CN102072940B - Method for measuring contents of coumarin and safrole in flavor and fragrance for cigarette - Google Patents

Abstract

The invention provides a method for determining the content of coumarin and safrole in flavors and fragrances for tobacco. It disperses the coumarin and safrole in the flavors and fragrances for tobacco in the aqueous solution, extracts them with organic reagents, and uses gas chromatography-mass spectrometry to perform instrumental analysis and determine the content after sampling and filtering. Select phenylethyl propionate as the internal standard, select ions with mass-to-charge ratio m/z of 104, 146, and 162 in the mass spectrogram of phenylethyl propionate, coumarin, and safrole for quantitative analysis, and use the internal standard The standard curve was drawn by the method, and the sample was fitted with the standard curve to obtain the content of coumarin and safrole in the sample. This method can not only extract coumarin and safrole in tobacco flavors and fragrances more completely, but also greatly remove interfering substances during the extraction process, so that the sample spectrum can be purified, reducing the impact of the sample on the instrument inlet and The pollution of the chromatographic column can meet the needs of large-scale sample detection in actual work.

Description

Determination method of coumarin and safrole in tobacco flavors and fragrances

technical field

The invention relates to a method for determining the content of coumarin and safrole, in particular to a method for determining the content of coumarin and safrole in flavors and fragrances for tobacco.

Background technique

At present, coumarin and safrole have been proved to have extremely strong toxic side effects on animals, which may cause damage to internal organs and even induce cancer. Coumarin and safrole are mainly derived from plants. In the manufacturing process of tobacco flavors and fragrances, coumarin and safrole may be introduced, which poses a potential threat to the health of consumers. Therefore, it is necessary to control the content of coumarin and safrole in flavors and fragrances for tobacco.

Compared with other types of flavors and fragrances such as food and daily necessities, tobacco flavors and fragrances have their own characteristics. Tobacco flavors and fragrances have complex components and a wide range of sources, mostly in liquid or paste state. The coumarin and safrole are insoluble in water and easily soluble in organic solvents.

At present, the sample processing methods of coumarin and safrole include direct extraction, ultrasonic extraction, simultaneous distillation extraction, steam distillation and so on. These pretreatment methods all have deficiencies in varying degrees. Direct extraction and ultrasonic extraction are faster, but more interfering substances will be extracted; at the same time, distillation extraction and steam distillation are more work-intensive and time-consuming. , and due to the higher temperatures used in the extraction process, there is a potential for chemical loss.

Contents of the invention

The purpose of the present invention is to provide a method for determining the content of coumarin and safrole in tobacco flavors and fragrances. The earth removes interfering substances, so that the sample spectrum is purified, and at the same time, it also reduces the pollution of the sample to the instrument inlet and chromatographic column, which can meet the needs of large-scale sample detection in actual work.

The invention provides a method for determining the content of coumarin and safrole in flavors and fragrances for tobacco, comprising the following steps:

1) Extraction preparation: take an appropriate amount of phenylethyl propionate, and prepare a dichloromethane solution containing phenylethyl propionate as the extraction solution;

2) Preparation of standard working solution: Weigh coumarin and safrole, dissolve and dilute the extract into a volumetric flask as a standard stock solution, and prepare 1000ng of coumarin and safrole respectively by serially diluting the standard stock solution /mL, 500ng/mL, 200ng/mL, 100ng/mL, 50ng/mL standard working solution;

3) Sample treatment: According to the amount of adding 5mL of saturated sodium chloride aqueous solution per gram of tobacco flavor and fragrance, take tobacco flavor and fragrance and add saturated sodium chloride aqueous solution, shake, so that the flavor and fragrance are evenly dispersed in saturated sodium chloride aqueous solution, Then add the extract, the volume ratio of the saturated sodium chloride aqueous solution and the extract is 1:2, fully oscillate and let it stand, after layering, take the lower layer of liquid and filter it as the sample to be tested;

4) Determination of samples: Take 0.5~1 μL of the sample to be tested and inject it into the GC-MS instrument. The ions of phenylethyl propionate, coumarin and safrole are quantified at m/z of 104, 146 and 162 respectively, and drawn by the internal standard method Standard curve, sample and standard curve fitting, draw sample coumarin and safrole content.

The concentration of phenylethyl propionate in the extract was 100ng/mL.

In step 3), a centrifuge can be used to speed up the layering speed during static layering.

In step 4), the chromatographic conditions of the GC/MS instrument: use a non-polar chromatographic column, the carrier gas is high-purity helium, the column flow rate is 2mL/min constant flow, splitless, the inlet temperature is 320°C, and the temperature is programmed The initial column temperature is 70°C, keep it for 1min, raise it to 160°C at a rate of 10°C/min, keep it for 0min, then raise it to 300°C at a rate of 40°C/min, and keep it for 10min;

The mass spectrometry conditions of the GC-MS: the interface temperature is 280°C, the ion source temperature is 280°C, the quadrupole temperature is 200°C, the ionization energy is 70eV, the solvent delay time is 6min, and the time event is 12min. The scanning mass range is 50-200 amu, selected ion monitoring mode: the m/z of safrole is 162, 135, 131, the m/z of phenylethyl propionate is 104, 91, 77, coumarin The mass-to-charge ratio m/z is 146, 118, 90.

The non-polar chromatographic column is DB-35MS.

The present invention takes the sample numbered 10712 (safrole has not been detected in this sample) as an object, adds medium-concentration coumarin and safrole, performs a standard addition recovery rate test, and measures 6 times in parallel. The results are shown in Table 1.

Table 1 The recovery rate of the method

The recovery rate of coumarin was 82.6%~110.7%, with an average value of 95.6%, and the recovery rate of safrole was 94.4%~103.2%, with an average value of 98.2%. Calculating the precision of the method with the same test result, the relative standard deviations of the six results were 5.6% for coumarin and 3.3% for safrole. Therefore, the present invention has better method reproducibility and high accuracy, and can meet the needs of mass sample detection in actual work.

Beneficial effects of the present invention: compared with the direct liquid-liquid extraction method, the spectrogram background obtained by the indirect liquid-liquid extraction method is lower, the peak shape is clean, and the spectrogram is beautiful. Because during the extraction process, the interfering substances can be greatly removed, the samples entering the instrument can be purified, the pollution of the sample to the instrument inlet and chromatographic column can be reduced, and the life of the instrument and consumables can be extended. This method can more completely extract the coumarin and safrole in the flavors and fragrances used for tobacco, the experimental result is basically consistent with the direct extraction method, the method quantification limit of calculating coumarin is 0.13mg/kg, the method quantification limit of safrole is 0.09mg/kg, which can meet the needs of large-scale sample testing in actual work. The method is simple and quick to operate, and the result is stable and reliable, and is more suitable for the detection of some samples that are difficult to disperse in the direct extraction method.

Description of drawings

Figure 1 is a comparison chart of TIC for the same sample treated by direct liquid-liquid extraction and indirect liquid-liquid extraction.

Fig. 2 is the standard curve of the safrole obtained by the prepared standard working solution.

Figure 3 is the standard curve of coumarin obtained from the prepared standard working solution.

Detailed ways

The present invention is further illustrated below by specific examples:

Example 1

Reagents: dichloromethane, sodium chloride, phenylethyl propionate, coumarin, safrole, purified water.

Instruments: HP7890A-5975C gas chromatography-mass spectrometry (Agilent, USA); AL204 electronic balance (accuracy 0.1 mg, Mettler-Toledo); oscillator.

Chromatographic conditions of GC/MS: Chromatographic column: DB-35MS (30m×0.32mm×0.25μm); carrier gas: high-purity helium; column flow rate: 2mL/min constant flow; split ratio: splitless; inlet temperature : 320°C; Injection volume: 1 μL; Programmed temperature: The initial column temperature is 70°C, keep for 1min, raise to 160°C at a rate of 10°C/min, hold for 0min, then rise to 300°C at a rate of 40°C/min, Keep it for 10min.

Mass spectrometry conditions of GC-MS interface temperature: 280°C; ion source temperature: 280°C; quadrupole temperature: 200°C; ionization (EI) energy: 70eV; solvent delay time: 6min; ;Mass range of full scan: 50~200amu; Selected ion monitoring mode: m/z of safrole is 162, 135, 131, m/z of phenylethyl propionate is 104, 91, 77, fragrance The mass-to-charge ratio m/z of beanin is 146, 118, 90.

Preparation of the extract: Take phenylethyl propionate and add it to the dichloromethane solution to prepare a dichloromethane solution containing 100 ng/mL of phenylethyl propionate as the extract.

Preparation of standard working solution: Weigh 50 mg each of coumarin and safrole, dissolve and dilute to a 50 mL volumetric flask with the extract, as a standard stock solution. Standard working solutions containing 1000 ng/mL, 500 ng/mL, 200 ng/mL, 100 ng/mL and 50 ng/mL of coumarin and safrole were prepared by serially diluting the standard stock solution.

Sample treatment: Take 2.00 grams of tobacco flavors and fragrances, add 10mL saturated sodium chloride aqueous solution, oscillate, so that the flavors and fragrances are evenly dispersed in saturated sodium chloride aqueous solution, then add 20mL extract, shake fully and let stand until layering Afterwards, the liquid in the lower layer was passed through a 0.22 μm microporous membrane as the sample to be tested. When it is difficult to stratify, a centrifuge can be used to speed up the stratification.

Sample determination: Take 1 μL of the sample to be tested and inject it into the GC-MS. The retention time deviation between the sample and the standard is less than ±0.1min. Qualitative ions: the m/z of safrole is 162, 135, 131, the m/z of phenylethyl propionate is 104, 91, 77, the m/z of coumarin is 146, 118, 90. Phenylethyl propionate, coumarin, and safrole were quantified with m/z 104, 146, and 162 ions, respectively. Use the internal standard method to draw a standard curve, and the sample is fitted with the standard curve to obtain the contents of coumarin and safrole in the sample.

The prepared standard working solutions of 5 concentration gradients were entered into the instrument respectively, with phenylethyl propionate as the internal standard, and the standard curve was set to pass through the origin forcibly, and the standard curves of safrole and coumarin obtained by the prepared standard working solutions were respectively shown in 2. As shown in Figure 3, safrole y=0.3548x, correlation coefficient 0.99948; coumarin y=0.2662x, correlation coefficient 0.99926.

This method was used to detect 18 flavor and fragrance samples, and each sample was measured twice in parallel. Two samples were detected to be coumarin, and no sample was detected to be safrole. During the detection process, no samples were found not applicable to this method.

comparative example

Taking the sample with the same number as the experimental object, using the pretreatment methods of direct liquid-liquid extraction and indirect liquid-liquid extraction respectively, and using the same instrument under the same conditions to obtain two total ion current (TIC) diagrams, the two spectra Stacked together for comparison, as shown in Figure 1.

The spectrum a is the spectrum obtained by the direct liquid-liquid extraction method, and the spectrum b is the spectrum obtained by the indirect liquid-liquid extraction method. Compared with the two methods, the spectral background of the indirect liquid-liquid extraction method is lower, and the peak shape is clean, indicating that this treatment method can keep a large amount of interfering substances in the water phase, so that the sample entering the instrument can be purified, so it is generally more beautiful. At the same time, purer samples can also reduce the contamination of the instrument and prolong the life of the instrument and consumables.

Claims (6)

1. cumarin and safrole Determination on content method in the essence spice for cigarette may further comprise the steps:

1) extract is prepared: phenylethyl propionate is joined be mixed with extract in the dichloromethane solution;

2) preparation of standard operation liquid: take by weighing cumarin and safrole, in volumetric flask,, standard reserving solution is contained the standard operation liquid of cumarin and safrole through the stepwise dilution preparation as standard reserving solution with extract dissolving constant volume;

3) processing of sample: the amount that adds the 5mL saturated sodium-chloride water solution according to every gram essence spice for cigarette; Get essence spice for cigarette and add saturated sodium-chloride water solution, vibration is dispersed in the saturated sodium-chloride water solution essence and flavoring agent; Add extract again; The volume ratio of saturated sodium-chloride water solution and extract is 1:2, fully leaves standstill after the vibration, treats to take off layer liquid filtering as treating test sample after the layering;

4) mensuration of sample: get 0.5 ~ 1 μ L and treat test sample; Inject gas chromatograph-mass spectrometer; Select phenylethyl propionate, cumarin and the mass-to-charge ratio m/z of safrole in mass spectrogram to be respectively 104,146,162 ion and carry out quantitative test; Use internal standard method drawing standard curve, sample and typical curve match draw sample cumarin and safrole content.

2. according to cumarin in the said essence spice for cigarette of claim 1 and safrole Determination on content method, it is characterized in that: the concentration of phenylethyl propionate is 100ng/mL in the said extract.

3. according to cumarin in the said essence spice for cigarette of claim 1 and safrole Determination on content method, it is characterized in that: in step 3), use hydro-extractor to accelerate to divide interval velocity during standing demix.

4. according to cumarin in the said essence spice for cigarette of claim 1 and safrole Determination on content method, it is characterized in that: in step 3), take off layer liquid after the layering and use filtering with microporous membrane.

5. according to cumarin in the said essence spice for cigarette of claim 1 and safrole Determination on content method, it is characterized in that: in step 4), the chromatographic condition of gas chromatograph-mass spectrometer: use nonpolar chromatographic column, carrier gas is a high-pure helium; Column flow rate is the 2mL/min constant current, does not shunt, and injector temperature is 320 ℃; Temperature programme is 70 ℃ of post initial temperatures, keeps 1min, rises to 160 ℃ with the speed of 10 ℃/min; Keep 0min, the speed with 40 ℃/min rises to 300 ℃ then, keeps 10min;

The mass spectrum condition of gas chromatograph-mass spectrometer: interface temperature is 280 ℃, and ion source temperature is 280 ℃, and the quadrupole rod temperature is 200 ℃; Ionizing energy is 70eV, and the solvent delay time is 6min, and time-event is to close detecting device in 12 minutes; The full scan mass range is 50-200amu; Select the ion monitoring pattern: the mass-to-charge ratio m/z of safrole is 162,135,131, and the mass-to-charge ratio m/z of phenylethyl propionate is 104,91,77, and the mass-to-charge ratio m/z of cumarin is 146,118,90.

6. according to cumarin in the said essence spice for cigarette of claim 5 and safrole Determination on content method, it is characterized in that: said nonpolar chromatographic column is DB-35MS.

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