Author’s Accepted Manuscript

Simultaneous voltammetric determination of

vanillin and caffeine in food products using an
anodically pretreated boron-doped diamond
electrode: Its comparison with HPLC-DAD

Hoshyar Saadi Ali, Abdullah A. Abdullah, Pınar

Talay Pınar, Yavuz Yardım, Zühre Şentürk
www.elsevier.com/locate/talanta

PII: S0039-9140(17)30462-9
DOI: http://dx.doi.org/10.1016/j.talanta.2017.04.037
Reference: TAL17490
To appear in: Talanta
Received date: 1 March 2017
Revised date: 12 April 2017
Accepted date: 15 April 2017
Cite this article as: Hoshyar Saadi Ali, Abdullah A. Abdullah, Pınar Talay Pınar,
Yavuz Yardım and Zühre Şentürk, Simultaneous voltammetric determination of
vanillin and caffeine in food products using an anodically pretreated boron-doped
diamond electrode: Its comparison with HPLC-DAD, Talanta,
http://dx.doi.org/10.1016/j.talanta.2017.04.037
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 1

Simultaneous voltammetric determination of vanillin and caffeine in food products

using an anodically pretreated boron-doped diamond electrode: Its comparison with

HPLC-DAD

Hoshyar Saadi Ali1, Abdullah A. Abdullah1, Pınar Talay Pınar2, Yavuz Yardım2*, Zühre Şentürk1*

1
Yüzüncü Yıl University, Faculty of Science, Department of Analytical Chemistry, 65080

Van, Turkey
2
Yüzüncü Yıl University, Faculty of Pharmacy, Department of Analytical Chemistry, 65080

Van, Turkey

yavuzyardim2002@yahoo.com

zuhresenturk@hotmail.com
*
Corresponding authors. Tel: 90 432 2251024; Fax: 90 432 2251188

Abstract

This paper describes an electroanalytical method for the simultaneous determination of

vanillin (VAN) and caffeine (CAF) using an anodically pretreated boron-doped diamond

electrode. Selective determination of one compound in the presence of other one was also

realized. Both compounds yielded a single irreversible oxidation peak using cyclic

voltammetry. The nature of the electrode reaction was found to be diffusion controlled with

contribution of adsorption. By using square-wave adsorptive stripping voltammetry after 60 s

accumulation under open-circuit voltage, method allowed simultaneous determination of

VAN and CAF in phosphate buffer, pH 2.5, with detection limits of 0.234 µg mL-1 (1.54×10-6

M) and 0.071 µg mL-1 (3.66×10-7 M), respectively. The proposed method was successfully

applied in the selective and simultaneous determination of VAN and CAF in commercial food
 2

and beverage samples. In addition, for the comparison, high-performance liquid

chromatographic method with diode-array detection was developed for the first time for their

simultaneous determination.

Keywords: Vanillin; Caffeine; Simultaneous voltammetric determination; Anodically

pretreated boron-doped diamond electrode; High-performance liquid chromatography;

Commercial food and beverage samples.

1. Introduction

Vanillin (4-hydroxy-3-methoxybenzaldehyde) (VAN) and Caffeine (1,3,7-trimethylxanthine)

(CAF) are two worldwide additives and their combination is often used in various foods,

beverages, and other products. As is well known, VAN is a phenolic aldehyde derived from

vanilla beans, which is indispensable smell additive usually in sweet foods such as instant

noodles, candies, pudding, chocolate, biscuits, ice creams. Besides, it has antioxidative and

antimicrobial property. On the other hand, CAF is a naturally occurring alkaloid which

represents a mild stimulant for central nervous system, muscle, heart and circular systems of

the human body. Owing to its effects, CAF may improve alertness, learning capacity and

exercise performance when moderately consumed. However, excessive ingestion of VAN and

CAF may cause many undesired consequences to their consumers. Considering VAN

overdose, headaches, nausea and vomiting may occur. It has been also reported potential

damage to human liver and kidney. Specifically, taking large amounts of CAF can lead

potentially adverse effects on health, including agitation, irritability, loss of appetite,

insomnia, hypertension, gastrointestinal problem, fever, delusions, tachycardia and even

death. [1-5].
 3

Consequently, monitoring the levels of VAN and CAF is required not only for the quality

(taste and health benefit of the product) control, but also to study the effects of these

compounds on the human body. To date, from the electroanalytical point of view, a number of

studies have been published for individual or simultaneous determination of VAN or CAF in

various matrices. Despite the fact that these organic molecules are electrooxidizable at several

electrodes, however, bare electrode materials have rarely been used for their analysis [6-10].

This is because oxygen evolution current interferes at high oxidation potential of CAF, or the

electrochemical oxidation of phenolic compound VAN produces phenoxy radicals that readily

polymerize to passivate the the electrode surface. A large number of papers for this purpose

involve the use of modified electrodes for quantification of VAN and CAF either alone or

simultaneously with other compounds [11-25]. However, up to now, only two methods have

been reported for the voltammetric determination of both compounds simultaneously [26, 27].

The first investigation has demonstrated the effectiveness a nitrogen-doped graphene/carbon

nanotubes nanocomposite by using square-wave voltammetry in 0.1 M H2SO4 to determine

VAN and CAF simultaneously in beverage and food samples [26]. The square-wave

voltammetric method in 0.1 M acetate buffer solution (pH 4.0) has been applied to their

simultaneous determination in energy drink and vanilla sugar samples using a poly(alizarin

red S) modified glassy carbon electrode [27].

The boron-doped diamond (BDD) is a relatively new carbon-based electrode material

which opens new possibilities for electroanalysis of organic compounds. It offers the

attractive electrochemical and mechanical properties, such as the widest usable

electrochemical potential window in aqueous solutions among all electrode materials, low and

stable background current, a high resistance to deactivation by surface fouling, corrosion

stability in highly aggressive media, low adsorption of polar molecules, a relative insensitivity

to dissolved oxygen, good mechanical robustness [28, 29]. However, it is important to point
 4

out that, for many analytes, these properties of BDD electrode are affected by its surface

termination (e.g. hydrogen or oxygen), which can be modified by appropriate electrochemical

pretreatment (anodic or cathodic pretreatment). These procedures enhance the particular

voltammetric signals and ensure repeatable and reproducible response of analytes [30-34].

The electrochemical determination of CAF on BDD electrode individiually [35, 36] or

simultaneously in combination with some other compounds [37-42] has been reported. In the

case of VAN, however, no literature data were found on the use of BDD electrode except in

previous work carried out in our laboratory dealing with its quantification of commercial

pudding powder [43].

Bearing in mind very new and limited data (both based on using modified electrodes) on

the simultaneous voltammetric determination of VAN and CAF, herein, we intend to verify

the capabilities of BDD electrode without the need of any chemical modifications for the

determination of these compounds separately as well as simultaneously. Based on this, the

analytical performance of BDD electrode will be demonstrated in analysis some food and

beverage products. From our knowledge it is important to remark that no attempts for the

simultaneous determination of both drugs using liquid chromatography have been published

yet to date. Considering this, the obtained results will be compared with those from high-

performance liquid chromatography (HPLC) which will be developed for the first time in the

present study.

2. Experimental

2.1. Apparatus

All voltammetric measurements were performed on a µAutolab type III (Metrohm Autolab

B.V., The Netherlands) potentiostat/galvanostat controlled with the GPES softwar (version

4.9). For a better and clearer identification of the original peaks, the experimental square-
 5

wave (SW) voltammograms presented were baseline-corrected using the Savicky and Golay

filter (level 2) and a moving average application (peak width = 0.01 V) of the GPES software.

A classical three-electrode cell of volume 10 mL was used with a BDD working electrode

(Windsor Scientific Ltd.; Ø: 3mm, diameter), a platinum wire auxiliary electrode and an

Ag/AgCl (3 M NaCl) reference electrode (Model RE-1, BAS, USA) to which all electrode

potentials herein after are referred.

At the beginning of each working day, the BDD electrode was electrochemically pretreated

in 0.5 M H2SO4 solution by applying a potential of +1.8 V (unless otherwise stated) during

180 s. An activation programme was used for 60 s under the same experimental conditions

between individual measurements. The pretreatment procedure was carried out in an

independent electrochemical cell.

The HPLC studies of VAN and CAF were carried out using an Agilent 1100 autosampler

system with a diode-array detector set at 207 nm. The separation of the compounds was

accomplished on a Nucleosil C18 (250 mm × 4.6 mm, 5 µm) chromatographic column at 25.0 oC.

All analysis were done at a flow rate of 1.0 mL min-1.

The pH was measured using a WTW inoLab pH 720 meter, employing a combined

electrode (glass-reference electrodes).

2.2. Chemicals and solutions

All chemicals employed in this work were of analytical grade, and aqueous solutions were

prepared with deionised water further purified via a Milli-Q unit (Millipore). VAN

(ReagentPlus®, 99%) and CAF (ReagentPlus®) were purchased from Sigma-Aldrich. Stock

solutions of VAN and CAF (both 1.0 mg mL-1) were prepared by dissolving in water/ethanol

mixture (50:50 v/v) and water, respectively, and then stored in refrigerator. On the day of the

experiment working solutions were prepared by diluting with a selected supporting electrolyte.
 6

Three different supporting electrolytes, namely acetate (0.1 M, pH 4.7), phosphate (0.1 M, pH

2.5 and 7.4) and Britton-Robinson (BR, 0.1 M, pH 2.0-7.0) buffer solutions were used.

For HPLC experiments, the mobile phase was acetonitrile/water mixture (25:75 v/v)

brought to pH 2.5 by the drop wise addition of an 85% (w/w) H3PO4 solution, while the

injection volume was 20 µL. All solutions were filtered using a vacuum filtration system

through 0.45 μm membrane filters from Agilent Technologies. The stock and standard

solutions of VAN and CAF were prepared in the mobile phase.

2.3. Measurement procedures

Cyclic voltammetry (CV) was employed for preliminary studies on the electrochemical

behavior of VAN and CAF. Square-wave adsorptive stripping voltammetry (SW-AdSV) was

used for the development of electroanalytical methodology for the selective and simultaneous

determination of VAN and CAF in real samples. The deaeration of the supporting electrolyte

was not necessary, since O2 presence does not cause any interference in the studied

experimental conditions.

After optimizing experimental parameters for the proposed method, analytical curves were

constructed by successive addition of aliquots of VAN and CAF standard solutions into to the

measurement cell containing 10 mL of supporting electrolyte. SW stripping voltammograms

were obtained after each aliquot addition of both compounds. All measurements were

performed in triplicate. The described method was validated for the parameters such as

linearity, limits of detection (LOD) and quantification (LOQ), precision and accuracy. The

LOD and LOQ values were calculated on using following equations: LOD = 3s/m; LOQ =

10s/m where s is the standard deviation of the peak current (three runs) of the lowest

concentration of the related linearity range, and m the slope of the related analytical curve. All

data were obtained at room temperature (~25 °C).

 7

2.4. Pretreatment of the commercial samples

Samples of commercially-available vanilla sugar, foamy instant coffee (with sugar and cream)

and cola soft drink were purchased from a local supermarket, and analyzed shortly after

opening. Solid samples (vanilla sugar and foamy instant coffee) taken from five package of

the same brand were previously mixed in a mortar. An accurately weighed portion of powder

(about 1 g of sample) was dispersed in 50 and 25 mL of water/ethanol mixture (50:50 v/v) for

vanilla and coffee samples, respectively. The mixture was left in an ultrasonic bath for 30

min, and then waited on the heater (70 oC) at about 2 h (solution A). In the case of the sample

of cola beverage, the content of three bottles was mixed thoroughly, and sonicated to to

remove CO2. An adequate volume of the resulting solutions was transferred to a voltammetric

cell already containing 10 mL of the selected supporting electrolyte (phosphate buffer, pH

2.5) and analyzed in the day of preparation.

For stripping voltammetric analysis of samples, pre-concentration of VAN and CAF onto

the surface of the previously treated BDD electrode was performed by adsorptive accumulation

at open-circuit condition for 60 s while the solution was stirred using a bar magnet (10 mm×3

mm) rotating at 500 rpm. After an equilibration time of 10 s, voltammograms were recorded,

while the potential was scanned from +0.2 to +1.8 V using the optimized SW modulation with

14 mV scan increment, 70 mV pulse amplitude, and a frequency of 50 Hz.

For HPLC experiments, solution of the solid samples (solution A) was diluted with mobile

phase. The final solution was filtered and injected into the HPLC apparatus. Liquid sample (cola

soft drink) was degassed by ultrasounds, filtered and injected directly in the HPLC system.

For the voltammetric analysis, the content of both compounds in all samples was

determined by standard addition method, when at least four standard additions were added.
 8

Concerning HPLC analysis, the calibration curve method from the related regression equation

was used for this purpose.

3. Results and discussion

3.1. Voltammetric behavior of VAN and CAF on BDD electrode

Initially, the oxidation behavior of these compounds was first studied by CV without an

accumulation step obtained with anodically pretreated BDD (here referred as APT-BDD)

electrode (see below for pretreatment studies). Individual representative CV curves of VAN

and CAF (both 50 μg mL-1) in phosphate buffer, pH 2.5 (optimized response) recorded within

the potential window from +0.0 and +1.8 V at a scan rate of 100 mV s-1 are shown in Fig.1.

As can be seen, VAN and CAF exhibit a well-defined irreversible oxidation peak at +1.15 and

+1.51 V, respectively. Multi-scan CV recordings (Fig. S1) revealed that the waves for both

compounds decreased upon the second and subsequent scans on the same electrode, pointing

to certain adsorption activity at the electrode surface. Moreover, it is noted that, at the

cathodic scan a very small reduction peak was observed at about +0.27 V for VAN. The CV

behavior of this compound in strong acidic solution was also investigated in our previous

report at a pencil graphite electrode [44]. In that case, a well-defined reduction peak coupled

with an anodic one on the second forward scan was obtained at lower positive potentials than

the main oxidation step. However, in the present investigation, the anodic peak of the redox

couple could not clearly be detected.

Next, the effect of scan rate on the voltammetric response of VAN and CAF oxidation at

an APT-BDD electrode was individually investigated by CV in phosphate buffer, pH 2.5 at

different scan rates within the range 10-400 mV s-1. The slight shift of peak potential towards

more positive potential was observed for both compounds as the scan rate increased thus

confirming the irreversible character of the electrode reaction of both them. On the other
 9

hand, the oxidation peak current (ip) varied linearly with the square root of scan rate (v1/2).

The linear dependence obeys the following equations:

ip (μA) = 0.448 v1/2 (mV s−1) + 0.172 (n=8, r = 0.993) (for VAN)

ip (μA) = 0.682 v1/2 (mV s−1) + 0.504 (n=8, r = 0.993) (for CAF)

Furthermore, there was a linear relationship between log ip and log v for both compounds

according to the following equations:

log ip (μA) = 0.485 log v (mV s-1) - 0.331 (n = 8, r = 0.995) (for VAN)

log ip (μA) = 0.469 log v (mV s-1) - 0.067 (n = 8, r = 0.997) (for CAF )

The slopes for both compounds are close to the theoretically expected values of 0.5 for a

diffusion controlled process.

However, the plot of ip versus v was also linear expressed by the equations:

ip (µA) = 0.018 ν (mV s-1) + 1.95 (n = 8, r = 0.985) (for VAN)

ip (µA) = 0.027 ν (mV s-1) + 3.82 (n = 8, r = 0.966) (for CAF)

These facts indicate that VAN and CAF are controlled by partial adsorption (more effective

for VAN) and partial diffusion.

Following these findings, the effect of pre-concentration/stripping conditions, such as

accumulation potential (Eacc) and time (tacc) on the oxidation process of 10 μg mL-1 VAN or

10 μg mL-1 CAF in single component solutions were carefully investigated using phosphate

buffer (pH 2.5) by SW-AdSV on APT-BDD electrode (results not presented). The stripping

peak currents of both compounds under open-circuit or over the potential range from +0.1 to

+0.5 V were measured for a tacc of 60 s in stirred solution to examine the influence of Eacc. It

was found that the maximum values for the stripping current were obtained at +0.3 and +0.4

V which were nearly equal to the value obtained at open-circuit voltage. The accumulation in

the rest of experiments was carried out under open-circuit, because the best baseline was

obtained. After fixing the Eacc at this value, the influence of tacc upon the analytical signal was
 10

examined between 0 and 360 s. The peak currents of VAN and CAF markedly increased to

about 60 s then rather slowly between 60 s and 360 s. However, considering the speed of the

measurement, 60 s was selected as tacc for all the AdSV experiments.

Prior to voltammetric quantification of VAN and CAF, the effect of pretreatment of BDD

electrode was investigated under the above experimental conditions (results not shown). The

absence of treatment leads to a decrease in current sensitivity and repeatability of the

measurements, and thus the electrode was either anodically or cathodically pretreated and the

stripping voltammetric signals of VAN and CAF were assessed. It was experimentally shown

that, their stripping peak potentials and intensities were almost similar in both cases. Due to

the fact that OH radicals produced during the initial stage of water and/or supporting

electrolyte discharge serves as a source of oxygen, the following experiments were carried out

using an anodic activation program (by applying activation potential of +1.8 V for 180 s) to

prevent fouling of BDD electrode. This pretreatment was repeated daily before starting the

voltammetric measurements. This pretreatment was always preceded by an anodic activation

procedure applying a shorter period (at +1.8 V for 60 s) before each voltammetric experiment

in order to guarantee a clean electrode surface.

Further work was dedicated towards analyzing the dependence of the AdSV performance

for both compounds on the solution pH using APT-BDD electrode. In Fig 2A and B, this

parameter was established in a series of BR buffer with pH 2.0-7.0 by carrying out stripping

measurements on 10 μg mL-1 VAN and 10 μg mL-1 CAF, respectively, in their single

component solutions, with an open-circuit accumulation at 60 s. For VAN oxidation, it was

observed one well-defined, sharp stripping peak at pH ≤ 3.0, and its splitting into two peaks in

less acidic solutions (pH 4.0 and 5.0). By raising the solution pH from 4.0 to 5.0, the first

peak occurred at more positive potential fell and the second increased. The splitting

disappeared again above pH 5.0, together with the increase of the peak currents. The
 11

evolution of peak currents with pH shows that this parameter reached the highest values at pH

7.0. Such behavior on APT-BDD electrode is in contrast to our earlier observation on the

same electrode pretreated by applying higher potential value (+3.0 V) at an analogous

polarization period (180 s) [43], in which peak potentials of compound shifted slightly

towards more positive values, together with the decrease of the anodic peak currents by

raising the solution pH. This demonstrates the importance of the anodic pre-treatment of BDD

electrode applying different potential values which is probably involved in the electrode

reaction. In the case of CAF, the peak potential and the magnitude of the peak current

practically got unchanged with the increase of pH from 2.0 to 3.0. At higher pH values of 3.0-

7.0, a slow enhancement of the peak current was observed. The peak potential was also

slightly displaced to less positive values. These facts are in good agreement with data found

on APT-BDD electrode (by setting an activation pragramme at potential of +2.0 V during 180

s) in our previous study [40]. Fig. 3C and D depicts the stripping voltammograms in various

supporting electrolytes including acetate (pH 4.7) and phosphate (pH 2.5 and 7.4) buffer

solutions for VAN and CAF, respectively. As seen from the figures, the results are nearly in

agreement with those in BR buffer. The only difference is that the two processes observed in

BR buffer at pHs 4.0 and 5.0 for VAN oxidation were fused into only one broad peak in

acetate buffer at about the same pH value (pH 4.7). This may be due to the differences of the

ionic species of the buffers.

Though the electrochemical mechanism of VAN and CAF is beyond the scope of the

present work, a comment can be made. The pKa value of VAN is reported to be 7.38 [43]. Thus,

in acidic environment, predominant form is neutral molecule in the solution. According to the

results in our previous works with regard to the oxidation of VAN on BDD [43] and pencil

graphite [44] electrodes, and considering the proposed oxidation mechanism for o-

methoxyphenol (guaiacol) [45] and its two derivatives, capsaicin and dihydrocapsaicin at
 12

carbon-based electrodes [44, 46], the electrochemical oxidation of VAN in acidic media could

be expected to occur by two-electron and two-proton process to the o-benzoquinone unit in the

structure of this compound through the formation of intermediate its phenoxonium cation. This

compound will be further reduced to the corresponding o-hydroxyphenol (catechol) part of

VAN molecule. At solution pH above the pKa value (in alkaline solutions), the hydroxyl of

VAN turns to predominantly its anionic form (o-methoxyphenate anion). According to previous

reports with regard to the oxidation of phenolic compounds in this condition (pH > pKa) [47], in

the first step, the oxidation of this anion is one-electron process to generate the phenoxy radical.

The first portion of the phenoxy radical can undergo dimerization, which is further oxidized,

probably to polymeric products. The second expected reactions of phenoxy radical are several

hydrolyses and oxidation steps ending with o-benzoquinone. On the other hand, CAF is a weak

base having a very low pKa value (~ 0.7), which corresponds to the protonation of imine

nitrogen (caffeinium ion) present in the molecule [48]. Taking into consideration the pKa value

of compound, it could be expected that imine nitrogen can only be protonated in very strong

acidic solutions. By decreasing the solution acidity, the unprotonated free base form of CAF

(neutral species) predominates in the supporting electrolytes. This is the reason why no

significant displacement in the Ep value was observed. Keeping the knowledge of earlier reports

in mind on the voltammetric methodology established by means of BDD electrode for the

determination of CAF, the overall oxidation process involves four electrons and four protons

[35, 42]. The first reaction step is a two-electron and two-proton oxidation leading to the 1,3,7-

trimethyluric acid. A further 2e−, 2H+ oxidation stage follows, leading to the the formation of

4,5-diol analogues of methylated uric acid, which rapidly fragments to the various products. The

proposed mechanism for the electrochemical oxidation of VAN (under the acidic conditions)

and CAF is presented in Fig 3.

 13

Considering all the data presented thus far, there are two possible oxidation process for

analytical use; one is in phosphate buffer pH 2.5, and the other is in BR buffer at pH 7.0, with

the highest magnitude of current responses for CAF and VAN, respectively (Fig. S2).

Better peak-potenial separation was achieved in pH 7.0, but highest response in terms of

magnitude of current for CAF and better repeatability for both compounds was obtained in pH

2.5. Following these facts, thus, phosphate buffer equal to pH 2.5 appeared to be best choice

in the following measurements with the aim of analytical application in real samples.

Before recording analytical curve for VAN and CAF determination, the optimization of

experimental parameters out such as frequency (f), scan increment (ΔEs) and pulse amplitude

(ΔEsw) that affect the SWV reponses was carried (results not shown). The frequency (f = 25-

120 Hz), scan increment (ΔEs = 6-16 mV), and pulse amplitude (ΔEsw = 20-80 mV) were

investigated in the determination of both compounds. For entire analysis the optimized values can

be summarized as follows: f, 50 Hz; ΔEs, 14 mV; and ΔEsw, 70 mV.

3.2. Analytical application of proposed method on BDD electrode

Based on the above results, APT-BDD electrode combined with SW-AdSV using optimized

accumulation time of 60 s at open-circuit voltage could allow to analyze of VAN and CAF in

phosphate buffer at pH 2.5. The stripping peaks presented a good peak potential separation

(more than 0.3 V), which clearly allows the selective and simultaneous determination of these

compounds.

In this respect, two cases were studied. Initially, the concentration of VAN was increased

linearly in the presence of a fixed concentration of CAF (Fig 4A) and vice versa (Fig. 4B).

These obtained results allow concluding that the change of concentration of one compound

did not have the significant effect on the stripping response of the other one, indicating that

their responses are independent. In the second case, both the molecules were determined by
 14

simultaneously increasing their concentrations (Fig. 5). As depicted in figure, the peak

currents of VAN and CAF at + 1.14 and +1.50 V, respectively, increase proportionally with

their concentrations. The statistical results are summarized in Table 1. It is clearly seen that

LOD values were almost identical when both the molecules have been analyzed selectively

and simultaneously.

The comparison of the analytical performance of the proposed method with previous

reported electrochemical methods for VAN and CAF determination is given in Table 2. It is

apparent that some reported papers declare lower sensitivity in terms of LOD, even the using

modified electrodes especially for CAF determination. Regarding VAN, the higher sensitivity

was achieved by modified electrodes reported in the literature. However, it should be pointed

out that the preparation of chemically modified electrodes is often time-consuming involving

various complicated steps and sometimes leading to non-reproducible results. In our

experiments, usage of BDD electrode without any modification (except for a simple anodic

pretreatment taking less than 5 min) decreases risk of measurement errors (a small number of

operations in analytical procedure), reduces expenses and operational skills of analyst.

The intra-day precision of the magnitude of stripping current was determined by successive

measurements (n = 10) of a 1 µg mL-1 VAN and CAF solution; corresponding relative

standard deviations (RSD) of 5.7 and 3.0% were obtained, respectively. Further, inter-day

precision was evaluated by measuring the current response for similar fresh solutions over a

period of 4 days. The RSDs were found to be 8.5 and 4.5% for VAN and CAF, respectively,

which are acceptable for practical applications.

Under the same experimental conditions, potential interferences such as some inorganic

ions and some biomolecules commonly existing in food samples were investigated by

addition of the compounds to mixture solution containing 10 µg mL-1 VAN and CAF. The

tolerance limit was defined as the maximum concentration of potential interfering substance
 15

that causes a relative error less than ±5% for the simultaneuos determination of VAN and

CAF. It was found that 100-fold excess of the common ions such as Cl-, NO3-, SO42-; Fe3+,

Mg2+, K+, Na+, Ca2+, Cu2+ and Zn2+ exhibited a negligible effect on the stripping peak of VAN

and CAF. No significant interferences were also observed in 10-fold excess of commonly

available biomolecules such as glucose, fructose, sucrose, salicylic acid and citric acid.

Chlorogenic acid (CGA) is a prominent polyphenol compound found in considerably amounts

in coffee beans and varying forms of coffee, so it is important to examine its interferences.

However, as it is seen from Fig.S3, good potential separation was observed in equal

concentration of CGA. Therefore, the experimental results confirmed the good selectivity of

the proposed method and offer the promising possibilities for simultaneous determination of

VAN and CAF.

In order to confirm the performance of the proposed methodology using APT-BDD

electrode in real samples, our effort was focused on its application for analysis of three

different samples of commercial products (vanilla sugar, foamy instant coffee, and cola)

frequently consumed in Turkey. Representative voltammograms of these samples are shown

in Fig. S4. As can be seen from the figure, in vanilla sugar and cola samples any peak could

not be observed corresponding to the oxidation of CAF and VAN, respectively, whereas the

coffee sample contained both compounds. Contents of VAN and CAF were obtained by the

standard addition method, and average results for three replicate measurements with standard

deviations (SD) are summarized in Table 3.

To evaluate the interference of matrix effects, the known amounts of VAN and/or CAF

standard was added to the diluted commercial samples and the recoveries were calculated.

Their values (range from 92.32-97.63% for VAN and 93.44-104.12% for CAF) suggest that

the proposed method is accurate, and has great potential for practical sample analysis.

3.3. HPLC as the comparison method

 16

Finally, the data obtained by the proposed voltammetric procedure for real samples were also

compared to those obtained by an isocratic HPLC technique coupled to diode-array detector.

Although various liquid chromatographic methods have been reported for VAN or CAF

quantification individually or simultaneously in combination with other compounds, there are

no works containing their simultaneous determination by an HPLC assay. In our case, the

chromatographic separation conditions were performed in according to previous work [49].

The reported method presents for the simultaneous determination of acetylsalicylic acid,

paracetamol, caffeine and phenobarbital, however it was successfully adopted for analysis of

VAN and CAF individually and simultaneously.

Typical chromatograms obtained in single component and binary mixture solutions of

VAN and CAF are illustrated in Fig. 6. The retention time (tR) values for analysis of

individual compounds (tR = 5.4 and 8.7 min for CAF and VAN, respectively) are slightly

higher than those obtained when analyzing compounds simultaneously in binary mixtures (tR

= 5.3 and 8.5 min for CAF and VAN, respectively).

The linearity of the detector response was determined by the calibration curves generated

by three repeated injections of standard solutions at 9 concentration levels in the range of 2.5-

150 µg mL-1 for both VAN and CAF. The calibration curves were obtained by plotting peak

area against concentration. Analytical parameters for chromatographic determination of both

compounds have been presented in Table 4. The results showed that the slopes of the standard

curves and LOD values obtained in simultaneous determination of VAN and CAF match well

with the case when both the molecules have been analyzed individually.

The developed chromatographic method was further used for the analysis of real samples,

including vanilla sugar (for VAN), cola (for CAF), and foamy instant coffe (for VAN+CAF).

Their chromatograms are shown in Fig. S5. It is clearly seen from the figure that there was no

interference observed during simultaneous method sample analysis. Concentrations of

 17

analyzed compounds present in the samples are shown in Table 3. The results reveal a good

agreement between those determined by these proposed method and the comparative one.

Besides, applying the Student′s t-test to the results obtained using both methods, the

calculated t values (0.96 for VAN in vanilla sugar, 2.19 for CAF in cola, and 1.48 for VAN

and 1.39 for CAF in foamy instant coffee) were smaller than the tabulated value (2.78, p =

0.05). These results indicate that there is no significant difference between the performance of

the proposed and comparison methods as regard to mean values at a confidence level of 95%.

On the basis of these results it can be stated that the proposed voltammetric procedure can

be applied to rapid, inexpensive and simultaneous determination of VAN and CAF in food

and beverage samples.

4. Conclusions

BDD electrode (without any electrode surface modification) was used in combination with

SW-AdSV technique for the simultaneous determination of VAN and CAF. The practical use

of the method is demonstrated by determining the concentration of these compounds in

commercial food and beverage samples and by comparing the obtained results with those

from a HPLC method which was the first attempt for the determination of VAN and CAF

simultaneously. Taking into account the results achieved in this study, the proposed method

may find possible applications as inexpensive alternative for future uses in food industry.

References

[1] A.K. Sinha, U.K., Sharma, N. Sharma. A comprehensive review on vanilla flavor: Extraction,

isolation and quantification of vanillin and others constituents, Int. J. Food Sci. Nutr. 59 (2008)

299-326.
 18

[2] P.L. Teissedre, A.L. Waterhouse. Inhibition of oxidation of human lowdensity lipoproteins by

phenolic substances in different essential oils varieties, J. Agric. Food Chem. 48 (2000) 3801-

3805.

[3] H.G Mandel. Update on caffeine consumption, disposition and action, Food Chem. Toxicol. 40

(2002) 1231-1234.

[4] P. Nawrot, S. Jordan, J. Eastwood, J. Rotstein, A. Hugenholtz, M. Feeley. Effects of caffeine on

human health, Food Addit. Contam. 20 (2003) 1-30.

[5] C.J. Reissig, , E.C. Strain, R.R. Griffiths. Caffeinated energy drinks-A growing problem, Drug

Alcohol Depend. 99 (2009) 1-10.

[6] E.G. Cookeas, C.E. Efstathiou. Preconcentration of organic compounds at a diphenyl ether

graphite paste electrode and determination of vanillin by adsorptive–extractive stripping

voltammetry, Analyst 117 (1992) 1329-1334.

[7] L. Agüi, J.E. Lopez-Guzman, A.Gonzalez-Cortes, P. Yanez-Sedeno, J.M. Pingarron. Analytical

performance of cylindrical carbon fiber microelectrodes in low-permittivity organic solvents:

Determination of vanillin in ethyl acetate, Anal. Chim. Acta 385 (1999) 241-248.

[8] J.L. Hardcastle, C.J. Paterson, R.G. Compton. Biphasic sonoelectroanalysis: Simultaneous

extraction from, and determination of vanillin in food flavoring, Electroanalysis 13 (2001)

899-905.

[9] A. Câmpean, M. Tertiş, R. Sǎndulescu. Voltammetric determination of some alkaloids and other

compounds in pharmaceuticals and urine using an electrochemically activated glassy carbon

electrode, Centr. Eur. J. Chem. 9 (2011) 688-700.

[10] R.N. Goyal, S. Bishnoi, B. Agrawal. Electrochemical sensor for the simultaneous

determination of caffeine and aspirin in human urine samples, J. Electroanal. Chem. 655 (2011)

97-102.
 19

[11] B.K. Chethana, S. Basavanna Y.A. Naik. Determination of vanillin in real samples using lysine

modified carbon paste electrode, J. Chem. Pharm. Res. 4 (2012) 538-545.

[12] S. Luo, Y. Liu. Poly(acid chrome blue K) modified glassy carbon electrode for the

determination of vanillin, Int. J. Electrochem. Sci. 7 (2012) 6396-6405.

[13] J. Peng, C. Hou, X. Hu. A graphene-based electrochemical sensor for sensitive detection of

vanillin, Int. J. Electrochem. Sci. 7 (2012) 1724-1733.

[14] L. Shang, F. Zhao, B. Zeng. Sensitive voltammetric determination of vanillin with an AuPd

nanoparticles–graphene composite modified electrode, Food. Chem. 151 (2014) 53-57.

[15] M.A. Xinying. Determination of vanillin in infant formula using poly valine modified

electrode, Int. J. Electrochem. Sci. 9 (2014) 3181-3189.

[16] P. Deng, Z. Xu, R. Zeng, C. Ding. Electrochemical behavior and voltammetric determination

of vanillin based on an acetylene black paste electrode modified with graphene-

polyvinylpyrrolidone composite film, Food Chem. 180 (2015) 156-163.

[17] J. Zhang, L.P. Wang, W. Guo, X.D. Peng, M. Li, Z.B. Yuan. Sensitive differential pulse

stripping voltammetry of caffeine in medicines and Cola using a sensor based on multi-walled

carbon nanotubes and Nafion, Int. J. Electrochem. Sci. 6 (2011) 997-1006.

[18] J.-Y Sun, K.-J. Huang, S.-Y. Wei, Z.-W. Wu. Application of cetyltrimethylammonium

bromide-Graphene modified electrode for sensitive determination of caffeine, Can. J. Chem. 89

(2011) 697-702.

[19] S. Guo, Q. Zhu, B. Yang, J. Wang, B. Ye. Determination of caffeine content in tea based on

poly (safranine T) electroactive film modified electrode, Food Chem. 129 (2011) 1311-1314.

[20] A.J. Jeevagan, S.A. John. Electrochemical determination of caffeine in the presence of

paracetamol using a self-assembled monolayer of non-peripheral amine substituted copper(II)

phthalocyanine, Electrochim. Acta 77 (2012) 137-142.

 20

[21] M. Amare, S. Admassie. Polymer modified glassy carbon electrode for the electrochemical

determination of caffeine in coffee, Talanta. 93 (2012) 122-128.

[22] X.C Lu., K.J. Huang, Z.W. Wu., S.F. Huang, C.X. Xu. Simultaneous determination of

acetaminophen and caffeine based on graphene oxide/cerium hexacyanoferrate modified glassy

carbon electrode, Chin. J. Anal. Chem. 3 (2012) 452-456.

[23] X.Q. Xiong, K.J. Huang, C.X. Xu, C.X. Jin, Q.G. Zhai. Glassy carbon electrode modified with

poly(taurine)/TiO2–graphene composite film for determination of acetaminophen and caffeine,

Chem. Ind. Chem. Eng. Q 19 (2013) 359-368.

[24] M. Tefera, A.Geto, M. Tessema, S. Admassie. Simultaneous determination of caffeine and

paracetamol by square wave voltammetry at poly(4-amino-3-hydroxynaphthalene sulfonic

acid)-modified glassy carbon electrode, Food Chem. 210 (2016) 156-162.

[25] A.I. Yiğit, Y. Yardım, M. Çelebi, A. Levent, Z. Şentürk. Graphene/Nafion composite film

modified glassy carbon electrode for simultaneous determination of paracetamol, aspirin and

caffeine in pharmaceutical formulations, Talanta 231 (2016) 688-695.

[26] L. Jiang, Y. Ding, F. Jiang, L. Li F. Mo. Electrodeposited nitrogen-doped graphene/carbon

nanotubes nanocomposite as enhancer for simultaneous and sensitive voltammetric

determination of caffeine and vanillin, Anal. Chim. Acta 833 (2014) 22-28.

[27] H. Filik, A.A. Avan, Y. Mümin. Simultaneous Electrochemical Determination of Caffeine and

Vanillin by Using Poly(Alizarin Red S) Modified Glassy Carbon Electrode, Food Anal.

Methods 10 (2017) 31-40.

[28] K. Peckova, J. Musilova, J. Barek, Boron-doped diamond film electrodes new tool for

voltammetric determination of organic substances, Crit. Rev. Anal. Chem. 39 (2009) 148-172.

[29] J.H.T. Luong, K.B. Male, J.D. Glennon, Boron-doped diamond electrode: synthesis,

characterization, functionalization and analytical applications, Analyst 134 (2009) 1965-1979.

 21

[30] H.B. Suffredini, V.A. Pedrosa, L. Codognoto, S.A.S. Machado, R.C. Rocha-Filho, L.A. Avaca,

Enhanced electrochemical response of boron-doped diamond electrodes brought on by a

cathodic surface pre-treatment, Electrochim. Acta 49 (2004) 4021-4026.

[31] H. Girard, N. Simon, D. Ballutaud, M. Herlern, A. Etcheberry, Effect of anodic andcathodic

treatments on the charge transfer of boron doped diamond electrodes, Diamond Relat. Materi.

16 (2007) 316-325.

[32] Y. Yardım, M.E. Erez. Electrochemical behavior and electroanalytical determination of indole-

3-acetic acid phytohormone on a boron-doped diamond electrode, Electroanalysis 23 (2011)

667-673.

[33] Y. Yardım, Z. Şentürk. Electrochemical behavior of folic acid at a boron-doped diamond

electrode: its adsorptive stripping voltammetric determination in tablets, Turk. J. Pharm. Sci., 11

(2014) 87-100.

[34] A. Yiğit, Y. Yardım, Ö. Selçuk Zorer, Z. Şentürk. Electrochemical determination of

pterostilbene at a cathodically pretreated boron-doped diamond electrode using square-wave

adsorptive anodic stripping voltammetry in cationic surfactant media, Sens. Actuator B-Chem.

231 (2016) 688-695.

[35] L. Švorc, P. Tomcik, J. Svítková, M. Rievaj, D. Bustin. Voltammetric determination of caffeine

in beverage samples on bare boron-doped diamond electrode, Food Chem. 135 (2012) 1198-

1204.

[36] C.A. Martínez-Huitle, N. Suely Fernandes, S. Ferro, A. De Battisti, M.A. Quiroz. Fabrication

and application of Nafion®-modified boron-doped diamond electrode as sensor for detecting

caffeine, Diamond Relat. Mater. 19 (2010) 1188-1193.

[37] B.C. Lourencão, R.A. Medeiros, R.C. Rocha-Filho, L.H. Mazo, O. Fatibello-Filho.

Simultaneous voltammetric determination of paracetamol and caffeine in pharmaceutical

formulations using a boron-doped diamond electrode, Talanta 78 (2009) 748-752.

 22

[38] B.C. Lourencão, R.A. Medeiros, R.C. Rocha-Filho, O. Fatibello-Filho. Simultaneous

differential pulse voltammetric determination of ascorbic acid and caffeine in pharmaceutical

formulations using a boron-doped diamond electrode, Electroanalysis 22 (2010) 1717-1723.

[39] E.O. Faria, A.C.V. Lopes Jr., D.E.P. Souto, F.R.F. Leite, F.S. Damos, R.C.S. Luz, A.S. Santos,

D.L. Franco, W.T.P. Santos. Simultaneous determination of caffeine and acetylsalicylic acid in

pharmaceutical formulations using a boron-doped diamond film electrode by differential pulse

voltammetry, Electroanalysis 24 (2012) 1141-1146.

[40] Y. Yardım, E. Keskin, Z. Şentürk. Voltammetric determination of mixtures of caffeine and

chlorogenic acid in beverage samples using a boron-doped diamond electrode, Talanta 116

(2013) 1010-1017.

[41] A. Yiğit, Y. Yardım, Z. Şentürk. Voltammetric sensor based on boron-doped diamond

electrode for simultaneous determination of paracetamol, caffeine, and aspirin in

pharmaceutical formulations, IEEE Sens. J. 16 (2016) 1674-1680.

[42] L. Švorc. Determination of Caffeine: A Comprehensive Review on Electrochemical

Methods, Int. J. Electrochem. Sci. 8 (2013) 5755-5773.

[43] Y. Yardım, M. Gülcan, Z. Şentürk. Determination of vanillin in commercial food product by

adsorptive stripping voltammetry using a boron-doped diamond electrode, Food Chem. 141

(2013) 1821-1827.

[44] Y. Yardım, Z. Şentürk. Electrochemical evaluation and adsorptive stripping voltammetric

determination of capsaicin or dihydrocapsaicin on a disposable pencil graphite electrode,

Talanta 112 (2013) 11-19.

[45] Y. Samet, D. Kraiem, R. Abdelhédi. Electropolymerization of phenol, o-nitrophenol and

o-methoxyphenol on gold and carbon steel materials and their corrosion protection effects,

Prog. Org. Coat. 69 (2010) 335-343.

 23

[46] Y. Yaa, L. Mo, T. Wang, Y. Fan, J. Liao, Z. Chen, K.S. Manoj, F. Fang, C. Li, J. Liang.

Highly sensitive determination of capsaicin using a carbon paste electrode modified

with amino-functionalized mesoporous silica, Colloids Surface B 95 (2012) 90-95.

[47] G. Arslan, B. Yazici, M. Erbil, The effect of pH, temperature and concentration on

electrooxidation of phenol, J. Hazard. Mater. B 124 (2005) 37-43.

[48] T. Katsu, Y. Tsunamoto, N. Hanioka, K. Komagoe, K. Masuda, S. Narimatsu. A

caffeine-sensitive membrane electrode: Previous misleading report and present

approach, Anal.Chim.Acta 620 (2008) 50-54.

[49] J.T Franeta, D. Agbaba, S. Eric, S. Pavkov, M. Aleksi, S. Vladimirov. HPLC assay of

acetylsalicylic acid, paracetamol, caffeine and phenobarbital in tablets, Farmaco 57 (2002) 709-

713.
 24

Captions to Figures:

Fig. 1. The cyclic voltammograms of individual solution of 50 μg mL-1 VAN and 50 μg mL-1

CAF. Electrode, APT-BDD; supporting electrolyte, phosphate buffer pH 2.5; scan rate, 100

mV s−1. Dashed lines represent background current.

Fig. 2. SW stripping voltammograms of 10 μg mL-1 VAN (A and C) and 10 μg mL-1 CAF (B

and D) in BR buffer at different pH values between pH 2.0-7.0 (A and B), and in various

supporting electrolytes (C and D) at the APT-BDD. tacc = 60 s at open circuit condition; SWV

parameters: frequency, 50 Hz; scan increment, 8 mV; pulse amplitude, 30 mV.

Fig. 3. Proposed electrode process diagrams of VAN (A) and CAF (B) at the APT-BDD.

Fig. 4. SW stripping voltammograms for various concentrations of VAN (A) and CAF (B) at

fixed concentrations of 10.0 µg mL-1 CAF and 10.0 µg mL-1 VAN, respectively. VAN

concentrations (1-8): 1.0-100.0 µg mL-1. CAF concentrations (1-9): 0.25-100.0 µg mL-1. Insets:

the respective analytical curves for VAN (A) and CAF (B). Electrode, APT-BDD; supporting

electrolyte, phosphate buffer pH 2.5; Preconcentration period, 60 s at open circuit condition;

SWV parameters: frequency, 50 Hz; step potential, 14 mV; pulse amplitude, 70 mV.

Fig. 5. SW stripping voltammogram of mixture solution of VAN and CAF obtained at their

equal concentrations (1-8): 1.0-100.0 μg mL-1. Insets: the respective analytical curves for

VAN and CAF. Other operating conditions as indicated in Fig.4.

Fig. 6. Chromatograms of the VAN (A) and CAF (B) standards, and VAN+CAF standard

mixture (C) obtained by HPLC. Chromatographic conditions: mobile phase, acetonitrile/water

mixture (25:75 v/v) adjusted to pH 2.5 with H3PO4 of 85 % (w/w) purity; diode-array detector

set at 207 nm; flow rate, 1 mL min-1

 25

Table 1. Analytical parameters for voltammetric determination of VAN and CAF obtained at
APT-BDD electrode.
LWR LRE r LOQ LOD
(A)

VAN 1.0-100 µg mL-1 ip (µA) = 0.202 C (µg mL-1) +0.027 0.999 0.743 µg mL-1 0.223 µg mL-1
(6.6×10-6–6.6×10-4 M) (4.90×10-6 M) (1.47×10-6 M)
(B)

CAF 0.25-100.0 µg mL-1 ip (µA) = 0.409 C (µg mL-1) +0.675 0.998 0.197 µg mL-1 0.059 µg mL-1
(1.3×10-6–5.2×10-4 M) (1.01×10-6 M) (3.04×10-7 M)
(C)

VAN 1.0-100 µg mL-1 ip (µA) = 0.169 C (µg mL-1) +0.467 0.995 0.779 µg mL-1 0.234 µg mL-1
(6.6×10-6–6.6×10-4 M) (5.13×10-6 M) (1.54×10-6 M)

CAF 1.0-100 µg mL-1 ip (µA) = 0.346 C (µg mL-1) +1.621 0.997 0.236 µg mL-1 0.071 µg mL-1
-6 -4 -6
(5.2×10 –5.2×10 M) (1.22×10 M) (3.66×10-7 M)
-1 -5
(A) Statistical data for VAN when the concentration of CAF is kept constant [10 µg mL (5.15×10 M)]
(B) Statistical data for CAF when the concentration of VAN is kept constant [10 µg mL-1 (6.6×10-5 M)]
(C) Statistical data for VAN and CAF simultaneously
LWR = linear working range; LRE = linear regression equation; r = correlation coefficient; LOQ, limit of
quantification; LOD = limit of detection

Table 2. Examples from the previously reported electrodes for voltammetric determination of VAN and CAF.
Target compounds Electrode Modifier Technique LOD (μM) Sample Ref.
Vanillin
VAN CF n.a. SWV 4.2 Puding powder [7]
VAN CPE Lysine DPV 2.88 Food samples [11]
VAN GCE Poly(Acid Chrome DPV 0.032 Food samples [12]
Blue K)
VAN GCE AuPd-GR DPV 0.02 Food samples [14]
VAN ABPE GR-PVP Deriv-LSV 0.01 Food samples [16]
Caffeine
CAF+some other EA- n.a. DPV 0.02 Drugs, Urine [9]
alkaloids GCE
CAF GCE Nafion/MWCNTs DP-AdSV 0.513 Cola, Drugs [17]
CAF GCE CTAB/GR DPV 0.091 Soft drink [18]
CAF GCE Poly(safranine T)/Nafion LSV 0.1 Tea [19]
CAF+PAR GCE CuIITAPcSAM DPV 0.03 Cola, Drugs, [20]
Serum
CAF+ASA GCE PT/TiO2-GR DPV 0.5 - [23]
CAF+PAR GCE Poly(AHNSA) SWV 0.79 Cola, Tea [24]
CAF+PAR+ASA GCE GR-Nafion SW-AdSV 0.038 Drugs [25]
Vanillin+Caffeine
 26

VAN+CAF GCE (NGR-NCNTs) SWV 0.0033+0.02 Food&beverage [26]

samples
VAN+CAF GCE Poly(Alizarin Red S) SWV 0.06+0.8 Food&beverage [27]
samples
VAN+CAF APT-BDD n.a. SW-AdSV 1.54+0.37 Food&beverage This
samples work
Compound: VAN, vanillin; CAF, caffeine; ASA, acetyl salicylic acid; PAR, paracetamol. Electrode: CF, carbon fiber; CPE,
carbon paste; GCE, glassy carbon; ABPE, acetylene black paste electrode; EA-BDD; electrochemically activated glassy carbon;
APT-BDD, anodically pretreated boron-doped diamond. Modifier: GR, graphene; PVP, polyvinylpyrrolidone; MWCNT, multi-
walled carbon nanotube; CTAB, cetyltrimethylammonium bromide; CuIITAPcSAM, self-assembled monolayer of non-peripheral
amine substituted copper(II) phthalocyanine; PT, poly(taurine); Poly(AHNSA), poly(4-amino-3-hydroxynaphthalene sulfonic
acid); NGR-NCNTs, nitrogen-doped graphene/carbon nanotubes. Technique: SWV, square-wave voltammetry; DPV, differential
pulse voltammetry; LSV, linear sweep voltammetry; AdSV, adsorptive stripping voltammetry. Other: LOD, limit of detection;
n.a., not applicable; Ref., reference

Table 3. Results of the analysis of VAN and CAF content in different food and beverage
products by using SW-AdSV (proposed) and HPLC (comparative) methods.
Samples Analyte SW-AdSVa HPLCa E1b (%)

S1 VANc 32.65± 1.22 31.88±0.68 2.42

S2 CAFd 106.25± 2.83 110.08±1.09 -3.48

S3 VANc 0.272±0.014 0.259±0.006 5.79

CAFc 2.58±0.11 2.49±0.05 3.61

S1, vanilla sugar; S2, cola; S3, foamy instant coffee
a
Mean ± SD (n = 3)
b
Relative error (%) = [(voltammetric value-HPLC value)/HPLC value] × 100
c
mg g-1
d
mg L-1
 27

Table 4. Analytical parameters for the determination of VAN and CAF obtained by HPLC method
LWR LRE r LOQ LOD
(A)

VAN 2.5-150 µg mL-1 y = 115.4x + 69.5 0.999 1.97 µg mL-1 0.59 µg mL-1
(1.6×10-5–9.9×10-4 (1.30×10-5 M) (3.90×10-6 M)
M)
CAF 2.5-150 µg mL-1 y = 150.9x + 407.1 0.999 1.53 µg mL-1 0.46 µg mL-1
(1.3×10-5–7.7×10-4 (7.90×10-6 M) (2.37×10-6 M)
M)
(B)

VAN 2.5-150 µg mL-1 y = 109.1x + 21.9 0.999 2.10 µg mL-1 0.63 µg mL-1
(1.6×10-5–9.9×10-4 (1.38×10-5 M) (4.14×10-6 M)
M)
CAF 2.5-150 µg mL-1 y = 146.2x + 415.1 0.999 1.57 µg mL-1 0.47 µg mL-1
-5 -4
(1.3×10 –7.7×10 (8.07×10 M) (2.42×10-6 M)
-6

M)
(A) Statistical data for individual molecules
(B) Statistical data for VAN and CAF simultaneously
LWR = linear working range; LRE = linear regression equation; r = correlation coefficient;
LOQ, limit of quantification; LOD = limit of detection;
y = peak area of compounds; x = concentration of compounds in µg mL-1

Highlights

• The usefulness of anodically pretreated boron-doped diamond electrode in

combination with the square-wave adsorptive stripping voltammetry for determination
of vanillin and caffeine in their mixture
• The first report on the simultaneous determination of vanillin and caffeine by high
performance liquid chromatography
• Practical applicability in food and beverage samples
 28

Figure 1
 29

Figure 2
 30

Figure 3
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Figure 4
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Figure 5
 33

Figure 6
 34

Graphical Abstract