Chemical Papers 69 (7) 901–910 (2015)

DOI: 10.1515/chempap-2015-0096

ORIGINAL PAPER

Voltammetric determination of B1 and B6 vitamins using

a pencil graphite electrode

Iulia Gabriela David, Mihai-Alexandru Florea, Oana Georgiana Cracea,

Dana Elena Popa, Mihaela Buleandra, Emilia Elena Iorgulescu, Vasile David,
Irinel Adriana Badea, Anton Alexandru Ciucu*

Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest,

Panduri Av. 90–92, District 5, Bucharest-050663, Romania

Received 31 October 2014; Revised 18 December 2014; Accepted 19 December 2014

Due to the importance of B1 and B6 vitamins for human health it is useful to develop new
cheap and rapid methods for their determination. Voltammetric behavior of these vitamins on
a pencil graphite electrode was investigated using cyclic voltammetry in different media. Direct
quantitative determination of the two vitamins, one in the presence of the other, was done by
differential pulse voltammetry. Vitamin B1 was electroactive only in a NaOH solution generating
two irreversible oxidation peaks; the first peak obtained at 250 mV is well-defined and was used in
quantitative determinations. In case of vitamin B6 , a well-defined oxidation peak was observed in
all investigated supporting electrolytes except for HCl. The linear concentration ranges were 10−5 –
10−3 M for vitamin B1 in a NaOH solution and 5 × 10−6 –10−3 M for vitamin B6 in an acetate
buffer solution. The obtained detection limits were 5.34 × 10−6 M and 2.81 × 10−6 M for vitamin
B1 and vitamin B6 , respectively. The developed method is simple and rapid and it was successfully
applied in the determination of the two vitamins in pharmaceuticals.

c 2015 Institute of Chemistry, Slovak Academy of Sciences

Keywords: pencil graphite electrode, B1 and B6 vitamins, voltammetry, pharmaceuticals

Introduction

Vitamins are biologically active compounds which

play different important roles in the normal function
of human body as they are involved in numerous
metabolic pathways (Marszall et al., 2005). Vitamin
B1 (VB1 ) existing as thiamine hydrochloride is water-
soluble and it is essential for the maintenance of car-
bohydrate metabolism, proper functioning of the ner-
vous and cardiovascular systems of the human body Fig. 1. Molecular structure of thiamine hydrochloride (vitamin
and for the prevention and treatment of the beriberi B1 ) (a) and pyridoxine (vitamin B6 ) (b).
disease (Tyszczuk-Rotko, 2012; Kamruzzaman et al.,
2013) (Fig. 1a).
Vitamin B6 (VB6 ) group contains six water-soluble first isolated form of VB6 , pyridoxine (Fig. 1b), is the
forms: pyridoxal, pyridoxine, pyridoxamine and phos- most stable and the best-known form of these com-
phate derivatives (pyridoxal 5
-phosphate, pyridoxine pounds being used in drug formulation as hydrochlo-
5
-phosphate and pyridoxamine 5
-phosphate). The ride. VB6 influences the activity of many enzymes, it

*Corresponding author, e-mail: anton ciucu@yahoo.com

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902 I. G. David et al./Chemical Papers 69 (7) 901–910 (2015)
is essential for the amino acids metabolism, gene and
immune modulation and it also has beneficial effects in
the treatment of HIV-1 and cancer (Pournaghi-Azar et
al., 2007; Desai et al., 2008; Ahmad et al., 2013; Antal
et al., 2013). Deficiencies of VB6 determine digestion
and skin problems, weakness, depressions, convulsions
and neurological diseases (Ahmad et al., 2013). Com-
mon sources of B vitamins uptake in humans are nat-
ural and enriched foods (Desai et al., 2008; Tyszczuk-
Rotko, 2012; Antal et al., 2013) and, if necessary, by
pharmaceutical preparations (Ghasemi, et al., 2004;
Chen et al., 2006; Desai et al., 2008; Tyszczuk-Rotko,
2012; Ahmad et al., 2013; Kamruzzaman et al., 2013).
Considering the importance of vitamins for human
health, it is necessary to develop sensitive and selec-
tive methods for their rapid determination in different
matrices like food, pharmaceuticals and biological flu-
ids.
Literature contains reports regarding the VB1 de-
termination using analytical methods based on high
performance liquid chromatography (Marszall et al,
2005; Chen et al., 2006), spectrophotometry (Ghasemi
et al., 2004), chemiluminescence (Kamruzzaman et al.,
2013) and electrochemistry (Antal et al., 2013), es-
pecially voltammetry (Aboul-Kasim, 2000; Tyszczuk-
Rotko, 2012; Brahman et al., 2012, 2013). Various an-
alytical methods have been developed also for the de-
termination of VB6 alone or in mixtures with other
vitamins (Ahmad et al., 2013) including spectropho-
tometry (Ghasemi et al., 2004; Ahmad et al., 2013),
spectrofluorimetry (Ahmad et al., 2013), chromatog-
raphy (Marszall et al, 2005; Chen et al., 2006; Ahmad
et al., 2013) and electrochemical methods (Mekon-
nen et al., 2014; Wu et al., 2005; Pournaghi-Azar et
al., 2007; Desai et al., 2008; Fonseca et al., 2011; Liu
et al., 2012; Antal et al., 2013; Ahmad et al., 2013).
For the above mentioned spectrometric methods, the
lower limits of the corresponding calibration ranges
were situated at values below 5 g mL−1 (Ghasemi
et al., 2004; Ahmad et al., 2013) whereas much lower
detection limits were obtained by chemiluminescence
(4.9 × 10−8 M, for VB1 ) (Kamruzzaman et al.,
2013), high-performance liquid chromatography (9.2
ng mL−1 for VB1 and 2.7 ng mL−1 for VB6 ) (Marszall
et al, 2005), or high-performance liquid chromatog-
raphy/electrospray ionization-mass spectrometry, e.g.
3 g L−1 for VB1 and 1 g L−1 for VB6 (Chen et
al., 2006). Despite their higher sensitivity, these meth-
ods are more complicated and more expensive; e.g.
the chemiluminescence method described by Kamruz-
zaman et al. (2013) involves the microchip fabrica-
tion by lithography and also the platinum nanoparti-
cles preparation. Performance characteristics of some
voltammetric methods reported in literature for VB1
and VB6 determination are presented in Table 1.
Moreover, voltammetric methods have some spe-
cific advantages like simplicity, rapidity and low
reagent consumption, which make them less expen-
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sive when compared with other analytical techniques.
They are also adequate for the investigation and de-
termination of pharmaceutically and biologically im-
portant compounds (Tanase et al., 1998; Litescu et al.,
2001; Duca et al., 2010; Balan et al., 2011; Patrascu
et al., 2011). A drawback of voltammetric analysis is
the contamination of the solid electrode surface dur-
ing the measurements due to the adsorption of differ-
ent compounds from the sample matrix or produced
through the electrochemical process. Therefore, a time
consuming electrode surface cleaning step is required
before each voltammetric recording in order to en-
sure reproducible measurements. The use of dispos-
able electrodes constitutes an advantageous alterna-
tive to the conventional solid electrodes. In this re-
spect, the pencil graphite electrode (PGE) combines
the graphite properties as a good electrode material
(e.g. chemical inertness, low background current and
large potential window) with other advantages as me-
chanical resistance, disposability, low price and com-
mercial availability. Bare (David et al., 2013, 2015)
or modified PGEs (Kuralay et al., 2011; Ensafi et al,
2012) were used in voltammetric analysis of various
types of compounds but also as super-capacitor mate-
rials (Arslan & Hur, 2014).
Searching for a simple and accurate electrochem-
ical method that can detect VB1 in the presence of
other vitamins (e.g. VB6 ) is of considerable interest.
A survey of the literature reveals that there is no re-
port regarding the electrochemical determination of
vitamin B1 and vitamin B6 using PGE. The present
paper describes the voltammetric behavior of VB1 and
VB6 at the PGE surface. A new approach to the de-
termination of the two vitamins one in presence of
the other has been realized on PGE using differential
pulse voltammetry (DPV). The problems related to
the surface contamination of this electrode have been
resolved by using a disposable electrode. A simple,
rapid and inexpensive method for accurate determi-
nation of VB1 and VB6 contents in pharmaceutical
formulations containing both vitamins has been de-
veloped.
Experimental
Reagents and solutions
Vitamin B1 (thiamine hydrochloride) (≥ 99
mass %, reagent grade, HPLC), vitamin B6 (pyri-
doxine hydrochloride) (≥ 98 mass %, reagent grade,
HPLC), HCl (37.0 mass %, ACS reagent), NaOH
(pellets), Na2 HPO4 · 2H2 O and KH2 PO4 (p.a., ACS
reagent), CH3 COOH (99.7 mass %, ACS reagent) were
purchased from Sigma–Aldrich (Germany). Stock so-
lutions of 10−2 M of VB1 and VB6 were freshly pre-
pared with deionized water; more diluted solutions of
concentrations from 10−6 M to 6 × 10−3 M VB1 and
VB6 , respectively, were obtained from the correspond-
 I. G. David et al./Chemical Papers 69 (7) 901–910 (2015) 903
Table 1. Analytical characteristics of voltammetric methods reported for the determination of VB1 and VB6 in pharmaceuticals

Electrode Modifier/technique Linear range LoD Reference

Vitamin B1

AgSA AgLAF/DPAdSV 0.01∼0.1 mg L−1 0.003 mg L−1 Baś et al. (2011)

Au Cys/SAM/SWV 1.1 × 10−8 ∼2.2 × 10−6 M 5.5 × 10−9 M Wan et al. (2002)
Au FI-FFT-CV 3.0 × 10−9 ∼2.2 × 10−7 M 1.0 × 10−12 M Norouzi et al. (2007)
CP MWCNT/DPAdSV 1.0 × 10−7 ∼1.0 × 10−6 M 1.1 × 10−10 M Brahman (2012)
CP MWCNT and ss-DNA/DPAdSV 0.0025∼0.80 g mL−1 1.1 ng mL−1 Brahman (2013)
GC Hg film/SWV 1.0 × 10−6 ∼4.0 × 10−3 M 1.0 × 10−7 M Jiang and Sun (2007)
GC PAR film 3.0 × 10−5 ∼1.1 × 10−3 M 8.0 × 10−6 M Khan et al. (2008)
GC Pb film/AdSWV 5.0 × 10−8 ∼1.0 × 10−6 M 2.0 × 10−8 M Tyszczuk-Rotko (2012)
Pt FI-FFT-SWV 5.0 × 10−10 ∼4.0 × 10−7 M 8.0 × 10−11 M Norouzi et al. (2010)

Vitamin B6

Au FI-FFT-CV 8∼42300 pg mL−1 2.8 pg mL−1 Ganjali et al. (2008)

BDD Ru(bpy)3−
3 /SWV 0.2775 × 10−6 ∼0.3686 × 10−3 M 6.32 × 10−8 M Wu et al. (2005)
CC MWCNT/DPV 2.5 × 10−7 ∼10 × 10−5 M 9.5 × 10−8 M Habibi et al. (2010)
CC PBNP/FI-CA 1.0 × 10−6 ∼8.0 × 10−5 M 0.87 × 10−6 M Razmi and Mohammad-Rezaei (2010)
CP CV 1.0 × 10−6 ∼2.0 × 10−4 M – Söderhjelm and Lindquist (1975)
CP VO(Salen)/LSV 4.5 × 10−4 ∼3.3 × 10−3 M 3.7 × 10−5 M Teixeira et al. (2004)
CP CuHCF/CV 1.2 × 10−6 ∼6.9 × 10−4 M 4.1 × 10−7 M Teixeira et al. (2003)
CP CoHCF/SWV 5.0 × 10−5 ∼2.6 × 10−5 M 1.72 × 10−7 M Mekonnen et al. (2014)
CP DB18C6/DPV 0.4∼100 g mL−1 0.4 g mL−1 Desai et al. (2008)
GC DPV 3.7 × 10−7 ∼2.0 × 10−4 M 1.0 × 10−7 M Wu and Song (2008)
GC CrHCF/CV 1.33 × 10−6 ∼1.32 × 10−5 M 1.05 × 10−6 M Cottica et al. (2009)
GC ssDNA/CV 0.1 × 10−3 ∼6.0 × 10−3 M 0.04 × 10−3 M Liu et al. (2012)
GC MWCNT/DPV 5.0 × 10−7 ∼1.0 × 10−4 M 2.0 × 10−7 M Qu et al. (2004)
GC AuNP-MWCNT/DPV 1.59∼102.74 g mL−1 0.53 g mL−1 Zhang and Wang (2011)
PGC DPV 2.5 × 10−6 ∼7.5 × 10−3 M 8.0 × 10−7 M Gu et al. (2001)
GPC SWV 2× 10−6 ∼18 × 10−6 M – Fonseca et al. (2011)

Vitamin B1 and vitamin B6

CP RuO-RuCN film/FI-CV VB1 : 0.5 × 10−6 ∼50 × 10−6 M 0.2 × 10−6 M Shaidarova et al. (2006)
VB6 : 0.2 × 10−6 ∼50 × 10−6 M 0.1 × 10−6 M

AgSA: silver solid amalgam annular band; Au: gold; BDD: boron doped diamond; CC: carbon-ceramic; CP: carbon paste; GC:
glassy carbon; PGC: pretreated glassy carbon; Pt: platinum; GPC: graphite-polyurethane composite; DPAdSV: differential pulse
adsorptive stripping voltammetry; SWV: square wave voltammetry; FFT-CV: fast Fourier transform continuous cyclic voltammetry;
AdSVW: adsorptive square wave voltammetry; FFT-SWV: fast Fourier transform square-wave voltammetry; CA: chronoamperom-
etry; LSV: linear sweep voltammetry; FI-CV: cyclic voltammetry in flow-injection conditions; AgLAF: silver liquid amalgam film;
Cys/SAM: self-assembled monolayer of L-cysteine; MWCNT: multiwall carbon nanotube; ssDNA: single-stranded DNA; Hg: mer-
cury; PAR: poly(4-(2-pyridylazo)-resorcinol); Pb: lead; Ru(bpy)3−

3 : ruthenium tris(2,2 )bipyridyl; PBNP: Prussian blue nanoparti-
cles; VO(Salen): N,N-ethylene-bis(salicylideneiminato) oxovanadium(IV) complex; CuHCF: copper(II) hexacyanoferrate(III); Co-
HCF: cobalthexacyanoferrate; DB18C6: dibenzo-18-crown-6; CrHCF: Cr(III)hexacyanoferrate(II); Au NP: gold nanoparticles; RuO–
RuCN: ruthenium(III) hexacyanoruthenate(II).

ing stock solution by its dilution with the appropriate
supporting electrolyte: 0.1 M HCl, acetate buffer so-
lution (ABS) of pH 4.03, phosphate buffer solution
(PBS) of pH 7.40 or 0.2 M NaOH, respectively.
Pharmaceutical samples consisting of 1 mL Mil-
gamma NA vials (Wörwag Pharma, Germany) con-
taining 100 mg of VB1 , 50 mg of VB6 and tartaric
acid were purchased from a local pharmacy.
Apparatus and procedures
All electrochemical measurements were performed
on an electrochemical workstation (BAS CV-50W,
Bioanalytical Systems, USA) coupled to a PC run-
ning the BAS CV-50W software to allow experimen-
tal control and data acquisition. A three electrode cell
consisting of a Pt, a glassy carbon (GC) or a pencil
graphite (PG) electrode as the working electrode, an
Ag/AgCl (3.0 M KCl) as the reference and a platinum
wire as the auxiliary electrode were employed. PGE
was obtained by cutting a 6 cm long 0.5 mm HB pen-
cil lead (Rotring) in half. Then, the 3 cm long pencil
lead was fixed with the cut edge into a Rotring Tikky
mechanical pencil acting as the holder. Electrical con-
tact with the pencil graphite lead was realized by a
metal wire soldered to the metallic part of the Rotring
pencil holder. Every time, 1.5 cm of the lead was left
outside the Rotring holder; 1 cm of the graphite lead
was introduced in the analyzed solution by keeping the
pencil in the upright position (Fig. 2). Each voltam-

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904 I. G. David et al./Chemical Papers 69 (7) 901–910 (2015)

Fig. 2. PGE fabrication steps.

Fig. 3. Cyclic voltammograms of 10−3 M VB1 in 0.2 M NaOH (a) and 10−3 M VB6 (b) in ABS, pH 4.03, obtained on Pt (– –),
GC (---) and PG (—) electrodes; scan rate: 100 mV s−1 .

metric recording was carried out on a new graphite
pencil lead (David et al., 2013, 2015). In case of solid
electrodes (GC and Pt), their surface was cleaned with
alumina powder, rinsed with deionized water and air
dried before each voltammetric recording.
All studies performed using cyclic voltammetry
(CV) were done in the potential range of 0 mV and
1000 mV at the scan rate of 100 mV s−1 unless oth-
erwise stated. Differential pulse voltammetry (DPV)
was applied in the potential range from 0 mV to
1200 mV (if not stated otherwise) using the follow-
ing optimized instrumental parameters: scan rate of
50 mV s−1 , pulse amplitude of 50 mV, pulse period
of 200 ms, pulse width of 50 ms and sampling width
of 17 ms. All experiments were carried out at room
temperature (25.0 ± 0.2) ◦C.
To test the application potential of the developed
DPV method on pharmaceutical samples, the Mil-
gamma NA formulation was used. The declared con-
tent for a vial of 1.0 mL is 100 mg of VB1 , 50 mg of
VB6 and tartaric acid. The whole content of a Mil-
gamma NA vial was diluted with deionized water in
a volumetric flask to 100 mL. An aliquot of 0.5 mL
of this solution was diluted with the proper support-
ing electrolyte (0.2 M NaOH for VB1 and ABS of pH
4.03 for VB6 determination, respectively) to the final
volume of 10 mL. DPVs were recorded for the diluted
pharmaceutical preparation solution before and after
three successive standard additions of 0.1 mL of the
corresponding vitamin solution. The obtained oxida-
tion peak currents were measured and further used to
calculate the VB1 and VB6 contents of the Milgamma
vials and the corresponding recoveries.
Results and discussion
Selection of optimum working conditions
In order to select the optimum electrochemical
working conditions, influence of the electrode mate-
rial (Pt, GC or PG) (Fig. 3) and of the supporting
electrolyte (0.1 M HCl; ABS: pH 4.03, PBS: pH 7.40
and 0.2 M NaOH) on the voltammetric behavior of
VB1 and VB6 (Fig. 4) was investigated.
CV studies emphasized that VB1 is electroactive
only in the NaOH medium presenting a poorly de-
fined anodic peak on Pt and GCE and two irre-
versible oxidation peaks at around 250 mV and a

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 I. G. David et al./Chemical Papers 69 (7) 901–910 (2015)
Fig. 4. Cyclic voltammograms of 10−3 M VB6 in different sup-
porting electrolytes: ABS, pH 4.03, (– –), PBS, pH 7.40,
(---) and 0.2 M NaOH (—) obtained on PGE; scan rate:
100 mV s−1 .
smaller and broader one at about 550 mV (Fig. 3a).
It is well-known that in an alkaline medium, thi-
amine is oxidized to thiochrome (Antal et al., 2013).
Considering the similarities between the anodic peak
potential obtained at around 250 mV in alkaline
medium for thiamine on PGE and that obtained on
the Cys/SAM/Au electrode (Wan et al., 2002), it can
be assumed that this peak is due to thiamine oxidation
to thiochrome.
CVs of VB6 in the 0.1 M HCl supporting elec-
trolyte obtained on all the investigated working elec-
trodes were electrochemically featureless. In all the
other investigated media (ABS: pH 4.03, PBS: pH 7.40
and 0.2 M NaOH) an oxidation peak was observed on
all tested working electrodes. The highest oxidation
peak for VB6 in ABS, pH 4.03, (Fig. 3b) was obtained
for the PGE surface at about 900 mV.
Moreover, CVs recorded in supporting electrolytes
with different pH values showed that the oxidation
peak potential of VB6 is shifted towards more posi-
tive values by 36 mV/pH when pH of the investigated
solution decreases, whereas the maximum peak cur-
rent does not change significantly (Fig. 4).
A similar pH dependence of the VB6 oxidation
peak potential was observed on GCE (Wu & Song,
2008) and on bare or modified carbon paste electrodes
(Desai et al., 2008), indicating that the electrode reac-
tion involves protons. This behavior can be explained
by the acid-base nature (pKa1 = 5 and pKa2 = 8.96)
of the pyridoxine molecule which has different forms
depending on the solution pH. Thus, according to lit-
erature data, pyridoxine at pH < pKa1 exists in the
cationic form with protonated pyridinic nitrogen and
the anionic form is predominant in a strong alkaline
solution (at pH > pKa2 ) due to the deprotonation
of the —OH group from the pyridine ring (Wang et
al., 2005; Habibi et al., 2010). According to literature
data, pyridoxine is oxidized to pyridoxal in an elec-
trode process involving two electrons and two protons
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905
Table 2. Regression equations of Ip = f (v1/2 ) and log(Ip ) =
f (log(v)) plots obtained from VB1 and VB6 CVs data
Analyte Ip = f (v1/2 ) log(Ip ) = f (log(v))
VBa
1 0.8561v1/2 + 0.1194 0.4645 log(v) + 0.0113
R2 = 0.9889 R2 = 0.9892
VB6 3.3677v1/2 + 3.1582 0.4355 log(v) + 0.7038
R2 = 0.9850 R2 = 0.9812
a) For the first peak with the peak potential at ≈ 250 mV.
(Pournaghi-Azar et al., 2007; Desai et al., 2008; Habibi
et al., 2010; Fonseca et al., 2011).
The possibility of using a pencil lead for several
measurements was studied by recording five succes-
sive DPVs on the same electrode in the same solution
of VB1 in 0.2 M NaOH or of VB6 in ABS, pH 4.03. In
both cases, the oxidation peak of VB6 and the two
peaks (with peak potentials of about 250 mV and
550 mV, respectively) corresponding to the oxidation
of VB1 were observed; however, the maximum cur-
rent decreased with the increasing repetitive number
of recordings. It is possible that this behavior is caused
by the electrode surface passivation by its coating with
some products of the electrochemical reaction (e.g.,
polymeric film) (Ferreira et al., 2006), which leads to
a hindered access of the analyte molecules to the elec-
trode surface. Thus, for further investigations, only
new pencil leads were used as the working electrode
for every voltammetric measurement.
Cyclic voltammetric behavior of VB and VB

on PGE
CVs recorded for VB1 and for VB6 on PGE in
0.2 M NaOH at different scan rates (v) ranging from
10 mV s−1 to 1000 mV s−1 present two anodic peaks
for VB1 (Ep1 ≈ 250 mV and Ep2 ≈ 550 mV) and one
for VB6 (Ep ≈ 600 mV). For both investigated com-
pounds, the anodic peak currents increased with the
increasing potential scan rate. Linear dependences of
the peak current intensities (Ip ) versus the square root
of the scan rate (v1/2 ) and the slope values (near 0.5)
of the log(Ip ) = f(log(v)) plots (Table 2) indicate that
electrochemical oxidation of VB1 and VB6 on PGE is
a diffusion controlled process (Brett & Oliveira-Brett,
1993).
Performance characteristics
The charging current contribution to the back-
ground current limits the analytical determination
of species using CV. Thus, the more sensitive DPV
was employed for the determination of the calibration
curves of the individual vitamins and for quantitative
assessments of VB1 and VB6 in a mixture.
906 I. G. David et al./Chemical Papers 69 (7) 901–910 (2015)

Table 3. Performance characteristics of the DPV method developed for VB1 and VB6 determination on PGE

Analyte/medium Linear range/M Calibration function SD SD R2 LoQ/M LoD/M

(slope) (intercept)

VB1 in 0.2 M NaOH 10−5 –10−3 Ip (VB1 ) = 19465c(VB1 ) – 0.2923 469 0.2197 0.9959 1.78 × 10−5 5.34 × 10−6
VB6 in 0.2 M NaOH 10−5 –2.5 × 10−3 Ip (VB6 ) = 21380c(VB6 ) + 2.0676 582 0.5281 0.9941 2.35 × 10−5 7.06 × 10−6
VB6 in ABS pH 4.03 5 × 10−6 –10−3 Ip (VB6 ) = 53889c(VB6 ) + 1.9417 2309 0.9331 0.9891 9.37 × 10−6 2.81 × 10−6

Fig. 5. Differential pulse voltammograms obtained on PGE for different concentrations of VB1 : 10−5 M; 2.5 × 10−5 M; 8 × 10−5
M; 10−4 ; 2.5 × 10−4 M; 5 × 10−4 M; 8 × 10−4 M; 10−3 M (a), and VB6 : 10−5 M; 2.5 × 10−5 M; 5 × 10−5 M; 10−4 M;
2.5 × 10−4 M; 5 × 10−4 M; 8 × 10−4 M; 10−3 M (b), in 0.2 M NaOH.

Linear range and limit of detection
Using PG as the working electrode and 0.2 M
NaOH as the supporting electrolyte, VB1 and VB6
(Fig. 5), respectively, gave signals only at concentra-
tions higher than 10−5 M. The maximum current of
the DPV peak obtained at around 250 mV shown by
VB1 and that of the peak at about 600 mV due to
VB6 , varied linearly with the analyte concentration in
the range of 10−5 M to 2.5 × 10−3 M (Table 3).
In ABS, pH 4.03, the oxidation peak of VB6 oc-
curring at 900 mV was narrow and well-defined. In
this case, the maximum peak current recorded for VB6
varied linearly with the analyte concentration in the
range of 5 × 10−6 –10−3 M VB6 (Table 3).
Although the determination coefficient (R2 ) is the
first calculated parameter when studying linearity, its
value is not a defining one. As it can be seen in Table 3,
comparing the results obtained for VB6 in NaOH and
in ABS, pH 4.03, the lowest R2 value is obtained for
the equation with the higher slope. It is wellknown
that a higher slope of the calibration graph indicates
higher sensitivity. In case of the VB6 determination in
ABS, pH 4.03, this fact was proved by an extension
of the linear range towards lower concentrations and
a better detection limit.
Data from the linearity study were used to calcu-
late the detection limit (LoD) and the quantification
limit (LoQ) according to: LoD = 3scmin /b and LoQ =
10scmin /b, where scmin is the standard deviation of the
lowest concentration from the calibration curve and b
is the calibration curve slope (González & Herrador,
2007). The obtained values of the detection and quan-
tification limits are listed in Table 3.
When compared to the data presented in Table 1,
detection limits of the method described in the present
paper for the two investigated analytes are not so low
as those presented in some of the previously published
works which used different chemically modified elec-
trodes. Nevertheless, linear ranges and detection lim-
its of DPV on bare PGE make the presented method
suitable for the application in pharmaceutical analysis
where the compounds exist as major components of a
formulation. Also, the determination of VB1 and VB6
by DPV on bare PGE has significant advantages over
the other electrochemical methods described in litera-
ture: simplicity, rapidity and low costs. These advan-
tages are due to the use of a commonly available, dis-
posable and cheap electrode material of PGE without
any surface modification, which eliminates the clean-
ing step of the electrode surface before each measure-
ment, reducing thus the analysis time significantly.
Repeatability
Series of ten successive measurements of 10−4 M

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 I. G. David et al./Chemical Papers 69 (7) 901–910 (2015) 907

Fig. 6. Differential pulse voltammograms obtained on PGE in 0.2 M NaOH for selected VB6 concentrations: 5 × 10−5 M; 7.5 × 10−5
M; 10−4 M; 2.5 × 10−4 M; 5 × 10−4 M; 7.5 × 10−4 M; 10−3 M in the presence of 10−4 M VB1 (a), and for selected VB1
concentrations: 2.5 × 10−5 M; 5 × 10−5 M; 7.5 × 10−5 M; 10−4 M; 2.5 × 10−4 M; 5 × 10−4 M; 7.5 × 10−4 M in the
presence of 10−4 M VB6 (b).

VB1 in NaOH and 10−4 M VB6 in NaOH and in ABS,
respectively, were performed in order to evaluate the
repeatability of the electrode response. Each voltam-
metric recording was done using a new PGE. Percent-
age relative standard deviations (RSD) of 4.70 % for
VB1 in NaOH, 4.06 % for VB6 in NaOH and 3.84 %
for VB6 in ABS were obtained. It is worth mentioning
that all the percentage RSDs are within the accepted
limits according to the respective concentration levels
(AOAC International, 2012).
Selective determination of VB and VB
in the
presence of the other
A more detailed evaluation was performed with re-
spect to the analytical performance of PG electrodes
for simultaneous measurement of VB1 and VB6 , which
has particular relevance for pharmaceutical analysis.
In order to study the possibility of voltammetric
determination of VB1 and VB6 , one in the presence of
the other, on PGE, DPVs were recorded for solutions
containing a fixed concentration (10−4 M) of one of
the vitamins (B1 or B6 ) and different concentrations
of the other vitamin (B6 and B1 , respectively) using
0.2 M NaOH as the supporting electrolyte (Fig. 6).
By inspecting the obtained DPVs it can be con-
cluded that the VB1 peak is constant in the presence
of various concentrations of VB6 , while the VB6 peak
is modified with the concentration of VB1 due to its
peak at a close potential value. Thus, using NaOH as
the supporting electrolyte, VB1 can be determined in
the presence of VB6 by the developed method with-
out any interference while VB6 cannot be quantified
directly by this method in the presence of VB1 .
Therefore, as VB1 does not present any oxidation
peak in ABS, pH 4.03, whereas VB6 shows a nar-
row and well-defined peak at more positive potentials
(around 900 mV), for the determination of both vita-
mins present in a mixture, different supporting elec-
trolytes should be used.
Analytical application on pharmaceutical for-
mulations
For the determination of both vitamins in a phar-
maceutical formulation (Milgamma, NA), the voltam-
metric behavior of VB1 and VB6 on PGE in different
supporting electrolytes was exploited; 0.2 M NaOH
was used as the supporting electrolyte to determine
the VB1 content and ABS, pH 4.03, was employed for
the voltammetric quantification of VB6 (Fig. 7). The
concentrations of VB1 and VB6 in the test sample
were evaluated using the standard addition method.
The obtained results are presented in Table 4. They
represent the average values acquired for three addi-
tions made for each of the four replicate samples; these
data were also used as the basis to calculate recovery
values. The latter are in the range of 91.94–102.09 %
for VB1 and of 91.43–103.67 % for VB6 , and the av-
erage values (Table 4) are within the expected limits
for these concentration levels (AOAC International,
2012).
The relative error values were calculated consider-
ing the value claimed by the manufacturer. For both
analyzed vitamins, these values were below 2 %, show-
ing a good agreement between the results obtained
using the proposed DPV method and the values de-
clared by the pharmaceutical producer. Thus, com-
parable and reliable results are produced when using
PGE.

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908 I. G. David et al./Chemical Papers 69 (7) 901–910 (2015)

Fig. 7. Differential pulse voltammograms obtained on PGE for a sample of Milgamma NA 2000 fold diluted in 0.2 M NaOH before
and after three additions of 0.1 mL 6 × 10−3 M VB1 solution each (a), and in ABS, pH 4.03, before and after three
additions of 0.1 mL 10−2 M VB6 solution each (b).

Table 4. Results of DPV on the PGE determination of VB1 and VB6 content of Milgamma NA vials (Wörwag Pharma) and the
corresponding recoveries

Vitamin B1 B6

Claimed content/(mg mL−1 ) 100.0 50.00

Found content by (DPV ± SD)/(mg mL−1 ) 101.5 ± 6.63 50.22 ± 0.61
RSD/% 6.53 1.21
Relative error/% 1.55 0.43

Recovery

Average (R ± SD)/% 98.24 ± 3.50 98.41 ± 3.98

RSDR /% 3.56 4.05

Note: SD – Standard deviation; RSD – relative standard deviation; R – recovery.

Conclusions but only from the difference of two recorded signals.

The present paper describes a simple and rapid References

PGE based DPV method which enables the assess-
ment of B1 and B6 vitamins in a mixture. VB1 is
electroactive only in a NaOH solution while VB6 is
electroactive in all investigated media. In the NaOH
medium, VB6 showed a large peak overlapping that
of VB1 . Thus, in order to determine the two vitamins
from a mixture, DPVs for VB1 have to be recorded in
a NaOH medium and those for VB6 in ABS, pH 4.03.
The new developed analytical method was successfully
applied in the determination of the two vitamins in a
pharmaceutical formulation. Moreover, the disposable
pencil graphite electrode used as the working electrode
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