Abstract. This study focused on using a central composite design (CCD) to optimize
the analytical method for simultaneous determination of choline, pantothenic acid
(vitamin B5), ascorbic acid (vitamin C), and niacin (vitamin B3) by capillary zone
electrophoresis (CZE). The optimization process was designed with 30 experiments,
including four factors: Tris concentration, pH, separation voltage and sample
injection time. The optimal condition was found: Tris concentration was 150
mmol/L, adjusted to pH = 9.5 by acetic acid with 10kV of applied voltage, and 60 s
of sample injection time. Analytical methods were evaluated through limit of
detection (LOD), limit of quantification (LOQ), linear range, repeatability, and
recovery. The results showed that the method had good LOD (0.22 - 0.45 mg/L),
extensive linear range (1 - 200 mg/L), high repetition with relative standard deviation
(RSD) for peak area and migration time were less than 4% and 0.5% respectively,
and relative recoveries ranged between 89 and 111%. The developed CZE-method
could be used in the quality control of food supplements.
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26
HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2017-0051
Chemical and Biological Science 2017, Vol. 62, Issue 10, pp. 26-35
This paper is available online at
USING A CENTRAL COMPOSITE DESIGN TO OPTIMIZE
THE ANALYTICAL METHOD FOR SIMULTANEOUS DETERMINATION
OF CHOLINE, PANTOTHENIC ACID, ASCORBIC ACID, AND NIACIN
IN FOOD SUPPLEMENTS BY CAPILLARY ZONE ELECTROPHORESIS
Nguyen Thanh Dam, Nguyen Manh Huy, Vu Minh Tuan,
Duong Hong Anh and Pham Hung Viet
VNU Key Laboratory of Analytical Technology for Environmental Quality and Food
Safety Control (KLATEFOS), University of Science, Vietnam National University, Hanoi
Abstract. This study focused on using a central composite design (CCD) to optimize
the analytical method for simultaneous determination of choline, pantothenic acid
(vitamin B5), ascorbic acid (vitamin C), and niacin (vitamin B3) by capillary zone
electrophoresis (CZE). The optimization process was designed with 30 experiments,
including four factors: Tris concentration, pH, separation voltage and sample
injection time. The optimal condition was found: Tris concentration was 150
mmol/L, adjusted to pH = 9.5 by acetic acid with 10kV of applied voltage, and 60 s
of sample injection time. Analytical methods were evaluated through limit of
detection (LOD), limit of quantification (LOQ), linear range, repeatability, and
recovery. The results showed that the method had good LOD (0.22 - 0.45 mg/L),
extensive linear range (1 - 200 mg/L), high repetition with relative standard deviation
(RSD) for peak area and migration time were less than 4% and 0.5% respectively,
and relative recoveries ranged between 89 and 111%. The developed CZE-method
could be used in the quality control of food supplements.
Keywords: Choline, vitamins, capillary electrophoresis, response surface
methodology, central composite design.
1. Introduction
Choline is a water-soluble vitamin-like essential nutrient. This compound is the
precursor molecule for the neurotransmitter acetylcholine, which is involved in many
functions including memory and muscle control. It has been found that intake of choline
during pregnancy can have long-term beneficial effects on memory for the child [6].
Nicotinic acid (niacin, vitamin B3), pantothenic acid (vitamin B5), and ascorbic acid
(vitamin C), which are in the water-soluble vitamins group, play an important role in human
health [5]. The deficiency of these compounds in the diet can cause serious health issues.
Received November 15, 2017. Revised December 8, 2017. Accepted December 16, 2017.
Contact Pham Hung Viet, e-mail address: phamhungviet@hus.edu.vn
Using a central composite design to optimize the analytical method for simultaneous determination
27
Thus, there is a need to have reasonable analytical methods for their quality control in
different types of food products. Several methods are used to measure choline [4, 7] and
vitamins [2] in supplements and food products. However, only a few methods dealt with
the simultaneous quantitative analysis of these compounds.
In recent years, capillary zone electrophoresis (CZE) has been expanded to the
simultaneous quantitative analysis of water-soluble vitamin in food products [5]. The
chemical properties of these compounds, their ionic nature and water solubility, make
them suitable for electrophoretic analysis. However, there are two main problems
encountered during the development of CZE-method for this aim: (i) different structures
and chemical properties of vitamins make the development of a single method for their
simultaneous determination difficult; and (ii) foods are complex matrices. These
difficulties can be overcome by performing an experimental design to choose the
optimized condition to analysis choline, pantothenic acid, ascorbic acid, and niacin.
Traditionally, optimization in analytical chemistry has been carried out by monitoring
the influence of one factor at a time on an experimental response (one-variable-at-a-time).
While only one parameter is changed, others are kept at a constant level. Its major
disadvantage is that it does not include the interactive effects of the variables studied.
Another disadvantage of this technique is the increase in the number of experiments
necessary to conduct the research, which leads to an increase of time and expenses as well
as an increase in the consumption of reagents and materials [3]. In order to overcome this
problem, the optimization of analytical procedures has been carried out by using
multivariate statistic techniques. Among the most relevant multivariate techniques used in
analytical optimisation is central composite design (CCD), a type of symmetrical second-
order experimental designs in response surface methodology (RSM). This paper describes
the development of a simple CZE-method for the simultaneous determination of choline,
pantothenic acid, ascorbic acid, and niacin, using an experimental design based on CCD
technique. The method was validated and can be used to quantitative these vitamins in
supplement foods.
2. Content
2.1. Experiment
2.1.1. Reagents and apparatus
All chemicals were of analytical or reagent grade and purchased from Tokyo
Chemical Industry (Japan) or Sigma-Aldrich (Germany), except lactic acid from
Guangdong (China). Stock solutions (1000 mg/L) of choline, pantothenic acid, ascorbic
acid, and niacin were used for the daily preparation of the standard solutions. Chemicals
used for the preparation of BGEs included: Acetic acid (Ace), histidine (His), 2-(N-
morpholino)ethanesulfonic acid (MES), lactic acid (Lac), tris(hydroxymethyl)
aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), N-Cyclohexyl-2-
aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS).
Deionized water, purified using a water purification system from Millipore - model
Simplicity UV (USA), was used for the preparation of all standard solutions and sample
dilution if required.
Nguyen Thanh Dam, Nguyen Manh Huy, Vu Minh Tuan, Duong Hong Anh and Pham Hung Viet
28
All experiments were performed on a hand-operated CE instrument in the VNU Key
Laboratory of Analytical Technology for Environmental Quality and Food Safety
Control (KLATEFOS), VNU University of Science. The electrophoresis section was
based on a high voltage power supply (Spellman CZE2000, UK) with ±30 kV maximum
output. Polyimide coated fused silica capillaries of 50 µm I.D. and 365 µm O.D. with 60
cm of total length and 49 cm of effective length (from Polymicro, USA) were used for
the separations. Before their use, the capillaries were pre-conditioned with 1 M NaOH
for 10 min and deionized water for 10 min, followed by flushing with the BGE.
Detection was carried out with a miniaturized high-voltage C
4
D cells built in-house that
is used a power supply of 12 VDC. The resulting signals were recorded with an e-corder
401 data acquisition system (eDAQ, Australia) connected to the USB-port of a personal
computer.
2.1.2. Methodology
* Design of experiment
The composition of BGE was investigated before the development of the
experimental design. Six different BGEs at pH 9.5 were evaluated: 100 mM Tris/Ace;
100 mM Tris/Lac; 60 mM Tris/40 mM CAPS; 100 mM Tris/5 mM MES; 100 mM Tris/5
mM CHES; 100 mM Tris/5 mM MOPS. After selecting the initial BGE component,
further optimization of analysis conditions by using a central composite design with four
independent variables (factors) was performed. The variables were: concentration of Tris
(30 ÷ 150 mmol / L), pH (8.5 ÷ 9.5), applied voltage (10 ÷ 20 kV), and injection time
(10 ÷ 60 s, Table 1). All statistical calculations were developed by the software Design
Expert, v.10.0.4 (USA).
Table 1. The values corresponding to -1, 0, +1, -2 (-α) and +2 (+α)
in the experimental design
Variables Levels
-2 (-α) -1 0 +1 +2 (+α)
X1 (Tris concentration, mmol/L) 30 60 90 120 150
X2 (pH) 8.5 8.75 9.0 9.25 9.5
X3 (applied voltage, kV) 10 12.5 15 17.5 20
X4 (injection time, s) 10 22.5 35 47.5 60
* Method validation
The method was validated using the following performance criteria: limit of detection
(LOD), limit of quantitation (LOQ), linearity, linear range, recovery and repeatability. LOD
includes instrument detection limit (IDL), the method detection limit (MDL). The IDL was
determined by measuring the standard solution (0.5 mg/L per each analyte), calculating
the analyte concentrations that produce a signal/noise (S/N) ratio = 3. MDL was
determined similarly but with a spiked sample of 2 mg/L per each analyte (on the real
sample without analytes). The corresponding LOQ values are determined by the
expression: LOQ = 3 LOD. The linearity, linear range were established through the
Using a central composite design to optimize the analytical method for simultaneous determination
29
calibration curve obtained by triplicate analysis of choline, pantothenic acid, ascorbic acid
and niacin at five concentration levels 2.0, 5.0, 10, 20, and 40 mg/L.
Repeatability was determined by intra-assay and inter-assay precision. The intra-
assay precision of the method expressed as the relative standard deviation (RSD) of peak
area and migration time measurements (n = 11), was evaluated through the results
obtained with the method operating over one day under the optimal condition, using
solution at concentration of 20 mg/L of each analyte. The inter-assay precision was
determined for the same solution but was performed for seven days (n = 7).
Recovery was determined by standard addition method on deionized water and real
samples at concentrations of 2.5 mg/L and 25 mg/L. Repeatability was determined by
repeated measurements of intraday (11 times) and daily (7 days) for the 20 mg/L standard
solution and calculated the RSD of the peak area and migration time.
2.2. Results and discussion
2.2.1. CZE method development
* Investigation of BGE’s composition
The BGE is an important factor that affects the detection and separation of the
analytes. Among the analytes, pantothenic acid, ascorbic acid and niacin all contain the
acid functional group (-COOH) in the molecule, so it would be preferable if they were
detected as anions. In contrast, choline is a quaternary ammonium salt and should be
detected in cationic form. For the simultaneous determination of four analytes in one run,
the use of reverse polarity technique proves to be most appropriate. At this point, the
positive voltage is applied to the electrode at the side that sample is injected while the
electrode on the detector side is grounded to produce a negative polarization (towards
the detector). It can be predicted that choline, in the form of cation, will move towards
the cathode. Interestingly, in reverse polarization, under the effect of electroosmotic
flow (EOF), the anion forms of vitamins will also move towards the cathode (in the
same direction as the cation of choline) and will be detected by the C
4
D detector. To do
this, the pH of the BGE must be relatively high to ensure that the EOF is powerful
enough to be able to bring vitamins, which are quite large compounds.
Figure 1. Investigation results of the background electrolytes solution
Nguyen Thanh Dam, Nguyen Manh Huy, Vu Minh Tuan, Duong Hong Anh and Pham Hung Viet
30
In six BGEs used in this investigation, the Tris/CHES (pH 9.5) and Tris/Ace (pH 9.5)
buffers gave the sharpest signal and highest peak area. However, in the case of the
Tris/CHES buffer, after the peak of niacin signal, there was a significant rise in the
background, which may affect the quantification of niacin (Figure 1). Thus, the Tris/Ace
buffer was chosen as the background electrolyte for subsequent optimization.
* Optimization design
The central composite design consists of three parts: (i) a full factorial or fractional
factorial design; (ii) an additional design, often a star design in which experimental points
are at a distance α from its center; and (iii) a central point. This design presents the
following characteristics:
(1) Require an experiment number according to N = 2
k
+2k+cp, where k is the factor
number and (cp) is the replicate number of the central point;
(2) α-values depend on the number of variables and can be calculated by α =2k/4. For
example, if there are four variables, the α-value will be 2.00;
(3) All factors are studied in five levels (−α, −1, 0, +1, + α).
In this work, an experimental design using CCD with four factors was conducted:
concentration of Tris (30 - 150 mmol/L), pH (8.5 - 9.5), voltage (10 - 20 kV), and
injection time (10 - 60 s). In CE method, these are the factors that affect the separation, in
which concentration of electrolyte and pH are the chemical factors while voltage and
injection time are factors of the device. The experimental design was constructed by the
use of a full 2
4
factorial design with six central and eight axial points. Therefore, the
overall matrix of CCD design involved 30 experiments.
This design was used to optimize and evaluate main effects, interaction effects, and
quadratic effects. The nonlinear computer-generated quadratic model is given as:
where Xi are the independent variables (factors); b0 is an intercept; bi, bii, bij are the
regression coefficients; and Y is the response function. In this study, the peak areas of
each target analyte are responses and denoted Y1, Y2, Y3, Y4 corresponding to the peak
area of choline, pantothenic acid, ascorbic acid, and niacin, respectively. The experiments
and the results of the model are presented in Table 2.
The separation of choline, pantothenic acid, ascorbic acid, and niacin under study is
achieved at a concentration of 150 mmol/L Tris, adjusted to pH 9.5 by acetic acid, voltage
10 kV, and 60 s of injection time. The responses of the model, R
2
values, were greater
than 0.87, implying that the model was well fitted by the data for the response of area of
peaks. The F-test for the models showed that the p-values of all responses were smaller
than 0.0001 while the lack of fit F-test had p-values that were greater than 0.1 (0.1155 - 0.8172).
This indicated a good correlation of the obtained regression equation, which shows that
the obtained models were consistent and had high statistical reliability. In addition, a low
coefficient of variation (CV%), less than 10% (1.21 - 9.48%), indicated that the
experiments had high repeatability (Table 3).
Using a central composite design to optimize the analytical method for simultaneous determination
31
Table 2. Experimental matrix and the results
Run X1 X2 X3 X4 Y1 Y2 Y3 Y4
mmol/L
kV s V.s V.s V.s V.s
1 30 9 15 35 0.005 0.027 0.015 0.024
2 60 8.75 12.5 22.5 0.006 0.022 0.010 0.020
3 60 8.75 12.5 47.5 0.008 0.044 0.022 0.048
4 60 8.75 17.5 22.5 0.004 0.014 0.007 0.013
5 60 8.75 17.5 47.5 0.006 0.030 0.015 0.030
6 60 9.25 12.5 22.5 0.005 0.022 0.010 0.019
7 60 9.25 12.5 47.5 0.008 0.046 0.019 0.041
8 60 9.25 17.5 22.5 0.004 0.017 0.007 0.015
9 60 9.25 17.5 47.5 0.006 0.032 0.013 0.028
10 90 8.5 15 35 0.005 0.022 0.012 0.024
11 90 9 10 35 0.007 0.035 0.021 0.035
12 90 9 15 10 0.003 0.012 0.005 0.013
13 90 9 15 35 0.006 0.028 0.009 0.022
14 90 9 15 35 0.006 0.027 0.014 0.028
15 90 9 15 35 0.007 0.028 0.009 0.025
16 90 9 15 35 0.006 0.027 0.009 0.028
17 90 9 15 35 0.006 0.027 0.008 0.030
18 90 9 15 35 0.006 0.026 0.008 0.026
19 90 9 15 60 0.009 0.052 0.024 0.057
20 90 9 20 35 0.004 0.018 0.010 0.018
21 90 9.5 15 35 0.005 0.031 0.017 0.029
22 120 8.75 12.5 22.5 0.005 0.017 0.006 0.018
23 120 8.75 12.5 47.5 0.009 0.038 0.012 0.041
24 120 8.75 17.5 22.5 0.003 0.012 0.004 0.013
25 120 8.75 17.5 47.5 0.005 0.024 0.007 0.026
26 120 9.25 12.5 22.5 0.005 0.023 0.008 0.022
27 120 9.25 12.5 47.5 0.010 0.051 0.015 0.046
28 120 9.25 17.5 22.5 0.004 0.015 0.005 0.015
29 120 9.25 17.5 47.5 0.006 0.037 0.011 0.032
30 150 9 15 35 0.006 0.026 0.010 0.029
Nguyen Thanh Dam, Nguyen Manh Huy, Vu Minh Tuan, Duong Hong Anh and Pham Hung Viet
32
Table 3. The results of analysis of variance and model testing
Response
Model Lack of fit
R
2
CV(%)
F p-value F p-value
Y1 38 < 0.0001 2.56 0.1520 0.92 9.48
Y2 285 < 0.0001 2.99 0.1155 0.99 1.21
Y3 24 < 0.0001 0.78 0.6826 0.87 3.88
Y4 95 < 0.0001 0.58 0.8172 0.97 8.23
Before proceeding to validate the CZE method for the determination the vitamins in
food supplements, the optimal conditions from the model was tested by comparing the
analysis results with the predicted value of the model. As shown in Table 4, the test
results were in the range predicted by the model and the deviation from the predicted
values of the model was less than 10%. Thus, the predictive values of the model were
highly accurate, in other words, the model was consistent.
Figure 2. The 3D response surface shows the effect of the pair
of variables on the peak area of choline
Using a central composite design to optimize the analytical method for simultaneous determination
33
Table 4. The results of the experiment used to test the analytical conditions
Peak area Predicted
range (V.s)
Predicted
value (V.s)
Analysis
results (V.s)
Choline 0.0110 - 0.0154 0.0130 0.0150
Pantothenic 0.0869 - 0.1234 0.1037 0.0777
Ascorbic 0.0303 - 0.1002 0.0560 0.0366
Niacin 0.0720 - 0.0890 0.0804 0.0740
2.2.2. Validation of the analytical method
The CZE method was validated for the analyses of analytes by evaluation of the
following parameters: LOD, LOQ, linear range, linearity, intra- and inter-assay precision
and accuracy. The results are summarized in Table 5. It can be seen that the method had
low LODs and LOQs. IDLs of choline, pantothenic acid, ascorbic acid and niacin were
0.12; 0.06; 0.13 and 0.09 mg/L, respectively. MDL and MQL were also low, MDL ranged
from 0.22 to 0.50 mg/L, and MQL varied from 0.74 to 1.68 mg/L; pantothenic acid had
the best, and ascorbic acid had the highest LOQ. The linear ranges of the analytes were
extensive, with the range of niacin was widest, from 0.98 to 500 mg/L, while choline gave
the narrowest linear range (1.17 to 200 mg/L). Repeatability for both peak area and
migration time were very good, RSD of peak areas and migration times for intra-assay
(n = 11) were lower than 4% and 0.5%, and for inter-assay (n = 7) were not greater than
10% and 4%, respectively.
Table 5. Quantitative features for choline, pantothenic acid, ascorbic acid and niacin
Parameter Choline Pantothenic acid Ascorbic acid Niacin
IDL (mg/L) 0.12 0.06 0.13 0.09
IQL (mg/L) 0.36 0.18 0.39 0.27
MDL (mg/L) 0.45 0.22 0.50 0.35
MQL (mg/L) 1.52 0.74 1.68 1.15
Linear range (mg/L) 1.17 - 200 0.6 - 250 1.29 - 250 0.98 - 500
Linearity (R
2
) 0.999 0.999 0.998 0.999
Intra-assay precision (20 mg/L, n = 11)
RSD
a
% 1.7 1.5 1.7 3.3
RSD
b
% 0.4 0.2 0.2 0.2
Inter-assay precision (20 mg/L, n = 7)
RSD
a
% 7.7 6.0 9.3 6.0
RSD
b
% 1.5 2.8 3.0 3.4
RSD
a
: relative standard deviation of peak area;
RSD
b
: relative standard deviation of migration time
Nguyen Thanh Dam, Nguyen Manh Huy, Vu Minh Tuan, Duong Hong Anh and Pham Hung Viet
34
The recovery of each analyte in standard solutions (deionized water background) at a
concentration of 2.5 mg/L and 25 mg/L were 94 - 100% and 93 - 96%, respectively. In real
sample background, recoveries were good at 89 to 111% and 91 to 109% for standard
additions of 2 mg/L and 10 mg/L, respectively (Table 6). According to AOAC, these values
are acceptable [1].
Figure 3. Electropherogram of choline, pantothenic acid, ascorbic acid, and niacin
Table 6. Recoveries (%) of