A method for simultaneously determining the contents of seven main volatile flavor components in maca
By using gas chromatography-mass spectrometry combined with the internal standard curve method, the problem of quantitative analysis of multiple volatile flavor components in maca was solved, enabling maca quality control and process improvement, and enhancing the accuracy and sensitivity of detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JING BRAND
- Filing Date
- 2026-05-29
- Publication Date
- 2026-07-10
AI Technical Summary
Existing technologies have failed to effectively quantify various volatile flavor components in maca, affecting the standardization of maca quality and the optimization of formulation processes.
The flavor compounds in maca were extracted by ultrasonic extraction with 95% ethanol using gas chromatography-mass spectrometry combined with the internal standard curve method. The maca was then analyzed by gas chromatography-mass spectrometry, and characteristic ion peaks were selected for quantification to eliminate systematic errors.
It has achieved accurate quantification of seven major volatile flavor components in maca, improving detection accuracy and sensitivity, and has the advantages of less interference, low detection limit, high response value and wide detection range.
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Figure CN122361675A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of component detection technology, specifically relating to a method for simultaneously determining the content of seven major volatile flavor components in maca. Background Technology
[0002] Maca (Lepidium meyenii walp, Maca) is a cruciferous plant native to South America. Rich in macamides, glucosinolates, and other beneficial components, it is known as "South American ginseng" due to its effects on strengthening the body, enhancing libido, improving fertility, combating benign prostatic hyperplasia, and protecting the nervous system. Since being listed as a new resource food in 2011, maca has been widely cultivated in Lijiang and Diqing Prefectures of Yunnan Province, my country. As a new resource food, maca has broad potential for medicinal development and promising future prospects.
[0003] However, maca's pungent and pungent odor, similar to dried radish, significantly reduces its appeal to consumers. Studies have shown that this distinctive odor is primarily caused by phenylacetonitrile, 3-methoxyphenylacetonitrile, benzyl isothiocyanate, and butyric acid, which contribute to the pungent and pungent smell, while acetic acid, benzaldehyde, and ethyl phenylacetate work synergistically to create maca's unique aroma. Currently, there are no publicly available reports on the accurate quantitative analysis of maca's volatile components using gas chromatography. This technological gap severely hinders the standardization of maca quality and the optimization of its formulation processes. Therefore, there is an urgent need to develop a rapid and accurate method for simultaneously quantifying the content of multiple volatile flavor components in maca. Summary of the Invention
[0004] The purpose of this invention is to provide a method for simultaneously determining the content of seven major volatile flavor components in maca, providing a material component detection solution for maca quality control and process improvement.
[0005] The technical solution adopted by this invention to solve its technical problem is: A method for simultaneously determining the content of seven major volatile flavor components in maca, wherein the major volatile flavor compounds include: acetic acid, benzaldehyde, butyric acid, ethyl phenylacetonitrile, phenylacetonitrile, benzyl isothiocyanate, and 3-methoxyphenylacetonitrile, comprising the following steps: 1) Dry the maca at a low temperature of 50℃~60℃, and then pulverize the sample after drying; 2) Weigh the maca powder obtained in step 1) and place it in a centrifuge tube. Add 6-12 times the amount of 95% ethanol, extract by ultrasonication for 0.5-1 h, filter through a microporous membrane, and obtain the sample solution to be tested for later use. 3) Accurately weigh acetic acid, benzaldehyde, butyric acid, ethyl phenylacetonitrile, phenylacetonitrile, benzyl isothiocyanate, and 3-methoxyphenylacetonitrile standards, add 95% ethanol to prepare standard stock solutions, and then prepare mixed standard solutions according to different concentration ratios. 4) Accurately weigh ethyl octanoate-d 15 Add 95% ethanol to prepare an internal standard solution; 5) Add 10-30 μl of the internal standard solution obtained in step 4) to the mixed solution of the sample solution to be tested in step 2) and the standard solution in step 3); 6) Take the organic phase from step 5) into a sample bottle, add anhydrous Na2SO4 to dehydrate it, and then obtain the sample and standard curve test solution for instrumentation. 7) Gas chromatography-mass spectrometry was used to determine the sample test solution and the standard curve test solution, respectively; 8) Construct a standard curve with the ratio of the peak area of the flavor compound to the internal standard in the test solution of the standard as the ordinate and the ratio of the mass concentration of the flavor compound to the internal standard as the abscissa. 9) Substitute the ratio of the peak area of volatile compounds in the sample test solution to that of the internal standard obtained in step 8) into the standard curve to calculate the mass concentration of flavor compounds in the sample to be tested.
[0006] Preferably, in step 1), the sample coarse powder passes through a 20-mesh sieve.
[0007] Preferably, in step 2), the extracted solution is filtered through a microporous membrane with a diameter of 0.22-0.45 μm.
[0008] Preferably, in step 4), ethyl octanoate-d 15 The concentration of the internal standard was 500 mg / L. Preferably, in step 7), a nitroterephthalic acid-modified polyethylene glycol capillary column with dimensions of 60×0.25mm×0.25μm is used, the carrier gas is high-purity N2 with a purity ≥99.999%, and the temperature program is as follows: initial temperature 40℃, hold for 5 min, increase to 240℃ at 3℃ / min, and hold for another 5 min; flow rate is 1.42ml / min; injection port temperature is 250℃, and the injection volume is 1μl, splitless.
[0009] Preferably, in step 7), the ionization method is electron impact source (EI) with an energy of 70 eV; the ion source temperature is 230°C; the quadrupole temperature is 150°C; the auxiliary channel heating temperature is 280°C; the mass spectrometry scanning mass range is 35–350 amu; and the mass spectrometry analysis uses the NIST20 spectral library.
[0010] Preferably, the calculation formula for the 7 flavor compounds in step 7 is as follows:
[0011] In the formula, Ci : The mass concentration of the i-th flavor compound in the maca sample, in μg / kg; Cf : Ethyl octanoate-d, the internal standard in the sample to be tested 15 The content is expressed in μg / L; Ai : The chromatographic peak area of the i-th flavor compound in the sample to be tested; Af : Ethyl octanoate-d, the internal standard in the sample to be tested 15 chromatographic peak area; k The slope of the standard curve; b The intercept value of the standard curve; c Mass fraction of dried maca in the extract mixture. The present invention has the following advantages over the prior art: This invention first employs ultrasonic extraction with 95% ethanol to extract flavor compounds from maca, improving the extraction efficiency of the target compounds. 95% ethanol is readily available, inexpensive, and environmentally friendly, and it offers high response values and detection sensitivity for the target peaks, thus enhancing detection accuracy to some extent. Quantification is achieved using an internal standard curve method to eliminate systematic errors. Analysis using gas chromatography-mass spectrometry avoids the poor resolution issues of liquid chromatography, and quantification by selecting characteristic ion peaks results in more stable and reliable results. The method of this invention can simultaneously detect seven major volatile flavor compounds in maca, offering advantages such as low interference, low detection limits, high response values, wide detection range, and high accuracy. Attached Figure Description
[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiment drawings of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0013] Figure 1 The chromatogram of the mixed standard solution and internal standard for the simultaneous determination of seven main odor components in maca (acetic acid, benzaldehyde, butyric acid, ethyl phenylacetate, phenylacetonitrile, benzyl isothiocyanate, and 3-methoxyphenylacetonitrile) in Example 1 is shown below. Figure 2 This is a standard curve of acetic acid in Example 1 of the present invention; Figure 3 This is a standard curve diagram of benzaldehyde in Example 1 of the present invention; Figure 4 This is a standard curve diagram of butyric acid in Example 1 of the present invention; Figure 5This is a standard curve of ethyl phenylacetate in Example 1 of the present invention; Figure 6 This is a standard curve diagram of phenylacetonitrile in Example 1 of the present invention; Figure 7 This is a standard curve diagram of benzyl isothiocyanate in Example 1 of the present invention; Figure 8 This is the standard curve of 3-methoxyphenylacetonitrile in Example 1 of the present invention; Figure 9 This is the chromatogram of sample 1 to be tested in Example 1 of the present invention; Figure 10 This is the chromatogram of the recovery experiment of sample 1 in Example 1 of the present invention; Figure 11 This is the chromatogram of sample 2 to be tested in Example 2 of the present invention; Figure 12 This is the chromatogram of sample 3 to be tested in Example 3 of the present invention. Detailed Implementation
[0014] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain. If any definition stated in this section is contrary to or otherwise inconsistent with a definition stated in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definitions listed here shall prevail over those incorporated herein by reference.
[0016] Unless otherwise specified, the methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, and can be obtained commercially by those skilled in the art.
[0017] Example 1 This embodiment provides a method for detecting the concentration of seven flavor compounds (including acetic acid, benzaldehyde, butyric acid, ethyl phenylacetate, phenylacetonitrile, benzyl isothiocyanate, and 3-methoxyphenylacetonitrile) in maca, as detailed below: 1. Reagents: 95% ethanol (analytical grade); 2. Samples to be tested and standards: Sample to be tested: Yellow Maca, purchased from Kunming Yuanzheng Biotechnology Co., Ltd.; Grade I; Acetic acid, benzaldehyde, butyric acid, ethyl phenylacetate, phenylacetonitrile, benzyl isothiocyanate, 3-methoxyphenylacetonitrile; Ethyl octanoate-d 15 Standards: purity greater than 98.0%, all purchased from Shanghai Anpu Experimental Technology Co., Ltd. 3. Preparation of standard solutions: 3.1 Preparation of standard stock solutions of volatile flavor compounds: Accurately weigh the above 7 flavor compound standards into volumetric flasks, add 95% ethanol to make up to 10 ml, and obtain the 7 flavor compound single standard stock solutions in sequence. The specific concentrations are shown in Table 1.
[0018] 3.2 Internal standard solution: Accurately weigh 5 mg of ethyl octanoate-d 15 The standard was dissolved in a 10 ml volumetric flask and diluted to volume with 95% ethanol to obtain a 500 mg / L internal standard working solution, which was stored at -20°C protected from light.
[0019] Table 1: Concentration of flavor compounds, both single and mixed standards
[0020] 4. Sample pretreatment: Weigh 2g of maca powder into a 50ml centrifuge tube, tighten the cap, add 20ml of 95% ethanol, extract by sonication for 30min, filter through a 0.22μm microporous membrane, add 20μl of internal standard solution and shake well; take the filtrate into a 1.5ml sample vial, dehydrate it with anhydrous Na2SO4, and then analyze the resulting extract using a gas chromatography-mass spectrometry (GC-MS) instrument.
[0021] 5. Sample testing: 5.1 Instrument conditions: Agilent 8890 gas chromatograph-mass spectrometer; nitroterephthalic acid modified polyethylene glycol capillary column (DB-FFAP: 60×0.25mm×0.25μm); carrier gas was high-purity N2 with a purity ≥99.999%.
[0022] Temperature program: Initial temperature 40℃, hold for 5 min, increase to 240℃ at 3℃ / min, hold for another 5 min; flow rate 1.42 ml / min; injection port temperature 250℃, injection volume 1 μl, splitless.
[0023] Mass spectrometry conditions: ionization mode was electron impact source (EI) with energy 70 eV; ion source temperature was 230℃; quadrupole temperature was 150℃; auxiliary channel heating temperature was 280℃; mass range of mass spectrometry scan was 35-350 amu; mass spectrometry analysis was performed using the NIST20 spectral library.
[0024] Detection method: Ion scanning acquisition; monitored ions are shown in Table 2.
[0025] Scanning method: First, a full scan mode (SCAN) was used for qualitative analysis to optimize the instrument parameters, determine the retention time and characteristic ions of each active ingredient and internal standard, and then a selected ion mode (SIM) was used for scanning, as shown in Table 2.
[0026] Table 2: Mass Spectrometry Parameters of 7 Major Flavor Compounds
[0027] 5.2 Preparation of Standard Curve: According to Table 1 in "3.1", accurately transfer appropriate amounts of the single standard stock solutions of the seven flavor compounds into the same 10ml volumetric flask and dilute to volume with purified water to prepare a mixed standard solution, which is standard sample 1. Then, dilute it 2.5 times to obtain seven series of gradient mixed standard solutions. Add 20μl of internal standard solution to each solution and shake well. Analyze the solutions using a gas chromatography-mass spectrometry (GC-MS). Plot the standard curve with the ratio of the peak area of the flavor compound to the peak area of the internal standard as the ordinate and the ratio of the content of the flavor compound in the series of standard solutions to the content of the internal standard as the abscissa.
[0028] The parameters of the standard curves for the seven flavor compounds are shown in Table 3.
[0029] The standard curves of the seven flavor compounds are as follows: Figures 2-8 As shown. Figure 1 Chromatographic peaks 1-8 in the middle are, in order, ethyl octanoate-d 15 (Internal standard), acetic acid, benzaldehyde, butyric acid, ethyl phenylacetonitrile, phenylacetonitrile, benzyl isothiocyanate, 3-methoxyphenylacetonitrile.
[0030] Table 3: Standard curve parameters of 7 major flavor compounds
[0031] 5.3 Sample Determination: The test sample and standard solution were tested under the same detection conditions. The peak areas Ai of the seven flavor compounds and the peak area Af of the internal standard were substituted into the standard curve to calculate the mass concentration of the corresponding flavor compounds in the test sample. The chromatogram of test sample 1 is shown below. Figure 9 As shown. The content of the seven flavor compounds in the sample to be tested is calculated using the following formula:
[0032] In the formula, Ci: The mass concentration of the i-th flavor compound in the dried maca sample, expressed in micrograms per kilogram (μg / kg); Cf: Ethyl octanoate as an internal standard in the sample to be tested. 15 The content is expressed in micrograms per liter (μg / L); Ai: The chromatographic peak area of the i-th methoxypyrazine compound in the sample to be tested; Af: Ethyl octanoate as an internal standard in the sample to be tested. 15 chromatographic peak area; k: The slope of the standard curve; b: Intercept value of the standard curve; c: Mass fraction of dried maca in the extract mixture. 6. Repeatability experiments: Six samples were taken and labeled as 1 to 6. The samples were processed according to the methods described in "4. Sample Pretreatment" and "5. Sample Detection". The content of flavor compounds was determined, and the repeatability of the determination results for the seven flavor compounds was calculated. The RSD values were all less than 5%, indicating good method repeatability. Specific results are shown in Table 4. Table 4: Results of repeatability tests on 7 flavor compounds in the test samples
[0033] 7. Accuracy Experiment: Six wine samples were taken and divided into three groups (A, B, and C), with two samples in each group. These were labeled as group A (Z1-1 and Z1-2), group B (Z2-1 and Z2-2), and group C (Z3-1 and Z3-2), respectively. The mixed standard stock solution b1 containing the seven flavor compounds described in section "3.1" was used. 300 μL was added to group A, 600 μL to group B, and 900 μL to group C. 20 μL of internal standard solution was added to each sample. The samples were processed according to the methods described in "4. Sample Pretreatment" and "5. Sample Detection," and the content of flavor compounds was determined. The chromatograms of the accuracy experiments for the wine samples are shown below. Figure 10 As shown in Table 5, the peak areas of the flavor compounds and internal standards were substituted into the standard curve to calculate the content and recovery rate of each flavor compound. The recovery rates of the seven flavor compounds were all between 83% and 106%, indicating that the method has good accuracy.
[0034] Table 5: Accuracy Experiment Results of 7 Flavor Compounds
[0035] 8. Limit of Detection (LOD) and Limit of Quantification (LOQ) The limit of detection (LOD) is the sample content corresponding to a signal intensity / baseline noise (S / N) ratio of 3:1; the limit of quantitation (LOQ) is the sample content corresponding to a signal intensity / baseline noise (S / N) ratio of 10:1. In this example, the LODs for the seven flavor compounds ranged from 3.051 μg / L to 19.249 μg / L, and the LOQs ranged from 3.051 μg / L to 64.164 μg / L. The specific calculation results are shown in Table 6.
[0036] Table 6: Results of Limits of Detection (LOD) and Limits of Quantification (LOQ) for 7 Flavor Compounds
[0037] Example 2 This embodiment provides a method for detecting the concentration of maca flavor compounds (including acetic acid, benzaldehyde, butyric acid, ethyl phenylacetate, phenylacetonitrile, benzyl isothiocyanate, and 3-methoxyphenylacetonitrile), as detailed below: 1. Reagents: Same as in Example 1.
[0038] 2. Samples to be tested and standards: Sample to be tested: Yellow Maca, purchased from Kunming Yuanzheng Biotechnology Co., Ltd.; Grade 1 product; Standard product: Same as in Example 1.
[0039] 3. Preparation of standard solutions: Same as in Example 1.
[0040] 4. Sample pretreatment: Weigh 2g of maca powder into a 50ml centrifuge tube, tighten the cap, add 12ml of 95% ethanol, and the subsequent treatment is the same as in Example 1.
[0041] 5. Sample testing: Same as in Example 1.
[0042] The determination conditions and methods were the same as in Example 1, and the chromatogram of the obtained sample is shown below. Figure 11 As shown in Table 7, the detection results of the seven main flavor compounds in the sample tested in this embodiment are shown in Table 7.
[0043] Table 7: Quantitative Detection Results of Seven Major Flavor Compounds in Samples
[0044] Example 3 This embodiment provides a method for detecting the concentration of maca flavor compounds (including acetic acid, benzaldehyde, butyric acid, ethyl phenylacetate, phenylacetonitrile, benzyl isothiocyanate, and 3-methoxyphenylacetonitrile), as detailed below: 1. Reagents: Same as in Example 1.
[0045] 2. Samples to be tested and standards: Sample to be tested: Black Maca (Kunming Yinka Maca Planting Technology Co., Ltd.); Standard product: Same as in Example 1.
[0046] 3. Preparation of standard solutions: Same as in Example 1.
[0047] 4. Sample pretreatment: Same as in Example 1.
[0048] 5. Sample testing: Same as in Example 1.
[0049] The determination conditions and methods were the same as in Example 1, and the chromatogram of the obtained sample is shown below. Figure 12 As shown in Table 8, the detection results of the seven main flavor compounds in the sample tested in this embodiment are shown in Table 8.
[0050] Table 8: Quantitative Detection Results of Seven Major Flavor Compounds in Samples
[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for simultaneously determining the content of seven major volatile flavor components in maca, characterized in that, The main volatile flavor compounds include: acetic acid, benzaldehyde, butyric acid, ethyl phenylacetonitrile, phenylacetonitrile, benzyl isothiocyanate, and 3-methoxyphenylacetonitrile, comprising the following steps: 1) Dry the maca at a low temperature of 50℃~60℃, and then pulverize the sample after drying; 2) Weigh the maca powder obtained in step 1) and place it in a centrifuge tube. Add 6-12 times the amount of 95% ethanol, extract by ultrasonication for 0.5-1 h, filter through a microporous membrane, and obtain the sample solution to be tested for later use. 3) Accurately weigh acetic acid, benzaldehyde, butyric acid, ethyl phenylacetonitrile, phenylacetonitrile, benzyl isothiocyanate, and 3-methoxyphenylacetonitrile standards, add 95% ethanol to prepare standard stock solutions, and then prepare mixed standard solutions according to different concentration ratios. 4) Accurately weigh ethyl octanoate-d 15 Add 95% ethanol to prepare an internal standard solution; 5) Add 10-30 μl of the internal standard solution obtained in step 4) to the mixed solution of the sample solution to be tested in step 2) and the standard solution in step 3); 6) Take the organic phase from step 5) into a sample bottle, add anhydrous Na2SO4 to dehydrate it, and then obtain the sample and standard curve test solution for instrumentation. 7) Gas chromatography-mass spectrometry was used to determine the sample test solution and the standard curve test solution, respectively; 8) Construct a standard curve with the ratio of the peak area of the flavor compound to the internal standard in the test solution of the standard as the ordinate and the ratio of the mass concentration of the flavor compound to the internal standard as the abscissa. 9) Substitute the ratio of the peak area of volatile compounds in the sample test solution to that of the internal standard obtained in step 8) into the standard curve to calculate the mass concentration of flavor compounds in the sample to be tested.
2. The method according to claim 1, characterized in that, In step 1), the sample coarse powder is passed through a 20-mesh sieve.
3. The method according to claim 1, characterized in that, In step 2), the extracted solution is filtered through a microporous membrane with a diameter of 0.22-0.45 μm.
4. The method according to claim 1, characterized in that, In step 4), ethyl octanoate-d 15 The concentration of the internal standard was 500 mg / L.
5. The method according to claim 1, characterized in that, In step 7), a nitroterephthalic acid-modified polyethylene glycol capillary column with dimensions of 60×0.25mm×0.25μm is used. The carrier gas is high-purity N2 with a purity ≥99.999%. The temperature program is as follows: initial temperature 40℃, hold for 5 min, increase to 240℃ at 3℃ / min, and hold for another 5 min. The flow rate is 1.42 ml / min. The injection port temperature is 250℃, and the injection volume is 1 μl. Splitless injection is used.
6. The method according to claim 1, characterized in that, In step 7), the ionization method is electron impact source (EI) with an energy of 70 eV; the ion source temperature is 230℃; the quadrupole temperature is 150℃; the auxiliary channel heating temperature is 280℃; the mass spectrometry scanning mass range is 35–350 amu; and the mass spectrometry analysis uses the NIST20 spectral library.
7. The method according to claim 1, characterized in that, The calculation formulas for the 7 flavor compounds in step 7 are as follows: In the formula, Ci : The mass concentration of the i-th flavor compound in the maca sample, in μg / kg; Cf : Ethyl octanoate-d, the internal standard in the sample to be tested 15 The content is expressed in μg / L; Ai : The chromatographic peak area of the i-th flavor compound in the sample to be tested; Af : Ethyl octanoate-d, the internal standard in the sample to be tested 15 chromatographic peak area; k The slope of the standard curve; b The intercept value of the standard curve; c Mass fraction of dried maca in the extract mixture.