WO2001098766A1 - Method for determining concentration of xanthin type compound and sensor for use therein - Google Patents

Abstract

A method for determining the concentration of a xanthin type compound represented by the formula (I), wherein R?1, R2 and R3¿ independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, which comprises providing a diamond electrode comprising electrically conductive diamond and a counter electrode, contacting the diamond electrode and the counter electrode with a sample to be tested, applying a voltage necessary for effecting an oxidation reaction on the diamond electrode between the diamond electrode and the counter electrode, measuring a current value under the voltage, calculating the concentration of the compound represented by the formula (I) in the sample to be tested from the resultant current value using a calibration curve prepared in advance. The method utilizes a property of a xanthin type compound represented by the formula (I) that it is specifically oxidized electrochemically at a diamond electrode. The method can be used for determining the concentration of a xanthin type compound such as caffeine, theophylline, theobromine and xanthin specifically in a short time and with ease.

Description

 Description Method for measuring the concentration of xanthine compounds and sensors used for the method

[Background of the Invention]

 Field of the invention

 The present invention relates to a method for measuring the concentration of xanthine-based compounds such as cuff-in, theophylline, theopromine, and xanthine, which are clinically or foodally important, using a diamond electrode, a sensor used therefor, and an apparatus therefor. About. Background art

Diamond is inherently the resistivity is an insulating material of about ¹ 0 1 ³ Ω cm, obtaining conductivity by doping with trace impurities thereof. This conductive diamond is expected to have various uses. One of them is utilization as an electrode for electrochemical use. When conductive diamond is viewed as an electrode for electrochemical use, it has the excellent features of having a wide potential window and extremely low background current. In addition, it is physically and chemically stable and has excellent durability. Electrodes having a conductive diamond (preferably a thin film thereof) have been commonly referred to as diamond electrodes.

 Pioneering research on diamond electrodes was performed by Iwaki et al. (Iwaki et al., Nuclear Instruments and Methods, 209-210, 1129 (1983)). They studied the properties of single-crystal diamond with surface conductivity imparted by implanting argon-nitrogen ions as an electrically conductive material. At the same time, a cyclic voltammogram in the electrolyte solution was also shown. Subsequently, the properties of polycrystalline diamond synthesized by gas phase using hot filaments have been reported (Pleskov et al., J. Electroanal. Chem., 228, 19 (1993)).

Some of the present inventors have previously reported the reduction of nitrogen oxides using a diamond electrode synthesized in a vapor phase (Tenne et al., J. Electroanal. Che., 347, 409 (1993)). In this study, a p-type semiconductor diamond with boron introduced as a dopant was used as the electrode. Then, boron is used as a diamond electrode. —Use of p-type semiconductors or highly conductive metal-like conductive diamonds as punts will become mainstream. In the 1990s, research on diamond electrodes was conducted by several groups, and from 1995 onwards, plasma CVD (PC Research on diamond electrodes obtained using the VD) instrument has also been found in the electrochemical field.

'On the other hand, various xanthine-based compounds having a xanthine structure play various physiological functions in animals and plants.

 For example, xanthine (3,7-dihydro-1H-purine-1,2,6-dione) belongs to purines and plays an important role as an intermediate in the degradation of adenine and guanine into uric acid. Is considered extremely important. In addition, xanthine is poorly soluble in water and xanthine, although very rare, can precipitate as aggregates due to metabolic abnormalities.

 Theophylline (3,7-dihydro-1,3, dimethyl-1H-purine-1,2,6-dione), Theopromine (3,7-dihydro-3,7-dimethyl-1H-purine-12,6-dione) And N-methyl derivatives of xanthine such as caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-1,2,6-dione) are widely distributed in plant products and beverages. Alkaloids are known to have many physiological effects such as gastric acid secretion, diuresis, and central nervous system stimulation. In particular, theophylline is useful as a bronchodilator and as a therapeutic agent for bronchial asthma.

 Therefore, if the abundance of these xanthine compounds in a living body or in a sample derived from a living body or in a food can be easily known, it is extremely significant in medical pharmacy or food production.

 [Summary of the Invention]

 The present inventors have now found that a diamond electrode is specifically sensitive to a xanthine-based compound, and that the abundance can be quantified from the current value at the oxidation potential.The present invention is based on this finding. is there.

Accordingly, an object of the present invention is to provide a method capable of easily and specifically measuring the concentration of a xanthine compound in a short time. Another object of the present invention is to provide a sensor used in the above method and a device therefor.

 According to a first aspect of the present invention, there is provided a method for measuring the concentration of a compound represented by the following formula (I) in a test sample, the method comprising:

(Wherein, RR ² and R ³ independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)

 A diamond electrode having conductive diamond and a counter electrode are prepared, and the diamond electrode and the counter electrode are brought into contact with a test sample,

 Applying a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode; measuring a current value under the voltage; and Calculating the concentration of the compound represented by the formula (I)

.

 According to another aspect of the present invention, there is provided a sensor for use in the above-mentioned measuring method, wherein the sensor comprises a diamond electrode having conductive diamond.

 Further, according to another aspect of the present invention, there is provided an apparatus for performing the above-mentioned measuring method, the apparatus comprising:

 A diamond electrode having a conductive diamond,

 A counter electrode,

 Means for bringing the diamond electrode and the counter electrode into contact with a test sample,

Means for applying a voltage that causes an oxidation reaction on the diamond electrode between the diamond electrode and the counter electrode; Means for measuring a current value under the applied voltage;

 Means for calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value;

At least.

 According to another embodiment of the present invention, a compound represented by the following formula (I) in a test sample:

(Wherein: ET, R ² and R ³ independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)

Using a diamond electrode with conductive diamond to measure the concentration of

[Brief description of drawings]

 FIG. 1 is a view showing the structure of a diamond electrode. FIG. 1 (a) is a cross-sectional view of a diamond electrode 1. This electrode comprises a conductive diamond thin film 3 formed on a substrate 2. (B) is a perspective view of the diamond electrode 1, comprising a conductive diamond thin film 3 formed on a substrate 2.

 FIG. 2 is a diagram showing a basic structure of a flow cell used in the measurement method according to the present invention.o

 FIG. 3 is a diagram showing a basic configuration of a measuring device according to the present invention.

FIG. 4 is a diagram showing the results of anodic voltammograms for a 5 OM theophylline solution at a sweep rate of 2 O mV s ¹ under the conditions of ρΗ1, 2, 3, 4, and 5.

Figure 5 is a calibration curve of caffeine concentration and peak current value. FIG. 6 is a calibration curve of theophylline concentration and the value of the peak current.

[Specific description of the invention]

 Test compound

 According to the method of the present invention, the concentration of the compound of the formula (I) in the sample solution can be selectively known. The present inventors have, as described above, that the compound of the formula (I) is specifically oxidized electrochemically on the conductive diamond electrode, and further, the conductive diamond is used as a working electrode, It was also confirmed that the current value generated between the electrode and the counter electrode was directly proportional to the concentration of the compound of the formula (I) in the system. As a result, it has become possible to quantitatively detect the compound of the formula (I) in the test sample.

 The test sample may be a biological blood, a body fluid or the like which is thought to contain the compound of the formula (I), or a food or a diluted solution or suspension of the food.

In the formula (I), the alkyl group having 1 to 6 carbon atoms represented by II ¹ , R ² and R ³ may be linear or branched, and may have a substituent. Is also good. In the present invention, the compounds of the formula (I) which are considered particularly biochemically important are as follows.

R ¹ R ^{2 3}

 Xanthine H H H

 Theophylline methyl methyl H

Theopromin H methyl methyl caffeine methyl methyl methyl xanthine is excreted in large quantities in the urine, for example in patients with xanthineuria. Precipitation of xanthine crystals can lead to so-called bladder stones. Therefore, the measuring method according to the present invention, which enables the urinary concentration of xanthine to be measured in a short time and simply, is useful for its diagnosis and treatment. In addition, theophylline is used clinically as a bronchodilator, a cardiotonic diuretic and the like, and it is useful to know its blood concentration and the like. Furthermore, theopromin has a cardiotonic effect and a utilization effect similar to theophylline, and it is significant that its blood concentration and the like can be known. Furthermore, caffeine is included in many foods besides pharmaceutical use, In terms of nutrition, it is significant to be able to easily know the concentration.

 Diamond electrode and its production

Diamond is inherently a good insulator. However, by adding Group 3 or Group 5 impurities, it becomes semiconductor-like or metal-like conductive. In the present invention, diamond having a semiconductor-metal-like conductive property is used as an electrode. Examples of the substance added to impart such a conductive property include elements of Groups 3 and 5 as described above, more preferably, boron, nitrogen, and phosphorus, and most preferably, boron or nitrogen. There. Amount of substance to be added to impart the conductivity may be appropriately determined within the range that can impart conductivity to the diamond, but give example ^{1 X 10_ 2 ~ 10- 6 Ω} cm approximately conductive It is preferable to add in an amount. In general, the amount of the substance added to impart the conductivity is controlled by the amount added in the process of producing the conductive diamond. The diamond electrode according to the present invention uses the conductive diamond as an electrode. This diamond electrode is extremely interesting because when the electrochemical reaction is carried out in water, oxygen is not generated by the electrolysis of water as an oxidation reaction, and specifically causes the oxidation reaction of the compound of the above formula (I). Had properties. This property is considered to be exhibited regardless of the surface condition of the conductive diamond. In other words, the surface of an unprocessed (as grown) diamond thin film manufactured using hydrogen gas as a carrier by the chemical vapor deposition method described later has a hydrogen atom as a terminal carbon atom. Is usually in a bonded state, but the conductive diamond in which the terminating hydrogen atom is replaced with another atom can also specifically cause the oxidation reaction of the compound of the above formula (I). Is considered to be possible.

Although the conductive diamond itself can be used as an electrode instead of supporting the base material, according to a preferred embodiment of the present invention, a conductive diamond thin film is formed on the base material, It is preferable to connect a conductive wire to form an electrode. As the substrate, S i (e.g., single crystal silicon), Mo, WヽNb, T is Fe, Au, Ni, Co, Ah0 3, S iC ;, S i 3 N4 Zr0 2, MgO, graphite, single Crystal diamond, cBNs quartz glass, etc., especially single crystal silicon, Mo, W, Nb, T i S The use of iC single crystal diamond is preferred.

 The electrode of this embodiment will be further described with reference to FIG. FIG. 1 (a) is a cross-sectional view of a diamond electrode 1, which comprises a conductive diamond thin film 3 formed on a base material 2, and furthermore, a conductive wire 5 Are connected, for example, via gold coating 4. FIG. 1 (b) is a perspective view of the diamond electrode 1, which is composed of a conductive diamond thin film 3 formed on a base material 2, and further has a metal core for using the conductive diamond thin film 3 as an electrode. Conductor 5 is connected via one ting 4.

 The thickness of the conductive diamond thin film is not particularly limited, but is preferably about 1 to 10θΛ6πι, more preferably about 5 to 50 m.

 Further, according to a preferred embodiment of the present invention, the diamond electrode according to the present invention can take the form of a micro-hole electrode. The concept of the microphone opening electrode is already known, and in the present invention, the microelectrode-shaped diamond electrode is obtained by cutting the end of a fine wire such as Pt sharply, further sharpening the end by electrolytic polishing, and then polishing the end surface. Means a structure in which a conductive diamond thin film is formed.

 According to a preferred embodiment of the present invention, the conductive diamond thin film is preferably produced by a chemical vapor deposition method. Chemical vapor deposition is a method in which a gaseous raw material is chemically reacted in a gas phase to synthesize a substance, and is generally called a CVD (Chemical Vapor Deposition) method. This method is widely used in the semiconductor manufacturing process, and can be used for the production of the conductive diamond thin film of the present invention with appropriate modification.

 Chemical vapor synthesis of diamond is performed by using a mixture of hydrogen and a carbon-containing gas such as methane as a raw material gas, exciting it with an excitation source, supplying it to a substrate, and depositing it.

 Excitation sources include hot filament, microwave, high frequency, DC glow discharge, DC arc discharge, and combustion flame. It is also possible to adjust the nucleation density by combining a plurality of them, and to increase the area and uniformity.

As a raw material, many types that contain a carbon, decomposition by the excitation source is excited, C, activated carbon, such as C _2, and CH, such as _{CH 2, CH 3, C 2} H 2 Charcoal Compounds that generate hydride radicals are available. Preferred examples include CH ₄ , C ₂ H ₂ C ₂ H ₄ , CioH ₁₆ s CO, CF ₄ as a gas, CH ₃ OH, C ₂ H ₅ OH, (CH ₃ ) ₂ CO as a liquid, graphite, fullerene, etc. Are mentioned.

In the gas phase synthesis method, the addition of a substance that imparts conductivity to diamond can be performed, for example, by placing a disk of the added substance in the system, exciting it in the same manner as the carbon source material, and introducing the added substance into the carbon gas phase, The method can be carried out by, for example, adding an additional substance to the carbon source in advance, introducing the additional substance into the system together with the carbon source, exciting with the excitation source, and introducing the additional substance into the carbon gas phase. According to a preferred embodiment of the present invention, the latter method is preferred. Especially, when using acetone, a liquid such as methanol as a carbon source, a method of dissolving the boron oxide (B ₂ 0 ₃₎ and boron source thereto, it is easy to control the concentration of boron, which is either One convenient This is preferred. For example, in the case of adding boron to a carbon source in a gas phase synthesis method, the amount is generally about 10 to 12,000 ppm, and preferably about 1,000 to 10,000 ppm.

 According to a preferred embodiment of the present invention, the production of the conductive diamond thin film is preferably performed by a plasma chemical vapor deposition method. This plasma-enhanced chemical vapor synthesis has the advantage that the activation energy for causing a chemical reaction is large and the reaction is fast. Furthermore, according to this method, it is possible to generate a chemical species that does not exist at a high temperature thermodynamically, and to perform a reaction at a low temperature. Several reports have already been made on the production of conductive diamond thin films by the plasma chemical vapor deposition method, including some of the present inventors (for example, Yano et al., J. Electrochem. Soc, 145 (1998)). 1870), preferably according to the method described in these reports.

 Measuring method and device therefor

In the present invention, the conductive diamond oxidizes the compound of the above formula (I) electrochemically and specifically, the conductive diamond is used as a working electrode, and the current value generated between the conductive diamond and the counter electrode in the system is Utilizing the property of being directly proportional to the concentration of the compound of the formula (I), the compound of the formula (I) in the test sample is quantitatively detected. Since the measurement is carried out by knowing the current value, the measuring time is short and simple, and the method according to the present invention is extremely advantageous in that the concentration of the compound of the formula (I) can be easily known in a short time. It is advantageous. Also, diamond electrodes have the potential to oxidize compounds other than the compound of formula (I), but are very specifically sensitive to compounds of formula (I). As far as the present inventors know, substances other than the compound of the formula (I) are present in a sample derived from a living body or a food in which the above-mentioned caffeine, theophylline, theopromine, and xanthine are expected to be present. Does not react with them, and the diamond electrode oxidizes only the compound of formula (I). Thus, the assay according to the invention makes it possible to specifically measure only compounds of the formula (I). When a mixture of the compound of the formula (I) or a substance capable of reacting with the diamond electrode is present in the test sample or is likely to be present, a pretreatment such as liquid chromatography is carried out, and the formula (I) It is needless to say that the compound of the formula (1) is preferably distinguished from other substances having a responsiveness to the diamond electrode and subjected to the measuring method according to the present invention.

 Further, since the oxidized voltage is different for each compound, that is, the peak voltage described later is different, it is possible to distinguish a plurality of compounds of the formula (I) by controlling the applied voltage.

 Further, according to the measuring method of the present invention, the compound of the formula (I) can be measured in a wide concentration range from about 1 / mo 1 to a saturated concentration.

 In the method for measuring the concentration of the compound of the formula (I) according to the present invention, the electrochemical system for quantifying the compound of the formula (I) is a general electrochemical system except that a conductive diamond is used as a working electrode. System.

That is, in the method for measuring the concentration of the compound of the formula (I) according to the present invention, a conductive diamond is used as a working electrode, brought into contact with a test sample together with a counter electrode, and placed between the two electrodes on a diamond electrode. Apply the voltage at which the oxidation reaction occurs, and measure the current value under this voltage. Then, the concentration of the compound represented by the formula (I) in the test sample is calculated from the obtained current value. As described above, the current value generated between the conductive diamond and the counter electrode by the oxidation of the compound of the formula (I) is directly proportional to the concentration of the compound of the formula (I) in the system. Therefore, once the relationship between the current value at a certain voltage value and the concentration of the compound of the formula (I) is determined, the relationship of the compound of the formula (I) in the test sample corresponding to the obtained current value is obtained from the relationship. The concentration can be easily known. That is, in a preferred embodiment of the present invention, the concentration of the compound represented by the formula (I) is By preparing a calibration curve with the current value in advance, and comparing this calibration curve with the obtained current value, the concentration of the compound of the formula (I) can be known.

 In the present invention, as the counter electrode, platinum, carbon, stainless steel, gold, diamond,

S n 0 ₂ use of the like are preferable.

 In the present invention, the voltage applied between the working electrode, the diamond electrode, and the counter electrode is not limited as long as an oxidation reaction of the compound of the formula (I) occurs on the diamond electrode. From the viewpoint of accuracy, the applied voltage is preferably a voltage that gives a peak current of oxidation of the compound of the formula (I). Here, the peak current can be determined as a voltage giving a maximum current value by, for example, cyclic voltage measurement. Further, according to another preferred embodiment of the present invention, the voltage giving the maximum current value can be determined by a rotating electrode method or a microelectrode method. The rotating electrode method or the microelectrode method is advantageous in that the possibility of measurement error due to measurement conditions and the like can be further eliminated.

 Further, according to a preferred embodiment of the present invention, the reference electrode is brought into contact with the test sample, and the absolute value of a voltage at which an oxidation reaction occurs on the diamond electrode is controlled between the diamond electrode and the counter electrode. It is preferable from the viewpoint of measurement accuracy. As the reference electrode, a known electrode can be used, and a standard hydrogen electrode, a silver / silver chloride electrode, a mercury / mercury chloride electrode, a hydrogen palladium electrode and the like can be used.

 In the measuring method according to the present invention, the electrochemical system can be a general electrochemical system except that conductive diamond is used as a working electrode. According to a preferred embodiment of the present invention, A flow injection method using a flow cell is preferable, in which a predetermined solution is caused to flow through the system as a carrier at a constant flow rate, and a test sample is injected into the carrier solution for measurement.

 The outline of the flow injection method using a flow cell is as shown in Fig. 2 (a). The carrier solution is injected into the flow cell 21 from the carrier solution reservoir 6 by the pump 7. A test sample inlet 8 is provided between the pump 7 and the flow cell 21 so that the test sample can be injected into the carrier solution. The carrier solution that has passed through the flow cell 21 is collected in the waste liquid reservoir 9.

The basic structure of the flow cell 21 is as shown in FIG. 2 (b). Flow cell Reference numeral 21 denotes a structure in which the diamond electrode 1, the counter electrode 22 and the reference electrode 23 are exposed in the flow channel 24 through which the carrier solution and the test sample flow, and can be brought into contact with the test sample. The diamond electrode 21 basically has the structure shown in FIG. 1. The diamond thin film 3 in FIG. 1 is exposed in the flow channel 24 and comes into contact with the carrier solution and the test sample. The carrier solution enters from the inlet 25 of the flow channel 24, flows as indicated by the arrow in the figure, and reaches the outlet 26. Between the conductive wire A of the diamond electrode 1 and the conductive wire B of the counter electrode 22, a voltage for causing the compound of the formula (I) to oxidize on the diamond electrode 1, preferably a voltage for generating a peak current, is applied.

 The measurement is performed as follows. First, only the carrier solution into which the test sample is not injected flows, so that the so-called background current is minimized and stabilized. Diamond electrodes are characterized by a low background current. Next, the test sample is injected from the test sample inlet 8. This injection may be performed continuously, or an amount that can measure the beak current may be injected at a time. When the compound of the formula (I) is contained in the test sample, the compound of the formula (I) is oxidized on the diamond electrode 1, and the current value accompanying the oxidation reaction can be measured. From this current value, the concentration of the compound of formula (I) is known.

 In the measurement method according to the present invention, it is preferable that the pH of the test sample is fixed to an arbitrary specific value before the measurement. For some of the compounds of formula (I), the pH that gives the peak current does not have a large pH dependence, but the ranges of the pH of the test object and the preferred test sample are as follows.

 Preferred range More preferred range

 Xanthine

 Theophylline 1-1-1 2 1-5

 Theopromin 1-5.3 1-3 1-3

 Caffeine 1-5. 3 1-3

 According to another preferred embodiment of the present invention, it is preferable that the diamond electrode 1 in the flow cell 21 is in the form of a microelectrode. According to another preferred embodiment of the present invention, the diamond electrode 1 may be a rotating electrode.

As described above, according to another aspect of the present invention, there is provided a method for measuring the concentration of a compound of the formula (I). However, according to another aspect of the present invention, there is provided a sensor for measuring the concentration of the compound of formula (I), comprising a diamond electrode having conductive diamond. Further, according to another aspect of the present invention, there is provided an apparatus for measuring the concentration of a compound of the formula (I) in a test sample. The basic configuration of this device is as shown in FIG. In the apparatus shown in FIG. 3, the power supply 電流 ammeter 31 is connected to the conductor A connected to the diamond electrode 1, the conductor B connected to the counter electrode 22, and the reference electrode 23 from the flow cell shown in FIG. Conductor C is connected. The power supply ammeter 31 serves as a means for applying a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode, and a means for measuring a current value under the applied voltage. is there. That is, a voltage that causes the compound of formula (I) to oxidize the compound of formula (I) on the diamond electrode 1, preferably a voltage that causes a peak current, is applied between the diamond electrode 1 and the counter electrode 22 by the power source and the ammeter 31. Further, the current value accompanying the oxidation reaction of the compound of the formula (I) on the diamond electrode 1 is measured. This current value is sent to the utility pole value comparison / concentration calculation device 32. The calibration curve data 33 is sent to this device 32. In this device, the current value sent from the power supply ammeter 31 is compared with the calibration curve data, and the compound of the formula (I) is compared. Calculate the concentration. That is, a means for calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value is provided. The obtained concentration of the compound of the formula (I) is displayed on the display device.

[Example]

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

 Preparation of diamond electrode

 Diamond thin films were prepared by a micro-mouth-wave plasma-assisted CVD method using a microwave CVD film forming apparatus manufactured by ASTeX. Specifically,

.

First, the surface of a silicon substrate (S i (100)) was polished with 0.5 μm diamond powder, and then set on a substrate holder of a CVD film forming apparatus. A mixture of acetone and methanol (mixing ratio 9: 1 (volume ratio)) was used as a carbon source. In addition, Boron in a mixture of oxide (B ₂ 0 _3), and the amount dissolve the 10 ⁴ ppm boron / carbon ratio. Pure hydrogen gas was passed through the mixture of carbon sources to replace dissolved gases. On the other hand, pure hydrogen gas was flowed through the chamber at a flow rate of 532 cc / min, and the pressure was set to 115 xtorr (115 x 133.322 Pa). Next, a 2.45 GHz microphone mouth wave was injected into the chamber and discharged, and the output was adjusted to 5 KW. After confirming that the equipment was stable, pure hydrogen gas as a carrier gas was flowed through the carbon source mixture at a flow rate of 15 cc / min, and introduced into the chamber to form a film. The deposition rate was 1 to 4 zm / hour. Film formation was performed until a thickness of about 3 was obtained. Although the substrate was not heated, it was observed that the temperature was about 850-950 ° C in the steady state.

When a Raman spectrum of the obtained diamond thin film was taken, only a single peak was observed at 1333 cm- ¹ . Further, the electric conductivity is about 10- ³ Ω cm, 0. 5M result of measuring the cyclic Bol evening thermogram in sulfuric acid, -1. 25 + 2. and 3 V (vs. SHE), wide potential It was confirmed that it had a window.

 Voltammetry measurement

Volumetric measurements were performed in a glass cell at room temperature using a Hokuto Denko HA-502 potentiometer, a Hokuto Denko HB-U function generator, and a Riken Denshi xy recorder. A diamond electrode having a diamond thin film obtained as described above was used as a working electrode (exposed area: 0.07 cm ² ), and a platinum foil was used as a counter electrode. A saturated calomel electrode (SCE) was used as a reference electrode.

 Experimental Example 1: Relationship between peak current value and concentration

 (a) Xanthine, theophylline (primary grade), theopromine, and caffeine (special grade) were obtained from Wako, and 1-400 zmo 1 using Milli-Q water (Millipore).

An aqueous solution was prepared in a concentration range of / 1. Incidentally, using air saturated Brrtton-Robinson buffer containing 0. lMNaC10 ₄ a (0. 04M) as the electrolyte, pH was measured using a conventional glass electrode.

(b) the pH of all of the aqueous solution and 1.8, the potential sweep rate performs por evening Nmetori one measurement of 2 OMVS ^1, was determined value of the peak current. Furthermore, the obtained peak power Investigation of the relationship between the stream value and the concentration yielded a linear relationship through the origin. Calibration curve obtained by the least-squares method: Concentration I = X Compound concentration M is as follows.

 Concentration (〃M) (j A / juM)

 Xanthine 1-1 00 00 04 6 0.9 9 9 9 7 Theophylline 1- 400 0.04 43 0.9 9 98 0 Theopromine 1-4 00 00 04 5 0 9 9 8 9 Caffeine 1-1 400 0.042 0.9 9 9 8 8 8 6 9 9 8 6 For xanthine, when the concentration exceeded about 150〃mo1 / 1, the peak current value lost its concentration dependence. This may be due to poor solubility of xanthine in the buffer.

 (c) Measurement was performed five times for solutions of several concentrations, and the reproducibility of the peak current value was confirmed as the deviation (%) of the beak current value.

 The results were as follows. Concentration ("M") Average peak current (A) Xanthine 2 0 1 0 3 5

 14 0 6 5 0 7 Theophylline 3 0 1 5 1 7

 1 0 0 4 6 0 6 2 00 8 48 0 3 Theopromine 3 0 1 6 1 2

 1 0 0 49 0 7 1 00 4.6 2 0 6 Cuffin 5 0.2 1 2 2

 2 2 0.97 0 7 1 5 0 6.29 0 3 Experimental example 2: Relationship between beak current value and pH

(a) For 50 zM theophylline solution, ρΗ 1, 2, 3, 4, and 5 Under conditions, it was recorded anode Bol evening Mogurafu in the 2 OMVS ¹ sweep rate. The results were as shown in Figure 4. From this result, it was observed that as the pH increased, the peak voltage for theophylline caused a negative shift.

 (b) Xanthine, theophylline, theopromine, and caffeine, each of which was prepared in a 50〃Μ solution, and the relationship between peak voltage and pH was examined. The results were as follows.

 ― _ _ _ΡΗ _Beak voltage _

 Xanthine 1.8-8.0 1.26-0.063ρΗ 0.9788

 8.0-12.0 0.95-0.043ρΗ 0.9643 Theophylline 1.8-9.0 1.41-0.059ρΗ 0.9782

 9.0-12.0 1.32-0.040ρΗ 0.9772 Tebromine 1.8-5.3 1.56-0.046ρΗ 0.9949 Caf 1.8in 1.8-5.3 1.51-0.023ρΗ 0.9909 A reproducible beak was observed over 12.0), but in the case of theopromine and caffeine, the oxidation peak tended to be less distinct at a pH value of about 5.5. Experimental example 3: Caffeine determination

 (a) Creating a calibration curve

The 0. I MHC 10 ₄ of pH l with a solvent, to prepare a solution of various concentrations of caffeine. For this solution, a voltammetric measurement was performed at a potential sweep rate of 2 OmVs- ¹ to obtain a relationship between the peak current value and the caffeine concentration, and a calibration curve was prepared. The calibration curves obtained were as shown in FIGS. 5 (a) and (b).

(b) Quantification of caffeine

Commercial cola beverage was 0. I MHC 10 ₄ of pH 1 and the solvent was diluted 125-fold. The peak current value of this diluted solution was determined by the flow injection method using the flow cell shown in FIG. The applied voltage was 1.5 V at the silver chloride electrode. The obtained peak current value was 0.5 l ^ A. From the above calibration curve, The caffeine concentration in the 125-fold diluted solution was calculated to be 8. lmmo 1Z1. The caffeine concentration in a commercial drink was determined to be 194.2 lmg / 1. Example 4: Determination of theophylline

 (a) Preparation of calibration curve

 In the same manner as in Example 3 (a), a calibration curve was created for theophylline by obtaining the relationship between the peak current value and the caffeine concentration. The obtained calibration curve was as shown in FIG.

 (b) Quantification of theophylline

The 0. I MHC 10 ₄ 31 Commercial theophylline 100111 tablets as a solvent, is first dissolved in the 5 OML, and further dissolve the solution 0. 04Ml in the solvent 2 OML. The peak current value was determined in the same manner as in Example 3 (b) except that the applied voltage was set to 1.4 V for this solution. The peak current value was 170 OnA, and the theophylline concentration in the solution to be measured was calculated to be 19.96 mmol / l from the above calibration curve. From this concentration, the amount of theophylline in the theophylline 10 Omg tablet was calculated to be 99.8 mg.

Claims

Scope of request 1. A method for measuring the concentration of a compound represented by the following formula (I) in a test sample,

(Wherein, RR ² and R ³ independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)

 A diamond electrode having a conductive diamond and a counter electrode are prepared, and the diamond electrode and the counter electrode are brought into contact with a test sample,

 Applying a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode; measuring a current value under the voltage;

 Calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value.

Comprising the method.

 2. The method according to claim 1, wherein the diamond electrode has a diamond thin film made conductive by mixing a Group 3 or 5 element.

 3. The method according to claim 1, wherein the diamond electrode has a diamond thin film that has been made conductive by the incorporation of one or more elements selected from the group consisting of boron, nitrogen, and phosphorus.

 4. The compound according to any one of claims 1 to 3, wherein the compound represented by the formula (I) is at least one selected from the group consisting of caffeine, theophylline, theopamine, and xanthine. The method described in.

5. The step of calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value includes the step of preparing the concentration of the compound represented by the formula (I) and the current value prepared in advance. And comparing the obtained current value with a calibration curve of The method according to any one of claims 1 to 4.

 6. The method according to any one of claims 1 to 5, wherein when the measurement object is theopromin or xanthine, the measurement is performed with the pH of the test sample being 5.5 or less.

 7. The method according to any one of claims 1 to 6, wherein a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode is a voltage that gives a peak current.

 8. The method further comprising: bringing a reference electrode into contact with a test sample; and controlling the absolute value of a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode. A method according to any one of claims 1 to 7.

 9. A sensor for measuring the concentration of a compound of formula (I) as defined in claim 1, comprising a diamond electrode having conductive diamond.

 10. The sensor according to claim 1, wherein the diamond electrode has a diamond thin film made conductive by the incorporation of boron.

 11. The sensor according to claim 9, wherein the compound represented by the formula (I) is at least one selected from the group consisting of caffeine, theophylline, theobromine, and xanthine.

 12. An apparatus for measuring the concentration of a compound of formula (I) as defined in claim 1 in a test sample,

 A diamond electrode having conductive diamond,

 A counter electrode,

 Means for bringing the diamond electrode and the counter electrode into contact with a test sample; means for applying a voltage causing an oxidation reaction on the diamond electrode between the diamond electrode and the counter electrode;

 Means for measuring a current value under the applied voltage;

 Means for calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value;

An apparatus comprising at least:

 1 3. It further comprises a reference electrode,

Means for bringing the reference electrode into contact with a test sample; Means for controlling an absolute value of a voltage at which an oxidation reaction occurs on the diamond electrode, between the diamond electrode and the counter electrode;

13. The device according to claim 12, comprising:

 14 4. The compound represented by the following formula (I) in the test sample:

(Wherein RR ² and independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)

For measuring the concentration, use of a diamond electrode having conductive diamond _c