CN102095816A - Method for determining alkaloid in coffee tincture - Google Patents
Method for determining alkaloid in coffee tincture Download PDFInfo
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- CN102095816A CN102095816A CN 201110002936 CN201110002936A CN102095816A CN 102095816 A CN102095816 A CN 102095816A CN 201110002936 CN201110002936 CN 201110002936 CN 201110002936 A CN201110002936 A CN 201110002936A CN 102095816 A CN102095816 A CN 102095816A
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Abstract
The invention relates to a method for determining alkaloid in coffee tincture. The method is characterized in that water is taken as sample diluting solvent, mixed solution of methyl alcohol and 0.1% methanoic acid aqueous solution is taken as a mobile phase, and the high performance liquid chromatography is adopted to simultaneously detect caffeine, trigonelline and nicotinic acid in coffee tincture, wherein a diode array detector is adopted. The method has the key points that the water is taken as the sample diluting solvent and the 0.1% methanoic acid aqueous solution is taken as the mobile phase. The water is taken as the sample diluting solvent, thus pigment in a sample which enters into a chromatographic column is reduced, a chromatographic column is effectively protected, a sample preprocessing solvent is environmentally friendly, and the chromatographic peak shape of alkaloid is not influenced. Compared with pure water taken as the mobile phase, the 0.1% methanoic acid aqueous solution is taken as the mobile phase, and the chromatographic peak shape can be effectively improved, thus being beneficial to accurate determination of nicotinic acid. The method has the advantages of simple preprocessing, good repeatability and high recovery rate and can be applicable to analysis of samples on a large scale.
Description
Technical field
The present invention relates to alkaloidal detection method, specifically is alkaloidal assay method in a kind of coffee tincture, can detect caffeine in the coffee tincture, trigonelline and 3 kinds of alkaloids of Nicotinicum Acidum simultaneously.
Background technology
Coffee tincture be with certain density ethanol hang oneself after the baking processing the lixiviate of coffee seed and make.Product has multiple alkaloid and other heterogeneous ring compound, also contains furfuryl alcohol, sulfur derivatives and a small amount of volatile acid.Have typical fragrance and bitter organoleptic attributes such as fragrance, add energy impart depth in the cigarette to, it is fragrant to increase baking, and the flavor change is bitter.
Main plant alkali in the coffee bean is caffeine, and the content in fresh coffee beans changes with the difference of kind, and it has tangible bitter taste, and human body is had significant physiological action.Trigonelline and niacin are other two kinds of alkaloids in the coffee bean.Trigonelline has the physiological action of low degree, can decompose to generate multiple compound in roasting stir-fry process, comprises niacin.The content of niacin in fresh coffee beans seldom from the decomposition of trigonelline, itself also can continue to decompose in roasting process.
At present, these 3 kinds alkaloidal mensuration are not simultaneously appeared in the newspapers as yet in the coffee tincture.
Summary of the invention
Purpose of the present invention just is being based on above-mentioned condition and three kinds of alkaloidal assay methods in a kind of coffee tincture of providing, has filled up in the coffee tincture analyzing and testing caffeine, trigonelline and 3 kinds of alkaloidal blank of Nicotinicum Acidum simultaneously.
The objective of the invention is to be achieved through the following technical solutions: alkaloidal assay method is as follows in the coffee tincture: be the diluted sample solvent with water, the mixed liquor of methyl alcohol and 0.1% aqueous formic acid is that moving phase adopts high performance liquid chromatography simultaneously the caffeine in the coffee tincture, trigonelline and Nicotinicum Acidum to be detected, and wherein detecting device is a diode array detector.
Concrete determination step is as follows:
(1) standard working curve is formulated: 4.25 * 10
-4Prepare the standard solution of at least five concentration of trigonelline standard items in the-0.17mg/mL scope with methyl alcohol, 3.33 * 10
-4Prepare the standard solution of at least five concentration of Nicotinicum Acidum standard items in the-0.26mg/mL scope with methyl alcohol, 2.75 * 10
-4Use the standard solution of methyl alcohol coffee preparation in the-0.22mg/mL scope because of at least five concentration of standard items, utilize high performance liquid chromatograph respectively the content of three kinds of standard items in the standard solution of above-mentioned all concentration to be detected again, and draw the standard working curve of three kinds of standard items respectively and calculate its regression equation according to the peak area and the corresponding concentration of three kinds of standard items;
(2) specimen preparation: take by weighing 0.100g coffee tincture dilute with water constant volume in the 10mL volumetric flask, ultrasonic 5min, behind 0.45 μ m water membrane filtration, use, utilize high performance liquid chromatograph to adopt the condition identical respectively sample solution to be detected again with step (1), and bring into respectively according to the peak area that obtains in the regression equation of standard working curve, calculate caffeine, trigonelline and Nicotinicum Acidum content in the sample.
The efficient liquid phase chromatographic analysis condition is as follows:
Chromatographic column: waters C18 analytical column (250 mm * 4.6 mm, 5 μ m);
Moving phase: mobile phase A is 0.1% formic acid water, and Mobile phase B is a methyl alcohol;
Condition of gradient elution: 2%B begins, 10%B behind the 5min, and 50%B behind the 12min, 100%B behind the 15min keeps 5min, and 21min is with 2%B balance pillar 4min.Analysis time is 25min altogether;
Flow velocity 1mL/min; 30 ℃ of column temperatures; Detect wavelength: 265nm.
In above-mentioned condition, be the diluted sample solvent with water and be that moving phase is the key of this method with 0.1% formic acid water.With water is the diluted sample solvent, not only can reduce pigment in the sample and enter amount in the chromatographic column, effectively protects chromatographic column, and sample pre-treatments solvent environment-protecting asepsis, and to the not influence of alkaloidal chromatographic peak profile.With pure water is that moving phase is compared, and 0.1% formic acid water effectively improves the chromatographic peak profile of Nicotinicum Acidum for flowing to be on good terms, and helps the accurately quantitative of Nicotinicum Acidum.
This method pre-treatment is simple, the direct dilute with water constant volume of coffee tincture, and ultrasonic 5min uses behind 0.45 μ m water membrane filtration, need not further to carry out the sample purification step.Utilize the inventive method that three kinds of alkaloids in the coffee tincture are measured, the repeatability and the recovery are good, are applicable to the analysis of batch samples.
Description of drawings
Fig. 1 is caffeine, trigonelline and Nicotinicum Acidum standard specimen chromatogram.
Fig. 2 is coffee tincture sample chromatogram figure.
Among the figure: 1 is trigonelline, and 2 is Nicotinicum Acidum, and 3 is caffeine.
Embodiment:
Below by embodiment the present invention is further specified:
Chromatographic condition: Agilent 1200 liquid chromatographies, the configuration diode array detector detects wavelength 265nm.Chromatographic column: waters C18 analytical column (250 mm * 4.6 mm, 5 μ m).Moving phase: 0.1% formic acid water (A), methyl alcohol (B); Gradient elution, 2%B begins, 10%B behind the 5min, 50%B behind the 12min, 100%B behind the 15min keeps 5min, and 21min is with 2%B balance pillar 4min.Flow velocity 1mL/min.Analysis time is 25min altogether.30 ℃ of column temperatures.
1) formulation of standard working curve:
Trigonelline, Nicotinicum Acidum and caffeine are prepared typical curve with methyl alcohol, and the concentration of mother liquor is respectively 1.7mg/mL, 13.3mg/mL and 11.0mg/mL.The standard curve range 4.25 * 10 of trigonelline
-4-0.17mg/mL, typical curve equation are y=20234x+0.0806, R
2=1; The standard curve range 3.33 * 10 of Nicotinicum Acidum
-4-0.26mg/mL, typical curve equation are y=29622x-13.269, R
2=1; The standard curve range 2.75 * 10 of caffeine
-4-0.22mg/mL, typical curve equation are y=32505x+5.484, R
2=1; Y is a chromatographic peak area, and x is alkaloid dense (mg/mL).Each calibration curve contains 7 check points.
2) sample detection:
Take by weighing 0.100g coffee tincture dilute with water constant volume in the 10mL volumetric flask, ultrasonic 5min.Behind 0.45 μ m water membrane filtration, use.
The standard model chromatogram as shown in Figure 1, the sample spectrogram as shown in Figure 2:
Measure caffeine, Nicotinicum Acidum and trigonelline content in the coffee tincture sample parallel 5 times according to the described method of experimental section, relative standard deviation (RSD) all sees Table 1.
The alkaloid standard specimen that adds three levels in the actual sample of coffee tincture is measured three kinds of alkaloids according to the described method of experimental section, and as shown in table 2, the recovery is all between 93%-108%.
This method pre-treatment is simple, good reproducibility, and recovery height can satisfy trigonelline in the coffee tincture sample in enormous quantities, Nicotinicum Acidum and three kinds of alkaloidal detections simultaneously of caffeine.
Claims (3)
1. alkaloidal assay method in the coffee tincture, it is characterized in that: be the diluted sample solvent with water, the mixed liquor of methyl alcohol and 0.1% aqueous formic acid is that moving phase adopts high performance liquid chromatography simultaneously the caffeine in the coffee tincture, trigonelline and Nicotinicum Acidum to be detected, and wherein detecting device is a diode array detector.
2. alkaloidal assay method in the coffee tincture according to claim 1 is characterized in that: concrete determination step is as follows:
(1) standard working curve is formulated: 4.25 * 10
-4Prepare the standard solution of at least five concentration of trigonelline standard items in the-0.17mg/mL scope with methyl alcohol, 3.33 * 10
-4Prepare the standard solution of at least five concentration of Nicotinicum Acidum standard items in the-0.26mg/mL scope with methyl alcohol, 2.75 * 10
-4Use the standard solution of methyl alcohol coffee preparation in the-0.22mg/mL scope because of at least five concentration of standard items, utilize high performance liquid chromatograph respectively the content of three kinds of standard items in the standard solution of above-mentioned all concentration to be detected again, and draw the standard working curve of three kinds of standard items respectively and calculate its regression equation according to the peak area and the corresponding concentration of three kinds of standard items;
(2) specimen preparation: take by weighing 0.100g coffee tincture dilute with water constant volume in the 10mL volumetric flask, ultrasonic 5min, behind 0.45 μ m water membrane filtration, use, utilize high performance liquid chromatograph to adopt the condition identical respectively sample solution to be detected again with step (1), and bring into respectively according to the peak area that obtains in the regression equation of standard working curve, calculate caffeine, trigonelline and Nicotinicum Acidum content in the sample.
3. alkaloidal assay method in the coffee tincture according to claim 1 is characterized in that: the efficient liquid phase chromatographic analysis condition is as follows:
Chromatographic column: waters C18 analytical column (250 mm * 4.6 mm, 5 μ m);
Moving phase: mobile phase A is 0.1% formic acid water, and Mobile phase B is a methyl alcohol;
Condition of gradient elution: 2%B begins, 10%B behind the 5min, and 50%B behind the 12min, 100%B behind the 15min keeps 5min, and 21min is with 2%B balance pillar 4min;
Analysis time is 25min altogether;
Flow velocity 1mL/min; 30 ℃ of column temperatures; Detect wavelength: 265nm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102353739A (en) * | 2011-09-01 | 2012-02-15 | 湖北中烟工业有限责任公司 | Rapid detection method of nicotine in tobacco |
| CN102539612A (en) * | 2012-01-04 | 2012-07-04 | 北京知蜂堂蜂产品有限公司 | High performance liquid chromatography (HPLC) quantitative determination method for natural caffeine in guarana fruit powder |
| CN103323419A (en) * | 2013-06-15 | 2013-09-25 | 广东医学院 | Method for testing nicotine alkaloid in tobacco and tobacco products |
| CN111999398A (en) * | 2020-07-09 | 2020-11-27 | 武汉迈特维尔生物科技有限公司 | Liquid chromatography-mass spectrometry analysis method for caffeine and metabolites thereof |
-
2011
- 2011-01-08 CN CN 201110002936 patent/CN102095816B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| 《Food Chemistry》 20001231 S. Casal，et al HPLC/diode-array applied to the thermal degradation of trigonelline, nicotinic acid and caffeine in coffee 第482页第2.2节－第2.4节 1－3 第68卷, * |
| 《饮料工业》 20031231 蔡瑞玲等 焙炒条件对咖啡风味影响的研究 参见的33页左栏第1.2节第2－4段 3 第6卷, 第6期 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102353739A (en) * | 2011-09-01 | 2012-02-15 | 湖北中烟工业有限责任公司 | Rapid detection method of nicotine in tobacco |
| CN102353739B (en) * | 2011-09-01 | 2013-05-15 | 湖北中烟工业有限责任公司 | Rapid detection method of nicotine in tobacco |
| CN102539612A (en) * | 2012-01-04 | 2012-07-04 | 北京知蜂堂蜂产品有限公司 | High performance liquid chromatography (HPLC) quantitative determination method for natural caffeine in guarana fruit powder |
| CN103323419A (en) * | 2013-06-15 | 2013-09-25 | 广东医学院 | Method for testing nicotine alkaloid in tobacco and tobacco products |
| CN111999398A (en) * | 2020-07-09 | 2020-11-27 | 武汉迈特维尔生物科技有限公司 | Liquid chromatography-mass spectrometry analysis method for caffeine and metabolites thereof |
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