U.S. patent application number 11/922265 was filed with the patent office on 2009-12-03 for detection of phenols.
This patent application is currently assigned to ISIS INNOVATION LIMITED. Invention is credited to Craig Edward Banks, Richard Guy Compton.
Application Number | 20090294298 11/922265 |
Document ID | / |
Family ID | 37054402 |
Filed Date | 2009-12-03 |
United States Patent
Application |
20090294298 |
Kind Code |
A1 |
Compton; Richard Guy ; et
al. |
December 3, 2009 |
Detection of Phenols
Abstract
According to the present invention, phenols may be detected
using an electrochemical sensor comprising a final compound, a
working electrode and an electrolyte in contact with the working
electrode, wherein the first compound operatively undergoes a redox
reaction at the working electrodes to form a second compound which
operatively reacts in situ with the phenol, wherein said redox
reaction has a detectable redox couple and wherein the sensor is
adapted to determine the electrochemical response of the working
electrode to the consumption of said second compound on reaction
with the phenol. The phenol may be, for example, cannabinoid or a
catechin compound.
Inventors: |
Compton; Richard Guy;
(Oxford, GB) ; Banks; Craig Edward; (Cheshire,
GB) |
Correspondence
Address: |
David W Hibler;VINSON & ELKINS
2500 First City Tower, 1001 Fannin Street
Houston
TX
77002
US
|
Assignee: |
ISIS INNOVATION LIMITED
Oxford
GB
|
Family ID: |
37054402 |
Appl. No.: |
11/922265 |
Filed: |
June 16, 2006 |
PCT Filed: |
June 16, 2006 |
PCT NO: |
PCT/GB2006/002219 |
371 Date: |
July 10, 2009 |
Current U.S.
Class: |
205/437 ;
204/400; 205/780.5; 205/782; 252/182.1 |
Current CPC
Class: |
G01N 27/28 20130101;
G01N 27/416 20130101; G01N 27/49 20130101; G01N 33/48714 20130101;
G01N 27/30 20130101; G01N 27/403 20130101 |
Class at
Publication: |
205/437 ;
204/400; 205/780.5; 205/782; 252/182.1 |
International
Class: |
C25B 3/02 20060101
C25B003/02; G01N 27/26 20060101 G01N027/26; G01N 27/30 20060101
G01N027/30; C25B 11/04 20060101 C25B011/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 16, 2005 |
GB |
0512282.5 |
Feb 3, 2006 |
GB |
0602203.2 |
Claims
1. An electrochemical sensor for the detection of a phenol, which
comprises a first compound, a working electrode and an electrolyte
in contact with the working electrode, wherein the first compound
operatively undergoes a redox reaction at the working electrode to
form a second compound which operatively reacts in situ with the
phenol, wherein said redox reaction has a detectable redox couple
and wherein the sensor is adapted to determine the electrochemical
response of the working electrode to the consumption of said second
compound on reaction with the phenol.
2. The sensor according to claim 1, wherein said first compound is
a 4-aminophenol.
3. The sensor according to claim 2, wherein said first compound is
a compound of the formula (I): ##STR00009## wherein m is 0, 1, 2, 3
or 4; each R.sup.1 is independently R.sup.2, or is hydrocarbyl or
heterocyclyl, either of which is optionally substituted with 1, 2,
3, 4 or 5 R.sup.2; each R.sup.2 is independently selected from
halogen, trifluoromethyl, cyano, nitro, oxo, .dbd.NR.sup.3,
R.sup.3, --OR.sup.35--C(O)R.sup.3, --C(O)OR.sup.3, --OC(O)R.sup.3,
--N(R.sup.3)R.sup.4, --C(O)N(R.sup.3)R.sup.4, --S(O).sub.1R.sup.3
and --C(R.sup.3).sub.3; R.sup.3 and R.sup.4 are each independently
hydrogen, or are selected from C.sub.1-6 alkyl,
--(CH.sub.2).sub.k-carbocyclyl and --(CH.sub.2).sub.k-heterocyclyl,
any of which is optionally substituted with 1, 2, 3, 4 or 5
substituents independently selected from halogen, hydroxy and
C.sub.1-6 alkyl; and l is 0, 1 or 2; and wherein the first compound
is operatively oxidised at the working electrode to form a second
compound which is of the formula (II): ##STR00010##
4. The sensor according to claim 3, wherein m is 0, 1 or 2.
5. The sensor according to claim 3, wherein each R.sup.1 is
independently selected from --NR.sup.3R.sup.4, halogen, C.sub.1,
C.sub.2, C.sub.3 or C.sub.4 alkyl, C.sub.1, C.sub.2, C.sub.3 or
C.sub.4 haloalkyl, C.sub.1, C.sub.2, C.sub.3 or C.sub.4 alkoxy, and
C.sub.2, C.sub.3 or C.sub.4 alkenyl, wherein R.sup.3 and R.sup.4
are each independently selected from hydrogen, --OH, C.sub.1,
C.sub.2, C.sub.3 or C.sub.4 alkyl, C.sub.1, C.sub.2, C.sub.3 or
C.sub.4 haloalkyl, C.sub.1, C.sub.2, C.sub.3 or C.sub.4 alkoxy, and
C.sub.2, C.sub.3 or C.sub.4 alkenyl.
6. The sensor according to claim 5, wherein each R.sup.1 is
halogen.
7. The sensor according to claim 6, wherein the first compound is
of the formula (IA): ##STR00011##
8. The sensor according to claim 3, wherein each R.sup.1 is
aryl.
9. The sensor according to claim 8, wherein the first compound is
of the formula (IB): ##STR00012##
10. The sensor according to claim 1, wherein the working electrode
is a screen printed electrode, a metallic electrode, an edge plane
pyrolytic graphite electrode, a basal plane pyrolytic graphite
electrode, a gold electrode, a glassy carbon electrode, a boron
doped diamond electrode, or a highly ordered pyrolytic graphite
electrode.
11. The sensor according to claim 1, wherein the sensor is adapted
to determine the current flow between the working electrode and a
counter electrode.
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. The sensor according to claim 1, wherein the electrolyte
comprises said first compound.
23. The sensor according to claim 1, wherein the working electrode
comprises said first compound.
24. A method of detecting a phenol in a sample, comprising: (a)
oxidising a first compound at a working electrode of an
electrochemical sensor to form a second compound which is
operatively reactive with the phenol; (b) contacting the phenol
with the second compound in the presence of an electrolyte, such
that the second compound reacts with the phenol; and (c)
determining an electrochemical response of the working electrode to
the consumption of the second compound on reaction with the
phenol.
25. The method according to claim 24, wherein the first compound is
a 4-aminophenol.
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. The method according to claim 24, wherein the working electrode
is a screen printed electrode, a metallic electrode, an edge plane
pyrolytic graphite electrode, a basal plane pyrolytic graphite
electrode, a glassy carbon electrode, a boron doped diamond
electrode or a highly ordered pyrolytic graphite electrode.
34. The method according to claim 24, wherein determination of the
electrochemical response comprises measuring the current flow
between the working electrode and a counter electrode to determine
the amount of the phenol.
35. The method according to claim 34, wherein the working electrode
is maintained at a constant voltage.
36. The method according to claim 34, wherein said current is
measured using linear sweep or cyclic voltammetry, square wave
voltammetry, or a pulsed voltammetry technique.
37. The method according to claim 24, wherein the phenol is a
para-substituted phenol.
38. The method according to claim 24, wherein the phenol is phenol,
4-phenoxyphenol, p-methylphenol, m-methylphenol, nitrophenol or
tetrahydrocannabinol.
39. The method according to claim 24, wherein the phenol is a
component or a metabolite of cannabis.
40. The method according to clam 24, wherein the phenol is a
natural or synthetic cannabinoid or a metabolite thereof.
41. The method according to claim 39, wherein the phenol is a
cannabis metabolite found in urine.
42. The method according to claim 39, wherein the phenol is
11-nor-9-carboxy-9-tetrahydrocannabinol.
43. The method according to claim 24, wherein the phenol is a
catechin.
44. The method according to claim 43, wherein the phenol is
(-)-epigallocatechin gallate (EGCG) or (-)-epigallocatechin
(ECG).
45. The method according to claim 24, wherein the electrolyte
comprises said first compound.
46. The method according to claim 24, wherein the working electrode
comprises said first compound.
47. A method of forming an indophenol compound comprising
electrochemically oxidising a 4-aminophenol compound to form a
benzoquinone compound, and reacting the benzoquinone compound with
a phenol to form an indophenol.
48. The method according to claim 47, wherein the 4-aminophenol
compound is of the formula (I): ##STR00013## wherein m is 0, 1, 2,
3 or 4; each R.sup.1 is independently R.sup.2, or is hydrocarbyl or
heterocyclyl, either of which is optionally substituted with 1, 2,
3, 4 or 5 R.sup.2; each R.sup.2 is independently selected from
halogen, trifluoromethyl, cyano, nitro, oxo, .dbd.NR.sup.3,
R.sup.3, --OR.sup.3, --C(O)R.sup.3, --C(O)OR.sup.3, --OC(O)R.sup.3,
--N(R.sup.3)R.sup.4, --C(O)N(R.sup.3)R.sup.4, --S(O).sub.1R.sup.3
and --C(R.sup.3).sub.3; R.sup.3 and R.sup.4 are each independently
hydrogen, or are selected from C.sub.1-6 alkyl,
--(CH.sub.2).sub.k-carbocyclyl and --(CH.sub.2).sub.k-heterocyclyl,
any of which is optionally substituted with 1, 2, 3, 4 or 5
substituents independently selected from halogen, hydroxy and
C.sub.1-6 alkyl; and l is 0, 1 or 2; and is oxidised to form a
benzoquinone compound of the formula (II): ##STR00014##
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. (canceled)
54. (canceled)
55. The method according to claim 47, wherein the phenol is phenol,
4-phenoxyphenol, p-methylphenol, m-methylphenol, nitrophenol,
tetrahydrocannabinol, a component or metabolite of cannabis, or a
natural or synthetic cannabinoid or a metabolite thereof.
56. The method according to claim 47, wherein the phenol is a
catechin.
57. The method according to claim 56, wherein the catechin is
(-)-epigallocatechin gallate (EGCG) or (-)-epigallocatechin
(ECG).
58. An electrode material comprising a 4-aminophenol compound.
59. The material according to claim 58, wherein the compound is
comprised on a surface of the material.
60. The material according to claim 58, wherein the compound is
comprised in the bulk of the material.
61. The material according to claim 58, wherein said material is
obtainable by screen printing.
62. The material according to claim 58, wherein said material
comprises a metallic material, edge plane pyrolytic graphite, basal
plane pyrolytic graphite, gold, glassy carbon, boron doped diamond
or highly ordered pyrolytic graphite.
63. The material according to claim 58, wherein the compound is a
compound of the formula (I): ##STR00015## wherein m is 0, 1, 2, 3
or 4; each R.sup.1 is independently R.sup.2, or is hydrocarbyl or
heterocyclyl, either of which is optionally substituted with 1, 2,
3, 4 or 5 R.sup.2; each R.sup.2 is independently selected from
halogens trifluoromethyl, cyano, nitro oxo, .dbd.NR.sup.3, R.sup.3,
--OR.sup.3, --C(O)R.sup.3, --C(O)OR.sup.3, --OC(O)R.sup.3,
--N(R.sup.3)R.sup.4, --C(O)N(R.sup.3R', --S(O).sub.1R.sup.3 and
--C(R.sup.3).sub.3; R.sup.3 and R.sup.4 are each independently
hydrogen, or are selected from C.sub.1-6 alkyl
--(CH.sub.2).sub.k-carbocyclyl and --(CH.sub.2).sub.k-heterocyclyl,
any of which is optionally substituted with 1, 2, 3, 4 or 5
substituents independently selected from halogen, hydroxy and
C.sub.1-6 alkyl; and l is 0, 1 or 2: and is oxidised to form a
benzoquinone compound of the formula (II): ##STR00016##
64. (canceled)
65. The material according to claim 63, wherein the compound is
2,6-diphenyl-4-aminophenol.
66. (canceled)
67. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and apparatus for
the detection and quantitative determination of analytes, in
particular phenols, phenolic compounds and phenol derivatives.
BACKGROUND TO THE INVENTION
[0002] The prevalence of driving while affected by cannabis is
rising. It has been shown that drugs are detected commonly among
those involved in motor vehicle accidents, various studies
reporting that up to 25% of drivers involved in accidents tested
positive for Illicit drugs, with cannabis being the most common
found, followed by benzodiazepines, cocaine, amphetamines and
opioids. It is apparent that drugs, when taken in combination with
alcohol, and multiple drugs, present an even greater risk; drug
driving is a significant problem, both in terms of a general public
health issue and as a specific concern for drug users.
[0003] The primary active component of cannabis is
.DELTA..sup.9-tetrahydrocannabinol (THC), the structure of which is
shown below:
##STR00001##
[0004] Studies have repeatedly shown that THC impairs cognition,
psychomotor function and actual driving performance. For example,
it has been reported that the degree of performance impairment
observed in experimental studies after doses up to 300 .mu.g per kg
of THC were equivalent to the impairing effect of a blood alcohol
concentration at the legal limit for driving under the influence in
most European countries. The combined use of THC and alcohol
produces severe impairment of cognitive, psychomotor, and actual
driving performance and increases the risk of crashing.
[0005] Cannabinoids (C.sub.21 compounds typical of and present in
cannabis, their carboxylic acids, analogues, and transformation
products) are routinely determined by gas chromatography-mass
spectrometry (GC-MS). This approach requires complex
instrumentation and all samples must be derivatized prior to
injection. High-performance liquid chromatography, utilising
electrochemical detection, has also been used. Low detection limits
are achievable but high potentials are required for the
electrochemical oxidation of cannabinoids. Typically, potentials of
up to 1.2 V are required, which is close to the decomposition of
water which increases the background current and introduces noise.
Backofen et al (2000, BioMed. Chrom., 14:49) recently addressed
this problem and explored non-aqueous electrolyte systems at
platinum and gold electrodes, observing reduced noise and allowing
a low detection limit of ca. 0.1 .mu.M. This limit is two orders of
magnitude lower than on-column UV detection and compares favourably
with GC-MS.
[0006] As mentioned above, electrochemical methodologies have been
employed as end of column detectors for THC. Typical sensing of THC
is based on the oxidation of the hydroxyl group. This technique is
not ideal since the electrochemical oxidation of phenols in aqueous
solution is plagued by irreversible adsorption of oxidation
reaction intermediates and products producing fouling of the
electrode surface. This leads to poor electrode response and
reproducibility, although this can be overcome to some extent by
using low phenol concentrations and/or elevated temperatures.
Alternative methods include the use of laser ablation to remove
such passivating electrolytically generated layers or high
overpotentials, which increase the anodic discharge of the solvent
generating hydroxyl radicals which degrade the adsorbed oligomeric
and polymeric products on the electrode surface.
[0007] A standard analytical technique for determining substituted
phenol compounds is via reaction with the Gibbs reagent, i.e.
2,6-dichloro-p-benzoquinone 4-chloroimine. Gibbs showed that
quinonechloroimides react with phenolic compounds producing
brightly coloured indophenol compounds, which can be conveniently
monitored via spectrophotometry. It was generally believed that the
position para to the hydroxyl must be unsubstituted (Gibbs, (1927)
J. Biol. Chem., 71:445; and Gibbs, (1927) J. Biol. Chem., 72:649).
Gibbs reported that the pH of the solution greatly affects the rate
of formation of the indophenol compound: at a pH of 10 the
beginning of indophenol blue formation was observed to occur within
two minutes, while at pH 8.5 this timescale was increased to 16
minutes. Dacre (1971, Anal Chem., 43:589) explored a large range of
phenolic compounds and concluded that the Gibbs reaction was
non-specific. A few substituted phenols were also reported as
giving a negative Gibbs reaction.
[0008] Josephy and Damme (1984, Anal. Chem., 56:813) explored the
Gibbs reaction with para-substituted phenols. The reaction
mechanism is shown in Scheme 1 below:
##STR00002##
[0009] The mechanism involves first the solvolysis of the Gibbs
reagent (1) which yields dichloro-benzoquinone monoamine (2). This
attacks the para position of the phenol resulting in an adduct (4)
which deprotonates with the resulting Intermediate (4) losing a
proton and R'', the para-substituted leaving group, to form
2,6-dichloroindophenol (6). Note that in the case R.dbd.H, (4) is
oxidised to (5) by reaction with a second molecule of (2). The
resulting indophenol is brightly coloured and can be easily
characterised via spectrophotometry. However in their work, Josephy
and Damme noted several exceptions which did not give a positive
Gibbs reaction. These Included halogen-substituted phenols (TCP,
TBP and TIP), hydroxybenzaldehydes and related compounds,
hydroxybenzyl alcohols and hydroxybenzoic acids. The reason why was
not elucidated.
[0010] Green tea (Camellia sinensis) is a rich source of polyphenol
compounds known as catechins. Catechins are effective anticancer
and anti-tumour agents and are claimed to have anti-mutagenic,
anti-diabetic, hypocholesterolemic, anti-bacterial and
anti-inflammatory properties. The most abundant catechins are
(-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (ECG)
the structures of which are shown below:
##STR00003##
[0011] EGCG and EGC are thought to be the most effective catechin
compounds, and the Important characteristics of green tea, e.g.
taste, nutritional values, palatability and pharmacological
effects, depend substantially on their polyphenol content.
[0012] Methods of detecting catechins include high performance
liquid chromatography using end of column detectors such as a
coulometric array, UV, mass spectrometry and electrochemical
detection. Caffeine, a major component in tea, can interfere with
the UV analysis of catechins. Chromatographic methods coupled with
electrochemical detection showed improved selectivity since
caffeine is electrochemically inactive. Such a technique is based
on simply holding an electrode at a suitably high potential which
corresponds to the electrochemical oxidation of the analyte of
interest. However, it is well documented that the electrochemical
oxidation of phenolic compounds results in deactivation of the
electrode surface (Pelillo et al., Food Chem. 87, (2004), 465; and
Wang et al, J. Electroanal. Chem. 313, (1991), 129); a passivating
polymeric film is produced which decreases the sensitivity and
degrades the reproducibility although this can be overcome to a
certain extent by using low phenol concentrations. The electrode
materials employed in electrochemical end of column detectors
include noble metals (Sano et al, Analyst 126, 2001, 816; and Yang
et al., Anal. BioChem. 283, 2000, 77) and glassy carbon (Kumamoto
et al, Anal. Sci., 16, 2000, 139; and Long et al., J. Chrom. B 763,
2001, 47) electrodes. Recently, Romani et al (J. Agric. Food Chem.
48, 2000, 1197) explored screen-printed electrodes modified with
tyrosinase enzyme as an electrochemical end of column sensor where
the disposable aspect overcomes electrode fouling and alleviates
the need to polish the electrode surface between runs.
[0013] In summary, the methods described above are limited by the
complexity of instrumentation, a need to derivatize samples,
unacceptable detection limits, high oxidation potentials or a lack
of specificity.
SUMMARY OF THE INVENTION
[0014] The present invention modifies or builds on the known Gibbs
reaction by electrochemically oxidising a p-aminophenol (PAP) to
form a benzoquinone monoamine (for example, a dichloro- or
diphenyl-benzoquinone monoamine), which then reacts with the
substituted phenol compound of interest, as in the classical Gibbs
reaction. Monitoring the reduction of an oxidised PAP provides an
indirect method of detecting phenols and phenolic compounds, for
example phenol, 4-phenoxyphenol, methylphenol (para and meta),
nitrophenol, cannabinolds (e.g. tetrahydrocannabinol) and catechins
(e.g. EGCG or ECG). The methodology according to the present
invention is attractive since it avoids the direct oxidation of the
phenol, which can lead to electrode passivation. The PAP may be
present in the electrolyte and/or on the surface or in the bulk of
the working electrode material.
[0015] According to a first aspect of the invention there is
provided an electrochemical sensor for the detection of a phenol,
which comprises a first compound, a working electrode and an
electrolyte in contact with the working electrode, wherein the
first compound operatively undergoes a redox reaction at the
working electrode to form a second compound which operatively
reacts in situ with the phenol, wherein said redox reaction has a
detectable redox couple and wherein the sensor is adapted to
determine the electrochemical response of the working electrode to
the consumption of said second compound on reaction with the
phenol.
[0016] According to a second aspect of the invention there is
provided a method of sensing a phenol in a sample, comprising:
[0017] (a) oxidising a first compound at the working electrode of
an electrochemical sensor to form a second compound which is
operatively reactive with the phenol; [0018] (b) contacting the
phenol with the second compound in the presence of an electrolyte,
such that the second compound reacts with the phenol; and [0019]
(c) determining the electrochemical response of the working
electrode to the consumption of the second compound on reaction
with the phenol.
[0020] According to a third aspect of the invention there is
provided a method of forming an indophenol compound comprising
electrochemically oxidising a 4-aminophenol compound to form a
benzoquinone compound, and reacting the benzoquinone compound with
a phenol to form an indophenol.
[0021] A further aspect of the invention is an electrode material
comprising a 4-aminophenol compound. The material may be present on
a surface and/or in the bulk of the electrode material.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1A shows the cyclic voltammogramic response to 1 mM
4-amino-2-,6-dichlorophenol at a scan rate of 5 mVs.sup.-1 in pH
3.4 buffer;
[0023] FIG. 1B shows the scan rate dependence of
4-amino-2,6-dichlorophenol from 25 to 100 mVs.sup.-1 in pH 3.4
buffer;
[0024] FIG. 1C compares the response of a gold macroelectrode
(dotted line) to 4-amino-2,6-dichlorophenyl with that of an edge
plane pyrolytic graphite (eppg) electrode at a scan rate of 100
mVs.sup.-1 in pH 3.4 buffer;
[0025] FIGS. 1D and 1E compare the response of eppg and gold
electrodes to 4-amino-2,6-dichlorophenyl recorded in a pH 10 buffer
at 25 mVs.sup.-1;
[0026] FIG. 1F shows the oxidation of 1 mM benzoquinone (BQ) to
hydroquinone (HQ) at an eppg electrode in a pH 10 buffer (dotted
line) compared with PAP at pH 10, both recorded at 100
mVs.sup.-1;
[0027] FIG. 2 shows cyclic voltammograms showing the response of
phenol additions to a pH 10 buffer solution containing 1 mM
4-amino-2,6-dichlorophenol, using a polished basal plane pyrolytic
graphite (bppg) electrode at a scan rate of 100 mVs.sup.-1. The
phenol additions were at 50, 100, 150, 200 and 250 .mu.M;
[0028] FIG. 3 is a voltammogram showing the oxidation of 1 mM
phenol in a pH 10 buffer solution recorded at 100 mVs.sup.-1 using
an eppg electrode;
[0029] FIG. 4A shows square wave voltammograms of phenol additions
to a 1 mM solution of PAP using an eppg electrode. The voltammetric
response is for additions of phenols at 99, 196, 291, 385 and 485
.mu.M respectively. The square wave parameters are: 10 s at +0.4V
followed by a potential sweep from +0.4V to -0.4V;
[0030] FIG. 4B is a graph of the peak height versus added phenol
concentration for voltammogram of FIG. 4A;
[0031] FIG. 5A is a voltammogram showing the response of the
addition of 4-phenoxyphenol to a pH 10 buffer solution using an
eppg electrode. The 4-phenoxyphenol additions were at
concentrations of 50, 100, 150, 200 and 250 .mu.M;
[0032] FIG. 5B is a voltammogram showing the response of the
addition of p-cresol to a pH 10 buffer solution using an eppg
electrode. The p-cresol additions were at concentrations of 100,
200, 300, 400 and 500 .mu.M;
[0033] FIG. 5C is a voltammogram showing the response of the
addition of m-cresol to a pH 10 buffer solution using an eppg
electrode. The m-cresol additions were at concentrations of 99,
196, 291 and 385 .mu.M;
[0034] FIG. 5D is a voltammogram showing the response of the
addition of p-nitrophenol to a pH 10 buffer solution using an eppg
electrode. The p-nitrophenol additions were at concentrations of
100, 200, 300, 400 and 500 .mu.M;
[0035] FIG. 6 shows cyclic voltammograms showing the response of
tetrahydrocannabinol (THC) additions to a pH 10 buffer solution
containing 1 mM PAP using an eppg electrode at a scan rate of 10
mVs.sup.-1. The THC additions were at concentrations of 100, 196,
291, 385 and 476 .mu.M respectively;
[0036] FIG. 7A shows voltammograms of additions of
tetrahydrocannabinol (THC) to a solution containing 1 mM
4-amino-2,6-dichlorophenol in pH 10 buffer using an eppg electrode.
The square wave parameters were: -0.4V (vs standard calomel
electrode) for 10 seconds followed by a potential sweep from +0.4V
to -0.4V. The additions of THC were at concentrations of 99, 196,
291, 385 and 476 .mu.M respectively;
[0037] FIG. 7B is a graph of the peak height versus added THC
concentration for voltammogram of FIG. 7A.;
[0038] FIG. 8 shows cyclic voltammetric responses due to the
additions of EGCG to a pH 10 buffer solution using an edge plane
pyrolytic graphite electrode. All scans were recorded at 100
mVs.sup.-1. The dotted scan is the initial response in the absence
of any EGCG. Additions of EGCG were at 1, 2, 3, 4 and 5 mM;
[0039] FIG. 9 shows an electrochemical sensing protocol in which
the reduction in magnitude of the reverse peak from the addition of
EGCG or EGC provides the analytical signal;
[0040] FIG. 10A shows the square-wave voltammetric response using a
bppg electrode modified with 4-amino-2,6-diphenylphenol in a pH 10
buffer solution to additions of 1.7 .mu.M EGCG. The square-wave
parameters were: +0.2 V for 5 seconds followed by potential sweep
from +0.2 to -0.4 V (vs. SCE);
[0041] FIG. 10B shows the analysis of the observed peak height
(from FIG. 10A) versus added EGCG concentration;
[0042] FIG. 11A shows the response of 1.7 .mu.M additions of EGC
into a pH 10 buffer solution using a bppg electrode modified with
4-amino-2,6-diphenylphenol. The modification procedure and
square-wave parameters are the same as for FIG. 10;
[0043] FIG. 11B shows the analysis of the observed peak height
versus added EGC concentration;
[0044] FIG. 12A shows typical square-wave voltammetric responses
using a bppg electrode modified with 4-amino-2,6-diphenylphenol
from analysis of a green tea sample, with 0.7 .mu.M additions of
EGCG and EGC made to the solution. The square-wave parameters were:
+0.2 V for 5 seconds followed by potential sweep from +0.2 to -0.4
V (vs. SCE); and
[0045] FIG. 12B shows the analysis of the observed peak height
(from FIG. 12A) versus added EGCG/EGC concentration.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0046] The term "hydrocarbyl" as used herein includes reference to
a moiety consisting exclusively of hydrogen and carbon atoms; such
a moiety may comprise an aliphatic and/or an aromatic moiety. The
moiety may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 carbon atoms. Examples of hydrocarbyl
groups include C.sub.1-6 alkyl (e.g. C.sub.1, C.sub.2, C.sub.3 or
C.sub.4 alkyl, for example methyl, ethyl, propyl, isopropyl,
n-butyl, sec-butyl or tert-butyl); C.sub.1-8 alkyl substituted by
aryl (e.g. phenyl) or by cycloalkyl; cycloalkyl (e.g. cyclopropyl,
cyclobutyl, cyclopentyl or cyclohexyl); aryl (e.g. phenyl, naphthyl
or fluorenyl) and the like.
[0047] The terms "alkyl" and "C.sub.1-6 alkyl" as used herein
include reference to a straight or branched chain alkyl moiety
having 1, 2, 3, 4, 5 or 6 carbon atoms. This term includes
reference to groups such as methyl, ethyl, propyl(n-propyl or
isopropyl), butyl(n-butyl, sec-butyl or tert-butyl), pentyl, hexyl
and the like.
[0048] The terms "alkenyl" and "C.sub.2-6 alkenyl" as used herein
include reference to a straight or branched chain alkyl moiety
having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at
least one double bond, of either E or Z stereochemistry where
applicable. This term includes reference to groups such as ethenyl,
2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl,
2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl and 3-hexenyl and the
like.
[0049] The terms "alkynyl" and "024 alkynyl" as used herein include
reference to a straight or branched chain alkyl moiety having 2, 3,
4, 5 or 6 carbon atoms and having, in addition, at least one triple
bond. This term includes reference to groups such as ethynyl,
1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl,
1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl and
3-hexynyl and the like.
[0050] The terms "alkoxy" and "C.sub.1-6 alkoxy" as used herein
refer to --O-alkyl, wherein alkyl is straight or branched chain and
comprises 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of
embodiments, alkoxy has 1, 2, 3 or 4 carbon atoms. This term
Includes reference to groups such as methoxy, ethoxy, propoxy,
isopropoxy, butoxy, tert-butoxy, pentoxy, hexoxy and the like.
[0051] The term "cycloalkyl" as used herein includes reference to
an alicyclic moiety having 3, 4, 5, 6, 7 or 8 carbon atoms. The
group may be a bridged or polycyclic ring system. More often
cycloalkyl groups are monocyclic. This term includes reference to
groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
norbornyl, bicyclo[2.2.2]octyl and the like.
[0052] The term "aryl" as used herein includes reference to an
aromatic ring system comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
or 16 ring carbon atoms. The group is often phenyl but may be a
polycyclic ring system, having two or more rings, at least one of
which is aromatic. This term includes reference to groups such as
phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the
like.
[0053] The term "carbocyclyl" as used herein includes reference to
a saturated (e.g. cycloalkyl or cycloalkenyl) or unsaturated (e.g.
aryl) ring moiety having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 or 16 carbon ring atoms. A carbocyclic moiety is, for example,
selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
norbornyl, bicyclo[2.2.2]octyl, phenyl, naphthyl, fluorenyl,
azulenyl, indenyl, anthryl and the like.
[0054] The term "heterocyclyl" as used herein includes reference to
a saturated (e.g. heterocycloalkyl) or unsaturated (e.g.
heteroaryl)heterocyclic ring moiety having from 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which
is selected from nitrogen, oxygen, phosphorus and sulphur. A
heterocyclic moiety is, for example, selected from thienyl,
furanyl, tetrahydrofuryl, pyranyl, thiopyranyl, benzofuranyl,
pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl,
benzimidazolyl, pyrazolyl, pyrazinyl and the like.
[0055] The term "heterocycloalkyl" as used herein includes
reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7
ring carbon atoms and 1, 2, 3, 4 or 5 ring heteroatoms selected
from nitrogen, oxygen, phosphorus and sulphur. The group may be a
polycyclic ring system but more often is monocyclic. This term
includes reference to groups such as azetidinyl, pyrrolidinyl,
tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazoildinyl,
imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl,
thiomorpholinyl, quinolizidinyl and the like.
[0056] The term "heteroaryl" as used herein includes reference to
an aromatic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 or 16 ring atoms, at least one of which is selected from
nitrogen, oxygen and sulphur. The group may be a polycyclic ring
system, having two or more rings, at least one of which is
aromatic, but is more often monocyclic. This term includes
reference to groups such as pyrimidinyl, furanyl,
benzo[b]thiophenyl, thiophenyl, pyrrolyl, imidazolyl, pyrrolidinyl,
pyridinyl, benzo[b]furanyl, pyrazinyl, indolyl, benzimidazolyl,
quinolinyl, phenothiazinyl, triazinyl, oxazolyl, isoxazolyl,
thiazolyl, isoindolyl, indazolyl, isoquinolinyl, quinazolinyl and
the like.
[0057] The term "halogen" as used herein includes reference to F,
Cl, Br or I.
[0058] Typically, electrochemical sensors are based upon the
configuration of an electrochemical cell, with an electrolyte and
at least two electrodes, for example. In potentiometric
measurements, there is no current passing through the cell, and
these two electrodes are sufficient. A signal is measured as the
potential difference (voltage) between the two electrodes.
[0059] Amperometric sensors are a type of electrochemical sensor,
in which measurements are made by monitoring the current in the
electrochemical cell between a working electrode (also called a
sensing electrode) and a counter electrode (also called an
auxiliary electrode) at a certain potential (voltage). These two
electrodes are separated by an electrolyte. A current is produced
when the sensor is exposed to a medium containing an analyte
because the analyte reacts within the sensor, either producing or
consuming electrons (e.sup.-). That is, the analyte is oxidized or
reduced at the working electrode. The oxidation or reduction of the
analyte will cause a change in current between the working and
counter electrodes, which will be related to the concentration of
the analyte. Complementary chemical reactions will occur at each of
the working electrode and counter electrode. In suitable
applications, these reactions can be accelerated by an
electrocatalyst, such as a platinum electrode or another material
on the surface of the electrodes, or there can be a sacrificial
electrode process in which the electrode material is consumed, for
example with Ag/AgCl electrodes. For amperometric sensors, in a
cyclic voltammetry experiment, an external potential is applied to
the cell, and the current response is measured. Precise control of
the external applied potential is required, but this is generally
not possible with a two-electrode system, due to the potential drop
across the cell due to the solution resistance and the polarization
of the counter electrode that is required to complete the current
measuring circuit. Better potential control is achieved using a
potentiostat and a three-electrode system, in which the potential
of one electrode (the working electrode) is controlled relative to
the reference electrode, and the current passes between the working
electrode and the third electrode (the counter electrode).
[0060] The choice of suitable sensor arrangement and materials is
important when considering the moiety to be sensed, temperature
range and electrochemical method to be used. Amperometric sensors
have been found to enable low cost of components, small size, and
lower power consumption than other types of sensor, and are ideal
for use in portable analysis systems. In the present invention,
amperometric sensing methodology is typically employed.
[0061] In the present invention, phenols are generally detected
indirectly. In particular, the present invention involves the use
of a compound which operatively undergoes a redox reaction at the
working electrode, wherein the reaction has a detectable redox
couple and wherein the product of said reaction operatively reacts
in situ with the phenol. The electrochemical response of the
working electrode to the consumption of the said compound on
reaction with the phenol is then determined. The phenol may be
contacted with the compound prior to, contemporaneously with or
subsequent to the oxidation of the compound, but is typically
admitted subsequent thereto.
[0062] The term `phenol` as used herein Includes reference to
phenols, phenolic compounds and derivatives thereof.
[0063] The phenol may be, for example, phenol, 4-phenoxyphenol,
p-methylphenol, m-methylphenol, nitrophenol, tetrahydrocannabinol,
a component or metabolite of cannabis, a natural or synthetic
cannabinoid or metabolite thereof, or a catechin such as EGCG or
EGC. An example of a cannabis or cannabinoid metabolite is a
metabolite found in urine, and especially
11-nor-9-carboxy-9-tetrahydrocannabinol. The phenol is preferably
para-substituted.
[0064] The first compound may be a 4-aminophenol (or
p-aminophenol). In one embodiment, the first compound is a compound
of the formula (I):
##STR00004## [0065] wherein [0066] m is 0, 1, 2, 3 or 4; [0067]
each R.sup.1 is independently R.sup.2, or is hydrocarbyl or
heterocyclyl, either of which is optionally substituted with 1, 2,
3, 4 or 5 R.sup.2; [0068] each R.sup.2 is independently selected
from halogen, trifluoromethyl, cyano, nitro, oxo, .dbd.NR.sup.3,
R.sup.8, --OR.sup.3, --C(O)R.sup.3, --C(O)OR.sup.3, --OC(O)R.sup.3,
--N(R.sup.3)R.sup.4, --C(O)N(R.sup.3)R.sup.4, --S(O).sub.1R.sup.3
and --C(R.sup.3).sub.3; [0069] R.sup.3 and R.sup.4 are each
independently hydrogen, or are selected from Con alkyl,
--(CH.sub.2).sub.k-carbocyclyl and --(CH.sub.2).sub.k-heterocyclyl,
any of which is optionally substituted with 1, 2, 3, 4 or 5
substituents independently selected from halogen, hydroxy and Cue
alkyl; and [0070] l is 0, 1 or 2.
[0071] A compound of formula (I) is generally oxidised at the
working electrode to form a compound of formula (II):
##STR00005##
[0072] In one embodiment, m is 0. In another embodiment, m is at
least 1 (e.g. 1 or 2).
[0073] R.sup.1 may be hydrocarbyl, for example Cue alkyl (e.g.
methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or
tert-butyl), Cue alkyl substituted by aryl (e.g. benzyl),
cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl or
cyclohexyl) and aryl (e.g. phenyl, naphthyl or fluorenyl), any of
which may be substituted with 1, 2, 3, 4 or 5 R.sup.2.
[0074] Alternatively, R.sup.1 may be heterocycyl, for example
heterocycloalkyl (e.g. tetrahydrofuranyl) and heteroaryl (e.g.
furanyl, pyranyl, thiophenyl, benzothiophenyl), either of which may
be substituted with 1, 2, 3, 4 or 5 R.sup.2.
[0075] In another class of compounds, each R.sup.1 is C.sub.1-6
alkyl (e.g. methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or
tert-butyl) or C.sub.1-6 alkoxy (e.g. methoxy or ethoxy), either of
which is optionally substituted with, for example, halogen or
hydroxyl.
[0076] In a further class of compounds, each R.sup.1 is
carbocyclyl, for example aryl, optionally substituted with 1, 2, 3,
4 or 5 R.sup.2. In particular, each R may be phenyl optionally
substituted 1, 2, 3, 4 or 5 R.sup.2.
[0077] R.sup.1 may be R.sup.2, in which case R.sup.2 is typically
selected from halogen (e.g. chlorine), hydroxy, cyano, nitro, oxo,
carboxy, amino, alkylamino, dialkylamino and C.sub.1-6 alkyl (e.g.
methyl or ethyl).
[0078] In certain compounds, each R.sup.1 is independently selected
from --NR.sup.3R.sup.4, halogen, C.sub.1, C.sub.2, C.sub.3 or
C.sub.4 alkyl, C.sub.1, C.sub.2, C.sub.3 or C.sub.4 haloalkyl,
C.sub.1, C.sub.2, C.sub.3 or C.sub.4 alkoxy, and C.sub.2, C.sub.3
or C.sub.4 alkenyl, wherein R.sup.3 and R.sup.4 are each
independently selected from hydrogen, --OH, C.sub.1, C.sub.2,
C.sub.3 or C.sub.4 alkyl, C.sub.1, C.sub.2, C.sub.3 or C.sub.4
haloalkyl, C.sub.1, C.sub.2, C.sub.3 or C.sub.4 alkoxy, and
C.sub.2, C.sub.3 or C.sub.4 alkenyl.
[0079] In one class of compounds, each R.sup.1 is halogen, in
particular chlorine.
[0080] In a particular embodiment, the compound of formula (I) is a
compound of formula (IA) or (IB):
##STR00006##
[0081] The first compound may be present in the electrolyte and/or
on the working electrode and/or in the working electrode. In a
particular embodiment, the electrolyte comprises the first
compound. A working electrode comprising the first compound may be
obtained by immobilising the compound on the electrode from
solution, using a compound having a low solubility in the solvent.
Solubility of the compound may be optimised by controlling its
molecular weight. By way of example, 2,6-diphenylamino-phenol can
be immobilised on an electrode substrate from a solvent such as
acetonitrile. Alternatively, the first compound may be comprised in
the bulk of the electrode material.
[0082] The working electrode may be a screen printed electrode, a
metallic electrode, an edge plane pyrolytic graphite electrode, a
basal plane pyrolytic graphite electrode, a gold electrode, a
glassy carbon electrode, a boron doped diamond electrode, or a
highly ordered pyrolytic graphite electrode. The working electrode
may be a microelectrode or a macroelectrode.
[0083] Determination of the electrochemical response of the working
electrode may comprise measuring the current flow between the
working electrode and a counter electrode to determine the amount
of the phenol or phenolic compound. It is particularly preferred
that the working electrode is operatively maintained at a constant
voltage.
[0084] In one embodiment, the current is measured using linear
sweep or cyclic voltammetry. In another embodiment, said current is
measured using square wave voltammetry. In an alternative
embodiment, the current is measured using a pulsed voltammetry
technique, in particular differential pulse voltammetry.
[0085] The following Examples illustrate the invention.
Materials and Methods
[0086] All chemicals were of analytical grade and used as received
without any further purification. These were
.DELTA..sup.9-tetrahydrocannabinol (HPLC grade, >90%, ethanol
solution), 2,6-dichloro-p-aminophenol, phenol, 4-phenoxyphenol,
methylphenol (para and meta), nitrophenol,
4-amino-2,6-dichlorophenol (>98% Sigma-Aldrich),
epigallocatechin gallate (minimum 97%, Sigma-Aldrich),
epigallocatechin (minimum 98%, HPLC grade, Sigma-Aldrich) and
4-amino-2,6-diphenylphenol (>98%, Sigma-Aldrich). The green tea
leaf sample (Xlamen Tea IMP, & EXP. CO., LTD) was purchased
from a local Chinese supermarket.
[0087] Solutions were prepared with deionised water of resistivity
not less than 18.2 M Ohm cm (Millipore Water Systems). Voltammetric
measurements were carried out using a .mu.-Autolab II potentiostat
(Eco-Chemie) with a three-electrode configuration. Edge and basal
plane pyrolytic graphite electrodes (Le Carbons Ltd.) were used as
working electrodes. In the former case, discs of pyrolytic graphite
were machined into a 4.9 mm diameter, which was oriented with the
disc face parallel with the edge plane, or basal plane as required.
The basal plane pyrolytic graphite electrode was prepared by
renewing the electrode surface with cellotape. This procedure
involves polishing the bppg electrode surface on carborundum paper
(P100 grade) and then pressing cellotape on the cleaned bppg
surface which is removed along with attached graphite layers. This
was then repeated several times. The electrode was then cleaned in
water and acetone to remove any adhesive. The counter electrode was
a bright platinum wire, with a saturated calomel electrode
completing the circuit. The EPPG electrodes were polished on
alumina lapping compounds (BDH) of decreasing sizes (0.1 to 5
.mu.m) on soft lapping pads.
[0088] All experiments were typically conducted at 20.+-.2.degree.
C. Before commencing experiments, nitrogen (BOC) was used for
deaeration of solutions. Stock solutions of the substituted phenols
were prepared by dissolving the required substituted phenol in
ethanol.
Initial Voltammetric Characterisation of 4-amino-2,6-dichlorothenol
(PAP)
[0089] First, the voltammetric response of an eppg electrode in pH
3.4 buffer solution containing 1 mM 4-amino-2,6-dichlorophenol
(PAP) was explored. The corresponding voltammetry is shown in FIG.
1A. The first cyclic (dotted line) shows an oxidation peak at
ca.+0.36 V (vs. standard calomel electrode; SCE) with a
corresponding reduction peak at ca.+0.24V (vs. SCE) which is due to
the redox system of p-aminophenol-quinoneimine (PAP-QI), i.e.:
H.sub.2N--C.sub.6H.sub.2Cl.sub.2OH-2H.sup.+-2eHN.dbd.C.sub.6H.sub.2Cl.su-
b.2=O
or, equivalently:
##STR00007##
[0090] The reduction wave is smaller than the corresponding
oxidation peak, which is due to an electrochemical mechanism
occurring in which the quinoneimine (QI) is slowly hydrolysed to
form a benzoquinone (BQ):
##STR00008##
[0091] This mechanism has been previously studied on platinum and
mercury electrodes. A small peak is observed on the cathodic scan
at ca.+0.06 V, (see FIG. 1A) which is due to the reduction of BQ to
hydroquinone (HQ) (Hawley et al, 1965, J. Electroanal. Chem.,
10:376). At ca.-0.27 V (vs. SCE) a new wave appears; it has been
shown that this is due to the benzoquinone rapidly reacting with
PAP via 1,4-addition reactions, with the main product being
2,5-bis(4-hydroxyanilino)-p-benzoquinone. On the second
voltammetric scan, (FIG. 1A), some new `bumps` have appeared on the
voltammogram, which is likely due to the fouling of the electrode.
These, and the new waves occurring from the electrochemical
mechanism are well resolved from the main redox features of the
voltammogram, indicating that the PAP-QI redox couple may be used
as a marker from which to monitor the loss of QI as it reacts with
phenols, phenolic compounds and phenol derivatives.
[0092] Next the variation of the peak potential with pH was
explored. The cathodic and anodic waves were observed to shift
toward more negative potentials from increasing the pH. A plot of
formal potential against pH was observed to be linear from pH 0.84
to pH 7 with the gradient found to be 61 mV per unit (Ep=0.061
pH+0.57; R2=0.998) which suggests an n-electron, n-proton process
where n is likely to be 2. Beyond pH 7 the plot of peak potential
vs. pH was non-linear which is attributed to a pKa of 7.3
(calculated using ACD/Labs Sloaris V4.67 software) and is in
agreement with previous studies (Hawley et al, 1965, J.
Electroanal. Chem., 10:376; Salavagione et al, 2004, J.
Electroanal. Chem., 565:375; and Bramwell et al, 1990, Analyst,
115:185).
[0093] Cyclic voltammograms were recorded over a range of scan
rates as shown in FIG. 1B, with analysis of the peak height
(oxidation) versus square root of scan rate revealing a linear
dependence indicating a diffusing species. From this plot the
diffusion coefficient of 2,6-dichloro-p-aminophenol was estimated
to be 4.4 (.+-.0.3).times.10.sup.-6 cm s.sup.-1 (in pH 3.4
phosphate buffer) for n=2, which is in agreement with
4.8.times.10.sup.-6 cm s.sup.-1 reported for
2,6-dichloro-p-aminophenol in 2 M sulphuric acid (Adams, 1969,
Electrochemistry at Solid Electrodes, Marcel Dekker, New York).
[0094] The response of a gold macroelectrode was next sought so as
to compare with that of the edge plane pyrolytic graphite electrode
at the same pH; the results are shown in FIG. 1C. Equivalent
responses are observed on the gold substrate and an eppg electrode.
Also similar peak-to-peak separations are observed: 78 mV (at 100
mVs.sup.-1) at the gold and 85 mV (at 100 mVs.sup.-1) at the eppg
electrode. Both these results indicate quasi-reversible electrode
kinetics on each substrate.
[0095] From the literature a range of pH values has been
recommended as suitable for carrying out the Gibbs reaction. Gibbs
(1927, J. Biol. Chem. 72:649), Baylis (1928, J. Am. Water Works
Assoc., 19:597), Ruchhoft (1948, Anal. Chem., 20:1191) and
Theriault (1929, Ind. Eng. Chem., 21:343) suggested pH values of
9.1 to 9.5, 9.6 to 10, 9 to 10 and 9.4 respectively. It therefore
appears that a pH range of from 9 to 10 is optimised for rapid
completion of the Gibbs reaction. This is due to the required
hydrolysis of the Gibbs reagent (species 1 of Scheme 1) to yield
the dichloro-benzoquinone monoamine (species 2 of Scheme 1) which
then undergoes the Gibbs reaction by attacking the substituted
phenol. However, in the present invention this not need be a
pre-requisite since the dichloro-benzoquinone monoamine is
electrochemically generated and then reacts with the target
compound. This means, usually providing that the voltammetry is
well-resolved, that a method of the present invention is applicable
over a range of pH values. Above, acidic conditions have been
considered; an exploration the oxidation of the PAP at pH 10 at
both eppg and gold electrodes follows.
[0096] In pH 10 the electrochemical oxidation wave of the PAP, as
shown in FIG. 1D, has shifted to ca.+0.059 V (vs. SCE) at the eppg,
having an identical voltammetric profile to that observed at pH
3.4, while the oxidation wave is observed at ca.+0.064 V on the
gold electrode (FIG. 1E) is similar except with a new voltammetric
reduction peak at ca.+0.23 V. This is likely to be due to electrode
filming and close inspection of FIG. 1A reveals this is also
observed on the eppg electrode although to a much lesser extent.
Given the similar voltammetric response and inherent low cost of
eppg electrodes compared with that of the gold and other electrode
substrates, the use of eppg is considered to be particularly
desirable and eppg electrodes are used throughout in the following
Examples.
Example 1
Detection of Phenol
[0097] The electrochemical adaptation of the Gibbs reaction for the
detection of substituted phenols underlies the present invention
and is discussed in more detail below. A pH 10 buffer solution
containing 1 mM PAP was prepared and using a polished basal plane
pyrolytic graphite electrode the initial cyclic voltammetric
response was obtained. Note that either a polished bppg or an eppg
electrode may be used since the edge plane sites are responsible
for fast heterogeneous electron transfer kinetics and polishing of
the bppg electrode leads to the formation of significant amounts of
edge plane defects. Additions of phenol were made over the range of
50 to 250 .mu.M to the solution with the observed response depicted
in FIG. 2. Three important features are evident the first is that
the reduction peak at ca.-0.06 V has decreased with increasing
phenol additions; second, the oxidation peak at ca.+0.05 V has
decreased with phenol additions; and thirdly there is a new
oxidation wave at ca.+0.46 V which slightly shifts in potential and
grows with each addition of phenol.
[0098] The new peak at ca.+0.46 V was explored by examining the
voltammetry of 1 mM phenol in a pH 10 buffer solution. FIG. 3 shows
that an oxidation wave is observed at ca.+0.5 V corresponding to
the electrochemical oxidation of phenol. On successive scans, the
peak diminishes. After the first scan, the background current has
increased which indicates that probably electrode passivation has
occurred. It is also likely that the new wave observed in FIG. 2 is
a combination of the direct oxidation of phenol and/or polymeric
species from the oxidation of aminophenol. In either case this
feature is well resolved from the PAP-QI redox couple. Returning to
FIG. 2, the analysis of the decreasing peak height (I.sub.H) at
ca.-0.06 V versus added phenol concentrations produced the
following linear regression data: I.sub.H=0.19
[(phenol/M)]+1.12.times.10.sup.-4; R.sup.2=0.98, N=5. This suggests
that the diminishing reduction wave can provide a simple analytical
methodology for the indirect detection of phenol and phenolic
compounds.
[0099] The response of PAP to increasing additions of phenol using
square-wave voltammetry (SWV) at an edge plane pyrolytic graphite
electrode was then explored, with a view to increasing the
sensitivity of the protocol. SWV was used because this technique
has an increased sensitivity over linear sweep (or cyclic
voltammetry) due to the fact that the former is a measure of the
net current, which is the difference between the forward and
reverse current pulses. Also, using SWV, only one peak is observed
allowing one to easily monitor the reduction of the voltammetry
peak on additions of the phenol compound. First, however, the
square-wave parameters were optimised. Using a pH 10 buffer
solution containing 1 mM PAP, the frequency and step potential were
each in turn changed to find the optimum peak height. This was
found to occur when the frequency was 8 Hz, the step potential 10
mV and the amplitude 25 mV.
[0100] Using these parameters the square-wave voltammetric response
from an eppg electrode was sought in a pH 10 buffer solution
containing 1 mM PAP. The voltammogram was cycled until the peak had
stabilised--which is typically after two cycles--after which phenol
additions were made to the solution. As depicted in FIG. 4A, the
well-defined voltammetric response was found to decrease with added
phenol concentrations. Analysis of the peak current versus added
phenol concentration was found to be highly linear from 50 to 480
.mu.M (I.sub.H=-0.198 [(phenol/M)]+1.26.times.10.sup.-4;
R.sup.2=0.997, N=10) which is also shown in FIG. 4B. From this a
limit of detection (3.sigma.) was found to be 15.3 .mu.M. Given the
simplicity of the SWV technique this was used throughout the
following.
[0101] Characterisation of the wave at ca.-0.2 V (FIG. 4A) was
investigated by exploring the voltammetry of hydroquinone in a pH
10 buffer solution. Using an eppg electrode, a well-defined redox
couple was observed, corresponding to the oxidation of HQ to BQ as
depicted in FIG. 1F. For clarity the voltammetric response of the
oxidation of PAP in pH 10 buffer at an eppg is overlaid. This also
helps `fingerprint` the new voltammetric features found when PAP is
oxidised in aqueous solution, according to the mechanism shown in
Scheme 2. Overall this demonstrates that the small voltammetric
wave at ca.-0.2 V in FIG. 4A is due to the reduction of
benzoquinone to hydroquinone formed via the hydrolysis of oxidised
PAP as described above.
[0102] The initial concentration of PAP was explored to see if it
was possible to extend the linear range of the phenol analysis or
increase the sensitivity of the technique. The above experiment was
repeated but with the concentration of PAP lowered to 0.1 mM with
phenol additions made to the solution over the same linear range.
Linear regression from analysis of the peak height versus added
phenol concentration (I.sub.H=-0.036
[(phenol/M)]+2.82.times.10.sup.-5 10; R.sup.2=0.987, N=10) revealed
that the sensitivity (gradient) was lower than that observed using
a initial 1 mM concentration of PAP. Conversely using an Initial 10
mM concentration of PAP, produced an identical sensitivity and
linear range as that seen using an initial 1 mM concentration.
[0103] As mentioned above, edge plane sites are responsible for
fast heterogeneous electron transfer kinetics, with polishing of
the bppg electrode leading to the formation of significant amounts
of edge plane defects meaning that either a polished bppg or an
eppg can be used as a sensor for the indirect determination of
substituted phenols and phenolic compounds. This is exemplified by
the following experiment.
[0104] A basal plane pyrolytic graphite electrode was prepared by
polishing with alumina lapping compounds, thereby exposing edge
plane sites. The polished bppg electrode was placed into a pH 10
buffer solution containing 1 mM PAP with the initial
SW-voltammetric response sought, after which additions of phenol
were made. Analysis of the peak height versus added phenol
concentration produced a linear response (I.sub.H=-0.188
[(phenol/M)]+1.74.times.10.sup.-4; R.sup.2=0.99, N=9) from 50 to
430 .mu.M, which is essentially identical to that observed above
using the edge plane pyrolytic graphite electrode. This reiterates
the notion that edge plane sites are responsible for the fast
electrode kinetics and consequently either a polished bppg or eppg
electrode can be used to monitor the voltammetric response.
[0105] A control experiment was performed where identical volume
sized additions were made of either water or ethanol to a pH 10
buffer solution containing 1 mM PAP without any phenol present. No
significant reduction in the PAP voltammetric peak was observed for
either the water or the ethanol additions. This indicates that
neither dilution effects nor reaction with ethanol were responsible
for the decrease in the voltammetric response of the PAP as
observed in FIG. 4A. Rather, the latter arises purely from the
Gibbs reaction of phenol with QI.
[0106] As described earlier, the Gibbs reagent has previously been
used spectrophotometrically to detect substituted phenols where it
has been observed that the most easily displaced substitutents
(good anionic leaving groups) give rise to high yields of
dichloroindophenol, while methylphenol and longer alkyl group
substitutions, such as hydroxybiphenyl, ethylphenol and
hydroxybenzoic acid, gave no detectable coloured product (Josephy
et al, supra).
[0107] It has been reported that phenol and phenoxyphenol give good
yields of coloured products (60 and 63% respectively), methylphenol
gives a low yield (18%) while nitrophenol produces no positive
result in a Gibbs reaction (Josephy et al, supra). However, this
technique is based on spectrophotometric observation of the product
of the Gibbs (or related) reaction. In the present invention it is
the loss of the benzoquinone monoamine as it reacts with the
substituted phenol of choice which is observed. Therefore, in the
following Examples, a range of substituted phenols is assessed for
use with the method of the invention.
Example 2
Detection of 4-phenoxyphenol
[0108] The method of Example 1 was repeated using SWV at an eppg
electrode for the detection of 4-phenoxyphenol. The SW-voltammetric
responses are shown in FIG. 5A with analysis. Analysis of the peak
height versus added 4-phenoxyphenol was found to produce a linear
range from 50 .mu.M to 244 .mu.M (I.sub.H=-0.51
([4-phenoxyphenol]/M)+1.2.times.10.sup.-4 A; R.sup.2=0.98, N=6).
The last voltammetric wave, as shown in FIG. 5A has disappeared
indicating complete reaction of the 4-phenoxyphenol with the
electrochemically generated dichloro-benzoquinone monoamine. In
comparison, spectrophotometric methods have reported a 63% yield of
dichloroindophenol (Josephy et al, supra). Finally, from the above
linear regression data, a limit of detection was found to be 34
.mu.M.
[0109] As discussed in section above, the Gibbs reaction requires
an optimised pH of 9-10 to facilitate the hydrolysis of the Gibbs
reagent to yield dichloro-benzoquinone monoamine (species 2 of
Scheme 1) which then undergoes the Gibbs reaction by attacking the
substituted phenol. Since the methodology of the present invention
does not require such a hydrolysis step the method is considered to
able to work at both basic and acidic conditions. This was further
explored as described below.
[0110] The above method for the indirect detection of
4-phenoxyphenol was repeated using SWV at an eppg electrode at pH
3.4. From analysis of the decreasing peak height versus added
phenoxyphenol, two linear ranges were observed. The first was found
to occur from 50 .mu.M to 291 .mu.M (I.sub.H=-0.15
([4-phenoxyphenol]/M )+1.6.times.10.sup.-4 A; R.sup.2=0.99, N=7),
while the second was from 130 .mu.M to 264 .mu.M (I.sub.H=-0.47
([4-phenoxyphenol]/M)+2.44.times.10.sup.-4 A; R.sup.2=0.998, N=6).
A similar gradient in comparison to the response obtained in pH 10
buffer is observed suggesting that the method of the invention can
be applied in both acidic and basic conditions.
Example 3
Detection of p-cresol and m-cresol (Methylphenol)
[0111] FIGS. 5B and C shows the response of additions of either
p-cresol and m-cresol respectively to a 1 mM solution of PAP in pH
10 buffer solution using SW-voltammetry at an eppg electrode. In
both cases, analysis of the decreasing wave versus additions of the
respective cresol were found to be linear (I.sub.H=-0.21
([p-cresol]/M)+1.08.times.10.sup.-4 A; R.sup.2=0.9895, N=8) from 99
.mu.M to 431 .mu.M for p-cresol, while m-cresol produced a linear
response from 100 .mu.M to 385 .mu.M (I.sub.H=-0.29
[(m-cresol/M)]+1.25.times.10.sup.-4; R.sup.2=0.99, N=8). Note that
in all cases, the addition of the substituted phenol of choice is
continued until the voltammetric peak stops diminishing indicating
that the reaction of the substituted phenol with the
electrogenerated dichloro-benzoquinone monoamine has ceased. From
the above linear regression data, the limit of detection (3.sigma.)
was found to be 32 .mu.M for p-cresol and 30 .mu.M m-cresol.
Example 4
Detection of p-Nitrophenol
[0112] The reaction of p-nitrophenol with the Gibbs reagent has
been reported spectrophotometrically not to occur, i.e. no
dichloroindophenol was observed using spectrophotometry (Josephy et
al, supra). However, as described above, the method of the
invention, although taking inspiration from the Gibbs reaction, is
different in that it is based on monitoring the loss of the
electrogenerated dichloro-benzoquinone monoamine as it reacts with
the substituted phenol of choice.
[0113] Using the method of the invention, the response of additions
of nitrophenol was explored. As depicted in FIG. 5D, the system
responds to additions of nitrophenol with a linear response from 50
.mu.M to 385 .mu.M (I.sub.H=-0.067
[(p-nitrophenol/M)]+1.29.times.10.sup.-4; R.sup.2=0.98, N=6), but
does not achieve a high sensitivity in comparison to the
substituted phenols studied above. This is likely to be due to the
presence of the poor NO.sub.2.sup.- leaving group. The limit of
detection (3.sigma.) was found to be 40 .mu.M.
[0114] In summary, it has been observed that the most easily
displaced leaving groups give rise to good sensitivities e.g.
(phenoxy) while for poor leaving groups e.g. (NO.sub.2) the
sensitivity is not so good. Nevertheless, in the latter case,
analysis by a method of the invention is still possible in
situation where the classical (calorimetric) Gibbs reaction
fails.
Example 5
Detection of Tetrahydrocannabinol (THC)
[0115] Using cyclic voltammetry, the electrochemical response at an
eppg electrode of the electrochemical oxidation of 1 mM PAP in a pH
10 buffer solution was established. Additions of THC were made over
the range of 100-476 .mu.M to the solution with the observed
response depicted in FIG. 6. As observed for the phenol additions
in the preceding Examples, the reduction peak decreased with
increasing THC additions, indicating that the protocol works as an
indirect methodology for the detection of THC, the active part of
cannabis. This result was quantified using square
wave-voltammetry.
[0116] Using a 1 mM solution containing 4-amino-2,6-dichlorophenol
in a pH 10 buffer solution, the initial SW voltammetric response
was obtained using an edge plane pyrolytic graphite electrode. The
response of additions of tetrahydrocannabinol was explored. As
depicted in FIG. 7A, the voltammetric peak was found to decrease
with increasing additions of THC. Analysis of the peak height
versus added THC concentrations revealed two linear parts of the
calibration curve (FIG. 7B). The first part was linear from 50 to
245 .mu.M (I.sub.H=0.148 [(THC/M)]+1.21.times.10.sup.-4;
R.sup.2=0.994, N=6) with the second from 290 to 476 M
(I.sub.H=-0.337 [(THC/M)]+1.71.times.10.sup.-4; R.sup.2=0.997,
N=5). From this, the limit of detection (3.sigma.) was found to be
25 .mu.M.
Example 6
Detection of EGCG and EGC at an eppg Electrode
[0117] A 1 mM solution containing 2,6-dichloro-p-aminophenol (AP)
in a pH 10 buffer was first prepared and examined with cyclic
voltammetry at an eppg electrode. The voltammetric response is
depicted in FIG. 8 (dotted line) which exhibits an oxidation and
reduction with peak potentials at ca.+0.08 V and ca.-0.07 V (vs.
SCE) respectively, which are due to the redox couple of the
aminophenol-quinoneimine (PAP-QI) system. A peak-to-peak separation
of 150 mV was observed (at 100 mVs.sup.-1), indicating
quasi-reversible electrode kinetics, 1 mM additions of EGCG were
then made to the buffer solution, which, as observed in FIG. 8,
results in the reduction peak of voltammetric profile to decrease.
This reduction in the size of the voltammetric peak is due to the
loss of the quinoneimine (QI), which reacts with EGCG to form an
adduct (see FIG. 9).
Example 7
Detection of EGCG and EGC at a Modified bppg Electrode
[0118] Bppg electrodes were modified with 2,6-diphenylamino-phenol
("diphenyl-AP") and subsequently used to detect EGCG and EGC.
Diphenyl-AP has the same required electrochemical functionalities
as other PAPs but has two diphenyl groups so greatly reducing the
compound's solubility in aqueous solutions. The diphenyl-AP
compound was immobilised onto the electrode surface by taking a
freshly prepared bppg electrode and immersing into a solution of 1
mM diphenyl-AP in acetonitrile for five minutes after which the
electrode was taken out and gently washed with distilled water.
Typically, the peak current after stabilisation was found to be an
average of 25 (.+-.10) .mu.A which likely reflects the variation in
surface roughness of the bppg electrode each time the electrode is
prepared.
[0119] The modified bppg electrode was placed into a pH 10 buffer
solution where, using square wave voltammetry, the potential was
held at +0.2 V (vs. SCE) for 5 seconds, followed by sweeping the
potential from +0.2 V to -0.4 V (vs. SCE). Square wave voltammetry
was chosen since it provides an easy way to monitor the loss of the
voltammetry peak on additions of the catechin compounds. A large
reduction wave having a peak maximum at ca.-0.11 V (vs. SCE) was
initially observed. The square wave protocol was continuously
repeated to assess the stability of the voltammetric peak. It was
found that 12 cycles were usually needed to stabilise the peak,
which was found to typically decrease by ca. 30% of its initial
value. The 12 cycles indicate that the modified electrode needs a
pretreatment to be applied which need last not longer than 60
seconds at +0.2 V (vs. SCE).
[0120] The response of the diphenyl-AP modified bppg electrode
toward EGCG additions in a pH 10 buffer solution was next
investigated. FIG. 10 shows the response of 1.7 .mu.M additions of
EGCG where analysis of the peak height (I.sub.H) versus added EGCG
concentration, as shown in FIG. 10B produces substantially linear
range from 3 .mu.M to 32 .mu.M with the following linear
regression: I.sub.H/A=-0.29 [(EGCG/M)]+2.5.times.10.sup.-5 A;
R.sup.2=0.98; N=19. The potential range was well resolved from that
of the direct electrochemical oxidation of EGCG (which occurs at
ca.+0.72 V vs. Ag/AgCl; see Kumamoto et al, Anal. Sci. 16, 2000,
139), thus removing any possibility of electrode fouling caused by
direct oxidation.
[0121] The response of 1.7 .mu.M additions of EGC into a pH 10
buffer solution using the diphenyl-AP modified bppg electrode was
then explored. FIG. 11A shows the square-wave voltammetic profiles,
which clearly diminish as EGG is added into the solution. Analysis
of the peak height versus added EGG concentration is depicted in
FIG. 11B, where two linear ranges are observed; the first from 1.7
.mu.M to 10 .mu.M (I.sub.H/A=-2.7 [(EGC/M)]+6.9.times.10.sup.-5 A;
R.sup.2=0.98; N=6) and the second from 10 .mu.M to 32 .mu.M
(I.sub.H/A=-0.9 [(EGG/M)]+5.1.times.10.sup.-5 A; R.sup.2=0.99;
N=8). The modified bppg electrode was explored with additions made
over the range 0.8 .mu.M to 8.3 .mu.M from a solution consisting of
both EGCG and EGG at the same concentration which produced the
following linear regression: I.sub.H/A=-0.85
[(EGCG+EGG/M)]+3.2.times.10.sup.-5 A; R.sup.2=0.97; N=10.
Comparison of this linear regression with that obtained from the
additions of EGCG and EGG reveal an identical response.
[0122] The above experiments demonstrate that the
diphenyl-AP-modified bppg electrode was successful in detecting the
anti-carcinogenic catechin compounds EGCG and EGG, obviating the
need to add the aminophenol into the solution. The modification of
the electrode avoids the need to dissolve the aminophenol compound
into the solution phase. For example, this methodology could be
utilised in end of column detectors thus obviating the need to
dissolve the electrochemical marker into the carrier solution.
Example 8
Detection of EGCG and EGC in Green Tea
[0123] EGCG and EGC were detected in a sample of green tea. A 1.97
g sample of green tea (Xiamen Tea IMP, & EXP. CO., LTD) was
placed into 100 mL of boiling distilled water, constantly stirred
and held at a rolling boil for 40 minutes to allow the tea to
infuse. The tea infusion was allowed to cool and was consequently
filtered. This solution was then diluted 1:1 with pH 10 buffer.
[0124] A phenyl-AP modified bppg electrode was prepared (see
Example 7) and placed into the tea sample. Using square-wave
voltammetry, the electrochemical response of additions of 0.7 .mu.M
EGCG and EGC (made up in the same solution) was explored. Typical
square-wave voltmmograms are depicted in FIG. 12, where the
additions of EGCG and EGC made to the green tea sample results in a
decrease in the voltammetric profile. A standard addition plot of
peak height versus added EGCG and EGC concentration is shown in
FIG. 12B. Analysis using the standard addition protocol reveals
that 180 (.+-.5) mg of EGCG and EGC exists in 1 g of green tea.
This content of catechins is in the same order of magnitude as that
reported by Pelillo et al above, who explored five green tea
samples with HPLC coupled with U and MS-electrospray detection.
They found that the total amount of catechins varied from 90 mg/g
to 760 mg/g depending on the source of the green tea and where the
total amount of EGCG and EGC were found to vary between 26 to 412
mg/g and 12 to 100 mg/g respectively in the green tea sample.
[0125] Throughout the description and claims of this specification,
the words `comprise` and "contain" and variations of the words, for
example "comprising" and "comprises", means "including but not
limited to", and is not intended to (and does not) exclude other
moieties, additives, components, integers or steps.
[0126] Throughout the description and claims of this specification,
the singular encompasses the plural unless the context otherwise
requires. In particular, where the indefinite article is used, the
specification is to be understood as contemplating plurality as
well as singularity, unless the context requires otherwise.
[0127] Features, integers, characteristics, compounds, chemical
moieties or groups described in conjunction with a particular
aspect, embodiment or example of the invention are to be understood
to be applicable to any other aspect, embodiment or example
described herein unless incompatible therewith.
* * * * *