Highly sensitive stripping voltammetric determination of a biomolecule, pyruvic acid in solubilized system and biological fluids

3.1.1 Cyclic voltammetric behavior

Pyruvic acid exhibited single well defined cathodic peak in the potential range −1.0 to −1.8 V, at all concentrations. No peak could be observed in the anodic direction of the reverse scans, suggesting the irreversible nature of the electrode process (Zeng and Huang, 2004). The peak potential shifted toward more negative values of applied potential with increasing scan rate, also confirming the irreversible nature of the reduction process. For a totally irreversible electrode process, the relationship between the peak potential (E_p) and scan rate (υ) is expressed as Brahman et al., 2012; Levent et al., 2009: $${\text{E}}_{\text{p}}=(2.303\text{RT}{\alpha }_{\text{n}}\text{F})\log ({\text{RTK}}_{\text{f}}/{\alpha }_{\text{n}}\text{F})-(2.303\text{RT}{\alpha }_{\text{n}}\text{F})\log \upsilon \text{.}$$ A straight line was observed when E_p was plotted against log υ at a particular concentration at pH 8.2 ± 0.01 and can be expressed as $$\text{y}({\text{E}}_{\text{p}})=0.036(\log \upsilon )+1.5596\text{,}{\text{r}}^2=0.99$$ From the slope of the straight line (ΔE/Δ log υ), the α_n value was calculated by the expression ΔE/Δ log υ = 30/α_n. The α_n value was found to be 0.20 and was taken for further calculation for the number of electron transferred. Fractional α_n values further confirmed the irreversible reduction of pyruvic acid.

The effect of scan rate (υ^1/2) on stripping peak current (i_p) was examined under the above experimental conditions (Fig. 1A). As the sweep rate was increased from 10 to 130 mV s⁻¹ at a fixed concentration of pyruvic acid, (i) the peak potential shifted cathodically, (ii) the peak current increased steadily, and (iii) the peak current function, i_p/ACυ^1/2, exhibited near-constancy.

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Figure 1

(A) The CVs of 0.004 mM pyruvic acid in the ammonia buffer of pH 8.2 ± 0.01 at HMDE, current range 10 μA. scan rates (mVs⁻¹) (1) 10, (2) 20, (3) 30, (4) 40, (5) 50, (6) 60, (7) 70, (8) 80, (9) 90, (10) 100 (11) 110, (12) 120 and 130. (B) Relationship between peak current (i_p) versus root of scan rate (υ^1/2).

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A straight line is obtained when i_p is plotted against υ^1/2 (Fig. 1B), which may be expressed by the equation $$\text{y}({\text{i}}_{\text{p}})=14.65{\upsilon }^{1/2}({\text{mV s}}^{-1})-36.31\text{,}{\text{r}}^2=0.974$$ All these facts pointed toward the diffusion-controlled nature of the electrode process. The plot of log i_p of the peak current in ammonia buffer of pH 8.2 ± 0.01, versus log υ (Fig. 2) was straight line with slope of 0.755, which is less than the theoretical value of 1.0 that is expected for an ideal reaction of surface species (Levent et al., 2009). For finding the adsorptive character of the drug at HMDE a cyclic voltammogram was recorded after 30 s preconcentration at −0.5 V and second cyclic voltammogram was also recorded at same mercury drop. Furthermore, a substantial decrease in the peak current value in subsequent scans was observed, which reached a steady state, indicating that pyruvic acid also shows adsorptive characteristics at mercury electrode. The lower experimental slope (0.755) than the theoretical one may be attributed to the partial involvement of the diffusive drug molecules in the electrode reaction of the adsorbed ones. The overall electrode process is mainly diffusion-controlled with adsorption of the pyruvic acid at the electrode surface.

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Figure 2

Relationship between log of peak current (log i_p) versus log of scan rate (υ).

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3.1.2 Differential pulse voltammetric behavior

In differential pulse voltammetry, pyruvic acid shows a single well defined, irreversible, diffusion controlled reduction peak in ammonia buffer solution of pH 8.2 ± 0.01 (Fig. 3), which is attributed to the reduction of the unsaturated C⚌O bond. When a solution of pyruvic acid (0.004 mM) in 0.1 M ammonia buffer of pH 8.2 ± 0.01, was electrolyzed using HDME as working electrode, it produced well defined reduction peak at −1.35 V.

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Figure 3

Differential pulse adsorptive stripping voltammogram of pyruvic acid (0.004 mM) in 0.1 M ammonia buffer and 4 mM CTAB (pH 8.2 ± 0.01) at HMDE, pulse amplitude −50 mV, scan rate 30 mVs⁻¹, current range 10 μA, accumulation time 30 s.

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3.1.3 Effect of CTAB concentration

Squarewave voltammetry shows, cathodic peak current increases steadily in the beginning with an increase in concentration of CTAB and reaches a maximum at 4 mM CTAB and after that decreases continuously (Fig. 4A). It may be interpreted that at 4 mM CTAB the adsorption behavior changes from monomer adsorption to monolayer adsorption with an increase in concentration of CTAB at the electrode surface. However peak current decreases with a further increase in CTAB concentration, it may be due to the inhibition of electron transfer by aggregates of micelles. Another reason for decrease in peak current is the increase of hydrophobicity of CTAB micelles that might decrease the electron transfer rate constant (Jain and Rather, 2011; Brahman et al., 2012; Tan et al., 1997) and result in the decrease of peak current at high CTAB concentration. Therefore, 4 mM concentration of CTAB was chosen as optimum one.

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Figure 4

(A) Plot of square wave adsorptive peak current vs. concentration of CTAB in ammonia buffer, pH 8.2 ± 0.01. (B) Influence of pH on the peak current response (using DPAdSV and SWAdSV) for 0.004 mM pyruvic acid in ammonia buffer (pH 7.5–11) and 4 mM CTAB after 30 s pre-concentration time; frequency (f) = 20 Hz, and pulse amplitude = 50 mV at Eacc = − 0.5 V.

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3.2.1 Effect of accumulation time and potential

Variation of accumulation time for 0.004 mM pyruvic acid solution was studied over 0–60 s. A remarkable enhancement for the SWAdSV peak current up to 30 s accumulation time was observed and then it became virtually curved due to the saturation of the surface of the working electrode. For further SWAdSV quantitative studies for pyruvic acid, an accumulation time of 30 s was selected as optimal value since it provided relatively high peak current with adequate practical time. In addition, when the influence of pre-concentration potential over the range of −1.2 V to +0.2 V at 30 s accumulation time, the peak current increased steadily over the positive direction till it reached its maximum value at −0.5 V where it decreased sharply thereafter. Hence, for optimal analytical sensitivity, this experimental parameter was maintained at −0.5 V.

3.2.2 Influence of frequency

The SWAdSV peak current is linearly dependent on the frequency 20–90 Hz, while a well defined peak was observed at 20 Hz. A linear relationship was observed between the stripping peak current and frequency. Although the response of pyruvic acid is increased with frequency, above 70 Hz the peak current was obscured by a large residual current. Thus the best peak was recorded using 20 Hz frequency.

3.2.3 Influence of scan increment and pulse amplitude

The effect of scan increment on adsorptive cathodic peak current of the pyruvic acid in ammonia buffer at pH 8.2 ± 0.01 revealed that the peak current increases upon the increase of scan increment (1–20 mV). The best peak resolution was recorded using 5 mV scan increment. Thus scan increment of 5 mV was used in the present study. At pulse amplitude of 50 mV, the peak current was found to be much more sharp and defined. Several instrumental parameters, those directly affect voltammetric response, were also optimized for e.g., mercury drop size, stirring rate and the rest period. The stripping was not significantly affected when varying the rest period, since it was observed that 10 s was sufficient for the formation of a uniform concentration of the reactant onto the mercury drop.

3.2.4 Effect of pH

The shape and characteristics of all voltammograms were strongly dependent on various electrolyte and pH of the medium. Britton–Robinson, acetate, borate, citrate, phosphate, Tris and ammonia buffer were used in the present study and the best results were obtained in ammonia buffer (0.1 M). Stripping peak potential shifted toward more negative potential with an increase in pH, indicating involvement of hydrogen ions in the electrode process. Variation of stripping peak potential of pyruvic acid as a function of pH (7.5–11) employing different voltammetric modes can be expressed by the following equations: $$\text{DPAdSV;pH}7.5-11.0:{\text{E}}_{\text{p}}(\text{V})=-0.0231-1.124\text{pH}:{\text{r}}^2=0.9829$$ $$\text{SWAdSV;pH}7.5-11.0:{\text{E}}_{\text{p}}(\text{V})=-0.021-1.102\text{pH}:{\text{r}}^2=0.9912$$ Studies for the dependence of stripping peak on pH for DPAdSV and SWAdSV (Fig. 4B) were carried out to determine whether the electro-active species participate in equilibria involving protons directly and to obtain the pH range for maximum signal. Sharp response and better peak shape with maximum current were observed at pH 8.2 ± 0.01. So this pH value was chosen as the working pH for further studies.

3.6.1 Linearity

In order to determine the effect of concentration of pyruvic acid on stripping peak current, voltammograms of pyruvic acid were recorded at HMDE. Under the optimum conditions, a very good linear correlation was obtained between the monitored voltammetric peak current and pyruvic acid concentration in the range of 0.004–0.036 mM. Least-square treatment of the calibration graph yielded the following regression equations: $$\text{SWAdSV}:{\text{i}}_{\text{p}}(\mu \text{A})=-29.83\text{x}+0.010{\text{r}}^2=0.995$$ $$\text{DPAdSV}:{\text{i}}_{\text{p}}(\mu \text{A})=12.4\text{x}+0.014{\text{r}}^2=0.999$$ where i_p is the peak current, x is the analyzed drug concentration and r² is the correlation coefficient.

3.6.2 Detection and quantification limits

Detection limit is calculated by equation LOD = 3s/m, where s is standard deviation of intercept and m is slope of the regression line. The calculated LOD value of pyruvic acid is 6.12 × 10⁻⁶ and 1.12 × 10⁻⁷ mM for DPAdSV and SWAdSV, respectively. The quantification limit (LOQ) is examined by the equation LOQ = 10s/m. The calculated LOQ value is 2.0 × 10⁻⁵ and 4.4 × 10⁻⁶ mM for DPAdSV and SWAdSV, respectively. Both LOD and LOQ values confirmed the sensitivity of the proposed methods.

3.6.3 Accuracy, precision and stability

The accuracy of the proposed method was checked by calculating the recovery of known amount of pyruvic acid (0.004 mM) added to ammonia buffer solution and analyzed via the optimized stripping voltammetric procedure. The value of the mean recovery obtained by the standard addition method was 100.9% with standard deviation of 1.2% (the analytical measurements repeated five times). The analytical precision of the developed method was verified from the reproducibility of 10 determinations of 0.004 mM pyruvic acid and the estimated relative standard deviation (RSD%) was 1.07%. Finally, when the SWAdSV signal of 0.004 mM pyruvic acid solution was monitored every fifteen minutes, it was found to be nearly stable for a period of at least 2 h.

3.6.4 Robustness

The robustness was examined by evaluating the influence of small variations of some of the most important procedure variables, including preconcentration potential, preconcentration time, and pH. The obtained results provided an indication of the reliability of the proposed procedure for the assay of pyruvic acid; hence, it can be considered as robust. The obtained mean percentage recoveries based on an average of five replicate measurements were not significantly affected within the studied range of variations of some operational parameters, and consequently the proposed procedure can be considered as robust.

3.6.5 Ruggedness

The ruggedness of the measurements is defined as the degree of reproducibility of results obtained by analysis of same sample under a variety of normal test conditions such as different laboratories and different lot of reagents, under the same operational conditions at different elapsed time by two different analysts. The methods were found to be rugged with the results of variation coefficients 0.85% and 0.91% for SWAdSV, 1.3% and 0.94% for DPAdSV methods for first and second analysts, respectively. The results show no statistical differences between different analysts.

3.6.6 Specificity/selectivity

Specificity is the ability of the method to measure the analyte response in the presence of all of the potential impurities. The selectivity of the optimized procedure for determination of pyruvic acid was examined in the presence of foreign species such as Ca²⁺, Mg²⁺, Zn²⁺, Al³⁺, Cu²⁺, glucose, valine, phenylalanine and lycine. Samples containing 0.004 mM bulk pyruvic acid and different concentrations of the excipient under evaluation were analyzed by means of the proposed method. The obtained mean percentage recoveries and RSD% values based on an average of five replicate measurements, 99.90 ± 0.54–100.10 ± 1.20 for SWAdSV and 99.21 ± 1.7–100.06 ± 0.90 for DPAdSV, showed no significant interference from excipients. Thus, the proposed procedure can be considered to be specific.

3.6.7 Comparison of the sensitivity of the proposed method with previously reported methods

Table 3 compares the detection limit of the proposed method with the other reported methods. It is obvious that the sensitivity of the proposed method is superior to all previously reported methods. The data in the table reveal that the detection limit of the method is lower than all previously reported methods.