JP2006308474A - Mass analyzing reagent for measuring heavy metal ion, and mass-analyzing method of heavy metal ion and mass analyzing system for analyzing heavy metal ion using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means which simply and highly sensitively measures heavy metal ions including trivalent heavy metal ions, without using any flame. <P>SOLUTION: A mass analyzing reagent for measuring the heavy metal ions is provided, which is composed of a compound with a specific ring structure having two anionic functional groups becoming monovalent negative ions in a solution, and which has a structure represented by following formula (IV), for example. The charge of the heavy metal ions is reduced by negative charge caused by the anionic functional groups, and a complex formed by complexing is observed as an almost single peak, whereby the heavy metal ions can be sensitively measured by a simple mass analysis using no flame. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a mass spectrometric reagent for measuring heavy metal ions, which is used for measuring heavy metal ions such as Pb ²⁺ , Cd ²⁺ , Hg ²⁺ , Fe ³⁺ and Cr ³⁺ by mass spectrometry.

  Causes of soil and groundwater contamination by heavy metals include leakage of raw materials including target substances and chemicals, leakage during storage and manufacturing processes, smoke drop, inadequate drainage of underground water or landfill disposal of waste. The behavior of heavy metals, etc. in soil varies depending on their physicochemical properties and the nature of the soil used as a medium. It exists in the soil near the surface of the earth and often does not diffuse deep. However, when a load exceeding the adsorption capacity of the soil occurs or a substance having high water solubility and high mobility such as hexavalent chromium, it may diffuse to the deep underground with underground penetration such as rainwater.

  Against this background, there is a need for an analysis method that can easily and accurately grasp the state of heavy metal contamination. Conventional methods include ICP and atomic absorption methods as high-sensitivity measurement methods, but they are dangerous and unfavorable at the site because of the use of frames, and problems such as complicated operation and high running costs have been pointed out. Yes. A colorimetric analysis method can be cited as a simple analysis method, but the problem of low sensitivity has been pointed out.

On the other hand, it has been reported that the complex formation ability of crown ether skeletons (including sulfur, nitrogen or oxygen atoms as heteroatoms) with heavy metal ions is measured by electrospray ionization mass spectrometry (ESI-MS) (non-patented) Reference 1). However, this method has a problem that the 1: 1 complex peak cannot be detected, the counter ion is added and detected, and the sensitivity is poor. When using a time-of-flight mass spectrometer, monovalent ions can be detected with higher sensitivity than multivalent ions. Conventionally, trivalent heavy metal ions such as Fe ³⁺ and Cr ³⁺ are monovalent. There is no known mass spectrometric reagent for measuring heavy metal ions to be used as an ion.

Sheldon M. et al., Anal. Chem. 2002, 74, 4423-4433

  An object of the present invention is to provide a means for measuring heavy metal ions including trivalent heavy metal ions easily and with high sensitivity without using a frame.

  As a result of diligent research, the inventors of the present application have developed a compound for mass spectrometry containing a specific structure that forms a complex with a heavy metal ion and two ionic functional groups that become anions in a solution in one molecule. By using it as a probe, it was found that trivalent heavy metal ions can be measured easily and with high sensitivity, and the present invention has been completed. Further, the present invention has been completed by finding that a compound having a specific ring structure can measure heavy metal ions particularly sensitively in the presence of iodine ions.

  That is, the present invention provides the following general formula [I]

(In the formula, R ¹ and R ² are independently an ionic functional group that becomes a monovalent negative ion in solution, R ³ and R ⁴ are each independently a hydrogen atom, and a straight chain having 1 to 10 carbon atoms. Type or branched alkyl group, amino group, nitro group or halogen, A ¹ and A ² may or may not be present independently of each other and, if present, represent any spacer moiety ),
The following general formula [II]

(However, in the formula, R ¹ and R ² are ionic functional groups that become monovalent negative ions in solution independently of each other, and A ¹ and A ² may or may not exist independently of each other. Often represents any spacer moiety if present),
Or the following formula [III]

(However, the carbon atom constituting the ring has ^one substituent represented by -A ¹ -R ¹ (the definitions of A ¹ and R ¹ are the same as those in the general formula [I]) for each ethylene chain. The following number may be combined)

A mass spectrometric reagent for measuring heavy metal ions having a structure represented by: The present invention also provides a heavy metal ion mass spectrometry method comprising reacting the reagent of the present invention with a heavy metal ion in a test sample, and mass-analyzing the product. Furthermore, the present invention aspirates the reagent container for storing the reagent of the present invention, the sample container for storing the test sample, the reagent stored in the reagent container and the test sample stored in the sample container. A suction means for suctioning the reagent housed in the reagent container and the test sample housed in the sample container via the tip of the suction means, and the middle of the suction pipe A first valve provided on the first valve, a liquid feed pump connected to the first valve via a liquid extrusion pipe, and a liquid mixing means connected to the first valve via a liquid feed pipe And a mass spectrometry system for heavy metal ion analysis, comprising a mass spectrometer connected downstream of the liquid mixing means.

When the reagent of the present invention is used, the negative charge resulting from the ionic functional group reduces the charge of heavy metal ions, and the complex formed by complex formation is observed as a substantially single peak. Heavy metal ions can be measured with high sensitivity by simple mass spectrometry without using. In particular, in the reagent having the structure represented by the above general formula [I] or [II], there are two ionic functional groups that become anions in a solution in one molecule, so Fe ³⁺ and Cr ^When combined with a trivalent heavy metal ion such as ³⁺ , it becomes a monovalent cation as a whole and can be analyzed with high sensitivity by time-of-flight mass spectrometry. In addition, in the reagent having the structure represented by the above general formula [III], mass spectrometry of divalent heavy metal ions such as Hg ²⁺ , Cd ²⁺ and Pb ²⁺ in the presence of iodine ions is particularly sensitive. it can. The present invention also provides a highly sensitive heavy metal ion mass spectrometry method and mass spectrometry system using the reagent of the present invention.

As described above, the mass spectrometric reagent for heavy metal ion measurement of the present invention has a structure represented by the general formulas [I] to [III]. Among these, the reagent having the structure represented by the general formula [I] or [II] is a reagent particularly suitable for mass spectrometry of trivalent heavy metal ions such as Fe ³⁺ and Cr ³⁺ . Further, among the reagents having the structure represented by the general formula [III], an unsubstituted reagent can be used for particularly sensitive mass spectrometry of divalent heavy metal ions such as Hg ²⁺ , Cd ²⁺ and Pb ^2+. It is what makes it possible.

In the general formulas [I] and [II], R ¹ and R ² are ionic functional groups that become anions, preferably monovalent anions, in a solution, preferably in an aqueous solution, independently from each other. As examples, a carboxyl group or a salt thereof, a sulfone group or a salt thereof, or a hydroxyl group or a salt thereof can be given. Among these, a carboxyl group or a salt thereof is particularly preferable. These preferred ionic functional groups, in particular carboxyl groups or salts thereof, not only reduce the charge of the heavy metal ion by a total of two valences due to the charge of the anion, but also a stable and selective complex with the heavy metal ion. It also contributes to the formation and, in turn, contributes to highly sensitive measurement of heavy metal ions.

In the general formula [I], R ³ and R ⁴ are independently of each other a hydrogen atom, a linear or branched alkyl group having 1 to 10 carbon atoms (preferably 1 to 4 carbon atoms), an amino group, A nitro group or halogen, preferably a hydrogen atom;

In the general formulas [I] and [II], A ¹ and A ² may be present independently of each other or may not be present (if they are not present, R ¹ and / or R ² is directly bonded to nitrogen. If present, it is an optional spacer portion. Since the reagent of the present invention is complexed with a heavy metal ion at the ring portion and the charge of the complex is changed from trivalent to monovalent by R ¹ and R ² , it is located between the ring and R ¹ or R ^2. A ¹ and A ² that can take an arbitrary structure. A ¹ and A ² do not exist, and an anionic functional group such as a carboxyl group may be directly bonded to a nitrogen atom. A ¹ and A ² are preferably absent, or a linear or branched alkylene group having 1 to 10 carbon atoms (more preferably 1 to 4 carbon atoms), a part of the alkylene group being an amino group, halogen, A substituted alkylene group substituted with an ether and / or carbonyl group, a phenylene group, or a substituted phenylene group in which a part of the phenylene group is substituted with an amino group, a halogen and / or a nitro group is preferable. The alkylene group substituted with ether means one in which a carbon atom in the alkylene chain is replaced with —O—. Similarly, an alkylene group substituted with a carbonyl group means one in which a carbon atom in the alkylene chain is replaced with —CO—. The number of substituents in the substituted alkylene group or substituted phenylene group may be one or more. Among these substituents, an alkylene group having 1 to 10 carbon atoms, more preferably 1 to 4 carbon atoms is preferable.

The heavy metal ion that can be measured by the reagent of the present invention having the structure represented by the general formula [I] or [II] is not particularly limited as long as it is a heavy metal ion that can form a complex with a ring portion, but trivalent. The cation is preferred. In recent years, time-of-flight mass spectrometers have been widely used as detectors for mass spectrometers. A time-of-flight mass spectrometer can detect monovalent ions with higher sensitivity than multivalent ions such as heavy metal ions. When the trivalent cation is complex-bonded with the reagent of the present invention having a monovalent anionic functional group, which is the preferred R ¹ and R ² , the charge of the heavy metal ion is reduced by two valences, and the total number of complexes formed The charge is monovalent and can be measured with high sensitivity by a time-of-flight mass spectrometer. Examples of preferable heavy metal ions measured using the reagent of the present invention include at least one selected from the group consisting of Fe ³⁺ and Cr ³⁺ . In the present specification and claims, “measurement” includes both detection (qualitative analysis) and quantitative analysis.

In the reagent of the present invention represented by the general formula [I] or [II], the structures of R ₁ , A and R ₂ are known and can be obtained by combining them. It can be synthesized based on conventional methods in the field, and preferred specific examples are described in detail in the following examples.

  When measuring heavy metal ions using the reagent of the present invention represented by the general formula [I] or [II], first, a complex of heavy metal ions and a reagent is formed by mixing with a sample containing heavy metal ions. The resulting complex is then subjected to mass spectrometry. Since the reaction between the reagent of the present invention and heavy metal ions proceeds well with a stoichiometric amount of 1: 1, the molar concentration of the reagent used is not particularly limited, but is about the same as the expected molar concentration of heavy metal ions. Good. Therefore, an upper limit of the expected molar concentration range of heavy metal ions or a slightly higher molar concentration reagent may be used. Alternatively, when examining whether or not the heavy metal ion concentration is less than or equal to the allowable reference value, a reagent having a molar concentration that is the same as or slightly higher than the molar concentration of the reference value can be used. The temperature of the complex formation reaction between the reagent and the heavy metal is not particularly limited, but since it reacts well at room temperature, it is convenient and preferable to carry out at room temperature. In addition, since the reaction takes place quickly, the reaction time is not particularly limited, and is usually about 1 to 30 seconds, and in many cases about 2 to 3 seconds. The reaction solvent is preferably water or an organic solvent mixed with water at an arbitrary ratio, such as methanol or ethanol, and most preferably methanol. The mixing is preferably performed with stirring.

On the other hand, the compound represented by the formula [III] (unsubstituted one) is a known compound called 1,4,7,10-tetraazadodecane (cyclen), and its production method is also known ( For example, the document Beilstein 26, 11, 24). Cyclene can also be used in the same manner as the reagent having the structure represented by the general formula [I] or [II]. Moreover, the inventors have, and cyclen, divalent heavy metal ions (M ^2+), the iodine ions coexist, M ²⁺ -I ^{- -} 1 monovalent complex of cyclen is formed, substantially single in mass spectrometry Since it was observed as a single peak, it was found that a highly sensitive analysis was possible using a mass spectrometer. In particular, when the M ²⁺ is Hg ²⁺ , the above monovalent complex is strongly formed, so that mass spectrometry of Hg ²⁺ can be performed with particularly high sensitivity in the presence of iodine ions.

  When measuring heavy metal ions using cyclen in the presence of iodine ions, first, a sample containing heavy metal ions, an iodine ion source, preferably iodine salt, and sacrene are mixed to obtain heavy metal ions and iodine ions. A complex with the cyclen is formed, and then the complex formed is subjected to mass spectrometry. The reaction between the cyclen, heavy metal ion and iodine ion proceeds well with a stoichiometric amount of 1: 1: 1 by molar ratio, so the molar concentration of the reagent used is not particularly limited, but the expected mole of heavy metal ion It may be about the same as the concentration. Therefore, an upper limit of the expected molar concentration range of heavy metal ions or a slightly higher molar concentration reagent may be used. Alternatively, when examining whether or not the heavy metal ion concentration is less than or equal to the allowable reference value, a reagent having a molar concentration that is the same as or slightly higher than the molar concentration of the reference value can be used. The temperature of the complex formation reaction between the reagent and the heavy metal is not particularly limited, but since it reacts well at room temperature, it is convenient and preferable to carry out at room temperature. In addition, since the reaction takes place quickly, the reaction time is not particularly limited, and is usually about 1 to 30 seconds, and in many cases about 2 to 3 seconds. The reaction solvent is preferably water or an organic solvent mixed with water at an arbitrary ratio, such as methanol or ethanol, and most preferably methanol. The mixing is preferably performed with stirring. The pH is preferably neutral to basic (pH 7.6 to 12.3).

The carbon atom constituting the cyclene has a substituent represented by -A ¹ -R ¹ (the definitions of A ¹ and R ¹ are the same as those in the general formula [I]) per ethylene chain constituting the ring. One or less number may be bonded. That is, when a substituent exists, the number of substituents is 1 to 4 in the entire ring. When such a substituent is present, an ionic functional group that becomes a monovalent negative ion in the solution is present, and thus the above-described iodine ion is unnecessary. In this case, the valence of the heavy metal ion to be measured is preferably a valence (that is, the number of substituents + 1) so that the valence of the whole complex in which the cyclen derivative and the heavy metal ion are complex-bonded is monovalent. The introduction of such a substituent into the cyclen can be easily performed by a conventional method of organic synthesis.

  When mass spectrometry is performed using a reagent having a structure represented by the formulas [I] to [III], the sample after the complex formation reaction can be directly subjected to mass spectrometry. Mass spectrometry itself can be performed by a conventional method using a commercially available mass analyzer. As the mass spectrometry, any known method may be used, but ESI-MS is preferable, and the detector is preferably a time-of-flight mass spectrometer.

  Since the molecular weight of the reagent to be used is known and the atomic weight of the heavy metal ion is also known, the heavy metal ion can be identified and measured by measuring m / z of the complex by mass spectrometry. In addition, as specifically shown in the following examples, even when multiple types of heavy metal ions are contained in the sample, since the atomic weight of each heavy metal ion is different, a plurality of m / z observed at different positions. Each heavy metal ion can be measured separately from the peak.

When the reagent of the present invention is used, the charge of the heavy metal ion can be offset by the anionic functional groups R ¹ and R ² or by the iodine ion, and the charge of the complex can be made monovalent. Mass spectrometry can be performed with high sensitivity using a meter. Further, when a reagent having a structure represented by the general formula [I] or [II] is used, when the above-described preferable functional groups are used as the anionic functional groups R ¹ and R ₂ , an anionic functional group is used. The group also contributes to complex formation, achieving stable and selective complex formation (ie, only one type of complex is formed for one type of heavy metal ion). For this reason, heavy metal ions in a sample can be accurately measured, and even when a plurality of types of heavy metal ions are contained in one sample, each heavy metal ion can be accurately and separately measured.

  The present invention also provides a method for mass spectrometry of heavy metal ions, which comprises reacting the reagent of the present invention with heavy metal ions in a test sample and mass-analyzing the product. Can be implemented as follows.

  The mass spectrometry method of the present invention can be performed manually using a general-purpose mass spectrometer using the reagent of the present invention, but the present invention is also suitable for automation using the reagent of the present invention. A mass spectrometry system for heavy metal ion analysis is also provided.

  This mass spectrometry system will be described with reference to FIGS. The system for mass spectrometry includes a reagent container 10 for storing a reagent and sample containers 12 and 12 'for storing a test sample. There may be one or a plurality of sample containers for storing the test samples. If there are a plurality of sample containers, a plurality of test samples can be analyzed. The mass spectrometric system includes suction means 14 for sucking the reagent contained in the reagent container and the test sample contained in the sample container. The suction means 14 is, for example, a syringe. A suction tube 16 is connected to the suction means 14, and the suction tube 16 sucks the reagent stored in the reagent container and the test sample stored in the sample container through its tip. A needle may be attached to the distal end portion of the suction tube 16, and in that case, a sample container or a reagent container film in which the top of the container is covered with a film is pierced with the needle to suck the liquid inside. Can do. A first valve 18 is provided in the middle of the suction pipe 16. A liquid feed pump 22 is connected to the first valve 18 via a liquid extrusion pipe 20. A liquid mixing means 26 is connected to the first valve 18 via a liquid feed pipe 24. The liquid mixing unit 26 is not limited as long as it is a unit that can mix the sample solution and the reagent solution. For example, a simple and preferable example can be a mixing column. The mixing column is a column in which glass beads are packed inside, and mixing of the two liquids occurs while the two liquids pass through them. A mass spectrometer is connected downstream of the liquid mixing means 26, which may be general purpose. Further, in a preferred specific example shown in FIGS. 18 and 19, a second valve 28 is provided in the middle of the liquid extruding pipe 20 between the liquid feed pump 22 and the first valve 18, In the middle of the suction pipe 16, a third valve 30 is provided between the suction means 14 and the first valve 18, and these are connected by a second liquid extrusion pipe 32. Yes.

  In operation, first, the aspirating means 14 is operated to aspirate the reagent solution of the present invention from the reagent container 10. At this time, the first valve 18 and the third valve 30 are set on the side where the liquid is sucked by the suction means 14. The liquid feed pump 22 is stopped. Next, in this state, the tip of the suction tube 16 (or a needle if a needle is attached) is moved to suck the sample liquid from the sample container 12 or 12 '. The fluid flow during suction is shown in FIG. 19 as a black triangle.

  When extruding the aspirated reagent liquid and test sample liquid into the mass spectrometer, the aspirating means 14 is stopped and the liquid feed pump 22 is operated to send out the solvent. As the solvent, like the reaction solvent, water or an organic solvent mixed with water at an arbitrary ratio such as methanol or ethanol is preferable, and methanol is most preferable. Further, the first, second and third valves are switched so that the fluid moves in the direction indicated by the black triangle in FIG. Then, the reagent solution and the test sample solution that are in the middle of the suction tube 16 are sent to the mass spectrometer through the liquid mixing means, as shown by the black triangles in FIG. As described above, the reagent solution and the test sample solution are mixed when passing through the liquid mixing means. When the reagent solution and the test sample solution are mixed, the above-described binding reaction occurs, and the binding product is subjected to mass spectrometry as described above.

EXAMPLES Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Synthesis of KHM-7 KHM-7, which is a reagent of the present invention, having a structure represented by the following formula [IV] was synthesized according to the following scheme.

Synthesis scheme

(1) Synthesis of 1,8-bis (ethoxycarbonylmethyl) -4,11-dimethyl-1,4,8,11-tetraazacyclotetradecane (7)
Into an Ar-substituted 200 ml three-necked eggplant flask, put 1,8-dimethyl-1,4,8,11-tetraazacyclotetradecane (6) (1 g, 4.4 mmol, 1 eq.) And acetonitrile (80 ml) Was added. Bromo-acetic acid ethyl ester (1.45 ml, 8.8 mmol, 2 eq.) And potassium carbonate (1.85 g, 13.0 mmol, 3 eq.) Were added, and the mixture was refluxed for 18 hours. After the reaction was stopped by adding H ₂ O, the solvent was distilled off under reduced pressure. The obtained solid was dissolved in ethyl chloroform and washed with H ₂ O, and then the organic layer was dried with sodium sulfate. The solvent was distilled off under reduced pressure, and purification was performed by column chromatography (Al ₂ O ₃ chloroform: methanol = 10: 1 v / v) to obtain a brown oily compound (Compound 7; 378.8 mg, 21.6%).

TLC (Al ₂ O ₃ ); R _f = 0.4 (chloroform: methanol = 10: 1 v / v)
ESI-TOFMS (+)
[M + 2H] ²⁺ = 201.2
¹ H-NMR (300 MHz, CD ₃ Cl, TMS, rt, δ / ppm), 1.27 (t, J = 7.1 Hz, 6H, -CH ₃ ), 1.64 (m, 4H, -CH _2- ), 2.21 (s, 6H, -CH ₃ ), 2.44 (m, 8H, -CH _2- ), 2.74 (m, 8H, -CH _2- ), 3.37 (s, 4H, -CH _2- ), 4.15 (m, J = 8.7 Hz, 4H, -CH _2- )

(2) Synthesis of 4,11-dimethyl-1,4,8,11-tetraazacyclotetradecane-1,8-diacetic acid (KHM-7)
Compound 7 (36.3 mg, 0.075 mmol) was placed in a 30 ml two-necked eggplant flask, ethanol (10 ml) and 10 wt% aq. NaOH (1 ml) were added, and the mixture was refluxed for 3 hours. After the reaction was stopped by adding H ₂ O, most of the solvent was distilled off under reduced pressure. 1M HCl (3 ml) was added to adjust the pH to 5, and then the solvent was distilled off under reduced pressure. Ethanol was added and the deposited precipitate was filtered off, and then the filtrate was concentrated under reduced pressure to obtain a white crystalline compound (compound KHM-7; 30.1 mg, 96.5%).

ESI-TOFMS (+)
[M + H] ⁺ = 345.5
¹ H-NMR (300 MHz, CD ₃ OD, TMS, rt, δ / ppm), 1.49 (m, 4H, -CH _2- ), 2.27 (s, 6H, -CH ₃ ), 2.36 (m, 8H, -CH _2- ), 2.46 (m, 8H, -CH _2- ), 3.30 (s, 4H, -CH _2- )

Synthesis of KHM-9 According to the following scheme, KHM-9 which is a reagent of the present invention having a structure represented by the following formula [V] was synthesized.

Synthesis scheme

(1) Synthesis of 1,7-bis (ethoxycarbonylmethyl) -4,10,13-trioxa-1,7-diazacyclopentadecane (9)
To a 200 ml three-necked eggplant flask substituted with Ar, 1,4,10-trioxa-7,13-diazacyclopentadecane 8 (0.5 g, 2.3 mmol, 1 eq.) Was added, and acetonitrile (50 ml) was added. Bromo-acetic acid ethyl ester (0.78 ml, 4.6 mmol, 2 eq.) And potassium carbonate (0.97 g, 6.9 mmol, 3 eq.) Were added and refluxed for 18 hours. After the reaction was stopped by adding H ₂ O, the solvent was distilled off under reduced pressure. The obtained solid was dissolved in chloroform and washed with H ₂ O, and then the organic layer was dried with sodium sulfate. The solvent was distilled off under reduced pressure, and purification was performed by column chromatography (Al ₂ O ₃ chloroform: methanol = 10: 1 v / v) to obtain a brown oily compound (Compound 9; 0.912 g, 70.1%).

TLC (Al ₂ O ₃ ); Rf = 0.3 (chloroform: methanol = 10: 1 v / v).
ESI-TOFMS (+)
[M + H] ⁺ = 391.5
¹ H-NMR (300 MHz, CD ₃ Cl, TMS, rt, δ / ppm), 1.27 (t, J = 7.1 Hz, 6H, -CH ₃ ), 2.95 (m, 8H, -CH _2- ), 3.48 (s, 4H, -CH _2- ), 3.60 (m, 12H, -CH _2- ), 4.14 (q, J = 7.2, 4H, -CH _2- )

(2) Synthesis of 4,10,13-trioxa-1,7-diazacyclopentadecane-1,8-diacetic acid (KHM-9)
Compound 9 (600.0 mg, 1.54 mmol) was placed in a 30 ml two-necked eggplant flask, ethanol (40 ml) and 10 wt% NaOH aqueous solution (5 ml) were added, and the mixture was refluxed for 3 hours. After the reaction was stopped by adding H ₂ O, most of the solvent was distilled off under reduced pressure. 1M HCl (7 ml) was added to adjust the pH to 5, and then the solvent was distilled off under reduced pressure. Ethanol was added and the deposited precipitate was filtered off. The filtrate was concentrated under reduced pressure to obtain a white crystalline compound (Compound KHM-9; 476.9 g, 92.8%).

ESI-TOFMS (+)
[M + H] ⁺ = 335.4
¹ H-NMR (300 MHz, CD ₃ OD, TMS, rt, δ / ppm),
2.58 (m, 8H, -CH _2- ), 3.31 (s, 4H, -CH _2- ), 3.56 (m, 12H, -CH _2- )

Using KHM-7 synthesized in Example 1, Cr ³⁺ ions were subjected to mass spectrometry as follows.

(1) Measurement conditions of mass spectrometer Fig. 1 shows the configuration of the apparatus. Using a syringe pump, the mobile solvent (MeOH) is constantly flowed at a flow rate of 10.0 μL / min. 20 μL of the sample solution is introduced from the injector using a microsyringe. The sample solution is directed to the mass spectrometer along the flow of the mobile solvent. The setting conditions of the mass spectrometer are as follows.
Model: Mariner manufactured by Applied Biosystems
Ionization method: Electrospray ionization detection method: Time-of-flight detection method Mobile solvent: Methanol spray tip potential: 3500 V
Nozzle potential: 90 V
Detector voltage: 2280 V
Quad RF voltage: 700 V
Flow rate of Nebulizer gas (N ₂ ): 0.25 L / min Flow rate of Auxiliary gas (N ₂ ): 1.0 L / min Temperature of the counter stream: 160 ℃

(2) Operation method Add 4.0 × 10 ^-5 M Cr (NO ₃ ) ₃ · 9H ₂ O and 4.0 × 10 ^-5 M KHM-7 to the screw tube (solvent is methanol) and stir at room temperature for 10 seconds. After that, measurement was performed with a mass spectrometer under the above measurement conditions.

  On the other hand, for comparison, mass spectrometry was performed in the same manner using KMH-1 having the following structure, which was previously developed by the present inventors, and compared with the case where KHM-7 was used.

The results of mass spectrometry performed using KMH-1 and KHM-7 are shown in FIGS. 2 and 3, respectively. As shown in FIGS. 2 and 3, when KMH-1 was added, no peak derived from the Cr ³⁺ complex was observed, but when KHM-7 was added, the peak of the Cr ³⁺ complex was observed. Was observed as the main peak.

The same operation as in Example 3 was performed except that KHM-9 synthesized in Example 2 was used instead of KHM-7 and FeCl ₃ .6H ₂ O was used as a test sample. Comparison with KMH-1 was performed in the same manner.

The results of mass spectrometry performed using KMH-1 and KHM-9 are shown in FIGS. 4 and 5, respectively. As shown in FIG. 4 and FIG. 5, when KMH-1 was added, no peak derived from the Fe ³⁺ complex was observed, but when KHM-7 was added, the peak of the Fe ³⁺ complex was observed. Was observed as the main peak.

Measurement of Bivalent Heavy Metal Ion Using Cyclen Cyclen was prepared by the method described in Beilstein 26, 11, 24. The grade methanol 1.0 x 10 ^-5 M of cyclen, divalent heavy metal ions ^{1.0 x 10 -5 M (Hg 2+} , Zn 2+, Ni 2+, Cu 2+, Cd 2+, Pb 2+) The results of mass spectrometry with the addition of salt are shown in FIGS. In all cases, peaks derived from the 1: 1 complex structure of cyclen and heavy metal ions could be detected with high sensitivity. This is probably because the four nitrogen atoms of the cyclene are coordinated to the heavy metal ion (four-coordinate), so that the complex formation constant of 1: 1 is high, while the 2: 1 complex is difficult to form.

Comparison between KMH-1 and cyclen
Each of the divalent heavy metal ions (Hg ²⁺ , Zn ²⁺ , Ni ²⁺ , Cu ²⁺ , Cd ²⁺ , Pb ²⁺ ) is coordinated by either the MS probe or the cyclone reagent created in the previous study. To compare easily, KMH-1, cyclen, and divalent heavy metal ions (Hg ²⁺ , Zn ²⁺ , Ni ²⁺ , Cu ²⁺ , Cd ²⁺ , Pb ²⁺ ) were added at a ratio of 1: 1: 1. It was measured which reagent and metal complex peak were obtained. The results of this experiment are summarized in Table 1 below. From these results, it was found that Hg ²⁺ , Cd ²⁺ , and Pb ²⁺ are easily coordinated with cyclen, and Zn ²⁺ , Ni ²⁺ , and Cu ²⁺ are easily coordinated with KMH-1. In this study, we focus on mercury, so when we look at mercury, cyclen is overwhelmingly easier to coordinate, so an improvement in the detection limit is expected.

Optimization of measurement conditions using cyclen The peak intensities of mass spectra when the measurement conditions were changed were compared using the most effective cyclen in mercury. In addition, benzyltriethylammonium iodide having a structure represented by the following formula was used as an internal standard substance. Since it was found that the mercury complex and I ⁻ were easily coordinated in the later counter anion experiment, a substance containing I ^{− in} the anion was used.

Change in strength by solvent All the above measurements were performed using special grade methanol as the solvent. Measurements were made using other organic solvents, special grade ethanol, special grade acetonitrile, and water for practical use. At this time, 100% special grade methanol, special grade ethanol, special grade acetonitrile, water, special grade methanol: water = 1: 1, special grade ethanol: water = 1: 1, special grade acetonitrile: water = 1: 1, special grade methanol: water = 1: 9. Special ethanol: water = 1: 9, special acetonitrile: water = 1: 9. When 100% organic solvent was used, almost the same result was obtained. However, when water was added, the strength decreased to 1/20.

Intensity change due to counter ion In the measurement of cyclen and mercury, water is added to the complex peak. In actual measurement, many ions are contained in water. Therefore, the influence of counter ions was investigated.

Reagents for investigating the effects of cyclen and mercury, cadmium, lead and counter ions, KBr, KI, KClO ₄ and KSCN were each 1: 1: 1, and all these reagents were added in the same molar amount.

KClO ₄ was not affected by counter ions, and the same metal complex peak [M + Hg + 2H ₂ OH] ⁺ was observed when this reagent was not added. In KSCN, [M + Hg + SCN] ⁺ coordinated with the counter ion was mainly observed. In KBr, [M + Hg + Br] ⁺ coordinated with a counter ion was observed. In KI, [M + Hg + I] ⁺ coordinated by a counter ion was observed. When all of these were added, [M + Hg + I] ⁺ was seen in the main. It can easily rank the peak counter ion coordinated from the experimental results I ^-> Br ^-> SCN ^- was. This depends on the size of the counter ion and the metal-counter ion binding energy.

With respect to the size of the counter ion, carbon - oxygen, chlorine - polyatomic molecular oxygen bonds chains to hide the optimization of the interaction of the holes of the binding of mercury and cyclen, halogen>> ClO ₄ ^- and becomes. In halogen, since large counter ions have low charge density, I ⁻ > Br ⁻ . The binding constants are shown in Table 2 below. The coupling constant is
^{Hg 2+ (aq) + X -} (aq) ⇔ HgX + (aq)
Where X ⁻ is a counter ion.
For cadmium and lead, no change in peak occurred with any reagent.

FIG. 12 shows a graph when the pH in methanol used as a solvent is changed. Since there is a possibility that a complex is formed when a buffer solution is used, the pH was adjusted using HCl and NaOH. Further, from the results of experiments counterion, I ^- because it was found to be susceptible coordinated to the complex of mercury, I ^- was obtained by adding the complexing peak of mercury added. The vertical axis represents the relative strength between the internal standard substance and the complex.

As can be seen from FIG. 12, it was found that optimum sensitivity was obtained in the neutral to basic region (pH 7.6 to 12.3). In the weakly acidic region, the peak of [cyclene + H] ⁺ is seen, so it seems that protons prevent the complex formation with mercury under acidic conditions. Moreover, it can be said that it is a reversible reaction because the same response was seen even when acidified with HCl and then neutralized with NaOH.

Quantification of heavy metal ion concentration From the above results, it was found that cyclen can detect mercury, cadmium and lead with higher sensitivity than KMH-1. Therefore, the correlation between heavy metal ion concentration and peak intensity was clarified by titration experiments, and the lower limit of detection was determined. Further, from the results of experiments counterion, I ^- because it was found to be susceptible coordinated to the complex of mercury, I ^- was added to the complexation peaks of mercury added. The vertical axis represents the relative intensity between the internal standard substance and the complex, and the horizontal axis represents mol obtained by multiplying the concentration of heavy metal ions by 20 μl of the injected volume.

Hg ²⁺ titration experiment The concentration of cyclen was 1.0 × 10 ⁻⁵ M, the concentration of KI was 2.0 × 10 ⁻⁵ M, the concentration of internal standard was 1.5 × 10 ⁻⁷ M, and Hg ²⁺ was 7.0 FIG. 13 shows the results of measurement in the concentration range from × 10 ⁻⁸ M to 2.0 × 10 ⁻⁵ M. From the measurement results, a good linear relationship was obtained in the concentration range of 7.0 × 10 ⁻⁷ M to 2.0 × 10 ⁻⁵ M.

Cd ²⁺ titration experiment The concentration of cyclen is 1.0 × 10 ⁻⁵ M, the concentration of internal standard is constant at 1.5 × 10 ⁻⁷ M, and Cd ²⁺ is 7.0 × 10 ⁻⁷ M to 2.0 × 10 ⁻⁵ The results of measurement in the concentration range up to M are shown in FIG. From the measurement results, a good linear relationship was obtained in the concentration range of 4.0 × 10 ⁻⁶ M to 1.0 × 10 ⁻⁵ M. The reason why the relative intensity was almost the same at 2.0 × 10 ⁻⁵ M is thought to be because the complex was already saturated at 1.0 × 10 ⁻⁵ M at the same concentration as the cyclen.

Pb ²⁺ titration experiment The concentration of cyclen was 1.0 × 10 ⁻⁵ M, the concentration of KI was 2.0 × 10 ⁻⁵ M, the concentration of internal standard was 1.5 × 10 ⁻⁷ M, and Pb ²⁺ was 4.0 FIG. 15 shows the results of measurement in the concentration range from × 10 ⁻⁷ M to 1.0 × 10 ⁻⁵ M. From the measurement results, a good linear relationship was obtained in the concentration range of 4.0 × 10 ⁻⁶ M to 1.0 × 10 ⁻⁵ M. However, the lower detection limit was higher than that of KMH-1. Therefore, the same measurement was performed without adding KI. The concentration of cyclen is 1.0 × 10 ⁻⁵ M, the concentration of internal standard is 1.5 × 10 ⁻⁷ M, and Pb ²⁺ is in the concentration range from 1.0 × 10 ⁻¹² M to 2.0 × 10 ⁻⁵ M. The measurement results are shown in FIG. From the measurement results, a good linear relationship was obtained in the concentration range of 4.0 × 10 ⁻⁶ M to 2.0 × 10 ⁻⁵ M.

Comparison between lower detection limit and emission standard / environmental standard In Table 3 below, the lower detection limit and environmental standard value of the measured heavy metal ions are summarized. With regard to mercury, we were able to improve the value to about 1/100 that of KMH-1 developed in the previous study, but it did not reach the standard value. Therefore, when using a micro TOF manufactured by Bruker Daltonics, a more sensitive device, it was possible to detect even the environmental standard value. From FIG. 17, it was proved that the measured metal complex peak and the isotope peak calculated by the software were almost equal in shape, so that the environmental standard value could be detected.

  Regarding lead, the lower limit of detection has become larger than that of KMH-1. Therefore, when I measured without using KI, I was able to improve the detection limit. Furthermore, the detection limit for cadmium was improved compared to KMH-1.

It is a figure which shows typically the structure of the mass spectrometer used in the Example of this invention. It is a figure which shows the result of having mass-analyzed Cr3 ⁺ with KMH-1 developed previously for the comparison. In the Example of this invention, it is a figure which shows the result of having mass-analyzed Cr3 ⁺ . It is a figure which shows the result of having mass-analyzed Fe3 ⁺ with KMH-1 developed previously for the comparison. In the Example of this invention, it is a figure which shows the result of having conducted mass spectrometry of Fe3 ⁺ . In the Example of this invention, it is a figure which shows the result of having conducted mass spectrometry of Hg < ²⁺ >. In the Example of this invention, it is a figure which shows the result of having mass-analyzed Zn2 ⁺ . In the Example of this invention, it is a figure which shows the result of having conducted mass spectrometry of Ni2 ⁺ . In the Example of this invention, it is a figure which shows the result of having conducted mass spectrometry of Cu2 ⁺ . In the Example of this invention, it is a figure which shows the result of having carried out mass spectrometry of Cd < ²⁺ >. In the Example of this invention, it is a figure which shows the result of having conducted mass spectrometry of Pb2 ⁺ . It is a figure which shows the relationship between pH and relative intensity | strength at the time of measuring Hg < ²⁺ > in the Example of this invention. It is a figure which shows the relationship between the Hg2 ⁺ density ^| concentration at the time of titrating Hg2 ⁺ in the Example of this invention, and the relative intensity of a peak. It is a diagram showing a relationship between Cd ²⁺ concentration and the relative intensities of the peaks at the time of titrating Cd ²⁺ in the embodiment of the present invention. Is a diagram showing the relationship between Pb ²⁺ concentration and the relative intensities of the peaks at the time of titrating Pb ²⁺ in the embodiment of the present invention. In the Example of this invention, it is a figure which shows the relationship between the Pb2 ⁺ density ^| concentration at the time of titrating Pb2 ⁺ without KI addition, and the relative intensity | strength of a peak. It is a figure which shows the isotope peak (left) and theoretical pattern (right) of mercury by microTOF. It is a figure which shows typically the state at the time of the liquid suction of the preferable specific example of the mass spectrometry system for heavy metal ion analysis of this invention. It is a figure which shows typically the state at the time of the liquid extrusion of the preferable specific example of the mass spectrometry system for heavy metal ion analysis of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Reagent container 12 Sample container 12 'Sample container 14 Suction means 16 Suction pipe 18 1st valve 20 Liquid extrusion pipe | tube 22 Liquid feed pump 24 Liquid feed pipe 26 Liquid mixing means 28 2nd valve 30 3rd valve 32 3rd 2 Liquid extrusion tube

Claims (17)

The following general formula [I]

(In the formula, R ¹ and R ² are independently an ionic functional group that becomes a monovalent negative ion in solution, R ³ and R ⁴ are each independently a hydrogen atom, and a straight chain having 1 to 10 carbon atoms. Type or branched alkyl group, amino group, nitro group or halogen, A ¹ and A ² may or may not be present independently of each other and, if present, represent any spacer moiety ),
The following general formula [II]

(However, in the formula, R ¹ and R ² are ionic functional groups that become monovalent negative ions in solution independently of each other, and A ¹ and A ² may or may not exist independently of each other. Often represents any spacer moiety if present),
Or the following formula [III]

(However, the carbon atoms constituting the ring have one or less substituents represented by -A ¹ -R ¹ (the definitions of A ¹ and R ¹ are the same as those in the general formula [I]) for each ethylene chain. May be combined)
A mass spectrometric reagent for heavy metal ion measurement having a structure represented by:   The mass spectrometric reagent for measuring heavy metal ions according to claim 1, which has a structure represented by the general formula [I] or [II]. The reagent according to claim 2, wherein R ¹ and R ^{2 in the} general formulas [I] and [II] are each independently a carboxyl group or a salt thereof, a sulfone group or a salt thereof, or a hydroxyl group or a salt thereof. A ¹ and A ² are not independently of each other, or are a linear or branched alkylene group having 1 to 10 carbon atoms, and a part of the alkylene group is an amino group, a halogen, an ether and / or a carbonyl group. The reagent according to any one of claims 2 to 4, which is a substituted phenylene group in which a substituted alkylene group, a phenylene group, or a part of the phenylene group is substituted with an amino group, a halogen and / or a nitro group. The reagent according to claim 4, wherein A ¹ and A ² are each independently an alkylene group. The reagent according to claim 5, which is a carboxylic acid having a structure represented by the following formula [IV] or [V] or a salt thereof.

  The reagent according to any one of claims 2 to 6, wherein the heavy metal ion is a trivalent cation. The reagent according to claim 7, wherein the trivalent cation is Fe ³⁺ and / or Cr ³⁺ .   The reagent of Claim 1 which has a structure represented by the said general formula [III].   The reagent according to claim 9, wherein there is no substituent on the carbon atom constituting the ring.   The reagent according to claim 10, wherein the heavy metal ion is a divalent cation. The reagent according to claim 11, wherein the divalent cation is at least one selected from the group consisting of Hg ²⁺ , Cd ²⁺ and Pb ²⁺ .   A method for mass spectrometry of heavy metal ions, comprising reacting the reagent according to any one of claims 1 to 12 with heavy metal ions in a test sample and mass-analyzing the product.   The method according to claim 12, comprising reacting the reagent according to any one of claims 10 to 11 with heavy metal ions in a test sample in the presence of iodine ions, and mass-analyzing the product.   A reagent container containing the reagent according to any one of claims 1 to 12, a sample container containing a test sample, a reagent contained in the reagent container, and a test sample contained in the sample container A suction means for aspirating the reagent, a suction tube connected to the suction means for sucking the reagent contained in the reagent container and the test sample contained in the sample container via the tip, and the suction A first valve provided in the middle of the pipe, a liquid feed pump connected to the first valve via a liquid extrusion pipe, and a liquid connected to the first valve via a liquid feed pipe A mass spectrometry system for heavy metal ion analysis, comprising: a mixing means; and a mass spectrometer connected downstream of the liquid mixing means.   A second valve is provided in the middle of the liquid extruding pipe and between the liquid feed pump and the first valve, and in the middle of the suction pipe, the suction means and the first valve The mass spectrometry system according to claim 15, wherein a third valve is provided between the second valve and the third valve, and the second valve and the third valve are connected by a second liquid extrusion tube. A mass spectrometry system using the heavy metal mass spectrometry method according to claim 13 or 14.

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