KR100537402B1 - Determination Method of Hydroxy-PCBs in Urine - Google Patents

Abstract

In order to simultaneously analyze hydroxy PCBs (hydroxy polychlorinated biphenyls) contained in urine samples, SPME fibers (fiber) are extracted continuously using a method of extracting using solid-phase microextraction (SPME). TMS derivatization on) characterized in that the analysis. In the present invention, the concentration of hydroxy PCBs is analyzed using gas chromatography or gas chromatography / mass spectrometry. When the urine sample is analyzed by the method of the present invention, it is possible not only to obtain excellent sensitivity, shorten analysis time, simplicity and economy, but also to prevent secondary contamination generated when using an extraction solvent.

Description

Determination Method of Hydroxy-PCBs in Urine

The present invention is a detection method for detecting hydroxy PCBs (hydroxy polychlorinated biphenyls) metabolites in urine samples, the hydroxy metabolites are extracted by adsorption on SPME fibers, and then trimethylsilyl (TMS) derivatization under a constant reaction time and temperature It is directed to a method of analyzing this using a gas chromatograph (GC) or gas chromatograph / mass spectrometer (GC / MS). The method of the present invention has advantages such as excellent sensitivity, shorter analysis time, simplicity, and economy by extracting and derivatizing on SPME fibers, and thus can be effectively used for monitoring hydroxy PCBs metabolites remaining in trace amounts in urine samples. .

PCBs are thermally stable, have good electrical insulation, chemically inert, resistant to acids and alkalis, and are widely used as insulating oils and heat mediums such as transformers and capacitors, but are classified and managed as toxic substances or pollutants.

In addition, PCBs are one of endocrine disruptors, and especially their metabolites, hydroxy PCBs, have a harmful effect on the human body, such as inhibiting the activity of estrogen in vivo.

Hydroxy PCBs to be analyzed in the present invention are representative metabolites of PCBs and are substances that have been continuously detected in the environment and in vivo until recently. When PCBs are introduced into a living body, hydroxy PCBs produced through metabolic processes may act as inhibitors of estrogen receptors in the body, inhibit metabolic processes of thyroid hormones, and interfere with oxidative phosphorylation of cells or mitochondria. It is known to act as a system barrier.

Analysis of trace amounts of hydroxy PCBs requires instrumentation with good sensitivity and separation, such as gas chromatography / mass spectrometry (GC / MS). However, since these substances have high polarity, it is difficult to directly analyze them by GC or GC / MS. To solve these problems, extraction, concentration and derivatization processes are required. As such, when the extraction and derivatization process is performed, it is time-consuming and requires a solid phase or an organic solvent, a derivatization reagent, and the like because it requires a complicated sample pretreatment process.

Therefore, there is a need for a method for detecting hydroxy PCBs that can be analyzed quickly, economically and in trace amounts.

Accordingly, the present invention enables the effective analysis of hydroxy PCBs metabolites in biological samples, especially urine samples, and provides an analytical method capable of exhibiting selective and excellent sensitivity by gas chromatography or gas chromatography / mass spectrometry. It is an object of the present invention to provide a method that can be applied to the analysis of biological samples, especially urine samples, which can satisfy the requirements of accuracy, accuracy, short analysis time, low cost and simplicity.

We adsorbed and extracted hydroxy PCBs in urine samples using SPME fiber, and then trimethylsilyl (TMS) derivatized immediately and analyzed by gas chromatography or gas chromatography / mass spectrometry The discovery that Roxy PCBs can be analyzed to trace concentrations has led to the present invention.

Hereinafter, the method of the present invention will be described in detail.

The method of the present invention comprises the steps of adsorbing hydroxy PCBs in a urine sample to fibers of SPME, derivatizing hydroxy PCBs adsorbed to SPME fibers, and trimethylsilyl (TMS) derivatization, and gasifying TMS derivatized hydroxy PCBs. Analysis by chromatography or gas chromatography / mass spectrometry.

In the present invention, SPME is used to extract and derivatize hydroxy PCBs from biological samples, particularly urine samples. Since hydroxy PCBs are highly polar substances, they are difficult to analyze in GC without derivatization. In addition, the extraction of substances contained in the urine sample requires several steps of extraction and impurity removal processes, and a large amount of highly toxic organic solvents are used. In the present invention, in order to compensate for these disadvantages, the organic solvent is used to directly adsorb the hydroxy PCBs without extracting the hydroxy PCBs to the SPME fiber, and then all TMS derivatized hydroxy PCBs adsorbed on the SPME fiber. Examples of such derivatization reactions are shown in Scheme 1 below.

Wherein X is Cl or NCOCF ₃ .

First, the adsorption time, adsorption temperature, type of fiber, etc. are very important in the adsorption process into SPME fibers. Adsorption time of about 60 minutes or more and adsorption temperature of about 90 ° C or more are suitable, and the fibers are all made of PDMS (polydimethylsiloxane), PA (polyacrylate) and PDMS-DVB (polydimethylsiloxane-divinylbenzene) fibers. Fibers can be used according to the invention, although PDMS-DVB fibers are most suitable.

In the TMS derivatization process, a reaction time of at least about 5 minutes, preferably about 5 to 30 minutes, at a temperature of about 60 ° C. is most suitable, and the reaction reagent is N, N-bis (trimethylsilyl) trifluoroacetamide (BSTFA) is used and the dosage is about 10 μl. In addition, in the case of a sample having a complex medium such as a urine sample, when the fiber is directly contacted with the sample, the fiber is rapidly contaminated. Therefore, in order to solve the problem, the fiber is placed in the headspace position as shown in FIG. The adsorption and derivatization are carried out.

The derivatized hydroxy PCBs are analyzed using gas chromatography or gas chromatography / mass spectrometry. Gases that can be used as mobile phases in the present invention in the analysis can be any gas generally used in gas chromatography, preferably gases which are inert to oxidation, ie nitrogen, helium and hydrogen can be used as the mobile phase. In addition, in the case of a column that can be used according to the present invention, there is no particular limitation on the length and the inner diameter of the column, but only a moderate polarity.

As described above, if the hydroxy PCBs are extracted, the hydroxy PCBs may be adsorbed onto the SPME fiber without using any organic solvent to complete the extraction. Also, the hydroxy PCBs may be directly converted into GC / MS by derivatizing the TMS on the SPME fiber. Analysis is possible. In case of derivatization using BSTFA, all solvents can be evaporated and then evaporated after extraction, but in this case, it must go through a separate extraction process and evaporation process, and the reaction is completed when the reaction time is over 30 minutes in a reactor of 60 ℃ or higher. However, according to the present invention, since all extraction and derivatization are terminated by reaction for about 5 minutes after adsorption for about 60 minutes, hydroxy PCBs can be analyzed very simply.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples in any way.

The reagents used in the examples below are as follows.

- standard solution of 11-hydroxy-PCBs: n- hexane each standard 10㎍, Accustandard four per 1㎖, (3- hydroxy-4,4'-dichloro-biphenyl (3-OH-4,4 ' -DCB), 2-hydroxy-2 ', 3,5'-trichlorobiphenyl (2-OH-2', 3,5'-TrCB), 2-hydroxy-2 ', 5,5'-trichloro Lobbyphenyl (2-OH-2 ', 5,5'-TrCB), 4-hydroxy-3,4', 5-trichlorobiphenyl (4-OH-3,4 ', 5-TrCB), 4- Hydroxy-2 ', 3', 4 ', 5'-tetrachlorobiphenyl (4-OH-2', 3 ', 4', 5'-TeCB), 4-hydroxy-2 ', 3,5 , 5'-tetrachlorobiphenyl (4-OH-2 ', 3,5,5'-TeCB), 4,4'-dihydroxy-3,3', 5,5'-tetrachlorobiphenyl ( 4,4'-DiOH-3,3 ', 5,5'-TeCB), 4-hydroxy-2', 3,3 ', 4', 5'-pentachlorobiphenyl (4-OH-2 ' , 3,3 ', 4', 5'-PeCB), 2-hydroxy-2 ', 3', 4 ', 5,5'-pentachlorobiphenyl (2-OH-2', 3 ', 4 ', 5,5'-PeCB), 4-hydroxy-2', 3,3 ', 4', 5,5'-hexachlorobiphenyl (4-OH-2 ', 3,3', 4 ' , 5,5'-HxCB), 4-hydroxy-2 ', 3,3', 5,5 ', 6-hexachlorobiphenyl (4-OH-2', 3,3 ', 5,5' , 6-HxCB))

- pyrene -d ₁₀ solution: n- hexane 10㎍, Aldrich Co. per 1㎖

N, N-bis (trimethylsilyl) -trifluoroacetamide (BSTFA): Aldrich

N- (tert-butyldimethylsilyl) -N-ethyltrifluoroacetamide (MTBSTFA): Aldrich

Pentafluoropropionic anhydride (PFPA): from Aldrich

Heptafluorobutyric anhydride (HFBA): from Aldrich

<Example 1>

This embodiment is for viewing the adsorption rate according to the adsorption temperature.

Four 4 ml vials were prepared, and 2 ml of blank sample (rat urine prior to PCB administration) was added to each vial, followed by 10 μl of standard solution of 11 hydroxy PCBs. After adding ₁₀ µl of the -d ₁₀ solution, the PDMS-DVB fiber holder was fixed in the headspace above the water of the solvent in the vial, and the fiber was removed from the needle of the fiber holder and exposed at a position of about 1 cm above the water. At this time, the temperature of the heater was adjusted to 20, 40, 60 and 90 ° C., respectively, and the vial was adsorbed while heating for 1 hour. The hydroxy PCBs adsorbed fibers were taken out and derivatized by exposing again in the headspace of a 10 μl solution of temperature controlled BSTFA in a 60 ° C. heater.

In order to check the adsorption degree, the derivatized hydroxy PCBs adsorbed fibers were injected into the inlet of the gas chromatography / mass spectrometer, and eleven hydroxy PCBs were separated by increasing the oven temperature of the gas chromatography. The degree of adsorption was compared according to the size of the selected ions of the substances shown in Table 1 below. The results are as shown in Figure 2 and showed the highest adsorption degree when the adsorption at 90 ℃ of the above four temperatures.

No. Target substance Ion (m / z) optional One 3-OH-4,4'-DCB 93 310 312 2 2-OH-2 ', 3,5'-TrCB 93 344 346 3 2-OH-2 ', 5,5'-TrCB 93 344 346 4 4-OH-3,4 ', 5-TrCB 173 344 346 5 4-OH-2 ', 3', 4 ', 5'-TeCB 365 380 378 6 4-OH-2 ', 3,5,5'-TeCB 365 363 380 7 4,4'-DiOH-3,3 ', 5,5'-TeCB 93 466 468 8 4-OH-2 ', 3,3', 4 ', 5'-PeCB 93 412 414 9 2-OH-2 ', 3', 4 ', 5,5'-PeCB 93 412 414 10 4-OH-2 ', 3,3', 4 ', 5,5'-HxCB 433 446 448 11 4-OH-2 ', 3,3', 5,5 ', 6-HxCB 93 446 448

<Example 2>

This embodiment is for viewing the adsorption rate according to the adsorption time.

Five 4 ml vials were prepared, each containing 2 ml of a blank sample (urine urine prior to PCB administration) and a standard solution of 11 hydroxy PCBs was added to each vial. Add 10µl and add 10µl each of the internal standard Pyrene-d ₁₀ solution, and fix the PDMS-DVB fiber holder to the headspace above the water of the solution in the vial, and then remove the fiber from the needle of the fiber holder. It was exposed at the position of degree. At this time, the temperature of the heater is adjusted to 90 ° C. and the adsorption time is changed to 10, 30, 45, 60, and 120 minutes, respectively, to adsorb the vials while heating, and the hydroxy PCBs adsorbed fibers are heated at a temperature of 60 ° C. Derivatization was performed by exposing again in the headspace of the adjusted BSTFA 10 μl solution.

As in Example 1, the degree of adsorption was checked by comparing the degree of adsorption according to the size of the selected ion of each material. The results are as shown in Figure 3, the adsorption increased up to 60 minutes, it can be seen that the adsorption does not increase above.

<Example 3>

This embodiment is for viewing the adsorption rate according to the adsorption fiber.

Prepare three 4 ml vials, add 2 ml of blank sample (urine urine before PCB administration) to each vial, add 10 µl of each standard solution of 11 hydroxy PCBs, and use the internal standard pyrene-d _10. After adding 10 µl of the solution, the PDMS, PA or PDMS-DVB fiber holder was fixed in the headspace above the water of the solution in the vial, and then the fibers were removed from the needle of the fiber holder and exposed at about 1 cm above the water surface. At this time, the temperature of the heater was adjusted to 90 ° C., and the vial was heated and the exposure time was adjusted to 60 minutes. Then, the hydroxy PCBs-adsorbed fibers were adsorbed again in a headspace of a 10 μl BSTFA solution temperature controlled in a 60 ° C. heater. Derivatization by exposure.

As in Example 1, the degree of adsorption was checked by comparing the degree of adsorption according to the size of the selected ion of each material. The results are as shown in Figure 4 and showed the highest adsorption when adsorbed using the above PDMS / DVB fiber.

<Example 4>

This example is to see the reaction rate according to the derivatization reagent.

Prepare 4 4 ml vials, add 2 ml of blank sample (urine urine before PCB administration) to each vial, add 10 µl of the standard solution of 11 hydroxy PCBs, and add the internal standard pyrene-d ₁₀ solution. After adding 10 μl each, the PDMS-DVB fiber holder was fixed in the headspace above the water of the solution in the vial, and then the fiber was removed from the needle of the fiber holder and exposed at about 1 cm above the water surface. The temperature of the heater was adjusted to 90 ° C. and the vial was heated and the exposure time was 60 minutes. Then, the hydroxy PCBs adsorbed fibers were adsorbed in a 10 µl solution of BSTFA, MTBSTFA, PFPA or HFBA, which were temperature-controlled in a 60 ° C. heater. Derivatization was performed in each of the headspace of.

As in Example 1, the degree of reactivity was confirmed by comparing the degree of adsorption according to the size of the selected ion of each material. The results are as shown in Figure 5 and showed the highest reactivity when TMS derivatization using BSTFA.

Example 5

The PDMS-DVB fiber was installed in accordance with the optimum conditions obtained in Examples 1-4, and the temperature of the heater was adjusted to 90 ° C., the adsorption time was adjusted to 60 minutes, and the hydroxy PCBs adsorbed fiber was kept at 60 ° C. After derivatization by exposure to a headspace of a 10 μl solution of temperature-controlled BSTFA in a heater, it was injected into a gas chromatography or a gas chromatography / mass spectrometer to show peaks and internal standards of 11 hydroxy PCBs as shown in FIG. 6. A chromatogram of was obtained. 7 is a mass spectrum of a TMS derivatized product of 4-hydroxy-3,4 ', 5-trichlorobiphenyl (4-OH-3,4', 5-TrCB), among 11 derivatized standards. Each of the derivatized molecular weights and characteristic ions are well represented.

The instrument used to determine the concentration of these 11 hydroxy PCBs was a Hewlett-Packard 5972 mass spectrometer and a 5890 plus gas chromatograph connected thereto. The ionization method used was Electron Impact (EI) and the temperature of the ion source was 200 ° C. Selected ion monitoring mode was used for quantification and the selected ions are shown in Table 1 above. The column used in the analysis was an ultra 2 column having a length of 25 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm, and helium as the mobile phase gas, and the flow rate was 0.8 ml / min. The temperature of the column was initially elevated to 200 ° C. at 100 ° C. at 10 ° C./min, held at 200 ° C. for 3 minutes, and then again at 300 ° C. at 10 ° C./min. The temperature of the sample inlet was 250 ° C., and the temperature of the gas chromatograph and mass spectrometer connector was 280 ° C.

<Example 6>

2 ml of urine sample of PCB-treated rat was added, 10 ml of internal standard pyren-d ₁₀ solution was added, and PDMS-DVB fiber was installed on the sample vial and the temperature of the heater was adjusted to 90 ° C. After adsorbing at 60 minutes, the hydroxy PCBs adsorbed fibers were derivatized by exposing them to 10 μl of BSTFA temperature controlled at 60 ° C. for 5 minutes. The adsorbed fibers were injected into the GC inlet and analyzed by gas chromatography / mass spectrometry. As a result, hydroxy PCBs metabolites were detected from urine samples in amounts ranging from 15 to 580 ng, depending on the time taken after administration.

As described above, the present invention enables the effective analysis of hydroxy PCBs in urine samples and provides selective and superior sensitivity for gas chromatography or gas chromatography / mass spectrometry. In addition, extraction and derivatization, which is a pretreatment required for analysis by gas chromatography / mass spectrometry, which is generally used for the analysis of substances contained in urine samples, can be performed very simply using the SPME apparatus.

Therefore, the present invention can satisfy the excellent sensitivity, accuracy, shortening of analysis time, low cost and simplicity, and thus can simultaneously analyze the hydroxy metabolites of PCBs, which are harmful to humans, in urine samples. It is widely applicable to.

1 is a schematic diagram of a device of solid-phase microextraction (SPME) used in the present invention.

Figure 2 is a graph showing the adsorption rate of 11 hydroxy PCBs (hydroxy polychlorinated biphenyls) according to the SPME adsorption temperature in Example 1.

Figure 3 is a graph showing the adsorption rate of 11 hydroxy PCBs according to the SPME adsorption time in Example 2.

4 is a graph showing the adsorption rate of 11 hydroxy PCBs according to the SPME adsorption fiber (fiber) in Example 3.

FIG. 5 is a graph showing the relative ratios of 11 hydroxy PCBs according to the SPME derivatization reagent in Example 4. FIG.

6 is a chromatogram of 11 hydroxy PCBs analyzed in a gas chromatography / mass spectrometer in Example 5. FIG.

7 is a mass spectrum of 4-hydroxy-3,4 ', 5-trichlorobiphenyl (4-OH-3,4', 5-TrCB) in hydroxy PCBs derivatized according to the present invention.

Claims (6)

Adsorbing hydroxy PCBs (urethane polychlorinated biphenyls) in urine to polydimethylsiloxane / divinylbenzene fiber, trimethylsilyl derivatizing hydroxy PCBs adsorbed on polydimethylsiloxane / divinylbenzene fiber, and Analyzing the trimethylsilyl derivatized hydroxy PCBs by gas chromatography or gas chromatography / mass spectrometry. The process of claim 1 wherein N, N-bis (trimethylsilyl) -trifluoroacetamide (BSTFA) is used to trimethylsilyl derivatize hydroxy PCBs adsorbed onto polydimethylsiloxane / divinylbenzene fiber. delete delete The method of claim 1, wherein the hydroxy PCBs are adsorbed at a headspace location for at least 60 minutes at a temperature of 90 ° C. or higher. The method of claim 1, wherein the derivatization is at a headspace position for 5 to 30 minutes at a temperature of 60 ° C. 3.