JP2014211433A - Quantification method of thiol compound and sulfide compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of quantifying easily with excellent reproducibility, a trace quantity of thiol compound and sulfide compound in an analyte.SOLUTION: A carrier carrying metal atoms is brought into contact with an analyte, to thereby separate a thiol compound or a sulfide compound from the analyte. An object component is recovered from the carrier, refined by solvent extraction, concentrated, and quantified with a gas chromatograph mass analyzer or the like.

Description

  The present invention relates to a method for quantifying thiol compounds and sulfide compounds.

  A thiol compound and a sulfide compound are a kind of aromatic components in food and drink. Foods and drinks contain a wide variety of components, and fragrance components and the like are extremely small amounts (for example, ng / L level). When trying to quantify such a component that is contained only in a trace amount in a specimen containing various components, it is necessary to specifically or selectively purify or concentrate the component to be detected.

  For example, Non-Patent Document 1 discloses that a complex formed by reversible binding of a thiol compound and p-hydroxymercuribenzoate is extracted with dichloromethane, and volatile thiol compounds in wine are extracted. It describes that it was purified and that the thiol compound was liberated with cysteine and the concentration of the thiol compound was measured by gas chromatography mass spectrometry.

Tominaga T. , Et al. , J .; Agric. Food Chem. 1998, 46, pp. 1044-1048.

  In the method described in Non-Patent Document 1, a large amount of waste liquid and waste (resin etc.) containing p-hydroxymercurybenzoic acid, which is a toxic substance, are generated, and thus there is a problem that the treatment is complicated. In addition, the operation is complicated in the first place, and in order to detect trace components, for example, it is often necessary to concentrate several hundred mL of samples to several tens of μL, and the number of samples that can be processed per day is limited. Therefore, there is a problem that the multipoint analysis cannot be performed. Thus, until now, there has been no method capable of quantifying trace amounts of thiol compounds and sulfide compounds in a specimen with high reproducibility and simple operation.

  Therefore, an object of the present invention is to provide a method capable of quantifying trace amounts of thiol compounds and sulfide compounds in a specimen with high reproducibility and simple operation.

  The present invention provides a method for quantifying thiol compounds and sulfide compounds, comprising a purification step comprising contacting a carrier carrying a metal atom with a specimen and separating the thiol compound or sulfide compound from the specimen.

  According to the present invention, a trace amount of a thiol compound and a sulfide compound in a specimen can be quantified with high reproducibility and a simple operation. In the purification step, these compounds can be separated from the specimen by binding a thiol compound or a sulfide compound via a sulfur atom to the metal atom of the carrier carrying the metal atom. Since a carrier carrying metal atoms is used, the operation is significantly simpler than the method described in Non-Patent Document 1. Further, the waste liquid does not contain a large amount of metal atoms, and the waste liquid can be easily treated.

  In the quantification method, the metal atom may be selected from metal elements belonging to Groups 4 to 15 of the periodic table. The metal atom is at least selected from copper (Cu), palladium (Pd), silver (Ag), platinum (Pt), gold (Au), mercury (Hg), chromium (Cr) and bismuth (Bi). One type is preferable. Thereby, the thiol compound or sulfide compound in the specimen can be more efficiently separated. Furthermore, the metal atom may be at least one selected from palladium (Pd), silver (Ag), platinum (Pt), gold (Au), and mercury (Hg). In this case, since the sulfide compound in the specimen can be separated more efficiently, it can be preferably used when separating the sulfide compound in the purification step.

  The quantitative method preferably further includes a step of reacting a thiol compound in a specimen with a maleimide compound represented by the following general formula (1) to obtain a thiol compound derivative before the purification step.

In general formula (1), R is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 5 to 12 carbon atoms, An arylalkyl group having 7 to 12 carbon atoms which may have a substituent or an aryl group having 6 to 12 carbon atoms which may have a substituent is shown.

  Since the maleimide compound has high reactivity with a thiol compound, a thiol compound derivative can be obtained quantitatively. In addition, by using a thiol compound derivative, a highly reactive thiol group can be protected and the thiol compound can be prevented from adsorbing to a plastic or the like. . Moreover, since adsorption | suction is suppressed, the influence by materials, such as a support | carrier and a container, can be reduced, for example, the support | carrier, container, etc. which are marketed can be used. Furthermore, since the molecular weight is increased and the boiling point is increased by using a thiol compound derivative, a highly volatile thiol compound can be quantified with higher reproducibility.

  When the step of obtaining the thiol compound derivative is performed, the thiol compound derivative can be extracted with an organic solvent while reacting the thiol compound with the maleimide compound, so that the components to be detected can be concentrated more efficiently.

  In addition, when the step of obtaining the thiol compound derivative is performed, there is also an advantage that an internal standard substance for analysis by gas chromatography mass spectrometry or the like can be easily prepared. For example, when an isotope of a thiol compound to be measured is used as an internal standard substance, it is necessary to prepare a stable isotope for each thiol compound to be measured, which is not easy. On the other hand, when performing the process of obtaining a thiol compound derivative, an internal standard substance can be easily produced by reacting a stable isotope of a thiol compound to be measured with a maleimide compound.

  Since the above quantification method is particularly suitable for quantification of thiol compounds and sulfide compounds that are contained in trace amounts in the sample, the concentration of the thiol compound or sulfide compound in the sample may be 1000 ng / L or less.

  In the quantification method, the carrier on which the metal atom is supported is preferably supported on a support. This simplifies operation. More preferably, the carrier carrying the metal atoms is packed in a column. This further simplifies the operation.

  The quantitative method may further include a step of quantitatively determining the thiol compound or the sulfide compound by gas chromatography mass spectrometry after the purification step.

  In addition, since the above quantification method is particularly suitable for quantification of thiol compounds and sulfide compounds that are contained only in trace amounts in samples containing a wide variety of components, the sample may be a food or drink, or a beverage. There may be.

  According to the present invention, it is possible to provide a method capable of quantifying a trace amount of a thiol compound and a sulfide compound in a specimen with high reproducibility and a simple operation.

1 is a schematic diagram illustrating a configuration of a two-dimensional gas chromatograph mass spectrometer used in Example 1. FIG. 2 is a calibration curve obtained in Example 1. 6 is a chromatogram obtained by two-dimensional gas chromatograph mass spectrometry in Test Example 3. 3 is a calibration curve obtained in Example 3. 6 is a chromatogram obtained by two-dimensional gas chromatograph mass spectrometry in Example 4.

  Hereinafter, modes for carrying out the present invention will be described. However, the present invention is not limited to the following embodiments.

  The method for quantifying a thiol compound and a sulfide compound according to this embodiment includes a purification step including contacting a carrier carrying a metal atom with a specimen to separate the thiol compound or sulfide compound from the specimen.

  Any metal atom may be used as long as it can bond to a thiol compound and a sulfide compound via a sulfur atom in these molecules (for example, coordination bond, hydrogen bond, ionic bond, covalent bond, adsorption), It may be selected from metal elements belonging to Groups 4 to 15 of the table.

  More specifically, as the metal atom, copper (Cu), palladium (Pd), silver (Ag), platinum (Pt), gold (Au), mercury (Hg), chromium (Cr) and bismuth (Bi) Is preferred. Since these metal atoms have high adsorbability of thiol compounds and sulfide compounds, it becomes possible to more efficiently separate thiol compounds and sulfide compounds. Further, from the viewpoint of improving the separation efficiency of the sulfide compound, palladium (Pd), silver (Ag), platinum (Pt), gold (Au), and mercury (Hg) may be used as the metal atom. There may be only one kind of metal atom supported on the carrier, or two or more kinds.

  Any carrier can be used as long as it can carry a metal atom. Examples thereof include hydrophilic polymers such as silica, carbon, alumina, florisil, synthetic resins, dextran, and agarose, and column chromatography carriers such as cellulose. Those usually used can be mentioned. These may be used alone or in combination of two or more. Moreover, in order to carry | support a metal atom, you may modify the surface.

  The carrier on which the metal atom is supported may be any material as long as the metal atom is supported on the carrier so that elution of the metal atom upon contact with the specimen is suppressed. Specifically, for example, a metal atom directly bonded to an ion exchange resin as a carrier (for example, coordination bond, ionic bond, hydrogen bond, etc.), a metal atom to the chelate compound on a carrier to which a chelate compound is bound And the like.

  The carrier on which the metal atom is supported is, for example, a method in which a solution containing a metal ion is brought into contact with a cation exchange resin and a metal atom is directly bonded to the cation exchange resin, and a chelate compound is bonded to the surface of the carrier (for example, (Covalent bond, ionic bond, hydrogen bond, adsorption), and a solution containing a metal ion is brought into contact therewith and a metal atom is chelate-bonded to a chelate compound. In these methods, the carrier before supporting the metal atoms may be supported by a support described later.

  The thiol compound to be detected is not particularly limited as long as it is a compound containing an —SH group. However, since the above quantification method is particularly suitable for quantifying a small amount of thiol compound, thiol contained in a sample at a low concentration. A compound is preferred. Examples of the thiol compound to be detected include, but are not limited to, for example, 4-mercapto-4-methyl-2-pentanone, 2-furfuryl mercaptan (furfuryl thiol), 3-mercaptohexyl acetate, Examples include 3-mercapto-1-hexanol.

  The sulfide compound to be detected is not particularly limited as long as it is a compound in which divalent sulfur is substituted with two organic groups, but the above quantification method is particularly suitable for quantifying a small amount of a sulfide compound. A sulfide compound contained in a sample at a low concentration is preferable. The sulfide compound to be detected is not limited to this. For example, a thiol compound derivative (a reaction product of a thiol compound and a maleimide compound represented by the general formula (1)), methional, dimethyl sulfide Etc.

  According to the above quantification method, a low-concentration thiol compound or sulfide compound can be quantified with good reproducibility, so that the concentration of the thiol compound or sulfide compound in the specimen may be 1000 ng / L or less. These concentrations may be, for example, 300 ng / L or less, 100 ng / L or less, or 50 ng / L or less. There is no particular lower limit to the concentration of the thiol compound or sulfide compound in the sample, and it may be 0 ng / L.

  The sample is not particularly limited, and any sample may be used. In addition, according to the above quantification method, since a low concentration thiol compound or sulfide compound can be quantified with high reproducibility even in a specimen containing a wide variety of components, food and drink may be used as the specimen. A variety of components are contained in food and drink, and thiol compounds and sulfide compounds contained in food and drink are often in very small amounts (for example, ng / L level). According to the above quantification method, the thiol compound or sulfide compound can be quantified with good reproducibility even when food or drink is used as a specimen.

  Examples of foods and drinks to be used as samples include beer, beer-taste beverages, fruit wines such as wine, sweet fruit wines, shochu, chuhai, cocktails, coffee, milk and fruit juice beverages, fruits and processed products thereof. It is done. When the food or drink is a solid, for example, the above quantitative method may be applied after pulverizing in a liquid such as water to form a liquid. When the food or drink is a beverage, the above quantification method can be basically applied as it is.

  In the above purification step, the carrier carrying the metal atom is brought into contact with the sample, and the thiol compound or sulfide compound is separated from the sample by utilizing the bond between the sulfur atom and the metal atom contained in the thiol compound or sulfide compound. Is done.

  Before contacting the carrier carrying the metal atoms with the specimen, for example, the specimen is extracted with an organic solvent such as dichloromethane, ether, pentane, ethyl acetate and chloroform, and the thiol compound or sulfide compound in the specimen is extracted with the organic solvent phase. The process of extracting to may be provided. Thereby, the thiol compound or sulfide compound in the specimen can be selectively concentrated. In this case, the purification step may be performed using the organic solvent phase as a specimen.

  When the carrier carrying the metal atom is brought into contact with the specimen, it is preferable that the carrier is supported by a support because the operation is simple. The support may be any support as long as it can hold the carrier when it contacts the sample, and examples thereof include a container such as a column and a cartridge, and a disk. Among them, it is preferable that the support is packed in a column because the operation is further simplified. That is, the sulfur atom contained in the thiol compound or sulfide compound and the metal atom can be bonded by passing the specimen through the column. In addition, it is easy to handle the waste liquid discharged through the column.

  The thiol compound or sulfide compound to be detected bound to the metal atom can be eluted from the carrier by contacting with an eluent containing a compound having a higher affinity for the metal atom than the thiol compound or sulfide compound, for example. it can. The eluent may be appropriately selected according to the type of thiol compound or sulfide compound to be detected, the type of metal atom, and the like, and examples thereof include cysteine solution, mercaptosuccinic acid, and thioglycerol.

  Prior to elution, the impurities may be removed while washing with an appropriate washing solution while the thiol compound and sulfide compound are bound to the carrier. The washing liquid may be appropriately selected according to the type of thiol compound or sulfide compound to be detected, the type of metal atom, etc., and examples thereof include thiol compound solutions such as dichloromethane, methanol, ultrapure water, and cysteine. It is done. As an example of the washing method, a method of washing in this order using dichloromethane, methanol and ultrapure water as washing liquids can be mentioned.

  The quantitative method preferably further includes a step of obtaining a thiol compound derivative by reacting a thiol compound in a specimen with a maleimide compound represented by the following general formula (1).

  In general formula (1), R is a hydrogen atom, an optionally substituted alkyl group having 1 to 12 carbon atoms, an optionally substituted cycloalkyl group having 5 to 12 carbon atoms, The arylalkyl group having 7 to 12 carbon atoms which may have a substituent or the aryl group having 6 to 12 carbon atoms which may have a substituent.

  In the present specification, the alkyl group may be linear or branched. Further, the alkyl group in the arylalkyl group may be linear or branched.

  Examples of the alkyl group having 1 to 12 carbon atoms in R include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, t-butyl group, 2-ethylhexyl group, nonyl group, A decanyl group and a lauryl group are mentioned. Moreover, the C1-C12 alkyl group in R may have a substituent.

  Examples of the cycloalkyl group having 5 to 12 carbon atoms in R include, for example, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a tricyclodecyl group, a bicyclooctyl group, a tricyclododecyl group, an isobornyl group, an adamantyl group, A tetracyclododecyl group is mentioned. Moreover, the C5-C12 cycloalkyl group in R may have a substituent.

  Examples of the arylalkyl group having 7 to 12 carbon atoms in R include benzyl group, 1-phenylethyl group, 2-phenylethyl group, 3-phenylpropyl group, and 6-phenylhexyl group. Moreover, the C7-12 arylalkyl group in R may have a substituent.

  Examples of the aryl group having 6 to 12 carbon atoms in R include a phenyl group and a naphthyl group. Moreover, the C6-C12 aryl group in R may have a substituent.

  Examples of the substituent include a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom), hydroxyl group, carboxyl group, amino group, formyl group, acetyl group, propionyl group, methyloxy group, and ethyloxy group. Is mentioned.

  The said maleimide compound should just select an appropriate thing according to the kind of thiol compound made into a measuring object. For example, the boiling point of the thiol compound derivative can be controlled by appropriately setting the molecular weight of the maleimide compound according to the molecular weight of the thiol compound to be measured. As a result, for example, gas chromatograph mass spectrometry can be easily performed with good reproducibility, and when the thiol compound to be measured is volatile, quantification with good reproducibility is possible by suppressing volatilization. It becomes. Further, for example, the hydrophobicity of the thiol compound derivative can be controlled by appropriately setting the hydrophobicity of the maleimide compound according to the hydrophobicity of the thiol compound to be measured. Thereby, for example, when extracting a thiol compound derivative from a specimen using an organic solvent, the extraction efficiency can be improved. When the thiol compound to be detected is 2-furfuryl mercaptan (furfuryl thiol), 3-mercapto-1-hexanol, 3-mercaptohexyl acetate, or the like, the effect of including a step of obtaining a thiol compound derivative Is remarkable.

Specific examples of the maleimide compound represented by the general formula (1) include, for example, N-ethylmaleimide (the following formula (3)), N-phenylmaleimide (the following formula (5)), N-trichlorophenylmaleimide ( The following formula (6) may be mentioned.

  For example, N-ethylmaleimide has a simple structure and is easier to handle. In addition, since N-phenylmaleimide has a high molecular weight, when the thiol compound to be detected has a low molecular weight, in addition to facilitating suppression of volatilization by derivatization, the degree of hydrophobicity increases by derivatization and depends on the organic solvent. Extraction efficiency is higher. Furthermore, since N-trichlorophenylmaleimide can be detected by a negative ion chemical ionization method (NCI), the detection sensitivity becomes higher.

  The reaction between the thiol compound in the sample and the maleimide compound is, for example, that the thiol compound is 2-furfuryl mercaptan (furfuryl thiol) (the following formula (2)), and the maleimide compound is N-ethylmaleimide (the following formula ( Taking the case of 3)) as an example, it is considered that the process proceeds as shown in the following equation. In this case, the obtained reaction product (the following formula (4)) becomes a sulfide compound.

  Since the reaction between the thiol compound and the maleimide compound easily proceeds, the reaction may be performed under any conditions. On the other hand, from the viewpoint of further increasing the reaction efficiency, for example, a 5% (w / v) maleimide compound solution is 0.5% (v / v) to 5% (v / v) with respect to the specimen. It is preferable to add, and it is more preferable to add so that it may become 0.5% (v / v)-2% (v / v). The reaction time can be, for example, 0.5 hours to 5 hours, preferably 0.5 hours to 2 hours, and more preferably 0.5 hours to 1 hour. The reaction temperature is not particularly limited, and may be room temperature, for example.

  The reaction between the thiol compound and the maleimide compound in the sample can be performed simultaneously with the above-described step of extracting the thiol compound or sulfide compound in the sample into the organic solvent phase. That is, the reaction may be performed while extracting the thiol compound (thiol compound derivative) derivatized with the maleimide compound into the organic solvent phase by extracting the thiol compound in the sample with the organic solvent while reacting with the maleimide compound. it can.

  By derivatizing the thiol compound with a maleimide compound, it is possible to protect the thiol group and improve the stability of the behavior of the thiol compound. Further, by increasing the boiling point, large volume injection into the gas chromatograph becomes possible. As a result, even if the final liquid volume in the above purification step is 100 μL, 50 μL of the final liquid volume can be injected. Therefore, even when 10000-fold concentration is performed, the sample volume can be reduced to 20 mL. Thereby, working efficiency can further be improved.

  The thiol compound or sulfide compound obtained in the purification step can be quantified using, for example, a gas chromatograph mass spectrometer, a gas chromatograph chemiluminescence sulfur detector, a gas chromatograph flame photometric detector, or the like. Among these, quantification by gas chromatography mass spectrometry is preferable because of the advantage that it can be specifically detected.

  Hereinafter, the present invention will be described in more detail based on examples and the like.

[Test Example 1: Evaluation of adsorption ability of thiol compound and sulfide compound (1)]
The adsorption ability of thiol compounds and sulfide compounds of various metal atoms was evaluated.

(Processing method)
8 mL of ultrapure water or 50 ppm of a metal solution was added to a 20 mL headspace vial, and furfuryl thiol (thiol compound) and methionol (sulfide compound) were added to each to 1 ppm and sealed. After the sealed vial was shaken at 40 ° C. for 15 minutes, an SPME fiber (Polydimethylsiloxane / Divynylbenzene 65 μm: manufactured by Spelco) was exposed to the head space in the vial. Volatile components were adsorbed on the fiber at 40 ° C. for 15 minutes, then desorbed at the inlet for 3 minutes and analyzed by GC / MS. The metal solution used was a commercially available (Wako Pure Chemical) 1000 ppm solution diluted with ultrapure water.

(GC / MS analysis)
The measurement conditions for GC / MS analysis are as follows.
・ Analytical instruments 6890N GC, 5973N MSD (Agilent Technologies)
Column HP-1MS, 30 m (length) × 0.25 mm (inner diameter), 1.0 μm (film thickness) (Agilent Technologies)
・ Injection mode Splitless injection ・ Flow rate 1mL / min (constant flow rate)
・ Inlet temperature 270 ℃
・ Oven temperature 40 ° C (3 minutes) → 5 ° C / minute → 200 ° C (0 minute: ultimate temperature) → 10 ° C / minute → 320 ° C (3 minutes)
MS detector SIM m / z 114 (furfurylthiol), 106 (methionol)

(result)
The determination result (area value) of furfurylthiol or methionol in a metal solution containing various metal atoms, and the determination result (area value) of furfurylthiol or methionol in ultrapure water (not including metal atoms) are 100. The relative value was obtained. The results are shown in Table 1.

  When furfuryl thiol (thiol compound) or methionol (sulfide compound) is adsorbed to a metal atom, the amount of furfuryl thiol or methionol transferred to the head space decreases. Therefore, it shows that adsorption capacity is so high that a fixed_quantity | quantitative_assay result (relative value) is small. For furfuryl thiol (thiol compound), the adsorption ability of Cu, Cr, Ag, Au, Pt, Pd, Hg and Bi was high (Table 1). Adsorption ability of Au, Pd, Ag, Pt and Hg was high for methionol (sulfide compound) (Table 1).

[Test Example 2: Evaluation of adsorption ability of thiol compound and sulfide compound (2)]
The adsorption ability of thiol compounds and sulfide compounds was evaluated using a column in which metal atoms were immobilized.

(Preparation of columns with immobilized metal atoms)
A column (metal-immobilized column) on which metal atoms were immobilized was prepared as follows.
-Ag fixed column The commercial item (MetSEP IC-Ag: GL Sciences Inc.) was used.
-Cu immobilization column Prepared by passing 50 mL of a 1000 ppm copper sulfate aqueous solution through a solid phase (MetaSEP IC-ME: GL Sciences) filled with a chelate resin.
Bi-immobilized column and Fe-immobilized column 50 mL of commercially available (Wako Pure Chemical Industries) 1000 ppm solution diluted 10-fold with ultrapure water was passed through a solid phase MetaSEP IC-ME (GL Sciences Inc.) filled with a chelate resin. Prepared.

(Processing method)
A metal-immobilized column conditioned with 10 mL of ultrapure water, 5 mL of methanol, and 5 mL of dichloromethane was loaded with 10 mL of a dichloromethane solution containing 1 ppm each of furfurylthiol and methionol. The loaded one was collected as it was and analyzed by GC / MS.

(GC / MS analysis)
The measurement conditions for GC / MS analysis are as follows.・ Analytical instruments 6890N GC, 5973N MSD (Agilent Technologies)
Column HP-5MS, 30 m (length) x 0.25 mm (inner diameter), 0.25 μm (film thickness) (Agilent Technologies)
・ Injection mode Splitless injection ・ Flow rate 1mL / min (constant flow rate)
・ Inlet temperature 250 ℃ ・ Oven temperature 50 ℃ (1 minute) → 25 ℃ / minute → 325 ℃ (0 minute: ultimate temperature)
MS detector SIM m / z 114 (furfurylthiol), 106 (methionol)

(result)
The quantification result (area value) was obtained as a relative value with the quantification result (area value) before column loading as 100. The results are shown in Table 2.

  As is clear from Tables 1 and 2, the thiol compound and sulfide compound adsorption ability of the metal atoms did not change even when the metal atoms were immobilized. Further, Cu and Ag are more preferable in that the fixing operation can be easily performed because the reagents (copper sulfate and silver nitrate, respectively) have high solubility in water.

[Example 1: Determination of thiol compound]
4-mercapto-4-methyl-2-pentanone (4MMP) in the standard solution was quantified by a simple standard addition method to prepare a calibration curve, and the validity of the quantification method was evaluated.

(Reagents)
4-Mercapto-4-methyl-2-pentanone (1% propylene glycol solution, INTERCHIM) was used as the thiol compound.

  Cysteine was used from Tokyo Kasei. Dichloromethane and methanol were used for residual agricultural chemicals of Wako Pure Chemicals, and other grades were used for special grades. As an internal standard substance, 1-pentadecanol (100 μg / L, Tokyo Chemical Industry Co., Ltd.) was used. For purification of the thiol compound, Discovery Ag-ION (SUPELCO) was used as a carrier carrying silver.

(Sample preparation)
20 mL of beer beverage (hereinafter referred to as “test brewed product X1”) was placed in a 50 mL glass centrifuge tube containing 6 g of sodium chloride, and 20 mL of dichloromethane was added. Three of these were prepared per sample, shaken for 15 minutes, extracted, and then centrifuged at 3000 rpm for 15 minutes. The three extracts were slowly transferred to a 200 mL separatory funnel without breaking the interface, and the lower layer was collected in a 100 mL Erlenmeyer flask. Further, the dichloromethane layer was recovered from the emulsion using a separation filter paper. After the dichloromethane layer was dehydrated with sodium sulfate, 50 mL was concentrated to 5 mL under a nitrogen stream using a turbobap. The whole amount was loaded onto Discovery Ag-ION (trade name, SUPELCO) conditioned with 5 mL of dichloromethane, washed with 10 mL of dichloromethane, 10 mL of methanol, and 50 mL of ultrapure water, and eluted with 20 mL of 1% cysteine solution. The eluate was transferred to a 50 mL glass centrifuge tube, 25 mL of dichloromethane was added, the mixture was shaken for 15 minutes, extracted, and then centrifuged at 3000 rpm for 15 minutes. Take the lower layer in a 50 mL Erlenmeyer flask, dehydrate with sodium sulfate, take 20 mL into a concentration container, add 1 mL of internal standard substance (100 μg / L, 1-pentadecanol), and then concentrate to 200 μL under a nitrogen stream with a turbobap. It was used for measurement.

(Create a calibration curve)
A calibration curve was prepared by measuring a sample brewed product X1 with 4 MMP added to 25 ng / L, 50 ng / L, and 100 ng / L, treated in the same manner as in the preparation of the sample.

(Two-dimensional gas chromatograph mass spectrometry)
The prepared sample for measurement was analyzed by two-dimensional gas chromatograph mass spectrometry (hereinafter sometimes referred to as “2D-GC-MS”). In addition, the prepared standard solution was similarly analyzed, and a calibration curve was prepared. A two-dimensional gas chromatograph mass spectrometer was set up as shown in FIG. 1 and analyzed under the following measurement conditions.
・ Analytical instruments 7890N GC, 5975C MSD (Agilent Technologies)
Column First dimension (1D) DB-WAX, 30 m (length) × 0.25 mm (inner diameter), 0.25 μm (film thickness) (Agilent Technologies):
Second dimension (2D) DB-5, 10 m (length) × 0.18 mm (inner diameter), 0.4 μm (film thickness) (Agilent Technologies)
・ Inlet PTV inlet, single baffle with glass wool:
Solvent vent, 0 kPa (0.35 min), purge flow rate 50 mL / min:
Split vent flow rate 50 mL / min (2 min):
10 ° C (0.65 minutes) → 600 ° C / minute → 300 ° C (3 minutes)
・ Inlet pressure 252.28kPa
・ Injection volume 10μL (injection speed 3000μL / min)
・ Oven temperature 40 ° C (1 minute) -10 ° C / minute-180 ° C-25 ° C / minute-250 ° C (13 minutes)
LTM temperature: 40 ° C (15 minutes) -25 ° C / minute-140 ° C (6 minutes) -25 ° C / minute-300 ° C (2 minutes)
・ AUX temperature 250 ℃
Deans pressure 100 kPa (0.35 minutes)-208.37 kPa
・ Deans valve Valve 1 ON (13 minutes)-OFF (13.7 minutes)-ON (23 minutes)-OFF (24 minutes)
FID detector 250 ° C., H ₂ 40 mL / min, Air 450 mL / min, Make up He 45 mL / min
MS detector gain factor 2, SIM m / z 132 (4MMP), 182 (internal standard), dwell 100

(result)
The standard solution was measured by two-dimensional gas chromatography mass spectrometry. A calibration curve was created by plotting the 4MMP concentration against the ratio of the obtained m / z 132 area value and m / z 182 area value {internal standard ratio: (m / z 132 area value) / (m / z 182 area value)}. (FIG. 2). As a result, the linearity of the calibration curve was very good (R ² value 0.99).

[Test Example 3: Evaluation of quantitative method]
(Evaluation of recovery rate)
A sample obtained by adding 4MMP to a commercial beverage X1 not containing 4MMP so as to be 50 ng / L and 100 ng / L was prepared in the same manner as in the preparation of the sample, and a purified and concentrated sample was used as a measurement sample. The obtained sample for measurement was measured and quantified using the slope of the calibration curve in FIG. 2 to obtain the recovery rate. The results are shown in Table 3. As shown in Table 3, 4MMP in the specimen could be extracted with a recovery rate of nearly 60%.

(Evaluation of parallel accuracy)
The test brewed product X1 was subjected to the same operation as the above sample preparation, and a purified and concentrated sample was used as a measurement sample. The obtained measurement sample was independently quantified four times (n = 4), and the parallel accuracy was determined. Table 4 shows the results. As shown in Table 4, the parallel accuracy (coefficient of variation, CV) was very good at 4.0%. In addition, although FIG. 3 is the chromatogram obtained by 2D-GC-MS, it was confirmed from this result that selectivity is favorable.

[Example 2: Analysis of test brewed products and commercial beverages]
4MMP in test brewed products and commercial beverages was quantified. As the test brewed products, different beer beverages (hereinafter referred to as “test brewed product X1” to “test brewed product X5”, respectively) were used.

  The sample preparation and the calibration curve were prepared in the same manner as in Example 1. The measurement conditions of 2D-GC-MS are the same as in Example 1. The results are shown in Table 5.

[Example 3: Determination by derivatization of thiol compound]
A calibration curve was prepared by quantifying furfurylthiol in the standard solution and the specimen, and the validity of the quantification method was evaluated.

(Reagents)
2-furfuryl mercaptan (furfuryl thiol) (Tokyo Kasei) was used as the thiol compound, and N-ethylmaleimide (Tokyo Kasei, the following formula (3)) was used as the maleimide compound.

  Cysteine was purchased from Tokyo Kasei, and N-ethylmaleimide-d5 (ETHYL-D5, 98%) was purchased from Cambridge Isotop Laboratories, Inc. Dichloromethane and methanol were used for residual agricultural chemicals of Wako Pure Chemicals, and other grades were used for special grades. For purification of the thiol compound derivative, Discovery Ag-ION (SUPELCO) was used as a carrier carrying silver.

(Preparation of internal standard)
To a 10 mL glass centrifuge tube, ultrapure water 2 mL, sodium chloride 0.6 g, 1% N-ethylmaleimide-d5 ethanol solution 0.2 mL, 1000 mg / L furfurylthiol ethanol solution 20 μL was added and stirred gently. After extraction by adding 2 mL of dichloromethane and shaking for 30 minutes, the lower layer was collected by centrifugation at 3000 rpm for 15 minutes. The collected lower layer diluted 50 times with dichloromethane was used as an internal standard stock solution, and further diluted 100 times with dichloromethane as an internal standard solution.

(Sample derivatization)
Commercial beer (hereinafter referred to as “commercial product A”) and commercial product A with 50 ng / L and 100 ng / L furfurylthiol added to each 50 mL glass centrifuge tube containing 6 g of sodium chloride. A sample was used. 400 μL of a derivatization reagent (5% (w / v) N-ethylmaleimide ethanol solution) was added to the specimen and lightly stirred. After adding 20 mL of dichloromethane and shaking for 30 minutes for extraction, the mixture was centrifuged at 3000 rpm for 15 minutes. 18 mL of the lower layer was collected in a 20 mL volumetric flask containing 2 mL of internal standard solution in advance. Discovery Ag-ION (trade name, SUPELCO) conditioned with 5 mL of dichloromethane was loaded with 18 mL of the collected specimen, washed with 10 mL of dichloromethane, 10 mL of methanol, and 50 mL of ultrapure water, and eluted with 40 mL of 1% cysteine solution. The eluate was transferred 20 mL each to two 50 mL glass centrifuge tubes, 6 g sodium chloride and 20 mL dichloromethane were added, shaken for 15 minutes, extracted, and then centrifuged at 3000 rpm for 15 minutes. The lower layer for two centrifuge tubes was placed in a 50 mL Erlenmeyer flask, dehydrated with sodium sulfate, and concentrated to 100 μL under a nitrogen stream. The sample subjected to the derivatization treatment thus obtained was used as a measurement sample.

(Preparation of standard solution)
To a 50 mL centrifuge tube, 400 μL of a derivatization reagent (5% N-ethylmaleimide ethanol solution) was added to 20 mL of a 1 mg / L furfurylthiol aqueous solution and lightly stirred. After adding 20 mL of dichloromethane and shaking for 30 minutes for extraction, the mixture was centrifuged at 3000 rpm for 15 minutes. The lower layer was collected and diluted with dichloromethane to prepare 1 μg / L, 2.5 μg / L, 5 μg / L, and 10 μg / L solutions. A mixture of 9 mL of these and an internal standard stock solution 1 mL was used as a standard solution.

(Two-dimensional gas chromatograph mass spectrometry)
The prepared measurement specimen was analyzed by 2D-GC-MS. In addition, the prepared standard solution was similarly analyzed, and a calibration curve was prepared. The measurement conditions of 2D-GC-MS are as follows.
・ Analytical instruments 6890N GC, 5973N MSD (Agilent Technologies)
Column 1st dimension (1D) DB-5MS, 15 m (length) × 0.25 mm (inner diameter), 0.25 μm (film thickness) (Agilent Technologies):
Second dimension (2D) HP-INNOWAX, 30 m (length) × 0.25 mm (inner diameter), 0.25 μm (film thickness) (Agilent Technologies)
・ Inlet PTV inlet, multi-baffle liner:
Solvent vent, 0 psi (0.6 minutes), purge flow 150 mL:
Split vent flow rate 50 mL / min (2 min):
40 ° C (0.6 minutes) → 600 ° C / minute → 320 ° C (3 minutes)
・ Inlet pressure 44.1 psi ・ Injection volume 50 μL (injection rate 100 μL / min)
・ Oven temperature 50 ℃ → 6 ℃ / min → 182 ℃ (1 minute) → 15 ℃ / min → 250 ℃ (13 minutes)
・ AUX temperature 250 ℃
FID detector 250 ° C., H 2 40 mL / min, Air 450 mL / min, Make up He 45 mL / min MS detector SIM m / z 239 (furfurylthiol derivative), 244 (internal standard), dwell 50:
MS OFF (36 minutes)

(result)
The standard solution was measured by two-dimensional gas chromatography mass spectrometry. The furfurylthiol concentration was plotted against the ratio {(m / z239 area value) / (m / z244 area value)} of the obtained area value of m / z239 and m / z244 to create a calibration curve. (FIG. 4). As a result, the linearity of the calibration curve was very good. Furthermore, the measurement was similarly performed on the measurement sample, and the quantitative value of furfurylthiol was determined by an internal standard method. As a result, as shown in Table 6, by subjecting the specimen to derivatization using N-ethylmaleimide, furfurylthiol in the specimen could be extracted with a recovery rate of almost 100%.

[Test Example 4: Evaluation of quantitative method]
A sample obtained by adding 20 ng / L furfurylthiol to a commercially available black beer-taste beverage was used as a sample, and the amount was independently determined five times (n = 5), and the parallel accuracy and the recovery rate were determined.

(Evaluation of concurrency accuracy and recovery rate)
Table 7 shows the results. As shown in Table 7, the concurrency accuracy (coefficient of variation, CV) was very good at 2.9%.

[Example 4: Analysis of commercial beverage]
The furfuryl thiol in the commercial beverage (black beer or black beer-taste beverage) was quantified. Commercially available black beer taste drink (hereinafter referred to as “commercial product B”), commercially available black beer (hereinafter referred to as “commercial product C”), and commercially available black beer different from the commercial product C include commercial beverages. (Hereinafter referred to as “commercial product D”). The standard solution (10 ng / L) was also quantified in the same manner.

  Quantification of furfuryl thiol was performed as follows. Except that the addition amount of 5% (w / v) N-ethylmaleimide ethanol solution to the sample was 1% (v / v) and the sample was derivatized under the condition that the reaction time was 0.5 hour, The sample was derivatized by the same method as in Example 3 to prepare a measurement sample. The sample for measurement was analyzed by 2D-GC-MS in the same manner as in Example 3, and the peak area values derived from the furfurylthiol derivative under each condition were compared.

  FIG. 5 shows the result of 2D-GC-MS. FIG. 5A is a 2D-GC-MS chromatogram of a standard solution. FIGS. 5B, 5C, and 5D are 2D-GC-MS chromatograms of a commercial product B, a commercial product C, and a commercial product D, respectively. As shown in FIG. 5, it was considered that the commercially available beverages did not have a great influence on the quantitative value of furfurylthiol, although a contamination peak was observed. The furfuryl thiols contained in the commercial product B, the commercial product C, and the commercial product D were 6 ng / L, 7 ng / L, and 13 ng / L, respectively.

Claims (12)

  A method for quantifying a thiol compound and a sulfide compound, comprising a purification step comprising contacting a carrier carrying a metal atom with a specimen and separating the thiol compound or sulfide compound from the specimen.   The quantification method according to claim 1, wherein the metal atom is at least one selected from metal elements belonging to Groups 4 to 15 of the periodic table.   The metal atom is at least one selected from copper (Cu), palladium (Pd), silver (Ag), platinum (Pt), gold (Au), mercury (Hg), chromium (Cr) and bismuth (Bi). The quantification method according to claim 1 or 2, wherein   The quantification method according to claim 1 or 2, wherein the metal atom is at least one selected from palladium (Pd), silver (Ag), platinum (Pt), gold (Au), and mercury (Hg).   The quantification method according to claim 4, wherein in the purification step, a sulfide compound is separated from the specimen. Prior to the purification step, the method further comprises a step of obtaining a thiol compound derivative by reacting a thiol compound in the specimen with a maleimide compound represented by the following general formula (1). The quantitative method according to item.

[In General Formula (1), R is a hydrogen atom, a C1-C12 alkyl group which may have a substituent, or a C5-C12 cycloalkyl group which may have a substituent. And an arylalkyl group having 7 to 12 carbon atoms which may have a substituent or an aryl group having 6 to 12 carbon atoms which may have a substituent. ]   The quantification method according to any one of claims 1 to 6, wherein the concentration of the thiol compound or the sulfide compound in the specimen is 1000 ng / L or less.   The quantification method according to any one of claims 1 to 7, wherein the carrier on which the metal atom is supported is supported by a support.   The quantification method according to any one of claims 1 to 8, wherein the carrier carrying the metal atoms is packed in a column.   The quantification method according to any one of claims 1 to 9, further comprising a step of quantifying the thiol compound or the sulfide compound by gas chromatography mass spectrometry after the purification step.   The quantification method according to claim 1, wherein the specimen is a food or drink.   The quantification method according to any one of claims 1 to 10, wherein the specimen is a beverage.