Determination of vitamin B6 (pyridoxine hydrochloride) based on a novel BZ oscillating reaction system catalyzed by a macrocyclic complex

1. Introduction Metabolism, a set of life-sustaining chemical reactions, involves enzymes or co-enzymes which catalyze reactions allowing organisms to grow and reproduce [1]. Vitamin B6, which serves as an enzyme or co-enzyme, plays an important role in amino acid metabolism, gluconeogenesis and liquid metabolism. Vitamin B6 has many medical applications such as treatment of vomiting during pregnancy and radiation sickness, and it can also help people preventing and recovering from anemia [2]. Thereby, a rapid and convenient determination method is required.

An oscillatory chemical system, which is maintained far-from equilibrium by keeping it open, has already been used as a tool to analyze organic compounds and inorganic ions. Among all oscillating systems, the Belousov-Zhabotinskii (BZ) reaction, the catalytic oxidation of organic compounds by the acidic bromate [3,4], is the most widely studied for analyte determination purposes and is modeled by the FKN mechanism [5]. The analyte pulse perturbation technique (APP) [6], which is based on a pulse perturbation on the chemical system (mainly the BZ system), has become a subject in modern quantitatively analytic methods since it was first proposed by Tichonova et al. in 1978 [7]. There

Central European Journal of Chemistry

Determination of vitamin B6 (pyridoxine hydrochloride) based on a novel BZ oscillating reaction system catalyzed by a macrocyclic complex

* E-mail: [email protected]

Received 3 July 2013; Accepted 28 October 2013

Abstract:

© Versita Sp. z o.o.Keywords: Determination • Oscillating reaction • Macrocyclic complex • Vitamin B6

1Department of Chemistry, Anhui University, Hefei 230601, P. R. China 2Institute of Applied Chemistry, East China Jiaotong University, Nanchang 330013, P. R. China

Qingling Zeng1, Lulu Chen1, Xianyi Song1, Gang Hu1*, Lin Hu2

Research Article

This paper reports a new method for determination of VB6 (pyridoxine hydrochloride) by its perturbation effects on a novel Belousov-Zhabotinskii (BZ) oscillating system. This novel BZ system, in which malic acid serves as the substrate, contains an enzyme-like complex, macrocyclic complex {[CuL](ClO4)2}, as catalyst. The ligand L in the complex is 5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-4,11-diene. Results show that the addition of pyridoxine hydrochloride can perturb the oscillation amplitude and period, and the change of the oscillation amplitude is linearly proportional to the concentration of pyridoxine hydrochloride in the range of 5×10-7- 2.5×10-4 M. The obtained RSD with seven samples is 3.073%. An assay of pharmaceutical tablets of vitamin B6 was evaluated. Some foreign ions were studied with respect to their possible influence on the determination of pyridoxine hydrochloride. The factors which influence this reaction include the concentration of reactant, the temperature of the reaction, property of catalyst, etc. Furthermore, the possible reaction mechanism has been proposed using the Field-Körös-Noyes (FKN) model.

Cent. Eur. J. Chem. • 12(3) • 2014 • 325-331DOI: 10.2478/s11532-013-0383-4

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Determination of vitamin B6 (pyridoxine hydrochloride) based on a novel BZ oscillating reaction system

catalyzed by a macrocyclic complex

are many reports using this perturbation technique to determine the quantity of tracing amounts of some metal ions and species etc. [8-10], while the majority of the catalysts being used in such BZ systems are Ce3+, Mn2+, Fe(phen)3

2+, or Ru(bipy)32+ [11-14].

Compared to these catalysts, macrocyclic complexes could be regarded as novel catalysts in the BZ system because of the presence of the extended p-system in the macrocyclic ligands, which ensure a high rate for reactions involving electron transfer at individual steps of the oscillating process [15]. This character favors their application as catalysts in B-Z system for determination of analytes [16-21], because such macrocyclic complexes-catalyzed BZ systems have lower activation energies, higher oscillating frequencies [20] and are vulnerable to external perturbations. From the viewpoint of analytical determination, the stronger the response is, the higher the sensitivity is.

During the oscillation process, the macrocyclic copper(III) complex [CuL]3+ gets an electron to form macrocyclic copper (II) complex [CuL]2+ and back to this state in a cycle. The reduced macrocyclic copper(II) complex [CuL]2+ is red in color and the oxidized macrocyclic copper(III) complex [CuL]3+ is orange. During the oscillation, the system undergoes periodic changes from red to orange then back to red due to the changes in [CuL]2+ and [CuL]3+, and hence it can be recorded by an electrochemical instrument grounded on the potential changes over time. By using macrocyclic copper(II) complex-catalyzed BZ system, we have already measured catechol [16], alizarin red S [17], pyrogallol [18], calcium pantothenate [19], Ag+ [20], and paracetamol [21].

Apart from serving as the essential component of two enzymes for amino acids metabolism, vitamin B6 also serves as a co-enzyme for many reactions and can help facilitate decarboxylation, transamination, racemization, elimination, replacement and beta-group interconversion reactions [22]. So it is meaningful to develop an efficient determination method to study its physiological function and diagnosis in some diseases in clinical medicine. Some methods for the determination of pyridoxine hydrochloride have been reported [23-25], and these methods need complex instruments like Continuous stirred-tank reactor (CSTR) or their limits

of detection are not at μM level but at mM level. Here we used macrocyclic copper (II) complex-catalyzed BZ system, as a tool for determination of pyridoxine

hydrochloride. Our quantitative analytical method, with a simple instrument, provides a new method for precise determination of pyridoxine hydrochloride, which can be detected in the range of 5×10- 7 M to 2.5×10- 4 M.

2. Experimental procedure

2.1. Reagents The catalyst [CuL] (ClO4)2 (Scheme 1) was synthesized according to literature [26] and was identified by its IR spectrum and elemental analysis. The ligand L in the catalyst [CuL] (ClO4)2 is 5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-4,11-diene. Pyridoxine hydrochloride was obtained from Aladdin Chemistry Co.Ltd; pharmaceutical vitamin B6 tablets (containing pyridoxine hydrochloride) were purchased from Nanjing Baijingyu Pharmaceutical Co., Ltd. and NaBrO3, malic acid and sulfuric acid were obtained commercially. All reagents were of analytical quality without further purification. Solutions of 0.6 M NaBrO3, 2 M malic acid, 0.0221 M [CuL] (ClO4)2 were separately prepared in 1 M sulfuric acid. Solutions of 0.1 M pyridoxine hydrochloride and 0.1 g per 5 mL (0.09725 M) pharmaceutical Vitamin B6 tablets were made immediately before the experiment. Solutions with lower concentrations were prepared prior to use. Double distilled water was used in all cases.

2.2. ApparatusOscillating experiments were carried out in a 50 mL vessel thermostated at 18 ± 0.5ºC with a Model 79-3 magnetic stirrer (Jiangsu, China) and stirring rate was kept at 500 rpm. A 213 type platinum electrode (Shanghai, China) monitored the temporal oscillations, using a Model 217 saturated calomel electrode (Shanghai, China) connected via a salt bridge containing 1 M Na2SO4 as the reference electrode. Potentials of the electrode as a function of time were recorded with a PHS-25B digital voltmeter (Shanghai, China) connected with a Model XWTD-204 Y-t recorder (Shanghai, China) to record kinetic curves of the reaction.

3. Results and discussion

3.1. Behavior of the completely unperturbed and perturbed oscillatorUnperturbed oscillator was prepared in the following order: 28.6 mL of 1.0 M H2SO4, 1.3 mL of 0.6 M NaBO3, 3.6 mL of 2.0 M malic acid and 6.5 mL of 0.0221 M [CuL]

2ClO4-

NH N

HNNCu2+

Scheme 1. The structure of [CuL] (ClO4)2.

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(ClO4)2 while perturbed BZ mixture had 0.6 mL of different concentration of pyridoxine hydrochloride added into the blank mixture using a pipette. Recordings of Potential (Pt) vs. time in the absence (Fig. 1a) and presence (Fig. 1b) of pyridoxine hydrochloride perturbations is shown in Fig. 1. During the oscillation, the periodic change of solution color (red–orange–red) was observed. This can be explained by a one-electronic transfer process:

[CuL] 2+ (red) ⇔ [CuL] 3+ (orange)

Several injection points were tested for the sake of the experiment accuracy and repeatability. When pyridoxine

hydrochloride was injected at the minimum of the cycle, the oscillation system went into a new oscillatory state with the shorter period and reduced amplitude. The changes in oscillation amplitude are proportional to the concentration of pyridoxine hydrochloride, so a new method could be expected to exploit this behavior for determining pyridoxine hydrochloride.

3.2. Influence of experimental variablesThe behavior of an oscillating chemical reaction is easily influenced by the experimental variables in the system. In order to obtain the optimum value for working conditions, the influences of the experimental variables on the proposed oscillating reaction were studied. According to Jiménez-Prieto et al. [27], the optimum value for working conditions were selected with three criteria: (a) maximizing the stability of the oscillating system over time, which enhances the reproducibility of the results; (b) maximizing the oscillation amplitude, which ensures maximal sensitivity for the determination of the analyte; (c) ensuring that the oscillation period allowed the effect of the analyte perturbation to be accurately determined. Accordingly, we selected changes in oscillation amplitude, ∆A, as one of the measured parameters. ΔA = |A – A0|, where A0 and A are the oscillation amplitude before and after the injection, respectively. Other parameters like oscillation period are also monitored.

The influence of changing the concentration of sulfuric acid (0.8-1.3 M) on the changes in oscillation amplitude of the system is shown in Fig. 2a. With increasing initial concentration of sulfuric acid, the changes in oscillation amplitude (∆A) decreased first and increased again at concentration of 0.95 M. A concentration of 1.0 M was chosen as the optimum point because the system has a good profile in both perturbed and unperturbed oscillation.

Changes in the catalyst [CuL] (ClO4)2 concentration over the range 0.001 M to 0.0045 M had a significant

effect on the behavior of the oscillation system. From Fig. 2b, it could be easily noted that with increasing [CuL] (ClO4)2 concentration, ∆A almost decreased till it reached a minimum at 0.00359 M. A concentration of 0.00359 M was chosen according to the above three criteria.

The effect of the sodium bromate was studied over the range 0.01 to 0.028 M. The change of the amplitude with increase in sodium bromate concentration is similar to the [CuL] (ClO4)2 and the effects are illustrated in Fig. 2c. As we can see, the changes in the oscillation amplitude (∆A) decreased to a minimum at the concentration of 0.0195 M and then increased with the further increase of sodium bromate concentration. A concentration of 0.0195 M was finally adopted as optimal as the system oscillated uniformly.

We have studied the effects of malic acid concentration from 0.15 to 0.21 M, and the results are shown in Fig. 2d. As the concentration increased, the curve of changes in oscillation amplitude (∆A) first decreased to a minimum and then reached a maximum slowly, after which it decreased again. A 0.18 M malic acid concentration was finally selected as optimal since it maximizes the stability of the oscillating system over time, which enhances the reproducibility of the results.

Figure 1. Typical oscillation profiles for the proposed oscillation system in the absence (a) and presence (b) of 2×10-5 M pyridoxine hydrochloride perturbation using a platinum electrode. Common conditions: [NaBrO3] =1.95×10-2 M, [H2SO4] =1.0 M, [malic acid] =0.18 M, [CuL] (ClO4)2 =3.595×10-3 M. T=18.3ºC. (a) [pyridoxine hydrochloride] = 0.000 M; (b) [pyridoxine hydrochloride] = 2×10-5 M.

327



The temperature also had a strong influence on the oscillating system, as it was reported in the literature by Körös et al. [28]. Temperature dependence and temperature compensation were reported recently [29]. High temperature was found to decrease the amplitude of the oscillation, the oscillation period and the life of the oscillation. Moreover, the mixture turned muddy and no oscillation happened at a higher temperature. This could be explained by each reaction in the chain having a different sensitivity to temperature. Increasing the temperature accelerates the reaction process and reduces the induction time. To obtain an exact and recurrent oscillating system, the temperature was maintained at 18°C.

The influences of foreign species and ions in the perturbed B-Z mixture were also investigated.

The oscillating system was perturbed with a sample, which contained pyridoxine hydrochloride and variable amounts of interferences. More than 10 foreign ions causing an error of less than 5% in the determination of 2×10–5 pyridoxine hydrochloride were studied (Table 1). It can be concluded that some species such as Ag+ and iodide has a strong effect on the determination of analyte, but large amounts of Cu2+ and most common ions have little effect on the determination.

3.3. Determination of pyridoxine hydrochlorideWe performed perturbation experiments under the optimal experimental conditions described above. The response to the pyridoxine hydrochloride perturbation was tested by employing changes in oscillation amplitude (ΔA) for the cycle following the sample injection as the measured parameter.

A fitted straight line was obtained by plotting changes in oscillation amplitude (Δ A) vs. concentration of pyridoxine hydrochloride, as it is shown in Fig. 3.

As can be seen from Figs. 1 and 3, the concentration of pyridoxine hydrochloride in the mixture has to be at μM level. (5×10- 7 M—2.5×10-4 M) The calibration data obtained obey the following linear regression equation:

ΔA = 0.96874 + 173423.18683 [VB6] (r = 0.99929, n = 9)

The precision (%RSD, relative standard deviation), calculated from seven perturbations of 2×10-5 M pyridoxine hydrochloride, was 3.073%. The detection

Figure 2. Influence of the concentrations of (a) Sulfuric acid; (b) [CuL] (ClO4)2; (c) Sodium bromate; (d) malic acid on the pyridoxine hydrochloride perturbed oscillation system. Common conditions: T = 18.3±0.5ºC. [VB6] = 2×10-5 M. (a) [NaBrO3] = 1.95×10-2 M, [malic acid] = 0.18 M, [CuL] (ClO4)2 = 3.595×10-3 M. (b) [NaBrO3] = 1.95×10-2 M, [H2SO4] = 1.0 M, [malic acid] = 0.18 M. (c) [H2SO4] = 1.0 M, [malic acid] = 0.18 M, [CuL] (ClO4)2 = 3.595×10-3 M. (d) [NaBrO3] = 1.95×10-2 M, [H2SO4] = 1.0 M, [CuL] (ClO4)2 = 3.595×10-3 M.

Table 1. Influence of Foreign Ions and Species on the Determination of 2×10-5 M Pyridoxine Hydrochloride.

Foreign ions and species Tolerated ratio

Al3+, NH4+, Li+, Na+ 2000:1

Ni2+, Zn2+, Cu2+ 1000:1

K+ 200:1

CO32- 100:1

glucose 10:1

Cl-, Mn2+, C2O42- 1:1

Ag+, Fe2+, Fe3+, I- 0.1:1

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limit obtained is 5×10-7 M. Such a precision is quite acceptable.

In order to assay the suitability of the proposed method, the same procedure with known amounts of the analytical solution of the pharmaceutical vitamin B6 tablets was applied. The recovery experiments indicate that the accuracy of the proposed method is in the range 93.5% to 108.8%. From above experimental results, it is clear that this method is suitable for practical sample analysis (Table 2).

3.4. Mechanism of action of pyridoxine

hydrochloride on the oscillating systemThe shape of the oscillation amplitude (ΔA) vs. concentration of pyridoxine hydrochloride is similar to that perturbed by calcium pantothenate [19]. This strongly suggests that the main features of the reaction

mechanism in the B-Z reaction perturbed by pyridoxine hydrochloride are the same as those perturbed by calcium pantothenate.

Based on the well known Field-Körös-Noyes (FKN) [5,30,31] mechanism, the overall reactions are simplified into the following nine processes:

HOBr + Br– + H+ Br2 + H2O (1)

HBrO2 + Br– + H+ 2HOBr (2)

BrO3–+ Br–+ 2H+ HOBr + HBrO2 (3)

2HBrO2 BrO3– + HOBr + H+ (4)

HBrO2 + BrO3– + H+ Br2O4 + H2O (5)

Br2O4 2BrO2· (6)

Br2 + HOOCCHOHCH2COOH → → Br–+ H+ + HOOCCHOHBrCHCOOH (7)

BrO2·+ [CuL] 2+ + H+ → [CuL] 3+ + HBrO2 (8)

HOOCCHOHBrCHCOOH + 6[CuL] 3+ + 3H2O → → 6[CuL] 2++ Br–+ 2HCOOH+ 2CO2 + 7H+ (9)

Pyridoxine hydrochloride could be oxidized to 4-pyridoxic acid, according to the literature [32,33]. So it is reasonable for us to believe in the occurrence of pyridoxine hydrochloride oxidation by oxidizing species in such a system, which can be expressed as follows:

(10)

The oxidizing species in the system are either initial reagents or intermediates that cannot be quantitatively measured. These are BrO3

–, HBrO2, HOBr, BrO2•, Br2O4,

or [CuL]3+ [30,31]. Pyridoxine hydrochloride could be oxidized by one, two or more of those oxidizing species. Standard redox potentials of bromine are shown in Scheme 2, according to the literature [5]. We assumed that HBrO2 is the most probable candidate that could participate in the oxidation of pyridoxine hydrochloride, for the potential of HBrO2/HOBr (1.74 V) is the highest among the above potentials.

When pyridoxine hydrochloride is injected into the oscillation system, the direct reaction of pyridoxine hydrochloride with HBrO2 causes a reduction of HBrO2 concentration according to Reaction 10. According to Reaction 5 Br2O4 concentration would decrease as a result of the decrease of HBrO2 concentration. As the Br2O4 concentration decreases, the BrO2· concentration

Figure 3. Plotting of changes in amplitude (∆A) vs. concentration of pyridoxine hydrochloride in the range 5×10-7 to 2.5×10-4 M. (Common conditions: [NaBrO3] = 1.95×10-2 M, [H2SO4] = 1.0 M, [malic acid] = 0.18 M, [CuL] (ClO4)2 = 3.595×10-3 M. T = 18.3oC).

Table 2. Determination Results and Recovery of Vitamin B6 Tablets in Water Sample.

Sample Determined/ 10–6mol dm–3

Found/10–6mol dm–3

Recovery (%)

1 5.97 6.5 108.8

2 9.72 9.23 94.9

3 9.95 10.2 102.5

4 14.58 15.26 104.6

5 19.45 18.2 93.5

Scheme 2. Standard redox potentials of bromine.

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will decrease accordingly due to Reaction 6. The decrease in BrO2·concentration causes the decrease in [CuL] 3+ concentration according to Reaction 8; as only little parts of the [CuL]2+ concentration could be oxidized into [CuL]3+, [CuL]2+concentration would remain unaffected. Therefore, the value of ln {[CuL]3+/[CuL]2+} decreases accordingly. As a result, the decrease in the maxim potential (corresponds to maxim concentration of [CuL]3+) was obtained. As the [CuL]2+concentration does not change, the minimum potential (corresponds to maxim concentration of [CuL]2+) would not change either. Therefore, a decrease in oscillation amplitude (from minimum potential to maxim potential) could be observed (Fig. 1b).

After the injection of pyridoxine hydrochloride, the shorter period length observed may be ascribed to an increased bromination reaction. According to Reaction 10, the concentration of HBrO2 (oxidant) would decrease because it is consumed when it reacts with pyridoxine hydrochloride. Decrease in HBrO2 would make the equilibrium of Reaction 3 shift to the right, and such a shift causes an increase in the concentration of HOBr. There will be an increase in Br2 concentration as a result of the increase in the concentration of HOBr, according to Reaction 1. Increase in Br2 concentration would cause an increased bromination reaction, according to Reaction 7. As a result, shorter period length could be observed (Fig. 1b).

4. ConclusionsThe results of our present paper illustrate that pyridoxine hydrochloride can be detected using the macrocyclic Cu(II) complex-catalyzed BZ reaction, composed of malic acid/ BrO3

- / [CuL](ClO4)2 / H+, as a tool. Moreover, larger linear range ( ca. 5×10-7 M—2.5×10-4 M) and lower detection limit (ca. 10-7 M) was found here. In order to obtain the optimum working conditions for quantitative analysis, the effects of concentrations of sodium bromate, sulfuric acid, malic acid and [CuL] (ClO4)2 were examined. A proper explanation was that pyridoxine hydrochloride was oxidized into 4-pyridoxic acid. Following from the experimental results presented here, such a perturbation technique based on BZ oscillating reaction is a suitable analytical method for measuring vitamin B6.

AcknowledgmentThe authors gratefully acknowledge funding of this work by the National Science Foundation of China (21171002).

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Determination of vitamin B6 (pyridoxine hydrochloride) based on a novel BZ oscillating reaction system catalyzed by a macrocyclic complex