Spectroscopic study on the inclusion complexes of β-cyclodextrin with selected metabolites of catecholamines

Article abstract of DOI:10.1016/j.molstruc.2016.08.010

Inclusion complexes formed between β-cyclodextrin (β-CD) and metabolites of catecholamines, i.e. vanillylmandelic acid (VMA), homovanillic acid (HVA) as well as vanillin (VA) were studied using NMR spectroscopy. Due to the importance of these compounds fo

Full text of DOI:10.1016/j.molstruc.2016.08.010

Accepted Manuscript
Spectroscopic study on the inclusion complexes of β-cyclodextrin with selected
metabolites of catecholamines
Anna Korytkowska-Wałach, Beata Dubrawska, Monika Śmiga-Matuszowicz, Tadeusz
Bieg
PII:
S0022-2860(16)30840-7
10.1016/j.molstruc.2016.08.010
MOLSTR 22836
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 June 2016
Revised Date: 28 July 2016
Accepted Date: 6 August 2016
Please cite this article as: A. Korytkowska-Wałach, B. Dubrawska, M. Śmiga-Matuszowicz, T. Bieg,
Spectroscopic study on the inclusion complexes of β-cyclodextrin with selected metabolites of
catecholamines, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.08.010.
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Kassoc
Kassoc
Kassoc

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Spectroscopic study on the inclusion complexes of
β-cyclodextrin with selected metabolites of catecholamines
Anna Korytkowska-Wałach1*, Beata Dubrawska1, Monika Śmiga-Matuszowicz2,
Tadeusz Bieg1
Address: 1Department of Organic Chemistry, Bioorganic Chemistry and
Biotechnology, Silesian University of Technology, Krzywoustego 4, 44-100 Gliwice,
Poland
2Department of Physical Chemistry and Technology of Polymers,
Silesian University of Technology, Strzody 9, 44-100 Gliwice, Poland
* Corresponding author
Email addresses:
anna.walach@polsl.pl (Anna Korytkowska-Wałach)
bkubarska@gmail.com (Beata Dubrawska)
monika.smiga-matuszowicz@polsl.pl (Monika Śmiga-Matuszowicz)
tadeusz.bieg@polsl.pl (Tadeusz Bieg)
Abstract
Inclusion complexes formed between β-cyclodextrin (β-CD) and metabolites of
catecholamines, i.e. vanillylmandelic acid (VMA), homovanillic acid (HVA) as well as
vanillin (VA) were studied using NMR spectroscopy. Due to the importance of these
compounds for the diagnosis tumours of the sympathoadrenal system, hydrogels
containing β-CD moieties for enhancing entrapping metabolites of catecholamine
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from aqueous solutions are located in the area of our interest. Stoichiometry and
association constants of the complexes of β-CD with VMA, HVA and VA respectively
were determined by using continuous variation and 1H NMR titration methods.
Significant discrepancies were pointed out depending on used referencing method. In
this study water solution of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt as
an external reference was used to avoid errors in the determination of association
constants. β-CD formed the most stable complexes with VA and HVA molecules
whilst smallest value of association constant was determined for the VMA/β-CD
complex. Two-dimensional rotating-frame Overhauser effect spectroscopy (2D
ROESY) allowed to establish definite information on the molecular structures of the
complexes formed. Geometry of the latter was proposed basing on contour plots of
the 2D ROESY spectra, which also indicated two possibilities of complexed molecule
arrangement into β-cyclodextrin interior. The values of determined association
constants are in good agreement with postulated geometry of the complexes.
Value of association constant determined for inclusion complexes of β-cyclodextrin
with homovanillic acid an vanillin indicates the strongest binding of molecules among
investigated complexes, so it was finally concluded that β-CD moiety introduced into
hydrogel network could be effective for homovanillic acid and vanillin entrapping.
Keywords:
β-cyclodextrin; metabolite of catecholamine; inclusion complex; association constant;
ROESY
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1. Introduction
Cyclodextrins (CDs) are known to form inclusion complexes with variety of
guest compounds, having hydrophobic moieties of appropriate size. Inclusion
complexes of cyclodextrins with 1:1 or 1:2 guest-host stoichiometry are the most
extensively studied but non-inclusion complexes have also been described [1].
An inclusion phenomenon of CDs originates from their structure. Cyclodextrins are a
group of cyclic oligomers, composed of six or more α-D-glucopyranose units. In this
study, we have used β-cyclodextrin (β-CD) containing 7 units (Scheme 1). Due to the
chair conformation of the glucopyranose units, the CDs have truncated cone shape.
The primary hydroxyl groups are oriented towards the narrower cone exterior whilst
secondary hydroxyl groups towards the wider edge. Interior of cyclodextrins is
somewhat hydrophobic thus water molecules in CD cavity are energetically
unfavoured and therefore can be readily substituted by less polar guest molecules.
2OH 3
OH
H 2
H4
H 3
OH
6
H 1
O
H2
H5
H1
H 5
HO
OH
H 4
H3
O
7
6
H2
OH 6
Scheme 1
A wide variety of analytical methods was employed for characterization of
inclusion complexes formed between the guest and cyclodextrin molecules both in
the solid and solution state. The most direct evidence for the inclusion of a guest into
a CD cavity in solution is obtained by NMR spectroscopy [2]. The NMR techniques
enable determining stoichiometry, association constants, structural characterization
of CDs complexes and allow to analysis of their dynamics (for review, see [3,4]).
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Cyclodextrins, due to their ability to form inclusion complexes with selective
molecules, achieved great significance in molecular recognition processes.
Moreover, CDs are chiral and exhibit chiral recognition [5]. Native, modified CDs or
CDs moieties introduced into polymers have been used as masking agent and
stabilizer in food and cosmetics [6], in chromatographic separations, drug delivery
systems (as drug solubilizing agent), hydrogels formed in situ [7], molecularly
imprinted polymers [8].
We have considered the use of hydrogels with β-CD moieties for enhancing
entrapping metabolites of catecholamine from aqueous solutions. Catecholamines
are biogenic amines which play an important role in the nervous systems. Some
catecholamines as well as their metabolites are used as markers of certain types of
tumours. Exemplary quantification of catecholamines or their metabolites [dopamine,
norepinephrine, epinephrine, vanillylmandelic acid (VMA) (I, Scheme 2) and
homovanillic acid (HVA) (II, Scheme 2)] in biological fluids is important in the
diagnosis of neuroblastoma as well as the other catecholamine secreting malignancy
such as pheochromocytomas. Neuroblastoma is a cancer of the symphathetic
nervous system and is the most common solid tumor in childhood. More than 90 % of
patients afflicted with neuroblastoma exhibit elevated levels of dopamine, VMA and
HVA in serum and urine [9]. Pheochromocytomas are endocrine tumours and are
characterized by elevated levels of urinal metanephrines (norepinephrine and
epinephrine) and vanillylmandelic acid [10]. Moreover, excretion rate of VMA with
urine is also informative and of great interest in characterization the effects of many
drugs.
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OH
O
H3CO
HO
O
H3CO
HO
O
H3CO
HO
H
OH
OH
III
I
II
Scheme 2
Materials composed of N-isopropylacrylamide, methacrylic acid or itaconic acid and
crosslinking agent were demonstrated recently as materials for dopamine recognition
[11]. Designing hydrogels with β-CD moieties for entrapping VMA and HVA is the
subject of our present study. In this paper we report a NMR study of the β-CD
inclusion complexes with VMA and HVA, formed in water. Additionally, inclusion
complexation of vanillin (VA) (III, Scheme 2) by β-CD was studied because of that
vanillylmandelic acid readily undergoes oxidation in mild condition to vanillin. In the
case of obtaining effective materials for binding vanillin, indirect determination of
VMA could be also possible.
The inclusion of vanillin by β-cyclodextrin in water was confirmed by NMR by
Divakar [12] and Pîrnău group [13]. Complexes of β-cyclodextrin with homovanillic
acid and other natural polyphenols were also investigated by NMR, thermodynamic
and molecular modeling studies by A. Rescifina group [14]. Association constants
were determined from NMR analysis in all abovementioned cases. L. A. Kartsova et
al. used Capillary Zone Electrophoresis for association constants determination of
complexes of i.a. β-CD with VMA and HVA [15].
In this article complexes of β-CD with selected metabolites of catecholamines and
vanillin were characterized. Their geometry and stability in water solution were
studied in details in order to evaluate the usefulness of the β-CD moieties in binding
process of the VMA, HVA and VA molecules in aqueous environment.
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2. Experimental
2.1. Materials
Vanillylmandelic acid, homovanillic acid and vanillin were supplied by Acros Organics
(Chemiatrade, Gliwice, Poland). Deuterium oxide was obtained from The
Radioisotope Production and Distribution Centre (Świerk, Poland). β-cyclodextrin and
3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP) were purchased from
Sigma-Aldrich Co. (Poznań, Poland).
All chemicals and solvents were of commercial grade and used as received.
2.2. NMR experiments
NMR spectra were recorded at 25 °C with the aid of UNITY/INOVA 300 MHz or 600
MHz spectrometers (Varian). All tested solutions were prepared one day prior the
measurements.
2.2.1. Job’s plot
Equimolar solutions (10 mM) of the β-CD and one of complexed molecule (CM):
VMA, HVA or VA in D2O were prepared respectively and distributed among 11 NMR
tubes in such a way that the molar fractions of β-CD varied between 0:1 and 1:0 (with
a constant sample volume of 0.6 mL). Solution of 3-(trimethylsilyl)propionic-2,2,3,3-d4
acid sodium salt (TSP) in D2O placed in melt-point capillary was used as an external
reference. Standard 1H NMR spectra were acquired with 48 scans and the relaxation
delay of 3s. The widths at half-height of TSP signals were less than 1 Hz. Values of
the molar fractions were recalculated from appropriate 1H NMR signals integration.
2.2.2. Titration studies for evaluation of association constants
Two stock solutions were prepared in D2O: first containing the β-CD (10 mM), second
containing the β-CD (10 mM) and the VMA, HVA or VA respectively (50 mM). These
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stock solutions were mixed to a constant volume (6 mL) keeping the β-CD
concentration equal to 10 mM and ranging the CM/β-CD nominal ratio from 1.0 to
5.0. Solution of TSP in D2O placed in capillary was used as an external reference.
Standard 1H NMR spectra were acquired with 48 scans and the relaxation delay of
3s. The widths at half-height of TSP signals were less than 1 Hz. Experiments were
carried out in triplicate. Values of molar ratio of CM to β-CD were recalculated from
appropriate 1H NMR signals integration.
2.2.3. 2D ROESY
For two-dimensional rotating-frame Overhauser effect spectroscopy (2D ROESY)
measurements solutions of β-CD (15 mM) and the VMA, HVA or VA (60 mM) were
prepared. No reference was added and chemical shifts were set based on the
residue water signal at 4.790 ppm [16]. ROESY experiments were carried out on 600
MHz spectrometer using a mixing time of 500 ms.
3. Results and Discussion
3.1. NMR chemical shift referencing
One of the fundamental and most important factor for a correct analysis of β-
cyclodextrin complexes in aqueous solution by the NMR technique is the selection
and properly using chemical shift reference. This aspect has been discussed in the
literature [17,18]. More than twenty years ago Q.X. Guo et. al. signalized that
tetramethylsilane (TMS) and sodium salt of 3-(trimethylsilyl)propane-1-sulfonic acid
(DSS) cannot be used as the internal reference, and suggested to use DSS water
solution rather than TMS as the external reference for cyclodextrin species in
aqueous solution for NMR measurement [17]. Meanwhile one can meet up with
practice of using DSS, TSP or TMS as internal reference, despite the fact that β-CD
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can form an inclusion complexes with all these standards. In the case of TMS
molecule complexation by β-CD increase the solubility of TMS in water threefold [18].
Frequently chemical shifts are referenced to the residual water signal which is very
temperature depended and sensitive to acidic impurities [19]. We decided to use
external reference (TSP solution in D2O) rather than residual water signal referencing
for stoichiometry and association constant determination. The reason is that we have
observed upfield shift of the HOD signal relative to TSP solution as external
reference with increasing complexed molecule content. In our case the sample and
reference are both diluted solutions in the same solvent, so the correction for the
change of bulk magnetic susceptibility is negligible [20].
3.2. Stoichiometry and association constants of the complexes
The inclusion complexes: VMA/β-CD, HVA/β-CD and VA/β-CD were
characterized in D2O by 1H NMR spectroscopy. In these systems, a fast exchange
regime between the complexed and non-complexed species was observed in the
NMR time-scale. In this case observed chemical shift (δobs) is the mole fraction
weighted average of the shifts observed in the free and complexed molecule [21].
Stoichiometry of complexes of β-CD with VMA, HVA and VA respectively were
determined by the Job’s method of continuous variation method. This is commonly
used procedure for determining the composition of complexes in solutions which
requires preparation of series of solution containing host and guest molecules. The
mole ratios of total concentration of complexed molecule (CM), i.e.: xVMA, xHVA and
xVA respectively, is setting between 0 and 1. Job’s plots prepared on the basis of
chemical shifts of the signals of the β-CD’s H3 are presented in Figure 1. The
maximum visible for equimolar composition of β-CD and appropriate complexed
molecule indicated 1:1 stoichiometry in all cases.
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0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0.000
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.000
0.025
VMA
VA
HVA
0.020
0.015
0.010
0.005
0.000
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
xVMA
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
xHVA
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
xVA
Figure 1: Job’s plots for the interaction between β-CD and VMA, HVA and VA
respectively in D2O by monitoring chemical shifts of the H3 signal of the β-CD
Consequently, for the determination of the association constants (Kassoc) the following
equilibrium was assumed:
[ꢄꢅ/βꢆꢄꢇ]
CM + β-CD = CM/β-CD
Kꢀꢁꢁꢂꢃ
=
(1)
[
]
ꢄꢅ [βꢆꢄꢇ]
Vanillylmandelic acid, homovanillic acid and vanillin have better solubility in
water than β-cyclodextrin. This is why the concentration of the β-CD was keeping
constant during experiments whilst VMA, HVA or VA was varied. Complexation
induced chemical shift δobs was traced and Kassoc values were calculated basing on
the H3 and H5 protons of β-CD signal in 1H NMR spectra respectively. The
aforementioned signals were selected because of the highest change of the chemical
shift among all protons. In our study we have assumed similarly to L. Fielding [21]
that the guest molecule is the NMR observed species. Therefore in such an
approach, commonly used terms for guest and host are switched and β-CD is treated
as guest molecule (G) whilst symbol (H) denotes to the one of the complexed
molecules (VMA, HVA or VA).
The association constants of the β-CD with VMA, HVA or VA were calculated by
using curve fitting method, which requires no approximations and allow unrestricted
distribution of reagents. The data obtained from the titration experiments were fitted
to a 1:1 binding model, using WinEQNMR computer program [22]. Exemplary data
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fitting for the 1H NMR titration of β-CD with VMA, HVA and VA respectively with
residuals showing deviation from the best fit are collected figure 2.
VMA
VA
HVA
H3
H5
Figure 2: Exemplary plots with experimental data and calculated chemical shifts of
signals of the H3 (upper) and H5 (bottom) of the β-CD together with the residues in
magnified form (WinEQNMR program)
The corresponding values of association constants (Kassoc) of the β-CD with VMA,
HVA and VA respectively, calculated by WinEQNMR are summarized in Table 1.
Table 1: The association constants for VMA, HVA and VA 1:1 complexes with β-CD
Kassoc ± SD [M-1]
complexed
based on δobs of H3 of CD (G)
based on δobs of H5 of CD (G)
molecule (H)
VMA
HVA
VA
29.3 ± 1.8
30.0 ± 2.9
43.1 ± 4.1
54.7 ± 3.7
73.7 ± 5.1
105.0 ± 3.6
152.0 ± 3.6
171.8 ± 3.3
VA*
*values determined using HOD proton signal at 4.790 ppm as an internal reference
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Each value of Kassoc is expressed as the mean ± standard deviation (SD) from three
independent experiments (in which association constant was determined with errors
below 10%). Values of association constants are arranged in series: Kassoc of VMA/β-
CD < Kassoc of HVA/β-CD < Kassoc of VA/β-CD. From presented data it can be
observed that the type of substituent at C1 position affects the inclusion of the
complexed molecule. VMA having largest group at C1 is bound least. At this position
vanillin has group which is smallest and with less tendency to forming hydrogen
bonds thus VA forms the most stable complex with β-CD.
As it was mentioned earlier, upfield chemical shift of residual water signal was
observed with increasing CM content. Presumably these chemical shift changes are
related to the release of water molecules included in CD cavity upon the β-CD/CM
complexes formation. Table 1 shows supplementary values of Kassoc evaluated for VA
when HOD signal was used as internal reference. It is seen that values of Kassoc
evaluated for VA/β-CD differ significantly depending on method of referencing.
In the Table 2 values of association constants for VMA/β-CD, HVA/β-CD and VA/β-
CD complexes reported in the literature are collected. Values of association constant
determined under similar conditions should be independent of the applied
methodology [23,24]. Meanwhile differences between association constants given for
the same complexes are significant even when the same method is used. Available
literature data for the association constant of VA/β-CD complex are derived from
NMR analysis. Our value of Kassoc for β-cyclodextrin complex with vanillin is about
50% smaller than literature values whilst association constants for VMA/β-CD and
HVA/β-CD determined in this study are in agreement with those obtained by L. A.
Kartsova group that used Capillary Zone Electrophoresis [15].
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Table 2: The literature data concerning determination of Kassoc of VMA, HVA or VA
1:1 complexes with β-CD
analytical method /conditions [ref.]
VMA HVA
VA
Capillary Zone Electrophoresis/ borate buffer
solution (25 °C) [15]
27 ± 2 56 ± 6
NMR titration / D2O solution (25 °C), in the
presence of DSS as internal standard [14]
137 [M-1]
NMR titration / D2O solution (20 °C), no
information about applied referencing method
[12]
1.11 x 104 ± 1800 [M-1]
170.2 [M-1]
NMR titration /D2O solution (25 °C), chemical
shifts measured relative to TMS [13]
3.3. Structure of the complexes
Two-dimensional rotating-frame Overhauser effect spectroscopy experiments
were performed for characterization of the geometry of β-CD complexes formed with
the VMA, HVA and VA respectively. The fourfold excess of complexed molecule
relative to β-CD was used in order to shift the equilibrium of the reaction (1) toward
formation of the complex. 2D ROESY experiments detect both intra- and
intermolecular spatial proximity among protons.
Partial contour plot of 2D ROESY spectrum for β-CD/VMA complex is shown in the
Figure 3. Strong 1H-1H cross signals of the methoxy group protons with aromatic
proton H2’ within VMA molecule is observed. Much less intense signals came from
intermolecular spatial coupling of aromatic protons of VMA with β-CD protons. NOE
cross-peaks of protons H5’ and H2’ with H3 of β-CD indicates binding VMA molecule
via the wider rim of cyclodextrin. Additionally, weak spatial coupling between α-CH of
VMA and H3 of β-CD (data not shown) proves formation the complex of proposed
structure. Simultaneously from the cross-peaks H5’/H5 and H2’/H5 and especially
H6’/H6 we concluded that the partial inclusion occurs also by the inclusion into
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cyclodextrin cone through its narrower rim. Presumably in both cases only partially
inclusion takes place due to steric hindrance and additionally owing to hydrogen
bonding between carboxylic group of VMA and hydroxyl groups of the β-CD.
OH3
-OCH3 (VMA)
H6
OH2
H2
H3
H2
H4
H5
H3
H1
H5
H4
H6’
H5’
6
H2
OH6
OH3
O
HO
OH2
C
H6'
H2
H1
HC
5'H
HO
H2’
OH
H3
H2'
OCH3
H5
H4
δ [ppm]
6
H2
OH6
Figure 3: Partial contour plot of the two-dimensional ROESY spectrum of 4:1 VMA/β-
CD ratio and postulated geometry of the complexes
Substituent in the 1-position of the phenyl ring in HVA is smaller than that of
VMA therefore one can expect that HVA molecule will entered the interior of
cyclodextrin deeper than VMA molecule. However analogical to VMA/ß-CD,
hydrogen bonding can also slightly hindrance the inclusion of HVA into cyclodextrin
cavity. Indeed all aromatic protons (H6’, H5’, H2’) of VMA show more intensive
intermolecular NOE cross-peaks with H5 and H3 protons of β-CD and concurrently
there is not observed any spatial coupling with proton H6 (Figure 4). Presented
ROESY cross-peak pattern for characterized complex HVA/β-CD proves both modes
of inclusion of the complexed molecules. For HVA molecule strong intramolecular
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NOE cross-peaks are also observed which correspond to the spatial coupling of
protons of –OCH3 and –CH2- groups with aromatic protons.
OH3
OH2
-OCH3 (HVA)
H6
-CH2- (HVA)
H4
H2
H3
H3
H5 H2
H1
H5
H4
6
H2
OH6
H6’
OH3
OH2
H2
H1
H5’
H2’
H3
H5
H4
6
H2
OH6
δ [ppm]
Figure 4: Partial contour plot of the two-dimensional ROESY spectrum of 4:1 HVA/β-
CD ratio and postulated geometry of the complexes
In the case of both: VMA/β-CD and HVA/β-CD complexes, values of Kassoc
determined basing on chemical shifts of H3 and H5 (Table 1) are almost the same
and suggest similar contacts between aromatic protons of the complexed molecules
and hydrogens of the β-CD located inside the cavity (H3, H5).
The spatial coupling of aldehyde proton of vanillin with H5 and H3 of β-CD
(Figure 5) shows that VA molecule enters most deeply into the cyclodextrin interior
among the three tested complexed molecules. Furthermore, vanillin/β-CD complex
can occur with two different inclusion modes. Visible cross-peaks: H5’, H6’ and
probably H2’ with H3 (superimposing with intramolecular cross-peak –OCH3/H2’) and
H5’, H2’ of VA with H5 of β-CD, additionally prove the concomitance of two complexes
VA/β-CD of different geometry. Values of Kassoc determined from δ of H5 signal are
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slightly higher than determined from chemical shift of the H3 proton and indicate that
the bottom structure presented in the Figure 5 is somewhat probable.
-OCH3 (VA)
H6
OH3
OH2
H2
H3
H2
H
4
H5
H3
H1
H5’
H2’
H5
H4
H6’
H2
OH6
6
OH3
OH2
H2
H1
H3
H5
H4
-CHO
6
H2
OH6
δ [ppm]
Figure 5: Partial contour plot of the two-dimensional ROESY spectrum of 4:1 VA/β-
CD ratio and postulated geometry of the complexes
4. Conclusions
The synthesis of polymeric materials with ability of effective binding of selected
molecules can be rationalized by cognition of the interaction between the functional
monomer and the targeted molecule. In this study NMR technique was chosen for
stoichiometry and association constant determination as well as for finding
arrangement of VMA/β-CD, HVA/β-CD and VA/β-CD complexes. The use of external
standard allows for elimination the errors associated with interaction of the chemical
shift reference with β-cyclodextrin.
2D ROESY spectra showed two binding modes between β-CD and complexed
molecules which can exist simultaneously in the aqueous solution.
The values of association constants are consistent with a depth of penetration of
complexed molecules into β-cyclodextrin. The smallest value of Kassoc exhibits a
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complex formed between β-CD and VMA where only partially inclusion takes place.
Inclusion complex stability in this case may be insufficient for effective binding of
vanillylmandelic acid from aqueous solutions. For this complexed molecule
presumably more favourably will be to use complexing agent with basic moieties.
Alternatively vanillylmandelic acid may be subjected oxidation to vanillin for the
indirect indication of VMA.
2D ROESY spectra showed that vanillin molecules are completely tucked into the β-
cyclodextrin cavities. Value of Kassoc determined for VA/ β-CD indicates the strongest
binding of molecules among investigated complexes. The HVA/ β-CD complex
characterizes slightly lower Kassoc, so we finally conclude that β-CD moiety introduced
into hydrogel network could be effective for homovanillic acid and vanillin entrapping.
Acknowledgments
This study was supported by the Polish Budget Founds for Scientific Research in
2015 as a core funding for statutory R&D activities in the Silesian University of
Technolgy No. BK-206/RCh2/2015.
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Highlights
Vanillylmandelic and homovanillic acid have importance in medical diagnosis
β-cyclodextrin forms inclusion complexes with these compounds as well with vanillin
Stability constant of complexes were calculated and geometry was established from NMR
Water solution of TSP as an external reference ensures more reliable values of Kassoc
β-cyclodextrin is able for effective entrapping of homovanillic acid and vanillin