A method for simultaneously determining multiple index components in traditional Chinese medicine lily by HPLC

Abstract

The present application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a detection method for simultaneously detecting multiple index components in traditional Chinese medicine lily. According to the pharmacological and pharmacodynamic research progress of traditional Chinese medicine lily, the index components that can be used as the quality markers of traditional Chinese medicine lily include ferulic acid, p-coumaric acid, gallic acid, dioscin and wangbaihe glycoside B, and a simple and practical HPLC quality control method for simultaneously detecting multiple index components in traditional Chinese medicine lily is urgently needed. The present application realizes the beneficial effect that the same sample processing method and the same liquid chromatography method can be used to simultaneously detect ferulic acid, p-coumaric acid, gallic acid, dioscin and wangbaihe glycoside B under the condition of 210 nm and 300 nm dual-wavelength.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a detection method for simultaneously determining multiple indicator components in the traditional Chinese medicine lily. Background Technology

[0002] Lily bulb is one of the nine traditional Chinese medicinal herbs of Hunan province, and it is cultivated throughout Hunan. Lily bulb refers to the dried, fleshy bulbs of several plants in the Liliaceae family, including *Lilium lancifolium* Thunb., *Lilium broumii* FEBrown var. *viridulum* Baker, and *Lilium pumilum* DC. It contains polysaccharides, gallic acid, p-coumaric acid, and other saponins. According to the *Compendium of Materia Medica*, lily bulb is sweet and slightly bitter in taste, and neutral in nature. It is believed to have the effects of nourishing the heart and lungs, strengthening the kidneys, tonifying the brain, clearing the heart, calming the mind, sedating, moistening the lungs, regulating the spleen and stomach, invigorating qi, clearing heat and relieving cough, nourishing yin and stopping bleeding, and relieving summer heat.

[0003] Literature reports that the key indicator components that can serve as quality markers for the traditional Chinese medicine lily mainly include ferulic acid, p-coumaric acid, gallic acid, diosgenin, and lily glycoside B. The 2020 edition of the Chinese Pharmacopoeia only addresses the quality control of lily medicinal materials / processed slices through morphological identification, thin-layer chromatography, and polysaccharide content determination. Therefore, in-depth research on the quality control of the traditional Chinese medicine lily is highly necessary.

[0004] Yuan Zhiying [HPLC Determination of p-coumaric acid and gallic acid content in lily. China Journal of Traditional Chinese Medicine Information. May 2018, Vol. 25, No. 5] used a dual-wavelength HPLC method to determine the content of p-coumaric acid and gallic acid in lily; Li Qian [Determination of content of index components in lily and correlation analysis of their characteristic chromatogram quality characterization. Global Traditional Chinese Medicine. April 2018, Vol. 11, No. 4] determined ferulic acid and diosgenin. However, her method of using 100 times the amount of 80% methanol and heating and refluxing for 60 minutes to extract the effective components from lily consumed too much organic solvent, and the sample processing method was cumbersome, time-consuming, and costly. Furthermore, her method of gradient elution for 90 minutes was even more... The methods are inefficient and unsuitable for industrial application. Shi Yanxia's study, "Determination of Diosgenin Content in Medicinal Lily by HPLC. Journal of Hunan University of Traditional Chinese Medicine. 2009, 29(02)", measured diosgenin at a wavelength of 203 nm. However, the peak shape of diosgenin was poor, with severe end absorption at 203 nm, affecting the determination of its content. Chinese invention patent CN109817344A further employed four independent high-performance liquid chromatography methods to determine four indicators: lily glycoside BA, coumaric acid, gallic acid, and lily polysaccharide. Because multiple indicators could not be measured simultaneously, the detection was time-consuming and labor-intensive, making it unsuitable for industrial application. The above studies primarily focus on the quality control of the medicinal herb lily, targeting only one or two effective components. Some detection methods also suffer from drawbacks such as complex sample processing and time-consuming testing. There is currently no detection method that can simultaneously determine the main indicator components in the Chinese herbal medicine lily, including ferulic acid, coumaric acid, gallic acid, diosgenin, and lily glycoside B.

[0005] Based on the progress in pharmacological and efficacy research on the Chinese medicinal herb lily, the indicative components that can be used as quality markers for lily include ferulic acid, p-coumaric acid, gallic acid, diosgenin, and lily glycoside B. There is an urgent need for a simple and practical HPLC quality control method that can simultaneously detect multiple indicative components in lily. Summary of the Invention

[0006] Current technologies for quality control of lily bulbs mainly focus on detecting one or two active ingredients. There is currently no detection method that can simultaneously determine the main indicator components in lily bulbs, such as ferulic acid, p-coumaric acid, gallic acid, diosgenin, and lily glycoside B.

[0007] This invention provides an HPLC method for simultaneously determining four indicator components in the traditional Chinese medicine lily: The HPLC method is used to simultaneously determine the contents of gallic acid, p-coumaric acid, ferulic acid, and lily glycoside B in lily.

[0008] The detection method was as follows: C18 column; mobile phase: acetonitrile (A) - 0.2% glacial acetic acid (B), gradient elution: 0 min - 12 min, 8% - 15% A; 12 min - 20 min, 15% - 30% A; detection wavelength for gallic acid, p-coumaric acid, ferulic acid, and limonin B was 300 nm ± 1 nm; flow rate: 1.0 mL / min; column temperature: 30℃ - 40℃; ultraviolet detector or diode array detector was used; reference solution and test solution were injected for analysis, and the contents of gallic acid, p-coumaric acid, ferulic acid, and limonin B in lily medicinal materials were calculated by external standard method.

[0009] This invention also provides an HPLC method for simultaneously determining five indicator components in the traditional Chinese medicine lily. The method employs a dual-wavelength HPLC approach to simultaneously determine the contents of gallic acid, p-coumaric acid, ferulic acid, lily glycoside B, and diosgenin in the lily medicinal material.

[0010] The detection method was as follows: C18 column; mobile phase: acetonitrile (A) - 0.2% glacial acetic acid (B), gradient elution: 0 min - 12 min, 8% - 15% A; 12 min - 20 min, 15% - 30% A; 20 min - 42 min, 30% - 60% A; detection wavelengths for gallic acid, p-coumaric acid, ferulic acid, and diosgenin B were 300 nm ± 1 nm, and the detection wavelength for diosgenin was 210 nm ± 1 nm; flow rate: 1.0 mL / min; column temperature: 30℃ - 40℃; ultraviolet detector or diode array detector was used; reference solution and test solution were injected for analysis, and the contents of gallic acid, p-coumaric acid, ferulic acid, diosgenin B, and diosgenin in lily medicinal materials were calculated by external standard method.

[0011] The preparation method of the test solution is as follows: Take 1g-10g of lily powder, place it in a container, add 5mL-50mL of 70% ethanol, weigh it, soak it for 1h, perform ultrasonic extraction in a water bath at 70℃-95℃ for 30min-60min, take it out and cool it, make up the weight with 70% ethanol, filter it, and take the filtrate for detection and analysis.

[0012] Furthermore, the preparation method of the test solution is as follows: Take 10g of lily powder, place it in a container, add 50mL of 70% ethanol, weigh it, soak it for 1h, extract it by ultrasonication in an 80℃ water bath, ultrasonicate for 30min, take it out and cool it, make up the weight with 70% ethanol, filter it, and take the filtrate for detection and analysis.

[0013] The preparation method of the reference solutions is as follows: Gallic acid, p-coumaric acid, ferulic acid, limonin B, and diosgenin reference stock solutions are prepared separately with methanol, and diluted with 70% ethanol to prepare reference standard positioning solutions and mixed reference solutions. The concentrations of gallic acid, p-coumaric acid, ferulic acid, limonin B, and diosgenin in the mixed reference solution are 0.00501 mg / ml, 0.00505 mg / ml, 0.5003 mg / ml, 0.0494 mg / ml, and 0.0499 mg / ml, respectively.

[0014] Furthermore, the column temperature was 35°C and the injection volume was 20 μL.

[0015] The detection wavelength for gallic acid, p-coumaric acid, ferulic acid, and lily glycoside B was 300 nm, and the detection wavelength for diosgenin was 210 nm.

[0016] The chromatographic column model is XDB C18 (Agilent, Zorbax Eclipse, 250 mm × 4.6 mm, 5 μm) or Kromasil C18 (4.6 mm × 250 mm × 5 μm).

[0017] Preferably, the chromatographic column is an XDB C18 (Agilent, Zorbax Eclipse, 250 mm × 4.6 mm, 5 μm).

[0018] Furthermore, the gradient elution was performed as follows: 0 min-12 min, 8%-15% A; 12 min-20 min, 15%-30% A; 20 min-42 min, 30%-60% A; 42 min-50 min, 60%-8% A; and at 50 min, the elution returned to the initial mobile phase of 8% A. This enabled the determination of the contents of five index components in lily medicinal materials—ferulic acid, p-coumaric acid, gallic acid, diosgenin, and lily glycoside B—under dual-wavelength conditions.

[0019] Based on the extraction and dissolution properties of five indicator components, this invention conducted extensive exploratory experiments. Ultimately, by coordinating ultrasonic time and temperature, it achieved the beneficial effect of simultaneously detecting five indicator components—ferulic acid, p-coumaric acid, gallic acid, diosgenin, and diosgenin B—under dual-wavelength conditions of 210 nm and 300 nm, using the same sample treatment method and the same liquid chromatography method. That is, only one sample treatment and one injection of the sample solution are required to simultaneously determine five indicator components—ferulic acid, p-coumaric acid, gallic acid, diosgenin B, and diosgenin (210 nm)—under dual-wavelength conditions of 210 nm and 300 nm. The method has been validated and can be applied to the quality control of lily medicinal materials production. This method can be applied to liquid chromatographs coupled with a DAD detector, as well as liquid chromatographs coupled with a UV detector with dual-wavelength detection capabilities, demonstrating good versatility. Another beneficial effect of this invention is that by using glacial acetic acid, which is less corrosive and volatile, as the mobile phase, the damage to the chromatographic column is less than that to highly corrosive phosphoric acid (which is not volatile and damages the chromatographic column more) and formic acid, thus effectively extending the service life of the chromatographic column. Attached Figure Description

[0020] Figure 1 Specific chromatograms (300 nm) for ferulic acid, p-coumaric acid, gallic acid, and lily glycoside B: A is the chromatogram of the mixed reference solution; B is the chromatogram of the test solution.

[0021] Figure 2 The specific chromatograms (210 nm) of diosgenin are as follows: A is the chromatogram of the diosgenin reference solution; B is the chromatogram of the test solution. Detailed Implementation

[0022] Example 1: Sample Processing

[0023] The sample processing method directly affects the dissolution of active ingredients, and the amount of active ingredients dissolved is directly related to the quality control of lily medicinal materials.

[0024] Previous experimental studies have found that 70% ethanol can effectively extract ferulic acid, p-coumaric acid, and gallic acid, but its extraction efficiency for diosgenin and diosgenin B is not high. Huang Jiangjian et al. [HPLC-ELSD Determination of Diosgenin Content in Lily from Different Origins. Chinese Journal of Experimental Traditional Medical Formulae. Vol. 17, No. 5, March 2011] reported using 150 mL of 8.5 times the volume of 80% ethanol, refluxed in a water bath for 3 h, refluxed twice, extracted twice with petroleum ether, and then extracted four times with water-saturated n-butanol. The extract was then evaporated to dryness. The method reported in this literature is too complex and unsuitable for widespread application. It is also prone to operational errors in certain steps, leading to inaccurate content determination.

[0025] The sample solution processing method used in this invention is simple, easy to operate, has a low probability of operational error, and achieves a high recovery rate. The specific method is as follows:

[0026] Take 10g of lily powder, place it in an Erlenmeyer flask, add 50mL of 70% ethanol, weigh it, soak it for 1 hour, and perform ultrasonic extraction in a water bath at 70℃-95℃ for 30-60 minutes. After extraction, remove it and cool it, add 70% ethanol to make up the weight, filter it, and pass the filtrate through a 0.45μm filter membrane for detection and analysis.

[0027] Example 2: Investigation at different temperatures and ultrasound durations

[0028] Water at 60℃, 70℃, 80℃, 90℃, and 95℃ (boiling water was added to the ultrasonic instrument; the conical flask containing the powder and 70% ethanol was preheated to boiling and kept boiling in a water bath, with the actual temperature approximately 95℃) was poured into the ultrasonic instrument. The conical flask containing the powder and 70% ethanol was heated to the corresponding temperature in a water bath, and ultrasonicated for 30 min, 45 min, and 60 min respectively. After cooling, the weight was made up with 70% ethanol, filtered, and the filtrate was filtered through a 0.45 μm filter membrane for detection and analysis. The detection and analysis methods were carried out according to Examples 4 and 5, with ferulic acid and diosgenin as evaluation indicators. The results are shown in Table 1.

[0029] Table 1. Investigation of the effect of ultrasonic time on water bath temperature

[0030]

[0031] The table above shows that, under the same 45-minute sonication period, the extraction yield of diosgenin continuously increases with increasing water bath temperature; at the same temperature, the longer the sonication time, the greater the extraction yield of diosgenin. With increasing water bath temperature, under the same 45-minute sonication period, the ferulic acid content first increases and then decreases, indicating that excessively high water bath temperatures affect the extraction efficiency of ferulic acid. Furthermore, with increasing water bath temperature, the sonication time tends to decrease.

[0032] In general, water bath ultrasonic extraction within the range of 60℃-95℃ for 30-60 minutes effectively extracts ferulic acid and diosgenin. Further optimization of water bath ultrasonic extraction within the range of 70℃-95℃ for 30-60 minutes yields even more effective extraction of ferulic acid and diosgenin. Considering that higher water bath temperatures and longer ultrasonic times may lead to a loss of ferulic acid content, extraction is initially preferred at 80℃ for 30 minutes.

[0033] Since coumaric acid, gallic acid, and ferulic acid are all phenolic acids, they may exhibit similar trends to ferulic acid in their changes with temperature and ultrasound time. Similarly, lily glycoside B and diosgenin are both saponins and may have similar effects.

[0034] This invention further investigated the water bath ultrasonication temperature for p-coumaric acid and gallic acid, ultrasonicating for 30 minutes at three different water bath temperatures: 90℃, 80℃, and 60℃. It was found that the measured amounts of p-coumaric acid and gallic acid at water bath ultrasonication temperatures of 80℃ and 60℃ were higher than those at 90℃, while the measured amounts at 80℃ and 60℃ showed no significant difference. Furthermore, the extraction rate of lily glycoside B was highest at 90℃ for 30 minutes, followed by 80℃.

[0035] Therefore, considering the effective extraction of each indicator component, the extraction of the above 5 indicator components was carried out by ultrasonication in an 80℃ water bath for 30 minutes.

[0036] Comparative Example 3:

[0037] Specific method: Take 10g of lily powder, place it in an Erlenmeyer flask, add 50mL of 70% ethanol, weigh it, soak it for 1 hour, perform ultrasonic extraction in a water bath at normal water temperature, after ultrasonication for 45 minutes, take it out and cool it, make up the weight with 70% ethanol, filter it, and take the filtrate to pass through a 0.45μm filter membrane for detection and analysis.

[0038] The results of the analysis were: ferulic acid 0.0706%; diosgenin 0.00139%. This indicates that water bath ultrasonic extraction within the range of 70℃-95℃ can effectively extract phenolic acid components such as ferulic acid and saponin components such as diosgenin. In particular, the extraction temperature has a significant impact on the extraction of saponin components such as diosgenin.

[0039] Example 4: HPLC method for simultaneous detection of multiple indicator components in the traditional Chinese medicine lily.

[0040] 1.1 Instruments and reagents: Shimadzu-20A high performance liquid chromatograph (UV detector), Mettler electronic analytical balance (1 / 100,000), ultrasonic cleaner; the purity of gallic acid, p-coumaric acid, ferulic acid, lily glycoside B, and diosgenin reference standards was not less than 98%, meeting the quantitative requirements; three batches of lily slices were purchased from different pharmacies; other reagents were commonly used in the laboratory.

[0041] 1.2 Chromatographic conditions: An XDB C18 column (Agilent, Zorbax Eclipse, 250 mm × 4.6 mm, 5 μm) was used. The mobile phase was acetonitrile (A) - 0.2% glacial acetic acid (B), with gradient elution: 0 min - 12 min, 8% - 15% A; 12 min - 20 min, 15% - 30% A; 20 min - 42 min, 30% - 60% A; 42 min - 50 min, 60% - 8% A. The flow rate was 1.0 mL / min, the column temperature was 35℃, the detection wavelength was 300 nm, the injection volume was 20 μL, and the theoretical plate number calculated based on the ferulic acid peak should be no less than 3000.

[0042] 1.3 Preparation of test solution: Take 10g of lily powder, place it in an Erlenmeyer flask, add 50mL of 70% ethanol, weigh, soak for 1h, extract by ultrasonication in an 80℃ water bath for 30min, remove and cool, make up the weight with 70% ethanol, filter, and take the filtrate through a 0.45μm filter membrane as the test solution for detection and analysis.

[0043] 1.4 Preparation of reference solutions: Gallic acid, p-coumaric acid, ferulic acid, diosgenin B, and diosgenin reference stock solutions were prepared separately with methanol. These were then diluted with 70% ethanol to prepare reference standard positioning solutions and mixed reference solutions. The concentrations of gallic acid, p-coumaric acid, ferulic acid, diosgenin B, and diosgenin in the mixed reference solution were 0.00501 mg / ml, 0.00505 mg / ml, 0.5003 mg / ml, 0.0494 mg / ml, and 0.0499 mg / ml, respectively.

[0044] 1.5 Specificity Examination: such as Figure 1 As shown, based on the elution times of each reference standard in the reference standard positioning solution, the elution times of gallic acid, p-coumaric acid, ferulic acid, and limonene glycoside B in the mixed reference standard solution were 7.550 min, 9.312 min, 10.019 min, and 18.997 min, respectively. There were no factors in the test solution that would affect the target peaks of gallic acid, p-coumaric acid, ferulic acid, and limonene glycoside B, nor were there any cases where the resolution between adjacent peaks was less than 1.5, indicating that the method has good specificity.

[0045] 1.6 Linearity Assessment: Accurately measure 0.4, 0.5, 0.6, 1, 2, and 10 mL of the mixed reference solution into 10 mL volumetric flasks, add 70% ethanol to the mark, and mix well. Accurately inject 20 μL of each solution into the liquid chromatograph. Measure the peak area. Plot a calibration curve with the concentration of the reference standard (μg / mL) as the x-axis and the peak area as the y-axis. The regression equation, correlation coefficient, and linear range are shown in Table 2. The table shows that gallic acid, p-coumaric acid, ferulic acid, and limonene glycoside B exhibit good linearity within their respective concentration ranges.

[0046] Table 2. Results of linear relationship investigation of four index components in lily medicinal material

[0047] gallic acid y = 4.92899x - 36.12023 r＝0.99973 0.2ug / ml-5ug / ml p-coumaric acid y = 15.15486x - 33.25604 r＝0.99998 20ug / ml-500ug / ml ferulic acid y = 7.55867x - 13.25564 r＝0.99993 2ug / ml-50ug / ml Wang Baihe Glycoside B y = 7.65864x - 50.56001 r＝0.99992 2ug / ml-50ug / ml

[0048] 1.7 Precision Test

[0049] The same test solution was injected six times, 20 μL each time. The RSDs of the peak areas of gallic acid, p-coumaric acid, ferulic acid, and lily glycoside B were 0.89%, 0.56%, 0.77%, and 1.02%, respectively, which met the requirements of the precision test, indicating that the instrument has good precision.

[0050] 1.8 Repeatability Test

[0051] Take an appropriate amount of lily medicinal material, crush it, and prepare 6 test solutions repeatedly according to the method under "1.3". Measure each solution, record the peak area of ​​the chromatogram, and calculate the contents of gallic acid, p-coumaric acid, ferulic acid, and lily glycoside B. The RSDs are 1.50%, 1.22%, 1.38%, and 1.75%, respectively, indicating that the method of the present invention has good repeatability.

[0052] 1.9 Stability Test

[0053] The same test solution was taken and measured at 0h, 2h, 4h, 8h, 12h and 24h respectively. The RSD of the peak area of ​​gallic acid, p-coumaric acid, ferulic acid and lily glycoside B were 0.10%, 0.78%, 0.92% and 1.03% respectively, indicating that the four index components in the sample were stable within 24 hours.

[0054] 2.0 Recovery Test

[0055] Weigh 1g of each lily sample powder with known content, accurately add gallic acid, p-coumaric acid, ferulic acid, and limonin B reference solutions, prepare the test solution according to the method in section "1.3", add 5mL of 70% ethanol, perform subsequent operations, and determine according to the chromatographic conditions in section "1.2". The calculated recoveries of gallic acid, p-coumaric acid, ferulic acid, and limonin B were 97.32%, 97.61%, 98.02%, and 96.38%, respectively, with RSDs of 1.58% (n=6), 1.74% (n=6), 2.10% (n=6), and 2.35% (n=6), respectively, indicating good recovery rates.

[0056] 2.1 Durability Test

[0057] For the chromatographic conditions under section 1.2, the test solution was analyzed using column temperature ±5℃ (i.e., 30℃, 40℃), detection wavelength ±1nm (i.e., 299nm, 301nm, 209nm, 211nm), and different types of chromatographic columns (Kromasil C18 (4.6mm×250mm×5μm)). Within the specified range, the above three conditions did not affect the effective determination of the five index components in lily medicinal materials: gallic acid, p-coumaric acid, ferulic acid, lily glycoside B, and diosgenin. The resolution between the five target peaks and adjacent impurities was greater than 1.5, indicating good method robustness.

[0058] 2.2 Sample Determination

[0059] Three portions of lily medicinal material from different batches were accurately weighed, pulverized (approximately 10g per portion), and test solutions were prepared according to the method described in section "1.3". Chromatographic determination was performed under the conditions described in section "1.2". The resolution of gallic acid, p-coumaric acid, ferulic acid, and limonin B from adjacent impurity peaks was greater than 1.5. The contents of gallic acid, p-coumaric acid, ferulic acid, and limonin B in the samples were calculated using the external standard method. The results are shown in Table 3.

[0060] Table 3. Results of content determination of four indicator components in lily medicinal materials

[0061] 202203 0.000562％ 0.000435％ 0.1002％ 0.0026％ 202111-1 0.000533％ 0.000406％ 0.0997％ 0.0024％ 202202-1 0.000538％ 0.000411％ 0.0998％ 0.0024％

[0062] Example 5: Study on the detection of diosgenin

[0063] The sample processing method and chromatographic conditions are the same as in Example 4. The detection wavelength is set to 210 nm (the Shimadzu HPLC-UV used in this invention has dual-wavelength detection function, and the HPLC-DAD detector has the function of simultaneous detection of dual wavelengths. Other brands of HPLC-UV may also have dual-wavelength detection function). The test solution and diosgenin reference solution are injected separately for analysis.

[0064] The results are as follows Figure 2 As shown, the elution time of diosgenin was 37.525 min. In the chromatogram of the test solution at a wavelength of 210 nm, the peak shape of diosgenin was good, no obvious terminal absorption was observed, and the resolution with adjacent impurity peaks was greater than 1.5.

[0065] Method validation was performed by preparing a series of reference solutions using diosgenin reference standard stock solutions. Diosgenin showed linearity in the range of 2 μg / ml to 50 μg / ml, with the linear equation being y = 6.85245x - 23.45844 and R = 0.99993. Diosgenin reference solutions remained stable at room temperature for 24 hours. The recovery rate (95.7%, RSD = 1.59%, n = 6), precision, and repeatability tests met the requirements for method validation in the Chinese Pharmacopoeia.

[0066] Under a wavelength of 210 nm, the diosgenin content in the three batches of test solutions described in 2.1 was 0.00603%, 0.00584%, and 0.00591%, respectively. Methodological verification showed that the content of diosgenin in multiple batches of lily medicinal materials could be determined.

[0067] This study employed the same sample processing method and the same liquid chromatography method to achieve the beneficial effect of simultaneously detecting five index components—ferulic acid, p-coumaric acid, gallic acid, diosgenin, and diosgenin B—at dual wavelengths of 210 nm and 300 nm. Specifically, only one sample processing and injection of the sample solution were required, enabling the simultaneous determination of these five index components (ferulic acid, p-coumaric acid, gallic acid, diosgenin B, and diosgenin at 210 nm) at dual wavelengths of 210 nm and 300 nm. This method can be applied to the quality control of lily medicinal materials production. This method can be used in liquid chromatographs coupled with a DAD detector, as well as in liquid chromatographs coupled with a UV detector with dual-wavelength detection capabilities, demonstrating good versatility. Another beneficial effect of this invention is that using glacial acetic acid, which is less corrosive and volatile, as the mobile phase causes less damage to the chromatographic column compared to highly corrosive phosphoric acid (which is less volatile and more damaging to the column) and formic acid, effectively extending the column's lifespan.

[0068] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Obviously, other related modifications can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. An HPLC method for simultaneously determining five indicator components in the traditional Chinese medicine lily, characterized in that: Five index components in lily, namely gallic acid, p-coumaric acid, ferulic acid, lily glycoside B and diosgenin, were measured simultaneously using dual wavelengths with the same sample pretreatment and single injection. Chromatographic conditions: C18 column; mobile phase: acetonitrile A - 0.2% glacial acetic acid B, gradient elution: 0 min - 12 min, 8% - 15% A; 12 min - 20 min, 15% - 30% A; 20 min - 42 min, 30% - 60% A; 42 min - 50 min, 60% - 8% A; return to the initial mobile phase of 8% A at 50 min. Detection wavelengths: Gallic acid, p-coumaric acid, ferulic acid and diosgenin B were detected at 300 nm ± 1 nm, and diosgenin was detected at 210 nm ± 1 nm; the separation degree between the five index components and adjacent impurities was greater than 1.

5. The flow rate was 1.0 mL / min, and the column temperature was 30℃-40℃; a UV detector or a diode array detector was used. The preparation method of the test solution is as follows: Take 10 g of lily powder, place it in a container, add 50 mL of 70% ethanol, weigh it, soak it for 1 h, extract it by ultrasonication in an 80℃ water bath for 30 min, take it out and cool it, make up the weight with 70% ethanol, filter it, and take the filtrate for detection and analysis. The preparation method of the reference solutions is as follows: Gallic acid, p-coumaric acid, ferulic acid, limonin B, and diosgenin reference stock solutions are prepared separately with methanol, and diluted with 70% ethanol to prepare reference standard positioning solutions and mixed reference solutions. The concentrations of gallic acid, p-coumaric acid, ferulic acid, limonin B, and diosgenin in the mixed reference solution are 0.00501 mg / ml, 0.00505 mg / ml, 0.5003 mg / ml, 0.0494 mg / ml, and 0.0499 mg / ml, respectively. Inject the reference solution and the test solution into the sample for analysis. Complete the determination within 50 minutes. Calculate the contents of gallic acid, p-coumaric acid, ferulic acid, lily glycoside B, and diosgenin in lily medicinal materials using the external standard method.

2. The method according to claim 1, characterized in that: The column temperature was 35℃ and the injection volume was 20ul.

3. The method according to claim 1, characterized in that: The detection wavelength for gallic acid, p-coumaric acid, ferulic acid, and diosgenin B was 300 nm, and the detection wavelength for diosgenin was 210 nm.

4. The method according to claim 1, characterized in that: The chromatographic column model is XDB C18 or Kromasil C18.

5. The method according to claim 3, characterized in that: The chromatographic column model is XDB C18.

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