CN104090042A - Method for measuring safrole in mainstream smoke of cigarettes - Google Patents

Abstract

The invention discloses a method for measuring safrole in mainstream smoke of cigarettes. The method comprises the steps of preparation of an internal standard solution, a standard working solution and a sample solution, gas chromatography-tandem mass spectrometry analysis, calculation of measurement results and the like. According to the invention, N-heptadecane is used as an internal standard substance for quantifying, the optimized detection method is extremely sensitive in response and accurate in quantitative analysis and effectively reduces the interference caused by complex sample matrix; the adopted chromatography and mass spectrometry conditions enable the target substance to have good signal response and good linear correlation, the detection limit is 0.015ng/cigarette, the relative standard deviation (RSD) is 6.35%, and the adding standard recovery is 87.78-110.9%, thus the method is high in sensitivity, good in repeatability and high in recovery rate and is suitable for quantitative analysis of the safrole serving as a trace target substance in the complex matrix of the smoke.

Description

A kind of method of measuring safrole in cigarette mainstream flue gas

Technical field

The invention belongs to cigarette physical and chemical index detection technique field, be specifically related to a kind of method of measuring safrole in cigarette mainstream flue gas.

Background technology

Safrole (Safrole) is colourless or light yellow liquid.There is camphorwood smell, be soluble in ethanol, can be miscible with chloroform, ether, water insoluble and glycerine.Relative density is 1.096, approximately 11 ℃, solidifying point, 232～234 ℃ of boiling points, index of refraction 1.5383,97 ℃ of flash-points, median lethal dose (rat, per os) 1950mg/kg.

Safrole be much edible natural essence as yellow camphor tree essential oil, aniseed essential oil and camphor main body of oil, account for 80% of yellow camphor tree essential oil.Safrole also has a small amount of existence in the essence made from spices such as nutmeg, Japanese asarum, California bay trees.Yellow camphor tree essential oil is often used as the local flavor adding ingredient of beer and other wine, in addition, contains more safrole in rotten ginger.Food and Drug Administration (FDA) studies show that, safrole is the hepatocarcinogen of white mouse and mouse.In the feed of mouse, add 0.04～1% safrole, within 150d to 2 year, can produce liver cancer by inducing mouse.Safrole is that active carcinogenic process is more clearly at present through metabolic conversion, safrole in Mice Body first metabolism be phenylethyl alcohol form, then be activated and be converted into acetate or sulfate, become final carcinogenic substance, two keys of the latter, because its electrophilicity and hereditary material DNA react, finally can cancerogenicly occur.In view of the above results, in the U.S., no longer allow safrole as food additive.

Cigarette, as a kind of fast-moving consumer goods, will add some essence feed liquid adjuvants in process of production.Due to may unexpected introducing safrole in cigarette additive, therefore, for the monitoring management of safrole, just seem particularly necessary.Patent 201010539732.X provides the assay method of cumarin and safrole content in a kind of essence spice for cigarette.After it is first scattered in aqueous solution by cumarin in essence spice for cigarette and safrole, use organic reagent to be extracted, after sampling filtering, use gas chromatography-mass spectrography carry out instrumental analysis and measure content.Patent 201210332468.1 provides the assay method of cumarin and safrole content in a kind of essence and flavoring agent.With being added with target ethanol solution dilute sample in methyl eugenol, after sample filtering, use gas chromatography/mass spectrometry to select ion method to measure after dilution the content of cumarin and safrole in sample.

For the analyzing and testing of safrole, existing method is mainly studied the safrole in plant, cosmetics and essence and flavoring agent, and the instrument analytical method adopting mainly contains GC, GC/MS, and HPLC.The Sample Pretreatment Technique adopting, according to the difference of sample substrate, mainly contains solvent extraction, Solid-Phase Extraction, steam distillation, supercritical fluid extraction, solid-phase microextraction etc.But, for cigarette sample, be limited in flue gas safrole burst size level low, and the impact of flue gas complex matrices on the factor such as the Analysis interference of object is serious, the burst size of Accurate Determining safrole is the work that has challenge of one, flue gas analysis field.

Summary of the invention

Goal of the invention: for the deficiencies in the prior art, the object of this invention is to provide a kind of method of measuring safrole in cigarette mainstream flue gas, to accurately detecting the content of safrole in cigarette mainstream flue gas, measurement result is accurate, matrix interference is few.

Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention is as follows:

A method of measuring safrole in cigarette mainstream flue gas, comprises the following steps:

(1) preparation of inner mark solution: take heptadecane as internal standard compound, make solvent with ethanol, prepare inner mark solution;

(2) preparation of standard operation solution: take safrole as standard items, make solvent with ethanol, be prepared into standard inventory solution through stepwise dilution, then add respectively quantitative inner mark solution, be prepared into standard operation solution;

(3) preparation of sample solution: collected the object in cigarette smoke, accurately added quantitative inner mark solution and extraction solvent, mechanical shaking extraction; Shift whole extracts, through Solid-Phase Extraction column purification, after scavenging solution is concentrated, obtain sample solution;

(4) gas chromatography-Tandem Mass Spectrometry Analysis: standard operation solution and sample solution are detected to analysis with gas chromatography-tandem mass spectrometer (GC-MS/MS); Chromatographiccondition is: adopt DB-5MS (30m * 0.25mm * 0.25 μ m) capillary chromatographic column; Carrier gas is helium, and constant current speed is 1mL/min; Sample size is 1 μ L; Injector temperature is 250 ℃; Transmission line temperature is 280 ℃; Adopt pulse Splitless injecting samples, pulse is 25psi; Heating schedule is that initial temperature is 60 ℃, is increased to 160 ℃, then is increased to 300 ℃ with the speed of 10 ℃/min with the speed of 4 ℃/min.Its mass spectrophotometry condition is: ionization mode is EI source, positive ion mode; Ion source temperature: 230 ℃; Quadrupole rod temperature: be 150 ℃; Collision gas: nitrogen, flow velocity 1.5mL/min, carrier gas (helium) flow velocity is 2.25mL/min; Multiple-reaction monitoring (MRM) pattern, detail parameters is in Table 1;

The MRM parameter list of table 1 object

(5) drafting of standard working curve and the calculating of sample result: the safrole in standard operation solution of take is horizontal ordinate with the ratio of interior target concentration, the safrole in chromatogram of take is ordinate with the ratio of interior target peak area, carry out linear regression analysis, obtain standard working curve.By safrole in the sample solution recording under the same terms and interior target chromatographic peak area ratio, substitution standard working curve, converts and tries to achieve the content of safrole in cigarette sample main flume.

The preparation of described inner mark solution specifically comprises the following steps: mark storing solution in (1): accurately take 200mg internal standard compound heptadecane, to 100mL, obtaining concentration is the interior mark storing solution of 2000 μ g/mL with ethanol constant volume.(2) inner mark solution: accurately pipette mark storing solution in 0.1mL, to 100mL, obtain the inner mark solution that concentration is 2 μ g/mL with ethanol constant volume.

The preparation of described standard operation solution specifically comprises the following steps: (1) primary standard storing solution: accurately take 100mg safrole, with ethanol constant volume, to 100mL, obtaining concentration is the primary standard storing solution of 1000 μ g/mL.(2) secondary standard storing solution: accurately pipette 0.1mL primary standard storing solution, to 100mL, obtaining concentration is the secondary standard storing solution of 1 μ g/mL with ethanol constant volume.(3) standard operation solution: accurately pipette respectively secondary standard storing solution 20 μ L, 50 μ L, 100 μ L, 400 μ L and 1000 μ L, and each 100 μ L of inner mark solution, with ethanol, as solvent, be settled in the volumetric flask of 100mL, obtain series standard working solution.This series standard solution concentration is respectively 0.2ng/mL, 0.5ng/mL, 1ng/mL, 4ng/mL and 10ng/mL, and wherein interior mark concentration is 2ng/mL.

The preparation of described sample solution specifically comprises the following steps: (1) cigarette smoking: the TPM of collecting 20 cigarette according to the requirement of GB/T19609 standard.(2) sample extraction: the trapping that upper step is obtained has the glass fiber filter of TPM to be positioned over that in 100mL conical flask, (filter disc is answered full wafer to put into conical flask and paved, can not shred or tear up), accurately add 40 μ L inner mark solutions, and add 40mL normal hexane, be placed in Clothoid type oscillator oscillation extraction 40min under 160 turn/min conditions, standing a moment.(3) extract purifies: extract is all transferred to solid-phase extraction column (silica gel that filler is 500mg), until extract toward be displaced downwardly to extraction pillar top after, divide and add for 3 times 10mL normal hexane to carry out drip washing, drip washing speed remains on 1～2mL/min, collects all leacheates.(4) leacheate is concentrated: the concentrated bottle that fills all leacheates is connected on Rotary Evaporators; rotary evaporation under 80 ℃, high pure nitrogen protection (or under 55 ℃, vacuum condition rotary evaporation); be concentrated to 1mL left and right, obtain sample feeding liquid, pending GC-MS/MS analyzes.

In step (5), the regression equation corresponding with standard working curve is Y=0.309X+0.0008, coefficient R ²be 0.9998, detect and be limited to 0.015ng/ and prop up, concentration range is 0.2138～10.69ng/mL.

In step (5), the computing formula of trying to achieve the content of safrole in cigarette sample is as follows:

$W=\frac{(\frac{A}{\mathrm{As}}-b)\×\mathrm{Ms}}{a\×M}$

In formula: W is the content of safrole in cigarette sample, unit is that ng/ props up; A is the peak area of safrole; As is the peak area of interior mark heptadecane; B is the intercept of standard working curve; Ms is interior target quality in sample solution, and unit is ng; A is the slope of standard working curve; M is the quantity of cigarette, and unit is for propping up.

Beneficial effect: compared with prior art, the present invention has following excellent results:

(1) the inventive method is measured the content of safrole in cigarette mainstream flue gas first based on gas chromatography-tandem mass spectrometer, has solved in flue gas safrole burst size level low, and the impact of flue gas complex matrices on factors such as the Analysis interference of object are serious.

(2) to have selected peak shape and the best HP-5MS post of separating effect be separating column to the inventive method, and adopt the GC-MS/MS (MRM) that can simultaneously provide quantitative and qualitative analysis information as detecting pattern, the sensitivity that has improved response signal, has guaranteed precision of analysis.

(3) the inventive method is utilized inner mark method ration, accurate constant volume, and can reduce the error of being brought by pre-treating method reappearance and instrument precision problem.

Accompanying drawing explanation

Fig. 1 is the process flow diagram of assay method of the present invention;

Fig. 2 is the extraction ion pair chromatogram of safrole in standard operation solution; Wherein, upper figure is 162-> 104 ion pairs, and figure below is 162-> 131 ion pairs;

Fig. 3 is the extraction ion pair chromatogram of safrole in sample solution; Wherein, upper figure is 162-> 131 ion pairs, and figure below is 162-> 104 ion pairs;

Fig. 4 is the extraction ion pair chromatogram of internal standard compound in standard operation solution; Wherein, upper figure is 240-> 71 ion pairs, and figure below is 240-> 85 ion pairs;

Fig. 5 is the extraction ion pair chromatogram of internal standard compound in sample solution; Wherein, upper figure is 240-> 71 ion pairs, and figure below is 240-> 85 ion pairs.

Embodiment

Below in conjunction with specific embodiment, the present invention is described further.

Embodiment 1

Measure a method for safrole in cigarette mainstream flue gas, process flow diagram as shown in Figure 1, comprises the steps:

(1) preparation of inner mark solution

Interior mark storing solution: accurately take 0.202g internal standard compound heptadecane, to 100mL, obtaining concentration is the interior mark storing solution of 2020 μ g/mL with ethanol constant volume; Inner mark solution: accurately pipette mark storing solution in 0.1mL, to 100mL, obtain the inner mark solution that concentration is 2.02 μ g/mL with ethanol constant volume.

(2) preparation of standard operation solution

Primary standard storing solution: accurately take 0.1069g safrole, to 100mL, obtaining concentration is the primary standard storing solution of 1069 μ g/mL with ethanol constant volume; Secondary standard storing solution: accurately pipette 0.1mL primary standard storing solution, to 100mL, obtaining concentration is the secondary standard storing solution of 1.069 μ g/mL with ethanol constant volume; Standard operation solution: accurately pipette respectively secondary standard storing solution 20 μ L, 50 μ L, 100 μ L, 400 μ L and 1000 μ L, and each 100 μ L of inner mark solution, with ethanol, as solvent, be settled in the volumetric flask of 100mL, obtain series standard working solution.This series standard solution concentration is respectively 0.2138ng/mL, 0.5345ng/mL, 1.069ng/mL, 4.276ng/mL and 10.69ng/mL, and wherein interior mark concentration is 2.02ng/mL.

(3) preparation of sample solution

1. cigarette smoking: the TPM of collecting 20 cigarette according to the requirement of GB/T19609 standard.2. sample extraction: the trapping that upper step is obtained has the glass fiber filter of TPM to be positioned over that in 100mL conical flask, (filter disc is answered full wafer to put into conical flask and paved, can not shred or tear up), accurately add 40 μ L inner mark solutions, and add 40mL normal hexane, be placed in Clothoid type oscillator oscillation extraction 40min under 160 turn/min conditions, standing a moment.3. extract purifies: extract is all transferred to solid-phase extraction column (silica gel that filler is 500mg), until extract toward be displaced downwardly to extraction pillar top after, divide and add for 3 times 10mL normal hexane to carry out drip washing, drip washing speed remains on 1～2mL/min, collects all leacheates.4. leacheate is concentrated: the concentrated bottle that fills all leacheates is connected on Rotary Evaporators; rotary evaporation under 80 ℃, high pure nitrogen protection (or under 55 ℃, vacuum condition rotary evaporation); be concentrated to 1mL left and right, obtain sample feeding liquid, pending GC-MS/MS analyzes.

(4) gas chromatography-Tandem Mass Spectrometry Analysis

Standard operation solution and the sample solution to be measured of getting respectively 5 variable concentrations carry out gas chromatography-Tandem Mass Spectrometry Analysis.Its chromatographiccondition is: adopt DB-5MS (30m * 0.25mm * 0.25 μ m) capillary chromatographic column; Carrier gas is helium, and constant current speed is 1mL/min; Sample size is 1 μ L; Injector temperature is 250 ℃; Transmission line temperature is 280 ℃; Adopt pulse Splitless injecting samples, pulse is 25psi; Heating schedule is that initial temperature is 60 ℃, is increased to 160 ℃, then is increased to 300 ℃ with the speed of 10 ℃/min with the speed of 4 ℃/min.Its mass spectrophotometry condition is: ionization mode is EI source, positive ion mode; Ion source temperature: 230 ℃; Quadrupole rod temperature: be 150 ℃; Collision gas: nitrogen, flow velocity 1.5mL/min, carrier gas (helium) flow velocity is 2.25mL/min; Multiple-reaction monitoring (MRM) pattern, detail parameters is listed in table 1.The extraction ion pair chromatogram of safrole in standard operation solution, as shown in Figure 2; The extraction ion pair chromatogram of safrole in sample solution, as shown in Figure 3; The extraction ion pair chromatogram of internal standard compound in standard operation solution, as shown in Figure 4; The extraction ion pair chromatogram of internal standard compound in sample solution, as shown in Figure 5.

The MRM parameter list of table 1 object

First, the safrole in standard operation solution of take is horizontal ordinate with the ratio of interior target concentration, the safrole in chromatogram of take is ordinate with the ratio of interior target peak area, carry out linear regression analysis, obtain standard working curve, get the standard operation solution of least concentration, do 9 Parallel testing analyses, calculate its standard deviation, the concentration corresponding to standard deviation of 3 times of take is detection limit.Wherein, the regression equation corresponding with standard working curve is Y=0.309X+0.0008, coefficient R ²be 0.9998, detect and be limited to 0.015ng/ and prop up, concentration range is 0.2138～10.69ng/mL.

Then, by safrole in the sample solution recording under the same terms and interior target chromatographic peak area ratio, substitution standard working curve, tries to achieve the content of safrole in cigarette sample, and computing formula is as follows:

$W=\frac{(\frac{A}{\mathrm{As}}-b)\×\mathrm{Ms}}{a\×M}$

In formula:

W---the content of safrole in cigarette sample, unit is every of nanogram (ng/ props up);

The peak area of A---safrole;

As---the peak area of interior mark heptadecane;

The intercept of b---standard working curve;

Ms---interior target quality in sample solution, unit is nanogram (ng);

The slope of a---standard working curve;

The quantity of M---cigarette, unit is for propping up.

The present embodiment adopts 2 different brands cigarette samples, carries out safrole content detection, the results are shown in following table 2.

Table 2 sample detection result

Sample number into spectrum	Cigarette type	Detected value (ng/ props up)
			Cigarette A	Mixed type	0.189
Cigarette B	Fire-cured tobacco type	0.176

Embodiment 2

Take a cigarette sample as analytic target, adopt the content of the method mensuration object of embodiment 1, carry out Precision Experiment, measure altogether 6 times, calculate respectively the relative standard deviation (RSD) of 6 replicate determination results, measurement result is in Table 3.In table, result shows, the repeated RSD<10% of this experimental technique.For trace materials analyzing and testing, the method has good precision.

The precision of table 3 method

Sequence number	Measurement result (ng/ props up)
		1	0.176
2	0.192
		3	0.190
4	0.191
		5	0.177
6	0.209
		RSD(％)	6.35

Take a cigarette sample as object, carry out the mark-on of basic, normal, high three variable concentrations levels and process, then according to above-mentioned standard pre-treatment step, process, carry out again GC-MS/MS analysis, the content of measuring object, calculates its recovery of standard addition, the results are shown in following table 4.From following table, can find out, the recovery of object is between 87.78%～110.09%.On the whole, for the object for content in nanogram level, the method has the recovery relatively preferably.

The recovery of table 4 method

The present embodiment standard solution used only be take one of them concentration and is described as example, typical curve and regression equation that the prepared standard solution of other concentration value obtains through gas chromatography-Tandem Mass Spectrometry Analysis are same as the previously described embodiments, at this, are not enumerating.Illustrated embodiment is method for a better understanding of the present invention just, does not have any restriction, and said method or the method that is equal to above-mentioned situation are all included in the protection domain of technical scheme of the present invention.

Claims (6)

1. a method of measuring safrole in cigarette mainstream flue gas, is characterized in that, comprises the steps:

(1) preparation of inner mark solution: take heptadecane as internal standard compound, make solvent with ethanol, prepare inner mark solution;

(2) preparation of standard operation solution: take safrole as standard items, make solvent with ethanol, be prepared into standard inventory solution through stepwise dilution, then add respectively quantitative inner mark solution, be prepared into standard operation solution;

(3) preparation of sample solution: collected the object in cigarette smoke, accurately added quantitative inner mark solution and extraction solvent, mechanical shaking extraction; Shift whole extracts, through Solid-Phase Extraction column purification, after scavenging solution is concentrated, obtain sample solution;

(4) gas chromatography-Tandem Mass Spectrometry Analysis: standard operation solution and sample solution are detected to analysis with gas chromatography-tandem mass spectrometer; Chromatographiccondition is: adopt DB-5MS capillary chromatographic column; Carrier gas is helium, and constant current speed is 1mL/min; Sample size is 1 μ L; Injector temperature is 250 ℃; Transmission line temperature is 280 ℃; Adopt pulse Splitless injecting samples, pulse is 25psi; Heating schedule is that initial temperature is 60 ℃, is increased to 160 ℃, then is increased to 300 ℃ with the speed of 10 ℃/min with the speed of 4 ℃/min; Mass spectrophotometry condition is: ionization mode is EI source, positive ion mode; Ion source temperature: 230 ℃; Quadrupole rod temperature: be 150 ℃; Collision gas: nitrogen, flow velocity 1.5mL/min, flow rate of carrier gas is 2.25mL/min; Multiple-reaction monitoring pattern, detail parameters is in Table 1;

The MRM parameter list of table 1 object

(5) drafting of standard working curve and the calculating of sample result: the safrole in standard operation solution of take is horizontal ordinate with the ratio of interior target concentration, the safrole in chromatogram of take is ordinate with the ratio of interior target peak area, carry out linear regression analysis, obtain standard working curve; By safrole in the sample solution recording under the same terms and interior target chromatographic peak area ratio, substitution standard working curve, converts and tries to achieve the content of safrole in cigarette sample main flume.

2. the method for safrole in mensuration cigarette mainstream flue gas according to claim 1, it is characterized in that: in step (1), the preparation of described inner mark solution specifically comprises the following steps: mark storing solution 1): accurately take 200mg internal standard compound heptadecane, with ethanol constant volume, to 100mL, obtaining concentration is the interior mark storing solution of 2000 μ g/mL; 2) inner mark solution: accurately pipette mark storing solution in 0.1mL, to 100mL, obtain the inner mark solution that concentration is 2 μ g/mL with ethanol constant volume.

3. the method for safrole in mensuration cigarette mainstream flue gas according to claim 1, it is characterized in that: in step (2), the preparation of described standard operation solution specifically comprises the following steps: (1) primary standard storing solution: accurately take 100mg safrole, with ethanol constant volume, to 100mL, obtaining concentration is the primary standard storing solution of 1000 μ g/mL; (2) secondary standard storing solution: accurately pipette 0.1mL primary standard storing solution, to 100mL, obtaining concentration is the secondary standard storing solution of 1 μ g/mL with ethanol constant volume; (3) standard operation solution: accurately pipette respectively secondary standard storing solution 20 μ L, 50 μ L, 100 μ L, 400 μ L and 1000 μ L, and each 100 μ L of inner mark solution, with ethanol, as solvent, be settled in the volumetric flask of 100mL, obtain the series standard working solution that concentration is respectively 0.2ng/mL, 0.5ng/mL, 1ng/mL, 4ng/mL and 10ng/mL, wherein interior mark concentration is 2ng/mL.

4. the method for safrole in mensuration cigarette mainstream flue gas according to claim 1, it is characterized in that: in step (3), the preparation of described sample solution specifically comprises the following steps: 1) cigarette smoking: the TPM of collecting 20 cigarette according to the requirement of GB/T19609 standard; 2) sample extraction: the trapping that upper step is obtained has the glass fiber filter of TPM to be positioned in 100mL conical flask, accurately add 40 μ L inner mark solutions, and add 40mL normal hexane, be placed in Clothoid type oscillator oscillation extraction 40min under 160 turn/min conditions, standing a moment; 3) extract purifies: extract is all transferred to solid-phase extraction column, until extract, toward being displaced downwardly to behind the top of extraction pillar, divides and add for 3 times 10mL normal hexane to carry out drip washing, drip washing speed remains on 1～2mL/min, collects all leacheates; 4) leacheate is concentrated: the concentrated bottle that fills all leacheates is connected on Rotary Evaporators, and rotary evaporation under 80 ℃, high pure nitrogen protection, or rotary evaporation under 55 ℃, vacuum condition, be concentrated to 1mL left and right, obtains sample feeding liquid.

5. the method for safrole in mensuration cigarette mainstream flue gas according to claim 1, is characterized in that: in step (5), the regression equation corresponding with standard working curve is Y=0.309X+0.0008, coefficient R ²be 0.9998, detect and be limited to 0.015ng/ and prop up, concentration range is 0.2138～10.69ng/mL.

6. the method for safrole in mensuration cigarette mainstream flue gas according to claim 1, is characterized in that: in step (5), the computing formula of trying to achieve the content of safrole in cigarette sample is as follows:

$W=\frac{(\frac{A}{\mathrm{As}}-b)\&times;\mathrm{Ms}}{a\&times;M}$

In formula: W is the content of safrole in cigarette sample, unit is that ng/ props up; A is the peak area of safrole; As is the peak area of interior mark heptadecane; B is the intercept of standard working curve; Ms is interior target quality in sample solution, and unit is ng; A is the slope of standard working curve; M is the quantity of cigarette, and unit is for propping up.