KR101028042B1 - Method for determination of volatile organic compounds metabolites in urine - Google Patents

Abstract

A method for analyzing volatile organic compound (VOC) metabolite concentration in a biological urine sample, wherein the pH of the biological urine sample is adjusted to acid to extract volatile organic compound metabolites from the biological urine sample, and anion is obtained from the acid controlled bio urine sample. A method of solid phase extraction of volatile organic compound metabolites using an exchange cartridge is provided. By using the analytical method according to an embodiment of the present invention, it is possible to simultaneously and accurately analyze the concentration of the major VOC metabolites in the urine sample using a small amount of biological urine samples. By using the concentration of the analyzed VOC metabolites, it can be used as an indicator for determining the effects of VOC influx in the human body.

Urine samples, volatile organic compounds, metabolites, solid phase extraction

Description

Methods for determination of volatile organic compounds metabolites in urine

The present invention relates to a method for analyzing the concentration of volatile organic compound metabolites in a urine sample.

Volatile organic compounds (VOCs) are very harmful to the human body as well as the ecosystem, and cause many chronic and acute side effects such as central nervous system disorders, respiratory disorders, and leukemia. It mainly occurs in petrochemical, steel, food industry, etc. In particular, VOC has a large effect on the human body by generating photochemical oxides, which are secondary pollutants such as ozone, through photochemical reactions. When the VOC is introduced into the human body is mainly discharged to the urine through the metabolic process, by measuring the concentration of the VOC metabolite in the urine sample, it can be seen as the effect of VOC introduced into the human body when the concentration rises.

In order to use the present invention as an indicator for determining the effects of VOC influx in the human body, a method of analyzing the concentration of VOC metabolites in a biological urine sample, specifically, can accurately analyze the concentrations of major VOC metabolites simultaneously. I want to provide a way.

The present invention comprises the steps of extracting volatile organic compound metabolites from the urine sample; And quantitatively analyzing the extracted volatile organic compound metabolites using gas chromatography-mass spectrometry (GC-MS), wherein the extracting step comprises: Adjusting the pH of the biological urine sample to acidic; And solid phase extraction of volatile organic compound metabolites from an acid-controlled biological urine sample using an anion exchange cartridge.

By using the analytical method according to an embodiment of the present invention, it is possible to simultaneously and accurately analyze the concentration of the major VOC metabolites in the urine sample using a small amount of biological urine samples. By using the concentration of the analyzed VOC metabolites, it can be used as an indicator for determining the effects of VOC influx in the human body.

The present invention relates to a method for analyzing the concentration of VOC metabolites in the urine sample in order to use as an indicator for determining the effects of VOC influx into the human body, specifically, the concentration of the main VOC metabolites can be analyzed simultaneously It is about how it can be.

According to one embodiment of the present invention, the pH of the biological urine sample is adjusted to acidic, solid phase extraction of the volatile organic compound metabolites from the acidic controlled urine sample using an anion exchange cartridge, and then the extracted volatile organic compound metabolism. Sieves can be quantitatively analyzed using gas chromatography-mass spectrometry (GC-MS) to analyze the concentration of volatile organic compound metabolites in biological urine samples. By adjusting the pH of the biological urine sample to acidic, it is possible to ionize a substance in the form of a dimer. In one embodiment of the present invention, hydrochloric acid (HCl) may be used for pH adjustment. In one embodiment of the invention, the pH can be adjusted to 1.0 ~ 2.0.

In one embodiment of the present invention, the indicator of the influence of toluene, 2-xylene, 3-xylene, 4-xylene, benzene, and styrene in the human body, which is about 80% released into the urine as the most representative harmful VOC in the atmosphere To obtain the metabolite of toluene, hipuric acid (hippuric acid, HA), 2-xylene metabolite, 2-methylhippuric acid (2-MHA), 3-xylene metabolite 3-methylhippuric acid (3-MHA), 4-xylene metabolite 4-methylhippuric acid (4-MHA), benzene metabolite trans, trans-mu The metabolite mandelic acid (MA) of choline (trans, trans-muconic acid, t, t-MU) and styrene was analyzed simultaneously.

Hippuric acid (HA) is represented by Chemical Formula 1, 2-methylhippuric acid (2-MHA) is represented by Formula 2, 3-methylhippuric acid (3-MHA). Formula 4, 4-methylhippuric acid (4-MHA) is formula 4, trans, trans-muconic acid (t, t-MU) is formula 5, mandelic acid ( mandelic acid (MA) has the structure of Formula 6.

In one embodiment of the present invention, the solid phase extraction of volatile organic compound metabolites from the biological urine sample is injected into the anion exchange cartridge, washed with 2 to 3% ammonia water to make the cartridge base In order to remove the basic matrix, the cartridge may be washed with methanol, followed by elution with methanol containing 2 to 3% formic acid. The wash may be washed once with ammonia water and then with methanol.

In one embodiment of the present invention, in order to effectively extract the VOC metabolite in the urine sample, the conditions of the washing solvent and the elution solvent were confirmed. Unlike the commonly used reversed phase cartridges, it was confirmed that the base level of the cartridge is most suitable for extraction when using about 2-3% ammonia water as the washing solvent. In addition, when methanol containing 2 to 3% of formic acid is used as the elution solvent, it is a background compared to other concentrations. It was confirmed that the most suitable as it was possible to secure the recovery rate while reducing the matrix.

In one embodiment of the present invention, 0.2-0.5% ascorbic acid is added to an acid-controlled biological urine sample to prevent denaturation due to oxidation of analytes, especially MA and t, t-MU. Thus, the analyte can be accurately analyzed during quantitative analysis.

In one embodiment of the present invention, the volume of the biological urine sample may be 0.1 ~ 1mL, specifically 0.5mL. In the conventional VOC metabolite analysis, the urine sample required about 1 mL to 10 mL. However, when using the analytical method of the present invention, a small amount of urine can be effectively extracted and the interfering substance can be effectively removed. Simultaneous analysis of VOC metabolites is possible with samples.

After pretreatment of the biological urine sample and extraction of the VOC metabolites, the concentration of the VOC metabolites is quantitated through gas chromatography-mass spectrometry (GC-MS).

In one embodiment of the present invention, an internal standard method may be used for quantitative analysis. In the internal standard method, first, the standard material is analyzed in various concentration ratios, and the calibration curve is made by using the ratio of the peak height (width) and the concentration ratio, and then the internal standard is added to the unknown sample with the correct amount of the internal standard material. It is a quantitative analysis method that measures the ratio of the peak height of the sample and the concentration ratio of the unknown sample and the internal standard material on the previously prepared calibration curve. As internal standard according to the embodiment of the present invention, hippuric -d ₅ - acid (HA-d _5), mandelate -d _5-acid (MA-d _5), trans, _trans-4 myukon -d-acid (t, t-MU-d ₄ ) can be used. Hippuric -d ₅ - acid (HA-d ₅₎ the following formula (7), mandelate -d _5-acid (MA-d ₅₎ are formula (8), trans, trans-myukon -d _4-acid (t, t-MU- d ₄ ) has the structure of Formula 9.

In one embodiment of the present invention, it can be quantitatively analyzed in the selective ion monitoring (SIM) mode of the GC-MS. SIM mode can improve the precision and accuracy of quantitative analysis by monitoring the mass-to-charge ratio (m / z) for only a few selected ions. A time program for elution time ions is possible for the analysis of several target components.

Hereinafter, specific examples are provided to help understanding of the present invention, but the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

[Example]

1. Bio-Urine Sample Preparation

To a 0.5 mL urine sample containing the internal standards HA-d ₅ , MA-d ₅ and t, t-MU-d ₄ (C / D / N isotope, Quebec, Canada) at a concentration of 100 ng / mL, 2 mL of distilled water was passed through a Milli-Q system (Millipore, Milford, Mass., USA) to activate the cartridge. Adjust the pH by adding 100 µl of 6N-HCl, add 50 µl of 0.2% ascorbic acid (Sigma-Aldrich, Steinheim, Germany) solution to prevent oxidation of the analyte, and shaker (Maxi Mix II Thermolyne). , Barnstead International, Dubuque, USA) and shake for about 20 seconds.

The sample was then injected into an anion exchange cartridge (Waters, Mildford, MA, USA) sufficiently washed with 2 mL methanol and 2 mL distilled water, washed with 1 mL ammonia water and 1 mL methanol, followed by methanol containing 2% formic acid. Elution three times with 1 mL, received in a test tube, evaporated and dried.

For high concentration HA analysis in urine samples, the eluate was dissolved in 4 mL of methanol, diluted, and 50 μl of HA was analyzed. The rest was 2-MHA, 3-MHA, 4-MHA, t, t-MU. It was used for and MA analysis. After the test tube taken 50㎕ HA-d ₅ gave into the HA-d ₅ at a concentration of 1000ng / mL and completely dried again using N ₂ Turbo Vap.

To detect by GC-MS, 50 µl of a mixed solution of MSTFA / TMCS / TMSI 100: 5: 2 (v / v / v) (Sigma-Aldrich, Steinheim, Germany) was added and reacted for 15 minutes at 60 ° C for derivatization. 2 μl were injected into GC-MS.

2. Measurement of creatinine

In quantitative analysis of urine samples, creatinine concentrations were measured together (corrected by Jaffe reaction method) to correct for intrinsic differences in each subject. Creatinine, like urea and uric acid, is a waste product of proteins used as energy in the body. Creatinine is used as energy in the muscles, then formed into creatinine or creatine phosphate, which is released into the blood and excreted from the kidneys into urine. In other words, the measurement of creatinine concentration is intended to be used as an index to determine whether the kidney function is normal and to correct for differences in individuals.

In the measurement method, 4.5 mL of distilled water was added to 200 μL of urine samples, and then 100 μL of 10% sodium tungstate (Na ₂ WO ₄ ) and 200 μL of 0.6NH ₂ SO 4 were added. Mix evenly with a shaker, separate using a centrifuge, and take 2 mL of the supernatant. 1 mL of 0.036 M picric acid (Allied Chemical, Gujarat, India) and 1 mL of 0.75 N NaOH were added to the supernatant, and left to stand for 20 minutes using a UV spectrophotometer (DU 530 UV spectrometer, Beckman, Califonia, USA). Absorbance was measured at λMAX 520 nm.

3. GC-MS Analysis

The GC-MS instrument used in one embodiment of the present invention used an Agilent 5975c mass selective detector connected directly to an Agilent 7890 Plus gas chromatograph of Agilent Technology (Santa Clara, Calif., USA). Samples were injected into the GC using an Agilent 7683B Series Injector.

(1) 2-MHA, 3-MHA, 4-MHA, MA, t, t-MU analysis

The separation tube used DB-1701 ((14% -Cyanopropyl-phenyl) methylpolysiloxan, length 30m, inner diameter 0.25mm ID, 0.25㎛ film thickness), the volatile temperature programming for 1 minute at 150 ℃ initial temperature after 270 ℃ Samples were analyzed by raising to 15 ° C./min and holding for 2 minutes. The injection amount was 2 μl, helium gas with a purity of 99.999% was flowed at a flow rate of 1.2 mL / min, and set to split mode (ratio 10: 1). The electron energy used for ionization was 70 eV, and a method of selecting and detecting only a few specific ions of the analyte (selected ion monitoring, SIM mode) was used. GC-MS operating conditions are summarized in Table 1.

(2) HA analysis

The separation pipe used DB-1701 ((14% -Cyanopropyl-phenyl) methylpolysiloxan, length 30m, inner diameter 0.25mm ID, 0.25㎛ film thickness), and the temperature programming for volatilization was carried out at 150 ℃ for 1 minute after initial temperature at 150 ℃. Samples were analyzed by raising them to 15 ° C./min until 20 ° C./min to 270 ° C. and holding them for 1 minute. The injection amount was 2 μl, helium gas with a purity of 99.999% was flowed at a flow rate of 1.2 mL / min, and set to split mode (ratio 10: 1). The electron energy used for ionization was 70 eV, and a method of selecting and detecting only a few specific ions of the analyte (selected ion monitoring, SIM mode) was used. GC-MS operating conditions are summarized in Table 2.

4. Analysis result

The analyte VOC metabolite MA, t, t-MU, 2-MHA, 3-MHA, 4-MHA, HA assay range was determined according to the concentration detected in human urine samples. The concentration range of the calibration curve is measured according to the method of the embodiment of the present invention by selecting concentrations of 1, 5, 10, 50, 100, 500, and 1000 ng / mL with respect to the mixture of five VOC metabolite standards excluding HA. To create a calibration curve. The calibration curve concentration range of HA was selected from 0.1, 0.5, 1, 2, 5, 10 and 20 µg / mL. The detection limit was measured using a signal to noise (S / N) method, and the concentration of the signal-to-noise ratio of 10 or more was expressed as a quantitative limit (LOQ). The test range and linearity correlation coefficient of each compound are summarized in Table 3. All calibration curves showed linearity (r ² > 0.996) within the quantitative range.

In addition, the concentrations of 30 and 200 ng / mL were measured and measured for the accuracy and precision of the five VOC metabolite standard liquid mixtures. For concentrations of 30 and 200 ng / mL, the results of five repeated treatments of the same concentration sample and the same concentration of the sample for seven days are expressed in accuracy (accuracy,%) and precision (Table 4). Shown in Accuracy means a degree close to a true value, and precision means a degree where scattering of measured values is small. According to Table 4, values of 85 to 127.9% accuracy and 4.4 to 24.8% accuracy were obtained. Therefore, the validity of the analysis result was verified.

Peaks were identified by comparing the retention times of each analyte using an internal standard and a standard and contrasting the height ratio of the peak of the analyte VOC metabolite to the internal standard peak. Total ion chromatograms (TICs) of the six VOC metabolites and ion chromatograms of HA metabolites are shown in FIGS. 1 and 2. VOC metabolites that were not separated on the chromatogram also exhibited different m / z values, which were sufficiently distinguished in SIM mode. Specific ions on the mass spectrum for SIM mode analysis are summarized in Table 5.

1 shows a total ion chromatogram (TIC) of six VOC metabolites, each peak being as follows. 1.MA-d ₅ , 2.MA, 3.t, t-MU-d ₄ , 4.t, t-MU, 5.HA-d ₅ , 6.HA, 7.2-MHA, 8. 3 -MHA, 9. 4-MHA

2 shows an ion chromatogram of HA metabolites, peak 1 shows HA-d ₅ peak 2 shows HA.

Claims (5)

Extracting volatile organic compound metabolites from a biological urine sample; And A volatile organic compound metabolite concentration analysis method comprising quantitating extracted volatile organic compound metabolites using gas chromatography-mass spectrometry (GC-MS), wherein the extracting step comprises: Adjusting the pH of the biological urine sample to acidic; And A solid phase extraction of volatile organic compound metabolites from an acid controlled biological urine sample using an anion exchange cartridge. The method of claim 1, wherein the solid phase extraction of volatile organic compound metabolites from the biological urine sample, Injecting the biological urine sample into an anion exchange cartridge; Washing with 2-3% ammonia water and then washing with methanol; And A method of analyzing volatile organic compound metabolite concentration in a biological urine sample, comprising eluting with methanol containing 2-3% formic acid. The method of claim 1, A method for analyzing the concentration of volatile organic compound metabolites in a urine sample further comprising adding 0.2-0.5% ascorbic acid to the acid-controlled urine sample. The method of claim 1, The volatile organic compound metabolites are metabolite of toluene (hippuric acid (HA)), 2-xylene metabolite 2-methylhippuric acid (2-MHA), 3-xylene Metabolite 3-methylhippuric acid (3-MHA), 4-xylene metabolite 4-methylhippuric acid (4-MHA), benzene metabolite trans, trans A method for analyzing the concentration of volatile organic compound metabolites in a biological urine sample, comprising: metaconic acid mandelic acid (MA) of styrene and trans, trans-muconic acid (t, t-MU). The method of claim 1, The volume of the biological urine sample is 0.1 ~ 1mL, volatile organic compound metabolite concentration analysis method in a biological urine sample.

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