CN106053659A - Method for measuring ratio of nicotine carbon, hydrogen and nitrogen stable isotopes in tobacco - Google Patents

Method for measuring ratio of nicotine carbon, hydrogen and nitrogen stable isotopes in tobacco Download PDF

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CN106053659A
CN106053659A CN201610518583.6A CN201610518583A CN106053659A CN 106053659 A CN106053659 A CN 106053659A CN 201610518583 A CN201610518583 A CN 201610518583A CN 106053659 A CN106053659 A CN 106053659A
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nicotine
tobacco
hydrogen
delta
isotope ratio
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韩书磊
陈欢
马潇
付亚宁
刘彤
侯宏卫
胡清源
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National Tobacco Quality Supervision and Inspection Center
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/025—Gas chromatography

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Abstract

A method for measuring ratio of nicotine carbon, hydrogen and nitrogen stable isotopes in tobacco is characterized in that extracting nicotine from tobacco by using an n-hexane and sodium hydroxide solution system, performing liquid sample injection, separating the nicotine from other impurities in extract by using a gas chromatograph fitted with a capillary chromatographic column, analyzing carbon stable isotope ratio Delta 13C, hydrogen stable isotope ratio (Delta D) and nitrogen stable isotope ratio Delta 15N in the nicotine by using a gas chromatography and isotope ratio mass spectrograph (GC-IRMS), and calibrating and calculating the values Delta 12C, Delta D and Delta 15N of the nicotine in tobacco according to measured values Delta 12C, Delta D and Delta 15N in nicotine isotope standard matters. By using GC-IRMS technology, nicotine carbon, hydrogen and nitrogen stable isotope ratio in tobacco can be measured quickly and accurately, and a technique is provided for tracing tobacco products.

Description

Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon, hydrogen, the assay method of nitrogen Stable isotope ratio
Technical field
The invention belongs to Stable Isotopic Analysis technical field, be specifically related to a kind of Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon, hydrogen, nitrogen stably same The assay method of position element ratio.
Background technology
Nicotiana tabacum L. is the basis of China's production of cigarettes, and the quality of Nicotiana tabacum L. interior quality directly affects the quality of cigarette product.It is subject to Home environment, such as the impact of the factors such as longitude and latitude, height above sea level, humiture, precipitation, soil types, different growing area quality of tobacco Widely different, style and features is totally different.The suction taste feature of various places Nicotiana tabacum L. is made full use of, prominent cigarette brand in cigarette tobacco leaf formulation Style and features and flavor quality, for do big, do specially, cigarette product of running business into strong one significant.But, up to now, it is difficult to Accomplishing the suction taste feature of various places Nicotiana tabacum L. is carried out quantitative analysis and description, tobacco productive corporation also is difficult to provide Nicotiana tabacum L. supplier Raw tobacco material carry out place of production judgement and identification.Therefore, adulterate, fill Nicotiana tabacum L. that is excellent and that obscure the place of production occur now and then with bad, Cause the instability of cigarette enterprise product quality and direct economic loss.Therefore, the differentiation to the Nicotiana tabacum L. place of production is studied and is had weight Want meaning, it is necessary to method for distinguishing can be sentenced in the Nicotiana tabacum L. place of production by research one.
Currently, the research differentiated the Nicotiana tabacum L. place of production both at home and abroad is relatively fewer, mainly has a sense organ assessment method, infrared spectrometry, Characteristic chemical componential analysis etc..Wherein, sensory evaluation method is subjective, infrared spectrometry and characteristic chemical componential analysis Research the most, but its preciseness and accuracy still Shortcomings.In the method traced to the source in the food place of production, stablize coordination Element technology is the analysis means of a relative efficiency.Due to the isotopics climate in organism, environment and biological metabolism The impact of the factors such as type, in the raw-food material in variety classes and different geographical source, isotope natural abundance there are differences, profit Different types of product and possible area of source thereof can be distinguished by this species diversity.
Stable isotope technology is relatively fewer in the research of Nicotiana tabacum L. and differentiation field, Nicotiana tabacum L. original producton location.Jamin etc. utilize steady Determine isotope technology, successfully the Nicotiana tabacum L. from South America, Europe, South Asia region and Africa is carried out subregion (Plant, Cell Environ. 1997, 20(5): 589-599.), but, this method needs complicated nicotine to extract and purification process, and Only carbon and two kinds of elements of nitrogen to Performance Liquid Chromatography Analysis for Nicotine in Tobacco carries out isotope ratio pH-value determination pH.This patent provides one and utilizes GC-IRMS Method quickly, efficiently measures Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon, hydrogen, the method for nitrogen Stable isotope ratio, for tracing back of Nicotiana tabacum L. and tobacco product Source provides technical support and scientific basis.
Summary of the invention
The purpose of the present invention is based on above-mentioned technical deficiency, establishes a kind of Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon, hydrogen, nitrogen stablize coordination The assay method of element ratio.This method is without locating before the sample pretreatment process, and a sample such as complicated nicotine extraction, purification Reason process can measure the carbon of Performance Liquid Chromatography Analysis for Nicotine in Tobacco, hydrogen, nitrogen Stable isotope ratio simultaneously, have easy and simple to handle, efficiency is high, repeat The advantage that property is good.
Hereinafter this method is described in detail.
It is an object of the invention to be achieved through the following technical solutions: a kind of Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon, hydrogen, nitrogen stablize coordination The assay method of element ratio, after the method uses normal hexane/sodium hydrate aqueous solution system to be extracted by the nicotine in Nicotiana tabacum L. Carry out Liquid sample introduction, use the gas chromatograph being furnished with capillary chromatographic column to be separated with other impurity in extract by nicotine, Gas chromatogram isotope mass spectrometer (GC-IRMS) is utilized to analyze the carbon stable isotope ratio delta of Performance Liquid Chromatography Analysis for Nicotine in Tobacco13C, stabilized hydrogen Isotopic ratio (δ D) and nitrogen Stable isotope ratio δ15N, further according to the δ in nicotine isotopic standard material12C, δ D and δ15N Measured value, the δ of calibration calculations Performance Liquid Chromatography Analysis for Nicotine in Tobacco12C, δ D and δ15N value.
More specifically, the inventive method comprises the following steps:
(1) using normal hexane/sodium hydrate aqueous solution system to be extracted by the nicotine in Nicotiana tabacum L., concrete mode is for weighing about 1 G tobacco sample, is placed in 100 mL triangular flasks, is separately added into 20 mL sodium hydroxide solutions (5 mol/L) and 20 mL normal hexane, It is placed in oscillation extraction 60 min on agitator.Take upper organic layer after static and carry out GC-IRMS analysis.
(2) gas chromatograph with capillary gas chromatographic column is used to be divided with other impurity in extract by nicotine Open；
(3) with online burner, the C in nicotine is changed into CO2, the N in nicotine is changed into N2, use isotope mass spectrometer (IRMS) measure nicotine and produce CO2In δ13C and N2In δ15N value；
(4) online burner is switched to thermal transition pattern, measure nicotine with isotope mass spectrometer (IRMS) and produce H2In δ D value (i.e. δ2H).
(5) δ demarcated by standard substance13C, δ D and δ15N value, obtains the δ of Performance Liquid Chromatography Analysis for Nicotine in Tobacco13C, δ D and δ15N value.
The instrumental conditions of described method is:
(1) GC conditions:
Chromatographic column: middle polarity capillary chromatographic column (30 mm × 0.25, m × 0.32 μm)；Detector temperature: 250 ℃；Injector temperature: 250 DEG C；Carrier gas: nitrogen (purity >=99.999%), constant current flow velocity: 2.0 mL/min；Sample size: 1 μ L, split ratio: 10:1(measure δ D and δ15Not shunt mode is used) during N；Heating schedule: initial temperature 80 DEG C, keeps 1 min, With the speed of 15 DEG C/min to 260 DEG C, keep 6 min.
(2) online burner condition:
Measure δ13C or δ15N: using oxidizing fire pipe, arranging temperature is 1000 DEG C.Nickel/nickel oxide (Ni/NiO) is loaded in pipe, Weak oxidant made by nickel oxide, and the C in sample is oxidized to CO2, N in sample is oxidized to NxOy, meanwhile, nickel oxide is converted into nickel Simple substance, is filled with O2Renewable nickel oxide, it addition, nickel simple substance can make special reducing agent, by NxOyIt is converted into N2, sample end reaction Generate CO2And N2.Oxidizing fire pipe should inject O before using2 Regeneration for oxidation tube in one hour.
Measure δ15N, need to use liquid nitrogen cold trap to freeze to remove CO2High-solidification point gas, prevents CO2Enter ion source and get broken Sheet ion [CO+] m/z 28,29 is to [N2+] m/z 28,29 interferes.
Measure δ D: using Pintsch process pipe, arranging temperature is 1450 DEG C.Pintsch process pipe should inject CH before using4One Hour for reaction tube utter misery.
(3) Mass Spectrometry Conditions is:
Ion source vacuum is 1.8x 10-6 MBar, voltage 3.04 kV, electric current 1.50 mA.
The method of the present invention is without sample pretreatment process such as complicated nicotine extraction, purification, and a sample pretreatment process The carbon of Performance Liquid Chromatography Analysis for Nicotine in Tobacco, hydrogen, nitrogen Stable isotope ratio can be measured simultaneously, have easy and simple to handle, efficiency is high, reproducible Advantage, provides technical support and scientific basis for Nicotiana tabacum L. and tracing to the source of tobacco product.
Accompanying drawing explanation
Fig. 1 is δ in GC-IRMS bioassay standard solution nicotine13The ion flow graph spectrum of C, upper figure vertical coordinate is ratio (Ratio), figure below vertical coordinate is intensity (Intensity [mV]);
Fig. 2 is δ in GC-IRMS bioassay standard solution nicotine15The ion flow graph spectrum of N, upper figure vertical coordinate is ratio (Ratio), figure below vertical coordinate is intensity (Intensity [mV]);
Fig. 3 is the ion flow graph spectrum of δ D in GC-IRMS bioassay standard solution nicotine, and upper figure vertical coordinate is ratio (Ratio), Figure below vertical coordinate is intensity (Intensity [mV]).
Detailed description of the invention
The present invention is further described by specific examples below, but is not intended to the present invention.
Embodiment 1:
1, instrument and reagent
Isotope mass spectrometer (AS1310 automatic sampler, Trace GC Ultra gas chromatograph, GC IsoLink burning and heat Reforming unit (burner is used for measuring the carbon in nicotine and nitrogen, and heat conversion device is used for measuring the hydrogen in nicotine), Delta V Advantage stable isotope compares mass spectrograph.Thermo Fisher company of the U.S.)；AE163 electronic balance (sensibility reciprocal: 0.0001 G, Mettler company of Switzerland)；High-purity helium (99.999%, carrier gas)；Nicotine standard substance comes from indiana ,US university Isotopic laboratory, #2(δ13C=7.72 ± 0.02 ‰, δ15N=-5.94 ± 0.15 ‰), #3(δ13C=-30.05 ± 0.02 ‰, δ15N=-33.62 ± 0.18 ‰), #4(δ13C=-2.06 ± 0.02 ‰, δ15N=-15.49 ± 0.13 ‰), #5(δ13C =-29.63 ± 0.01 ‰, δ15N=--6.03 ± 0.04 ‰, δ D=-161.30 ± 1.7 ‰)；Normal hexane (chromatographically pure, TEDIA company of the U.S.)； Sodium hydroxide (analytical pure, traditional Chinese medicines group)；Ultra-pure water (Milli-Q Integral ultrapure water system system), Y-8 velocity-modulated oscillator (Changzhou Guohua Electric Appliance Co., Ltd.).
2, sample pre-treatments
Weigh about 1 g tobacco sample, be placed in 100 mL triangular flasks, be separately added into 20 mL sodium hydroxide solutions (5 mol/L) and 20 mL normal hexane, are placed in oscillation extraction 60 min on agitator.Take upper organic layer after static and carry out GC-IRMS analysis.
3, instrumental conditions
(1) GC conditions:
Chromatographic column: middle polarity capillary chromatographic column (30 mm × 0.25, m × 0.32 μm)；Detector temperature: 250 ℃；Injector temperature: 250 DEG C；Carrier gas: nitrogen (purity >=99.999%), constant current flow velocity: 2.0 mL/min；Sample size: 1 μ L, split ratio: 10:1(measure δ D and δ15Not shunt mode is used) during N；Heating schedule: initial temperature 80 DEG C, keeps 1 min, With the speed of 15 DEG C/min to 260 DEG C, keep 6 min.
(2) burning conversion device condition:
Measure δ13C or δ15N: using oxidizing fire pipe, arranging temperature is 1000 DEG C.Nickel/nickel oxide (Ni/NiO) is loaded in pipe, Weak oxidant made by nickel oxide, and the C in sample is oxidized to CO2, N in sample is oxidized to NxOy, meanwhile, nickel oxide is converted into nickel Simple substance, is filled with O2Renewable nickel oxide, it addition, nickel simple substance can make special reducing agent, by NxOyIt is converted into N2, sample end reaction Generate CO2And N2.Oxidizing fire pipe should inject O before using2 Regeneration for oxidation tube in one hour.
Measure δ15N, need to use liquid nitrogen cold trap to freeze to remove CO2High-solidification point gas, prevents CO2Enter ion source and get broken Sheet ion [CO+] m/z 28,29 is to [N2+] m/z 28,29 interferes.
Measure δ D: using Pintsch process pipe, arranging temperature is 1450 DEG C.Pintsch process pipe should inject CH before using4One Hour for reaction tube utter misery.
(3) Mass Spectrometry Conditions is:
Ion source vacuum is 1.8x 10-6 MBar, voltage 3.04 kV, electric current 1.50 mA.
4, precision measures
After tobacco sample dilution extraction, by the δ of GC-IRMS technology replication Performance Liquid Chromatography Analysis for Nicotine in Tobacco of the present invention13C, δ D And δ15N value (n=6), the relative standard deviation (RSD) of measurement result respectively 0.83%, 0.42% and 0.93%(table 1), measure Precision all can reach requirement.
The precision of table 1. tri-kinds of elemental stable isotopes ratios of Performance Liquid Chromatography Analysis for Nicotine in Tobacco C, H, N measures
According to above-mentioned Gas Chromatographic Method, select a kind of tobacco sample A, record the δ of wherein nicotine13C, δ D and δ15N is respectively For-31.41 ,-211.80 and-7.51.
Embodiment 2
According to the assay method described in embodiment 1, select a kind of tobacco sample B, record the δ of wherein nicotine13C, δ D and δ15N divides Wei-31.17 ,-211.44 and-6.83.
Embodiment 3
According to the assay method described in embodiment 1, select a kind of tobacco sample C, record the δ of wherein nicotine13C, δ D and δ15N divides Wei-32.97 ,-229.05 and-5.51.

Claims (5)

1. a Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon, hydrogen, the assay method of nitrogen Stable isotope ratio, it is characterised in that: use normal hexane/hydrogen Aqueous solution of sodium oxide system carries out Liquid sample introduction after being extracted by the nicotine in Nicotiana tabacum L., use the gas being furnished with capillary chromatographic column Chromatography by other impurity in nicotine and extract separately, utilizes gas chromatogram isotope mass spectrometer GC-IRMS to analyze The carbon stable isotope ratio delta of Performance Liquid Chromatography Analysis for Nicotine in Tobacco13C, hydrogen stable isotope ratio delta D and nitrogen Stable isotope ratio δ15N, then root According to the δ in nicotine isotopic standard material12C, δ D and δ15N measured value, the δ of calibration calculations Performance Liquid Chromatography Analysis for Nicotine in Tobacco12C, δ D and δ15N value.
Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon the most according to claim 1, hydrogen, the assay method of nitrogen Stable isotope ratio, its feature exists In: specifically comprise the following steps that
(1) normal hexane/sodium hydrate aqueous solution system is used to be extracted by the nicotine in Nicotiana tabacum L.；
(2) use the gas chromatograph with capillary gas chromatographic column by other impurity in nicotine and extract separately；
(3) with online burner, the C in nicotine is changed into CO2, the N in nicotine is changed into N2, use isotope mass spectrometer (IRMS) measure nicotine and produce CO2In δ13C and N2In δ15N value；
(4) online burner is switched to thermal transition pattern, measure nicotine with isotope mass spectrometer IRMS and produce H2In δ D Value；
(5) δ demarcated by standard substance13C, δ D and δ15N value, obtains the δ of Performance Liquid Chromatography Analysis for Nicotine in Tobacco13C, δ D and δ15N value.
Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon the most according to claim 2, hydrogen, the assay method of nitrogen Stable isotope ratio, its feature exists In: the GC conditions in step (2) is:
Chromatographic column: middle polarity capillary chromatographic column, mm × 0.25, specification 30 m × 0.32 μm；Detector temperature: 250 ℃；Injector temperature: 250 DEG C；Carrier gas: nitrogen, constant current flow velocity: 2.0 mL/min；Sample size: 1 μ L, split ratio: 10:1, surveys Determine δ D and δ15Not shunt mode is used during N；Heating schedule: initial temperature 80 DEG C, keeps 1 min, with the speed of 15 DEG C/min To 260 DEG C, keep 6 min.
Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon the most according to claim 2, hydrogen, the assay method of nitrogen Stable isotope ratio, its feature exists In: the online burner condition used by step (3), (4) is:
Measure δ13C or δ15N: using oxidizing fire pipe, arranging temperature is 1000 DEG C, loads nickel/nickel oxide (Ni/NiO) in pipe, Weak oxidant made by nickel oxide, and the C in sample is oxidized to CO2, N in sample is oxidized to NxOy, meanwhile, nickel oxide is converted into nickel Simple substance, is filled with O2Renewable nickel oxide, it addition, nickel simple substance can make special reducing agent, by NxOyIt is converted into N2, sample end reaction Generate CO2And N2, oxidizing fire pipe should inject O before using2 Regeneration for oxidation tube in one hour；
Measure δ15N, need to use liquid nitrogen cold trap to freeze to remove CO2High-solidification point gas, prevents CO2Enter ion source get fragment from Son [CO+] m/z 28,29 is to [N2+] m/z 28,29 interferes；
Measuring δ D: using Pintsch process pipe, arranging temperature is 1450 DEG C, Pintsch process pipe should inject CH before using4One hour with In reaction tube utter misery.
Performance Liquid Chromatography Analysis for Nicotine in Tobacco carbon the most according to claim 2, hydrogen, the assay method of nitrogen Stable isotope ratio, its feature exists In: the extracting mode in step (1), particularly as follows: weigh about 1 g tobacco sample, is placed in 100 mL triangular flasks, is separately added into 20 ML concentration is sodium hydroxide solution and the 20 mL normal hexane of 5 mol/L, is placed in oscillation extraction 60 min on agitator；After static Take upper organic layer and carry out GC-IRMS analysis.
CN201610518583.6A 2016-07-05 2016-07-05 Method for measuring ratio of nicotine carbon, hydrogen and nitrogen stable isotopes in tobacco Pending CN106053659A (en)

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CN109470776A (en) * 2017-09-08 2019-03-15 上海市合成纤维研究所有限公司 A kind of discrimination method that regenerated celulose fibre pulp is traced to the source
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CN114755324A (en) * 2021-12-22 2022-07-15 中国科学院地球环境研究所 Organic monomer compound carbon isotope analysis system and corresponding analysis method

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CN108445072A (en) * 2018-03-01 2018-08-24 天津师范大学 The assay method of carbon and nitrogen stable isotope in a kind of high carbon-nitrogen ratio sample
CN108445072B (en) * 2018-03-01 2020-08-07 天津师范大学 Method for determining carbon-nitrogen stable isotope in high carbon-nitrogen ratio sample
CN111307923A (en) * 2020-02-06 2020-06-19 中国食品发酵工业研究院有限公司 Method for measuring stable hydrogen isotope ratio on propionic acid non-exchangeable hydrogen site
CN114755324A (en) * 2021-12-22 2022-07-15 中国科学院地球环境研究所 Organic monomer compound carbon isotope analysis system and corresponding analysis method

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