Analytical Letters, 42: 1321–1327, 2009

Copyright # Taylor & Francis Group, LLC

ISSN: 0003-2719 print=1532-236X online
DOI: 10.1080/00032710902954490

PHARMACEUTICAL ANALYSIS

FT-IR Spectrophotometric Analysis of Ascorbic

Acid and Biotin and their Pharmaceutical
Formulations

Andrei A. Bunaciu,1 Elena Bacalum,2 Hassan Y. Aboul-Enein,3

Gabriela Elena Udristioiu,4 and Şerban Fleschin4
1
Cromatec Plus SRL, Analytical Research Department,
Bucharest, Romania
2
National Research and Development Institute for Chemistry
and Petrochemistry, Bucharest, Romania
3
Pharmaceutical and Medicinal Chemistry Department,
Pharmaceutical and Drug Industries Research Division,
National Research Center, Cairo, Egypt
4
Department of Analytical Chemistry, Faculty of Chemistry,
University of Bucharest, Bucharest, Romania

Abstract: Fourier transform–infrared (FT-IR) spectrometry was used for the

rapid, direct measurement of ascorbic acid (vitamin C) and biotin (vitamin H)
in different pharmaceutical products. Conventional KBr spectra were compared
for the best determination of active substances in drug preparations. The Beer–
Lambert law and chemometric approaches were applied in data processing.

Received 8 January 2009; accepted 12 March 2009.

A. A. B. thanks the administration of Cromatec Plus SRL for the financial
support.
Address correspondence to Hassan Y. Aboul-Enein, Pharmaceutical and
Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research
Division, National Research Center, Dokki, Cairo 12311, Egypt. E-mail: enein@
gawab.com

1321
1322 A. A. Bunaciu et al.

Keywords: Ascorbic acid, biotin, FT-IR analysis, pharmaceutical analysis,

simultaneous drug analysis

INTRODUCTION

Infrared (IR) spectrometry provides a useful way to identify drugs

(United States Pharmacopeia 1990; Moffat 1986; Ciuczak and Drennen
2001; McClure 1992). However, the traditional techniques employed to
obtain the IR spectra, such as alkali halides disks, mulls, and thin films,
are sometimes not adequate for quantitative analysis. Fourier transform
(FT)–IR permits continuous monitoring of the spectral baseline and
simultaneous analysis of different components of the same sample
(Garrigues, Gallignani, and de la Guardia 1992; Miller, Danielson, and
Katon 1988).
Ascorbic acid, also known as vitamin C [2-oxo-L-threo-hexono-1,4-
lactone-2,3-enediol or (R)-3,4-dihydroxy-5-((S)-1,2-dihydroxyethyl)-
furan-2(5H)-one; Fig. 1] is an essential nutrient for a large number of
higher primate species (McClusky 1985), representing 20% of all mamma-
lian species, and a small number of other species, such as the guinea pig
and a few species of birds and fish. The presence of ascorbate is required
for a range of essential metabolic reactions in all animals and plants. It is
biosynthesized by almost all organisms except humans. It is widely
known as the vitamin whose deficiency causes scurvy in humans
(Li, Byers, and Walvekan 2008; Velandia et al. 2008; Moyad and Combs
2007).
Biotin, also known as vitamin H or B7 (coenzyme R, Biopeiderm;
Fig. 1), is a water-soluble B-complex vitamin composed of an ureido
(tetrahydroimidizalone) ring fused with a tetrahydrothiophene ring.
A valeric acid substituent is attached to one of the carbon atoms of the
tetrahydrothiophene ring. Biotin is important as a cofactor in the cataly-
sis of essential metabolic reactions to synthesize fatty acids, in gluconeo-
genesis, and in metabolising leucine (Holmberg et al. 2005).

Figure 1. Chemical structures of ascorbic acid and biotin.

FT-IR Spectrophotometric Analysis of Ascorbic Acid and Biotin 1323

The techniques used for the assay of ascorbic acid and biotin are
high-performance liquid chromatography (HPLC; Gioia et al. 2008;
Chaistain, Bowers-Komrod, and McCormick 1985), spectrophotometry
(Meng et al. 2008; Ozyurek et al. 2007), and solid-phase 125I-avidin assay
techniques (Tolymat and Mock 1989). There are some studies regarding
FT-IR analysis of ascorbic acid (Lohmann, Pagel, and Penka 1984) and
biotin (Swamy, Heimburg, and Marsh 1996).
The aim of the present study is to investigate the potential of FT-IR
spectrometry to quantify vitamin C and vitamin H in pharmaceutical pre-
parations. The proposed analytical method in this study was performed
on standards of vitamin C and vitamin H and four different pharmaceu-
tical formulations (namely Biotin, sample A, containing about 1000 mg of
vitamin H per tablet; Cevarol, sample B, containing about 500 mg of
vitamin C per tablet; Supradyn, sample C, containing about 150 mg
of vitamin C and 0.25 mg of vitamin H per tablet).
The main objective of this work was to develop a chemometric proce-
dure for the fast and accurate determination of the two active principles
under study. The Beer-Lambert law and=or principal component regres-
sion plus (PCRþ) and partial least squares (PLC) approaches, reducing
the sample pretreatment, and providing direct FT-IR measurement.

EXPERIMENTAL

Apparatus

Data acquisition was performed using a Spectrum 100 Systems FT-IR

spectrometer equipped with Spectrum for Windows version 5.01 (Perkin-
Elmer Co., Beaconsfield, UK). The commercial software used to generate
analysis for the components analysis were Spectrum Beer’s Law version
2.01 and Spectrum QUANTþ respectively (Perkin-Elmer).

Reagents and Materials

Ascorbic acid and biotin standards used for this study were provided
by Sigma (St. Louis, MO) and Merck (Darmstadt, Germany) respec-
tively. The pharmaceuticals were manufactured by Nature Bounty,
Inc., Bohemia, NY, USA (BIOTIN1 1000 mmg=tablet, sample A), by
Memphis Co. for Pharmaceutical and Chemical Industries Cairo, Egypt
(CEREVOL1 500 mg=tablet ascorbic acid, sample B), and by Euro-
pharm Braşov Romania (SUPRADYN1 150 mg of ascorbic acid and
250 mg biotin per tablet, sample C), respectively. Spectral grade KBr from
Merck was used for disk preparation.
1324 A. A. Bunaciu et al.

Recommended Procedures

Conventional fused KBr disk spectra were recorded between 4000

and 350 cm
1 by averaging 32 scans for each spectrum with a resolution
of 4 cm
1 (data point resolution=interval cm
1) with a deutrated trigly-
cine sulfate (DTGS) detector from samples prepared by compressing
2.0 mg of drug sample with spectral grade KBr, while the background
was spectral grade KBr. For calibration, the spectra were recorded with
the same detector from samples prepared by compressing the standard
substance, vitamin C (0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, and 0.6 mg, respec-
tively) and vitamin H (0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, and 0.6 mg, respec-
tively) in spectral-grade KBr.

RESULTS AND DISCUSSION

Figures 2 and 3 show the spectra of standard ascorbic acid and biotin
as well as for the pharmaceutical preparations using the KBr disk
method.

Figure 2. FT-IR standard spectra for ascorbic acid and biotin, respectively.
FT-IR Spectrophotometric Analysis of Ascorbic Acid and Biotin 1325

Figure 3. FT-IR spectra for pharmaceutical preparations studied: biotin,

Cevarol, and Supradyn, respectively.

We studied the possibility of using the Beer–Lambert law for the

quantitative determination of vitamins C and H in pharmaceutical
products at 1675 cm
1 for vitamin C, corresponding to C ¼ C stretching,
and at 3420 cm
1 for vitamin H, corresponding to COOH stretching.
The measurements, carried out in the previously mentioned conditions,
provide a typical calibration line corresponding to

Table 1. Quantitative determination of ascorbic acid and biotin using

spectrophotometric Beer’s law
Sample

Parameter Cevarol (A) Biotin (B) Supradyn (C)

Vitamin C content (mg=tablet) 487.2 — 153.6

RSD (%) (n ¼ 5) 2.15 2.83
Biotin content (mg=tablet) — 995.8 256.8
RSD (%) (n ¼ 5) 2.69 2.95
1326 A. A. Bunaciu et al.

Figure 4. Calibration graphs for FT-IR determination: A, ascorbic acid

(vitamin C), and B, biotin (vitamin H).

T ¼ 72:22 þ 5:222Cðmg VitCÞ

with a regression coefficient, R, of 0.9921 and

T ¼ 57:28 þ 3:209Cðmg VitHÞ

with a regression coefficient, R, of 0.9936.

Figure 4 (a and b) presents the calibration curves for the two active
principles studied using the KBr disk method, and Table 1 shows the
results obtained by the Beer’s law method, where A, B, and C represent
the pharmaceuticals formulations used.
As can be seen in Table 1, the results are satisfactory with the claimed
amount of the active ingredients, and therefore we suggest the use of the
Beer’s law method, because of the smaller value of RSD (<3.0%).
We also applied the chemometric methods (PCRþ and PLS appro-
aches) for the quantitative determination of vitamins C and H in pharma-
ceutical products, but the results obtained are greater than the theoretical
values, so we believe Beer’s law is better for the simultaneous quantitative
analysis of these vitamins.

CONCLUSIONS

It is clear that FT-IR spectrometry is capable of direct determination of

vitamins C and H in several formulations. The method proposed is sim-
ple, precise, and not time-consuming compared to the chromatographic
methods that exist in literature. Quantification could be done in about
5–10 min, including sample preparation and spectral acquisition.
FT-IR Spectrophotometric Analysis of Ascorbic Acid and Biotin 1327

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