Method for simultaneously determining contents of six polyphenol compounds in fructus alpiniae oxyphyllae shell based on UPLC-QQQ-MS / MS technology

Abstract

The invention provides a detection method for simultaneously determining the content of six polyphenol compounds in fructus alpiniae oxyphyllae shells based on a UPLC-QQQ-MS / MS technology, and belongs to the technical field of food detection and analysis. According to the detection method, the content of six substances including chrysin, poplatin, procyanidine B2, protocatechuic acid, rutin and epicatechin in the fructus alpiniae oxyphyllae shells is determined. The method provided by the invention is simple to operate and short in analysis time, is good in precision, repeatability and stability through methodology investigation, and has the characteristics of strong specificity, high sensitivity and the like. Quantification is completed, and meanwhile, compound ion pair and other information can be accurately given, so that the effect of component identification is achieved. The method can provide technical support for clinical application and quality control of alpinia oxyphylla shell polyphenol.

Description

Technical Field

[0001] The invention provides a method for simultaneously determining the contents of six polyphenol compounds in Alpinia oxyphylla shells based on UPLC-QQQ-MS / MS technology, belonging to the technical field of food detection and analysis. Background Art

[0002] Alpinia oxyphylla is the dried mature fruit of the plant Alpinia oxyphylla of the Zingiberaceae family. It has the effects of warming the spleen, stopping diarrhea, and controlling saliva, warming the kidneys, reducing urine, and consolidating semen. It is known as one of the "four major southern medicines" in my country, along with Areca nut, Amomum villosum, and Morinda officinalis. Its effective active ingredients mainly include polyphenols, volatile oils, diphenylheptane, sesquiterpenes, etc. In clinical practice, the Alpinia oxyphylla kernel is often used as a medicine after removing impurities and shells. However, the Alpinia oxyphylla shell accounts for about one-third of the weight of the Alpinia oxyphylla fruit and is often discarded as waste, resulting in a waste of Alpinia oxyphylla resources.

[0003] Studies have shown that polyphenol compounds have a variety of pharmacological activities, such as anti-inflammatory, antioxidant, and anti-cancer, and natural polyphenols extracted from plants are often added to food as antioxidants. The fruit of Alpinia oxyphylla is rich in polyphenols and is a rare source of natural antioxidants. According to the laws revealed by plant resource chemistry, different parts of the same plant often contain the same or similar chemical components. Existing research mainly focuses on the chemical composition and pharmacological activity of Alpinia oxyphylla kernels, and there is a lack of systematic research on the material basis of the efficacy of Alpinia oxyphylla shells, and there is no quality control research on polyphenols in Alpinia oxyphylla shells. Therefore, establishing a method for the determination of polyphenol content in Alpinia oxyphylla shells is of great significance to improving the quality control of Alpinia oxyphylla shells.

[0004] Ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry has the advantages of short analysis time, good separation, high sensitivity, etc., and has been widely used in food ingredient detection, drug content determination, pesticide residues, etc. In recent years, the present invention optimizes the detection method to establish a simple, efficient and accurate method, which can simultaneously determine 6 polyphenol compounds in Alpinia oxyphylla shells using UPLC-QQQ-MS / MS, better meeting the needs and development trends of detection and analysis work. Summary of the invention

[0005] In order to solve the above problems, the present invention adopts UPLC-QQQ-MS / MS technology to establish a method for simultaneously determining the contents of 6 polyphenolic compounds in Alpinia oxyphylla shell extract. The method has short detection time and high accuracy and sensitivity.

[0006] The present invention adopts the following technical solution:

[0007] (1) Preparation of mixed reference solution:

[0008] Weigh 10 mg of chrysin, chrysin, proanthocyanidin B2, protocatechuic acid, rutin, and epicatechin reference substances, add methanol to dissolve and dilute to a 10 mL volumetric flask, shake well, and obtain a 1 mg / mL single standard stock solution. Take an appropriate amount of the standard stock solution for dilution to obtain mixed reference solutions with concentrations of 1, 2, 10, 20, 50, 100, 200, 500, and 1000 ng / mL.

[0009] (2) Preparation of test solution:

[0010] Accurately weigh 10g of Alpinia oxyphylla shell powder, according to the solid-liquid ratio of 1:30, the volume fraction of the extraction solvent is 50% ethanol, the extraction temperature is 30℃, ultrasonic extraction is performed for 40min, and the above steps are repeated for 3 times. Centrifuge at 10,000rpm for 10min, and the supernatant is combined. Concentrate the supernatant to a certain volume, pass it through an AB-8 macroporous resin column, first remove macromolecules such as proteins with 10 column volumes of distilled water and 5 column volumes of 10% ethanol water, and then elute with 5 column volumes of 60% ethanol. After rotary evaporation of the eluate to a certain volume, freeze-dry it with a vacuum freeze dryer and store it in a desiccator for later use. Redissolve it with 60% methanol, centrifuge it at 10,000rpm for 10min, and take the supernatant as the test solution.

[0011] (3) Determination method:

[0012] UPLC-QQQ-MS / MS was used to determine the contents of six chemical components in the mixed standard and test solution:

[0013] Chromatographic conditions: The chromatographic column was a ZORBAX Eclipse Plus C18 column (Agilent) with a specification of 3×150 mm, 1.8 μm. The mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, the flow rate was 0.3 mL / min, the column temperature was 35°C, and gradient elution was performed. The elution program was: 0-3 min: 10-10% B, 3-12 min: 10-80% B, 12-15 min: 80-80% B, 15-17 min: 80-10% B, 17-21 min: 10-10% B.

[0014] The mass spectrometry conditions were as follows: ESI positive and negative scanning modes were used for detection, the scanning mode was multiple reaction monitoring mode (MRM), and the parameters were as follows: nitrogen was used as drying gas, collision gas, and nebulizer gas. The drying gas temperature was 290°C, the drying gas flow rate was 14 L / min, the sheath gas temperature was 250°C, the sheath gas flow rate was 11 L / min, the nebulizer gas pressure was 35 psi, and the capillary voltage was 5500 V (+), 4500 V (-).

[0015] The determination method in step 3 above includes the determination of linear relationship, detection limit and quantification limit: take 9 series of reference solution with different mass concentrations prepared in step 1. Detect under test conditions, record the chromatographic peak areas of 6 chemical components, draw a standard curve with the mass concentration of the reference as the horizontal axis (X) and the peak area as the vertical axis (Y), and perform linear regression. The mass concentration when the signal-to-noise ratio (S / N) is 3 is used as the detection limit (LOD), and the mass concentration when the signal-to-noise ratio (S / N) is 10 is used as the quantification limit (LOQ).

[0016] The determination method in step 3 above includes the determination of precision: accurately pipette an appropriate amount of mixed reference solution, configure it into three concentration gradients of low, medium and high, and continuously inject it 6 times on the same day under the same conditions to determine its intra-day precision; continuously measure for 3 days to examine its inter-day precision. Record the peak area of ​​each component to be measured and calculate the RSD.

[0017] The determination method in step 3 above includes repeatability determination: taking the same batch of Alpinia oxyphylla shell samples, preparing 6 samples in parallel according to the preparation method of the test solution, determining them under the test conditions, recording the peak areas of the 6 chemical components and calculating the RSD.

[0018] The determination method in the above step 3 includes the determination of stability: take the test solution, place it at room temperature, inject and analyze it according to the determination conditions at 0h, 2h, 4h, 8h, 12h, and 24h, determine the peak area and calculate the RSD.

[0019] The determination method in step 3 above includes the determination of sample recovery rate: accurately pipette an appropriate amount of Alpinia oxyphylla shell sample with a known content, add low, medium and high concentration mixed reference solution to 6 parallel portions, perform determination according to the determination conditions, and calculate the average recovery rate and RSD.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention is based on UPLC-QQQ-MS / MS technology to simultaneously determine the contents of 6 polyphenolic compounds in Alpinia oxyphylla shells. The method is simple, rapid, accurate, sensitive, and has a low detection limit. It is suitable for the qualitative and quantitative analysis of polyphenolic compounds in Alpinia oxyphylla shells and has good application prospects.

[0022] (2) Mass spectrometry has the characteristics of high sensitivity and high selectivity. It can accurately give the mass number of each component on the premise of accurately determining the component content, thereby achieving simultaneous identification of each component. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the chromatogram of the six compounds in the mixed standard and the Alpinia oxyphylla shell sample

[0024] Figure 2is the chemical structural formula diagram of the six components to be tested in the present invention, wherein

[0025] A: protocatechuic acid; B: proanthocyanidin B2; C: epicatechin; D: rutin; E: chrysin; F: chrysin. DETAILED DESCRIPTION

[0026] The above contents of the present invention are further described in detail below in conjunction with the accompanying drawings. However, the implementation of the present invention is not limited to the following embodiments.

[0027] Example 1: Method for simultaneously determining the contents of six polyphenolic compounds in Alpinia oxyphylla shells based on UPLC-QQQ-MS / MS technology:

[0028] 1. Materials, Reagents, and Instruments

[0029] 1.1 Materials: Alpinia oxyphylla (Hainan Danzhou Lvfuxian Agricultural Development Co., Ltd.), Alpinia oxyphylla kernels and Alpinia oxyphylla shells were separated to obtain Alpinia oxyphylla shells.

[0030] 1.2 Drug testing

[0031] Acetonitrile was of chromatography grade and purchased from Beijing Myrida Technology Co., Ltd.; formic acid was of chromatography grade and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; protocatechuic acid (purity ≥ 98%), proanthocyanidin B2 (purity ≥ 98%), epicatechin (purity ≥ 98%), rutin (purity ≥ 98%), chrysin (purity ≥ 98%), and jasminoides (purity ≥ 98%) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the remaining reagents were of analytical grade.

[0032] 1.3 Instruments

[0033] Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (G6495C, Agilent, USA); ultrasonic cleaning machine (XM-P10H, Xiaomei Ultrasonic Instrument Co., Ltd.); high-speed desktop centrifuge (H1850, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); rotary evaporator (RE-52AA, Shanghai Yarong Biochemical Instrument Factory); freeze dryer (SJIA-18N, Ningbo Shuangjia Instrument Co., Ltd.)

[0034] 2. Methods and Results

[0035] 2.1 Preparation of mixed reference solution

[0036] Weigh 10 mg of chrysin, chrysin, proanthocyanidin B2, protocatechuic acid, rutin, and epicatechin reference substances, add methanol to dissolve and dilute to a 10 mL volumetric flask, shake well, and obtain a 1 mg / mL single standard stock solution. Take an appropriate amount of the standard stock solution for dilution to obtain mixed reference solutions with concentrations of 1, 2, 10, 20, 50, 100, 200, 500, and 1000 ng / mL.

[0037] 2.2 Preparation of test solution

[0038] Accurately weigh 10g of Alpinia oxyphylla shell powder, according to the solid-liquid ratio of 1:30, the volume fraction of the extraction solvent is 50% ethanol, the extraction temperature is 30℃, ultrasonic extraction is performed for 40min, and the above steps are repeated for 3 times. Centrifuge at 10,000rpm for 10min, and the supernatant is combined. Concentrate the supernatant to a certain volume, pass it through an AB-8 macroporous resin column, first remove macromolecules such as proteins with 10 column volumes of distilled water and 5 column volumes of 10% ethanol water, and then elute with 5 column volumes of 60% ethanol. After rotary evaporation of the eluate to a certain volume, freeze-dry it with a vacuum freeze dryer and store it in a desiccator for later use. Redissolve it with 60% methanol, centrifuge it at 10,000rpm for 10min, and take the supernatant as the test solution.

[0039] 2.3 Determination conditions

[0040] 2.3.1 Chromatographic conditions: The chromatographic column model is ZORBAX Eclipse Plus C18 column (Agilent), the specification is 3×150 mm, 1.8 μm, the mobile phase A is 0.1% formic acid water, the mobile phase B is acetonitrile, the flow rate is 0.3 mL / min, the column temperature is 35°C, and gradient elution is performed. The gradient elution program is shown in Table 1 below. The chromatograms of the six compounds in the mixed standard and the Alpinia oxyphylla shell sample are shown in Figure 1 .

[0041] Table 1 Gradient elution program

[0042]

[0043] 2.3.2 Mass spectrometry conditions: ESI positive and negative scanning mode was used for detection, scanning mode: multiple reaction monitoring mode (MRM), parameters are as follows: nitrogen as drying gas, collision gas, nebulizer gas. Drying gas temperature 290℃, drying gas flow rate 14L / min, sheath gas temperature 250℃, sheath gas flow rate 11L / min, nebulizer gas pressure 35psi, capillary voltage: 5500V(+), 4500V(-). The mass spectrometry optimization parameters of each component to be tested are shown in Table 2.

[0044] Table 2 Mass spectrometry optimization parameters for each component to be analyzed

[0045]

[0046]

[0047] 2.4 Methodological Review

[0048] 2.4.1 Investigation of linear relationship and limit of detection and limit of quantification: Take an appropriate amount of standard stock solution and dilute it to obtain mixed reference solution with concentrations of 1, 2, 10, 20, 50, 100, 200, 500, and 1000 ng / mL. Use the above-mentioned method "2.3" to detect, record the chromatographic peak areas of the 6 chemical components, use the mass concentration of the reference substance as the horizontal axis (X) and the peak area as the vertical axis (Y), draw a standard curve, and perform linear regression. The mass concentration when the signal-to-noise ratio (S / N) is 3 is used as the limit of detection (LOD), and the mass concentration when the signal-to-noise ratio (S / N) is 10 is used as the limit of quantification (LOQ). The results are shown in Table 3, which shows that the linear relationship of each component is good in the range of 1-1000 ng / mL.

[0049] Table 3 Linear equations, detection limits and quantification limits of each component to be tested

[0050]

[0051] 2.4.2 Precision investigation: Accurately pipette the mixed reference solution and prepare it into three concentration gradients of low, medium and high. Under the same conditions, inject the sample 6 times continuously on the same day to determine its intra-day precision; measure it continuously for 3 days to investigate its inter-day precision. Record the peak area of ​​each component to be measured and calculate the RSD. The results are shown in Table 4. The RSD is less than 5%, indicating that the instrument has good precision.

[0052] Table 4 Precision test results

[0053]

[0054] 2.4.3 Repeatability test

[0055] Take the same batch of Alpinia oxyphylla shell samples and inject them 6 times continuously according to the conditions in item "2.3". Record the peak areas of the 6 chemical components and calculate the RSD. The results are shown in Table 5, indicating that the method has good repeatability.

[0056] 2.4.4 Stability test

[0057] Take the test solution, place it at room temperature, and analyze it according to the conditions in "2.3" at 0h, 2h, 4h, 8h, 12h, and 24h, measure the peak area and calculate the RSD. The results are shown in Table 5, indicating that the test solution is stable within 24h.

[0058] 2.4.5 Sample recovery test

[0059] Accurately pipette an appropriate amount of Alpinia oxyphylla shell sample with a known content, add low, medium and high concentration mixed reference solution to 6 parallel samples, and perform the determination according to the conditions under "2.3", and calculate the average recovery rate and RSD. The results are shown in Table 5.

[0060] Table 5 Stability, repeatability and spike recovery test results

[0061]

[0062] 2.4.6 Content determination

[0063] Accurately weigh the Alpinia oxyphylla shell sample, prepare the test solution according to the method in "2.2", and then measure it. Calculate the contents of the six components in the sample according to the external standard method. The results are shown in Table 6.

[0064] Table 6 Sample content

[0065]

[0066] 3. Discussion

[0067] The UPLC-QQQ-MS / MS method was used to analyze the contents of six chemical components in Alpinia oxyphylla shell. Taking into account the structural and polar characteristics of different components, the MRM monitoring mode was used for simultaneous detection in positive and negative ion modes. Each component had a good response in different modes. The mobile phase was optimized to achieve effective separation of the components. Finally, acetonitrile-water was used as the mobile phase system for gradient elution. The six chemical components could be effectively separated and the peak shape was good. This mobile phase system improved the experimental efficiency, saved reagents, and had practical economic significance.

[0068] 4. Conclusion

[0069] The present invention establishes and optimizes a rapid, efficient and sensitive UPLC-QQQ-MS / MS system MRM mode qualitative and quantitative method for polyphenol compounds in Alpinia oxyphylla shells. The present invention successfully achieves effective separation and precise quantification of 6 polyphenol compounds in Alpinia oxyphylla shells within 21 minutes, and the results are accurate, highly sensitive and stable, which can provide an experimental basis for the subsequent quality control of polyphenol compounds in Alpinia oxyphylla shells.

Claims

1. A method for simultaneously determining the contents of six polyphenolic compounds in Alpinia oxyphylla shells using UPLC-QQQ-MS / MS, characterized in that The following steps are involved: (1) Preparation of mixed reference solution: Weigh 10 mg of chrysin, chrysin, proanthocyanidin B2, protocatechuic acid, rutin, and epicatechin reference substances, add methanol to dissolve and dilute to a 10 mL volumetric flask, shake well, and obtain a 1 mg / mL single standard stock solution. Take an appropriate amount of the standard stock solution for dilution to obtain mixed reference solutions with concentrations of 1, 2, 10, 20, 50, 100, 200, 500, and 1000 ng / mL. (2) Preparation of test solution: Accurately weigh 10g of Alpinia oxyphylla shell powder, according to the solid-liquid ratio of 1:30, the volume fraction of the extraction solvent is 50% ethanol, the extraction temperature is 30℃, ultrasonic extraction is performed for 40min, and the above steps are repeated for 3 times. Centrifuge at 10,000rpm for 10min, and the supernatant is combined. Concentrate the supernatant to a certain volume, pass it through an AB-8 macroporous resin column, first remove macromolecules such as proteins with 10 column volumes of distilled water and 5 column volumes of 10% ethanol water, and then elute with 5 column volumes of 60% ethanol. After rotary evaporation of the eluate to a certain volume, freeze-dry it with a vacuum freeze dryer and store it in a desiccator for later use. Redissolve it with 60% methanol, centrifuge it at 10,000rpm for 10min, and take the supernatant as the test solution. (3) Determination method: UPLC-QQQ-MS / MS was used to determine the contents of six chemical components in the mixed standard and test solution: Chromatographic conditions: The chromatographic column was a ZORBAX Eclipse Plus C18 column (Agilent) with a specification of 3×150 mm, 1.8 μm. The mobile phase A was 0.1% formic acid in water, the mobile phase B was acetonitrile, the flow rate was 0.3 mL / min, the column temperature was 35°C, and gradient elution was performed. The elution program was: 0-3 min: 10-10% B, 3-12 min: 10-80% B, 12-15 min: 80-80% B, 15-17 min: 80-10% B, 17-21 min: 10-10% B. The mass spectrometry conditions were as follows: ESI positive and negative scanning modes were used for detection, the scanning mode was multiple reaction monitoring mode (MRM), and the parameters were as follows: nitrogen was used as drying gas, collision gas, and nebulizer gas. The drying gas temperature was 290°C, the drying gas flow rate was 14 L / min, the sheath gas temperature was 250°C, the sheath gas flow rate was 11 L / min, the nebulizer gas pressure was 35 psi, and the capillary voltage was 5500 V (+), 4500 V (-).

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