CN102495162B - Method for determining content of eugenol in formulated cut tobacco - Google Patents
Method for determining content of eugenol in formulated cut tobacco Download PDFInfo
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- CN102495162B CN102495162B CN2011103627088A CN201110362708A CN102495162B CN 102495162 B CN102495162 B CN 102495162B CN 2011103627088 A CN2011103627088 A CN 2011103627088A CN 201110362708 A CN201110362708 A CN 201110362708A CN 102495162 B CN102495162 B CN 102495162B
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Abstract
The invention relates to a method for determining the content of eugenol in formulated cut tobacco. The method comprises the steps of: preparation of an extraction reagent and a sample solution, liquid chromatogram analysis and detection result calculation, etc. The optimized detection method has the advantages of short detection time, simple operation, high sensitivity, and good repeatability, etc. The chromatographic condition of the method in the invention makes the chromatographic peak of eugenol and the chromatographic peak of impurities well separated. And the method of the invention also has good linear range, the detection limit is 0.3 microgram/g, the average recovery rate of standard addition is 101.4%, and the average relative standard deviation is less than 2.4%.
Description
Technical field
The present invention relates to a kind of improvement assay method of the middle eugenol content that cuts filler, belong to the physical and chemical inspection technical field of cigarette with raw material.
Background technology
Eugenol, Coryophyllic acid, colourless to weak yellow liquid, turn brown in air, strong cloves smell is arranged.Very easily water-soluble, be dissolved in ethanol, ether, chloroform and essential oil.Be used for preparation carnation type essence and isoeugenol processed and vanillic aldehyde etc. as flavouring agent.Also as pesticide and antiseptic, antioxidant, carminative and be used for anodyne in dentistry.In the carcinogenic list that international cancer research institution (IARC) announces, eugenol is listed in Group 3.The cloves cigarette is the distinctive a kind of cigarette in Indonesia Malaysia, good to eat pure and fresh the driving away summer heat of its taste fragrance.
On September 14th, 2009, U.S. food and drug administration (FDA) issue open letter, remind industry to note forbidding that according to " federal food drug and cosmetic act, medicine and cosmetics bill " and " family's smoking prevention is controlled bill with tobacco " production and selling contains the cigarette of specific fragrance from September 22.The US President Barack Obama had once signed " family's smoking prevention is controlled bill with tobacco " June 22, authorized the production and selling of FDA management and control tobacco product.According to (A) chapter regulation of this bill the 907th (a) (1), cigarette or its ingredient (comprising tobacco, filter tip or cigarette paper) must not contain or add any artificial, natural perfume material, medicinal herbs and the flavouring except the tobacco menthol, thereby make cigarette or smog contain strawberry, grape, orange, the fragrance such as cloves.This standard is applicable to all tobacco products and comprises cigarette, cigar and some other tobacco products.The present domestic regulation that does not also have in this respect.
At present less about the report of the mensuration of eugenol, the mensuration of eugenol in meat soup that adopted liquid chromatography for measuring such as Li Yong-Hong wherein; Cut filler the mensuration of middle eugenol due to the complicacy of matrix, rarer report, the eugenol in the tobacco that wherein adopted external standard-liquid chromatography for measuring in Canadian method T-314, because the boiling point of eugenol is lower, volatility is larger, external standard method is lost larger in the sample preparation process, therefore be necessary to develop the internal mark method determination method that reduces the sample pretreatment process loss; In T-314, the liquid chromatographic detection time is 20min in addition, and chromatographic condition also has necessity of further optimization.
Summary of the invention
Purpose of the present invention is intended to overcome the prior art defect, the assay method of eugenol content in a kind of pipe tobacco is provided, content for detection of eugenol in pipe tobacco, by the optimization to liquid phase chromatogram condition, make the liquid phase minute be reduced to 10min, effectively raise determination efficiency, make the chromatographic peak of eugenol separate better with the impurity chromatographic peak, have the characteristics such as sensitivity height.
The objective of the invention is to be achieved through the following technical solutions:
The cut filler assay method of middle eugenol content comprises the following steps:
(1) contain the preparation of interior mark extractant: for containing the ethanol solution of anethole, the concentration of anethole is 0.1-0.2mg/mL;
(2) preparation of standard solution: take the eugenol standard items, with containing interior mark extractant, be mixed with the eugenol standard operation solution with concentration gradient;
(3) preparation of sample solution: use and contain interior mark extractant extraction sample, then prepare sample solution after centrifugal filtration;
(4) liquid-phase chromatographic analysis: utilize liquid chromatograph respectively standard solution and sample solution to be detected analysis;
(5) calculating of eugenol content in pipe tobacco.
The described preparation that contains interior mark extractant is to take 0.4 g anethole in 4000 mL ethanol solutions, and wherein the concentration of anethole is 1mg/mL.
The preparation of described standard solution is to take approximately 0.2g eugenol, is accurate to 0.0001g, be transferred in the 100mL volumetric flask with containing after interior target extractant dissolves, and constant volume, be decided to be standard reserving solution.Pipette respectively 0.01mL, 0.1mL, 0.5mL, 1.0mL, 2.5mL, 5.0mL standard reserving solution in the 10mL volumetric flask, use the extractant constant volume, the concentration of eugenol is 2.0 μ g/mL, 20 μ g/mL, 100 μ g/mL, 200 μ g/mL, 500 μ g/mL, 1000 μ g/mL.
The preparation method of described sample solution specifically comprises and takes 0.2 g tobacco sample (being accurate to 0.1 mg) in 50 mL tool plug triangular flasks, adds 50mL to contain interior target absolute ethyl alcohol, with sealant sealing oscillation extraction 2h.At 10000rmp centrifugal 10 minutes if necessary.After standing, pipette the 2.0mL supernatant liquor with the 0.45 organic membrane filtration of μ m, obtain sample solution.
Described chromatographiccondition is chromatographic column adopting Nova-pak C18 liquid-phase chromatographic column, and chromatogram column temperature is 30 ℃, and sample size is 10 μ L, and flow velocity is 1.0 ml/min; Detect wavelength: 280nm, mobile phase: A is water, and B is acetonitrile, adopts the Gradient elution program, and namely water is 30%, and methyl alcohol is 70%, and elution time is 10min.
The specification of described chromatographic column is 3.9 * 150mm, 5 μ m particle diameters.
The calculating of eugenol content: at first with the detected object of standard solution and its respective concentration of comparing of interior target chromatographic peak area, carry out regretional analysis, obtain typical curve; The ratio of the object that then sample solution is detected and interior target chromatographic peak area, the substitution typical curve, namely obtain the eugenol concentration in sample, can calculate thus the content of eugenol in pipe tobacco.
Detection method of the present invention is optimized disposal route and the chromatographic condition of sample, has reached following effect:
(1) detection time is short: adopting the present invention to measure the eugenol content cycle in pipe tobacco only needed about 10 minutes;
(2) the present invention have advantages of easy and simple to handle, highly sensitive, the recovery is high and good reproducibility: the chromatographic condition of the inventive method makes the chromatographic peak of eugenol separate better with the impurity chromatographic peak, and has linear dependence preferably, detection is limited to 0.3 μ g/g, average recovery of standard addition is 101.14%, and mean relative deviation is less than 2.4%.
Description of drawings
Fig. 1 is the process flow diagram of assay method of the present invention;
Fig. 2 is eugenol typical curve linear equation and linear regression coeffficient coordinate diagram;
Fig. 3 is the chromatogram of standard solution;
Fig. 4 is the chromatogram of sample solution.
Embodiment
The present invention is described further below in conjunction with embodiment (accompanying drawing):
Embodiment 1
The present embodiment is to the detection method of eugenol content in pipe tobacco following (process flow diagram of described detection method as shown in Figure 1)
(1) contain the preparation of interior mark extractant: contain the ethanol solution of debita spissitudo internal standard compound (anethole), concentration is generally 0.1mg/mL～0.2mg/mL;
(2) preparation of standard solution: take approximately 0.2g eugenol, be accurate to 0.0001g, with containing after interior target extractant dissolves, be transferred in the 100mL volumetric flask, and constant volume, be decided to be standard reserving solution;
Pipette respectively 0.01mL, 0.1mL, 0.5mL, 1.0mL, 2.5mL, 5.0mL standard reserving solution in the 10mL volumetric flask, use the extractant constant volume, the concentration of eugenol is 2.0 μ g/mL, 20 μ g/mL, 100 μ g/mL, 200 μ g/mL, 500 μ g/mL, 1000 μ g/mL; Mark liquid lucifuge 4 ℃ of preservations, be positioned over while taking under normal temperature, can use after reaching normal temperature.Do weekly graticule one time;
(3) preparation of sample solution: take 0.2 g tobacco sample (being accurate to 0.1 mg) in 50 mL tool plug triangular flasks, add the 50mL absolute ethyl alcohol, with sealant sealing oscillation extraction 2h.At 10000rmp centrifugal 10 minutes if necessary.After standing, pipette the 2.0mL supernatant liquor with the 0.45 organic membrane filtration of μ m, obtain sample solution, to be analyzed in 4 ℃ of preservations;
(4) stratographic analysis: chromatographiccondition is chromatographic column adopting Nova-pak C18 liquid-phase chromatographic column, and chromatogram column temperature is 30 ℃, and sample size is 10 μ L, and flow velocity is 1.0 ml/min; Detect wavelength: 280nm, mobile phase: A is water, and B is methyl alcohol, adopts the Gradient elution program, and namely water is 30%, and methyl alcohol is 70%, and elution time is 10min; The stratographic analysis result of standard solution as shown in Figure 3; The stratographic analysis result of sample solution as shown in Figure 4;
(5) the cut filler calculating of middle eugenol content
At first carry out regretional analysis with the detected object of standard solution and its respective concentration of comparing of interior target chromatographic peak area, obtain typical curve (as shown in Figure 2); The data such as the regression equation corresponding with typical curve, related coefficient are as shown in table 1.
The typical curve of eugenol and detectability in table 1 pipe tobacco
Annotate: 1. detectability calculates with 3 times of signal to noise ratio (S/N ratio)s (S/N=3).
Then the ratio that detects object and interior target chromatographic peak area that sample is recorded, the substitution typical curve, namely obtain the eugenol concentration in sample, calculates thus the content of eugenol in pipe tobacco, and computing formula is as follows
In formula:
The content of eugenol in m-every gram pipe tobacco, unit are the every gram of microgram (μ g/g);
The concentration of eugenol in A-sample (μ g/mL);
The S-liquor capacity;
N-the take quality (g) of sample.
By table 1 and Fig. 4 as can be known, the chromatographic condition that adopts makes the chromatographic peak of eugenol separate better with the impurity chromatographic peak, and has correlativity preferably, and detectability is at 0.3 μ g/g.The content of the eugenol of the present embodiment sample is 865.7 μ g/g.
Embodiment 2
The present embodiment is as follows to the detection method of the repeatability of the inventive method and recovery of standard addition
Adopt the test of sample recovery of standard addition, add respectively 20.8 μ g/g in sample, 208.0 μ g/g, 1040.0 the standard solution of three variable concentrations of μ g/g carries out the recovery of standard addition test, each sample is measured respectively 6 times, the condition of stratographic analysis is with embodiment 1, and according to the relative standard deviation of measured value after the recovery of standard addition of eugenol in Analysis result calculation this method pipe tobacco and mark-on, result is as shown in table 2;
The recovery of eugenol and repeatability (n=6) in table 2 pipe tobacco
As can be seen from Table 2, on 3 mark-on levels, the average recovery rate that utilizes the method to detect eugenol in pipe tobacco is 101.14%, and the average relative standard deviation of sample test result, less than 2.4%, illustrates that the recovery of this law is higher, and repeatability better.
Claims (3)
1. assay method of middle eugenol content that cuts filler is characterized in that: comprise the following steps:
(1) contain the preparation of interior mark extractant: containing interior mark extractant is the ethanol solution that contains anethole, and the concentration of anethole is 0.1-0.2mg/mL;
(2) preparation of standard solution: take the eugenol standard items, with containing interior mark extractant, be mixed with the eugenol standard operation solution with concentration gradient;
(3) preparation of sample solution: use and contain interior mark extractant extraction sample, then prepare sample solution after centrifugal filtration; Concrete grammar is: take 0.2 g tobacco sample in 50 mL tool plug triangular flasks, add 50mL to contain interior target absolute ethyl alcohol, with sealant sealing oscillation extraction 2h; At 10000rmp centrifugal 10 minutes if necessary, standing after, pipette the 2.0mL supernatant liquor with the 0.45 organic membrane filtration of μ m, obtain sample solution;
(4) liquid-phase chromatographic analysis: utilize liquid chromatograph respectively standard solution and sample solution to be detected analysis; Described chromatographiccondition is chromatographic column adopting Nova-pak C18 liquid-phase chromatographic column, and specification is 3.9 * 150mm, 5 μ m particle diameters, and chromatogram column temperature is 30 ℃, and sample size is 10 μ L, and flow velocity is 1.0 ml/min; Detect wavelength: 280nm, mobile phase: A is water, and B is methyl alcohol, adopts the Gradient elution program, and namely water is 30%, and methyl alcohol is 70%, and elution time is 10min;
(5) calculating of eugenol content in pipe tobacco: at first with the detected object of standard solution and its respective concentration of comparing of interior target chromatographic peak area, carry out regretional analysis, obtain typical curve; The ratio of the object that then sample solution is detected and interior target chromatographic peak area, the substitution typical curve, namely obtain the eugenol concentration in sample, can calculate thus the content of eugenol in pipe tobacco.
2. the assay method of the middle eugenol content that cuts filler according to claim 1, it is characterized in that: the described preparation that contains interior mark extractant is to take 0.4 g anethole in 4000 mL ethanol solutions, and wherein the concentration of anethole is 1mg/mL.
3. the assay method of the middle eugenol content that cuts filler according to claim 1, it is characterized in that: the preparation of described standard solution is to take the 0.2g eugenol, be accurate to 0.0001g, be transferred in the 100mL volumetric flask with containing after interior target extractant dissolves, and constant volume, be decided to be standard reserving solution; Pipette respectively 0.01mL, 0.1mL, 0.5mL, 1.0mL, 2.5mL, 5.0mL standard reserving solution in the 10mL volumetric flask, use the extractant constant volume, the concentration of eugenol is 2.0 μ g/mL, 20 μ g/mL, 100 μ g/mL, 200 μ g/mL, 500 μ g/mL, 1000 μ g/mL.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103336085B (en) * | 2013-07-10 | 2014-10-22 | 江苏中烟工业有限责任公司 | High performance liquid chromatography-based method used for detection of eugenol in cigarette main stream smoke |
| CN103344734B (en) * | 2013-07-10 | 2015-01-21 | 江苏中烟工业有限责任公司 | Method for measuring eugenol in edible flavor and fragrance based on gas chromatography-mass spectrography |
| CN103399089B (en) * | 2013-07-10 | 2015-02-25 | 江苏中烟工业有限责任公司 | Method for measuring eugenol in edible flavour and fragrance based on high performance liquid chromatography |
| CN107192767A (en) * | 2016-03-15 | 2017-09-22 | 中国水产科学研究院 | The method that isotopic dilution gaschromatographic mass spectrometry determines eugenol in aquatic products |
| CN105954434B (en) * | 2016-07-19 | 2018-08-07 | 吉林烟草工业有限责任公司 | A kind of detection method of phenols fragrance |
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