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2 AUTHORS:
Nail Altunay
Ramazan Grkan
Cumhuriyet University
Cumhuriyet University
21 PUBLICATIONS 19 CITATIONS
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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
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-
MoO3 Ox
(a)
0.6
0.5
Absorbance at 627 nm
0.4
0.3
0.2
2
0.1
MoO3 Ox
3
0.0
1
pH
(b)
0.8
0.7
Absorbance at 627 nm
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
4
MoO4
H MoO3 OH
5a
5b
(a)
1.4
1.2
1.0
Absorbance at 627 nm
0.8
0.6
0.4
0.2
2+
-1
TBH concentration,mol L
(b)
1.6
1.4
1.2
Absorbance at 627 nm
*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
(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
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
(b)
1.2
1.0
0.8
Absorbance at 627 nm
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
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
(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
Oxalate concentration,g L
2-
-3
50
100
150
200
Oxalate concentration,g L
250
300
-1
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
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)
Table 2
Intermediate precision
(interday)
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
Interfering species
Tolerance limits
Recovery (%)
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
1 (50)c, 5 (100)d
98.1104
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)
By the independent
kinetic method
Foundd
RSD % Recoveryb (%) Founde
1
(g kg )
(g kg1)
Total oxalate
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
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
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
1/100
Foundf
(g kg1)
By the independent
kinetic method
Foundd
RSD % Recoveryb (%) Founde
(g kg1)
(g kg1)
Total oxalate
Leek
Samples
Table 4 (continued)
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
Spinach
0.7 mg L1
2180 mg L1
Catalytic spectrophotometry
4.20 mg g1
IE-HPLC
1.95 mol L1
(quantification
limit, S/N 10)
301 mol L1
Average
030 mol L1
3 mg per 100 g
(quantification
limit)
mol L
0.5 mol L1
510
CE and IC
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
210 to 210
mol L1
225 mol L1
DPP
o-Phenylenediamine
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
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
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
Wu et al. (1999)
Li et al. (2014)
Judprasong et al.
(2006)
References
1.2240 g L1
6.4, 7.0
Vegetable samples
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
0.5 mg L1
Detection tool
Complexing reagent
or other remarks
Matrix type
Table 5 (continued)
RSD %
Average
Linear range
Published data
References
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.
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