A method for quality detection of areca processing by-products

Abstract

The application discloses a quality detection method for betel nut processing byproducts, and comprises qualitative analysis of chemical components in the betel nut processing byproducts; the qualitative analysis comprises the following steps: using a methanol solution containing an internal standard to extract the betel nut processing byproducts, centrifuging, and collecting supernatant; using a UPLC-Q-Exactive Orbitrap-MS / MS method to qualitatively analyze the chemical components in the supernatant; the UPLC-Q-Exactive Orbitrap-MS / MS analysis method has the advantages of high resolution, good mass accuracy, strong qualitative capacity and the like, and can widely and accurately detect the chemical components in complex traditional Chinese medicine compounds.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a method for quality detection of areca nut processing by-products. Background Technology

[0002] Areca nut is the fruit of the palm family, Areca palm. Areca catechu The dried, mature seeds of L. are a traditional medicinal resource in southern China, possessing properties such as anthelmintic, digestive, qi-regulating, and diuretic effects. They have high application value in the development of traditional Chinese medicine and related products. my country's areca nut resources are mainly distributed in Hainan, Yunnan, and Taiwan, with Hainan being one of the major areca nut producing areas, possessing a good foundation for raw material supply and favorable conditions for industrial development.

[0003] With the development of areca nut cultivation and primary processing industries, a certain amount of by-products are generated during areca nut processing, including areca nut processing wastewater, waste areca nut seeds, and areca nut stems. These by-products may still contain various plant-derived components such as alkaloids, polyphenols, flavonoids, organic acids, polysaccharides, fatty acids, terpenes, and steroids, which have the potential value for further development into medicinal, feed, functional plant resources, or other high-value products.

[0004] Currently, most areca nut processing byproducts are directly discarded, incinerated, discharged, or subjected to simple treatment in actual production, which not only wastes plant resources but may also increase environmental pressure. As the areca nut industry transforms from traditional primary processing to intensive processing, resource utilization, and full-industry chain development, how to effectively evaluate, screen, and control the quality of areca nut processing byproducts has become an important foundation for promoting their subsequent development and utilization.

[0005] Existing research and standards on areca nut quality control mainly focus on the raw areca nut or areca nut preparations themselves. The testing targets are mostly whole raw materials, processed slices, or finished preparations, often using the content determination of a few indicative components as the evaluation basis. However, areca nut processing by-products differ significantly from raw areca nut in terms of source, processing method, moisture content, matrix composition, and component distribution. Directly applying existing testing methods for raw areca nut or preparations cannot comprehensively and accurately reflect the quality characteristics of areca nut processing by-products.

[0006] Furthermore, the chemical composition and content of areca nut by-products from different sources, batches, and processing stages can vary significantly. Without a unified, stable, and repeatable quality testing method, it will be difficult to achieve quality grading, raw material screening, batch consistency evaluation, and subsequent product development control of areca nut processing by-products. This also hinders their standardized application in pharmaceutical, animal feed, or functional products.

[0007] In the existing technology, the quality detection of areca nut processing by-products still has the following shortcomings: First, the detection objects are mostly concentrated on areca nut raw materials or finished preparations, and there are few specific detection methods for processing by-products; Second, the detection indicators are relatively simple and it is difficult to comprehensively reflect the overall quality characteristics of multiple active ingredients in the by-products; Third, some detection methods have complex pretreatment steps and insufficient repeatability, making it difficult to meet the needs of rapid detection of large batches of samples; Fourth, there is a lack of systematic detection methods that can be used for quality comparison, stability evaluation and standardized control of different batches of by-products.

[0008] Therefore, it is necessary to establish a quality testing method applicable to areca nut processing by-products. Through reasonable sample pretreatment, characteristic component detection, content determination or fingerprint analysis, etc., the quality of areca nut processing by-products can be evaluated stably, accurately and repeatedly, providing technical support for the resource utilization, product development and quality standard establishment of areca nut by-products. Summary of the Invention

[0009] The purpose of this invention is to provide a method for quality testing of areca nut processing by-products. This method can perform extensive and accurate testing of the chemical composition of complex areca nut by-products.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0011] The first aspect of the present invention provides a method for quality detection of areca nut processing by-products, the method comprising qualitative analysis of chemical components in areca nut processing by-products;

[0012] The qualitative analysis includes the following steps:

[0013] (a) The areca nut processing byproducts were extracted and centrifuged using a methanol solution containing an internal standard, and the supernatant was collected;

[0014] (b) The chemical components in the supernatant were qualitatively analyzed by UPLC-Q-Exactive Orbitrap-MS / MS. In step (b), the chromatographic conditions of UPLC-Q-Exactive Orbitrap-MS / MS were as follows: ACQUITY UHPLCHSS T3 column; column temperature 35℃; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution; injection volume was 10 μL; flow rate was 0.3 mL / min.

[0015] Preferably, in step (b), the gradient elution program is as follows: from 0 to 10 min, mobile phase B increases from 0% to 30%; from 10 to 25 min, mobile phase B increases from 30% to 40%; from 25 to 30 min, mobile phase B increases from 40% to 50%; from 30 to 40 min, mobile phase B increases from 50% to 70%; from 40 to 45 min, mobile phase B increases from 70% to 100%; from 45 to 60 min, mobile phase B is maintained at 100%; from 60 to 60.5 min, mobile phase B decreases from 100% to 0%; from 60.5 to 70 min, mobile phase B is maintained at 0%.

[0016] Preferably, in step (b), the mass spectrometry conditions in the UPLC-Q-Exactive Orbitrap-MS / MS method are as follows:

[0017] Full MS-dd-MS2 mode was used for acquisition, with simultaneous scanning of positive and negative ions; the scanning range was [missing information]. m / z 100-1200; MS1 resolution is 70000, MS2 resolution is 17500; spray voltage is 3.2kV; capillary temperature is 320℃; auxiliary gas heating temperature is 350℃; sheath flow rate is 40L / min, auxiliary flow rate is 15L / min; AGC target is 1×10 6 TopN 5; MS2 uses stepped collision energies of 30, 40, and 50.

[0018] Preferably, in step (a), the internal standard content in the methanol solution containing the internal standard is 5 μg / mL, and the methanol solution concentration is 80%; the internal standard is nicodipine.

[0019] Preferably, the quality detection method further includes quantitative analysis of arecoline, norarecoline, norarecoline and arecoline in areca nut processing byproducts:

[0020] The quantitative analysis includes the following steps:

[0021] (1) Extract the areca nut processing byproducts with methanol solution, centrifuge, and collect the supernatant;

[0022] (2) The contents of arecoline, norarecoline, norarecoside and arecoside in the supernatant were determined by liquid chromatography; the liquid chromatography conditions are as follows:

[0023] An SCX column was used as the packing material, with strong cation exchange bonded silica gel as the mobile phase, and acetonitrile-0.01 mol / L ammonium dihydrogen phosphate solution as the mobile phase, wherein the volume ratio of acetonitrile to ammonium dihydrogen phosphate solution was 35:65. An ultraviolet detector was used with a detection wavelength of 215 nm. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the injection volume was 10 μL.

[0024] Preferably, in step (1), the concentration of the methanol solution is 70%.

[0025] Preferably, in step (2), the pH value of the ammonium dihydrogen phosphate solution is 2.2.

[0026] Preferably, the areca nut processing byproducts include areca nut processing wastewater, areca nut stems, and areca nut seeds.

[0027] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0028] This invention employs UPLC-Q-Exactive Orbitrap-MS / MS to qualitatively analyze the chemical components of areca nut processing byproducts, including areca nut processing wastewater, areca nuts, and areca nut calyxes. High-performance liquid chromatography (HPLC) is then used to determine the content of arecoline, norarecoline, norarecoside, and arecoside in these byproducts. The UPLC-Q-Exactive Orbitrap-MS / MS method offers advantages such as high resolution, good quality accuracy, and strong qualitative capabilities, enabling the broad and accurate detection of chemical components in complex traditional Chinese medicine formulas. HPLC provides highly accurate, sensitive, and reproducible results; it is simple to operate, has passed method validation, exhibits good linearity, and meets requirements for precision, stability, repeatability, and recovery. This combination of qualitative and quantitative analysis lays the foundation for establishing quality standards for areca nut processing byproducts. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0030] Figure 1 The total ion chromatograms of areca nut processing wastewater in positive and negative ion modes, and areca nut seeds and stems in positive and negative ion modes in Example 1 of the present invention;

[0031] Figure 2 The chromatograms are of the blank solution, reference solution, and test solution in Example 2 of this invention;

[0032] Figure 3 This is the standard curve of norarecoline, arecoline, norarecoline, and arecoline reference standards in Example 3 of the present invention. Detailed Implementation

[0033] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0034] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0035] This invention provides a method for quality detection of areca nut processing by-products, the method including qualitative analysis of chemical components in areca nut processing by-products;

[0036] The qualitative analysis includes the following steps:

[0037] (a) The areca nut processing byproducts were extracted and centrifuged using a methanol solution containing an internal standard, and the supernatant was collected;

[0038] (b) The chemical components in the supernatant were qualitatively analyzed by UPLC-Q-Exactive Orbitrap-MS / MS. In step (b), the chromatographic conditions of UPLC-Q-Exactive Orbitrap-MS / MS were as follows: ACQUITY UHPLCHSS T3 column; column temperature 35℃; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution; injection volume was 10 μL; flow rate was 0.3 mL / min.

[0039] In one embodiment, in step (b), the gradient elution program is as follows: from 0 to 10 min, mobile phase B increases from 0% to 30%; from 10 to 25 min, mobile phase B increases from 30% to 40%; from 25 to 30 min, mobile phase B increases from 40% to 50%; from 30 to 40 min, mobile phase B increases from 50% to 70%; from 40 to 45 min, mobile phase B increases from 70% to 100%; from 45 to 60 min, mobile phase B is maintained at 100%; from 60 to 60.5 min, mobile phase B decreases from 100% to 0%; from 60.5 to 70 min, mobile phase B is maintained at 0%.

[0040] In one embodiment, the mass spectrometry conditions in step (b) of the UPLC-Q-Exactive Orbitrap-MS / MS method are as follows:

[0041] Full MS-dd-MS2 mode was used for acquisition, with simultaneous scanning of positive and negative ions; the scanning range was [missing information]. m / z 100-1200; MS1 resolution is 70000, MS2 resolution is 17500; spray voltage is 3.2kV; capillary temperature is 320℃; auxiliary gas heating temperature is 350℃; sheath flow rate is 40L / min, auxiliary flow rate is 15L / min; AGC target is 1×10 6 TopN 5; MS2 uses stepped collision energies of 30, 40, and 50.

[0042] In one embodiment, in step (a), the methanol solution containing the internal standard has an internal standard content of 5 μg / mL and a methanol solution concentration of 80%; the internal standard is nicodipine.

[0043] In one embodiment, the quality detection method further includes quantitative analysis of arecoline, norarecoline, norarecoside, and arecoside in areca nut processing byproducts:

[0044] The quantitative analysis includes the following steps:

[0045] (1) Extract the areca nut processing byproducts with methanol solution, centrifuge, and collect the supernatant;

[0046] (2) The contents of arecoline, norarecoline, norarecoside and arecoside in the supernatant were determined by liquid chromatography; the liquid chromatography conditions are as follows:

[0047] An SCX column was used as the packing material, with strong cation exchange bonded silica gel as the mobile phase, and acetonitrile-0.01 mol / L ammonium dihydrogen phosphate solution as the mobile phase, wherein the volume ratio of acetonitrile to ammonium dihydrogen phosphate solution was 35:65. An ultraviolet detector was used with a detection wavelength of 215 nm. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the injection volume was 10 μL.

[0048] In one embodiment, in step (1), the concentration of the methanol solution is 70%.

[0049] In one embodiment, in step (2), the pH value of the ammonium dihydrogen phosphate solution is 2.2.

[0050] In one embodiment, the areca nut processing byproducts include areca nut processing wastewater, areca nut stems, and areca nut seeds.

[0051] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0052] Example 1

[0053] This embodiment is a quality detection method for areca nut processing by-products, the quality detection method including qualitative analysis of chemical components in areca nut processing by-products;

[0054] The qualitative analysis includes the following steps:

[0055] (a) Preparation of the test solution:

[0056] Wastewater from betel nut processing, waste betel nut seeds, and betel nut calyxes generated during the betel nut chewing process were added to an 80% methanol solution containing the internal standard nicodipine (5 μg / mL) to prepare a sample solution with a final concentration of 10 mg / mL. After vortexing and ultrasonic extraction for 10 min, the solution was centrifuged at 14000 rpm for 10 min. 0.8 mL of the supernatant was transferred to a new centrifuge tube, centrifuged again, and the supernatant was transferred to a sample vial to obtain the test solution.

[0057] (b) UPLC-Q-Exactive Orbitrap-MS / MS analysis:

[0058] The above-mentioned test solutions were analyzed using UPLC-Q-Exactive Orbitrap-MS / MS; the blank control samples were processed simultaneously using the same procedure.

[0059] Chromatographic and mass spectrometric conditions

[0060] The chromatographic column was an ACQUITY UHPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution were used as the mobile phases, respectively, at a flow rate of 0.3 mL / min, a column temperature of 35℃, and an injection volume of 10 μL. Gradient elution was performed, and the steps are shown in Table 1. Mass spectrometry data acquisition was performed using UPLC-Q-Exactive Orbitrap-MS / MS high-resolution mass spectrometry. The detection mode was Ful1MS-dd-MS2, with simultaneous scanning in both positive and negative ion modes. The scanning range was [not specified]. m / z 100~1200, MS1 and MS2 resolutions are 70000 and 17500 respectively, spray voltage is 3.2 kV, capillary temperature is 320℃, auxiliary gas heating temperature is 350℃, sheath gas and auxiliary gas flow rates are 40 L / min and 15 L / min respectively, and AGC target is 1×10 6 The TopN value is 5, and MS2 uses stepped collision energy with collision energies of 30, 40, and 50.

[0061] Table 1

[0062] .

[0063] UPLC-Q-Exactive Orbitrap-MS / MS was used to acquire mass spectrometry information of areca nut by-products in both positive and negative ion modes. The total ion chromatogram is shown below. Figure 1 ( Figure 1 The total ion chromatograms of areca nut processing wastewater under positive (A) and negative (B) ion modes, and areca nut waste seeds and stems under positive (C) and negative (D) ion modes; based on the mzCloud online database, the self-built mzVault Traditional Chinese Medicine Natural Products Database, and Xcalibur qualitative software, combined with the molecular ions and secondary fragment ions of the compounds, a total of 19 compounds of areca nut by-products were identified, including 5 flavonoids, 2 alkaloids, 2 sugars, 1 phenol, 1 amino acid, 1 coumarin, and 7 ketone / aldehyde / acid compounds (see Table 2).

[0064] Table 2 Chemical composition information of areca nut by-products

[0065] .

[0066] Example 2

[0067] This embodiment is a quality detection method for arecaine, norarecaine, norarecaine and arecaine in arecaine processing by-products.

[0068] The quantitative analysis includes the following steps:

[0069] (1) Preparation of test solution, blank solution, and reference solution:

[0070] Take an appropriate amount of areca processing wastewater, about 5 mL, or an appropriate amount of waste areca seeds / areca stems, about 0.2 g. Weigh them accurately and place them in a stoppered conical flask. Add 25 mL of 70% methanol accurately, stopper tightly, weigh, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Let cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and take the filtrate to obtain the test solution.

[0071] Blank solution: 70% methanol solution;

[0072] Reference solution: Take appropriate amounts of arecoline reference standard, arecoline reference standard, arecoline reference standard, and arecoline hydrobromide reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 170 μg of arecoline, 70 μg of arecoline, 20 μg of arecoline, and 70 μg of arecoline hydrobromide per 1 ml (arecoline weight = arecoline hydrobromide weight / 1.5214).

[0073] (2) The test solution, blank solution, and reference solution were analyzed by liquid chromatography; the liquid chromatography conditions were as follows:

[0074] An SCX column was used as the packing material, with strong cation exchange bonded silica gel as the mobile phase, and acetonitrile-0.01 mol / L ammonium dihydrogen phosphate solution as the mobile phase, wherein the volume ratio of acetonitrile to ammonium dihydrogen phosphate solution was 35:65. An ultraviolet detector was used with a detection wavelength of 215 nm. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the injection volume was 10 μL.

[0075] Liquid chromatogram as shown Figure 2 As shown, Figure 2 In the image, from top to bottom, are the chromatograms of the blank solution, the reference solution, and the test solution.

[0076] Example 3

[0077] This embodiment focuses on the study of linear range:

[0078] Solutions of arecoline, arecoline, arecoline, and arecoline hydrobromide were prepared separately and analyzed by liquid chromatography according to the method in Example 2. Linearity plots were generated with peak area as the ordinate (Y) and solution concentration (μg / mL) as the abscissa (X). The linear equations for each reference solution were obtained, and the results are shown in Table 3. Figure 3 This indicates that the linear relationship of each reference solution is good.

[0079] Table 3 Linearity and Range

[0080] .

[0081] Example 4

[0082] This example is a precision test:

[0083] The mixed reference solution was injected six times consecutively using the liquid chromatography method of Example 2. The RSD of the peak areas of norarecan, arecoline, norarecan, and arecoline in the mixed reference solution was less than 2% among the six results, indicating that the instrument precision of this method is good.

[0084] Example 5

[0085] This example is a stability test:

[0086] The mixed reference solution was analyzed by high performance liquid chromatography (HPLC) at 0h, 2h, 4h, 6h, 8h, 12h, and 24h after standing, according to the HPLC method in Example 2. The arecoline content and total arecoline content were stable within 24 hours, and the test solution showed good stability within 24 hours.

[0087] Example 6

[0088] Repeatability test in this embodiment

[0089] The mixed reference solution was analyzed using the liquid chromatography method in Example 2. The sample repeatability was good, and the determination method was accurate and reliable.

[0090] Example 7

[0091] This example is a sample recovery test:

[0092] Accurately transfer known stock solutions of norarecanine, arecoline, norarecanine, and arecoline to a certain volume of the mixed sample solution. Prepare nine parallel solutions and perform HPLC analysis according to the method in Example 2. The results are shown in Tables 4 and 5. The average recoveries of norarecanine, arecoline, norarecanine, and arecoline were 100.97%, 100.16%, 99.87%, and 99.91%, respectively, and the average recovery of total arecoline was 100.66%, indicating that the method of the present invention has good accuracy.

[0093] Table 4 Results of the sample recovery test

[0094]

[0095] Table 5 Results of the sample recovery test

[0096] .

[0097] Example 8

[0098] This example demonstrates content determination.

[0099] Ten parallel test solutions were prepared and analyzed by liquid chromatography according to Example 2. The results are shown in Table 6, and the total alkaloids were calculated to be 35.39 mg / g.

[0100] Table 6. Content determination results

[0101] .

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for quality testing of areca nut processing by-products, characterized in that, The quality testing method includes qualitative analysis of the chemical components in areca nut processing by-products; The qualitative analysis includes the following steps: (a) The areca nut processing byproducts were extracted and centrifuged using a methanol solution containing an internal standard, and the supernatant was collected; (b) The chemical components in the supernatant were qualitatively analyzed by UPLC-Q-Exactive Orbitrap-MS / MS. In step (b), the chromatographic conditions of UPLC-Q-Exactive Orbitrap-MS / MS were as follows: ACQUITY UHPLCHSS T3 column; column temperature 35℃; mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution; injection volume was 10 μL; flow rate was 0.3 mL / min.

2. The quality inspection method according to claim 1, characterized in that, In step (b), the gradient elution program is as follows: from 0 to 10 min, mobile phase B increases from 0% to 30%; from 10 to 25 min, mobile phase B increases from 30% to 40%; from 25 to 30 min, mobile phase B increases from 40% to 50%; from 30 to 40 min, mobile phase B increases from 50% to 70%; from 40 to 45 min, mobile phase B increases from 70% to 100%; from 45 to 60 min, mobile phase B is maintained at 100%; from 60 to 60.5 min, mobile phase B decreases from 100% to 0%; from 60.5 to 70 min, mobile phase B is maintained at 0%.

3. The quality inspection method according to claim 1, characterized in that, In step (b), the mass spectrometry conditions for the UPLC-Q-Exactive Orbitrap-MS / MS method are as follows: Full MS-dd-MS2 mode was used for acquisition, with simultaneous scanning of positive and negative ions; the scanning range was [missing information]. m / z 100-1200; MS1 resolution is 70000, MS2 resolution is 17500; spray voltage is 3.2kV; capillary temperature is 320℃; auxiliary gas heating temperature is 350℃; sheath flow rate is 40L / min, auxiliary flow rate is 15L / min; AGC target is 1×10 6 TopN 5; MS2 uses stepped collision energies of 30, 40, and 50.

4. The quality inspection method according to claim 1, characterized in that, In step (a), the internal standard content in the methanol solution containing the internal standard is 5 μg / mL, and the methanol solution concentration is 80%; the internal standard is nicodipine.

5. The quality inspection method according to claim 1, characterized in that, The quality testing method also includes the quantitative analysis of arecoline, norarecoline, norarecoside, and arecoside in areca nut processing by-products: The quantitative analysis includes the following steps: (1) Extract the areca nut processing byproducts with methanol solution, centrifuge, and collect the supernatant; (2) The contents of arecoline, norarecoline, norarecoside and arecoside in the supernatant were determined by liquid chromatography; the liquid chromatography conditions are as follows: An SCX column was used as the packing material, packed with strongly cation-exchange bonded silica gel; the mobile phase was acetonitrile-0.01 mol / L ammonium dihydrogen phosphate solution, with a volume ratio of acetonitrile to ammonium dihydrogen phosphate solution of 35:65; an ultraviolet detector was used with a detection wavelength of 215 nm; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the injection volume was 10 μL.

6. The quality inspection method according to claim 5, characterized in that, In step (1), the concentration of the methanol solution is 70%.

7. The quality inspection method according to claim 5, characterized in that, In step (2), the pH value of the ammonium dihydrogen phosphate solution is 2.

2.

8. The quality inspection method according to claim 1 or 5, characterized in that, The areca nut processing byproducts include areca nut processing wastewater, areca nut stems, and areca nut seeds.

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