KR102228072B1 - A method for detecting methanol in a biospecimen - Google Patents

Abstract

In a methanol detection method for identifying methanol contained in a biopsy specimen according to the present invention, the method comprises: a step of collecting the biopsy specimen from a subject (a biopsy specimen extraction step); a step of forming a first specimen mixture by mixing the biopsy specimen, an internal standard aqueous solution, a saturated NaCl aqueous solution, and a methanol standard solution (a specimen mixing step); a step of derivatizing methanol and an internal standard material in the biopsy specimen (a derivatization step); and a step of extracting and analyzing the derivatized methanol and the derivatized internal standard (an extraction analysis step).

Description

A method for detecting methanol in a biospecimen}

The present invention relates to a method for detecting methanol in a biological sample, and more particularly, by derivatizing methanol contained in a biological sample and then analyzing it by GC/MS (Gas chromatography-Mass Spectrometry) to determine whether methanol is included in a biological sample. It relates to a method for detecting methanol in a biological sample that can be quickly identified.

In general, methanol (methanol) is used in the manufacture of solvents, pharmaceuticals, pesticides, fuels, resins, electronic products, etc., and can be easily contacted in the surroundings, so a number of human damages have been reported. Methanol absorbed into the body is metabolized to produce highly toxic formaldehyde and formic acid, leading to blindness and death. After metabolizing in the liver, formaldehyde is formed by alcohol decomposition enzyme (ADH). And the converted formaldehyde is metabolized to formic acid by aldehyde degrading enzyme (ALDH). At this time, formic acid causes toxicity to the retina and nerves and destroys the body by blocking energy metabolism of mitochondria. The amount of poisoning and lethality is not constant depending on individual differences, but the lethal dose is generally known as 30-100 ml, and the blood concentration is When it reaches 0.033%, it is reported that blindness and death in 0.114% or more.

Death from methanol poisoning may be caused by smuggling, misuse of medicines, or pesticides drunk for suicide. In the event of a methanol poisoning accident, first aid measures include gastric lavage and ethanol administration. Since methanol entering the body is in a competitive relationship with ethanol in the process of metabolism, rapid emergency treatment is most important in the case of occurrence of methanol poisoning, and for that purpose, rapid detection is more important than anything else.

Methanol analysis is generally performed with Gas Chromatography-Flame Ionization Detector (GC-FID). As shown in Fig. 1, methanol in blood was analyzed by GC-FID, and methanol with a retention time of 0.96 minutes (Fig. 1(A)) and tertiary-butyl alcohol (t- butyl alcohol) two peaks can be identified, but it was difficult to specify the methanol detected only by the analysis result using GC-FID. Because the 0.96 minute retention time peak may be derived from methanol, but may be acetaldehyde, a metabolite of ethanol. Especially, if methanol is consumed while drinking, the retention time overlaps and the 0.96 minute peak is derived from methanol. There was a problem in that information on whether it was derived from acetaldehyde, a metabolite, or not, after drinking ethanol, could not be accurately presented.

For this reason, methanol analysis by GC-FID, which is analyzed only with retention time, has difficulty in qualitative and quantification. To overcome this, it is possible to analyze using GC/MS (Gas chromatography-Mass Spectrometry), but as shown in FIG. 2, the molecular weight of methanol is _{similar to that of oxygen (O 2} ), especially in the headspace. When collected and injected, there is a problem in that column bleeding may occur due to moisture in the sample.

Republic of Korea Patent Publication No. 10-2015-0109995 (2015.10.02) Republic of Korea Patent Publication No. 10-2009-0131560 (2009.12.29) Republic of Korea Patent Publication No. 10-2040820 (2019.10.30)

The present invention has been proposed to solve the problems of the prior art as described above. By derivatizing methanol contained in a biological sample and then analyzing it by GC/MS, it is possible to quickly check whether methanol is contained in a biological sample. The purpose of this study is to provide a method for detecting methanol in biological samples that can be applied to detect methanol for emergency treatment in the event of methanol intoxication, as well as forensic and scientific events, so that a person can quickly and accurately identify that poisoning by methanol.

In the methanol detection method for identifying methanol contained in a biological sample according to the present invention,

Collecting a biological sample from the subject (biological sample extraction step);

A step in which a first sample mixture is formed by mixing a biological sample, an aqueous solution of an internal standard substance, a saturated NaCl aqueous solution, and a methanol standard solution (sample mixing step);

Derivatizing methanol and an internal standard in the biological sample (derivatization step);

It provides a method for detecting methanol in a biological sample, comprising the step of extracting and analyzing the derivatized methanol and the derivatized internal standard material (extraction analysis step).

In the above, the biological sample is blood, and the aqueous solution of the internal standard substance is an aqueous ethanol solution substituted with 0.1% deuterium;

In the sample mixing step, the mixing ratio of blood: an aqueous solution of an internal standard substance: an aqueous NaCl solution: a standard solution of methanol is 1:1:2:1.

In the above, in the derivatization step, methanol and an internal standard are derivatized by adding a pyran-based compound and an acid catalyst.

In the above, in the derivatization step, an acid catalyst is added after the compound is added to the first sample mixture;

The pyran-based compound is characterized in that 0.1v/v% to 100v/v% of the volume of the biological sample is mixed.

In the above, the pyran-based compound is characterized in that DHP (3,4-Dihydro-2H-pyran).

In the above, the acid catalyst is characterized in that one of concentrated hydrochloric acid, sulfuric acid, and nitric acid.

In the above, the acid catalyst, concentrated hydrochloric acid, is characterized in that 0.1v/v% to 50v/v% of the volume of the biological sample is mixed.

In the above, the extraction analysis step is characterized in that the extraction is performed by a solvent extraction method or a solid state fine extraction method (SPME).

In the above, the solid state fine extraction method (SPME) is characterized by consisting of one of Carboxen/PDMS, polyacrylate (PA), PDMS, and carbowax/DVB.

In the above, the extraction analysis step is characterized in that it is made of the Carboxen / PDMS extraction method.

In the above, it characterized in that it further comprises a formate (formate) analysis step.

The method for detecting methanol in a biological sample according to the present invention is economical and can easily and quickly determine whether methanol is contained in a biological sample. By applying this to a forensic case, there is an effect that it can more quickly and accurately identify that the victim is poisoned by methanol.

1 is a graph showing the analysis result of a biological sample by GC-FID,
2 is a graph showing the analysis result of methanol using GC/MS,
3 is a diagram showing a reaction equation (A) of methanol and DHP and a reaction equation (B) of ethanol substituted with deuterium (D5-ethanol) and DHP,
4 is a graph showing the analysis results of derivatized methanol and ethanol (D5-ethanol) substituted with derivatized deuterium by GC/MS,
5 is a graph showing a mass spectrum result of derivatized methanol ions,
6 is a graph showing a mass spectrum result of an ethanol (D5-ethanol) ion substituted with derivatized deuterium,
7 is a diagram showing the molecular structures of a base ion (A), a derivatized methanol ion (B), and an ethanol ion (C) substituted with derivatized deuterium,
FIG. 8 is a graph showing a GC/MS chromatogram of blood of a consortium to which a standard methanol solution was added,
9 is a graph showing a calibration curve plotting the area ratio of derivatized methanol to the derivatized internal standard,
10 is a graph showing the results of analyzing formate using ion chromatography,
11 is a graph showing a correction curve of formate by ion chromatography,
12 is a diagram for explaining an application method of the present method.

Hereinafter, a method for detecting methanol in a biological sample according to the present invention will be described in detail with reference to the drawings.

3 is a diagram showing the reaction formula (A) of methanol and DHP and the reaction formula (B) of ethanol (D5-ethanol) and DHP substituted with deuterium, and FIG. 4 is a diagram showing the reaction formula (B) of derivatized methanol and derivatized deuterium. It is a graph showing the analysis result of ethanol (D5-ethanol) by GC-FID, FIG. 5 is a graph showing the mass spectrum result of derivatized methanol ion, and FIG. 6 is a graph showing the result of derivatized deuterium-substituted ethanol ( D5-ethanol) is a graph showing the result of the mass spectrum of the ion, and Figure 7 shows the molecular structure of the base ion (A), the derivatized methanol ion (B) and the ethanol ion (C) substituted with the derivatized deuterium Fig. 8 is a graph showing a GC/MS chromatogram of blood of a convicted person to which a standard methanol solution has been added, and Fig. 9 is a plot of the area ratio of derivatized methanol to the derivatized internal standard. A graph showing a correction curve, FIG. 10 is a graph showing a result of analysis of formate using an ion chromatogram, and FIG. 11 is a correction curve of a formate by ion chromatography. It is a graph, and FIG. 12 is a diagram for explaining an application method of the present method.

The method for detecting methanol in a biological sample according to the present invention is a method of derivatizing methanol and then analyzing using GC/MS (Gas Chromatography-Mass Spectrometry). GC/MS analysis for methanol in biological samples is performed by standard addition method.

The method for detecting methanol in a biological sample consists of a biological sample extraction step (ST-100), a sample mixing step (ST-200), a derivatization step (ST-300), and an extraction analysis step (ST-400) ( 12).

In the biological sample extraction step (ST-100), a biological sample is extracted from the subject. Hereinafter, the biological sample will be described using blood as an example.

In the sample mixing step (ST-200), a first sample mixture is formed by mixing blood extracted from the subject, an aqueous solution of an internal standard substance, a saturated NaCl aqueous solution, and a standard methanol solution.

The internal standard is for quantification of methanol contained in blood, and ethanol (D5-EtOH) substituted with five deuteriums as shown in (B) of FIG. 3 is used. Sigma-Aldrich (Darmstadt, Hessen, GER) was used as the internal standard material.

The internal standard substance is not only extremely unlikely to exist in a general natural environment, but also has the advantage of being similar to the behavior of methanol, and is particularly applicable to quantitative analysis of ethanol in the blood of drinkers containing ethanol.

The NaCl aqueous solution is for increasing the volatility of the analyte in the gas phase by increasing the ionic strength of the sample solution.

In the first sample mixture, a mixing ratio of blood: an aqueous solution of an internal standard substance: a saturated aqueous NaCl solution: a standard solution of methanol is 1:1:2:1. At this time, the aqueous solution of the internal standard substance is an aqueous solution of ethanol (D5-EtOH) substituted with 0.1% deuterium, and the concentration of the aqueous NaCl solution is a saturated aqueous solution of NaCl at that temperature .

The first sample mixture is formed by mixing 100 µl of blood, 100 µl of an internal standard solution, 200 µl of NaCl aqueous solution, and 100 µl of a methanol standard solution in a 10 ml vial. As the methanol standard solution, an analytical standard grade manufactured by Supelco (Bellefonte, PA, USA) was used.

In the first sample mixture, the concentration of the methanol standard solution is 0.025%, 0.05%, 0.10%, 0.15%, and 0.20%. For each concentration of the methanol standard solution, blood, an aqueous solution of an internal standard substance, and NaCl The aqueous solution is mixed in each of 5 vials.

In the derivatization step (ST-300), in order to derivatize methanol, DHP (3,4-Dihydro-2H-pyran) is added to each vial to homogenize it, and then an acid catalyst is added to derivatize it. The DHP is a compound that is also applied as a protecting group for alcohol in organic synthesis.

The DHP is added in the range of 0.1v/v% to 100v/v% of the volume of the biological sample. DHP is preferably added in the range of 25v/v% to 50v/v%, and more preferably 35v/v% of the biological sample is added. In the following, 35 µl of DHP is added per 100 µl of a biological sample. remind TCI (Kita-ku, Tokyo, JPN) was used as the DHP.

The acid catalyst consists of one of concentrated hydrochloric acid, sulfuric acid, and nitric acid. As the acid catalyst, concentrated hydrochloric acid is most preferred. When concentrated hydrochloric acid is selected as the acid catalyst, concentrated hydrochloric acid is added in the range of 0.1v/v% to 50v/v% of the volume of the biological sample. Concentrated hydrochloric acid is preferably added in the range of 15v/v% to 35v/v% of the biological sample, more preferably 25v/v% of the biological sample. Below, concentrated hydrochloric acid is added 25 µl per 100 µl of a biological sample.

This reaction for derivatization was carried out through a nucleophilic addition reaction, and as a result of the reaction, methanol formed 2-methoxytetrahydropyran (Fig. 3(A)), and internal standard The material formed 2-D5-ethoxytetrahydropyran (FIG. 3(B)).

In the extraction analysis step (ST-400), various pretreatment techniques are used for GC/MS analysis of 2-methoxytetrahydropyran produced as a result of the reaction. Pretreatment techniques include liquid-liquid extraction, solid phase extraction, and solid phase micro extraction (SPME). In the present invention, extraction was performed using a solid state fine extraction method (SPME) in the headspace of the vial.

SPME fibers for pretreatment sample extraction include Carboxen-polydimethylsiloxane (CAR/PDMS), polyacrylate (PA), polydimethylsiloxane (PDMS), and Carbowax/divinylbenzene (Carbowax/DVB). Among them, SPME fiber composed of CAR/PDMS is used. It is most desirable to do it.

The SPME fiber includes CAR/PDMS, PDMS, PA and carbowax/DVB from Supelco (Bellefonte, PA, USA), and in the present invention, it is preferable to use a 75 μm SPME fiber composed of CAR/PDMS.

In the extraction analysis step (ST-400), 2-methoxytetrahydropyran produced in the derivatization step (ST-300) is extracted using SPME, and then immediately analyzed by GC/MS.

Analysis by GC/MS was performed using a 7890B GC and 5977B MSD system from Agilent Technologies (Foster City, CA, USA), and DB-5MS UI capillary column (30m length×0.23㎜ id, 0.25 μm film thickness, J&W Scientific, Folsom, CA, USA) using 99.999% He carrier gas (carrier gas) was made with a constant flow of 1.0 ml / min.

The injector temperature was 260°C, the interface temperature was 280°C, and the oven temperature was maintained at 40°C for 3 minutes, and then the temperature was raised to 250°C at 10°C/min and maintained for 3 minutes. The solvent delay time was set to 4.2 minutes to exclude the influence of the derivatization reagent DHP, and the mass spectrum was analyzed in an EI mode of 70 eV at a split ratio of 10:1.

Through the above analysis, it was possible to confirm 2-methoxytetrahydropyran, which is methanol derivatized at a retention time of 5.9 minutes, as shown in the analysis result shown in FIG. 4, and to 2-D5-, which is an internal standard derivatized at 7.3 minutes. Toxytetrahydropyran could be confirmed.

In addition, the mass spectrum for each component can be confirmed through FIGS. 5 and 6. In FIGS. 5 and 6, the molecular weight of the base ion is m/z 85, which is the same as the molecular structure of FIG. 7(A), and the molecular weight of the 2-methoxytetrahydropyran ion by methanol is m/z 115. It is the same as the molecular structure of Fig. 7(B), and the molecular weight of the 2-D5-ethoxytetrahydropyran ion by the internal standard is m/z 134, which is the same as the molecular structure of Fig. 7(C).

GC/MS analysis results obtained by derivatizing the blood as a biological sample by standard addition of methanol in a concentration range of 0 to 0.20% in the sample mixing step (ST-200) are shown in FIG. A calibration curve as shown in FIG. 9 was derived by applying an area ratio of ions of m/z 134 of the internal standard to the ion area of z 115. For the linearity of the calibration curve, the r ² value was very good at 0.9999, the detection limit was 0.56 µg/ml, and the quantification limit was 1.7 µg/ml. For the limit of detection and limit of quantification, the S/N ratio of 10 blank samples was calculated as a concentration exceeding 3 and 10.

In the method for detecting methanol in a biological sample according to the present invention, a formate analysis step (ST-500) may be further included.

The formate (formate) is a metabolite generated when drinking methanol. The formate is analyzed using IC (Ion Chromatography).

As shown in FIG. 10(A), blood, which is a biological sample, was diluted 50 times with demineralized water and analyzed. As a result, formate was detected at a retention time of 3.7 minutes, and 25 µg/ml of formate was used to confirm this. By adding 1:1 to the sample diluted with demineralized water, it was confirmed that the peak identified at the retention time of 3.7 minutes in FIG. 10(A) as shown in FIG. 10(B) was formate.

For the quantification of formate, formate was diluted in demineralized water so that the formate concentration was 5-50 µg/ml, and then analyzed (see Fig. 10(C)) to derive a calibration curve as shown in Fig. 11 as an area, and r ² It showed excellent linearity of =0.9997.

Thermo Fisher Scientific (Sunnyvale, CA, USA) was used as an anion eluent for IC analysis in the formate analysis step (ST-500), and a standard format used for quantification of formate contained in blood Mate was used by diluting 100 ppm standard stock solution of AccuStandard (New Haven, CT, USA) with demineralized water.

IC analysis of formate was analyzed by injecting 5 µl using the ICS 5000 system of Dionex ((part of Thermo Scientific) Sunnyvale, CA, USA). To prevent column contamination, an IonPac AG-11 4 mm guard column of Thermo Scientific (Sunnyvale, CA, USA) was used, and the IonPac AS-11 4 mm column was separated. Eluent is an electric conductivity by applying a current of 26 ㎃ to a Thermo Scientific ASRS 4 mm suppressor at a 1 ml/min isocratic flow rate of 4.5 mM/1.5 mM carbonate/bicarbonate eluent of Thermo Scientific. It was analyzed by a detector (conductivity detector).

In the method for detecting methanol in a biological sample according to the present invention, the methanol contained in the biological sample is derivatized and then analyzed by GC/MS to quickly and easily determine whether the blood contains methanol. By checking whether to drink methanol, the cause of death from methanol can be identified, as well as quick emergency treatment for methanol drinkers.

Claims (11)

In the methanol detection method for identifying methanol contained in a biological sample,
Collecting a biological sample from the subject (ST-100; extracting a biological sample);
A step in which a first sample mixture is formed by mixing a biological sample, an internal standard aqueous solution, a saturated NaCl aqueous solution, and a methanol standard solution (ST-200; sample mixing step);
Derivatizing methanol and an internal standard in the biological sample (ST-300; derivatization step);
A method for detecting methanol in a biological sample comprising the step of extracting and analyzing the derivatized methanol and the derivatized internal standard material (ST-400; extraction analysis step). The method of claim 1, wherein the biological sample is blood, and the aqueous solution of the internal standard substance is an aqueous ethanol solution substituted with 0.1% deuterium;
In the sample mixing step (ST-200), the mixing ratio of blood: an aqueous solution of an internal standard substance: an aqueous NaCl solution: a standard solution of methanol is 1:1:2:1. The method of claim 1 or 2, wherein in the derivatization step (ST-300), methanol and an internal standard are derivatized by adding a pyran-based compound and an acid catalyst. The method of claim 3, wherein in the derivatization step (ST-300), an acid catalyst is added after the pyran-based compound is added to the first sample mixture;
The method for detecting methanol in a biological sample, wherein the pyran-based compound is mixed with 0.1v/v% to 100v/v% of the volume of the biological sample. The method of claim 4, wherein the pyran-based compound is DHP (3,4-Dihydro-2H-pyran). The method of claim 3, wherein the acid catalyst is one of concentrated hydrochloric acid, sulfuric acid, and nitric acid. The method of claim 6, wherein the acid catalyst, concentrated hydrochloric acid, is mixed with 0.1v/v% to 50v/v% of the volume of the biological sample. The method for detecting methanol in a biological sample according to claim 1 or 2, wherein in the extraction analysis step (ST-400), the extraction is performed by a solvent extraction method or a solid phase fine extraction method (SPME). The method of claim 8, wherein the solid state micro-extraction method (SPME) comprises one of Carboxen/PDMS, polyacrylate (PA), PDMS, and carbowax/DVB. The method of claim 9, wherein the extraction analysis step (ST-400) comprises a Carboxen/PDMS extraction method. The method of claim 1, further comprising a formate analysis step (ST-500).

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