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European Journal of Applied Sciences – Vol. 13, No. 1

Publication Date: February 25, 2025

DOI:10.14738/aivp.131.18346.

de Silva, D. S. P. C. N., Nonis, P. C. M., & Jayasundara, U. K. (2025). Method Development and Validation of Vitamin C Formulations

Using Zeroth Order UV-visible Spectrophotometry. European Journal of Applied Sciences, Vol - 13(1). 473-485.

Services for Science and Education – United Kingdom

Method Development and Validation of Vitamin C Formulations

Using Zeroth Order UV-visible Spectrophotometry

1D.S.P.C.N de Silva, 2P.C.M Nonis, 3,*U.K Jayasundara

1Department of Chemistry, Institute of Chemistry Ceylon, Rajagiriya, Sri Lanka 10100;

2Department of Chemistry, University of Bonn, Regina-pacis-Weg 3, 531113 Bonn, Germany;

3Department of Chemistry, College of Arts and Science, University of Maine at Presque Isle,

Presque Isle ME, USA 04769;

udaya.jayasundara@maine.edu

ABSTRACT

Vitamin C, also known as ascorbic acid (AA), is an antioxidant and one of the

essential nutrients and used as a treatment during the COVID-19 pandemic. In this

research, an accurate, precise, reproducible, and cost-effective UV-visible

spectrophotometric method has been developed based on the International

Conference on Harmonization guidelines to determine the AA content of vitamin C

in tablet formulations. The method validation study was conducted by evaluating

and optimizing parameters such as linearity, range, limit of detection, limit of

quantification, repeatability, inter-day precision, accuracy, and stability. A series of

standard stock solutions (6 ppm to 18 ppm) was prepared by using HCl as the co- solvent and measuring the absorbance at 242 nm using a double-beam UV-Vis

spectrophotometer. The calibration plot of the standard solutions resulted in a

linear graph with a correlation coefficient (R2) of 0.9997 which obeyed the Beer’s

Law in the selected range. The stability studies confirmed that the samples were

stable for up to 7 days in the refrigerator conditions and 3 days in room

temperature conditions. The accuracy was tested with the spike recovery method

which showed the mean recoveries occurred from 91.15% to 105.23%. In addition,

sample analysis was performed using the developed method.

Keywords: Beer-Lambert law, repeatability, inter-day precision, ICH Guidelines, Healthy

life, and Ascorbic acid.

INTRODUCTION

Health is the wealth. Hence those who maintain healthy lives will have a long-life span as well

as be financially stable. To maintain healthy living and well-being, the human body requires a

trace amount of substances called vitamins. The body does not constitute vitamins; therefore,

vitamin supplements must be provided from the diet or other nutrients. Based on the age and

body mass index, the vitamin requirement could be different from person to person. Vitamins

are mainly organic compounds that are barely detectable in natural meals and may degrade

due to conventional food processing technologies such as pasteurization, thermal treatment,

and freezing.[1-4]

Table 1: Physical and chemical properties of AA

Physical properties Chemical properties

White crystalline powder. [5,6] Dibasic acid with pKa values of 4.2 and 11.6. [7]

Both enolic hydroxyl groups can dissociate.

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European Journal of Applied Sciences (EJAS) Vol. 13, Issue 1, February-2025

Among the available vitamins, vitamin C is considered to be an essential nutrient for health.

The active ingredient of vitamin C is the ascorbic acid (AA). The IUPAC name of AA is ((5R)-

[(1s)-1,2-Dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one) and it has a structure as shown in

figure 1. Determination of properties of drug components has become more convenient with

the introduction of in-silico studies which support the identification of chemical and physical

properties as shown in Table 1.[5-14] The human body requires vitamin C to perform various

functions, including wound healing processes ,formation of blood vessels, muscle, cartilage, and

collagen in bones.[15] Since the vitamin C is an antioxidant, it acts as a shield to protect human

cells from free radicals generated from harmful molecules due to tobacco smoke, x-rays, or

other radiations from the sun.[16] These free radicals can lead to cancer, heart disease, and

arthritis. In addition, vitamin C is essential for the growth, development, and repair of body

tissues. Vitamin C is one of the main nutrients that support for human immune system and

absorbs iron.[15]

Vitamin C can be found naturally in plants, fruits ( oranges, lemons, grapefruit, watermelon,

papaya, strawberries, mango, pineapple, raspberries, and cherries) tomatoes, green leafy

vegetables, etc.[17] However, cooking may convert vitamin C into molecules that the human

body does not need.[16,18] According to in vivo studies, gluconolactone oxidase which is the

terminal enzyme of biosynthetic pathway of ascorbic acid synthesizing in mammals, is absent

in humans. Therefore, vitamin C is unable to be produced and stored in the human body since

it is a water-soluble compound, Hence, it is necessary to include vitamin C in the everyday

diet.[7] Sometimes healthcare professionals recommend getting vitamin C externally since the

vitamin C deficiency may result in scurvy (which causes bleeding gums, teeth loss, and weak

tissue growth and wound healing), infections, allergies, osteoarthritis, cardiovascular disease,

stress, and cancer.[18] However, vitamin C is commercially available as tablets and syrups as

an over-the-counter drug. However, there is no guarantee that vitamin C contains the desired

amount of active pharmaceutical ingredient (API). Therefore, it is important to quantitatively

analyze whether the label claim is present in vitamin C. The API concentration analysis is a

crucial stage in the dose formulation because each patient needs a certain dose based on their

age, weight, and health condition. Both overdosing and underdosing are extremely dangerous

and sometimes life-threatening.[19] According to the United States Department of Health and

Human Services, the recommended daily intake of vitamin C based on age and health conditions

is given in Table 2.[18] Similar to other drug substances, vitamin C may have side effects such

as nausea, vomiting, and diarrhea, stomach pain or bloating, fatigue, sleepiness, insomnia, a

headache, and flushed skin. Taking large doses of vitamin C supplements orally may lead to

kidney stones for some persons.[7] Further, taking over 2,000 mg of vitamin C supplements

orally per day for an extended period may raise the risk of serious adverse effects.[7,18]

Density: 1.65 g/cm3. [8] Act as a mild reducing agent and antioxidant.

Soluble in water, ethanol, methanol, glycerol,

etc.

Oxidizes to dehydroascorbic acid (DHA) via radical

intermediate by losing of the second electron.

Insoluble in diethyl ether, chloroform, benzene,

petroleum ether, oils, and fats.

Ascorbate radical is stable and less in reactivity.

Molar mass: 176.12 g/mol. [8] Available as sodium and calcium salt.

A nucleophilic attack on ascorbic acid on a proton result in

1,3-diketone.