CN116046933A

CN116046933A - Method for determining phthalate in essence

Info

Publication number: CN116046933A
Application number: CN202211668095.5A
Authority: CN
Inventors: 杨飞; 孙莹莹; 孟冬玲; 路军丽; 唐纲岭; 王颖; 刘珊珊; 刘洋; 周芸; 陈志燕; 邓惠敏
Original assignee: China Tobacco Guangxi Industrial Co Ltd; National Tobacco Quality Supervision and Inspection Center
Current assignee: China Tobacco Guangxi Industrial Co Ltd; National Tobacco Quality Supervision and Inspection Center
Priority date: 2022-12-23
Filing date: 2022-12-23
Publication date: 2023-05-02

Abstract

The invention relates to a method for determining phthalate in essence, and belongs to the technical field of physical and chemical inspection. Phthalic acid in essence of the inventionAn ester assay comprising the steps of: analyzing phthalate in the essence sample by adopting a supercritical fluid chromatography-tandem mass spectrometry method; the chromatographic column of supercritical fluid chromatography in the supercritical fluid chromatography-tandem mass spectrometry is virdis HSS C18SB, the elution mode is gradient elution, and the mobile phase A is supercritical CO ₂ Mobile phase B is an alcoholic solvent. The method for determining the phthalate in the essence is environment-friendly, high in specificity and high in separation speed, and can shorten the chromatographic separation time to 6.0min and achieve good separation degree (baseline separation) by optimizing the detection conditions of an instrument.

Description

Method for determining phthalate in essence

Technical Field

The invention relates to a method for determining phthalate in essence, and belongs to the technical field of physical and chemical inspection.

Background

As a plasticizer with wide application, phthalate plays a role similar to female hormone in human and animal bodies, can interfere endocrine, and limits the application range and the application amount of the phthalate in various countries in the world. Currently, methods for detecting phthalate plasticizers mainly include gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, and liquid chromatography-mass spectrometry. For example, zhang Xiaotao (journal of analytical science, 2013, 29 (6): 806-810) is reported in the method of simultaneously measuring 16 phthalate compounds in the essence and the spice for cigarettes by high performance liquid chromatography-tandem mass spectrometry, and the phthalate compounds in the essence and the spice are directly analyzed by sample injection after ultrasonic extraction and centrifugation. In addition, as Ruan Xiaojiao, a method for simultaneously detecting the residual quantity of 22 phthalic acid esters (PAFs) in the essence by combining a solid-phase extraction method with a meteorological chromatograph-triple quadrupole tandem mass spectrum is established in gas chromatograph-triple quadrupole tandem mass spectrum (food technology, 2018,39 (22): 235-240, 246). The method has the common problems that the detection time is relatively long, a large amount of toxic and harmful solvents are required to be consumed, the method is not green and environment-friendly, and the like in the actual detection process.

Disclosure of Invention

The invention aims to provide an efficient and environment-friendly method for determining phthalate in essence.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a method for determining phthalate in essence comprises the following steps: analyzing phthalate in the essence sample by adopting a supercritical fluid chromatography-tandem mass spectrometry method; the chromatographic column of supercritical fluid chromatography in the supercritical fluid chromatography-tandem mass spectrometry is virdis HSS C18SB, the elution mode is gradient elution, and the mobile phase A is supercritical CO ₂ Mobile phase B is an alcoholic solvent.

The method for determining phthalate in essence of the invention adopts supercritical fluid chromatography-tandem mass spectrometry to analyze phthalate in essence samples, selects a Viridis HSS C18SB chromatographic column, and uses supercritical CO during chromatographic separation ₂ And alcohol solvents are used as mobile phases for gradient elution, so that the method is environment-friendly, high in specificity and high in separation speed, chromatographic separation time can be shortened to 6.0min by optimizing instrument detection conditions, and separation degree is good (baseline separation).

Further, the supercritical fluid chromatography-tandem mass spectrometry is adopted to analyze phthalate esters in the essence sample, and the sample solution to be detected is obtained by adding an isotope internal standard, an extraction solvent and a purification material into the essence sample for extraction, and then carrying out solid-liquid separation. The purification material is preferably spherical carbon and/or multiwall carbon nanotubes. The method integrates the extraction and purification of the sample, and is simpler and faster to operate. Further, the preparation method of the sample solution to be tested further comprises the following steps: the solution obtained by solid-liquid separation was filtered.

Further, the purification material is spherical carbon, the spherical carbon can play a good role in adsorbing and purifying polar and nonpolar non-target objects in the essence, and the loss of components to be detected caused by the concentration and purification processes of the sample is reduced. Can ensure that the spherical carbon has larger surface area, realizes effective adsorption to non-target substances, realizes separation, shortens the extraction time of the sample solution to be detected, and further has the particle diameter of 0.4-1.0 mu m and the specific surface area of 80-120m ² And/g. For example, spherical carbon is a product available from Beijing micro label technology Co., ltd., product No. JH-Y-028.

The dosage of the extraction solvent and the spherical carbon is adjusted within a proper range, so that the components to be detected of the essence sample can be effectively extracted, the use of an organic solvent is reduced, the cost is reduced, and the pollution to the environment is reduced. Further, the volume of the extraction solvent used for each 0.18-0.22g of essence sample is 9-11mL. The mass of the purification material used for each 0.18-0.22g of essence sample is 5mg, for example, the mass of the purification material used for each 0.2g of essence sample is 50mg.

Further, the phthalate comprises one or any combination of di (2-butoxy) ethyl phthalate (DBEP), dicyclohexyl phthalate (DCHP), diphenyl phthalate (DPHP). For example, the phthalates include di (2-butoxy) ethyl phthalate, dicyclohexyl phthalate, and diphenyl phthalate.

The effective separation of the components to be tested is facilitated by selecting chromatographic test conditions and gradient elution modes, and the accuracy of test results is improved. Further, the chromatographic conditions of the supercritical fluid chromatography in the supercritical fluid chromatography-tandem mass spectrometry are as follows: the column temperature is 45-50 ℃; the sample injection amount is 2-3 mu L; in the elution mode, the total flow rate of the mixed mobile phase A and mobile phase B is 0.7-1.0mL/min. Further, the volume ratio of the mobile phase A is reduced from 97-99% to 88-90% during gradient elution, and then is restored to 97-99%. Further, the volume ratio of the mobile phase A is 99 percent, 1 to 2.5 minutes, the volume ratio of the mobile phase A is reduced to 96 percent, 2.5 to 3.2 minutes, the volume ratio of the mobile phase A is reduced to 90 percent, 3.2 to 4.0 minutes, the volume ratio of the mobile phase A is kept 90 percent, 4.0 to 4.5 minutes, the volume ratio of the mobile phase A is increased to 99 percent, 4.5 to 6.0 minutes, and the volume ratio of the mobile phase A is kept 99 percent; the volume ratio of the mobile phase A is uniformly changed; as shown in table 1. The mobile phase B is preferably ethanol.

TABLE 1 gradient elution procedure

Further, the specification of the column was 3.0mm×100mm, and the particle size was 1.8. Mu.m.

Further, the isotopic internal standard is diphenyl phthalate-D4 (DBEP-D4). The isotope internal standard is added into the essence sample by adding the internal standard working fluid prepared from the isotope internal standard. The concentration of the isotope internal standard substance in the internal standard working fluid is 0.01mg/mL. The solvent of the internal standard working fluid is preferably ethanol. The preparation steps of the internal standard working solution are as follows: weighing 10mg of diphenyl phthalate-D4 in a 10mL volumetric flask, and fixing the volume after ethanol is dissolved to obtain an internal standard stock solution; taking 100 mu L of internal standard stock solution in a 10mL volumetric flask, and fixing the volume by ethanol to obtain an internal standard working solution.

Further, the mass spectrometry conditions in the supercritical fluid chromatography-tandem mass spectrometry are as follows: the scanning mode is positive ion scanning; an electrospray ion source; the temperature of the ion source is 140-150 ℃; the capillary voltage is 2.6-2.8kV; the air flow of the taper hole is 70-80L/h; the flow rate of the desolventizing agent is 650-700L/h; the desolventizing gas temperature is 310-330 ℃; and adopting MRM mode acquisition. Further, the MRM parameters are shown in Table 2. It will be appreciated that the MRM parameters for the corresponding compounds may be selected from Table 2 depending on the compound being tested.

Table 2 MRM parameters of mass spectrometry conditions

In table 2, ions were quantified.

Further, the extraction solvent is an alcohol solvent. The alcohol solvent is preferably ethanol.

Further, the extraction is an oscillation extraction. The proper oscillation rate and the proper extraction time can improve the extraction effect of the spherical carbon on the components to be detected and save the time cost, and furthermore, the oscillation rate of the oscillation extraction is 200-250r/min, and the oscillation extraction time is 8-12min.

Drawings

FIG. 1 is a flow chart of the measurement method of example 1 of the present invention;

FIG. 2 is a selective ion chromatogram of DBEP, DPHP, DCHP and DPHP-D4 obtained in step 4) of example 1 of the present invention;

FIG. 3 is a chromatogram of a sample to be tested having phthalate added in comparative example 1 in the experimental example each having a phthalate content of 5.0. Mu.g/g.

Detailed Description

The technical scheme of the invention is further described below in connection with the specific embodiments.

The reagent specifications and instrument models involved in the examples are as follows:

a mixed solution of phthalates (available from Shanghai Annotation technology Co., ltd.) is specifically bis (2-butoxy) ethyl phthalate, dicyclohexyl phthalate, diphenyl phthalateMixing ethanol solutions, wherein the concentration of each phthalate is 100 mug/mL; ethanol is chromatographic grade; spherical carbon (particle diameter of 0.4-1 μm, specific surface area of 80-120 m) ² /g, available from Beijing micro label technology Co., ltd., cat# JH-Y-028).

Waters supercritical fluid chromatography tandem mass spectrometer; AE 163 electronic balance with a feel of 0.0001g and AE 166 electronic balance with a feel of 0.01g (Mettler company, switzerland).

Example 1

The flow of the method for measuring phthalate in the essence of the embodiment is shown in fig. 1, and the method comprises the following steps:

1) Weighing 0.2g essence sample in a 20mL triangular flask, sequentially adding 100 μL internal standard working solution, 10mL ethanol and 50mg spherical carbon, and oscillating at a rate of 200r/min for 10min to obtain an extract; then centrifuging and filtering to obtain a sample solution to be detected;

the preparation steps of the internal standard working solution are as follows: weighing 10mg of DBEP-D4 in a 10mL volumetric flask, and fixing the volume after dissolving in ethanol to obtain an internal standard stock solution; taking 100 mu L of internal standard stock solution in a 10mL volumetric flask, and fixing the volume by ethanol to obtain 10 mu g/mL of internal standard working solution, and preserving the internal standard working solution at the temperature of minus 18 ℃ in the dark.

2) Preparation of mixed standard working solution: transferring 1mL of phthalate mixed solution into the same 10mL volumetric flask, and diluting with ethanol to a certain volume to prepare 10 mug/mL of mixed standard stock solution; respectively transferring a certain volume of mixed standard stock solution into 6 10mL volumetric flasks, adding 100 mu L of internal standard working solution, and fixing the volume by using ethanol to prepare mixed standard working solutions with different concentrations, wherein the concentration sequences are as follows: 20. 50, 100, 200, 500, 1000ng/mL;

3) Standard curve acquisition: and (3) injecting the prepared mixed standard working solutions with different concentrations into SFC-MS/MS, and quantitatively analyzing the phthalate esters by an internal standard method, namely, performing linear regression analysis on the peak areas of the standard samples, the corresponding internal standard peak area ratio (y) and the concentration (x) of the standard samples to obtain standard curves of the phthalate esters, wherein the correlation coefficient is more than 0.996. Wherein, the selective ion chromatogram obtained when the mixed standard working solution with the concentration of 100ng/mL is measured is shown in figure 2.

4) Analysis of the sample solution to be tested: and (3) injecting the sample solution to be detected obtained in the step (1) into SFC-MS/MS, measuring the chromatographic peak area ratio of each phthalate and the internal standard, and substituting the chromatographic peak area ratio into a corresponding standard curve to obtain the content of each phthalate in the sample.

The chromatographic conditions for the analysis of phthalates using SFC-MS/MS in this example were: the column was a virdis HSS C18SB (3 mm x 100mm,1.8 μm, waters company, usa); the column temperature is 45 ℃; the sample injection amount is 2 mu L; the elution mode is gradient elution, and the mobile phase A is supercritical CO ₂ Mobile phase B is ethanol; the flow rate after mixing the two phases was 0.7mL/min, the gradient elution procedure is shown in Table 1, and the volume ratio of mobile phase A was uniformly varied.

TABLE 1 gradient elution procedure

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	99	1.0
1.0	99	1.0
			2.5	96	4.0
3.2	90	10
			4.0	90	10
4.5	99	1.0
			5.5	99	1.0

In this example, mass spectrometry conditions for analysis of phthalates using SFC-MS/MS were: the scanning mode is positive ion scanning; an electrospray ion source; the ion source temperature is 140 ℃; the capillary voltage is 2.6kV; the air flow of the taper hole is 70L/h; the flow rate of the desolventizing agent is 650L/h; the desolventizing gas temperature is 310 ℃; MRM parameters are shown in Table 2 using MRM mode acquisition.

Table 2 MRM parameters of mass spectrometry conditions

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In table 2, ions were quantified.

Example 2

The method for measuring phthalate in essence of this example is different from that of example 1 only in that: in the extraction of the sample solution to be detected, 0.18g of the sample to be treated is extracted by using 9mL of ethanol, and the spherical carbon is 45mg.

Example 3

The method for measuring phthalate in essence of this example is different from that of example 1 only in that: when the sample solution to be tested is extracted, 0.22g of the sample to be treated is extracted by adopting 11mL of ethanol, and the spherical carbon is 55mg.

Example 4

The method for measuring phthalate in essence of this example is different from that of example 1 only in that: the oscillation extraction rate was 250r/min.

Example 5

The method for measuring phthalate in essence of this example is different from that of example 1 only in that: the total flow rate after mixing of the two phases was 1.0mL/min.

Example 6

The method for measuring phthalate in essence of this example is different from that of example 1 only in that: when SFC-MS/MS is separated, the column temperature is 50 ℃; the sample loading was 3. Mu.L.

Example 7

The method for measuring phthalate in essence of this example is different from that of example 1 only in that: the temperature of the ion source is 150 ℃; the capillary voltage is 2.8kV; the air flow of the taper hole is 80L/h; the flow rate of the desolventizing agent is 700L/h; the desolventizing gas temperature was 330 ℃.

Example 8

The method for measuring phthalate in essence of this example differs from the method for measuring in example 1 only in that: the purification material used in this comparative example was a multi-walled carbon nanotube (particle size about 0.5 to 1 μm, available from Shanghai Annotation science and technology Co., ltd.), and specifically, the spherical carbon of example 1 was replaced with the multi-walled carbon nanotube.

Comparative example 1

The method for detecting phthalate in essence of this comparative example differs from the method for detecting in example 1 only in that: the sample solution to be measured for measurement of the comparative example is obtained by adopting a method reported in high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) for simultaneously measuring 16 phthalate compounds in tobacco flavor and fragrance (Zhang Xiaotao, etc., analytical science journal, 2013, 29 (6): 806-810). Because of low separation efficiency and poor purification effect, the pretreatment and chromatographic separation time of the comparative example are long, the method is not suitable for detecting daily mass samples, and the recovery rate cannot be well corrected without pretreatment of an internal standard.

Comparative example 2

The method for detecting phthalate in essence of this comparative example was the same as that of example 1, and the method for detecting the sample solution to be detected was the same as that reported in "gas chromatography-triple quadrupole tandem mass spectrometry while measuring 22 phthalate in essence" (Ruan Xiaojiao et al, food industry science and technology, 2018,39 (22): 235-240). The comparative example has too long chromatographic separation time to be suitable for detection of daily mass samples.

Comparative example 3

The method for measuring phthalate in essence of this comparative example is different from the method for measuring in example 1 only in that: the column used in this comparative example was a Viriddis BEH (2.1 mm. Times.100 mm,1.7 μm). The selective ion chromatograms of the resulting DBEP, DPHP and DCHP are shown in fig. 3, with poor phthalate peak shape and separation.

Experimental example

1) Sensitivity of

Phthalate mixed solutions with different concentrations are added into blank essence samples, and after extraction and purification, SFC-MS/MS is injected to generate concentration with 3 times of signal to noise ratio (S/N=3) as a method detection Limit (LOD), and concentration with 10 times of signal to noise ratio (S/N=3) as a method quantification Limit (LOQ). As shown in Table 3, the detection limits of the phthalic acid esters measured by the measurement method of example 1 of the present invention were 0.07 to 0.26. Mu.g/g and the quantitative limits were 0.25 to 0.98. Mu.g/g, respectively.

Table 3 recovery and repeatability (n=3) of the measurement method of example 1

Analyte(s)	Correlation coefficient	LOD(μg/g)	LOQ(μg/g)	Recovery (%)	RSD(％)
						DBEP	0.9996	0.26	0.98	90.3-97.8	3.6
DCHP	0.9987	0.19	0.69	89.4-96.5	4.4
						DPHP	0.9991	0.07	0.25	89.8-95.2	3.7

2) Accuracy and repeatability

To determine the reproducibility of the assay of the present invention, a 10. Mu.g/mL phthalate mixture solution (obtained by diluting the phthalate mixture obtained from Shanghai Ann Spectroscopy Co., ltd.) was added to a blank essence sample (not containing all analytes to be measured) so that the phthalate content in the sample was 5.0mg/kg, to obtain a sample to be measured. The labeled test samples were then measured according to the methods of example 1, example 8 and comparative examples 1 to 2, respectively, and the experiment was repeated 5 times, and the recovery rate was calculated according to the scalar addition and the measured value, and the results are shown in Table 4. Wherein the recovery of phthalate esters by the assay of example 1 is between 89.4% and 97.8%, and the mean Relative Standard Deviation (RSD) is less than 4.5%. The method has the advantages of high recovery rate, stable method and good repeatability.

Table 4 pretreatment time, recovery and repeatability (n=5)

Comparative examples 1 and 2 have lower recovery rates and longer separation times than example 1, and consume more toxic and harmful solvents. Example 8 has a lower recovery than example 1, indicating that the spherical carbon has a better purification effect on interfering substances in the essence, less extract matrix and more accurate results. The pretreatment method can better remove impurities and reduce the interference to the detection result.

The DBEP content of 5.0mg/kg of the standard sample measured by the detection method of example 1 and comparative example 2 was 4.84mg/kg and 4.75mg/kg, respectively. The accuracy of the measurement method of example 1 of the present invention was shown to be good. The ion flow diagram of the sample to be tested obtained by analyzing the sample to be tested with the phthalate content of 5.0 μg/g added in comparative example 1 is shown in fig. 3, and the separation effect of the three samples to be tested by virdis BEH is poor as shown in fig. 3.

3) Matrix effect

Extracting and purifying a blank essence sample according to the method of the embodiment 1 to obtain a blank essence sample extracting solution, respectively adding an object to be detected, an object to be detected and an internal standard substance into the blank essence sample extracting solution, analyzing the object to be detected by adopting a supercritical fluid chromatography-tandem mass spectrometry, and respectively analyzing the object to be detected by adopting a supercritical fluid chromatography-tandem mass spectrometry on ethanol solvent added with the object to be detected, the object to be detected and the internal standard substance; the chromatographic conditions and mass spectrometry conditions of the supercritical fluid chromatography-tandem mass spectrometry were the same as in example 1, and the results are shown in Table 5.

Table 5 shows that the samples to be tested have strong matrix effect in the essence matrix, and the response ratio of the samples to be tested to the internal standard in the blank essence sample extracting solution and the response ratio of the samples to be tested to the internal standard in the ethanol solvent have no obvious difference after single internal standard correction, so that the standard working solution prepared by the ethanol solvent can be used for quantitative analysis.

TABLE 5 response ratio after correction for matrix effect and internal standard

As can be seen from the measurement results of the examples and the comparative examples, the measurement method of the invention is efficient, economical and environment-friendly. And aiming at the characteristics of essence, spherical carbon is selected as a purifying material, so that not only can the interfering substances in the matrix be well removed, but also the recovery rate is better, and the performance is stable. As can be seen from the measurement results of example 1 and comparative example, the pretreatment conditions used in the measurement method of the present invention are simple and convenient, and the extraction and purification are integrated.

Claims

1. A method for determining phthalate in essence is characterized in that: the method comprises the following steps: analyzing phthalate in the essence sample by adopting a supercritical fluid chromatography-tandem mass spectrometry method; the chromatographic column of supercritical fluid chromatography in the supercritical fluid chromatography-tandem mass spectrometry is virdis HSS C18SB, the elution mode is gradient elution, and the mobile phase A is supercritical CO ₂ Mobile phase B is an alcoholic solvent.

2. The method for determining phthalate in essence according to claim 1, characterized in that: the preparation method of the sample solution to be detected adopted in the process of analyzing phthalate in the essence sample by adopting a supercritical fluid chromatography-tandem mass spectrometry comprises the following steps: adding an isotope internal standard, an extraction solvent and a purification material into an essence sample for extraction, and then carrying out solid-liquid separation; the purification material is spherical carbon and/or multi-wall carbon nano-tube.

3. The method for determining phthalate in essence according to claim 2, characterized in that: the purification material is spherical carbon with the particle diameter of 0.4-1.0 μm and the specific surface area of 80-120m ² /g。

4. A method of determining phthalate esters in fragrances according to claim 2 or 3, characterized in that: each 0.18-0.22g essence sample has a volume of 9-11mL corresponding to the extraction solvent, and a mass of 50mg corresponding to the purification material.

5. A method of determining phthalate esters in fragrances according to claim 1 or 2 or 3, characterized in that: the phthalate esters include di (2-butoxy) ethyl phthalate, dicyclohexyl phthalate, and diphenyl phthalate.

6. A method of determining phthalate esters in fragrances according to claim 1 or 2 or 3, characterized in that: the chromatographic conditions of supercritical fluid chromatography in the supercritical fluid chromatography-tandem mass spectrometry are as follows: the column temperature is 45-50 ℃; the sample injection amount is 2-3 mu L; and during gradient elution, the total flow rate of the mixed mobile phase A and mobile phase B is 0.7-1.0mL/min.

7. The method for determining phthalate in essence according to claim 6, characterized in that: the volume ratio of the mobile phase A is reduced from 97-99% to 88-90% during gradient elution, and then the mobile phase A is recovered to 97-99%.

8. A method of determining phthalate esters in fragrances according to claim 1 or 2 or 3, characterized in that: the mass spectrum conditions in the supercritical fluid chromatography-tandem mass spectrometry are as follows: the scanning mode is positive ion scanning; an electrospray ion source; the temperature of the ion source is 140-150 ℃; the capillary voltage is 2.6-2.8kV; the air flow of the taper hole is 70-80L/h; the flow rate of the desolventizing agent is 650-700L/h; the desolventizing gas temperature is 310-330 ℃; and adopting MRM mode acquisition.

9. A method of determining phthalate esters in fragrances according to claim 2 or 3, characterized in that: the extraction solvent is an alcohol solvent.

10. A method of determining phthalate esters in fragrances according to claim 2 or 3, characterized in that: the extraction is oscillation extraction; the oscillation rate of the oscillation extraction is 200-250r/min, and the time of the oscillation extraction is 8-12min.