WO2016036334A1

WO2016036334A1 - Voltametric methods to determine alpha-amanitin and phalloidin

Info

Publication number: WO2016036334A1
Application number: PCT/TR2015/050073
Authority: WO
Inventors: Yücel ŞAHİN; Mustafa YAMAÇ; Ayşe Betül KARADUMAN; Hakan GÖRÇAY
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-09-03
Filing date: 2015-08-20
Publication date: 2016-03-10
Anticipated expiration: 2017-03-03

Abstract

The method of determining α-amanitin and phalloidine toxins individually or together in samples such as mushroom or body fluids (blood, urine, gastric juice etc.) with the use of operation steps of examining electrochemical behaviours of α-amanitin and phalloidine toxins and optimization of the electrochemical determination conditions.

Description

VOLTAMETRIC METHODS TO DETERMINE ALPHA-AMANITIN AND

PHALLOIDIN

The Related Art

The invention relates to a method used for determining a-amanitin and phalloidine toxins.

The Prior Art

Nowadays, only 200 to 300 of the known more than 20,000 macrofungus species can be safely consumed by people. 50 to 100 of these have toxic effect on people. Toxins found in toxic fungi are categorized into two groups as early-acting and late-acting toxins according to the time they present their toxic effects. Late-acting macrofungus toxins have higher toxicity than the early-acting ones. Therefore, the toxic effect of late-acting macrofungus toxins on humans may lead to dangerous results. As a result, the ability of determining the presence of late-acting toxins in human body and/or the amount thereof in a quick and practical manner is a significant necessity for saving human life.

Most of the deaths caused by mushroom poisoning are due to the members of the group containing cyclopeptide toxins. This group of toxins is mostly common in macrofungus species of Amanita, Galerine, and Lepioia genus. When macrofungus-originated poisoning cases are examined, the most toxic and the most fatal mushroom species is found to be Amanita phalloides, while the most toxic mushroom toxins are found to be amanitin and phalloidine found in this species.

In the known status of the art, various methods are used in determining amanitin and phalloidine toxins that have fatal effect on human. However, each one of these methods has various problems. For instance; in the operating principle of the Wieland/Meixner test found in the known status of the art, specimen taken from a poisoned patient is poured on a paper drop wise and then left to drying. Afterwards, concentrated HCI solution is added drop wise onto the same region. If amanitin is present in the specimen, then a blue-green colour is formed on the paper. However, Wieland/Meixner test is not safe, since it gives false positive or negative values under sunlight or high temperature.

The competitive immunological method (ELISA) found in the known status of the art comprises a or γ-amanitin-specific polyclonal antibody and makes analysis based on streptavidin-biotin complex formation. The colour formed as a result of the reaction that occurs in the presence of amanitin is spectrophotometrically analyzed. However, the method has disadvantages such as having short shelf life, high cost, and not being found/used commonly in all health institutions. Moreover, some kits require quite long time before they give result.

Amanitin amount can also be determined with the Radio Immuno Assay (RIA) known in the prior art, which is a standardized method in rabbit serum. However, this method also has drawbacks such as short shelf life and high cost.

High performance liquid chromatography (HPLC) known in the prior art is based on the principle of dissolving the analyte in a solvent and forcing it to pass through a chromatography column under high pressure. However, this method also has drawbacks such as the availability of the analysis device, its cost, and the lack of trained personnel who can use the device.

Liquid chromatography-mass spectrometry (LC-MS) is a method used for determining analyte amount formed of high performance liquid chromatography and mass spectrometry units. However, the drawbacks of the high performance liquid chromatography device used in the known status of this method are also valid.

In order to determine the amount of analytes in blood and urine samples with the capillary electrophoresis (CE) method known in the prior art, first of all, they are required to be isolated from body fluids (blood, urine). When the urine sample pH value is brought to pH 2, it is treated with n-butanol and following phase separation, n-butanol is evaporated and the precipitation that contains toxins is dissolved in phosphate buffer and determined by capillary electrophoresis. In the blood samples, after the pH value is brought to pH 2, they are treated with acetonitrile, centrifuged, acetonitrile is evaporated, and the remaining precipitate is dissolved in phosphate buffer, so that the toxin content can be determined. However, this method also has the same drawbacks stated for high performance liquid chromatography due to applicability with devices. Also, the possibility of not being able to reproduce the results is another drawback.

In the prior art patent document with the Application No. RU228784, a set is disclosed, by which amanitin and phalloidine toxins can be determined by mushroom gatherers while gathering mushrooms. However, reliable results can not be obtained either with the method disclosed in the relevant document.

Table 1 : Some measurement methods used for toxins having cyclopeptide structure Brief Description of the Invention

The purpose of the invention is to develop an electrochemical method to determine α-amanitin and phalloidine among macrofungus toxins.

The method of the invention comprises the optimum tools and conditions determined for the electrochemical method used for determination of α-amanitin and phalloidine toxins in an electrochemical mechanism consisting of an electrolyte solution (mixture of substance(s) providing electrical conductivity and solvent), a measurement circuit (potentiostat/galvanostat device, connection cables, and container (glass, ceramic, plastic etc.)), and electrodes (working, reference, and counter electrodes).

The electrolyte used in the electrochemical determination method of α-amanitin and phalloidine according to the invention has electroinactive characteristics and does not create an interference effect against the response of the analyte to be measured.

In the electrochemical determination method, more reliable solutions can be obtained in shorter time periods with the optimization of conditions. In this method, the α-amanitin and phalloidine toxins having the most toxic effect on humans among the macrofungus toxins, are determined reliably in a short time by being applied on analytes taken from blood, urine, and stomach etc. Obtaining reliable results in a short time with this method is of great importance in saving lives of people who are exposed to a-amanitin and phalloidine toxins. The invention relates to a method of determining a-amanitin and phalloidine individually or together in samples such as mushroom gathered from the nature or body fluids (blood, urine, gastric juice etc.) with the use of operation steps of examining electrochemical behaviours of α-amanitin and phalloidine toxins and optimization of the electrochemical determination conditions.

The step of optimization of the electrochemical determination conditions for a-amanitin and phalloidine toxins according to the present invention comprises:

- pH optimization

- Voltage scanning rate optimization

- Calibration graph preparation

In an embodiment of the method according to the invention, electrolyte concentration optimization can also be performed between the steps of voltage scanning rate optimization in the optimization step and the calibration graph preparation step.

In the method, a working electrode found in the mechanism is selected from non-modified carbon (graft, penpoint graphite, glassy carbon, graphene, carbon felt, carbon foam, carbon nanotubes etc.), metal electrodes (platinum, gold, steel etc.), modified carbon, platinum, gold, mercury, or modified steel electrodes.

A reference electrode found in the mechanism used in the method of the invention is formed by selecting an element or compound such as Ag/AgCI, Ag, Carbon, calomel,

A counter electrode found in the mechanism used in the method of the invention is formed by selecting from non-modified and modified platinum, carbon, gold, steel etc. metals.

The electrolyte used in the mechanism of the invention is formed by selecting from phosphate, acetate, Britton-Robinson etc. buffer solutions; KCI, NaCI, L1CO4 etc. inorganic salt solutions; HCI, HNO3, H2SO4 etc. inorganic acid solutions; NaOH, KOH, NH3 etc. inorganic base solutions; and organic acid and base solutions. In the electrochemical mechanism according to the method of the invention, water and acetonitrile, alcohol, tetrahydrofuran, acetone etc. solvents and their aqueous mixtures are selected as solvents.

In order to determine the a-amanitin and phalloidine toxins, differential pulse voltametry and potentiometry, polarography, linear and alternate voltametry, amperemetry, coulometry, normal pulse and square wave voltametric methods can be applied to the electrochemical mechanism. Electrochemical behaviours of a-amanitin and phalloidine toxins are determined with glassy carbon and penpoint graphite working electrodes by using alternating voltametry.

Electrochemical behaviours of different α-amanitin and phalloidine toxin concentrations are determined with glassy carbon and penpoint graphite working electrodes by using alternating voltametry.

In the basic embodiment of the invention, during the pH optimization step, analytes having constant concentration are determined by differential pulse voltametry in 0.01 - 0.50 M phosphate buffer saturation ranges with different pH values.

In a preferred embodiment of the method according to the invention, during the pH optimization step, analytes having constant concentration are determined by differential pulse voltametry in 0.05 M phosphate buffer media with different pH values.

In the pH optimization step applied at 20 mV/s scanning rate under pre-determined optimum conditions, the pH is determined to be in the range of pH 6 - 9, for instance pH = 7.4 for α-amanitin, and in the range of pH 3 - 8, for instance pH = 5.0 for phalloidine.

In the method of the invention, the oxidation peak potential for α-amanitin at pH= 7.4 is determined as 0.252 V. Similarly, the oxidation peak potential for phalloidine at pH= 5.0 is determined as 0.675 V.

In the voltage scanning rate optimization step according to the method, oxidation peak current values are compared and the optimum scanning rate is determined with the values in the range of 5 - 100 mV/s. In relevant value range, the oxidation peak current values in the voltage scanning rate optimization step are compared and the optimum scanning rate is determined as 20 mV/s.

Calibration graphs for α-amanitin and phalloidine are obtained under optimized working conditions. Detailed Description of the Invention Figure 1 : Electrochemical mechanism Description of References

In the mechanism of the method according to the invention, the parts found in the attached Figure are enumerated respectively and the corresponding reference numbers for these parts are given below.

I . Electrochemical mechanism 10. Working electrode

I I . Reference electrode

12. Counter electrode

13. Measurement circuit

14. Electrolyte

The electrochemical mechanism (1 ) performing the electrochemical determination method of the invention comprises a working electrode (10), a reference electrode (1 1 ), a counter electrode (12), a measurement circuit (13), and an electrolyte (14).

In the voltametric methods used in electrochemical analysis, a three-electrode system comprising the reference electrode (1 1 ), counter electrode (12), and working electrode (10) is used. In the voltametric method, the current between the counter electrode (12) and the working electrode (10) is measured by applying voltage between the working electrode (10) and the reference electrode (1 1 ). The concentration of the analyte can be determined by making use of the measured current. In alternating voltametry, a voltage scan is made that changes linearly in time. In this way, both the oxidation and the reduction behaviours of the analyte to be measured can be monitored simultaneously. Monitoring both the oxidation and reduction behaviours of the analyte to be measured, enables the observer to understand whether the electrochemical reaction occurring on the electrode surface is reversible or not.

In voltametric methods, the electrode where electron transfer and electrochemical reaction occurs is called as working electrode (10). Oxidation or reduction of an organic or inorganic substance on the surface of the working electrode (10) causes a mass transfer between the electrode surface and the substance, as a result of application of a suitable potential, and thus a current is formed.

In the basic application of the invention, the working electrode (10) to be used in the electrochemical mechanism (1 ) is selected from non-modified carbon (graft, penpoint graphite, glassy carbon, graphene, carbon felt, carbon foam, carbon nanotubes etc.), metal electrodes (platinum, gold, steel etc.), modified carbon, platinum, gold, mercury, or modified steel electrodes.

The modification of the working electrode (10) enables more sensitive and selective determination of a-amanitin and phalloidine toxins.

The modification of the working electrode (10) can be made by choosing among substances such as conjugated polymers, conjugated molecules, metal complexes, carbon nanotubes, graphene, inorganic and/or organic oxides, organic or inorganic dyes etc. The reference electrode (1 1 ) can be formed by choosing an element or compound such as Ag/AgCI, Ag, Carbon, calomel, Hg/HgSO4 etc. Reference electrodes are electrodes that can not be ideally polarized. They have a constant electrode potential and change of this value is not wanted. In electrochemical methods, voltage is applied between reference electrodes and working electrodes.

Counter electrode (12) can be formed by choosing from non-modified and modified platinum, carbon, gold, steel etc. metals. In electrochemical methods with three electrodes, a counter electrode is used in addition to the working and reference electrodes. In electrochemical methods, current is measured between the counter electrode and the working electrode.

The measurement circuit (13) comprises a container (glass, ceramic, plastic etc.) in which the analyte to be measured is found, a potentiostat / galvanostat device, and connection cables.

Electrolyte (14) is used in order to provide electrical conductivity in the solution. Interference of electrolytes via amanitin and phalloidine oxidation and reduction peaks is not desired, and they are desired to be electro-inactive. The electrolyte can be selected from phosphate, acetate, Britton-Robinson etc. buffer solutions; KCI, NaCI, L1CO4 etc. inorganic salt solutions; HCI, HNO3, H2SO4 etc. inorganic acid solutions; NaOH, KOH, NH3 etc. inorganic base solutions; and organic acid and base solutions.

In the electrochemical mechanism (1 ) according to the method of the invention, water and acetonitrile, alcohol, acetone, tetrahydrofuran, and dichlorometane etc. solvents and their aqueous mixtures are used as solvents. Determination of a-amanitin and phalloidine is made under inert media such as air and nitrogen gas.

The method of the invention comprises the steps of:

- examination of the electrochemical behaviours of α-amanitin and phalloidine toxins,

- optimization of the electrochemical determination conditions of α-amanitin and phalloidine toxins, and thus determine α-amanitin and phalloidine.

The step of optimization of the electrochemical determination conditions for a-amanitin and phalloidine toxins comprises the below given steps:

- pH optimization,

- Voltage scanning rate optimization,

- Calibration graph preparation.

In the step of examining the electrochemical behaviours of α-amanitin and phalloidine toxins according to the method of the invention, modified and non-modified glassy carbon (GCE), penpoint graphite (PGE), and different metal electrodes are tested for determining the suitable working electrode (10). In order to determine the effective electrode, different concentrations of α-amanitin and phalloidine toxins are used for taking alternating voltamograms via glassy carbon (graph 2a and graph 2b) and penpoint electrode (graphs 3a and graph 3b).

Graph 2(a): Alternating voltamograms of α-amanitin in phosphate buffered medium (Working electrode: glassy carbon, Ref. Electrode: Ag/AgCI, v=50 mV/s, 10^-5 M toxin concentration,

Graph 2(b): Alternating voltamograms of phalloidine in phosphate buffered medium (Working electrode: glassy carbon, Ref. Electrode: Ag/AgCI, v=50 mV/s, 10^-5 M toxin concentration,

Graph 3 (a): Alternating voltamograms of a-amanitin in phosphate buffered medium (Working electrode: penpoint graphite, Ref. Electrode: Ag/AgCI, v=50 mV/s, 10^-5 M toxin concentration,

Graph 3(b): Alternating voltamograms of phalloidine in phosphate buffered medium (Working electrode: penpoint graphite, Ref. Electrode: Ag/AgCI, v=50 mV/s, 10^-5 M toxin concentration,

In this step, the electrochemical potential operating range of the analytes to be measured is determined. Moreover, reduction and oxidation characteristics of each analyte (a-amanitin and phalloidine) are also examined. Alternating voltamograms of the phosphate buffer are also taken in order to understand whether the oxidation peaks are caused by the analytes or the electrolyte (14) solution.

As a result of these studies made by GCE and PGE electrodes, it is found out that: a) α-amanitin and phalloidine have irreversible characteristics, b) α-amanitin and phalloidine each have one basic oxidation peak, c) the oxidation peaks of α-amanitin and phalloidine have different potentials from each other, d) the oxidation peak potential of α-amanitin is 0.556 V and the oxidation peak potential of phalloidine is 0.781 V (alternating voltametry, conditions: pH 3.0, 0.05 M phosphate buffer, reference electrode: Ag/AgCI) e) the electrolyte solution does not have an interference effect on the oxidation peaks of α-amanitin and phalloidine toxins, f) the performance of penpoint graphite electrodes is higher than glassy carbon electrodes. Since the oxidation potentials of a-amanitin and phalloidine toxins are different from each other, these two toxins have low possibility of making an interference effect for each other when they are found together. This situation also provides obtaining accurate and sensitive results with the method of the invention in conditions where more than one cyclopeptide toxins are found together.

In this step of the present invention method, alternating voltamograms applied on toxins in 10^"5 - 10^-7 M concentration with glassy carbon (GCE) and penpoint graphite (PGE) electrodes show that, PGE electrodes give better results than GCE electrodes in determining lower saturation toxins. These results are shown in graph 4(a), graph 4(b), graph 5(a) and graph 5(b).

Graph 4 (a): Alternating voltamograms of glassy carbon working electrode and α-amanitin (v=50 mV/s, against Ag/AgCI electrode, phosphate buffer,

toxin concentration,

Graph 4(b): Alternating voltamograms of glassy carbon working electrode and phalloidine (v=50 mV/s, against Ag/AgCI electrode, phosphate buffer, 10^-5 - 10^-7 M toxin concentration:

Graph 5 (a): Alternating voltamograms of penpoint graphite working electrode and a-amanitin (v=50 mV/s, against Ag/AgCI electrode, phosphate buffer, 10^-5 - 10^-7 M toxin concentration:

Graph 5(b): Alternating voltamograms of penpoint graphite working electrode and phalloidine (v=50 mV/s, against Ag/AgCI electrode, phosphate buffer,

toxin concentration:

As a result, PGE is preferred as the working electrode in electrochemical determination of amanitin and phalloidine toxins.

In the step of determining the electrochemical behaviours of α-amanitin and phalloidine toxins according to the method of the invention, alternating voltametry (CV) can be used in order to determine the reduction and oxidation behaviours of the analyte to be measured.

In order to perform determination of low concentration α-amanitin and phalloidine toxins, differential pulse voltametry method can be used. Moreover, polarography, ampereometry, pulse voltametry (such as square wave) etc. methods can also be used.

The step of optimization of the electrochemical determination conditions for a-amanitin and phalloidine toxins according to the method of the invention comprises steps, where the measurements made for determining that the method is suitable for the aims, is performed under optimum determination conditions. In this step, the oxidation characteristics of the analytes (α-amanitin and phalloidine) under various parameters such as pH, scanning rate, and electrolyte concentration etc. are analyzed with differential pulse voltametry method using penpoint graphite working electrode. pH optimization is one of the most important factors affecting the reduction and oxidation behaviours of analytes. Due to the electron exchange differences of analytes in acidic or basic media, shifts may occur in their peak potentials and increase or decrease may be observed in their peak current values. In the pH optimization step, analytes having constant concentration are determined by differential pulse voltametry in 0.05 M phosphate buffer media with different pH values.

When the current response obtained as a result of 20 mV/s scanning rate applied under said constant conditions, during pH optimization, the pH value for a-amanitin is determined to be between pH 6 and 9 with the optimum pH = 7.4, and the pH value for phalloidine is determined to be between pH 3 and 8 with the optimum pH = 5.0. Moreover, for a-amanitin, the oxidation peak potential at pH = 7.4 is determined as 0.252 V and the current value is determined as 1 .462 pA. For phalloidine, the oxidation peak potential at pH = 5.0 is determined as 0.675 V and the measured current value is determined as 2.681 pA. These results are shown in graph 6(a), graph 6(b), graph 7(a) and graph 7(b).

Graph 6 (a): Voltamograms obtained at different pH values under phosphate buffer medium containing α-amanitin (a) in 10^"5 M concentration (Working electrode:

Penpoint graphite, v=20 mV/s, against Ag/AgCI electrode,

Graph 6(b): Voltamograms obtained at different pH values at 10^-5 M concentration under phosphate buffer medium (0.05 M) containing phalloidine (b) (Working electrode,

Graph 7 (a): Graph showing current responses obtained against α-amanitin in 10^"5M concentration at different pH values

Graph 7 (b): Graph showing current responses obtained against phalloidine in 10^"5M concentration at different pH values

Voltage scanning rate optimization is another significant factor affecting the reduction and oxidation behaviours of analytes in the method according to the invention. The effect of voltage scanning rate on the electrochemical behaviours of analytes is analyzed using differential pulse voltametry (DPV) method. In this step, voltamograms are taken in different scanning rates between 5-100 mV/s range under constant analyte concentrations. Optimum scanning rate is determined as 20 mV/s by comparing oxidation peak current values obtained from different scanning rates. These results are shown in graph 8(a), graph 8(b), graph 9(a), and graph 9(b).

Graph 8 (a): Voltamograms obtained at different pH values under phosphate buffer medium containing a-amanitin (a) in 10^"5 M concentration (Working electrode:

Penpoint graphite, v=20 mV/s, against Ag/AgCI electrode,

Graph 8(b): Voltamograms obtained at different pH values in 10^-5 M concentration under phosphate buffer medium (0.05 M) containing phalloidine (a) (Working electrode Penpoint graphite, v=20 mV/s, against Ag/AgCI electrode,

Graph 9(a): Graph showing current responses obtained against α-amanitin in 10^-5M concentration at different scanning rates

Graph 9(b) Graph showing current responses obtained against phalloidine in 10^-5M concentration at different scanning rates

In an embodiment of the invention, the electrolyte concentration optimization is performed between the steps of pH optimization and voltage scanning rate optimization. In the step of supporting electrolyte concentration optimization, voltamograms are taken by adding different electrolyte (14) concentrations into the buffer solution with the optimum pH value, which is determined by applying differential pulse voltametry under constant analyte concentration conditions. The effect of electrolyte (14) concentration on the current response is analyzed by comparing the oxidation peak potentials and peak current values of analytes in the voltamograms obtained by using 0.02, 0.1 , 0.2, 1 .0, and 2.0 M KCI solutions as supporting electrolyte (14). It is observed that the KCI solution used as supporting electrolyte (14) in different concentrations does not have an increasing effect on the current response of a-amanitin and phalloidine toxins.

Calibration graph preparation: The calibration graphs of a-amanitin and phalloidine toxins are formed under the determined optimum conditions by using peak current values against different toxin concentrations. The calibration graphs are prepared by using different toxin concentrations such as in the range of 0.01 to 10 μΜ for α-amanitin and 0.01 to 2.5 μΜ for phalloidine. During the stage of preparing calibration graph, the region or regions where the working electrodes (10) operate linearly with regard to the toxin concentrations is/are determined. These results are shown in graph 10 for α-amanitin and in graph 1 1 (a) and graph 1 1 (b) for phalloidine.

Graph 10: Calibration graph for a-amanitin in 0.01 to 10 μΜ concentrations optimized working conditions.

Graph 11 (a): Calibration graph for phalloidine in 0.01 to 2.5 μΜ concentrations under optimized working conditions.

Graph 11 (b): Calibration graph for phalloidine in 5 to 10 μΜ concentrations under optimized working conditions.

By means of applying differential pulse voltametry with the electrochemical method of the invention, it is possible to determine a-amanitin and phalloidine toxins individually and/or together in blood, urine, gastric juice etc. body fluids or mushroom samples via penpoint graphite working electrode under optimum conditions such as at 20 mV/s scanning rate in 0.05 M phosphate buffer solution medium, at pH = 7.4 (oxidation peak potential 0.252 V, current value 1 .462 pA) for α-amanitin and at pH = 5.0 (oxidation peak potential 0.675 V, current value 2.681 pA) for phalloidine. The measured current values change linearly with the changing concentrations of α-amanitin and phalloidine toxins.

The method and the electrochemical mechanism (1 ) of the invention enables determining α-amanitin and phalloidine toxins in a quicker, easier, and portable manner in laboratory or in field.

By means of determining these toxins in a quick manner with the electrochemical mechanism (1 ) applying the method of the invention, it is possible to diagnose and treat a patient who has mushroom poisoning.

The method of the invention (100) and the electrochemical mechanism (1 ) applying this method, can be applied under air, nitrogen gas etc. inert media for mushroom and body fluid (blood, urine, gastric juice etc.) samples from which analytes are obtained during determination of a-amanitin and phalloidine.

The invention is not limited to the above disclosed embodiments and persons skilled in the related art can easily form other embodiments of the invention. These embodiments shall be evaluated according to the scope of protection claimed in the claims part of the invention.

Claims

1) A method of determining a-amanitin and phalloidine toxins, characterized in that; for the purpose of determining a-amanitin and phalloidine individually or together in samples such as mushroom gathered from the nature or body fluids (blood, urine, gastric juice etc.), it comprises the operation steps of:

- examining the electrochemical behaviours of α-amanitin and phalloidine toxins, and

- optimizing the electrochemical determination conditions of α-amanitin and phalloidine.

2) A method according to Claim 1 , characterized in that; the step of optimization of the electrochemical determination conditions for α-amanitin and phalloidine toxins comprises the steps of:

- pH optimization,

- Voltage scanning rate optimization, and

- Calibration graph preparation.

3) A method according to Claim 2, characterized in that; electrolyte concentration optimization is performed between the steps of voltage scanning rate optimization and the calibration graph preparation.

4) A method according to Claim 2 or Claim 3, characterized in that; a working electrode (10) used in the in the mechanism (1 ) of the method is chosen from non-modified carbon (graphite, penpoint graphite, glassy carbon, graphene, carbon felt, carbon foam, carbon nanotubes etc.), metal electrodes (platinum, gold, steel etc.), modified carbon, platinum, gold, mercury, or modified steel electrodes.

5) A method according to Claim 4, characterized in that; a reference electrode (1 1 ) found in the mechanism (1 ) used in the method is formed by choosing an element or compound from Ag/AgCI, Ag, Carbon, calomel, and Hg/HgSO4 etc.

6) A method according to Claim 5, characterized in that; a counter electrode (12) found in the mechanism (1 ) used in the method of the invention is formed by choosing from non-modified and modified platinum, carbon, gold, and steel etc. metals.

7) A method according to Claim 6, characterized in that; the electrolyte (14) used in the mechanism (1 ) of the method is formed by choosing from phosphate, acetate, and Britton-Robinson etc. buffer solutions; KCI, NaCI, and L1CO4 etc. inorganic salt solutions; HCI, HNO3, and H2SO4 etc. inorganic acid solutions; NaOH, KOH, NH3 etc. inorganic base solutions; and organic acid and base solutions.

8) A method according to Claim 7, characterized in that; in the electrochemical mechanism (1 ) where the method of the invention is applied, water and acetonitrile, alcohol, tetrahydrofuran, acetone etc. solvents and their aqueous mixtures are selected as solvents.

9) A method according to Claim 8, characterized in that; in order to determine the a-amanitin and phalloidine toxins, differential pulse voltametry and potentiometry, polarography, linear and alternate voltametry, amperemetry, coulometry, normal pulse and square wave voltametric methods are applied to the electrochemical mechanism

(1 )-

10) A method according to Claim 9, characterized in that; in the pH optimization step, analytes having constant concentration are determined by differential pulse voltametry in phosphate buffer with different pH values in a concentration range of 0.01 to 0.50 M.

11) A method according to Claim 10, characterized in that; in the pH optimization step, analytes having constant concentration are determined by differential pulse voltametry in 0.05 M phosphate buffer media with different pH values.

12) A method according to Claim 10 or 1 1 , characterized in that; in the 20 mV/s scanning rate applied under pre-determined optimum conditions, during the pH optimization step, optimum pH for a-amanitin in the pH 6 - 9 range is determined as pH = 7.4 and for phalloidine in the pH 3 - 8 range is determined as pH = 5.0.

13) A method according to Claim 12, characterized in that; the oxidation peak potential for α-amanitin is determined as 0.252 V at pH = 7.4.

14) A method according to Claim 12, characterized in that; the oxidation peak potential for phalloidine is determined as 0.675 V at pH = 5.0.

15) A method according to Claim 14, characterized in that; by comparing the oxidation peak current values in the voltage scanning rate optimization step, the optimum scanning rate is determined to be in the range of 5 - 100 mV/s.

16) A method according to Claim 15, characterized in that; by comparing the oxidation peak current values in the voltage scanning rate optimization step, the optimum scanning rate is determined as 20 mV/s.