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A Simple, Low-Cost, and Useful


Preconcentration Method for Quantification of
Soluble, Insoluble, and Total Oxalate in Selected
Vegetables Through Spectrophotometry
ARTICLE in FOOD ANALYTICAL METHODS JULY 2015
Impact Factor: 1.96 DOI: 10.1007/s12161-015-0265-9

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Nail Altunay

Ramazan Grkan

Cumhuriyet University

Cumhuriyet University

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Food Anal. Methods


DOI 10.1007/s12161-015-0265-9

A Simple, Low-Cost, and Useful Preconcentration


Method for Quantification of Soluble, Insoluble, and Total
Oxalate in Selected Vegetables Through Spectrophotometry
Nail Altunay 1 & Ramazan Grkan 1

Received: 4 May 2015 / Accepted: 14 July 2015


# Springer Science+Business Media New York 2015

Abstract The amount of dietary oxalate from food may be an


important risk factor in the development of idiopathic calcium
oxalate nephrolithiasis. The main aim of this research was to
develop an accurate and reliable method by cloud point extraction (CPE) combined with spectrophotometry to measure
the oxalate contents of selected vegetables. The method is
based on ion association of stable anionic complex, which is
produced by the reaction of oxalate with Mo(VI), with Toluidine blue (TBH2+), and then by extraction into micelles of
octylphenol ethoxylate (Triton X-45) in the presence of
NH4F as a salting-out agent at pH 6.0. Using the optimized conditions, the calibration graph was highly linear in the range of 1.2240 g L1. The detection limit
of the method (LOD (3blank/m) was 0.36 g L1, and
the relative standard deviation (RSD %) as a measure of
precision was in the range of 1.15.3 % (n=5 for 5, 25,
and 50 g L1). The method was successfully applied
to the speciative determination of soluble and total oxalate in vegetables after ultrasonic-assisted extraction
and dilution at suitable ratios. The amount of insoluble
Highlights The method is simple, low cost, sensitive, and eco-friendly.
The accuracy was validated by comparing the results to those of the
independent kinetic method.
TBH2+ was successfully used in the preconcentration of oxalate in the
presence of Mo(VI) at pH 6.0.
The low-cost method with good accuracy can be an alternative to other
techniques in the literature.
An accurate and reliable determination of soluble, insoluble, and total
oxalate in vegetables has been successfully developed in this study.
* Nail Altunay
naltunay@cumhuriyet.edu.tr
1

Department of Chemistry, Faculty of Science,


University of Cumhuriyet, TR-58140 Sivas, Turkey

oxalate was calculated from the analytical difference between total oxalate and soluble oxalate contents of samples with and without acidic extraction under ultrasonic
power (300 W, 50 Hz) for 15 min at 60 C. The accuracy of the method was intrinsically controlled and verified by comparing the results obtained with those of an
independent kinetic method as well as recoveries of
spiked samples.
Keywords Oxalate . Toluidine blue . Mo(VI) . Cloud point
extraction . Vegetable samples . Spectrophotometry

Introduction
The main sources of dietary oxalate are plants and herbal
products (Hnow and Hesse 2002). Oxalate is widely distributed in plant foods in a readily water-soluble form as potassium, sodium, and ammonium oxalates and as insoluble calcium oxalates (Holloway et al. 1989). Oxalate forms strong
chelates with dietary calcium, thus rendering the complex unavailable for absorption and assimilation. It precipitates as
insoluble salts accumulating in the renal glomeruli and contributes to the development of renal disorder. While other
factors have to be considered in the development of renal
disorder, it is being recommended to limit the intake of
oxalate-rich foods, specifically for individuals at risk for kidney stone formation (Bsc 1999; Savage et al. 2000, 2009).
After oxalate is absorbed, it is transported to the kidneys and
excreted in urine as a waste product (Noonan and Savage
1999). It can be said that the daily consumption of oxalate is
50200 mg day1 (Noonan and Savage 1999). When this value is exceeded, the harmful effects in terms of human health
can be seen. Therefore, the analysis of oxalate is of great
importance in vegetables because of its effect on the human

Food Anal. Methods

body. Due to the harmful effect of oxalate species, there is a


great need to develop a simple, applicable, sensitive, selective,
and inexpensive method for the determination and continuous
monitoring of trace levels of oxalate species in vegetables.
At present, there are a number of spectrophotometric
methods for the determination of oxalate in real matrices.
The determination methods of oxalate species are mainly
based on flow injection spectrofluorimetric determination
(FISD) (Prez-Ruiz et al. 1995), gas chromatography-mass
spectrometry (GC-MS) (March et al. 2003),
spectrofluorimetry (Cha et al. 2002), amperometry (Pundir
et al. 2011), chemiluminescence (Prez-Ruiz et al. 2005),
ion chromatography (IC) with chemiluminescence detection
(Maya et al. 2011), high-performance liquid chromatography
(HPLC) (Hnow and Hesse 2002; Judprasong et al. 2006),
linear sweep voltammetry (LSV) (ljuki et al. 2007), differential pulse polarography after derivatization with ophenylenediamine (DPP) (Rodrigues and Barros 1993), capillary zone electrophoresis (CZE) on a chip with conductivity
detection (Masar et al. 2003), CZE (Guan et al. 2012), HPLC
(Judprasong et al. 2006), headspace gas chromatography (HSGC) (Li et al. 2014), catalytic spectrophotometry (Safavi and
Banazadeh 2007), HPLC with chemiluminescence detection
(Wu et al. 1999), HPLC-ER (Hnow et al. 1997), ion suppression RP-HPLC (Lian et al. 1999), enzymatic assays using
oxalate oxidase (Kasidas and Rose 1980), capillary electrophoresis (CE) and ion chromatography (IC) (Holmes and
Kennedy 2000), and ion-exchange high-performance liquid
chromatography (IE-HPLC) (Nguyn and Savage 2013) until
now. Determination of oxalate via these methods such as GCMS and flow injection spectrofluorimetry that have very high
analytical sensitivity is expensive and time consuming. Also,
a wide variety of other techniques, based on CE and liquid
chromatography (LC) interfaced with techniques of mass
spectrometry, known as hyphenated techniques, CE-MS, and
LC-MS/MS (Keevil and Thornton 2006) have been proposed
for the separation and determination of oxalate species from
interfering matrix ions. The primary advantage of these approaches is the unequivocal species separation and specific
online detection. However, these hyphenated techniques
based on in situ/online separation and detection are considerably more difficult to use. Hence, we described a method
which is sensitive, less time consuming, and relatively inexpensive. UV-Vis spectrophotometry as a tool in the analysis of
real samples is still widely used in analytical research laboratories of undeveloped and developing countries with low budget. Also, this technique is traditionally used to determine
anion, cation, and metal species in water, food, and vegetable
samples. Moreover, the device has advantages such as being
simple, inexpensive, accurate, selective, and rapid and does
not require user-operating skills compared to other techniques.
In addition, UV-Vis spectrophotometry is the most widely
used method for oxalate determination due to its simplicity

and excellent limit of detection. Also, the technique has successfully been applied to the determination of cationic and
anionic species like chromium as a beneficial species essential
for organisms at low concentrations (Ulusoy et al. 2012), cyanide as an environmental pollutant (Grkan and Ylmaz
2013), and sulfite as a food additive (Altunay et al. 2015) by
our research group.
Oxalate can efficiently be used after extraction of the sample matrix in which amounts of oxalate in the samples are low.
Therefore, a separation and preconcentration method should
be applied prior to analysis. Among the separation and
preconcentration methods, the cloud point extraction (CPE)
method has gained intense attention. This is due to the fact
that CPE offers many advantages over traditional liquid-liquid
extraction, such as not easily flammable and being simple and
inexpensive, does not use organic solvents with high capacity
to concentrate a wide variety of analytes with higher recovery
efficiency and large preconcentration factor, and generates a
few laboratory residues (Grkan et al. 2015). CPE has been
successfully applied to the preconcentration of various types
of chemical species by our research group. Moreover, there is
a good agreement between the results obtained using this
method. This is an important practical requirement for routine
monitoring or any large-scale investigations of oxalate contents in vegetables.
The main aim of the present work was to develop a rapid,
accurate, and reliable CPE method for the quantification of
oxalate species. The method developed is based on selective
ion association of anionic complexes, MoO 3 (Ox) 2 ,
Mo2O5(Ox)2, and/or Mo2O5(OH)(Ox)23, produced by the
reaction of oxalate with Mo(VI), with cationic thiazine dye,
Toluidine blue (TBH2+), and then extraction into micelles of
octylphenolethoxylate (Triton X-45) as an extracting agent in
the presence of NH4F as a salting-out agent, at pH 6.0. The
CPE/UV-Vis method was successfully applied to the analysis
of selected vegetables to determine whether or not the method
could be suggested for real sample analysis.

Experimental
Instrumentation
Absorbance measurements at 627 nm after CPE were made on
a double beam UV-Visible spectrophotometer (Shimadzu UV1800 PC, Kyoto, Japan) equipped with 1.0-cm quartz cells. A
centrifuge (Universal-320, Hettich Centrifuges, England) was
used to accelerate the phase separation process. A thermostatic
water bath (EPC 4420, Termal, Istanbul, Turkey) was used to
maintain the temperature in CPE experiments. A pH meter
(pH-2005 model, JP Selecta, Spain) was used for pH measurements. Eppendorf pipettes (10100 and 2001000 L) were
used to deliver accurate volumes. An ultrasonic cleaner (UCS-

Food Anal. Methods

10 model, Jeiotech, Co., Ltd., Seoul, South Korea) was used


to assist the fast and efficient extraction of the analyte from
vegetable samples. A refrigerator was used to keep the samples fresh and cool till the analysis.
Chemicals and Reagents
All chemicals and reagents used were of analytical reagent
grade or higher purity. Ultrapure water with a resistivity of
18.2 M cm was prepared using a Labconco (USA) water
purification system. All solutions were prepared with water.
Stock standard solutions of 500 mg L1 Mo(VI) and oxalate
were prepared from sodium molybdate and sodium oxalate,
respectively (Sigma, St. Louis, MO, USA). Working solutions
were prepared daily by appropriate dilution of water. A 1.0
104-mol-L1 aqueous solution of TBH+2 was also prepared
fresh daily by dissolving the reagents in ethanol (Merck) and
diluting with water. The solutions of 5.0 % (v/v) of nonionic
surfactants (Sigma) were prepared by dissolving an appropriate amount of 2.5 mL of surfactants with 50 mL of water. A
0.04-mol-L1 Britton-Robinson (BR) buffer was used to keep
the pH of the solutions. The buffer consists of a mixture of
0.04 mol L1 H3PO4 (Merck), 0.04 mol L1 H3BO3 (Merck),
and 0.04 mol L1 CH3COOH (Merck) that has been titrated to
the desired pH with 0.2 mol L1 NaOH. The vessels and
pipettes used for trace analysis were kept in 10 % (w/v)
HNO3 for at least 24 h and subsequently washed five times
with water.
Preparation to Analysis of Vegetable Samples
All of the vegetable samples such as cress, dill, parsley, broccoli, celery, etc., collected for analysis, were supplied from a
local produce store in Sivas, Turkey. Firstly, all the glassware
and other mineralization containers used were washed in 10 %
(v/v) HNO3 to avoid contamination.
To determine the soluble and total oxalate, all vegetable
samples were carefully washed with ultrapure water. Then,
the edible parts such as the leaves and roots were cut, crushed,
and finally frozen at 20 C. The oxalate species in selected
vegetables were determined according to the method as described by Savage et al. (2000). To the extraction of the total
oxalate, 3.0-g samples of finely ground and dried vegetable
samples were placed into a beaker of 100 mL. Then, 30 mL of
0.2 mol L1 HCl was added and the mixture was degassed and
digested under ultrasonic power (300 W, 50 Hz) at 60 C for
15 min. The mixture was allowed to cool and then filtered by
using a membrane filter of 0.45 m into a 100-mL volumetric
flask, and the final volume was diluted to 100 mL with ultrapure water before analysis.
To determine the soluble oxalate, the same procedure was
followed as in the extraction for total oxalate except that ultrapure water was used to extract the soluble oxalates under

ultrasonic power (300 W, 50 Hz) for 15 min at 60 C. The


soluble and total oxalate contents of all vegetable samples
were determined by using the standard addition method at
three different concentration levels within the working range
by spectrophotometry at 627 nm after CPE under the optimized conditions. The content of the insoluble oxalate was
calculated from the difference between soluble and total oxalate contents. Each point in the optimization step and calibration curves with and without preconcentration with CPE was
run in triplicate, and the results were indicated with error bars.
The one- and two-paired ANOVA tests in optimization and
analysis step of samples were conducted for 237 statistical
comparisons.
The General CPE Procedure
A specific portion of a sample solution containing oxalate in
the range of 1.2240 g L1 is added to a volumetric tube of
50 mL. Then, 1.5 mL of 0.04 mol L1 pH 6.0 BR buffer,
1.25 mL of 10 mg L 1 Mo(VI), 0.75 mL of 1.0
104 mol L1 Toluidine blue (TBH2+), 1.5 mL of 20.0 % (v/
v) NH4F, and 1.2 mL of 5.0 % (v/v) Triton X-45 were mixed.
The mixture was diluted to a volume of 50 mL with water.
Then, the solutions were mixed well and kept in a thermostatic
water bath for 10 min at 50 C. The phase separation was
accelerated by centrifuging at 4000 rpm for 5 min. Then, the
resulting mixtures were cooled in a freezer for 5 min to increase the viscosity of the surfactant-rich phase and make the
extraction of the aqueous phase easy. Then, the aqueous phase
was easily separated from the surfactant-rich phase by
inverting the tube. The surfactant-rich phase was dissolved
and diluted to 0.75 mL with ethanol acidified with
0.1 mol L1 HCl and transferred into a 1.0-mL quartz cell prior
to spectrophotometric detection at 627 nm. Finally, the
amounts of soluble, insoluble, and total oxalate present in
selected vegetable samples after dilution of pretreated and
digested samples with and without 30 mL of 0.2 mol L1
HCl at 60 C for 15 min were reliably determined by the
analysis of the samples spiked at levels of 5, 25, and
50 g L1 in order to suppress the possible matrix effect.
The Modified Independent Comparison Method
For comparison purposes, due to the lack of a CRM related to
oxalate in vegetable matrices including higher order of instruments like CZE, HPLC, and ion-exchange HPLC in our research laboratory, it was initially decided to use an independent kinetic method based on the catalytic effect of oxalate in
order to ensure the reliability of the results. In this context, the
oxalate levels of the samples, which similarly were pretreated,
were spectrophotometrically measured at 525 nm and comparatively evaluated by the independent kinetic method based
on the signal-enhancing effect of oxalate to permanganate

Food Anal. Methods

produced by oxidation of Mn(II) ions with periodate using


approaches of a fixed time of 3 min and an induction period
of 12 min in 330 min in an ultrasonic bath (300 W, 50 Hz) at
35 C, in which Mn(III) ions form thermally unstable complexes, MnC2O4+, Mn(C2O4)2, and Mn(C2O4)33, depending
on the oxalate concentration. The analysis of selected vegetables by means of the standard comparison method (Hassouna
and Elsuccary 2002) was carried out as follows: The reagent
solutions, water, and 10 mL volumetric flasks were kept at
35 C in the ultrasonic bath (300 W, 50 Hz). One milliliter
of Mn(II) solution (200 g mL1) was transferred into a
10-mL measuring flask, added with 0.75 mL of H3PO4
(0.2 mol L1), 1.3 mL of CH3COONa (0.1 mol L1), and
oxalate solution in the range of 0.520 mg L1, and then
diluted to approximately 8 mL with water. After that, the solution was sonicated in the ultrasonic bath (35 C) for 10 min
to attain the reaction temperature, then added with 2 mL of
KIO4 (3.0103 mol L1), and made up to the mark with
water. The reaction mixture was efficiently induced and mixed
with the help of an ultrasonic wave and immediately transferred to the spectrophotometric cell, and the increase in absorbance was recorded at 525 nm against water with increases
of 3 min during 30 min from the initiation of the reaction.
Similarly, a blank analysis not containing oxalate was conducted. In order to remove the effect of potential interfering
ions present in the digested vegetable samples such as Cu2+,
Fe2+,3+, Sn2+,4+, Ni2+, Co2+, Cd2+, Cr3+, and V4+,5+, sample
solutions were also passed through a cation-exchange resin
such as Amberlite IR 120 prior to analysis when necessary.
When a regression analysis (n:=6, independently) for both the
fixed-time method and induction period method is conducted
for a serial standard oxalate solutions in the range of 0.05
1.25 mg L1 oxalic acid in a suitable pH range forming H3PO4
and CH3COONa, according to the modified standard comparison method, a good enhancement in regression data with
increasing sensitivity was obtained as follows:
(i) (A)=Asample Ablank =0.34150.012 [oxalate, mg
L1](3.720.15)103 with limits of detection and
quantification of 4.60 and 15.3 g L1 (r2 0.9978, n=6)
and
(ii) 1/tinduction (min1)=0.03650.0024 [oxalate, mg L1]+
0.06250.0135 with limits of detection and quantification of 1.16 and 3.85 g L1 (r2 0.9976, n=6)

Results and Discussion


General Considerations Related to Method Development
The method is based on ion association of stable anionic complex, which is produced by reaction of oxalate with Mo(VI),

with cationic thiazine dye, Toluidine blue (TB+), at pH 6.0 and


then by extraction into micelles of Triton X-45 in the presence
of NH4F as a salting-out agent. The extracted surfactant-rich
phase is diluted to 0.75 mL with acidic ethanol, and its absorbance of hydrophobic complex, which is linearly related to
oxalate concentration, is spectrophotometrically measured at
627 nm. Therefore, as a result of the highly stable, sensitive,
and selective complexation of oxalate with Mo(VI) in the
presence of TBH2+, the ternary complex assisted by Triton
X-45 micelles can easily be extracted into the micellar phase
by the CPE procedure. Thus, for further applications, the different variables affecting CPE efficiency were optimized in
order to achieve the maximum sensitivity. In the voltammetric
determination of molybdenum(VI) using carbon paste electrodes modified in situ with cetyltrimethylammonium bromide (CTAB) (Stadlober et al. 1997), it has been observed
that in the presence of oxalate, the reduction peak is
shifted to more negative potentials, whereas the oxidation peak is shifted to more positive potentials; thus, the
electrochemical redox reaction becomes more irreversible. It is well known that molybdate ions react in acidic aqueous solutions with a large number of polydentate
organic ligands, such as oxalate (Ox). It has been observed that a stable complex between Mo(VI) and oxalate at ratio of 1:1 is formed with a formation constant
of 6.31013 mol3 L3 according to Eq. (1) (Arkan and
Tunay 2006).
MoO4 2 Ox2 2H MoO3 Ox2 H2 O

Also, it was implied that, in a study conducted by Stadlober


et al. (1997) as a consequence of the stable complex formation
between molybdenum(VI) and oxalate, two more complexes,
[Mo2O5(Ox)2]2 and [Mo2O5(OH)(Ox)2]3, exist in a solution
in the pH range of 27; it has been found that the main species
at pH 2 is [Mo2O5(Ox)2]3 in equimolar solution of molybdenum and oxalate. Toluidine blue (TB+/TBH2+), a kind of phenothiazine with a pKa value of 7.2, is generally used as a
polymerization inhibitor, complexing agent, biological sensitizer, and oxygen radical inactivator in an organisms body
avoiding pathological changes (Liu et al. 2010). It is well
known that phenothiazine drugs are a kind of typical and
useful organic compound with an N atom, which makes it
easy to lose one electron to form the cation radicals (Nascentes
and Arruda 2003). At pH 7.5 Tris-HCl buffer, it was observed
that TB+ strongly interacted with surfactin, which is a
cyclic lipoheptapeptide containing seven amino acids
and a -hydroxyl fatty acid, with a blue shift from
626 to 590 nm and formed a stable ion-pairing complex
with a binding constant (Kb) of 3.91104 above a critical micelle concentration (CMC) of 1.1105 mol L1
at 20 C, in which surfactin has two negatively charged
amino acid residues L-Glu and L-Asp, which can be

Food Anal. Methods

MoO3 Ox

TBH TBH MoO3 Ox


2

be seen from Fig. 1a, the maximum absorbance was


obtained at pH 6.0 with the BR buffer system. The
complexation at pH values lower than 6.0 is quantitatively incomplete because a complex formation is inadequate as a measure of protonation of oxalate with pKa
values of 1.25 and 4.14 and molybdenum, Mo(VI), with
pKa values of 2.4 and 3.8. From cyclic voltammetric
and ultraviolet-Vis spectral data (Merc et al. 2006), it
has been observed that in fact, molybdenum, Mo(VI),
acts as an anion, MoO42 (in which molybdate anion,
M o O 4 2 , u nde rg oe s t he fo rma tion o f d iff ere nt
polyanions in acidic solutions, particularly at pH 4.1),

(a)
0.6

0.5

Absorbance at 627 nm

partially ionized as the anionic surfactant in Tris-HCl


buffer solution of pH 7.5.
Due to this property, it is clear that the reagent, TB+
or TBH2+, tends to give a stable hydrophobic complex
with an anionic complex, produced in the presence of
Mo(VI) at pH 6.0, so that it will form an ion association complex. Because of its high solubility in aqueous
micellar media, from prior studies, it was observed that
the ion-pairing complex could easily be extracted into a
surfactant-rich phase with an optimum concentration of
0.0106 % (w/w) above CMC (0.009 %, w/w) of the
nonionic surfactant, Triton X-45. To further improve
the calibration sensitivity and selectivity of the method,
CPE has been explored using different nonionic surfactants in the presence of NH4F as a salting-out agent to
enhance the binding of a hydrophobic complex to the
surfactant-rich phase.
CPE can efficiently be used when the target species
are hydrophobic in nature. Though the ion-pairing complex is water soluble, it has successfully been extracted
into the surfactant-rich phase in the presence of a
resonance-stabilized reagent, TBH2+ or TB+, at pH 6.0.
The mechanism proposed for CPE of oxalate in aqueous
micellar medium assisted by Triton X-45 micelles can
be postulated as follows:

0.4

0.3

0.2

2
0.1

MoO3 Ox

2TB TB2 MoO3 Ox

3
0.0
1

pH

The Effect of pH on Extraction Efficiency

(b)
0.8
0.7

Absorbance at 627 nm

The pH of the sample solution is an important factor


that needs to present sufficient hydrophobicity to be
extracted into the small volume of the surfactant-rich
phase; therefore, the effect of pH on the separation
and preconcentration of 25 g L1 of C2O42 by CPE
in micellar media was extensively investigated in the
range of 2.08.0 using different buffers. The best calibration sensitivity was obtained by using universal BR
buffer. In order to adjust and stabilize the desired pH
during analysis, monohydrogen phosphate containing
buffers like BR buffer is broadly used in the pH range
of 212, due to its weak complexing capacity. Due to
the varying calibration sensitivity of the system in different buffers, in a broader pH range for analyzing the
pH dependence of analyte or ion-pairing reagent
(TBH2+), BR buffer consisting of more than two components was preferably used for accurate and reliable
pH measurements, so that for buffers, two further
criteria will be considered, the ionic strength and concentration and the nature of buffer components. As can

0.6
0.5
0.4
0.3
0.2
0.1
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

p H 6.0 BR buffer concentration, mmol L-1

Fig. 1 Effect of a pH and b BR buffer concentration on CPE efficiency.


Other experimental conditions are described under the proposed CPE
procedure

Food Anal. Methods

MoO3 H2 Lin the presence of bidendate ligandMoO2 L H2 O

4
MoO4

H MoO3 OH

MoO3 OH H 2H2 OMoOH6 or MoO2 OH2 H2 O2

5a
5b

Perhaps, it may be either degradation of the ion-pairing


reagent by one electron reduction of TBH2+ to radical ion or
dimerization of Mo(VI) in the forms of Mo 2 O 3 2+ and
Mo2O34+ at acidic pH values. Also, at pH values higher than
6.0, the reason for the decrease in absorbance can be the deprotonation or aggregation of the ion-pairing reagent at low
concentrations. Hence, a pH of 6.0 was selected as an optimal
value in order to give the highest sensitivity.
Furthermore, the effect of buffer concentration in the final
volume of 50 mL on the analytical signal at pH 6.0 was investigated in the range of 0.22.0 mmol L1 in Fig. 1b. As a
result of repeated experiments, the best analytical signal was
obtained by using a BR buffer concentration of 1.2 mmol L1.

was investigated in the range of 0.45.0 mol L1, and the


results are shown in Fig. 2a. It could be seen that the analytical
signal for oxalate increased rapidly as the concentration
of TBH2+ increased from 0.0 to 1.6 mol L1 and then
gradually decreased with increasing TBH2+ concentration from 1.6 to 5.0 mol L1. The decrease in signal
may be due to aggregation of the dye. Thus, a TBH2+
concentration of 1.6 mol L1 was selected as optimal
for further studies.
The variation of the analytical signal as a function of molybdenum concentration in the presence of 25 g L1 oxalate

(a)

1.4

1.2

1.0
Absorbance at 627 nm

only after pH around 4.0, whereas at pH values higher


than 4.0, it chemically acts as a cation, MoO22+, according to Eqs. 4 and 5a, b, presented as follows:

0.8

0.6

0.4

0.2

Effect of the Ion-Pairing Reagent and Metal


Concentrations on Extraction Efficiency

where Ci symbolizes the concentration of oxalate in the initial


sample of volume Vi, Cc symbolizes the concentration of oxalate in the aqueous phase of volume Vc, and Cs symbolizes
the concentration of oxalate in the surfactant-rich phase of
volume Vs. One factor that increases the extraction efficiency
also is the effect of the ion-pairing reagent. TB+ and/or TBH2+
as an ion-pairing agent in the present study was selected for
oxalate species in the presence of Mo(VI) and Triton X-45 at
pH 6.0. TBH2+ is a basic metachromatic dye with resonancestabilized planar geometry, especially due to its phenothiazine
group containing hetero-N and S atoms including NH2 and
N(CH3)2 groups.
Hence, the effect of TBH 2+ concentration of 1.0
4
10 mol L1 on the analytical signal and extraction efficiency

2+

-1

TBH concentration,mol L

(b)

1.6

1.4

1.2
Absorbance at 627 nm

CPE efficiency depends on the hydrophobicity of the ligand


and the complex formation. The extraction efficiency of oxalate by Triton X-45 surfactant from the aqueous sample was
calculated as follows:


C s V s
*100
Extraction efficiency %
C i V i


C i V i C c V c

*100
6
C i V i

0.0

1.0

0.8

0.6

0.4

0.2

0.0
0.0

0.1

0.2

0.3

0.4

0.5

-1

Mo(VI) concentration, mg L

Fig. 2 Effect of a TBH2+ and b Mo (VI) concentrations on CPE efficiency. Other experimental conditions are described under the proposed
CPE procedure

Food Anal. Methods

(a)

0.6
NaCl

0.5

KCl
NH4Cl

0.4
Absorbance at 627 nm

was investigated in the range of 0.040.46 mg L1. The experimental results in Fig. 2b indicated that the signal intensity
of the analyte linearly increases with molybdenum concentration up to 0.24 mg L1. The maximum signal intensity gradually decreased with increasing slope at higher concentrations.
This decrease in signal may be due to ion-pairing of Mo(VI) in
the form of MoO(OH)5 or MoOF5 based on electrostatic
interaction or redox reaction with TBH2+ in the absence of
oxalate, so as to cause an increase in blank signal. So, a molybdenum concentration of 0.24 mg L1 was selected as optimal for further studies.

NH4F

0.3

0.2

0.1

Effect of the Addition of Different Electrolytes


as Salting-Out Agent on Extraction Efficiency

Effect of Nonionic Surfactant Type and Volume


on Sensitivity
In CPE, choosing a suitable surfactant is important because
the temperature corresponding to cloud point is related with
the hydrophilic property of a surfactant. The concentration of
the nonionic surfactant not only affected the volume of the
surfactant-rich phase but also the extraction efficiency. A successful CPE should maximize the extraction efficiency by
minimizing the phase volume, thus increasing its concentrating capability. The effect of the nonionic surfactant concentrations on extraction efficiency was investigated in the range of
0.020.2 % (v/v). As can be seen from Fig. 3b, the best

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Electrolyte concentration, % (w/v)

(b)
1.2

1.0

0.8
Absorbance at 627 nm

Generally, the addition of salt increases the distribution of


analyte into the surfactant-rich phase. Also, studies on the
effects of inorganic electrolytes on the cloud point behavior
of nonionic surfactants have been reported (Komaromy-Hiller
and Wandruszka 1996). It was observed that the presence of
electrolytes decreased the cloud point, resulting in a more
efficient extraction. The lower cloud point is attributed to
electrolytes promoting dehydration of the poly(oxyethylene)
chains. Because of all these reasons, different inorganic salts
like NaCl, KCl, NH4Cl, and NH4F in the range of 0.11.2 %
(w/v) were added to investigate the electrolyte effect on the
extraction efficiency. From the results obtained, it was found
that the phase separation ability of salts is in the following
order: NH4F > NaCl > NH4Cl >KCl. As can be seen from
Fig. 3a, a high extractability and absorbance for oxalate was
also obtained using 0.6 % (w/v) NH4F as a sensitivity enhancement agent. The absorbance considerably decreased
with increasing NH4F concentration in the range of 0.6
1.2 % (w/v). This effect might be explained by the additional
surface charge when the NH4F concentration is very high,
thus changing the molecular architecture of the surfactant
and, consequently, the micelle formation process. It is necessary to emphasize that different blank solutions were also
evaluated and no significant signal was obtained.

0.0

0.6

0.4

0.2

Triton X-45
Triton X-100

0.0

Triton X-114
Ponpe 7.5

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22

Nonionic surfactant concentration, % (v/v)

Fig. 3 Effect of a electrolyte and nonionic surfactant concentrations b on


CPE efficiency. Other experimental conditions are described under the
proposed CPE procedure

extraction efficiency was obtained using Triton X-45 concentration of 0.12 % (v/v). Therefore, Triton X-45 was preferably
chosen as a micellar system for further studies. Also, this
value corresponds to a maximum concentration of 0.0106 %
(w/w) above the CMC of 0.009 % (w/w) for Triton X-45.
Effects of Equilibrium Temperature and Incubation Time
In the completion of the complex formation and efficient separation of phase in CPE, it is desirable to have the lowest
equilibration temperature and shortest incubation time. Hence,
a series of experiments was investigated in the range of 30

Food Anal. Methods


Fig. 4 The calibration curves of
trace oxalate at a low and b high
concentrations with changing
sensitivities under optimized
reagent conditions

(a)

(b)
0.45

1.6
Calibration points

0.40

1.4

Linear fit

Calibration points
Linear fit

Absorbance at 627 nm

Absorbance at 627 nm

0.35

0.30

0.25

1.2

1.0

0.8

0.20

0.10

0.6

Regression equation,

0.15

-1

2-

Regression equation,

10

12

14

0.4

-1

Table 1

-1

Abs.: 4.48x10 [C 2O4 , g L ] + 0.4465, r : 0.9932

Oxalate concentration,g L

70 C for the optimization of temperature in the CPE system.


As a result of the experimental studies, the extraction efficiency was found to gradually increase when the temperature increased from 30 to 50 C and then decreased slightly above
50 C in the CPE. The solutions became turbid as soon as the
solutions were put into the water bath at 50 C. Keeping the
equilibrium temperature of 50 C, the influence of incubation
time on CPE was investigated in the range of 230 min. It has
been found that 10 min is adequate for the quantitative extraction of oxalate. Thus, 50 C and 10 min were chosen as the
equilibrium temperature and incubation time for the CPE procedure, respectively. In addition to these experiments, centrifugation time and rate have been optimized because they are

2-

-3

Abs.: 0.0232 [C2O4 , g L ] + 0.140, r : 0.9974

50

100

150

200

Oxalate concentration,g L

250

300

-1

very necessary to preconcentrate trace amounts of oxalate


with high efficiency in a short time. From the results, it has
been observed that centrifugation for 5 min at 4000 rpm leads
to the maximum recovery and sensitivity for oxalate.
Selection of the Diluting Agent
After separation and preconcentration with CPE, the
surfactant-rich phase obtained is very viscous. Thus, its viscosity can be reduced using diluting agents prior to detection
by UV-Vis spectrophotometry. In order to investigate the selectivity and extraction efficiency of the method, it is very
important to choose the suitable diluting agent. The effect of

Analytical features of the proposed CPE/spectrophotometric method

Parameters

Analytical species
Linear ranges
Slopes (m)
Intercepts (b)
Regression coefficient, r2
Precision, RSD % (n=5; 5, 25, and 50 g L1)
Recovery (%) (n=5; 5, 25, and 50 g L1)
Detection limita (n=10, LOD, 3blank/m), g L1
Quantification limita (n=10, LOQ, 10blank/m), g L1
Sensitivity enhancement factorb
Preconcentration factorc

Analytical features
After preconcentration at 627 nm

Before preconcentration at 645 nm

C2O42, g L1
1.212, 12240
0.0232, 4.48103
0.140, 0.4465
0.9974, 0.9915
1.15.3
97103
0.36

C2O42, g L1
90750
3.2104
0.142
0.9862

25.4

1.20
72.5
65.8

84.7

The limits of detection and quantification corresponding ratio of three- and tenfold of standard deviations of ten replicate blank analysis to slope of
calibration curve obtained after preconcentration with CPE

The value calculated as the ratio of slopes of calibration curves obtained with and without preconcentration with CPE

The value calculated as the analyte concentration giving the same analytical signal with and without preconcentration by means of CPE

sm and sb are the standard deviations of the slope and intercept of the matrix-matched calibration curve (n=7) obtained in selected vegetable mixtures in 10200 mg kg1 , respectively

95.6 (4.5)
93.5 (5.2)
92.3 (6.5)
97.5 (4.3)
95.6 (4.6)
94.1 (5.8)
5.42
1.63
10200
OxalateA mixture of three vegetables y=(4.800.32)103 C
0.994
(2 g, 2:1:1, w/w)
(g kg1)+(8.50.26)102

Linearity, r2 Linear range, MDL, MQL, Recovery and RSD (%) (n=3)
g kg1
g kg1 g kg1
Repeatability (intraday)
Regression equation,
y=(msm) x+(bsb)a
Analyte Sample matrix

CPE/spectrophotometric method validation results obtained in the selected vegetable matrices (from the matrix-matched calibration curve)

After preconcentration with CPE, a series of experiments has


been carried out to evaluate the proposed method. The calibration curves obtained under the optimized conditions are
shown in Fig. 4a, b. The calibration curves have been obtained
at low and high oxalate concentration regions with changing
sensitivities after the standard solutions used in the CPE/UVVis system. The calibration curves were constructed by measuring the difference between signals of the sample and blank
for the surfactant-rich phase as a function of serial oxalate
concentrations in the range of 1.2240 g L1 at 627 nm after
preconcentration with CPE. Also, the characteristic performance data of the proposed method are summarized in Table 1.
As seen from Tables 1 and 2, the limits of detection and quantification defined as 3blank/m and 10blank/m (where blank is
the standard deviation of 12 replicate measurements of the
blank and m is the slope of the calibration curve at low concentrations), LOD and LOQ, respectively, have been 0.36 and
1.2 g L1 for oxalate. The sensitivity enhancement factor
(EF), defined as the ratio of the slopes of the calibration curves
with and without preconcentration, has been determined to be
72.5 for oxalate at 627 nm. The preconcentration factor (PF),
calculated as the analyte concentration giving the same analytical signal with and without preconcentration, has been determined as 65.8 at 627 nm. The accuracy and precision of the
method were established by five replicate measurements of
oxalate at levels of 5, 25, and 50 g L1. From the results
obtained, it has been observed that the relative standard deviations as a measure of precision and the percent recoveries are
in the range of 1.15.3 and 97103 %, respectively.
In terms of reliability, the proposed method was also validated by evaluating the analytical curve, linearity, sample matrix effect, method detection and quantification limits (MDL,
MQL), accuracy (recovery), and precision (repeatability and
intermediate precision) in accordance to FDA guidelines for
the analysis of a mixture prepared from vegetables with low
oxalate contents at known ratios (a mixture of three vegetable1.0 g dill, 0.5 g cauliflower, and 0.5 g broccoli) by spectrophotometric analysis at 627 nm after preconcentration with

Table 2

Analytical Figures of Merits

Intermediate precision
(interday)

various diluents such as acetone, acetonitrile, THF, methanol,


ethanol, acidic methanol, and ethanol in the range of 0.5
1.5 mL was investigated by dissolving the surfactant-rich
phase and reducing its viscosity. From the results, the best
analytical sensitivity, m/s and the regression coefficient, r2,
for each diluent, in which m and s are the slope of the calibration curves and the standard deviation of three replicate measurements, respectively, was obtained in the presence of
0.75 mL ethanol acidified with 0.1 mol L1 HCl from calibration curves obtained for oxalate at levels of 5, 25, and
50 g L1.

25 g kg1 50 g kg1 100 g kg1 25 g kg1 50 g kg1 100 g kg1

Food Anal. Methods

Food Anal. Methods

ultrasonic-induced CPE. A calibration curve was obtained for


the concentration levels of 10, 25, 50, 75, 100, 150, and
200 g L in acidic ethanol and in the matrix blank extract,
corresponding to a range of 10200 g kg1 in the sample
with three replicates each (n=3). To evaluate the possible matrix effect in the spectrophotometric analysis, the slopes of the
curves in acidic ethanol and in the matrix blank extract were
compared, and a significant difference with an increase of
7.1 % in the slope of the matrix-matched calibration curve
was quantitatively not observed in terms of the matrix effect.
The real MQL was based on the lowest spike level that meets
the requirements of accuracy (recovery from 70 to 120 %) and
precision (RSDRSDsed on tMDL was statistically calculated dividing this value by 3). The real accuracy of the method
was evaluated by carrying out extraction and analysis of three
replicates of blank samples spiked at levels of 25, 50, and
100 g kg1, to estimate the accuracy, expressed as recovery
(%), and precision, in terms of repeatability (intraday). Due to
the complexity of the studied sample matrix, the same experiment at three spiked levels was repeated in different days in
order to estimate the intermediate precision (interday). The
results are given in Table 2 in detail.

Matrix Effect
In order to evaluate the selectivity and extraction efficiency of
the method, the effects of foreign ions on the determination of
40 g L1 of oxalate under the optimal experimental conditions according to the optimized procedure was investigated.
The foreign ions, potentially existing in sample matrices, may
commonly affect the preconcentration and quantitative determination of oxalate with CPE. The tolerance limit was identified as the concentration of added ion that caused more than
5.0 % relative error in the absorbance of the sample. As
Table 3 The effect of possible
interfering species on
determination of 40 g L1 of
oxalate

understood from the results in Table 3, there is no significant


interference by the ions commonly found in vegetable samples. But the interfering effects of interfering anionic and cationic species such as Ni2+, Sn2+, Zn2+, tartrate, and NO3 were
efficiently removed by the addition of suitable masking agents
to the solution prior to preconcentration with CPE. As will be
clearly understood from the results, the application of the
method permits the interference-free determination of oxalate
in the selected samples.

The Analysis Results of Vegetable Samples


The accuracy of the method was verified in two ways: firstly,
through the spiked recoveries and, secondly, by analysis of the
modified independent kinetic method of similarly pretreated,
extracted, and diluted vegetable samples for comparison purposes. The results can be seen in Table 4. It has been observed
that the results obtained from five replicate measurements of
samples by the developed CPE-UV-Vis method are statistically in good agreement with those of the independent comparison method. It can be concluded that the method is accurate
and free from systematic error. Also, in order to control the
accuracy of the proposed method, a recovery study was performed by the addition of known amounts of oxalate into
aqueous solutions of vegetable samples (5, 25, and
50 g L1). From the results of five replicate measurements,
it has been found that the recoveries of spiked samples are
highly quantitative in the range of 96103 % with an RSD
ranging from 1.0 to 4.6 % for total oxalate. Moreover, as can
be seen from Table 4, Students t test for comparison of the
mean values and their RSDs demonstrated that there was no
significant difference between the mean values obtained by
both the developed method and the independent kinetic method at the significance level of 0.05. Because the calculated t

Interfering species

Tolerance limits

Recovery (%)

Cl, CH3COO, HPO42, and HCO3


Na+, K+, NH4+, and Mn2+
Cr3+, Ag+, Pb2+, and Co2+
Br, S2, S2O32, and NO2
Cd2+, SO42, and citrate
Mg2+, Al3+, Fe2+, Ca2+, and Sn4+
Bi3+ and Sb3+
Ni2+a, Sn2+b, and Zn2+b

1000
5001000
250500
150250
100150
75100
2575
10 (100)a, 15 (125)b

97.3102
97.9103
96.599.8
97.1102
98.6103
96.3104
95.8103
97.8104

Tartratec and NO3d

1 (50)c, 5 (100)d

98.1104

After pretreatment with 0.5 mL of 0.001 mol L1 Na2S2O3 solution

After pretreatment with 1.0 mL of 0.01 mol L1 NH4F solution

After pretreatment with 1.0 mL of 0.02 mol L1 sulfite or bisulfite solutions

After reduction of NO3 to NO2 with a mixture of Cu(II) and Zn(II) in alkaline media

1/100

1/100

1/100

1/100

1/100

1/100

1/100

1/100

1/100

Dill

Parsley

Cauliflower

Broccoli

Celery

Black cabbage

Red radish

Lettuce

41.31.4
59.61.6
85.51.8

50

78.22.0
35.21.3

50

25

33.91.2
53.21.5

63.81.5
28.51.0

50

25

18.10.7
38.01.2

72.81.6
13.50.6

50

47.01.3

25

25

22.40.8
27.90.9

25.60.9
51.11.5

25

5.80.3

50

51.51.3
0.90.02

50

26.70.8

7.00.3

88.02.3
1.900.1

50

25

43.31.6
62.02.0

51.61.5
37.61.5

50

25

6.10.2
25.90.8

75.41.5
1.300.05

50

50.11.2

25

25

24.80.9
29.31.0

2.1

2.7

3.4

3.7

2.6

2.8

3.5

3.5

2.4

3.2

3.9

4.4

2.2

2.8

3.2

3.6

2.9

3.7

4.8

5.1

2.5

3.0

4.3

5.3

2.6

3.2

3.7

4.0

2.9

3.1

3.3

3.8

2.0

2.4

3.4

3.6

100

98.8

103

99.6

99.4

102

101

98.7

97.8

101

99.2

102

100

98.8

98.3

99.2

99.3

102

101

99.0

102

101

98.5

96.8

101

101

98.3
66.01.6

50.81.3

77.02.0

61.02.1

19.10.9
40.21.3
65.01.9

14.60.6

67.22.3

20.5

22.20.9
42.31.2

17.60.7

56.51.3

10.9

11.40.4
31.41.0

6.80.3

35.21.5

6.7

10.60.4
15.30.7

25.31.2
50.62.1

11.8

5.60.4

0.50.01

51.01.3

0.4

6.20.2
25.90.7

1.120.05

0.8

31.81.1
51.91.5

26.51.0

11.1

5.50.2
25.40.8

0.60.03

41.91.2

0.7

16.50.7
21.80.8

8.3

4.2

2.9

3.2

3.7

4.1

1.6

2.4

3.5

4.0

2.3

3.2

3.5

4.4

2.1

3.0

3.5

3.8

3.0

3.8

4.9

5.3

2.5

2.7

3.2

4.5

2.6

2.9

3.5

3.8

2.6

3.1

3.6

5.0

2.4

2.9

3.7

101

101

96.9

99.4

99.2

98.2

99.5

98.5

96.6

101

99.7

98.6

100

99.2

102

99.8

99.2

102

101

101

10

100

99.2

98.2

99.2

10

103

16.20.6

14.550.5

17.50.6

6.70.3

10.50.4

ndg

1.100.04

26.10.9

ndg

35.11.2

28.41.1

13.30.5

22.20.7

ndg

1.930.1

37.11.3

1.320.06

24.51.0

Foundf
(g kg1)

Soluble oxalate Total oxalate

By the independent
kinetic method

Foundd
RSD % Recoveryb (%) Founde
1
(g kg )
(g kg1)

Insoluble oxalate Soluble oxalate

Founda (g kg1) RSD % Recoveryb (%) Foundc


(g kg1)

Total oxalate

Dilution ratio Added oxalate By the developed CPE/spectrophotometric


method
(g kg1)

Determination of soluble, insoluble, and total oxalate contents of vegetable samples and recovery rates of spiked samples (n=5)

Cress

Samples

Table 4

0.35, 0.40

0.42, 0.68

1.60, 1.90

1.18, 1.10

1.33, 1.95

1.58, 1.86;

1.60

2.0, 1.39

The Students t test

Food Anal. Methods

101

36.11.1
60.31.6

4.7

2.3

3.0

3.8

99.5

101

103

10.70.4

28.80.9

0.93, 0.98

The insoluble oxalate values obtained by calculating the analytical difference between total oxalate and soluble oxalate contents of samples

Not detected due to below the method detection limit

The average plus standard deviation of five replicate measurements for total oxalate contents of samples with the independent kinetic method

The average plus standard deviation of five replicate measurements for soluble oxalate contents of samples with the modified independent kinetic method

In order to compare the mean values of soluble and total oxalate of vegetable samples with equal sample size obtained by using both the developed CPE/spectrophotometric and the independent kinetic
method, the statistical t value for the degree of freedom of n1 +n2 2=8 at the probability level of 0.05 is 2.31

The average plus standard deviation of five replicate measurements for soluble oxalate after pretreatment without 30 mL of 0.2 mol L1 HCl under ultrasonic power (300 W, 50 Hz) at 60 C 15 min

2.1

101

10.60.5
16.00.6

18.4

79.71.7

50

2.6

102

The percent recoveries obtained for five replicate measurements of spiked oxalate at three different concentration levels of 5, 25, and 50 g L1

54.61.4

25

3.4
3.5

The average plus standard deviation of five replicate measurements for total oxalate after pretreatment with 30 mL of 0.2 mol L1 HCl under ultrasonic power (300 W, 50 Hz) at 60 C 15 min

34.51.2

29.01.0

The Students t test

1/100

Foundf
(g kg1)

Soluble oxalate Total oxalate

By the independent
kinetic method

Foundd
RSD % Recoveryb (%) Founde
(g kg1)
(g kg1)

Insoluble oxalate Soluble oxalate

Founda (g kg1) RSD % Recoveryb (%) Foundc


(g kg1)

Total oxalate

Dilution ratio Added oxalate By the developed CPE/spectrophotometric


(g kg1)
method

Leek

Samples

Table 4 (continued)

Food Anal. Methods

Oxidation with 0.2


mol L1 H2SO4/
BrO3 for 4 min
at 70 C
Extraction by using 0.2
mol L1 H3PO4 for
CE assays or 0.2 mol
L1 HCl or 0.1 mol
L1 H3PO4 containing
400 mg L1 NaCl at
60 C, conductivity
detection/UV detection
at 254 nm
Extraction with 2.0 mol
L1 HCl or water for
15 min at 21 C for
total and soluble oxalate

3.24 mg g1
5.1, 18.6 mg/100 g soluble,
total oxalate

6.2106 mol L1
1.20105 mol L1

1.5 mol L1

(0.014)103
mol L1

Complex matrices

Fruits

Spinach

(0.051.5)102
mol L1

Separation by anion
exchange column
at pH 5.0, amperometric
detection of H2O2
enzymatically produced
by immobilized oxalate
oxidase
Separation by anion exchange
column at pH 5.0, amperometric
detection of H2O2 enzymatically

HPLC-ER

Electrocatalytic oxidation
in 0.1 mol L1 H2SO4
HPLC-ER

HPLC with chemiluminescence


detection

0.25 mol/L Ru(phen)32+/2.0


mmol/L Ce(SO4)2/0.08
mol/L H2SO4
MWNTs/GC electrode

Spinach

0.7 mg L1

2180 mg L1

Catalytic spectrophotometry

Victoria blue 4R, bichromate

1340, 8.1, 470980, and


190380 mg/100 g
carrots, pear, cocoa

4.20 mg g1

61.3, 56.1 mg for total;


9.53, 11 mg for soluble
and 51.8, 45.2 mg insoluble/
100 g green and golden
kiwifruit
18.8, 5.6, and 6.3 mg L1

IE-HPLC

794, 24.7, and 0.68


mg/100 g spinach,
potatoes, and apple
with IC; 774, 22.7,
and 0.59 mg/100 g
spinach, potatoes, and
apple with CE

0.2 mg per 100 g


(quantification
limit, a S/N 10)

1.95 mol L1
(quantification
limit, S/N 10)

22.3 mg soluble, 31.6


mg insoluble, and
52.6 mg total/100
g leaf vegetables;
23.3 mg soluble,
30.4 mg insoluble,
and 53.4 mg total/
100 g flower or fruit
consumed vegetables;
37.9 mg soluble, 37.6
mg insoluble, and 74
mg total/100 g legume
seeds
51.7690.9 mg/100 g

301 mol L1

Average

0.24 mg per 100 g

030 mol L1

3 mg per 100 g
(quantification
limit)

mol L

0.5 mol L1

510

Detection limit Oxalate concentration

CE and IC

Spinach, beet, and


mushroom

Two different cultivars


of kiwifruit

Food samples

HS-GC

0500 g mL1

HPLC

Foods

Different vegetable
samples

Aspartate/bis-tris propane/
methyl hydroxyethyl
cellulose at pH 3.8
Using an isocratic elution
at 0.5 mL min1 with
0.0125 mol L1 sulfuric
acid as a mobile phase,
UV detection at 210 nm

Different beer samples

210 to 210
mol L1
225 mol L1

DPP

o-Phenylenediamine

Corks, beer, and spinach

Linear range

CZE

Detection tool

Complexing reagent
or other remarks

Comparison of analytical performance of the proposed method in similar matrices with those of other detection techniques

Matrix type

Table 5

2.26.7, 3.78.6 for


intraday and interday
precision

4.5 (0.015 mol L1,


n=5)
4.76, 3.87 for soluble
and total oxalate

5.6 (1103 mol L1,


n=5)

2.14, 0.52 for fraction


of skin; 0.69, 1.97
for fraction of pulp;
1.33, 2.71 for fraction
of seeds
0.7

0.8, 9.3, and 4.0 spinach,


potatoes, and apple
with IC; 10.2, 9.4,
and 26.8 spinach,
potatoes, and apple
with CE

0.84 (662.1 mg/100


g, n=5)

5.5, 8.4

3.4

RSD %

0.1185.6, 0.3345.7
mg/100 g soluble,
total oxalate

0.1185.6, 0.3345.7
mg/100 g soluble,
total oxalate

28.1 and 38.1 mg, 14.4,


15.2 mg and 82.6 and
81.3 mg for fractions
of skin, pulp/100 g
green and golden kiwifruit

0.313 mg/100 g broccoli,


potatoes from 5.530
mg/100 g potatoes,
and 58524 mg/100
g wheat bran

3110 mg soluble,
3135 mg insoluble,
and 3161 mg total/
100 g leaf vegetables;
3163 mg soluble,
3105 mg insoluble,
and 3222 mg total/
100 g flower or fruit
consumed vegetables;
3118 mg soluble,
3145 mg insoluble,
and 3204 mg total/
100 g legume seeds
Relative difference of
2.26 % with those of IC

168501 mol L1

Published data

Hnow et al. (1997)

Hnow and Hesse


(2002)

Zheng et al. (2009)

Wu et al. (1999)

Safavi and Banazadeh


(2007)

Nguyn and Savage


(2013)

Holmes and Kennedy


(2000)

Li et al. (2014)

Judprasong et al.
(2006)

Masar et al. (2003)

Rodrigues and Barros


(1993)

References

Food Anal. Methods

The present study


0.9376 mg/100 g total
oxalate, 0.5265
mg/100 g soluble
oxalate, 0.4205
mg/100 g insoluble
oxalate
2.05.1 for total oxalate;
1.65.3 for soluble
oxalate
0.36 g L1
CPE/spectrophotometry
at 627 nm

1.2240 g L1

19.5 mg/100 g total oxalate,


10.5 mg/100 g soluble
oxalate, 3.30 mg/100 g
insoluble oxalate

6.4, 7.0

Vegetable samples

Tartaric and malic acids

Isocratic elution at pH
2.102.15 with
HClO4, UV detection
at 210 nm
Toluidine blue in the
presence of Mo(VI)
at pH 6.0

Ion suppression RP-HPLC

0.5 mg L1

powder, and rhubarb


(as total oxalate);
0.971.65 mg/100 ml
fennel tea
28, 25 mg L1
produced by immobilized
oxalate oxidase

Detection tool
Complexing reagent
or other remarks
Matrix type

Table 5 (continued)

RSD %
Average

Linear range

Detection limit Oxalate concentration

Published data

References

Lian et al. (1999)

Food Anal. Methods

values in the range of 0.352.00 and 0.401.95 for soluble


oxalate and total oxalate, respectively, are lower than the tabulated t value of 2.31, it can be concluded that, statistically,
there isnt a significant difference between the mean values
obtained by both methods. It is clear that the method has a
good reproducibility with lower RSD than 5.3 % for the analysis of both soluble and total oxalate contents.
Also, we investigated the applicability of the CPE
methodology by determining soluble, insoluble, and total oxalate species in vegetable samples. The samples
were prepared as described in the section BPreparation
to Analysis of Vegetable Samples.^ The prepared sample solutions (3.0 mL) were transferred into volumetric
tubes of 50 mL, individually. Then, the method was
successfully applied to determine the amounts of total
and soluble oxalate after pretreatment with and without
30 mL of 0.2 mol L1 HCl under ultrasonic power
(300 W, 50 Hz) at 60 C by adopting the standard
addition method in the range of 6180 g L1, respectively. The insoluble oxalate level was calculated from
the difference between soluble and total oxalate levels.
The received samples were analyzed for the presence of
oxalate species in the pretreated and diluted samples
with the proposed method. The results are given in
Table 4. In any event, the calibration was attained using
the aqueous standard calibration curves. It can be seen
that the recoveries from spiked solutions are quantitatively varied in the range of 96.6103 % for vegetable
samples. The precision of five replicate determinations
is typically better than maximum RSD of 5.3 % for
both soluble oxalate and total oxalate.

Comparison of the Proposed Method with Other


Preconcentration Techniques
When some of the past studies based on the analysis of similar
matrices in the literature were considered in Table 5, a good
sensitivity improvement and extraction efficiency, in terms of
detection limit (LODs) and preconcentration and enhancement factors (PF and EF), were achieved by the proposed
method according to those of different separation and detection techniques like DPP (Rodrigues and Barros 1993), CZE
on a chip with conductivity detection (Masar et al. 2003), CZE
(Guan et al. 2012), catalytic spectrophotometry (Safavi and
Banazadeh 2007), HPLC with chemiluminescence detection
(Wu et al. 1999), HPLC-ER (Hnow et al. 1997), ion suppression RP-HPLC (Lian et al. 1999), HPLC (Judprasong et al.
2006), HS-GC (Li et al. 2014), enzymatic assays using oxalate
oxidase (Kasidas and Rose 1980), and IC (Holmes and
Kennedy 2000) without preconcentration. The detection
limits, LODs (0.36 g L1), obtained in the present study
are either better than or comparable to those of the reported

Food Anal. Methods

detection methods. Also, it has relatively a wider working


range (1.2240 g L1) with a lower RSD than 5.3 % at low
concentrations. It has a good preconcentration factor (65.8)
and sensitivity enhancement factor (72.5) when compared to other preconcentration procedures such as
CZE with online sample preconcentration (Suzuki
et al. 2005) and indirect FI-FAAS with online
preconcentration (Esmadi and Attiyat 1994). Moreover,
the precision as RSDs is better than that in previous
results with HPLC (Judprasong et al. 2006), IE-HPLC
(Nguyn and Savage 2013), HPLC-ER (Hnow et al.
1997), ion suppression RP-HPLC (Lian et al. 1999),
HPLC with chemiluminescence detection (Wu et al.
1999), and CE and IC (Holmes and Kennedy 2000)
separation techniques with UV detection. Moreover, it
is considerably more difficult to use these techniques
as well as time-consuming and poor precision. As a
result, the developed method provides advantages of
wider linear range, low detection limit, high selectivity,
good precision, adequate accuracy, quantitative recovery,
and high sensitivity enhancement factor for the spectrophotometric detection of trace oxalate species in vegetable samples.

Conclusions
In the current study, a new CPE/UV-Vis system was
developed for the preconcentration and determination
of soluble, insoluble, and total oxalate in vegetable samples. TBH 2+ giving a stable ternary complex in the
presence of Mo(VI) using Triton X-45 as an extracting
agent and NH4F as a salting-out agent at pH 6.0 was
for the first time studied for the quantification of the
oxalate species in vegetable samples. Under the optimized conditions, the method permits oxalate detection
at 0.36 g L1 levels in the range of 1.2240 g L1 at
627 nm. Moreover, ultrasonic induction was successfully used to assist the fast and efficient extraction of the
analyte from the sample matrix before separation/
preconcentration with CPE. In terms of apparatus, UVVis spectrophotometry is a comparatively simple, economical, and versatile detection tool, which can be
available in nearly every research laboratory. Also, the
power of this technique can be improved at increasing
ratios in terms of sensitivity and selectivity when
selecting a suitable photometric probe in the visible region such as TBH2+. For these reasons, the proposed
method can be considered as an alternative tool to the
sensitive but expensive, time-consuming, and necessitating an experienced user hyphenated analytical techniques such as CE-MS and LC-MS/MS as well as IC,

HPLC, CE, CZE and GC with conductivity, UV, fluorescence, and flame ionization detection.

Compliance with Ethical Standards


Acknowledgments Thanks are also due to Prof. Dr. Mehmet Akay for
his meaningful help and contributions in the critical evaluation and publication of the presented research article.
Conflict of Interest The authors, Nail Altunay and Ramazan Grkan,
have no financial relationship with the organization that sponsored the
research.
Ethics Approval This article does not contain any studies with human
or animal subjects.
Informed Consent According to the authors, informed consent was
obtained from all individual participants included in the study.

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