Electrochemical Detection of Some Water Soluble and Fat Soluble Vitamins by Using Bare Pencil Graphite Electrode

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Electrochemical Detection of Some Water Soluble and Fat Soluble Vitamins by Using Bare Pencil Graphite Electrode

https://doi.org/10.21203/rs.3.rs-2080842/v1

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Vitamins are a group of organic compounds needed in small quantities by the body to perform its essential functions like growing, regulating metabolism and maintain a healty immune system. In recent years, substantial efforts have been focused on development of simplified, fast and cost-effective approaches for vitamin analysis. Aim of this study was electrochemical detection of some water and fat-soluble vitamins individualy and simultaneously by using bare pencil graphite electrode (PGE). Vitamins of C, B1, B6, B12, A, D3, and E were selected and electrochemically analyzed in pharmaceutical preparates. Simultaneous analysis of water-soluble vitamins by using DPV with PGE can be promising technique for the determination of vitamin C and B6 or vitamin B1, B12 and B6. In the case of fat-soluble vitamins, resolution between vitamin D3 and E was not satisfactory. However, vitamin A and D or vitamin A and E could be detected in a single run. Limit of detection of the selected vitamins were quite low (4-548 µg/ml).

Fat-soluble vitamins

water-soluble vitamins

vitamin analysis

electrochemical methods

differential pulse voltammetry

Pencil Graphite Electrode

There are thirteen vitamins important for human nutrition and these can be divided into two groups according to their solubility (Ball, 2006). Most of the vitamins are absolutely essential in human nutrition, because the tissues in the human body cannot synthesize them (Combs & McClung, 2017).

There are two notable exceptions that are vitamin D and niacin. The cutaneous synthesis of vitamin D depends on adequate exposure of the skin to sunlight; niacin synthesis depends on adequate intake of tryptophan, which is protein-bound amino acid precursor (Cagetti et al., 2020). While various diseases can occur when vitamins are taken below recommended daily intake dose (RDI) and toxic effects can be seen above these RDI amounts.

Best approach to adopt a balanced diet is that we get a variety of vitamins and minerals in the proper amount. This involves ultimate emphasis on fruits and vegetables, whole grains, beans and legumes, low-fat protein and dairy products. Unfortunately, this is far from being achievable every where, since it requires universal access to adequate food and appropriate dietary habits (Bakre et al., 2015; Carlucci et al., 2013), and also some losses of certain vitamins during food processing are inevitable. Another point to consider is that natural variations in the vitamin content of a raw food material may effect the content of vitamins in the final product more than the processing itself (Ball, 2006). From these stand points, food fortification can have advantage to deliver nutrients to large segments of the population without requiring radical changes in food consumption patterns.

Therefore, it is essential to depelope quick, easy, efficient and safe analytical methods to determine raw and final food product nutritional quality, hence to be able to use vitamins reasonably in our diets, and to combat micro nutrient deficiencies (Pardakhty et al., 2018).

Great progress in analytical chemistry has been achieved and some new analytical instruments have been developed since 2010 (Zhang et al., 2018; Chauhan et al., 2019). Considering few comprehensive reviews of pretreatment and determination of vitamins has been published systematically. Ultrasonic assisted extraction (UAE), supercritical fluid extraction (SFE), SPE, LLE, dispersive liquid-liquid microextraction (DLLME) and various analysis methods, including chromatographic methods, electrophoretic methods, microbiological assays, immunoassays, biosensors and others have been reported and used to analyze vitamins (Zhang et al., 2018).

Among many techniques, electrochemical methods using carbon paste electrode and pencil graphite electrode have drawn attention due to its easy operation, sensitivity and low cost (Parvin et al., 2018; Galeano et al., 2004).

David et al., (2015) studied voltammetric analysis of B1 and B6 vitamins using a pencil graphite electrode, because of the importance of vitamins for human health. In the presence of each other, they made it by quantitatively determining the two vitamins directly with differential pulse voltammetry. B1 was just electroactive inside a NaOH solution that produced two irreversible oxidation peaks; the first peak at 250 mV is finely described as well as used for quantitative determinations.

Sys et al., (2017) studied the amount of vitamin E in margarine and edible oils. Their system was consisting of a glassy carbon paste electrode as a processing electrode, Ag & AgCl with 1M KCL salt bridge as a reference electrode, as well as platinum wire as a counter electrode and they used square wave anodic stripping voltammetry (SWASV) on 0.2 M HNO3 for detection. The method used by the researchers is to use a lipophilic binder of the glassy carbon paste electrode in silicone oil extraction and biologically active compounds (Sys et al., 2016).

Falat & Cheng (1982) reported cyclic voltammetry results of L-(+) ascorbic acid on a graphite/epoxy working electrode at pH 7.4. Oni & Nyokong (2001) studied detection of vitamin B1 on modified carbon paste electrodes at pH = 10.

Lovander et al., (2018) reported that lipid soluble vitamins have formal potentials at potentials positive (A, D, E) and negative (A, K) out of the water window, but none have potentials within the water window.

Voltammetric studies have been undertaken for the two forms of vitamin D, vitamin D2 and D3. Characterization by cyclic voltammetry yields and EC_İ mechanism of an electron transfer followed by a fast, irreversible chemical step (Hart and Norman, 1992; Chan et al., 2014; Cincotto et al., 2014; Liu et al., 2001).

In many voltammetric studies of fat soluble vitamins were carried of by using organic solvents such as dichloromethane, acetonitrile, benzene, methanol in acetate buffer (Webster, 2007; Webster, 2012; Mariovski et al., 1975).

Aim of this study was to investigate possibilities of analyzing some water-soluble and fat-soluble vitamins either as single vitamin or in a mixture by using electrochemical methods with bare pencil graphite electrode. For this purpose, vitamin C, B1, B6, B12, A, D3, and E were selected and were electrochemically analyzed in pharmaceutical preparates in acetate buffer (ACB) or phosphate buffer (PBS) depending on solubility of vitamins.

2.1. Equipments

Cyclic voltammetric and differential pulse voltammetric measurements were performed using AUTOLAB PGSTAT 204 (Utrecht, The Netherlands) potentiostat with NOVA 2.1.2 software. Reference electrode (RE) was an Ag/AgCl with 3M KCl, counter electrode (CE) was platinum wire and working electrode (WE) was pencil graphite lead. 0.5mm Pentel P205 model pencil and Tombow HB 0.5mm pencil leads obtained from book store and used as pencil graphite electrode (PGE). Pencil leads are composed of ∼65% graphite, ∼30% clay, and a binder (wax, resins, or high polymer).

2.2. Reagents

In this study, 7 very common vitamins were used namely vitamin A (standard of 30.000 I.U. per capsule by KOÇAK FARMA), D3 (standard of 300.000 I.U. per capsule by KOÇAK FARMA), E (standard of 200 I.U. per capsule by KOÇAK FARMA), B₁ + B₆ (standard of 33.333 I.U. per capsule by KOÇAK FARMA), B12 (standard of 4000 I.U. per capsule by KOÇAK FARMA), and C (standard of 99% purity by SIGMA). Vitamin standards as pharmaceutical pure preparats were obtained from drug stores and stored at room temperature. Working solutions of water-soluble vitamins were prepared in 0.1 M phosphate buffer solution (PBS) containing 1N KH₂PO₄ (pH, 8.0). 0.1 M acetate buffer solution (ACB) were prepared by using glaciel acetic acid, pH of diluted acetic acid was adjusted with 1N NaOH to 4.8 and then NaCl added (0.02 M) for the detection of fat soluble vitamins. Ionized water was used in all solutions.

3.3.1. Preparation of pencil graphite electrode (pge) and chrono amperometry

3 cm long graphite pencil tip 0.5 HB was placed in the Rotring Tikky Model pencil which is used as a holder for graphite lead. One end of pencil tip was connected to a copper wire for electrical contact, and a 1.5cm pencil graphite tip was inserted into the buffer solution.

Chrono amperometry was applied to bare PGE at + 1.4V for 30 seconds in 0.5M acetate buffer solution (ABS) or 0.1 M phosphate buffer solution (PBS) for activation and cleaning its surface. These leads were then used for vitamin analysis.

3.3.2. Cyclic voltammetric (cv) and differantial pulse voltammetric (dpv) measurements

The electrochemical behavior of vitamins was examined by using cyclic voltammetry in negative and positive region in acetate buffer for fat soluble vitamins and phosphate buffer for water soluble vitamins. Cyclic voltammetry (CV) measurements were performed with 1.5 V of upper vertex potential − 1.5V of lover vertex potential, 0.005 V of step potential and 10mV/s scan rate.

Based on CV results, selected fat-soluble and water-soluble vitamins, and their calibration standards were analyzed using differential pulse voltammetry techniques in acetate buffer or phosphate buffer systems respectively. Differential pulse voltammograms were performed by potential range of -0.2 to + 1.2 V with 0.005 V step potential and 10 mV/s scan rate.

3.3.3. Electrochemical detection of fat-soluble vitamins by coating method

200 µL of vitamin samples from the liquid capsule content were directly transfered into Eppendorf tubes. PG tips were dipped into the vitamin solution, and kept for 30 minutes. At the end of this period, tips were taken from solution and air-dried for 30 minutes. These coated and dryed tips were then used for differential pulse voltammetric detection of fat-soluble vitamins. 30 min was chosen as optimum immobilization time from preliminary studies.

3.3.4. Electrochemical detection of water-soluble vitamins by immersion method

For the voltammetric detection of water-soluble vitamins, working solutions of vitamins were prepared in PBS (pH 8) and electrochemical behaviours of vitamins were evaluated in this medium by using bare PGE.

4.1. Individual and simultaneous detection of selected vitamins using cyclic voltammetry and differential pulse voltammetry

First, cyclic voltammetry was used to obtain electrochemical finger print of each vitamin. All the vitamins were electroactive (Fig. 1).

Based on cyclic voltammetry studies of vitamins, differential pulse voltammetry measurements were performed in the range of-0.2-1.2V, in ACB medium for fat-soluble vitamins and in PBS medium for water-soluble vitamins. Oxidation signals of vitamins in simultaneous detection can be seen in Fig. 2. Oxidation potentials of fat soluble vitamins map between 0.40 and 0.93 V. Vitamin D3 and E oxidation waves were overlapping each other in ACB. Oxidation potentials of water soluble vitamins in PBS are between 0.14 V and 0.70 V. Vitamin C, B1 and B12 have similar peak positions (Table 1 and Fig. 2).

Table 1

Peak Positions, LOD and coefficient of correlation (R²) values of vitamins A, D3, E, C, B1, B6, B12.
Vitamin	Peak Position (V)	Correlation Coefficient (R²)	Limit of Detection (µg/ml)
A D3 E C B1 B6 B12	0.93 ± 0.02 0.48 ± 0.05 0.40 ± 0.04 0.17 ± 0.02 0.14 ± 0.02 0.70 ± 0.02 0.14 ± 0.01	0.998 0.999 0.999 0.999 0.999 0.998 0.999	19.6 4.0 32.0 548.7 5.7 5.0 4.6

4.2. Linearity and limit of detection (LOD) values of vitamin A, D3, E, C, B1 + B6 and B12

In method development studies, linearity and LOD are significant parameters to be determined. Using the calibration curves is an easy way to estimate the linearity and LOD. In the present study, linearity for vitamin A, D3, E, C, B1 + B6 and B12 were determined through calibration experiments. Correlation Coefficient (R²) and LOD values were calculated from calibration curve by using a template. Peak Positions, LODs and Coeffients of Correlation of vitamins have been summarized in Table 1 and Fig. 3, 4-a & 4-b

A rapid, cost-effective and sensitive voltammetric detection of some fat and water-soluble vitamins was performed by using bare pencil graphite-based ‘’ single use’’ sensor.

Total of 7 selected vitamins were analyzed individually and simultaneously by using differential pulse voltammetry (DPV). It was found that in simultaneous analysis of vitamins, fat-soluble and water-soluble vitamins cannot be detected in a single run, since pH dependence of their reactions are different and fat-soluble vitamins fall outside the water-soluble window. Simultaneous analysis of water-soluble vitamins by using DPV with PGE can be a promising technique for the determination of vitamin C and B6 or vitamin B1, B12, B6, since vitamin C, B1 and B12 voltammetric waves are overlapping each other. Vitamin A and D3 or vitamin A and E can be detected in a single run. However, all three fat-soluble vitamins cannot be simultaneously detected in a single run, since voltammetric waves of vitamin D3 and E are overlapping each other.

The perspective on vitamins, electrochemical behaviors developed here may serve as a basis for using other sensing techniques and further validation studies in pharmaceutical and food samples.

Ethical approval is not applicable, because this article does not contain any studies with human or animal subjects.

There is no Conflict of Interest.

Authors' contributions; Tolga Saruhan and Asst.Prof.Dr. Perihan Adun wrote the main manuscript text and Asst.Prof.Dr. İhsan Erol Özçil edited the article. All authors reviewed the manuscript.

There is no funding.

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.

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No competing interests reported.

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Electrochemical Detection of Some Water Soluble and Fat Soluble Vitamins by Using Bare Pencil Graphite Electrode

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Equipments

2.2. Reagents

3. Methods

3.3.1. Preparation of pencil graphite electrode (pge) and chrono amperometry

3.3.2. Cyclic voltammetric (cv) and differantial pulse voltammetric (dpv) measurements

3.3.3. Electrochemical detection of fat-soluble vitamins by coating method

3.3.4. Electrochemical detection of water-soluble vitamins by immersion method

4. Results And Discussion

4.1. Individual and simultaneous detection of selected vitamins using cyclic voltammetry and differential pulse voltammetry

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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