Morphological Studies of Disposable Graphite and its Effective utilization for Vitamin B12 Analysis in Pharmaceutical Formulations

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ScienceDirect Materials Today: Proceedings 18 (2019) 3314–3320

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ICMPC-2019

Morphological Studies of Disposable Graphite and its Effective utilization for Vitamin B12 Analysis in Pharmaceutical Formulations Rajasree G. Krishnana, Greeshma Sa, Shivon Da’ Morris, Suryasree S. Rameshana, Beena Sa*. Department of Chemistry, Amrita School of Arts and Sciences, Amritapuri, Clappana P.O, Kollam, 690525, India.

Abstract Electrochemical sensor for the determination of Vitamin B 12 was fabricated using a disposable pencil graphite electrode. The main highlight of this work is that without any electrode modification and analyte pre-concentration, direct electrochemical detection of Vitamin B 12 was made possible. Morphological and structural studies were incorporated. Electrochemical studies were done using cyclic voltammetry and differential pulse voltammetry. Electrochemical reduction of Vitamin B 12 was observed at -0.82 V. Sensor exhibited a linear range of 2.5-30 nM with a sensitivity of 1.96 µA/nM/cm2. The viability of the sensor towards practical use was tested in commercial Vitamin B 12 tablet. © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the 9th International Conference of Materials Processing and Characterization, ICMPC-2019 Keywords: Vitamin B 12; Micronutrient, Dementia, Electrochemical sensor; Voltammetry; Pharmaceutical analysis.

1. Main text Vitamin B 12 is an essential micronutrient required for the various physiological functions in our body. It has got a corrin ring which is in close resemblance to the porphyrin ring in the heme which is further centered by a cobalt ion [1]. The function of Vitamin B 12 includes the development of red blood cells, healthy maintenance of nerve cells [2], co-factor in DNA synthesis etc. Its deficiency is more prone in old age population, pregnant women, and among vegetarian community. Normal levels of Vitamin B 12 in blood is 200-900 pg/mL [3]. Abnormal levels of Vitamin B 12 can end up in fatigue, dementia [4],

* Corresponding author. Tel.: +919495188217 E-mail address: [email protected].

2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the 9th International Conference of Materials Processing and Characterization, ICMPC-2019

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pernicious anemia [5] etc. Recent studies have shown that elevated Vitamin B 12 levels can serve as a biomarker for hepatocellular carcinoma [6]. Sources of vitamin B 12 includes tablets, fortified foods, supplements, and animal derived foods such as fish, meat, eggs etc. [7]. Since vitamin B 12 can only be supplied through these diet and tablets, it demands for the monitoring of the levels of vitamin B 12 in tablets, supplements, food materials etc. Many methods like high-performance liquid chromatography (HPLC) [8], chemiluminescence [9], atomic absorption spectroscopy, radioimmunoassay etc. [2] have been employed for the quantification of vitamin B 12 in different matrices. Even though these methods have several advantages, need of sophisticated instrumentation, time consumption, and high cost etc. stands as a bottleneck in the facile determination of Vitamin B 12. Unlike other methods, electrochemical detection offers high sensitivity, less reagents, lower response time, cost-effectiveness, room for miniaturization etc. Apart from that, vitamin B 12 being an electroactive molecule, electrochemical ways of detection is highly sensible. Scientists have utilized both the metallic centre [10] as well as the corrin ring [11] to tune the electrochemical detection of vitamin B 12. Different research groups have developed electrochemical sensors for Vitamin B12 in pharmaceutical formulations, food materials, urine samples etc. Peter et al. has employed a carbon paste electrode modified with trans 1,2-dibromocyclohexane as the reactive material [12]. They utilized the reduction of Co (III) to Co (I) which in turn reacted with the catalyst with which the determination of Vitamin B 12 was made possible. Many unmodified electrodes like boron doped diamond [1], bismuth film electrode [13], graphite screen printed electrode [10] were also used for the detection of vitamin B 12. Apart from this modified pencil graphite electrodes were also excellent candidates for the electrochemical sensing of vitamin B 12 [14] [15]. Pencil graphite electrode being cost effective and versatile, it has been applied for the detection of many analyte like ascorbic acid [16], 4-aminophenazone [17] etc. Here we present a disposable unmodified pencil graphite electrode for the detection of vitamin B 12 in NaOH medium. It is for the first time a highly alkaline medium is used for the quantification of vitamin B 12. 2. Materials and Methods 2.1. Reagents Vitamin B 12 was obtained from Sigma Aldrich. Sodium hydroxide was obtained from loba chemie. Pencil lead with a diameter of 0.7 mm (Cello Fine Leads Pvt. Ltd.) was purchased from a stationary store. All other chemicals are of analytical grade and used without further purification. Millipore water (18.2 MΩ cm) was used to prepare all solutions. 2.2. Instrumentation All electrochemical experiments were performed with CHI 610E electrochemical workstation (CH Instruments, USA). Conventional three electrode electrochemical cell was used. A pencil lead served as the working electrode, platinum wire as the counter electrode and silver/silver chloride (Ag/AgCl (1 M KCl)) as the reference electrode. 0.1 M NaOH was used as electrolyte for all electrochemical experiments after performing the optimisation studies. Morphological studies were done using field emission scanning electron microscope (Hitachi model S3000H, Hitachi Ltd, Japan) and structural characterization of the electrode surface was performed with the aid of X-ray diffractometer (XPERT-PRO, PANalytical, Netherlands). 2.3. Fabrication of the electrode A length of 0.2 cm was exposed by covering rest of the pencil lead with a Teflon tape. Thus a working area of 0.0439 cm2 was obtained finally. Electrochemical characterization was done using cyclic voltammetry. A potential window of -0.6 V to -1 V was used. Quantification of Vitamin B 12 was performed using differential pulse voltammetry. The pulse amplitude was 0.05 V and pulse width was 0.05 s. Real sample analysis was carried out using complete Vitamin B 12 tablets obtained from a local medical store. DPV was used to carry out real sample analysis.

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3. Results and Discussions 3.1. FE-SEM analysis FE-SEM analysis shows that the surface of pencil graphite is highly buildup of graphite. The flake like structures in Fig. 1 clearly depicts the chief component of pencil lead as graphite.

Fig. 1. FE-SEM image of the pencil graphite surface

3.2. XRD analysis XRD analysis of the pencil graphite surface proves that the prime component is graphite. Fig. 2 shows the XRD pattern for the pencil graphite electrode. The well intense peak at 26.70 indicates the material is of very good crystallinity. The peak at 26.70 and 54.80 corresponds to the (002) and (004) plane of the graphite respectively.

Fig. 2. XRD of the pencil graphite surface

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3.3. Electrochemical characterization Fig. 3 shows the cyclic voltammogram obtained for the pencil graphite electrode with and without Vitamin B 12 in 0.1 M NaOH. It depicts that under the scanned potential window of -0.6 V to -1 V, no characteristic peaks were observed for the pencil graphite electrode in 0.1 M NaOH. A new irreversible reduction peak was observed at a potential of -0.85 V in the voltammogram on addition of 15 nM Vitamin B 12 to the same NaOH solution. Evidence of any anodic peaks was not observed in the backward scan. This newly formed cathodic peak was ascribed to the reduction of Vitamin B 12 in alkaline medium.

Fig. 3. Cyclic voltammogram of the pencil graphite electrode at 100 mV/s in 0.1 M NaOH containing 15 nM Vitamin B 12.

3.4. Optimization of NaOH concentration Optimization studies of the electrolyte were carried out using DPV. Different concentrations of NaOH were prepared and tested. The parameters like peak current and peak potential for 2.5 nM of Vitamin B 12 in different NaOH concentrations were compared and shown in Table 1. Higher peak current and lower reduction potential was obtained for 0.1 M NaOH and hence this was fixed as the final electrolyte concentration throughout the studies. Table 1. Optimization of NaOH concentration. Concentration of NaOH (M)

Current (µA)

Potential (V)

0.001

No peak

No peak

0.005

0.216

-0.880

0.01

0.384

-0.867

0.05

0.413

-0.868

0.1

0.760

-0.826

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3.5. Effect of scan rate The effect of scan rate towards the reduction behavior of Vitamin B 12 was investigated by CV. It was found that the cathodic peak currents obtained for 10 nM of Vitamin B 12 were directly proportional to the square root of the scan rate from 100-900 mV/s (Fig. 4 A). A linear plot was developed by plotting current vs. square root of scan rate and obtained a linear regression equation of Ipc (µA) =13.45+1.05*ν1/2 (s-1) with R2=0.999 and a standard deviation of 0.3318 for N=9 points (Fig. 4 B). This suggests that the electrode process is diffusion-controlled in nature.

Fig. 4. (A) CV of the pencil graphite electrode with scan rates ranging from 100-900 mV/s. (B) Linear plot of square root of scan rate vs. cathodic peak current containing 10 nM Vitamin B 12 in 0.1 M NaOH.

3.6. Voltammetric determination of Vitamin B 12 on bare pencil graphite electrode As for signal generation we focus on faradaic current resulted from the electron transfer phenomenon, DPV is the best technique to accomplish the quantification of Vitamin B 12. DPV measures only the current difference at the beginning and end of each pulse thereby nullifying the background current generated from the solution resistance etc. [18]. Thus the electrochemical response of the bare pencil graphite electrode to the Vitamin B 12 concentrations was illustrated using DPV. A potential scan from 0 to -1 V was performed in 0.1 M NaOH. No discernible peaks were observed for pencil graphite electrode under the applied potential window and exhibited a distinct cathodic peak at an applied potential of -0.82 V when 2.5 nM of Vitamin B 12 was added. This well-defined cathodic peak was attributed to the reduction of the metallic centre Co(III) to Co(II) of Vitamin B 12 in alkaline medium [19]. The cathodic peak currents were found to be increasing with successive Vitamin B 12 concentrations. A calibration plot was constructed with the peak currents against the concentrations of Vitamin B 12 and found to be linear in the range of 2.5 nM-30 nM with a linear equation of Ipc (µA) =0.7522+0.0862*C (nM) and R2=0.9901. A standard deviation of 0.1375 was obtained for N=7 points. Fig. 5 (A) shows the DPV and the corresponding calibration plot Fig. 5 (B) for different concentrations of Vitamin B 12 in 0.1 M NaOH.

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Fig. 5 (A) DPV of the pencil graphite electrode for different concentrations of Vitamin B 12 in 0.1 M NaOH. (B) Calibration plot for the current obtained to the varying Vitamin B 12 concentrations in the range of 2.5-30 nM.

3.7. Sensitivity and Reproducibility. The sensitivity of the pencil graphite electrode for the determination of Vitamin B 12 was found to be 1.96 µA/nM/cm2. Reproducibility studies for the sensor were performed with 6 different pencil graphite electrodes with 2.5 nM Vitamin B 12 in NaOH at -0.82 V. The variation obtained was found to be less than 5% which describes the sensor is highly reproducible in nature. 3.8. Real sample analysis The developed sensor was employed for testing its practical use in complete Vitamin B 12 tablets. Vitamin B 12 tablets were grinded to obtain a homogeneous powder and 1 µM stock solution was prepared. An aliquot of the tablet solution was subjected to DPV and the current response was noted from which the concentration of the tablet was calculated. The obtained concentration was compared with the amount declared by the manufacturer and obtained an error percentage of 9.1. Table 2 shows the results obtained for the real sample assay for complete Vitamin B 12 tablets with the developed sensor. Since the results are in agreement with the Vitamin B 12 content declared by the manufacturer, the sensor showed its potentiality in determining the Vitamin B12 content from real Vitamin B1 2 tablets. Table 2. Real sample analysis in complete Vitamin B 12 tablet matrix. Sample

Amount added

Amount determined

Amount declared by manufacturer

Percentage error (%)

Complete B 12 tablet

0

1091.42 mcg/pill

1000 mcg/pill

9.1

3.9. Comparison of the developed sensor performances with other reported sensors Table 3 elucidates the comparison of the developed sensor with other electrochemical sensors for vitamin B 12 reported in the literature. Our sensor outperforms most of the other sensors in terms of the nanomolar detection. Need of no modification, pre-concentrations steps etc. makes our sensor superior to many other sensors for the determination of Vitamin B 12.

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Table 3. Comparison data of the performance of different electrochemical sensors for Vitamin B 12. Electrode

Electrolyte

Linear

and pH

range (nM)

Au/PPy/FMNPs@TD

B.R, 7

4-500

[7]

Mn-dsDNA-CPE

Acetate buffer, 5.2

2.7-174

[20]

SWCNT-PGE

PBS, 2

5-100

[15]

PNT-PGE

PBS, 2

200-9500

[14]

Modified electrodes

Reference ..

Unmodified electrodes BDD

B.R, 2

2000-35000

[1]

SPE

PB, 3

0.1-8

[10]

Bi-film

B.R, 12

100-1000

[13]

PGE

NaOH, 13

2.5-30

This work

4. Conclusion We report a simple and selective electrode for determining Vitamin B 12 concentrations in highly alkaline medium. A disposable, cost-effective and unmodified pencil graphite was used for the same. Unlike many other sensors, no pre-concentration of Vitamin B12 was needed. Vitamin B 12 was found to exhibit a reduction peak at a potential of -0.82 V. The reduction current was taken to construct the calibration plot for different concentrations of Vitamin B 12. Linearity in the range of 2.5 nM-30 nM was obtained with a sensitivity of 1.96 µA/nM/cm2. The sensor was effective in monitoring the concentration of Vitamin B 12 in real tablet matrix. Acknowledgements Authors acknowledge AmritaVishwa Vidyapeetham, Amritapuri campus for the internal support provided to carry out the research work. References [1] D.M. Stanković, D. Kuzmanović, D. Manojlović, K. Kalcher, G. Roglić, Journal of The Electrochemical Society 163(7) (2016) B348-B351. [2] O. Karmi, A. Zayed, S. Baraghethi, M. Qadi, R. Ghanem, IIOAB J 2(2) (2011) 23-32. [3] S. Hanna, L. Lachover, R. Rajarethinam, Primary care companion to the Journal of clinical psychiatry 11(5) (2009) 269. [4] N. Goebels, M. Soyka, The Journal of neuropsychiatry and clinical neurosciences 12(3) (2000) 389-394. [5] E. Andrès, N.H. Loukili, E. Noel, G. Kaltenbach, M.B. Abdelgheni, A.E. Perrin, M. Noblet-Dick, F. Maloisel, J.-L. Schlienger, J.-F. Blicklé, Canadian Medical Association Journal 171(3) (2004) 251-259. [6] E. Öksüz, M. Öksüz, T. Egesel, G. Özgür, G. Saydaoğlu, Eastern Journal Of Medicine 21(3) (2016) 113. [7] M.H. Parvin, E. Azizi, J. Arjomandi, J.Y. Lee, SENSORS AND ACTUATORS B 261(1) (2018) 335-344. [8] F. Hasnat, H.A. Bhuiyan, M. Misbahuddin, Bangladesh Journal of Pharmacology 12(3) (2017) 251-255, Jul 12, 2017. [9] Z. Song, S. Hou, Analytica Chimica Acta 488(1) (2003) 71-79. [10] A. Michopoulos, A.B. Florou, M.I. Prodromidis, Electroanalysis 27(8) (2015) 1876-1882. [11] M. Kaumal, International Journal of Chemical and Pharmaceutical Analysis 4(3) (2017). [12] P. Tomčik, C.E. Banks, T.J. Davies, R.G. Compton, Analytical chemistry 76(1) (2004) 161-165. [13] G.L. Kreft, O.C. de Braga, A. Spinelli, Electrochimica Acta 83 (2012) 125-132. [14] B.B. Pala, T. Vural, F. Kuralay, T. Çırak, G. Bolat, S. Abacı, E.B. Denkbaş, Applied Surface Science 303 (2014) 37-45. [15] F. Kuralay, T. Vural, C. Bayram, E.B. Denkbas, S. Abaci, Colloids and Surfaces B: Biointerfaces 87(1) (2011) 18-22. [16] D. King, J. Friend, J. Kariuki, Journal of Chemical Education 87(5) (2010) 507-509. [17] J.I. Gowda, S.T. Nandibewoor, Industrial & Engineering Chemistry Research 51(49) (2012) 15936-15941. [18] T. Choudhary, G. Rajamanickam, D. Dendukuri, Lab on a Chip 15(9) (2015) 2064-2072. [19] J.A. Koza, C.M. Hull, Y.-C. Liu, J.A. Switzer, Chemistry of Materials 25(9) (2013) 1922-1926. [20] G. Dimitropoulou, S. Karastogianni, S. Girousi, Issues 2016 (2015) 2017.