Journal of Pharmacological and Toxicological Methods 73 (2015) 56–62

Contents lists available at ScienceDirect

Journal of Pharmacological and Toxicological Methods

journal homepage: www.elsevier.com/locate/jpharmtox

Original article

Simultaneous determination of iron (II) and ascorbic acid in

pharmaceuticas based on flow sandwich technique
Christina Vakh ⁎, Elena Freze, Alexsey Pochivalov, Ekaterina Evdokimova, Mihail Kamencev,
Leonid Moskvin, Andrey Bulatov
Department of Analytical Chemistry, Institute of Chemistry, Saint Petersburg State University, Saint Petersburg, pr. Universitetskij 26, 198504, Russia

a r t i c l e i n f o a b s t r a c t

Article history: The simple and easy performed flow system based on sandwich technique has been developed for the simulta-
Received 13 January 2015 neous separate determination of iron (II) and ascorbic acid in pharmaceuticals. The implementation of sandwich
Received in revised form 7 March 2015 technique assumed the injection of sample solution between two selective reagents and allowed the carrying out
Accepted 31 March 2015
in reaction coil two chemical reactions simultaneously: iron (II) with 1,10-phenanthroline and ascorbic acid with
Available online 8 April 2015
sodium 2,6-dichlorophenolindophenol. For achieving of excellent repeatability and considerable reagent saving
Keywords:
the various parameters such as flow rate, sample and reagent volumes, reaction coil length were also optimized.
Automation of pharmaceutical analysis The limits of detection (LODs) obtained by using the developed flow sandwich-type approach were 0.2 mg L−1
Flow analysis for iron (II) and 0.7 mg L−1 for ascorbic acid. The suggested approach was validated according to the following
Sandwich technique parameters: linearity and sensitivity, precision, recoveries and accuracy. The sampling frequency was 41 h−1.
Iron (II) © 2015 Elsevier Inc. All rights reserved.
Ascorbic acid

1. Introduction disease — iron-deficiency anemia (IDA) (Clark, 2008). The treatment of

IDA is provided by the antianemic drugs which contain iron (II) com-
Pharmaceuticals are the chemical substances containing one or sev- pounds and ascorbic acid as the mostly used excipient, which improves
eral pharmacologically active ingredients and excipients — substances the gastrointestinal absorption of iron (Killip, Bennett, & Chambers,
which are added to a prescription in order to confer a suitable consisten- 2007). However, such pharmaceuticals often cause side effects: nausea,
cy or form of the drug as well as to affect the bioavailability of the active anorexia, diarrhea and metal taste in the mouth (Beutler, Hoffbrand, &
ingredients (Jackson, Young, & Pant, 2000; Pifferi & Restani, 2003; Cook, 2003). Thus, it is important to carry out the quality control of
World Health Organization, 2007). The quality control of pharmaceuti- antianemic drugs and determine the concentrations of iron (II) and
cals is a key factor at all stages of their development and production. In ascorbic acid.
recent years, the international pharmacopoeia requirements and stan- Traditionally, most methods of pharmaceutical analysis for separate
dards related to the quality control of pharmaceuticals become stricter determination of iron (II) and ascorbic acid are focused on the spectro-
(Pimenta, Montenegro, Araujo, & Martınez Calatayud, 2006; World photometry (Davies & Masten, 1991; Guclu, Sozgen, Tutem, Ozyurek, &
Health Organization, 2007). This demands the modern pharmaceutical Apak, 2005; Karpinska & Kulikowska, 2002; Zargba & Hopkata, 1996)
analysis to develop rapid, reliable, selective and sensitive analytical and electrochemistry (Mahmoud, 2001; Mary Nancy, Anithakumary, &
methods for the determination of active ingredients and excipients in Kumara Swamy, 2014; Merli, Profumo, & Dossi, 2012; Thangamuthu,
pharmaceuticals (David & Webb, 2003). Senthil Kumar, & Chandrasekara Pillai, 2007) (Table 1). These methods
Nowadays, by the reason of the accelerated pace of life, hyponutrition, are characterized by high selectivity, sensitivity, rapidity and the possi-
people are exposed by a lacking of essential trace elements, among which bility of automation. However, these techniques as well as atomic ab-
the most common is iron deficiency. Iron is of vital importance in numer- sorption and emission spectrometry (Zachariadis & Michos, 2007;
ous biological processes, primarily involved oxygen binding and trans- Zachariadis, Raidou, Themelis, & Stratis, 2002) are primarily focused
port, muscle oxygen use and storage, gene regulation, cell growth and on the determination of iron. In turn, the chromatography (Gioia,
differentiation, enzyme reactions, neurotransmitter synthesis, and pro- Andreatta, Boschetti, & Gatti, 2008; Khuhawar & Lanjwani, 1998;
tein synthesis (Beard, 2001; Crichton, Wilmet, Legssyer, & Ward, 2001; Pengfei et al., 2012) and the capillary electrophoresis (Fotsing, Fillet,
Halliberg, 2001; Schumann, Ettle, Szegner, Elsenhans, & Sololmons, Bechet, Hubert, & Crommen, 1997; Lin Ling, Baeyens, Van Acker, &
2007). The lack of this element in the human body causes a widespread Dewaele, 1992) are focused on the determination of iron (II) or ascorbic
acid.
⁎ Corresponding author. Tel.: +7 929 110 0987; fax: +7 812 372 4421. Inevitably, automation plays an important role in pharmaceutical
E-mail address: [email protected] (C. Vakh). analysis, especially when a lot of samples have to be analyzed in the

http://dx.doi.org/10.1016/j.vascn.2015.03.006
1056-8719/© 2015 Elsevier Inc. All rights reserved.
 C. Vakh et al. / Journal of Pharmacological and Toxicological Methods 73 (2015) 56–62 57

Table 1
Reported methods for determination of iron (II) and ascorbic acid in pharmaceuticals.

Method of analysis Analyte Detection Matrix Linear range, LOD, Sample R.S.D., Reference
mg L−1 mg L−1 frequency %
(h−1)

Spectrophotometry Iron (II) With 4-(2-pyridylazo) resorcinol at λ = 537 nm, Tablets 0.025–0.2 0.008 10 2.2 Karpinska &
pH = 9.1 (borate buffer) Kulikowska
(2002)
With three azoderivatives of pyrocatechol at Tablets 0.05–2.11 0.01 10 3.0 Zargba &
λ = 490; 560; 600 nm, pH = 10.0; 0.05–0.89 0.01 2.7 Hopkata (1996)
0.05–1.00 0.01 1.9
Ascorbic acid With neocuproine at λ = 450 nm, pH = 7.0 Tablets 1–15 0.3 10 2.0 Guclu et al.
(2005)
With sodium 2,6-dichlorophenolindophenol at Tablets 0.3–20 0.08 10 1.8 Davies &
λ = 520 nm, pH = 4.2 Masten (1991)
Electrochemical Iron (II) Differential pulse voltammetry, pH = 7.0 Tablets 0.05–5 0.017 5–10 3.6 Merli et al.
methods (2012)
Potentiometric detection with ion-selective Tablets 0.028–560 0.006 5–10 2.6 Mahmoud
electrode, pH = 7.0 (2001)
Ascorbic acid Amperometric detection using glassy carbon Tablets 2–100 0.5 5–10 3.5 Thangamuthu
electrode modified by K4Mo(CN)8, pH = 7.0 et al. (2007)
Differential pulse voltammetry with glassy carbon Tablets 8–80 0.7 5–10 3.0 Mary Nancy
electrode modified by graphene, pH = 7.0 et al. (2014)
AAS/AES Iron (II) AES with inductively coupled plasma Tablets 0.01–0.5 0.002 10 2.3 Zachariadis &
Michos (2007)
AAS with flame atomization (air-acetylene flame) Tablets 0.5–4 0.15 10–20 1.1 Zachariadis
et al. (2002)
Chromatography Iron (II) Preliminary derivatization HPLC complexing agent Tablets 0.5–2.5 0.025 4 4.8 Khuhawar &
(2-thiophene aldehyde-4-phenyl-3-tiosemicarbazone), Lanjwani
stationary phase — C-18; mobile phase — a mixture of (1998)
methanol, acetonitrile, water, sodium acetate and
ammonium tetra butylbromide (78:10:10:1:1)
Ascorbic acid HPLC stationary phase — C-18; mobile phase — a Tablets 0.4–1.2 0.13 10 2.0 Pengfei et al.
mixture of phosphate buffer and acetonitrile (95:5) (2012)
Couple-ion reversed liquid chromatography; Injection 14–45 2 10 1.8 Gioia et al.
stationary phase — C-18, mobile phase — solutions (2008)
cetyltrimethylammonium bromide solution
Capillary Ascorbic acid Detection in the medium of a borate buffer solution Tablets 0.2–1 0.02 6 4.3 Fotsing et al.
Electrophoresis (pH = 8.5) (1997)
Detection in the medium of a phosphate buffer Tablets 10–1000 0.5 12 5.0 Lin Ling et al.
solution (pH = 5.0) (1992)
Flow analysis Iron (II) FIA, reagent — salicylic acid, λ = 520 nm, pH = 4.0 Tablets 1–20 0.3 100 3.4 Udnana et al.
(2004)
SIA, reagent — 1,10-phenanthroline,λ = 510 nm, Tablets 0.25–5 0.02 40 3.0 Tesfaldet et al.
pH = 5.5 Tablets 5.0–40 1.0 100 5.0 (2004)
SIA, reagent — 2,2-bipyridyl, λ = 523 nm, pH = 4.5 Oliveira &
Masini (2001)
Ascorbic acid FIA, reagent — ferrozine, λ = 562 nm, pH = 5.5 Tablets 0.1–20 0.005 90 1.9 (Molina-Diaz
et al., 1998)
FIA, reagent — tripiridilttriazin iron (III), λ = 593 Tablets 0.015–2 0.005 180 0.8 Kukoc-Modun
nm, pH = 3.6 et al. (2012)
SIA, reagent — KMnO4, λ = 525 nm Tablets 0.1–1000 0.03 60 2.0 Lenghor et al.
(2002)
Iron (II) and Ascorbic Flow sandwich technique, reagent for Iron (II) — Tablets 0.5–4 0.2 41 3.0 This
acid simultaneously 1,10-phenanthroline, λ = 510 nm, pH = 5.5 2–20 0.7 3.0 work
reagent for Ascorbic acid — sodium
2,6-dichlorophenolindophenol, λ = 512 nm, pH = 4.5

minimum of time. The most widespread in pharmaceutical analysis are Kukoc-Modun, Biocic, & Radic, 2012; Lenghor et al., 2002; Mervartova
the flow injection (FIA) (Ruzicka & Hansen, 1975) and sequential injec- et al., 2007; Molina-Diaz, Ortega-Carmona, & Pascual-Reguera, 1998;
tion (SIA) analysis (Ruzicka & Marshall, 1990). Coupling to FIA and SIA Mortatti, Krug, Pessenda, & Zagatto, 1982; Ruzicka & Hansen, 1975;
manifolds selective detectors make the developed approaches simple Ruzicka & Marshall, 1990; Tzanavaras & Themelis, 2007; Udnana et al.,
and easy implemented in pharmaceutical analysis (Horstkotte & 2004) and sequential injection (Lenghor et al., 2002; Oliveira & Masini,
Cerdà, 2009; Mervartova, Polasek, & Martınez Calatayud, 2007). Thus, 2001; Tesfaldet, van Staden, & Stefan, 2004) techniques were developed,
flow analysis with spectrophotometric detection offers practically which provide high sensitivity (Table 1) in combination with high sam-
endless possibilities to the automation of pharmaceutical analysis pling frequency (up to 180 h−1). However, they are focused only on the
procedures. Moreover it also provides miniaturization of analysis determination of iron (II) or ascorbic acid. To the best of our knowledge,
(Tzanavaras & Themelis, 2007). simultaneous separate determination of iron (II) and ascorbic acid
For the determination of iron (II) and ascorbic acid in pharmaceuticals based on flow methods has not been reported.
the flow injection (Alonso, Bartrolí, del Valle, & Barber, 1989; Alonso, This present work describes a new automated procedure for simul-
Bartroli, Delvalle, Escalada, & Barber, 1987; Alonso-Chamarro, Bartroli, & taneous separate determination of iron (II) and ascorbic acid in pharma-
Barber, 1992; Araujo, Lima, Alonso-Chamarro, Bartroli, & Poch, 1991; ceuticals. For this purpose the flow system based on sandwich
Cerda & Cerda, 2009; Falkova, Pushina, Bulatov, Alekseeva, & Moskvin, technique is implemented. At first the application of this technique in
2014; Horstkotte & Cerdà, 2009; ISO 5725-1, 1994; IUPAC, 1997; flow analysis was presented by Alonso and co-authors and was applied
58 C. Vakh et al. / Journal of Pharmacological and Toxicological Methods 73 (2015) 56–62

for the determination of iron (II) and total iron in a ground water sam- LS-1 and a USB 4000 spectrometer (Ocean Optics, USA) with a 50 mm
ples (Alonso et al., 1989). The sandwich technique provides the strict path length flow cell FIA-Z-SMA-50-TEF (Fialab, USA). The analyzer
order of reagents and sample injection into the flow system and was operated automatically by means a computer.
means the sample “sandwiching” between two selective reagents To carry out the reference analyses of pharmaceuticals a capillary
(Alonso et al., 1987; Alonso-Chamarro et al., 1992; Araujo et al., 1991). electrophoresis system Capel-105М (Lumex, Russia) and an atomic ab-
During the moving thought the reaction coil two analytical products sorption spectrometer АА-7000 (Shimadzu, Japan) were used. To carry
are formed. Despite the necessity to use the relatively large sample vol- out the sampling preparation of pharmaceuticals an ultrasonic bath
ume for preventing the overlapping of the colored zones, the sandwich Sapphire (Sapphire, Russia) was used.
technique is characterized by satisfactory sampling frequency because
of the high flow rate using. Moreover, because of the optimizing of phys- 2.3. Samples
ical parameters of the flow systems it become possible to achieve the
excellent repeatability of analysis and considerable reagent saving Three different samples of antianemic drugs at solid dosage form
(Cerda & Cerda, 2009). “Sorbifer Durules”, “Fenules” and “Tardyferon” were analyzed. All
these antianemic drugs contain ferrous sulfate as active ingredient and
2. Materials and methods ascorbic acid as excipient, with dosage form 500 mg.

2.1. Reagents and solutions 2.4. Sample preparation

All chemicals were of analytical reagent grade. Ultra pure water The sample preparation was performed by ultrasound-assisted dis-
from Millipore Milli-Q RG (Millipore, USA) was used throughout the solution of solid dosage form of antianemic drugs in the medium of
experiment. The stock solution of 0.045 M of iron (II) was prepared by 0.1 M HCl. Three tablets of antianemic drugs were grounded to a homo-
dissolving of the corresponding weight of FeSO₄ · (NH₄)2SO₄ · 6H2O geneous powder in a ceramic mortar. Then, 0.01 g of the powder was
in 0.1 M H2SO4. The stock solution of 0.1 M of ascorbic acid was prepared mixed with 1 g of potassium chloride. This step is carrying out because
by dissolving of the corresponding weight in the water. The working so- of the high content of iron (II) and ascorbic acid in antianemic drugs.
lutions of analytes were prepared by appropriate dilution of the stock Further, 0.01 g of the obtained mixture was placed in a glass vessel
solutions with the water. The solution of 1 μМ 1,10-phenanthroline and the 5 mL of 0.1 M HCl was added. Afterwards the dissolution of
and sodium 2,6-dichlorophenolindophenol were prepared by dissolv- antianemic drugs took place under ultrasonication (325 W, 35 kHz) at
ing of the corresponding weights of reagents in the acetate buffer solu- 20 °C for 10 min. The prepared solution was then analyzed by flow sys-
tion (рН = 4.5) and the water, respectively. The 1,10-phenanthroline tem based on sandwich technique. While the calculation of the content
solution was colorless, stable within a week when stored in the dark of the iron (II) and ascorbic acid in the tablet, the mass of the tablet
place. The solution of sodium 2,6-dichlorophenolindophenol was and the analyte content using the calibration curve were taken into
brightly colored, stable within a week when stored at temperature consideration.
from 2 to 5 °С. The working solutions of reagents were prepared by ap-
propriate dilution of the stock solutions with acetate buffer solution. 2.5. The procedure of flow determination of iron (II) and ascorbic acid

2.2. Manifold and apparatus To implement the flow system based on sandwich technique (Fig. 1)
the portions of sample and reagent solutions were sequentially deliv-
The flow system based on sandwich technique includes (Fig. 1): an ered into the reaction coil by movement of the peristaltic pump through
eight-port valve (Cole-Parmer, USA), a peristaltic pump MasterFlex L/S a eight-port valve in strict order: 75 μL of acetate buffer solution as the
(Cole-Parmer, USA) (flow rate 3.0 mL min−1), a reaction coil (80 cm carrier solution (g), 150 μL of 1,10-phenanthroline solution (b), 1 mL
length and 1 mm in i.d.), home-made PTFE filter prepared according of sample solution (a), 150 μL of sodium 2,6-dichlorophenolindophenol
to Falkova et al. (2014) for filtering a sample solution and communica- solution (d) and again 500 μL of carrier solution (g). The high flow rate
tion tubes (PTFE 1 mm in i.d.). It is equipped with a source of visible light (3.0 mL min−1) and a large volume of the sample eliminate overlapping

Fig. 1. The manifold of the flow system based on sandwich technique.

 C. Vakh et al. / Journal of Pharmacological and Toxicological Methods 73 (2015) 56–62 59

of the colored zones of reaction products. The analytical signals The absorbance spectra of complex of Fe (II) with 1,10-
were measured in the flow-stop condition for 3 s by means of the spec- phenanthroline and sodium 2,6-dichlorophenolindophenol are pre-
trophotometer at the wavelength 510 nm for iron (II) and 512 nm for sented in the Fig. 2.
ascorbic acid. The first analytical signal corresponds the complex of The reaction between ascorbic acid and sodium 2,6-
iron (II) with 1,10-phenanthroline, the second one — the product of dichlorophenolindophenol is strongly dependent on the acidity of
ascorbic acid with sodium 2,6-dichlorophenolindophenol. To avoid the the medium. However, solving the problem of choosing the optimal
memory effect the manifold was washed out with carrier solution. To pH of the reaction, it was necessary to take into account the presence
ensure the strict order of zones formation, sequence and time of of iron (II) in the antianemic drugs and its possible influence on the re-
performing of all analysis steps the special program was set up. action. According to Davies & Masten (1991), it was decided to carry out
the reaction in the presence of acetate buffer solution. In the pH range
from 4.0 to 4.5 the interference effect of Fe2+ ions is not found. Thus,
2.6. Procedure for the determination of iron (II) and ascorbic acid by
the acetate buffer solution (pH = 4.5) was used as an appropriate
reference methods
carrier-solution.
For the AAS determination of iron (II) in antianemic drugs the pre-
3.2. Optimization of parameters
treatment stage was performed by ultrasound-assisted dissolution of
powdered tablets in 50 mL of 1 M HNO3 for 10 min. Then, the mixture
The conditions for the determination of iron (II) and ascorbic acid
was filtered through the paper filter and farther analyzed by AAS with
were optimized by studying the influence of the various parameters
atomization in a continuous flame.
such as flow rate, sample and reagent volumes, reaction coil length
For the CE determination of ascorbic acid in antianemic drugs the
and reagent concentration. 4 × 10−5 M of iron (II) and ascorbic acid,
pre-treatment stage was performed by ultrasound-assisted dissolution
5 × 10− 3 M of 1,10-phenanthroline and 5 × 10− 4 M of sodium 2,6-
of powdered tablets in 5 mL of 0.1 M HCl for 10 min. For the stabilization
dichlorophenolindophenol were used to optimize these parameters. In
of ascorbic acid in the prepared solution 10 mL of 0.05 M EDTA was
all cases both the relative peak height and percentage of RSD were
added. Then, the mixture was filtered through the paper filter and far-
used as criteria for establishing the most appropriate parameters.
ther analyzed by capillary electrophoresis. The length of used quartz
The flow rate is a very important parameter to be optimized because
capillary was 50 cm with 75 μm in i.d. The measurements were per-
it influences to the products formation. The reactions between iron (II)
formed at 254 nm using Na2B4O7 — sodium dodecyl sulfate solution as
and 1,10-phenanthroline as well as ascorbic acid and sodium 2,6-
a supporting electrolyte.
dichlorophenolindophenol are rapid, resulting in an almost instant
products formation. The flow rate was varied between 0.5 and
3. Results and discussion 3.5 mL min−1 by changing the speed of the pump. The better repeatabil-
ity and lower consumption of carrier-solution was achieved at
3.1. Theoretical aspects 3.0 mL min−1 and this flow rate was chosen as optimum working con-
dition (Fig. 3A).
For the spectrophotometric determination of iron (II) a well-known The aim of optimization of sample and reagent volumes is to mini-
color-forming reagent 1,10-phenanthroline was used. This reaction pro- mize the consumption of sample and reagents, maintaining the best
ceeds in wide range of pH from 2.0 to 9.0 in a molar ratio 3:1 (R:Fe2+) sensitivity and repeatability and to prevent the mixing of products
(Mortatti et al., 1982). The complex absorbance maxima is observed at while moving them in the reaction coil into the detector. The injected
the 510 nm (ε510 = 1.1 × 104 L mol−1 cm−1). sample volume was varied from 0.5 to 1.5 mL and the volumes of
For the spectrophotometric determination of ascorbic acid the re- 1,10-phenanthroline and sodium 2,6-dichlorophenolindophenol
agent sodium 2,6-dichlorophenolindophenol was chosen, because of were varied from 50 to 250 μL. The results presented in the Fig. 3B
the reducing properties of the analyte. This reaction proceeds in a shown that the injected sample volume less than 1 mL leads to decreas-
molar ratio 1:1 according to the following schematic representation: ing of repeatability because product zone mixing is observed. It was also
found that optimal reagent volumes are 150 μL for both reagents
In the acidic medium the reagent solution has a purple color, which (Fig. 3C).
is discoloring during the interaction with ascorbic acid. Absorbance The reaction coil is usually kept as short as possible to avoid the
maximum of sodium 2,6-dichlorophenolindophenol is observed at the product dispersion but sufficient to provide the product formation.
512 nm (ε512 = 3.2 × 104 L mol−1 cm−1). The length of this coil was varied from 50 to 100 cm. The optimum
length of the reaction coil was 80 cm, which ensures the effective over-
lapping zones of reagents with the sample and prevent the mixing of
products zones (Fig. 3D).
The concentrations of the reagents were varied from 1 × 10− 3 to
1 × 10−2 M of 1,10-phenanthroline and from 1 × 10−4 to 1 × 10−3 М
of sodium 2,6-dichlorophenolindophenol. It was found that the
optimal concentrations of 1,10-phenanthroline and sodium 2,6-
dichlorophenolindophenol are 5 × 10−3 and 5 × 10−4 М, respectively.

3.3. Optimization of dissolution

Most of antianemic drugs are in a solid dosage form with a relatively

high content of iron (II) and ascorbic acid (13–20%). Therefore, it is im-
portant to provide the effective dissolution as well as dilution of the
sample. It is known that H2SO4 and HNO3 have strong oxidizing proper-
ties, which contribute to the destruction of ascorbic acid and oxidation
Fig. 2. Absorbance spectra of complex of Fe (II) with 1,10-phenanthroline (СR = 10−4 М,
of Fe2 + to Fe3 +. Thus for investigating of the sample dissolution the
CFe2+ = 10−4 М, рН = 4.5) and sodium 2,6-dichlorophenolindophenol (СR = 10−4 М, deionized water and HCl were used. The effects of temperature,
ascorbic acid = 10−4 М, рН = 4.5). thermostating and ultrasonication time were investigated to increase
60 C. Vakh et al. / Journal of Pharmacological and Toxicological Methods 73 (2015) 56–62

Fig. 3. Investigation of flow system based on sandwich technique experimental conditions: (A) effect of flow rate, (B) effect of sample volume, (C) effect of reagents volumes, (D) effect of
reaction coli length.

the efficiency of dissolution. Thus the temperature was varied from 20 between the relative peak height and concentrations of iron (II) as
to 50 °C and the thermostating and ultrasonication time — from 2 to well as ascorbic acid were given by the follow equations:
20 min. Two parallel experiments were performed — with and without
h i
ultrasound assistants. The results showed that effective dissolution of −1
Relative peak height ¼ 0:231  Concentration of Fe ðIIÞ; mg L
antianemic drugs is observed in the media of 0.1 M HCl at 50 °С 2
þ 0:062; r h¼ 0:996; i
for 10 min without ultrasonication and at 20 °C for 10 min with −1
Relative peak height ¼ −0:046  Concentration of ascorbic acid ðIIÞ; mg L
ultrasonication.
2
þ 0:0968; r ¼ 0:995:
3.4. Interference effect
The absorbance of colored products at the wavelength 510 nm and
The effect of potentially interfering components presented in 512 nm for iron (II) and ascorbic acid, respectively, obey the Beer's
antianemic drugs (Mg2 +, Co2 +, Cu2 +, SO24 − ions and riboflavin, law in the range of 0.5–4.0 mg L−1 for iron (II) and of 2.0–20 mg L−1
pyridoxine, thiamine and pantothenic acid) on the determination of for ascorbic acid.
analytes was investigated. It was performed by addition of known con- The sensitivity was characterized by the limit of detection (LOD). It
centration of each component into a model solution, containing iron (II) was measured by standard IUPAC method as 3 standard deviation of
ions and ascorbic acid at concentration of 4 × 10−5 М in 0.1 M hydro- the blank (3 s) (IUPAC, 1997). The LODs calculated from the calibration
chloric acid. The tolerable excess of each taken foreign species is consid- plots based on 3 s were 0.2 mg L−1 for iron (II) and 0.7 mg L−1 for ascor-
ered to be less than 5% of relative error in the signal. The greatest bic acid. The results indicated that the LODs both for iron (II) and ascor-
influence (less than 10-fold excess) on the product formation has ribo- bic acid are satisfied for their determination in antianemic drugs.
flavin, thiamine, pyridoxine and pantothenic acid, but in the antianemic
drugs a content of these components is much lower than the content of 3.5.2. Precision
iron (II) and ascorbic acid. Thus it should be concluded with the absence The precision of the method was evaluated with regard to its repeat-
of interference effect. ability and reproducibility (ISO 5725-1, 1994).
The repeatability of the developed approach was determined by an-
3.5. Validation alyzing 10 replicates of model solutions which contain iron (II) and
ascorbic acid in the range from 0.5 mg L− 1 to 4.0 mg L−1 and from
For validation, the following parameters were evaluated for linearity 2 mg L−1 to 20 mg L−1, respectively. It was studied that the developed
and sensitivity, precision, recoveries and accuracy. flow sandwich-type approach provides satisfactory results. The RSD
values were b3.0%. This level of precision of the proposed flow
3.5.1. Linearity and sensitivity sandwich-type approach was adequate for quality-control of antianemic
Under the optimized reaction condition according to the previously drugs.
described procedure, the calibration curves for iron (II) and ascorbic The reproducibility of the proposed methods was assessed by apply-
acid determination were constructed from ten data points using the ing the developed flow sandwich-type approach with the use of 2 differ-
standard solution of analytes (Fig. 4). The relationship obtained ent instruments in 2 different laboratories at 2 different times. Results
 C. Vakh et al. / Journal of Pharmacological and Toxicological Methods 73 (2015) 56–62 61

Fig. 4. Calibration curves and depiction of absorbance peaks obtained by suggested flow system based on sandwich technique.

obtained with the laboratory-to-laboratory and day-to-day variations used to test the equality of the means of the experimental data obtained
were found to be reproducible because the RSD values under conditions by developed method and by the reference method. Hypothesis about
were 5.0%. the equality of the means of the experimental data obtained by the de-
veloped method and by the reference method was taken as the null hy-
3.5.3. Recovery pothesis (H0). Hypothesis about the difference of mentioned means was
Recovery values were determined for each analyte at 2 mg and 5 mg taken as an alternative hypothesis (H1). The significance was taken
concentrations in dosage form of antianemic drugs. The investigation equal to the 0.05 level. Based on the obtained results, the observed
was performed for 3 replicate of three samples (“Sorbifer Durules”, differences were not contrary to the hypothesis H0 and the obtained
“Fenules”, “Tardyferon”). The standard additions of analytes were discrepancies with the 0.05 significance level could be considered
placed into the samples of antianemic drugs before dissolving. It is insignificant.
shown in Table 2 that recovery values for both analytes are between
98 and 102%. 4. Conclusion

3.5.4. Accuracy In comparison with the previously reported flow approaches

The available solid dosage form of antianemic drugs was analyzed by (Kukoc-Modun et al., 2012; Lenghor et al., 2002; Molina-Diaz et al.,
proposed approach and by reference methods: AAS for iron determina- 1998; Oliveira & Masini, 2001; Tesfaldet et al., 2004; Udnana et al.,
tion and CE for ascorbic acid determination. It should be pointed out 2004) the flow system based on sandwich technique has been proposed
that in the presence of ascorbic acid iron exists in the form of Fe (II). for the simultaneous separate determination of iron (II) and ascorbic
The accuracy of the approach was evaluated by the comparison of the acid in antianemic pharmaceuticals for the first time. Taking into ac-
results receiving by both approaches. The obtained results represent count high content of analytes (13–20%) in the pharmaceuticals the
no significant differences in iron (II) and ascorbic acid concentrations LOD values of the developed approach are quite satisfactory. The sample
obtained by suggested and reference methods (Table 2). Student's frequency of the developed approach is comparable with the previously
t-test of statistical hypotheses about the equality of the means was reported flow approaches, because during the one flow cycle it is

Table 2
The results of the iron (II) and ascorbic acid determination in antianemic pharmaceuticals (F-critical = 19.0, t-critical = 4.3, P = 0.95, n = 3).

Drug Labeled value, Added The developed ААS, mg/tablets F-value t-value Recovery, The developed CE, mg/tablets F-value t-value Recovery,
mg/tablets amount, method, % method, %
mg/tablets mg/tablets mg/tablets

Fe (II) AA Iron (II) Ascorbic acid

Sorbifer Durules 100 60 0 95 ± 5 96 ± 8 2.5 0.5 – 58 ± 4 57 ± 2 7.0 1.1 –

2 96 ± 5 97 ± 7 1.1 0.3 99 61 ± 5 60 ± 6 1.6 0.7 101
5 101 ± 5 100 ± 8 2.3 0.5 101 63 ± 5 62 ± 5 1.7 0.5 101
Fenules 55 50 0 58 ± 3 60 ± 5 4.3 1.8 – 66 ± 5 67 ± 3 5.2 0.5 –
2 61 ± 4 62 ± 6 2.7 0.6 98 69 ± 4 70 ± 4 1.0 0.8 99
5 64 ± 5 65 ± 6 1.3 0.8 99 72 ± 5 73 ± 4 4.0 0.8 99
Tardyferon 80 – 0 69 ± 4 70 ± 5 1.9 0.7 – 86 ± 4 87 ± 3 1.8 0.3 –
2 72 ± 4 71 ± 4 0.7 0.5 101 88 ± 4 90 ± 5 1.3 1.3 102
5 74 ± 5 75 ± 6 1.6 0.4 99 91 ± 4 93 ± 5 1.7 1.1 98
62 C. Vakh et al. / Journal of Pharmacological and Toxicological Methods 73 (2015) 56–62

possible to analyzed two analytes. The proposed procedure is of great Kukoc-Modun, L., Biocic, M., & Radic, N. (2012). Indirect method for spectrophotometric
determination of ascorbic acid in pharmaceutical preparations with 2,4,6-tripyridyl-
value in quality-control analysis of the developed pharmaceuticals, s-triazine by flow-injection analysis. Talanta, 96, 174–179.
because it improves automation, miniaturization, simplicity, sensitivity, Lenghor, N., Jakmunee, J., Vilen, M., Sara, R., Christian, G. D., & Grudpan, K. (2002). Sequen-
flexibility and low cost. tial injection redox or acid-base titration for determination of ascorbic acid or acetic
acid. Talanta, 58, 1139–1144.
Lin Ling, B., Baeyens, W. R. G., Van Acker, P., & Dewaele, C. (1992). Determination of
Acknowledgments ascorbic acid and isoascorbic acid by capillary zone electrophoresis: application to
fruit juices and to a pharmaceutical formulation. Journal of Pharmaceutical and
Biomedical Analysis, 10, 717–721.
This work was supported by the Russian Foundation for Basic Mahmoud, W. H. (2001). Iron ion-selective electrodes for direct potentiometry and
Research (project No. 13-03-00031-a). Scientific researches were potentiotitrimetry in pharmaceuticals. Analytica Chimica Acta, 436, 199–206.
performed at the Center for chemical analysis and materials research Mary Nancy, T. E., Anithakumary, V., & Kumara Swamy, B. E. (2014). Solar graphene
modified glassy carbon electrode for the voltammetric resolution and detection of
of St. Petersburg State University. dopamine, ascorbic acid and uric acid. Journal of Electroanalytical Chemistry,
720–721, 107–114.
Merli, D., Profumo, A., & Dossi, C. (2012). An analytical method for Fe (II) and Fe (III)
References
determination in pharmaceutical grade iron sucrose complex and sodium ferric
gluconate complex. Journal of Pharmaceutical Analysis, 2, 450–453.
Alonso, J., Bartrolí, J., del Valle, M., & Barber, R. (1989). Sandwich techniques in flow-
Mervartova, K., Polasek, M., & Martınez Calatayud, J. (2007). Recent applications of flow-
injection analysis: Part 2. Simultaneous determination of Iron (II) and total Iron.
injection and sequential-injection analysis techniques to chemiluminescence deter-
Analytica Chimica Acta, 219, 345–350.
mination of pharmaceuticals. Journal of Pharmaceutical and Biomedical Analysis, 45,
Alonso, J., Bartroli, J., Delvalle, M., Escalada, M., & Barber, R. (1987). Sandwich techniques
367–381.
in flow injection analysis. Part 1. Continuous recalibration technique for process con-
Molina-Diaz, A., Ortega-Carmona, I., & Pascual-Reguera, M. I. (1998). Indirect spectropho-
trol. Analytica Chimica Acta, 199, 191–196.
tometric determination of ascorbic acid with ferrozine by flow-injection analysis.
Alonso-Chamarro, J., Bartroli, J., & Barber, R. (1992). Sandwich techniques in flow injec-
Talanta, 47, 531–536.
tion analysis. Part3. Simultaneous determination of Cr (VI) in two concentration
Mortatti, J., Krug, F. J., Pessenda, L. C. R., & Zagatto, E. A. G. (1982). Determination of iron in
ranges. Analytica Chimica Acta, 261, 219–223.
natural waters and plant material with 1,10-phenanthroline by flow injection.
Araujo, A. N., Lima, J. L. F. C., Alonso-Chamarro, J., Bartroli, J., & Poch, M. (1991). Flow in-
Analyst, 107, 659–663.
jection system based on the sandwich technique for saving expensive reagents.
Oliveira, P. C. C., & Masini, J. C. (2001). Sequential injection of iron (II) in antianemic phar-
Clinica Chimica Acta, 203, 67–76.
maceutical formulations with spectrofotometric detection. Analytical Letters, 34,
Beard, J. L. (2001). Iron biology in immune function, muscle metabolism and neuronal
389–397.
functioning. The Journal of Nutrition, 131, 568–579.
Pengfei, J., Lufeng, X., Zheng, L., Ning, Ch., Ding, Z., & Xin, H. (2012). Rapid determination
Beutler, E., Hoffbrand, A. V., & Cook, J. D. (2003). Iron deficiency and overload, hematology,
of thiamine, riboflavin, niacinamide, pantothenic acid, pyridoxine, folic acid and
The Educational Prog. American Society of Hematology, 40–61.
ascorbic acid in vitamins with minerals tablets by high-performance liquid chroma-
Cerda, A., & Cerda, V. (2009). An introduction to flow analysis, SCIWARE, Palma de Mallorca,
tography with diode array detector. Journal of Pharmaceutical and Biomedical
66–67.
Analysis, 70, 151–157.
Clark, S. F. (2008). Iron deficiency anemia. Nutrition in Clinical Practice, 23, 128–141.
Pifferi, G., & Restani, P. (2003). The safety of pharmaceutical excipients II. Farmaco, 58,
Crichton, R. R., Wilmet, S., Legssyer, R., & Ward, F. R. (2001). Molecular and cellular mech-
541–550.
anisms of iron homeostasis and toxicity in mammalian cells. Journal of Inorganic
Pimenta, A. M., Montenegro, M. C. B. S. M., Araujo, A. N., & Martınez Calatayud, J. (2006).
Biochemistry, 91, 9–18.
Review. Application of sequential injection analysis to pharmaceutical analysis.
David, C. L., & Webb, L. M. (2003). Pharmaceutical analysis. Blackwell Publishing Ltd.
Journal of Pharmaceutical and Biomedical Analysis, 40, 16–34.
Davies, S. H. R., & Masten, S. J. (1991). Spectrophotometric method for ascorbic acid using
Ruzicka, J., & Hansen, E. H. (1975). Flow injection analyses: Part I. A new concept of fast
dichlorophenolindophenol: elimination of the interference due to iron. Analytica
continuous flow analysis. Analytica Chimica Acta, 78, 145–157.
Chimica Acta, 248, 225-221.
Ruzicka, J., & Marshall, G. D. (1990). Sequential injection: A new concept for chemical
Falkova, M. T., Pushina, M. O., Bulatov, A. V., Alekseeva, G. M., & Moskvin, L. N. (2014).
sensors, process analysis and laboratory assays. Analytica Chimica Acta, 237, 329–343.
Stepwise injection spectrophotometric determination of flavonoids in medicinal
Schumann, K., Ettle, T., Szegner, B., Elsenhans, B., & Sololmons, N. (2007). On risks and
plants. Analytical Letters, 47, 970–982.
benefits of iron supplementation recommendations for iron intake revisited. Journal
Fotsing, L., Fillet, M., Bechet, I., Hubert, Ph., & Crommen, J. (1997). Determination of six
of Trace Elements in Medicine and Biology, 21, 147–168.
water-soluble vitamins in a pharmaceutical formulation by capillary electrophoresis.
Tesfaldet, Z. O., van Staden, J. F., & Stefan, R. I. (2004). Sequential injection spectrophoto-
Journal of Pharmaceutical and Biomedical Analysis, 15, 1113–1123.
metric determination of iron as Fe (II) in multi-vitamin preparations using 1,10-
Gioia, M. G., Andreatta, P., Boschetti, S., & Gatti, R. (2008). Development and validation of a
phenanthroline as complexing agent. Talanta, 64, 1189–1195.
liquid chromatographic method for the determination of ascorbic acid, dehydroascorbic
Thangamuthu, R., Senthil Kumar, S. M., & Chandrasekara Pillai, K. (2007). Direct ampero-
acid and acetaminophen in pharmaceuticals. Journal of Pharmaceutical and Biomedical
metric determination of L-ascorbic acid (Vitamin C) at octacyanomolybdate-doped-
Analysis, 48, 331–339.
poly(4-vinylpyridine) modified electrode in fruit juice and pharmaceuticals. Sensors
Guclu, K., Sozgen, K., Tutem, E., Ozyurek, M., & Apak, R. (2005). Spectrophotometric deter-
and Actuators, B, 120, 745–753.
mination of ascorbic acid using copper (II) — neocuproine reagent in beverages and
Tzanavaras, P. D., & Themelis, D. G. (2007). Review of recent applications of flow injection
pharmaceuticals. Talanta, 65, 1226–1232.
spectrophotometry to pharmaceutical analysis. Analytica Chimica Acta, 588, 1–9.
Halliberg, L. (2001). Perspectives on nutritional iron deficiency. Annual Review of
Udnana, Y., Jakmunee, J., Jayasavati, S., Christian, G. D., Synovec, R. E., & Grudpan, K.
Nutrition, 21, 1–21.
(2004). Cost-effective flow injection spectrophotometric assay of iron content in
Horstkotte, B., & Cerdà, V. (2009). Coupling of flow techniques with capillary electropho-
pharmaceutical preparations using salicylate reagent. Talanta, 64, 1237–1240.
resis: review of operation principles, challenges, potentials, and applications. Journal
World Health Organization (2007). Quality assurance of pharmaceuticals: A compendium of
of Chromatographic Science, 47, 636–647.
guidelines and related materials, 2-nd edition, V 2, good manufacturing practices and in-
ISO 5725-1 (1994). Accuracy (trueness and precision) of measurement methods and
spection. India: WHO Press.
results — Part 1: General principles and definitions.
Zachariadis, G. A., & Michos, C. E. (2007). Development of a slurry introduction method
IUPAC (1997). Compendium of chemical terminology The “Gold Book” (2nd ed.).
for multi-element analysis of antibiotics by inductively coupled plasma atomic emis-
Jackson, K., Young, D., & Pant, S. (2000). Drug–excipient interactions and their affect on
sion spectrometry using various types of spray chamber and nebulizer configura-
absorption. Pharmaceutical Science & Technology, 3, 336–345.
tions. Journal of Pharmaceutical and Biomedical Analysis, 43, 951–958.
Karpinska, J., & Kulikowska, M. (2002). Simultaneous determination of zinc (II), manga-
Zachariadis, G. A., Raidou, E. S., Themelis, D. G., & Stratis, J. A. (2002). Determination of
nese (II) and iron (II) in pharmaceutical preparations. Journal of Pharmaceutical and
mineral content of active dry yeast used in pharmaceutical formulations. Journal of
Biomedical Analysis, 29, 153–158.
Pharmaceutical and Biomedical Analysis, 28, 463–473.
Khuhawar, M. Y., & Lanjwani, S. N. (1998). Liquid chromatographic determination of
Zargba, S., & Hopkata, H. (1996). Spectrophotometric determination of Fe (II) in pharma-
cobalt (II), copper (II) and iron (II) using 2-thiophenaldehyde-4-phenyl-3-
ceutical multivitamin preparations by azo dye derivatives of pyrocatechol. Journal of
thiosemicarbazone as derivatizing reagent. Talanta, 46, 485–490.
Pharmaceutical and Biomedical Analysis, 14, 1351–1354.
Killip, S., Bennett, J. M., & Chambers, M. D. (2007). Iron deficiency anemia. American
Family Physician, 75, 671–678.