KR101910442B1 - Simple extraction analysis methods for volatile polychlorinated biphenyls in high-fat foodstuff - Google Patents

Abstract

The present invention relates to a method for extracting volatile polychlorinated biphenyls (VPCBs) contained in high-fat foods using headspace solid-phase microextraction (HS-SPME) and a method for extracting volatile polychlorinated biphenyls Phenyl < / RTI >

Description

TECHNICAL FIELD The present invention relates to a simple extraction method for volatile polychlorinated biphenyls in high-fat food samples,

A method for extracting volatile polychlorinated biphenyls (VPCBs) from a high fat food sample is disclosed herein.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with acid and alkali, heat-stable and fat-soluble properties. The route of entry into the body is mainly known as food intake and is classified as Group 2A, a human carcinogen in the International Agency for Research on Cancer (IARC).

Polychlorinated biphenyls are biologically concentrated in the food chain by binding to lipid components of animal tissues because of their high solubility in fat. It may enter the body by food ingestion or breathing and accumulate in the body, resulting in damage of the liver, kidney and reproductive function, and diseases such as cancer. Because of the high fat solubility characteristics of polychlorinated biphenyls, there is a high possibility of contamination in meat and processed meat products containing a large amount of fat. Therefore, the contamination of polychlorinated biphenyls (PCBs) It is necessary to establish a matching regulatory standard.

Conventional methods for analyzing polychlorinated biphenyls include Soxhlet extraction or Solid-phase extraction (SPE), but the purification process of the multi-layered column is required. Therefore, this method consumes a lot of solvent, and it is troublesome and takes a very long time.

Levels of polychlorinated biphenyls (PCBs) in meat and meat products from the state of Rio Grande do Sul, Brazil, I. Costabeber et al., Food and Chemical Toxicology 44, 2006, 17. Determination of Polychlorinated Biphenyls in Brazilian Breast Milk Samples using Solid-Phase Microextraction and Gas Chromatography-Electron Capture Detection, Kowalski et al., 2010, Vol. 21, No. 3, 502-509.

In view of the above, it is an object of the present invention to provide a method for easily extracting, detecting and quantifying volatile polychlorinated biphenyls (VPCBs) contained in high-fat food samples.

According to an aspect of the present invention, there is provided a method of analyzing a sample, comprising: subjecting a sample to be analyzed to a nonpolar organic solvent and performing ultrasonic extraction using an ultrasonic wave;

Liquid-liquid extraction (LLE) from the same non-polar organic solvent;

Concentrating the extract obtained in the above step to obtain fat;

Adding the fat to methanol and further liquid-liquid extraction; And

And then extracting only the supernatant of the extract with a head-space vial followed by extraction using headspace solid-phase microextraction (HS-SPME) VPCBs, and Volatile Polychlorinated biphenyls).

Further, an embodiment of the present invention is a method for detecting volatile poly (biphenyls), which further comprises detecting or quantifying volatile polychlorinated biphenyls (VPCBs) contained in a sample to be analyzed by mass spectrometry of the extract obtained through the above extraction method A method for detecting or quantifying biphenyl chloride is provided.

Another embodiment of the present invention relates to a method for the preparation of a pharmaceutical composition comprising an ultrasonicator, a nonpolar organic solvent and methanol; And

Head-space vial and combi-PAL;

(VPCBs), comprising the steps of: (a) providing an extraction kit of volatile polychlorinated biphenyls (VPCBs).

Another embodiment of the present invention relates to a method for the preparation of a pharmaceutical composition comprising an ultrasonicator, a nonpolar organic solvent and methanol;

Head-space vial and combi-PAL; And

Mass spectrometer;

(Volatile Polychlorinated Biphenyls (VPCBs)), which comprises a volatile polychlorinated biphenyls (VPCBs).

The present invention can extract the fat by ultrasonic extraction and liquid-liquid extraction from a high-fat food sample and then extract it again in a headspace solid phase to effectively shorten the time required for the pretreatment of the food sample, Volatile polychlorinated biphenyls contained in high fat food samples can be extracted and quantified.

FIG. 1 is a diagram illustrating a sample preparation process LLE-SPME (liquid-liquid extraction-headspace solid-phase microextraction) according to an embodiment of the present invention and a liquid-liquid extraction (LLE)
FIG. 2 is a graph showing the results of volatile polychlorinated biphenyl analysis (LLE-SPME) and liquid-liquid extraction (LLE) in a sample according to an embodiment of the present invention. Fig.

Hereinafter, the present invention will be described in detail.

One embodiment of the present invention provides a method for extracting volatile polychlorinated biphenyls (VPCBs).

In one embodiment, the extraction method comprises the steps of subjecting a sample to be analyzed to a non-polar organic solvent and performing ultrasonic extraction using an ultrasonic wave; Liquid-liquid extraction (LLE) from the same non-polar organic solvent; Concentrating the extract obtained in the above step to obtain fat; Adding the fat to methanol and further liquid-liquid extraction; And extracting only the supernatant of the extract solution in a head-space vial, followed by extraction using headspace solid-phase microextraction (HS-SPME).

In one embodiment, the sample to be analyzed may include a food sample containing fat. The above-mentioned "food sample" is a concept covering all the samples capable of being consumed. Specifically, the food sample may include both a natural state sample not subjected to cooking and a cooked state sample. Specifically, the food sample may be a high fat food sample, and may specifically include meat and meat processed products, turbulence, cheese, milk, processed milk products, and the like.

Polychlorinated biphenyls are compounds in which 1 to 10 of the 10 hydrogen atoms of biphenyl in which two benzene rings are connected are substituted with chlorine atoms, and have various kinds of isomers depending on the number of chlorine atom or position thereof substituted. Information on 209 species of PCBs isomers can be found at https://en.wikipedia.org/wiki/PCB_congener_list. The volatile polychlorinated biphenyls according to one embodiment of the present invention may be polychlorinated biphenyls of PCB 101 or less. More specifically, the volatile polychlorinated biphenyls are PCBs (2, 4, 4'-trichlorobiphenyl), PCBs (2, 2 ', 5, 5'-tetrachlorobiphenyl) , 5'-pentachlorobiphenyl). At this time, the PCB 101 and the like are referred to as IUPAC, and PCB No. 101 and so on. PCBs theoretically have 209 isomers. In IUPAC, the order of 1 to 209 is specified according to the number and location of chlorine. Therefore, in the present specification, the term " below the PCB 101 " means that the PCB number of the PCB 1 to the PCB 209 is 101 or less, i.e., PCB 1 to PCB 101.

In one embodiment, the non-polar organic solvent used for the ultrasonic extraction and the liquid-liquid extraction may include a mixture of hexane, heptane, nonene or hexane and methylene chloride, Any solvent can be used without limitation.

In one embodiment, the ultrasound extraction step may include a step of inserting a sample to be analyzed and a mixed internal reference material into a test tube, adding a nonpolar organic solvent, and then extracting the sample using an ultrasonicator. The extraction time using the ultrasonic wave may be about 5 minutes to 15 minutes, and the re-extraction time using the shaker may be about 15 minutes to 25 minutes.

In one embodiment, the step of liquid-liquid extraction (LLE) of the ultrasound extract after the ultrasonic extraction may be performed using a shaker. In one embodiment, the step of obtaining the fat may include a step of centrifuging the extract obtained in the liquid-liquid extraction step, separating the organic solvent layer as the supernatant, and concentrating the organic solvent layer to obtain fat. More specifically, the centrifugation can be carried out at about 2200 rpm to 3200 rpm for 5 minutes to 10 minutes. In one embodiment, the step of concentrating the extract to obtain fat may include concentrating the extract using a nitrogen concentrator. The reason for obtaining the fat from the extract is that the polybrominated biphenyl is fat-soluble and is likely to accumulate in adipocytes of animal tissues.

In one embodiment, the headspace solid phase microextraction may further comprise an adsorption step of adsorbing the sample from the supernatant in the head-space vial and a desorption step. In one embodiment, the headspace solid-phase microextraction method may further include, before the adsorption step, immersing only the supernatant of the extract in a head-space vial, then adding an aqueous solution of sodium chloride and homogenizing.

More specifically, the headspace solid-phase microextraction method comprises: an adsorption step of agitating the supernatant contained in the head-space vial using a combi-PAL at 80 to 100 DEG C for 40 to 60 minutes; And a desorption step of injecting the solution into a column or a sample inlet at a temperature of 250 to 280 ° C. after the adsorption step and desorbing the solution for 2 to 5 minutes.

In one embodiment, the fibers of the headspace solid phase microextraction method may have an average diameter of 60 to 100 mu m, and the coating material may include at least one of polyacrylate and PDMS (Polydimethylsiloxane) . The combi-PAL according to one embodiment is one of the automatic sample injectors used in connection with a gas chromatograph. When the adsorption and desorption conditions are inputted numerically in software, the combi-arm automatically extracts the head space Automatic injection.

In one embodiment of the present invention, there is provided a method for producing a polyvinyl chloride copolymer, comprising the steps of: extracting volatile polychlorinated biphenyls by an extraction method of volatile polychlorinated biphenyls; And detecting or quantifying volatile polychlorinated biphenyls contained in the sample to be analyzed by mass spectrometry of the extract obtained in the above step. The present invention also provides a method for detecting or quantitatively analyzing volatile polychlorinated biphenyls.

In one embodiment, mass spectrometry can be performed using a mass spectrometer, such as gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry, As long as the concentration of the volatile polychlorinated biphenyl in the sample to be analyzed can be measured.

More specifically, the temperature condition of the mass spectrometry is, in one embodiment, from the initial 100 ° C to a temperature of 200 ° C at a rate of 10 ° C / min, a holding time of 3 minutes; The temperature was raised to 260 DEG C at 3 DEG C / min, and then the holding time was 0 minutes; And the holding time after heating to 300 DEG C at 40 DEG C / min may be set to 1 minute.

One embodiment of the present invention relates to an ultrasonicator, a non-polar organic solvent and a solvent selected from the group consisting of methanol; And volatile polychlorinated biphenyls (VPCBs) extraction kits including a head-space vial and a combi-PAL.

In addition, one embodiment of the present invention may be applied to an ultrasonic generator, a nonpolar organic solvent and methanol; Head-space vial and combi-PAL; And volatile polychlorinated biphenyls (VPCBs), including a mass spectrometer, as well as a kit for detecting or quantitatively analyzing volatile polychlorinated biphenyls (VPCBs).

The extraction kit and the detection or quantitation kit can be non-polar organic daily, comprising a mixture of hexane, heptane, nonene or hexane and methylene chloride as an example of a nonpolar organic solvent. In addition, in one embodiment, the kit may further include at least one of a shaker, a nitrogen concentrator, and a centrifuge.

In one embodiment, the mass spectrometer may include a gas chromatography-mass spectrometry or a liquid chromatography-mass spectrometry.

In one embodiment, the extraction kit may further include an instruction to describe the above-described extraction method of volatile polychlorinated biphenyl.

In one embodiment, the detection or quantitative analysis kit may further comprise an instruction describing the above-described detection or quantitative analysis method of volatile polychlorinated biphenyls.

Conventional testing standards for PCBs (polychlorinated biphenyls) include purification using a multilayer silica gel column after Soxhlet extraction. However, this method of sample preparation (extraction) 12 to 24 hours, and only about 9 to 10 analyzes are possible per week. However, according to the above-described embodiments of the present invention, since it takes about 5 hours to analyze one sample, and since multiple samples can be processed, about 160 analyzes can be performed in a week, so that the efficiency of detection and quantitative analysis is remarkably improved .

Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the following examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example 1] Extraction 1 of volatile polychlorinated biphenyl (LLE-SPME)

As a sample to be analyzed, pork belly was selected as a representative medium among the foods containing fat, and was homogeneously and smallly crushed using a blender.

5 g of the sample was taken with a chemical balance (Ohaus Scout Pro Portable Electronic Balance) and transferred to a 50 mL falcon tube. 25 μL of a mixed internal standard of 100 μg / L was added, and 25 mL of hexane as an extraction solvent was added thereto. The mixture was extracted with an ultrasonicator and a shaker for 10 minutes and 20 minutes, respectively. After extraction, the mixture was centrifuged at 2700 rpm for 5 minutes, and then the organic solvent layer was separated and concentrated using a nitrogen concentrator until only the fat remained. After concentrating, 1 mL of methanol was added, vortexed and centrifuged. And only the supernatant was transferred to a 20 mL head-space vial. 9 mL of a 25% aqueous solution of sodium chloride was added to the vial, the sample was sufficiently homogenized, and the mixture was stirred in an agitator at 90 DEG C for 50 minutes using a combi-PAL. Headspace solid-phase microextraction (HS-SPME) was desorbed for 2 minutes using 85 μm polyacrylate fiber at 280 ° C inlet conditions by gas chromatography.

[Comparative Example 1] Extraction 2 of volatile polychlorinated biphenyl (LLE)

A sample to be analyzed was prepared in the same manner as in Example 1, and was uniformly and finely pulverized using a blender.

5 g of the sample was taken with a chemical balance (Ohaus Scout Pro Portable Electronic Balance) and transferred to a 50 mL falcon tube. 25 μL of a mixed internal standard of 100 μg / L was added, and 25 mL of hexane as an extraction solvent was added thereto. The mixture was extracted with an ultrasonicator and a shaker for 10 minutes and 20 minutes, respectively. After extraction, the mixture was centrifuged at 2700 rpm for 5 minutes, and then the organic solvent layer was separated and concentrated using a nitrogen concentrator until only the fat remained. After concentrating, 1 mL of methanol was added, vortexed and centrifuged. Only the supernatant was transferred to a vial for gas chromatography.

[Example 2] Quantitative analysis of volatile polychlorinated biphenyls

An Agilent 7693 A series injector was used in a gas chromatography mass spectrometer (Agilent 5975 C mass selective detector directly connected to Agilent 7890 A gas chromatography of Agilent Technologies (Palo Alto, Calif., USA)).

The sample prepared in Example 1 was injected into a combi-arm in accordance with the procedure described in Example 1, using an 85 μm polyacrylate fiber, and headspace solid-phase microextraction (HS-SPME).

The separation tube used was ultra-2 (cross-linked 5% phenylmethyl silicone, length 25 m, internal diameter 0.2 mm, film thickness 0.33 μm) and ultrafast purity (99.9999%) helium at a flow rate of 1 mL / min Respectively. The temperature of the inlet was 280 ° C and a splitless injection mode was used. The oven conditions were set to be heated from 100 ° C to 300 ° C. The electron energy used for ionization was 70 eV, and selected ion monitoring (SIM mode) was used. Gas Chromatography Mass Spectrometer (GC-MS) Operating conditions are shown in Table 1 below.

Condition Separator Ultra-2
(Cross-linked 5% phenylmethyl silicone, 25 m x 0.2 mm ID, 0.33 um film thickness) Carrier gas A flow rate of 1.0 mL / min of helium Injection mode Non-dividing injection Acquisition mode SIM mode in GC-MSD Ionization EI at 70 eV Solvent delay time 11 minutes Total time 35 minutes Temperature programming Initial 100 ° C # Heating rate (° C / min) Temperature (℃) Time (minutes) One 10 200 3 2 3 260 0 3 40 300 One

FIG. 2 is a graph showing the results of measurement of volatile polychlorinated biphenyl low molecular weight volatile polychlorinated biphenyl PCB 28 (2, 4, 4'-trichlorobiphenyl), PCB 52 (2, 2 ', 5, tetrachlorobiphenyl) and PCB 101 (2, 2 ', 4, 5, 5'-pentachlorobiphenyl).

Liquid-liquid extraction (LLE) was obtained by directly injecting a sample after liquid-liquid extraction according to the method of Comparative Example 1, and LLE-SPME was subjected to headspace solid-phase microextraction The results of mass spectrometry after adsorption, desorption and injection of the sample. As a result, as shown in FIG. 2, according to one embodiment of the present invention, LLE-SPME using the headspace solid-phase microextraction method can extract a significantly larger amount of volatile polychlorinated biphenyl than LLE according to Comparative Example 1 , And it can be confirmed that excellent volatile polychlorinated biphenyl is detected and quantitatively analyzed.

[Experimental Example 1] Comparison of extraction methods

The high fat food samples were subjected to ultrasonic extraction and liquid-liquid extraction in the same manner as in Examples 1 and 2, followed by extraction using a head space solid-phase microextraction method and analysis with a gas chromatography mass spectrometer (GC-MS) Respectively. As a result, the detection limit of 0.015-0.2 ng / g was shown as shown in Table 2 below.

Compounds PCB 28 PCB 52 PCB 101 LOD (ng / g) 0.015 0.02 0.2

As a comparative example, a sample of meat or meat processed products obtained from Rio Grande do Sul state in Brazil was subjected to solid-phase extraction (SPE) using Florisil, and then the detection limit was analyzed after purification using a multilayer silica gel column. As a result, the detection limit of PCB 10, PCB 28, and PCB 52 was 0.2 ng / g as shown in Table 3 below.

Compounds PCB 10 PCB 28 PCB 52 LOD (ng / g) 0.2 0.2 0.2

As another comparative example, 20 breast milk samples obtained from a breast milk bank associated with a public health service in Brazil were subjected to headspace solid-phase microextraction (HS-SPME) without ultrasonic extraction and liquid-liquid extraction in Example 1, ), And then analyzed using gas chromatography electronic capture detector (GC-ECD). As a result, a detection limit of 0.54 to 0.74 ng / g was exhibited as shown in Table 4.

Compounds PCB 28 PCB 52 PCB 101 LOD (ng / g) 0.54 0.74 0.61

When all of PCB 28, PCB 52 and PCB 101, which are volatile polychlorinated biphenyls (PCBs) having a low molecular weight, are extracted using the headspace solid-phase microextraction (HS-SPME) in the final stage of pretreatment, The detection limit was 0.015 ~ 0.2 ng / g.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

As an extraction method of volatile polychlorinated biphenyls (VPCBs)
The extraction method comprises the steps of: subjecting a sample to be analyzed to a non-polar organic solvent and subjecting the sample to ultrasonic extraction using an ultrasonic wave, without sample pretreatment through Soxhlet extraction;
Liquid-liquid extraction (LLE) of the supersonic extract in the same non-polar organic solvent;
Concentrating the extract obtained in the above step to obtain fat;
Adding the fat to methanol and liquid-liquid extraction again; And
Extracting only the supernatant of the liquid extracted with the liquid-liquid into a head-space vial, and extracting using a headspace solid-phase microextraction (HS-SPME) method;
≪ / RTI > wherein the volatile polychlorinated biphenyl is extracted. The method of claim 1, wherein the non-polar organic solvent comprises a mixture of hexane, heptane, nonane or hexane and methylene chloride. The method of extracting volatile polychlorinated biphenyl according to claim 1, wherein the sample to be analyzed comprises a food sample containing fat. 4. The method of claim 3, wherein the food sample comprises at least one of the group consisting of meat, meat processed products, turbulence, cheese, milk and processed milk products. The method of claim 1, wherein the volatile polychlorinated biphenyl is polychlorinated biphenyl of PCB 1 to PCB 101. The method of claim 1, wherein the volatile polychlorinated biphenyls are selected from the group consisting of PCB 28 (2,4 ', 4'-trichlorobiphenyl), PCB 52 (2,2', 5,5'-tetrachlorobiphenyl) 4,5,5'-pentachlorobiphenyl). ≪ / RTI > The method of claim 1, wherein the fiber conditions of the headspace solid phase microtexture
An average diameter of 60 to 100 mu m; And
A coating material of one or more fibers of polyacrylate and PDMS (Polydimethylsiloxane);
Wherein the volatile polychlorinated biphenyl is extracted. The method of claim 1, wherein the headspace solid phase microextraction
Agitating the supernatant in the head-space vial using a combi-PAL at 80 to 100 DEG C for 40 to 60 minutes; And
A desorption step of injecting into a column at a temperature of 250 to 280 ° C. after the adsorption step and desorbing the solution for 2 to 5 minutes;
Wherein the volatile polychlorinated biphenyl is extracted. The method according to claim 1, wherein the step of obtaining the fat comprises centrifuging the extract obtained in the liquid-liquid extraction step, separating the organic solvent layer as the upper layer liquid, and concentrating the organic solvent layer to obtain fat, Extraction method of polychlorinated biphenyls. As a method for the detection or quantitative analysis of volatile polychlorinated biphenyls,
Wherein the detection or quantitative analysis method comprises the steps of: extracting volatile polychlorinated biphenyls according to the method of any one of claims 1 to 9 without sample pretreatment through Soxhlet extraction; And
Detecting or quantitating volatile polychlorinated biphenyls (VPCBs) contained in the sample to be analyzed by mass spectrometry of the extract obtained in the above step;
≪ / RTI > wherein the volatile polychlorinated biphenyl is selected from the group consisting of: Ultrasonicators, nonpolar organic solvents and methanol;
Head-space vial and combi-PAL; And
10. An extraction kit for volatile polychlorinated biphenyls (VPCBs), comprising: an instruction describing an extraction method according to any one of claims 1 to 9; delete Ultrasonicators, nonpolar organic solvents and methanol;
Head-space vial and combi-PAL;
Mass spectrometer; And
A detection or quantification kit for volatile polychlorinated biphenyls (VPCBs), comprising instructions for the detection or quantitation method of claim 10. delete

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