CN112782293B - Quantitative detection method for cocoa shell doped in cocoa powder - Google Patents
Quantitative detection method for cocoa shell doped in cocoa powder Download PDFInfo
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Abstract
The invention discloses a quantitative detection method for cocoa shell doped in cocoa powder, which is mainly used for detecting the content of cocoa shell in cocoa powder based on a characteristic marker quassinolide I (Nigakilactone I) of the cocoa shell. The quantitative detection method comprises the following steps: 1) Sample pretreatment: weighing a proper amount of sample, dissolving the sample in methanol, ultrasonically extracting the sample, centrifuging the sample to obtain supernatant, concentrating the supernatant for redissolving the supernatant, and filtering the supernatant by using a microporous filter membrane to prepare a sample solution; 2) Liquid phase chromatographic separation: fully separating the characteristic marker to be detected from other impurities in the sample solution; 3) Mass spectrum detection: establishing a linear standard curve between the peak area of the characteristic marker and the incorporation amount of the cocoa shell in the cocoa powder, determining the retention time and the accurate mass number, and quantifying by an external standard curve method. The method can be used for quantitatively detecting the cocoa shells doped with 1% or more (mass fraction) in the cocoa powder, has the characteristics of rapidness, accuracy, stability and the like, and can meet the quantitative detection requirement of cocoa powder market supervision.
Description
Technical Field
The invention relates to a quantitative detection method for cocoa shell doped in cocoa powder, in particular to a quantitative detection method for cocoa shell doped in cocoa powder based on a characteristic marker.
Background
The cocoa powder has strong and fragrant unique flavor and is rich in various nutrient elements, thereby being one of three favorite beverages in the world. However, the incorporation of cocoa shells is one of the common quality problems of cocoa powder, and the presence of cocoa shells not only affects the sensory experience, but can also cause contamination by mycotoxins, heavy metals or microorganisms. The cocoa shell in the cocoa powder is doped mainly in two reasons, namely, in order to obtain violence, some illegal enterprises import the cocoa shell on the name of feed, spray cocoa essence after crushing and processing the cocoa shell, and then mix the cocoa shell into the cocoa powder; secondly, the production process remains, and to a certain extent, the remaining of the cocoa shells in the cocoa processing process cannot be completely avoided. At present, no quality control standard for the cocoa shell incorporation amount in cocoa powder exists at home and abroad, and the limit value of the cocoa shell incorporation amount in cocoa pulp and cocoa cake is regulated to be 5% (m/m) by CXS 141-1983 published by the International Commission on food code Commission (CAC), which becomes the cocoa shell incorporation limit value generally accepted by the machinable industry.
Until now, methods for detecting the incorporation amount of cocoa shells in cocoa powder include microscopy, gravimetric, spectrophotometric, liquid chromatography, and near-infrared spectroscopy, but these methods are limited by stability, sensitivity, accuracy, and selectivity and do not provide reliable and repeatable results for samples of different origin, variety, and processing stages. Therefore, a rapid, accurate and stable quantitative detection method for the cocoa shell incorporation amount in cocoa powder needs to be established, and a new scientific basis is provided for the supervision and regulation of the cocoa powder market.
Disclosure of Invention
The invention provides a quantitative detection method for cocoa shell doped in cocoa powder, which is mainly used for detecting the content of cocoa shell in cocoa powder based on a feature marker of the cocoa shell, namely, the quassia lactone INigakilactone I, and comprises the following steps:
a) Sample pretreatment: weighing a proper amount of sample, dissolving the sample in methanol, ultrasonically extracting the sample, centrifuging the sample to obtain supernatant, concentrating the supernatant for redissolving the supernatant, and filtering the supernatant by using a microporous filter membrane to prepare a sample solution;
b) Liquid phase chromatographic separation: fully separating the characteristic marker to be detected from other impurities in the sample solution;
c) Mass spectrum detection: establishing a linear standard curve between the peak area of the characteristic marker and the incorporation amount of the cocoa shell in the cocoa powder, determining the retention time and the accurate mass number, and quantifying by an external standard curve method.
Wherein, the liquid phase chromatographic separation conditions are as follows: the chromatographic column is C18 or other equivalent chromatographic columns; the column temperature is 40 ℃; the mobile phase A is 5mmol/L ammonium acetate water solution, the phase B is acetonitrile, and gradient elution is carried out; the gradient elution ratio is 0-3.0min, A is 95%; 3.0-8.0min, A is 95% → 80%; 8.0-17.0 min, A is 80% → 72%; 17.0-22.0 min, A is 72% → 10%; 22.0-26.0 min, A is 10%; 26.0-26.1 min, A is 10% → 95%; 26.1-28.0min, A is 95%; the flow rate is 0.3mL/min; the sample size was 5. Mu.L.
The mass spectrum detection conditions are as follows: the ion source being ESI - (ii) a The ion source temperature is 100 ℃; the temperature of the desolventizing gas is 250 ℃; the desolventizing air flow rate is 600L/h; the air flow rate of the taper hole is 50L/h; the capillary voltage is 2.0kV; the voltage of the taper hole is 40V; the working mode is as follows: MSMS mode; the collision energy is 15eV to 40eV; the scan time was 0.2s.
The parent ion of the characteristic marker quassia lactone I is m/z 375.18, and the daughter ion is m/z 191.07, m/z289.07.
When the cocoa shell is added in the range of 1-50%, the linear relation between the peak area of the quassia lactone I and the cocoa shell is good, and the correlation coefficient R 2 Greater than 0.9999, detection limit of 0.3%, quantification limit of 1%.
The invention has the beneficial effects that: the method can quantitatively detect the cocoa shells doped with more than 1% in the cocoa powder, has the characteristics of rapidness, accuracy, stability and the like, and can meet the requirement of batch detection of cocoa powder market supervision.
Drawings
FIG. 1 MSMS spectrum of cocoa powder standard sample with cocoa shell
FIG. 2 MSMS spectrum of cocoa powder standard sample without cocoa shell
FIG. 3 is a standard curve of the peak area of quassinolide I and the content of cacao shell
FIG. 4 MSMS spectra of commercial samples of cocoa powder
Note: the retention time of the characteristic marker quassinolide I in FIGS. 1 and 4 is 16.31min and 16.30min, respectively.
Detailed Description
Example one
1. Sample pretreatment
Accurately weighing about 1g (accurate to 0.001 g) of sample in a 50mL centrifuge tube with a plug, adding about 20mL of methanol, uniformly mixing by vortex, and performing ultrasonic treatment for 20min; centrifuging at 8000r/min for 5min, collecting supernatant, adding 20mL methanol into residue, repeatedly extracting, and mixing supernatants; and (3) rotatably evaporating the supernatant at 40 ℃ until the supernatant is dry, adding 1mL of methanol for redissolving, and filtering through a 0.22-micron filter membrane to prepare a sample solution.
2. Conditions of liquid chromatography
The instrument comprises the following steps: ultra-high performance liquid chromatography; a chromatographic column: acquity
BEH C18 column (100 mm. Times.2.1mm, 1.7 μm); mobile phase: phase A is ammonium acetate aqueous solution (5 mmol/L), phase B is acetonitrile, and gradient elution conditions are shown in Table 1; flow rate: 0.3mL/min, sample size: 5 μ L, column temperature: at 40 ℃.
TABLE 1 gradient elution conditions
3. Conditions of Mass Spectrometry
The instrument comprises the following steps: quadrupole-time of flight mass spectrometry; an ion source: ESI-; ion source temperature: 100 ℃; desolvation gas temperature: 250 ℃; desolventizing air flow rate: 600L/h; taper hole air flow rate: 50L/h; capillary voltage: 2.0kV; taper hole voltage: 40V; the working mode is as follows: MSMS mode; scanning mode: a sensitivity mode; scanning range: m/z is 50-500; scanning time: 0.2s; collision energy: 15eV to 40eV; monitoring the parent ion: m/z 375.18, monitor daughter ion: m/z 191.07, m/z 289.07; real-time correction fluid: leucine enkephalin, m/z 554.2615.
4. Standard curve and detection limit
Cocoa shells with the mass fractions of 0, 1.00, 5.00, 10.00, 20.00 and 50.00 percent are added into cocoa powder to prepare a series of cocoa powder standard samples with different cocoa shell incorporation amounts, detection is carried out according to the method described in 1-3 of example I, the peak area of the picrasma quassioides lactone I is taken as the ordinate, the cocoa shell incorporation amount (%, m/m) in the cocoa powder is taken as the abscissa, a linear standard curve is fitted, the retention time is determined qualitatively, and the external standard curve is used for quantification. Results tableIt is clear that the linear relation between the peak area (Y) of the quassia lactone I and the doping amount (X) of the cocoa shell is good in the range of 1-50% of the cocoa shell doping amount, the linear equation is Y =187.82X +646.96, and the correlation coefficient (R =187.82X + 646.96) 2 ) Greater than 0.9999, detection limit (LOD, S/N > 3) of 0.3%, quantification limit (LOQ, S/N > 10) of 1%.
5. Blind sample validation and commercial sample detection
3 blind samples were prepared using cocoa shell and cocoa powder simulations and 4 cocoa powder samples were purchased from the market. The results of the tests conducted according to the methods described in examples 1 to 3 are shown in Table 2. The results show that: the detection content of the 3 blind samples mixed with the cocoa shells is more consistent with the actual mixing amount, and the relative error is between 3.89 and 7.81 percent; the content of cocoa shell in 4 commercial cocoa powder samples was 9.03%, 2.51%, 3.33% and 2.79%.
TABLE 2 detection of cocoa shell incorporation in blind and commercially available cocoa powder samples
Finally, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (5)
1. A quantitative detection method for cocoa shell doped in cocoa powder is characterized in that the content of cocoa shell in cocoa powder is detected mainly based on a feature marker of the cocoa shell, namely quassinolide I Nigakilactone I, and comprises the following steps:
a) Sample pretreatment: weighing a proper amount of sample, dissolving the sample in methanol, ultrasonically extracting the sample, centrifuging the sample to obtain supernatant, concentrating the supernatant for redissolving the supernatant, and filtering the supernatant by using a microporous filter membrane to prepare a sample solution;
b) Liquid phase chromatographic separation: fully separating the characteristic marker to be detected from other impurities in the sample solution;
c) Mass spectrum detection: establishing a linear standard curve between the peak area of the characteristic marker and the incorporation amount of the cocoa shell in the cocoa powder, determining the retention time and the accurate mass number, and quantifying by an external standard curve method.
2. The method for quantitatively detecting the cocoa shell adulteration in the cocoa powder according to claim 1, wherein the liquid chromatography separation conditions are as follows: the chromatographic column is a C18 chromatographic column; the column temperature was 40 ℃; the mobile phase A is 5mmol/L ammonium acetate water solution, the phase B is acetonitrile, and gradient elution is carried out; the gradient elution ratio is 0-3.0min, A is 95%; 3.0-8.0min, A is 95% → 80%; 8.0-17.0 min, A is 80% → 72%; 17.0-22.0min, A is 72% → 10%; 22.0-26.0 min, A is 10%; 26.0-26.1 min, A is 10% → 95%; 26.1-28.0min, A is 95%; the flow rate is 0.3mL/min; the amount of sample was 5. Mu.L.
3. The method for quantitatively detecting the cocoa shells doped in the cocoa powder according to claim 1, wherein the mass spectrometry detection conditions are as follows: the ion source is ESI-; the ion source temperature is 100 ℃; the temperature of the desolventizing gas is 250 ℃; the desolventizing air flow rate is 600L/h; the air flow rate of the taper holes is 50L/h; the capillary voltage is 2.0kV; the taper hole voltage is 40V; the working mode is as follows: MSMS mode; the collision energy is 15eV to 40eV; the scanning time was 0.2s.
4. The method for quantitatively detecting the cocoa shells doped in the cocoa powder according to claim 1, wherein the parent ion of the characteristic marker quassia lactone I is m/z 375.18, and the daughter ion is m/z 191.07, m/z289.07.
5. The method for quantitatively detecting the cocoa shells doped in the cocoa powder according to claim 1, wherein when the content of the cocoa shells is in the range of 1-50%, the linear relationship between the peak area of the quassinoid I and the content of the cocoa shells is good, and the correlation coefficient R is 2 Greater than 0.9999, detection limit of 0.3%, quantification limit of 1%.
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Citations (4)
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|---|---|---|---|---|
| CN102680607A (en) * | 2012-06-07 | 2012-09-19 | 江南大学 | Cocoa powder adulteration detection method based on fingerprints |
| CN102778517A (en) * | 2012-07-16 | 2012-11-14 | 无锡市产品质量监督检验中心 | Method for detecting cocoa powder adulteration based on lipid clustering analysis |
| CN103424499A (en) * | 2013-07-26 | 2013-12-04 | 广州白云山和记黄埔中药有限公司 | Method for detecting picrasma quassioides alkali content in picrasma quassioides |
| CN108169374A (en) * | 2017-12-25 | 2018-06-15 | 河南广电计量检测有限公司 | The detection method of synthetic coloring matter in a kind of Grain and its product |
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- 2020-12-17 CN CN202011531877.5A patent/CN112782293B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680607A (en) * | 2012-06-07 | 2012-09-19 | 江南大学 | Cocoa powder adulteration detection method based on fingerprints |
| CN102778517A (en) * | 2012-07-16 | 2012-11-14 | 无锡市产品质量监督检验中心 | Method for detecting cocoa powder adulteration based on lipid clustering analysis |
| CN103424499A (en) * | 2013-07-26 | 2013-12-04 | 广州白云山和记黄埔中药有限公司 | Method for detecting picrasma quassioides alkali content in picrasma quassioides |
| CN108169374A (en) * | 2017-12-25 | 2018-06-15 | 河南广电计量检测有限公司 | The detection method of synthetic coloring matter in a kind of Grain and its product |
Non-Patent Citations (2)
| Title |
|---|
| Food fingerprinting: Mass spectrometric determination of the cocoa shell content (Theobroma cacao L.) in cocoa products by HPLC-QTOF-MS;Nicolas Cain等;《Food Chemistry》;20190624;第298卷;第1-8页 * |
| 高效液相色谱法测定可可粉中可可壳含量;张九魁等;《食品与发酵工业》;20191231;第45卷(第3期);第261-265页 * |
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