INTRODUCTION:

Vitamin C (2-oxo-L-threo-hexono-1.4 -lactone -2.3-endiol,AA)¹ is a water soluble vitamin, which is very essential because itinvolves ina wide variety of biological events concerning electron transport reaction, hydroxylation the oxidativecatabolism of aromatic acid^2,3 in addition to this vitamin C plays an important role in improving macular degeneration, reducing inflammation and lowering the risk of cancer and cardiovascular disease^4-9.

However, itcannot be produced in enough quantities by the human body therefor the daily consumption in sufficient quantities is so important⁵.

Numerous analytical methods i.e., spectrophotometry^10-15, electrochemical^16-20, chromatographic^21-25, fluorescence ^26-29and kinetic³⁰ have been developed for the determination of vitamin C in pharmaceutical samples.

However, these methods have some flows such as,time consuming, the need to use expensive solvents, special equipment laboratories and skillful individual.Therefore, there is a need to develop a rapid, simple and inexpensive test for vitamin C determination ^5,31-34.

Prussian blue was probably synthesized for the first time by the paint makerJhon Jacob Dieshach in Berlin around 1706.The pigment is believed to have been accidentally created when Dieshach used potash tained with blood to create some red cochineal dye^35,36.

The detection principal of this method depends on the ability of vitamin C [C₆H₆O₆] to reduce FeCl₃and form Prussian blue complex³⁷.

The first step is the reduction of Fe^III by vitamin C to produce Fe^II in acidic medium of HCl.

Fe^III + C₆H₂O₆ → 2Fe^II + C₆H₆O₆ + 2H⁺ (1)

The second step is the formation of Prussian blue [KFe^III [Fe^II (CN)₆]) when Fe₂ reacted with potassium ferricyanide [K₂ [Fe^III (CN)₆]³⁷.

Fe^II + K₂[Fe^III (cn)₆] → K Fe^III[Fe^II(CN)₆] + 2K⁺ (2)

The UV/vis spectra of Prussian blue Showed a broad peak at lower energy wavelengths in the visible region near 700nm³⁸.

The aim of this paper is to validate this new, rapid and convenient spectrophotometric method and compareit with the classical titration method.

MATERIALS AND METHODS:

Pure vitamin C andpotassium ferricyanidewere obtained from Zien Pharma for pharmaceutical industry. Ferric chloride was obtained from Damascus University. HCl was obtained from Zienpharma laboratories.

All other chemicals were analytical reagent grade,all solution used in this work were prepared with distilled water.

Commercially available tablets were purchased from local markets in Syria in addition to instruments like UV-visible spectrophotometry and pH measurement.

General procedure for vitamin C determination:

The determination of vitamin C is performed based on the visible color of Prussian blue.1ml of different concentrations of standard vitamin c solution, 1ml of 0.2 mM ferric chloride and 1ml of 1mM potassium ferricyanide were mixed together by strong stirring for 8 min.

The reduction of FeCl₃by vitamin C and the production of Prussian blue as described in Eq 1 and 2 were obtained.The blue color of Prussian blue solution was obtained and can be observed clearly.

The color change was monitored by spectrophotometer at maximum wavelength of 697 nm. The optimization of the determination test conditionswas carried out to meet the highperformance. The affected parameters including ferric chloride concentration, potassium ferricyanide concentration, reaction time and pH were studied.

Real sample analyzes:

Commercial effervescent vitamin C tablets samples were obtained from the local markets in Syria. the samples were filtered and diluted with appropriate volume of distilled water to obtain the concentration between 0.3 and 5 mM. The samples were then analyzed by the developed spectrophotometric test.

Study of spectral characteristics of Prussian blue complex in distilled water after enabling the initial adjustments and blank correction, the absorption of vitamin C solution was then scanned in the range from 400 to 800 to obtain the wavelength of maximum absorption λmax

RESULTS AND DISCUSSION:

Spectrophotometric detection of vitamin C:

The design of the colorimetric sensor array was based on the noticing of the color change, Prussian blue reaction was used as an indicating reagent to determine vitamin C.

First FeCl₃ was reduced by a reducing agent of vitamin c in acidic medium of HCl to FeCl₂and then reacted with potassium ferricyanide.

Then color of this mixed solution was immediately changed to blue within 3 seconds.It was found that the product exhibiting λmax at 697nm as shown in fig 1

This was a result of transition from the ground state to an excited state upon which an electron is moved from a ground state Fe^III Fe^IIto an excited state Fe^II Fe^IIIform ⁵. While the absorbencies of the blank [FeCI₂ + K₂ [Fe (CN)₆was certainly weaker than Prussian blue in the range of 400-800 nm. So, all the following procedures were accomplished at 697 nm which resulted from Prussian blue.

Fig 1: UV-visible absorption spectra of FeCl₃0.2 mM with Vitamin C 0.1 Mm and K₃[Fe(CN)₆] 1 Mm as Prussian blue

Optimization of reaction conditions:

Effect of reaction time:

The reaction time and the stability of developed Prussian blue were studied by monitoring the absorbancechanged every two minutes for 90 min at the maximum wavelength 697nm using 0.1 mM of vitamin C.

Figure 2 shows the influence of reaction time as the absorbance increased rapidly when the reaction time increased from 1 to 8 min and reach the steady state at 8 to 60 min. This clearly demonstrates that Fe^III was completly reduced by vitamin C and reacted with excess potassium ferricyanide to form Prussian blue. The developed color of Prussian blue did not change and was constant after 8 min up to 60 min. Therefore, this method was convenient and powerful to develop as a rapid determination test for vitamin C determination.

Fig 2: Effect of reaction time on Prussian blue complex

Effect of ferric chloride concentration:

Different concentrations of FeCl₃from 0.01 – 1mM have been studied to obtain the optimum concentration that achieves the highest absorbance.

The concentration of vitamin C was constant 0.1mM, the results are shown in fig 3. The increase in ferric chloride concentration led to more production of Prussian blue compound and subsequently led to the increase of the absorbance. Theabsorbance reached the maximum value when the amount of ferric chloride was 0.2mm. At higher concentration the color resulting from Prussian blue cannot be defined because there was a precipitation solution. Therefore 0.2mM of ferric chloride was selected as optimum concentration.

Fig 3 : The effect of FeCl₃ concentration on Prussian blue complex

Effect of potassium ferricyanide concentration:

Different concentrations of potassium ferricyanide between 0.5 - 2mM were studied by maintaining the concentration of vitamin C and FeCl₂ constant at 0.1 and 0.2mm respectively.

As shown in fig 4 the highest absorbance value was marked at 1mM. The color of Prussian blue was clearly recognized and the absorbance can be measured without any perception of the Prussian blue compound by this concentration. So, the optimum concentration of potassium was chosen at 1mm.

Fig 4: The effect of potassium ferricyanide concentration on Prussian blue complex

Effect of pH:

Normally all tested samples are acidic as a result of adding two drops of HCL to speeds up the oxidation process. The pH range was between 1.2 and 2.2.

The influence of pH was examined by changing the pH between 3.4 and 5.8 during samples preparation using acetate buffer. The influence of pH was determined at constant concentration of vitamin C 0.1mM. As the pH Increased there was an increase in the absorbance as shown in fig 5, but above pH 3.7 the absorbance decreased because FeCl₃ precipitated.

Fig 5: The effect of pH on Prussian blue complex

Effect of temperature and the sequence of additions:

No changes in absorbance were observed after changing temperatures and the order of addition.

Analytical method validation:

Linearity:

The linearity of the proposed method was determined by measuring the absorbance of six concentrations covering the range 0.05 to 0.3mm fig 6.

Each concentration was measured in triplicate. Then the plot of observance against concentration was examined visually and statistically by calculating coefficient intercept and slop for the calibration data were calculated using the leastsquares method.(table1)

Table 1: Y = a + bX, where Y is the absorbance, a intercept, b slope and X concentration in mM

Spectral Data	Parameters
697	Max nmλ
0,05 -0.3	Beer’s law limits, Molarity
3.250 × 10³	Molar absorptivity, L mol^-1cm^-1
0.05459	Sandell’s sensitivity, μg/cm³
0.00001	Limit of detection ,μg/ml (LOD)
0.000032	Limit of quantitation, μg/ml (LOQ)
Y = 3240X + 0.03	Regression equation
3240	Intercept (a)
0.03	Slope (b)
0.9999	Correlation coefficient (r)

Fig 6 : Calibration curve for vitamin c

Sensitivity (LOD,LOQ):

The limit of detection LOD and the limit of quantitation LOQ for the developed method were calculated using the following equations

LOD =3.3 SD/S

LOQ =10 SD/S

SD is calculated as the standard deviation of the residuals around the regression line,S is the slope of calibrationcurve. LOQ and LOD are mentioned in table 1.

Sandell’ssensitivity is the concentration of the drug in ug/cm3 which will result in an absorbance of 0.001 in 1 cm path length.

Precision:

Interday and Intraday precision were evaluated by triplicate analysis of three different concentration 0.1 -0.2- 0.3mM.

Precision of the proposed method is expressed as SD or RSD of series of measurements by replicate determination of samples by the developed method.

The calculated relative standard deviation value was obtained to be very small (>1%) indicating good repeatability and precision of the developed method. The results and their statistical analyzes where shown in table 2.

Table 2: evaluation of precision of the proposed method

Statistical Parameters		0.1 mM	0.2 mM	0.3 mM
	1	0.0997	0.2005	0.3002
	2	0.0996	0.1995	0.3009
	3	0.1003	0.1997	0.2995
	Mean	0.099867	0.1999	0.3002
Intraday	Mean recovery %	99.8	99.95	100.06
	S.D	0.000379	0.000529	0.0007
	R.S.D %	0.3790994	0.2647075	0.2331779
	1	0.1007	0.2008	0.301
	2	0.1009	0.1996	0.3002
	3	0.0998	0.1994	0.2997
	Mean	0.1004667	0.1999333	0.3003
Interday	Mean recovery %	100.4	99.96	100.1
	S.D	0.0005859	0.0007572	0.0006557
	R.S.D %	0.5832248	0.3787201	0.2183629

Accuracy:

Accuracy of the proposed methodwas evaluatedby performing recovery studiesusing standard addition method.

A constant amount of drug from commercial samples was taken in 100ml volumetric flask to make 0.01m vitamin C solution and pure standard vitamin C at three different concentrationswithin Beer’s range was added to baseline amount of commercial tablets to reach the following concentrations 0.01 – 0.02 – 0.03.

These mixtures werethen transferred into separate 100 ml volumetric flasks and then 1ml of FeCl₃0.02 m was added to each flask followed by 1ml of potassium ferricyanide 0.1m and two drops of HCl. The contents of each flask were shaking for few minutes andeach mixture was diluted to 100ml with distilled water to reach the following concentration 0.1- 0.2-0.3mM respectively.

The absorbance of the new solutions was measured atλmax against the blankprepared in the same conditions without addition of vitamin C.

The determination of each concentration was in triplicate and averaged percent recovery of the added standard was calculated and results areshown in table 3. The results obtained showed excellent mean recovery percent value and low standard division value SD < 1 which means high accuracy of the developed spectrophotometric method.

Table 3: results of recovery study of vitamin C

Base level	Amount spiked	Amount recovered*	SD	Recovery %
0. 1 mM	0.1 mM	0.0997 mM	0.0008	99.7%
0.1 mM	0. 2 mM	0.1996 mM	0.0007	99.8%
0.1 mM	0. 3 mM	0.3001 mM	0.0006	100.04%

*Mean value of three concentrations

Specificity:

The specificity of the method was evaluated by noticing any interference from common excipients or drugs that may be added to vitamin C pharmaceutical forms.

To achieve this purpose two mixtures were prepared, the first one contained vitamin C 0.1 mM with excipients that are commonly found in tablets at their highest concentrations possible, in addition to Paracetamol 300mg as a common pharmaceutical ingredient with vitamin C in a pharmaceutical form,the second one contained all the previous substances except for vitamin C.

Then the absorbances of the resulting solutions were measured at λmax against the blank, we didn't observe any interaction between the regent and the addictions as it is shown in fig 9 which confirms the selectivity of the proposed methods and the results of the recovery basis on a standard sample of vitamin C at a concentration of 0.1 ml are shown in the table 4.

Fig 7: UV/vis spectra of a: vitamin C standard solution, b: vitamin C solution with the additions, c: the addition solution without vitamin

Table 4: recovery results for vitamin C with Paracetamol and common excipients in comparison with excipients and Paracetamol without vitamin C

Solutions	Absorbance	Recovery
STD	0.715	-
With vit c	0.714	99.80%
Without vit c	0.007	0.97%

Comparison between the proposed method and titration method:

Three solutions of vitamin C commercial samples were prepared in specific concentration 0.1 - 0.25 - 0.5mM respectively each one was prepared in triplicate and then was analyzed with the proposed method and the titration method.

The titration method is based on the reaction of vitamin C in acidic medium of sulfuric acid with an iodine 0.1 Nusing starch as an indicator for the endpoint of the titration method.

The concentration obtained from the two methods were compared as results shownin table 5 indicate that there is no difference between the twomethods, which refers to the possibility of using the proposed method in the titration of commercial forms of vitamin C.

Table 5: comparison of the vitamin C concentrations obtained from the developed spectrophotometric method and the titration method

Sample	Developed method	Titration method
0. 1mM	0. 107mM	0. 11mM
0. 25mM	0. 2497mM	0. 259mM
0.5mM	0. 502mM	0. 534mM

composition of Prussian blue complex:

a - Molar ratio method:

The stoichiometry of Prussian blue complex by molar ratio method according to the following equations λmax = K₃ [Fe(CN)₆]/ FeCl₂where the concentration of FeCl₃ is constant 0.2mM and the concentrations of potassium ferricyanide are changing from (0 to 2.2)mM, 1ml of vitamin C 0.1mM and 2 drops of HCl was added to all Solutions .

As it is shown in figure 8 the molar ratio method confirms that the ratio of Prussian blue complex is equal to 1.1.

Fig 8: Molar ratio method to calculate coupling ration for prussian blue comple

B- Continuous variation:

The nature of Prussian blue complex was determined using Job's method of continuous variation a series of solution of the studied complex were prepared in which the concentration of FeCl₃and potassium ferricyanide changed in each solutionso that the sum of their concentrations remained constant 1mM.

1ml of vitamin C 0.1mM and two drops of HCl were added to each solution . Then the absorbance of the resulting solution was measured at λmaxagainst the blank.A graph of absorbance then plotted versus mole fraction. The results of applying this method showed that the K₃ [Fe(CN)₆] FeCl₃ ratio was 1:1 as shown in fig 9.

Fig 9: Job’s method of continuous variation of Prussian blue complex

CONCLUSION:

A simple, accurate, cost effective and rapid spectrophotometric method was used and validated for the determination of vitamin C through Prussian blue complex.

Good LOD and LOQ were obtained. Preparation of samples for analysisis very simple. The assay involves the optimization of effective parameters and application in real samples. the proposed spectrophotometric method meets requirements of a reliable routine analyzes in the pharmaceutical industries because it is sensitive, selective and economic.

ACKNOWLEDGMENTS:

I wish to acknowledge the help provided by Zein Pharma Pharmaceutical Industry for the technical and support staff in quality control laboratory. I would also like to show my deep appreciation to Dr GhoufranKawas in Department of Chemistry Faculty of Science AL Andalus University, Syria.

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Received on 19.05.2022 Modified on 21.06.2022

Research J. Pharm. and Tech 2023; 16(4):1731-1737.

DOI: 10.52711/0974-360X.2023.00285