C at 70:10:20; within 0.5 to 8 minutes, linearly change the proportion of mobile phase A from 70% to 58%, the proportion of mobile phase B from 10% to 22%, and maintain the proportion of mobile phase C at 20%; within 8 to 21 minutes, linearly change the proportion of mobile phase A from 58% to 15%, the proportion of mobile phase B from 22% to 72%, and the proportion of mobile phase C from 20% to 13%; within 21 to 24 minutes, maintain the volume ratio of mobile phases A, B, and C at 15:72:13; within 24 to 25 minutes, restore the mobile phase ratio to 70:10:20 and maintain this ratio for 30 minutes.
6. The method of claim 1, wherein, In step S4, the chromatographic determination step, the column temperature of the chromatographic column is 35℃, the flow rate is 0.3mL / min, and the detection wavelength is 220nm.
7. A method of quality control of a gavac extract, characterized by, The content of the nine calvalactone components in the calva extract is simultaneously determined by the method described in any one of claims 1 to 6, and the content of each component is compared with a preset standard to evaluate the product quality.
8. An ultra-high performance liquid chromatography detection system for carrying out the method of any one of claims 1 to 6, characterized in that, include: Ultra-high performance liquid chromatograph, equipped with an octadecylsilane-bonded silica column and an ultraviolet detector; The system control unit is configured to control the chromatograph to perform the gradient elution program as defined in any one of claims 1-6.
9. A mixed control for quality testing of kava extract, characterized in that, It consists of a solvent and nine calovalin standards dissolved therein, namely: capsanthin, demethoxycapsanthin, dihydrocapsanthin, capsanthin, capsanthin, dihydrocapsanthin, yellow capsanthin A, yellow capsanthin B, and yellow capsanthin C. The concentration ratio of the nine standards is such that the peak area response values of each component are on the same order of magnitude when performing ultra-high performance liquid chromatography analysis.
10. The use of the method according to any one of claims 1 to 6 in the preparation of a kava extract formulation for relieving anxiety, promoting relaxation or improving sleep.