CN104280505A - High-performance liquid phase chromatography for simultaneously determining six forbidden and restricted additives in flavors and fragrances for cigarettes - Google Patents
High-performance liquid phase chromatography for simultaneously determining six forbidden and restricted additives in flavors and fragrances for cigarettes Download PDFInfo
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- CN104280505A CN104280505A CN201410591724.8A CN201410591724A CN104280505A CN 104280505 A CN104280505 A CN 104280505A CN 201410591724 A CN201410591724 A CN 201410591724A CN 104280505 A CN104280505 A CN 104280505A
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Abstract
The invention discloses a high-performance liquid phase chromatography for simultaneously determining six forbidden and restricted additives in flavors and fragrances for cigarettes. The high-performance liquid phase chromatography comprises the steps of firstly extracting the flavors and fragrances for the cigarettes in an oscillating manner by utilizing an extract solvent, standing the extract solution, and filtering the extract solution by utilizing a 0.45-micrometer organic phase filter membrane to obtain a to-be-tested sample solution; and preparing standard solutions of the six forbidden and restricted additives, detecting and analyzing the to-be-tested sample solution and the standard solutions by adopting a high-performance liquid phase chromatograph, carrying out the quantitative analysis in an external standard method, and calculating the content of the six forbidden and restricted additive in the flavors and fragrances for the cigarettes. By adopting the high-performance liquid phase chromatography, the six forbidden and restricted ingredients such as sesamol, coumarin, beta-naphthol, beta- asarone, safrole and musk xylene can be simultaneously detected, simplicity and rapidness in operation can be achieved, a satisfactory separation effect and qualitative detection limit can be acquired, and the reproducibility is good. The high-performance liquid phase chromatography can be used for rapidly, accurately and simultaneously detecting the six forbidden and restricted additives in the flavors and fragrances for the cigarettes, and a method reference can be provided for the supervision and inspection of the flavor and fragrance additives for the cigarettes.
Description
Technical field
The present invention relates to the detection method that in essence spice for cigarette, 6 kinds limit with adjuvant, refer to the high performance liquid chromatography that in a kind of Simultaneously test essence spice for cigarette, 6 kinds limit with adjuvant particularly.
Background technology
In cigarette, add essence and flavoring agent, its role is to improve the physicochemical property of cigarette, mellow and full fragrance, modification cigarette fragrance, improve the style of inhaling taste, establishing product.Essence spice for cigarette extracts from band Studies of The Aromatic Substances or obtain with artificial synthesis and for the general name of the flavor matter in tobacco, belongs to food additives category.Under normal circumstances, essence spice for cigarette to be allocated according to certain ratio and technique by multiple spices and appropriate solvent and other adjuvants to form, and is added in cigarette to play to improve cigarette pungency, cover assorted gas, improve the effects such as cigarette sensory quality.In adjuvant, because sesamol, cumarin, betanaphthol, beta-Asarone, safrole and musk xylene all have carcinogenesis, be therefore prohibited or limit use.
At present, in essence spice for cigarette, the detection method of these 6 kinds of adjuvants is based on GC-MS(gas chromatography-mass spectrography) (GC/MS), and tobacco business has promulgated the examination criteria of betanaphthol, beta-Asarone respectively, all adopts the method for GC/MS; Li Jing etc. utilize the method for GC-MS to determine sesamol in essence and flavoring agent; The detection of cumarin, safrole adopts the method for GC/MS a lot; Liao Huiyun etc. use the method for deuterated interior mark GC-MS to detect musk xylene in essence and flavoring agent respectively.Method due to GC/MS measures these 6 kinds and limits with adjuvant required time all longer, and the adjuvant boiling points such as cumarin are higher, easily cause residual.Therefore, need badly a kind of can simultaneously and detect the detection methods that in essence spice for cigarette, 6 kinds limit with adjuvant fast.
Summary of the invention
Object of the present invention is exactly to overcome the deficiency existing for prior art, and provide the high performance liquid chromatography that in a kind of Simultaneously test essence spice for cigarette, 6 kinds limit with adjuvant, the quality and safety control be intended to for essence spice for cigarette provides reference.
For achieving the above object, 6 kinds of high performance liquid chromatographies limited with adjuvant in Simultaneously test essence spice for cigarette provided by the present invention: first essence spice for cigarette extraction solvent is carried out oscillation extraction, cross 0.45 μm of organic phase filter membrane after leaving standstill, obtain testing sample solution; Prepare 6 kinds of standard solution limited with adjuvant simultaneously, adopt high performance liquid chromatograph to carry out detection to testing sample solution and standard solution respectively again to analyze, then carry out quantitative test with external standard method, calculate the content that in essence spice for cigarette, 6 kinds limit with adjuvant; Described 6 kinds to limit with adjuvant be sesamol, cumarin, betanaphthol, beta-Asarone, safrole and musk xylene; Wherein, the testing conditions of high performance liquid chromatograph:
Adopt chromatographic column to be Phenomenex Luna C18 post, it is of a size of 150mm × 4.6mmi.d., 5 μm,
column temperature is 30 DEG C; Determined wavelength is 214nm;
Adopt gradient elution, mobile phase A is water, and Mobile phase B is acetonitrile; Flow velocity is 1mL/min; Sample size: 20 μ L; Gradient elution program is as follows, and wherein following ratio is volume ratio:
During 0min, mobile phase A: Mobile phase B=60:40;
During 5min, mobile phase A: Mobile phase B=5:95;
During 12min, mobile phase A: Mobile phase B=5:95;
During 14min, mobile phase A: Mobile phase B=60:40;
During 15min, mobile phase A: Mobile phase B=60:40.
When essence spice for cigarette extraction solvent is carried out oscillation extraction by the present invention, described extraction solvent is methyl alcohol, ethanol and acetonitrile, and the speed of oscillation extraction is 200r/min, oscillation extraction time 20 ~ 30min; Preferably, extraction solvent is ethanol, and the extraction ratio of described essence spice for cigarette and extraction solvent is 0.05g/mL, oscillation extraction time 20min.
Beneficial effect of the present invention is: the present invention can detect sesamol, cumarin, betanaphthol, beta-Asarone, safrole and musk xylene 6 kinds in essence spice for cigarette sample simultaneously and limit with composition, simple and quick, obtain satisfied separating effect and qualitative detection limit, favorable reproducibility, the recovery is 91.9% ~ 107.5%, and relative standard deviation (RSD) is less than 10%.The present invention can to meet in essence spice for cigarette 6 kinds and limit and detect fast and accurately with adjuvant simultaneously, is the reference of essence spice for cigarette adjuvant supervision and inspection supplying method.
Accompanying drawing explanation
Fig. 1 is the liquid chromatogram of standard solution.
Fig. 2 is the liquid chromatogram of sample solution.
Embodiment
In order to explain the present invention better, below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, but they do not form restriction to the present invention.
Embodiment
The material that the present embodiment adopts, reagent and instrument are as follows:
Dionex P680 high performance liquid chromatograph (DIONEX company of the U.S.), joins DAD detecting device; AL204 electronic balance (sensibility reciprocal: 0.0001g, METTLER TOLEDO company of Switzerland); Milli-Q Advantage A10 ultrapure water instrument (Millipore company of the U.S.); HY-5A Clothoid type oscillator (city east, Jintan City Xin Rui instrument plant).
6 kinds of adjuvant standard items: sesamol (purity >=98.0%, Shanghai Nature Standard Biotechnology Co., Ltd.), betanaphthol (purity >=99.0%, Dr.Ehrenstorfer GmbH company of Germany), beta-Asarone (purity >=71.0%, Dr.Ehrenstorfer GmbH company of Germany, safrole (purity 99.4%, Chem Sevvice company of the U.S.); Cumarin (purity 99.4%, Sigma Co., USA); Musk xylene (the 100ng/ μ L cyclohexane solution of 1mL, German Dr.Ehrenstorfer GmbH company).
Methyl alcohol, ethanol, acetonitrile (CP, German CNW company), water is ultrapure water.
Assay method is as follows:
First take 0.5g essence spice for cigarette sample in 50mL conical flask, accurately add 10mL ethanol, on the oscillator with 200r/min rate oscillation extraction 20min, cross 0.45 μm of organic phase filter membrane after leaving standstill, obtain testing sample solution; Prepare 6 kinds of standard solution limited with adjuvant simultaneously, adopt high performance liquid chromatograph to carry out detection to testing sample solution and standard solution respectively again to analyze, then carry out quantitative test with external standard method, calculate the content that in essence spice for cigarette, 6 kinds limit with adjuvant; Wherein, the testing conditions of high performance liquid chromatograph:
Adopt chromatographic column to be Phenomenex Luna C18 post, it is of a size of 150mm × 4.6mmi.d., 5 μm,
column temperature is 30 DEG C; Determined wavelength is 214nm;
Adopt gradient elution, mobile phase A is water, and Mobile phase B is acetonitrile; Flow velocity is 1mL/min; Sample size: 20 μ L; Gradient elution program is as follows, and wherein following ratio is volume ratio:
During 0min, mobile phase A: Mobile phase B=60:40;
During 5min, mobile phase A: Mobile phase B=5:95;
During 12min, mobile phase A: Mobile phase B=5:95;
During 14min, mobile phase A: Mobile phase B=60:40;
During 15min, mobile phase A: Mobile phase B=60:40.
The trust adopting method of the present invention to have detected Hubei Province's tobacco product quality monitoring testing station acceptance detects sample and keeps sample, and does not all detect above-mentioned 6 kinds of adjuvants, as shown in Figure 2 in 19 essence spice for cigarette samples.Illustrate that tobacco business comparatively strictly controls limiting to have with adjuvant, sesamol, cumarin, betanaphthol, beta-Asarone, safrole and musk xylene these 6 kinds limits and compares specification with the use of adjuvant in tobacco business.
The selection of sample pre-treatments condition and optimization
1, the selection of extraction solvent
For selecting suitable extraction solvent, by adding calibration method in same sample, methyl alcohol, ethanol, acetonitrile is selected to be that extraction solvent is tested.Result shows, the extraction efficiency of methyl alcohol, ethanol and acetonitrile is suitable, and consider that the toxicity of methyl alcohol and acetonitrile is comparatively large, preferred alcohol of the present invention is extraction solvent.
2, the selection of volume is extracted
Extraction volume is investigated on the impact of experiment by adding calibration method in same sample, the ethanol (5,10,15 and 20mL) adding 4 kinds of different volumes respectively extracts, when finding to select 5mL, extract must be centrifugal, complex operation, when extracting volume and being larger, signal value is less, is subject to baseline noise impact, for the accuracy taking into account the complete of sample extraction and detect, the preferred extractant volume of the present invention is 10mL.
3, way choice is extracted
In essence spice for cigarette, adjuvant extraction mode is more, and the present invention investigates effect of extracting that is ultrasonic and vibration two kinds of modes in an experiment.Take ethanol as extractant, for selecting suitable extraction mode, choosing same sample, investigating by adding calibration method in the sample to which.Result shows, 25 DEG C time, vibration is suitable with ultrasonic extraction effect, and consider that ultrasonic extraction temperature is wayward, therefore, the present invention selects the mode of oscillation extraction.
4, the selection of extraction time
By adding calibration method in the sample to which, when other conditions are constant, the present invention has investigated duration of oscillation when being respectively 10,15,20,25 and 30min in an experiment, on the impact of extraction efficiency.Result shows, along with the increase of duration of oscillation, the concentration of the adjuvant be detected increases gradually, and after duration of oscillation reaches 20min, continuing increase time extraction efficiency does not have significant change.Therefore, the present invention selects duration of oscillation to be 20min.
The optimization of chromatographic condition
The present invention is that 200nm, 214nm, 254nm, 280nm and 298nm investigate to determined wavelength respectively, and find when 214nm, 6 kinds of adjuvant responses in standard model are higher.The present invention has also investigated the separation case of sample under the different systems such as acetonitrile-water, methanol-water, acetonitrile-acetic acid aqueous solution and ratio mobile phase, find when flow visualizing selects acetonitrile-water to be system gradient elution, 6 kinds of adjuvant separating effects the bests in standard model and actual sample.Under condition of gradient elution, 6 kinds of adjuvants have optimal separation effect, and respectively as shown in Figure 1, 2, wherein beta-Asarone is due to the existence of isomeride, and figure is bimodal, as shown in Figure 1 for the chromatogram of standard solution and the chromatogram of typical sample solution.
Working curve and detectability
Take ethanol as solvent, prepare the series standard mixed solution that 6 kinds of additive concentrations are respectively 0.1,0.2,0.5,1,2,5,10 and 20 μ g/ml, and respectively HPLC analysis is carried out to it.Adopt quantified by external standard method, with the peak area of each object (Y) to its respective concentration (X, μ g/ml) carry out regretional analysis, and qualitative detection limit (LOD) and the quantitative detection limit (LOQ) of each analysis thing is determined by 3 times of signal to noise ratio (S/N ratio)s (S/N) and 10 times of S/N, the equation of linear regression obtained, related coefficient and detectability, as shown in table 1.
The working curve of table 16 kinds of adjuvants and detection limit
As shown in Table 1, the linear relationship of the typical curve of 6 kinds of adjuvants is good, r=0.9994 ~ 0.9999; The LOD of 6 kinds of adjuvants is 0.05 ~ 0.47mg/kg, LOQ is 0.17 ~ 1.58mg/kg.In summary, the high-efficient liquid phase chromatogram condition adopted makes 6 kinds of adjuvants be separated very well, and typical curve has good linear relationship, and the sensitivity of method is higher, is suitable for quantitative test.
Repeatability and the recovery
6 parallel laboratory tests are carried out to same sample, adds the standard solution of 6 kinds of adjuvants of 2 μ g/mL, according to the relative standard deviation (RSD) of measured value after the recovery of standard addition of result computing method and mark-on, as shown in table 2.Table 2 result shows, the method recovery of the present invention is high, reproducible.
The recovery of table 2 method and repeatability
Claims (3)
1. in Simultaneously test essence spice for cigarette 6 kinds limit the high performance liquid chromatography with adjuvant, it is characterized in that: first essence spice for cigarette extraction solvent is carried out oscillation extraction, cross 0.45 μm of organic phase filter membrane after leaving standstill, obtain testing sample solution; Prepare 6 kinds of standard solution limited with adjuvant simultaneously, adopt high performance liquid chromatograph to carry out detection to testing sample solution and standard solution respectively again to analyze, then carry out quantitative test with external standard method, calculate the content that in essence spice for cigarette, 6 kinds limit with adjuvant; Described 6 kinds to limit with adjuvant be sesamol, cumarin, betanaphthol, beta-Asarone, safrole and musk xylene; Wherein, the testing conditions of high performance liquid chromatograph:
Adopt chromatographic column to be Phenomenex Luna C18 post, it is of a size of 150mm × 4.6mmi.d., 5 μm,
column temperature is 30 DEG C; Determined wavelength is 214nm;
Adopt gradient elution, mobile phase A is water, and Mobile phase B is acetonitrile; Flow velocity is 1mL/min; Sample size: 20 μ L; Gradient elution program is as follows, and wherein following ratio is volume ratio:
During 0min, mobile phase A: Mobile phase B=60:40;
During 5min, mobile phase A: Mobile phase B=5:95;
During 12min, mobile phase A: Mobile phase B=5:95;
During 14min, mobile phase A: Mobile phase B=60:40;
During 15min, mobile phase A: Mobile phase B=60:40.
2. in Simultaneously test essence spice for cigarette according to claim 16 kinds limit the high performance liquid chromatography with adjuvant, it is characterized in that: when essence spice for cigarette extraction solvent is carried out oscillation extraction, described extraction solvent is methyl alcohol, ethanol and acetonitrile, the speed of oscillation extraction is 200r/min, oscillation extraction time 20 ~ 30min.
3. in Simultaneously test essence spice for cigarette according to claim 16 kinds limit the high performance liquid chromatography with adjuvant, it is characterized in that: when essence spice for cigarette extraction solvent is carried out oscillation extraction, described extraction solvent is ethanol, the extraction ratio of described essence spice for cigarette and extraction solvent is 0.05g/mL, the speed of oscillation extraction is 200r/min, oscillation extraction time 20min.
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| EP1208754A1 (en) * | 2000-11-21 | 2002-05-29 | Givaudan SA | Particulate material |
| CN102998382A (en) * | 2012-10-17 | 2013-03-27 | 国家烟草质量监督检验中心 | Method for measuring contents of methanol, sec-butyl alcohol, sesamol and musk xylene in additive for cigarette |
| CN103149311A (en) * | 2013-01-30 | 2013-06-12 | 福建中烟工业有限责任公司 | Measuring method of sesame phenol content in tobacco essence perfume |
| CN103983725A (en) * | 2014-05-22 | 2014-08-13 | 广西中烟工业有限责任公司 | Quick measurement method for coumarin and safrole in essence and flavor |
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Patent Citations (4)
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| EP1208754A1 (en) * | 2000-11-21 | 2002-05-29 | Givaudan SA | Particulate material |
| CN102998382A (en) * | 2012-10-17 | 2013-03-27 | 国家烟草质量监督检验中心 | Method for measuring contents of methanol, sec-butyl alcohol, sesamol and musk xylene in additive for cigarette |
| CN103149311A (en) * | 2013-01-30 | 2013-06-12 | 福建中烟工业有限责任公司 | Measuring method of sesame phenol content in tobacco essence perfume |
| CN103983725A (en) * | 2014-05-22 | 2014-08-13 | 广西中烟工业有限责任公司 | Quick measurement method for coumarin and safrole in essence and flavor |
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| PAOLA CURRÒ ET AL: "Determination of β-asarone, safrole, isosafrole and anethole in alcoholic drinks by high-performance liquid chromatography", 《JOURNAL OF CHROMATOGRAPHY A》, vol. 404, 31 December 1987 (1987-12-31), pages 273 - 278 * |
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