International Journal of Cosmetic Science, 2014, 36, 494–504
12152
Factors affecting formulation characteristics and stability of
ascorbic acid in water-in-oil creams
M. A. Sheraz*, M. F. Khan*, S. Ahmed*, S. H. Kazi*, S. Rehman Khattak† and I. Ahmad*
*Baqai Institute of Pharmaceutical Sciences, Baqai Medical University, 51, Deh Tor, Toll Plaza, Super Highway, Gadap Road, Karachi 74600,
Pakistan and †Central Drug Laboratories, Ministry of Health, Karachi 74400, Pakistan
Received 12 March 2014, Accepted 25 June 2014
Keywords: ascorbic acid, citric acid, kinetics, pH, stability, water-in-oil creams
SYNOPSIS
OBJECTIVE: The present investigation is based on the formulation
of water-in-oil (w/o) cream preparations of ascorbic acid (AA) at
pH 4–6 using different emollients and humectants.
METHODS: The preparations were stored in the dark at 30°C for
a period of 3 months and were studied for their chemical and phys-
ical stability.
RESULTS: The pH of the creams appeared to influence the stability
of AA as the degradation was found to increase with an increase in
pH. The degradation of AA in w/o creams followed an apparent first-
order kinetics. Among all the preparations, the creams containing
castor oil as emollient and glycerine as humectant showed highest
viscosity and minimum degradation as compared with the creams
containing other excipients. This indicated that higher the viscosity
of the medium lower will be the degradation of AA. Citric acid (CT)
was found to decrease the rates of degradation of AA in all the cream
preparations.
CONCLUSION: The physical mixtures of AA and CT showed no
interaction between the two compounds by FTIR spectrometry indi-
cating that the reduction in rates of degradation is due to the anti-
oxidant activity of CT which protected AA to some extent from the
atmospheric oxygen.
Re
sume

OBJECTIF: La pr
esente
etude est bas
ee sur la formulation de pr
epara-
tions de cremes eau dans l’huile (w/o) contenant de l’acide ascorbique
(AA)  a pH 4-6 en utilisant diff
erents
emollients et des humectants.
METHODES: Les pr
eparations ont
et
e conserv
ees a  l’obscurit
e a

30°C pendant une p
eriode de trois mois, et ont
et
e
etudi
es pour
leur stabilit
e chimique et physique.


RESULTATS: Le pH des cremes semblait influencer la stabilit
e de AA
puisqu’il a
et
e constat
e que la d
egradation augmentait avec l’augmen-
tation du pH. La d
egradation de AA dans les cremes w/o suit une
cin
etique apparente de premier ordre. Parmi toutes les pr
eparations,
les cremes a base d’huile de ricin comme
emollient et avec l’humec-
tant comme la glyc
erine ont montr
e une d
egradation minimale par
rapport aux cremes contenant d’autres excipients. Ceci indique que
plus la viscosit
e est
elev
ee, moins rapide sera de la d
egradation de
Correspondence: Muhammad Ali Sheraz, Baqai Institute of Pharmaceu-
tical Sciences, Baqai Medical University, 51, Deh Tor, Toll Plaza, Super
Highway, Gadap Road, Karachi, 74600, Pakistan. Tel.: +92-21-
34410293; fax: +92-21-34410317; e-mail: ali_sheraz80@hotmail.com
494 © 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie
doi: 10.1111/ics.
l’AA. L’acide citrique (CT) a
et
e trouv
e capable de r
eduire le taux de
d
egradation des AA dans toutes les pr
eparations a  base de creme.
CONCLUSION: Les m
elanges de AA et CT n’ont montr
e aucune
interaction entre les deux compos
es par spectrom
etrie FTIR ce qui
indique que la r
eduction des taux de d
egradation est due a  l’activit
e
antioxydante de CT qui prot
egeait AA dans une certaine mesure de
l’oxygene de l’air.
Introduction
Ascorbic acid (AA), also known as vitamin C, is one of the most
commonly used antioxidants in both cosmetic and pharmaceutical
preparations. Due to these properties, it has been used as an anti-
ageing ingredient in cosmetic preparations and as a stabilizer in
various pharmaceutical dosage forms [1–6]. AA is not produced in
human body and, therefore, it is required from an external source
on daily basis in order to promote health and to prevent disease
conditions, like scurvy. Biologically AA is known to perform a
number of significant functions including the growth and mainte-
nance of teeth, gums, bones, ligaments, blood vessels, etc. [7].
AA is a highly sensitive compound and is degraded into dehy-
droascorbic acid (DAA) which on further degradation forms 2,3-dike-
to-L-gulonic acid (DKGA), oxalic acid, etc. in aqueous media [8–10].
DAA possesses biological activity similar to that of AA whereas
DKGA and other degradation products are biologically inactive [8,
10–12]. AA is relatively more stable in dry form as compared with
its aqueous solution [6, 8–10, 13]. Its degradation is affected by a
number of factors including oxygen, temperature, humidity, pH,
light and formulation ingredients [1–3, 10, 12, 14, 15].
In the recent past, interest in the topical use of AA has gained
considerable importance due to its scavenging activity against reac-
tive oxygen species (ROS) produced on exposure to sunlight and
thus delaying the effect of ageing [16]. Various workers have formu-
lated different AA topical preparations [1–4, 16–32] but still the sta-
bility of AA remains a major concern. Derivatives of AA like sodium
ascorbate, ascorbyl palmitate, etc. are also widely used as antioxi-
dants in cosmetics and pharmaceutical preparations [6, 33] but they
lack the biological activity similar to that of AA [16, 28]. Various
stabilizers like boric acid, citric acid, ferulic acid, sodium metabisul-
fite, sodium oxalate, sodium thiosulfate, tartaric acid, etc. are known
to inhibit or slow down the degradation of AA and have been
employed for its stabilization [3, 26, 34–37]. Citric acid (CT) was
found to be better than other stabilizers such as tartaric and boric
acids in slowing down the degradation of AA in o/w creams [3].
Stability of AA in water-in-oil creams M. A. Sheraz et al.

Previously, we have reported that various formulation factors

such as carbon chain length of the emulsifier, nature of stabilizer,
redox potentials, viscosity and pH of the medium play an important
role in the degradation/stabilization of AA in oil-in-water (o/w)
cream formulations [1–3]. However, in this study, an attempt has
been made to formulate different water-in-oil (w/o) creams of AA
as a potential topical or cosmetic preparation. Different factors such
as excipients, medium pH and viscosity have been considered in
the formulation of oily creams which may affect the physical as
well as chemical stability of AA during storage. Moreover, the
inhibitory effect of CT on the degradation of AA at pH 6 has also
been investigated in w/o creams with respect to the factors like
medium viscosity and formulation characteristics. A synergistic
effect between CT and AA is known, but no study to the best of
our knowledge has so far been conducted to explain any interac-
tion between the two compounds at molecular level. A study of
such factors may help a great deal in developing topical prepara-
tions of AA with optimum stability.
Preparation of creams
The emulsifiers were heated and melted in a glass jar immersed in
a water bath at 60–70°C. AA was separately dissolved in a small
portion of distilled water. Humectants were dissolved in the
remaining portion of water and mixed with the oily phase along
with the heated emollients (continuous phase) with constant stir-
ring until the formation of a homogenous emulsion. The homoge-
nous mass was cooled to 40°C and AA solution was added to it
with constant stirring. This mass was thoroughly mixed using a
mechanical mixer with a glass stirrer at 1000 rpm for 5 min. The
pH of the cream was adjusted to the desired value, and the con-
tents again mixed for 10 min at 500 rpm. The contents were pro-
tected from the atmospheric oxygen during mixing by positioning
the glass stirrer at the bottom of the container and covering it
during mixing to avoid air. All the creams were prepared under
uniform conditions to maintain their individual physical character-
istics.

Materials and methods

Materials Table I Composition of cream formulations (w/o) containing ascorbic acid

L-ascorbic acid (99%), cetyl alcohol (>96%), citric acid (99.5%),

glycerine (87%), propylene glycol (99.5%), butylene glycol (99%), Ingredients (%w/w)
acetic acid (99%), acetone (99.5%), acetonitrile (99.9%), benzene
(99.9%), ethanol (96%) and methanol (99.9%) were purchased from Cream
no. pH WF CA MO AO CO GL BG PG AA CT DW
Merck Chemicals (Darmstadt, Germany). The oils (mineral, almond
and castor oil), dehydroascorbic acid (80%) and phenylhydrazine
(>99%) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). 2, 1a 4 21 4 32

5

2
36
3-Diketogulonic acid was prepared by the hydrolysis of dehydroasc- 1b 5 21 4 32

5

2
36
orbic acid (DAA) according to the method of Homann and Gaffron 1c 6 21 4 32

5

2
36
1d 6 21 4 32

5

2 0.4 35.6
[38]. All other chemicals used in the study were of analytical grade
2a 4 21 4 32



5 2
36
obtained from Merck and Co. (Whitehouse Station, NJ, U.S.A.). 2b 5 21 4 32



5 2
36
Freshly boiled glass-distilled water was used throughout the study. 2c 6 21 4 32



5 2
36
2d 6 21 4 32



5 2 0.4 35.6
3a 4 21 4
32
5

2
36
Cream formulations 3b 5 21 4
32
5

2
36
3c 6 21 4
32
5

2
36
On the basis of the various cream formulations reported in the lit-
3d 6 21 4
32
5

2 0.4 35.6
erature [1–3, 39–43], the following basic formula was used for the 4a 4 21 4
32

5
2
36
preparation of w/o creams containing AA: 4b 5 21 4
32

5
2
36
4c 6 21 4
32

5
2
36
4d 6 21 4
32

5
2 0.4 35.6
Oil phase Percentage (w/w) 5a 4 21 4
32


5 2
36
5b 5 21 4
32


5 2
36
5c 6 21 4
32


5 2
36
Emulsifiers 5d 6 21 4
32


5 2 0.4 35.6
Lanolin (wool fat) 21 6a 4 21 4

32 5

2
36
Cetyl alcohol 4 6b 5 21 4

32 5

2
36
Emollients 6c 6 21 4

32 5

2
36
Mineral oil/Almond oil/Castor oil 32 6d 6 21 4

32 5

2 0.4 35.6
Aqueous phase 7a 4 21 4

32
5
2
36
Humectants 7b 5 21 4

32
5
2
36
Glycerine/propylene glycol/butylene glycol 5 7c 6 21 4

32
5
2
36
Active ingredient 7d 6 21 4

32
5
2 0.4 35.6
Ascorbic acid 2 8a 4 21 4

32

5 2
36
Vehicle 8b 5 21 4

32

5 2
36
Distilled water 36 8c 6 21 4

32

5 2
36
Stabilizer 8d 6 21 4

32

5 2 0.4 35.6
Citric acida 0.40 (1.90 9 10
2 M)

WF, lanolin; CA, cetyl alcohol; MO, mineral oil; AO, almond oil; CO, castor oil;
a GL, glycerine; BG, butylene glycol; PG, propylene glycol; AA, ascorbic acid; CT,
The stabilizer concentration used was found to be effective in the inhibition of
degradation of AA in cream formulations [3]. citric acid; DW, distilled water.

© 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie 495
International Journal of Cosmetic Science, 36, 494–504
Stability of AA in water-in-oil creams
In the case of creams containing CT, its required amount was
added directly to the AA solution, and the creams were prepared
by the same procedure as described above. The compositions of the
various cream formulations used in this study are given in the
Table I.
pH measurements
The pH measurements of the creams were carried out using a
digital pH meter (model-CP501; sensitivity 0.01 pH units, Elme-
tron, Poland). All measurements were made with a combination
glass electrode, and the temperature of the creams was recorded
by the help of a probe attached with the pH meter. The instru-
ment was calibrated using the commercially available buffer tab-
lets (Merck) of pH 4.0 and 7.0 dissolved in 100 mL of distilled
water.
The glass electrode was immersed directly into the cream, kept for
few seconds to equilibrate, and the pH was adjusted in the range of
4.0–6.0 with phosphoric acid/sodium hydroxide solution [1–3, 44].
Storage of creams in the dark
To determine the stability of various cream formulations, samples
were stored in a dark cupboard in tightly closed glass containers at
30  1°C for a period of 3 months. The samples were analysed
periodically for the content of AA and the presence of any degrada-
tion products.
Thin-layer chromatography
The following thin-layer chromatography (TLC) systems were used for
the separation and identification of AA and its degradation products:
Adsorbent (a) Silica gel GF 254 (250-lm) pre-coated plates (Merck)
Solvent systems S1: acetic acid-acetone-methanol-benzene
(5 : 5 : 20 : 70, v/v) [45]
S2: ethanol-10% acetic acid-water (90 : 10, v/v) [46]
S3: acetonitrile-butylnitrile-water (66 : 33 : 2, v/v) [47]
Temperature 25–27°C
Location of spots AA: UV light, 254 nm (Uvitec lamp, UK)
DAA and DKGA: Spray with a 3% aqueous
phenylhydrazine hydrochloride solution
Assay methods
UV spectrometric method for the assay of creams containing AA
All absorbance measurements and spectral determinations were
performed on Shimadzu UV-visible spectrometer (UV-1601) using
matched quartz cells of 10-mm path length. The instrument cali-
bration for the absorbance scale was checked according to the
method described in British Pharmacopoeia [44] by using 0.057–
0.063 g/L of potassium dichromate in 0.005 M sulphuric acid and
the wavelength scale was automatically calibrated by the instru-
ment. The baseline was corrected by the built-in baseline memory
at the initializing period. Auto-zero adjustment was made by a
one-touch operation.
The creams were thoroughly mixed, and a quantity of 2 g was
accurately weighed. The assay of AA was carried out by the UV
method of Zeng et al. [48] and Ahmed et al. [1–3]. The method was
496 © 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie
M. A. Sheraz et al.
slightly modified and involved the extraction of AA with methanol-
acetone mixture; 1 : 1, v/v (3 9 10 mL), followed by pH adjustment
to 2.0 with H3PO4. The absorbance measurement of the solution was
made at 245 nm using an appropriate blank. The concentration of
AA was calculated using 560 as the value of specific absorbance [A
(1%, 1 cm)] at the analytical wavelength [1, 13]. DAA has negligible
absorbance at 245 nm [49, 50] and, therefore, does not interfere
with the assay.
Iodimetric method for the assay of AA in creams
The assay of AA in creams was also carried out according to the
procedure of British Pharmacopoeia [44] as follows:
The cream (2 g) was transferred to a flask containing 40 mL of
distilled water and 10 mL of 1 M sulphuric acid. The solution was
mixed and titrated with 0.02 M iodine solution, using 1 mL of
starch solution as indicator until a persistent violet-blue colour was
obtained. Each mL of 0.02 M iodine solution is equivalent to
3.52 mg of AA. All experiments were performed in triplicate and a
mean value was calculated.
Retention kinetics of AA in w/o creams
The retention rate (r) can be defined as the ratio of measured AA
concentration (AAfound) to its initial concentration (AAinitial). The r
of AA in cream formulations was calculated by using the following
formula [23, 24]:
AAfound
r¼  100:
AAinitial
Viscosity measurements
The viscosity of the creams was measured using a digital viscome-
ter (Model NDJ-8SN; China). The instrument was calibrated using
the manufacturer’s viscosity standard. A 100 g quantity of the
cream was placed in a beaker and the spindle (No. 3) was dipped
into the cream up to the given mark. The spindle was rotated at a
speed of 3.0 rpm for 1 min, and the viscosity was recorded at
30  1°C. The test was repeated three times to account for the
experimental variability and the average viscosity was noted.
Creaming index
The physical stability of the prepared creams was observed visually
for any signs of physical instability such as change in colour, phase
separation, etc. The creaming index (CI) was determined by using
the ratio of the height of cream or aqueous layer in the container
(HC) to the initial or total height of the emulsion in the container
(HE) at various time intervals [51, 52]:
HC
CIð%Þ ¼  100:
HE
Fourier Transform Infrared spectrometry
Fourier transform infrared (FTIR) spectrometry was employed for
the observation of any possible interactions between AA and CT.
The AA–CT physical mixtures were prepared in different ratios,
that is, 1 : 1, 1 : 2 and 2 : 1. Each sample was thoroughly ground
and mixed in a mortar and pestle for 5 min before analysis.
All spectra were collected using a diamond crystal (Smart iTR)
International Journal of Cosmetic Science, 36, 494–504
Stability of AA in water-in-oil creams M. A. Sheraz et al.

sampling assembly attached to a Thermo Scientific Nicolet iS10 Table II Validation data for the analysis of ascorbic acid in w/o cream
spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.). formulations by the iodimetric methoda
Each spectrum was collected in a range of 4000–700 cm
1 by per-
forming 32 scans with a resolution of 4 cm
1. The spectral data Linearity
were analysed using the Omnic software (version 8.1, Thermo Range 1.25–40 mg
Fisher Scientific Inc.). Correlation coefficient 0.9999
Slope 5.74 9 10
1
Intercept
0.0356
Results and discussion SE of slope 0.0889
SE of intercept 0.0506
SD of intercept 0.1131
Formulation of the w/o creams
Recovery range (%)b 97.68–102.08
The details of the composition of w/o creams formulated in this Accuracy (%)c  SD 99.81  1.769
study are given in Table I. All creams were prepared with a 2% Precision (%RSD)d 1.768
Selectivity
concentration of AA. Each ingredient was selected on the basis of Recovery range (%)b 96.67–101.67
its common use as topical excipient in cream formulations as well Accuracy (%)c  SD 99.29  1.945
as its suitability for the compositions. The amount of emulsifying LODe (mg) 0.65
agents, lanolin and cetyl alcohol, are constant in all preparations LOQf (mg) 1.97
whereas different emollients (mineral, almond and castor oils) have
been used with each humectant (glycerine, propylene glycol and a
n = 5.
butylene glycol) of varying viscosity at pH 4–6 (Table I). The b
Recovery (%) = (amount found/amount added) 9 100.
creams with mineral oil and butylene glycol have not been studied c
Accuracy (%) = Mean recovery range.
due to immiscibility of the two components [6] resulting in immedi- d
%Relative standard deviation = (SD/Mean) 9 100.
ate separation of the two phases. e
Limit of Detection = 3.3 9 (SD of intercept/slope).
f
Limit of Quantification = 10 9 (SD of intercept/slope).

Appearance of the creams
The w/o creams were off-white to slight yellow in appearance proba-
bly due to the yellow colour of lanolin [6, 13, 44]. All creams after
final adjustment of the pH and uniform mixing were consistent in
their appearance, with no phase separation or any other signs of
instability. No signs of grittiness or gritty particles were observed
visually and after spreading them on the front of hands thus indicat-
ing uniform mixing and homogenization of all ingredients.
the storage of w/o cream formulations (pH 4–6) was carried out by
TLC using appropriate solvent systems (Materials and Methods). All
the formulations showed the presence of DAA, which was identified
by comparison of the Rf value and spot colour with those of the
standard compound. The formation of DAA in cream formulations
of AA has also been reported previously in o/w creams [1–3]. The
formation of DKGA has not been detected in these formulations,
which may be due to the fact that this compound is formed at
neutral and alkaline pH [1, 3, 8].

Assay of AA in w/o creams

The UV spectrometric method has previously been applied to the
analysis of AA in o/w creams [1–3]. However, in this case, the oily
nature of the excipients caused some problems in the extraction
and filtration of AA from the creams. Moreover, the excipients of
the formulations (e.g. lanolin, almond oil) were also found to inter-
fere with the spectrometric analysis of AA at 245 nm as they
showed strong absorption in the region of 200–300 nm. Therefore,
the iodimetric method given in the British Pharmacopoeia [44] was
employed for the analysis of AA in the creams.
The iodimetric method has previously been applied to the analy-
sis of o/w creams of AA by Ahmad et al. [2]. However, in this
study, the method has been applied to w/o creams and, therefore,
it was validated prior to analysis in the presence of all excipients
including stabilizer and degradation products. The method has
been found to be precise, accurate and selective for all w/o cream
formulations as the excipients and the degradation products do not
interfere with the assay of AA present in the creams (Table II).
Identification of the degradation products
AA is known to reversibly oxidize to DAA, which is further
degraded irreversibly to form DKGA in neutral or alkaline solu-
tions. It is also a sensitive compound and degrades by hydrolysis to
form other products like threonic acid, oxalic acid, etc. [8, 10–12].
The identification of the degradation products of AA formed during
Retention of AA in w/o creams
The amount of AA has been found to decrease with time in all the
cream formulations probably due to the oxidation of the vitamin.
The % loss of AA has been found to be different in each formula-
tion which indicates the role of pH and various formulation factors
on the degradation of AA. All the cream formulations on storage
for 3 months have been found to retain AA in the range of around
9–44% with the highest values of r (~44%) observed in formula-
tion 6 of pH 4 whereas the maximum loss (~90%) has been
observed in formulation 2 at pH 6 (Fig. 1). These values show bet-
ter stability than those previously reported for w/o emulsions of
AA containing soybean and moringa oil where the r values of 50%
and 30% were observed after 30 days of storage at 4 and 25°C,
respectively [23].
Kinetics of degradation of AA
The assay data on the degradation of AA in cream formulations
were subjected to a kinetic treatment, and the reaction was found
to follow first-order kinetics. The apparent first-order rate constants
(kobs) determined from the plots of log concentration vs. time
(R2 = 0.990–0.997) are reported in Table III. Various studies have
earlier reported a similar order of reaction for the degradation of
AA in o/w and w/o emulsions, multiple emulsions, etc. [1–4, 23,
24, 53]. It appears that the pH and formulation characteristics of

© 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie 497
International Journal of Cosmetic Science, 36, 494–504
Stability of AA in water-in-oil creams M. A. Sheraz et al.

Figure 1 Change in retention rate (r) of AA with time in w/o cream formu-
lations 2 (●) and 6 (▲) at pH 4–6.

Table III Apparent first-order rate constants (kobs) for the degradation of
ascorbic acid in w/o cream formulations in dark at pH 4–6

kobs 3 102 (day
1)a,b,c  SD

Cream
formulation pH 4.0 5.0 6.0d

1 1.27  0.10 1.46  0.08 2.14  0.14 (1.79  0.09)

2 2.08  0.19 2.29  0.16 2.68  0.18 (2.17  0.15)
3 1.15  0.08 1.31  0.09 1.53  0.13 (1.34  0.11)
4 1.21  0.13 1.38  0.11 1.62  0.12 (1.39  0.10)
5 1.30  0.09 1.49  0.12 1.73  0.11 (1.45  0.12)
6 0.94  0.05 1.10  0.07 1.39  0.09 (1.19  0.09)
7 1.11  0.09 1.25  0.09 1.51  0.13 (1.25  0.08)
8 1.24  0.12 1.40  0.09 1.62  0.11 (1.42  0.13)

a
The rate constants at pH 4.0–6.0 represent the values for formulations a–d of
each cream, respectively.
b
The values of rate constants are relative and depend on specific experimental
Figure 2 Plots of rate constants (kobs) for the degradation of AA vs. pH in
conditions including storage conditions.
c
n = 3. cream formulations (1–8). Cream 1 (mineral oil/glycerine), 2 (mineral oil/
d
The values in parenthesis are for the creams formulated with CT at pH 6. propylene glycol), 3 (almond oil/glycerine), 4 (almond oil/butylene glycol), 5
(almond oil/propylene glycol), 6 (castor oil/glycerine), 7 (castor oil/butylene
glycol), 8 (castor oil/propylene glycol).
creams greatly affect the rate of reaction, and hence these factors
are discussed in the following sections.

Effect of pH
Degradation of AA is highly influenced by the pH of the medium
as higher stability is observed in acidic solutions compared with
that of the neutral and alkaline solutions [1, 12, 54]. Similarly, an
increase in rate of AA degradation has been observed with an
increase in pH in all cream formulations, that is,
pH 6 [ 5 [ 4
This could be due to the fact that ionized form of AA (pKa 4.17)
[13] is more susceptible to oxidation as compared with the non-
ionized form of the molecule. The ionized form of AA accounts for
about 50–85% at pH 4–6 that facilitates the oxidative degradation
of AA with increasing pH as compared with that of the non-ionized
form [1, 55]. The degradation pattern of AA in w/o creams with
increasing pH is similar to that observed for o/w creams [1] as well
as for aqueous solutions of AA where the chemical oxidation takes
place by molecular oxygen [56]. The plots between rate constant
and pH indicate an increasing pattern with an increase in pH from
4 to 6 in all cases (Fig. 2). However, the difference in the rates
between pH 4 and 5 is less as compared with that of 5 and 6. This
could be due to a decrease in the redox potential of AA with
increasing pH, that is +0.166 V at pH 4 [13] to +0.096 at pH 6
[57]. Similar effects have also been observed in o/w creams of AA
[1] which indicate that the most suitable range for the preparation
of AA cream formulations is pH 4–5.
Effect of formulation characteristics
It is of utmost importance to consider formulation characteristics
before the preparation of any dosage form. Different combinations
of emollients and humectants have been employed in the current
study at different pH values to formulate the most compatible w/o
creams of AA with highest stability. It has been observed that the

498 © 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie
International Journal of Cosmetic Science, 36, 494–504
Stability of AA in water-in-oil creams
replacement of an excipient with another of the same class but
with different physicochemical properties also affects the stability of
AA in the cream formulations. The level of impurity in the ingredi-
ents may also play an important role in the stability of the active
drug as sometimes the impurities (such as heavy metals) present in
minor and/or trace amount may accelerate the degradation. The
changes observed with different emollients and humectants are
discussed as follows:
Effect of emollients
Emollients, also known as moisturizers, are an important class of
excipients that are incorporated in topical preparations due to their
softening and pliable effects [58]. They are often added in the
cream formulations to modify the characteristics of pharmaceutical
vehicles on skin for better penetration of the active drug [58, 59].
To observe the effect of emollients on the stability of AA, three dif-
ferent emollients namely, mineral oil (liquid paraffin), almond oil
and castor oil were selected for this study. These emollients have
been found to affect the stability of AA in w/o cream formulations.
This can be ascertained from the values of kobs of different creams
that show a change in the rate constant with a change in the nat-
ure of emollient (Table III). The formulations prepared with min-
eral oil (creams 1–2) are found to have the highest rate of
degradation (Table III) for AA followed by creams containing
almond oil (Creams 3–5). The preparations with castor oil (Creams
6–8) showed highest stability for AA. Thus the rates of degradation
of AA with respect to emollients are in the following order:
mineral oil [ almond oil [ castor oil
The reason for the highest rate of degradation in creams con-
taining mineral oil could be due to the fact that it also undergoes
oxidation which begins with the formation of peroxides during the
induction period [6]. Once the traces of peroxides are formed,
further oxidation of the mineral oil as well as AA would have
proceeded auto-catalytically.
The effect of viscosity should also be considered as it plays an
important role in the degradation of AA in cream formulations. It
has been reported that the more viscous the medium the lesser will
be the degradation of AA [1]. The most viscous oil among the
three emollients is castor oil (~1000 mPa s) followed by almond
(~600 mPa s) and mineral oils (~230 mPa s). The rates of degrada-
tion of AA are also in the similar order, whereas the minimum
rates have been observed in creams with castor oil and maximum
with mineral oil (Table III). The effect of cream viscosity is further
discussed in a later section (Effects of Humectants). This study thus
provides a better understanding of the use of emollients in the
stabilization of semisolid formulations of AA.
Effect of humectants
Humectants are water-soluble organic compounds that can imbibe
water and thus prevent the creams from drying out, improve spread-
ability and also avoid the formation of a crust when the cream is
packaged in a jar [60]. Humectants of varying viscosity are known
to affect the rate of degradation of AA in o/w creams [1]. In this
study, three humectants, that is glycerine (GL), butylene glycol (BG)
and propylene glycol (PG) with varying viscosity of ~935, ~105,
~41 mPa s, respectively, were employed for the preparation of w/o
creams. The creams thus formulated with different emollients and
humectants showed varying viscosities which are reported in
© 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie
International Journal of Cosmetic Science, 36, 494–504
M. A. Sheraz et al.
Table IV Viscositiesa,b of ascorbic acid cream formulations containing differ-
ent emollients and humectants at 25  1°C
Humectant
Glycerine Butylene glycol Propylene
Emollient (~935)c (~105)c glycol (~41)c
Mineral oil (~230)c 1130  13.2d – 595  15.0d
Almond oil (~600)c 1389  11.5d 949  12.5d 749  16.1d
Castor oil (~1000)c 1592  15.1d 1222  16.7d 932  14.5d
a
n = 3.
b
All values are in mPa s.
c
Values in parentheses is the viscosity of the individual substance.
d
The  values are the SD.
Table IV. A linear relationship has been observed between rate con-
stants of degradation vs. the reciprocal of viscosity at pH 4–6 (Fig. 3),
indicating that an increase in viscosity leads to a decrease in the deg-
radation of AA. The humectants have been found to affect the rates
of degradation of AA in the following order:
PG [ BG [ GL
This relationship indicates that AA is more stable under viscous
conditions due to lesser penetration of oxygen into the medium.
Physical stability of the cream formulations
The stabilization of AA in pharmaceutical, cosmetic and food prep-
arations is a great challenge due to its sensitivity to factors like
oxygen, light, pH, temperature, etc. The preparation of any formu-
lation showing both chemical and physical stability requires the
consideration of a number of factors. The w/o cream formulations
have been studied for any physical changes, such as colour
change, creaming, phase separation (breaking), during the storage
period. The colour changes observed in all the cream formulations
over this period are given in Table V. The appearance of the
creams has already been discussed earlier in the section on Appear-
ance of the Creams.
No considerable changes in colour have been observed for the
majority of the creams in the first month. The major colour change
has been observed after 2 months of storage in most cases (Table V).
The colour change also appeared to be affected by the factors respon-
sible for chemical degradation such as pH and viscosity, as the major
changes have been observed in formulations prepared with PG at pH
6 in all the creams (Table V). This is in agreement with the results of
degradation kinetics of AA, where greater degradation was observed
with an increase in pH (Table III) and decrease in the viscosity of the
medium (Table IV). Similarly, the creams prepared with castor oil
showed minimum colour changes as compared with the other cream
formulations (Table V), which is in accordance with the results
observed for chemical degradation.
Creaming is a natural phenomenon that takes place in biphasic
systems and gives an indication of physical instability of the prepa-
ration [51]. It causes inelegancy to the emulsion system but is not
a serious problem due to its reversible nature. A uniform redisper-
sion can be obtained by shaking the emulsion. Therefore, such type
of emulsions should be shaken adequately before use to avoid
499
Stability of AA in water-in-oil creams M. A. Sheraz et al.

Table V Colour changes in the w/o cream formulations of ascorbic acid

stored in dark at 30  1°C for 3 months

Colour change

Days

Cream
No. pH 10 20 30 40 50 60 90

1a 4



+ + +
1b 5



+ + +
1c 6


+ + + ++
1d 6



+ + ++
2a 4


+ + + ++
2b 5


+ + ++ ++
2c 6

+ + ++ ++ +++
2d 6


+ + ++ +++
3a 4



+ + ++
3b 5



+ + ++
3c 6

+ + ++ ++ +++
3d 6



+ + ++
4a 4



+ + ++
4b 5


+ + + ++
4c 6

+ + + ++ +++
4d 6



+ + ++
5a 4



+ + ++
5b 5


+ + + ++
5c 6
+ + + ++ ++ +++
5d 6


+ + ++ +++
6a 4





+
6b 5




+ +
6c 6




+ ++
6d 6





+
7a 4





+
7b 5




+ +
7c 6




+ ++
7d 6




+ ++
8a 4





+
8b 5




+ +
8c 6



+ + ++
8d 6




+ ++

, no change; +, slight change (off-white to yellow); ++, moderate change (yel-

low to dark yellow); +++, major change (dark yellow to rust).

Figure 3 Plots of kobs vs. reciprocal of viscosity in cream formulations (1–8)

containing glycerine (●), butylene glycol (▲) and propylene glycol (♦) at pH
4–6.
incorrect dosage. Creaming separates emulsion into two layers, one
of which is richer in the disperse phase than the other. It often
leads to phase separation or breaking of the emulsion [61–63]. The
results for % creaming index (%CI) and phase separation are
reported in Table VI. None of the formulations showed any signs of
creaming and phase separation for the initial 10 days, but with the
passage of time all formulations have been found to cream and sep-
arate out. The phase separation may separate AA in the aqueous
layer from the cream, which could result in increase chemical
breakdown. In majority of the formulations, the phase separation
has been observed after 2 months of storage (Table VI). No signifi-
cant differences have been observed in the creaming and phase sep-
aration of the w/o formulations after 3 months of storage, but still
the effect of pH and medium viscosity can be speculated from the
results. Comparatively, a better physical stability has been observed
in creams with pH 4 and 5. Similarly, the creams prepared with
castor oil are found to be most stable physically (Table VI). The
highest CI value after 3 months of storage has been observed for
formulation 2c whereas minimum CI value has been observed in
formulations 6a and 7a (Table VI). The appearance of creaming
and phase separation could be ascribed to the differences in the
specific gravities of the liquids used for the formulation of w/o
creams.
Effect of CT on the stability of AA in w/o cream formulations
CT is widely used in pharmaceutical formulations and food prod-
ucts as an acidifying, buffering and chelating agent, flavour enhan-
cer, preservative, etc. [6]. It is also used as an antioxidant and is
known to act synergistically with AA [2, 6, 36, 64]. The addition
of CT in nutritional supplements is known to inhibit the oxidation

500 © 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie
International Journal of Cosmetic Science, 36, 494–504
Stability of AA in water-in-oil creams M. A. Sheraz et al.

Table VI Percent creaming index (%CI) and phase separation (PS) of the w/ the absence of CT with almost similar viscosity. This indicates that
o cream formulations of ascorbic acid stored in dark at 30  1°C for along with appropriate selection of emollient and humectant it is
3 months also important to add a suitable stabilizer like CT to further
enhance the stabilization of AA in topical preparations. The colour
%CI/PS changes appeared to be lesser in creams with CT (Table V) and are
in accordance with the chemical degradation of AA (Table III).
Minimum colour changes have been observed in creams containing
Days castor oil and glycerine (Table V). The creaming index and phase
separation are slightly better in some formulations whereas similar
Cream
no. pH 10 20 30 40 50 60 90
results have been observed in the majority of cases (Table VI). This
could be due to the fact that creaming and phase separation are
more related to the compatibility between the ingredients and may
1a 4
/

/
1.0/
2.1/
4.3/
6.1/
12.5/PS also correlate to their specific gravities rather than the degradation
1b 5
/

/

/
1.8/
3.0/
5.4/
9.5/PS of AA. This indicates that although CT protects AA from chemical
1c 6
/

/
1.1/
2.3/
3.9/
6.4/PS 10.2/PS
degradation, it may not have an effect on the physical stability of
1d 6
/

/
/
1.2/
2.6/
5.0/
7.5/PS
2a 4
/

/
1.2/
2.2/
3.7/
7.5/PS 15.7/PS the creams.
2b 5
/

/
1.1/
2.4/
3.9/
7.4/PS 14.9/PS
2c 6
/

/
1.3/
2.9/
6.6/PS 10.1/PS 20.3/PS
2d 6
/

/
1.0/
3.0/
5.5/
7.1/PS 11.3/PS
3a 4
/

/
1.1/
3.2/
5.0/
6.6/PS 11.0/PS
3b 5
/

/
1.5/
5.2/
6.0/PS 9.1/PS 16.5/PS
3c 6
/
1.1/
2.2/
4.0/
6.7/PS 10.2/PS 17.7/PS
3d 6
/

/
1.4/
2.7/
4.4/
6.0/PS 9.9/PS
4a 4
/

/
1.0/
3.2/
5.7/
7.4/PS 12.5/PS
4b 5
/

/
1.5/
5.4/
7.1/PS 9.1/PS 14.5/PS
4c 6
/
1.3/
2.1/
5.9/
7.9/PS 10.1/PS 15.5/PS
4d 6
/

/
1.6/
4.5/
7.1/PS 9.0/PS 13.7/PS
5a 4
/

/
1.2/
3.0/
4.1/
5.6/
10.5/PS
5b 5
/

/
1.1/
2.8/
4.7/
6.0/
10.7/PS
5c 6
/
1.0/
2.1/
3.6/
6.5/PS 8.4/PS 12.7/PS
5d 6
/
1.3/
3.4/
4.9/
7.0/PS 8.6/PS 13.5/PS
6a 4
/

/

/

/
1.2/
3.2/
7.1/PS
6b 5
/

/

/

/
1.9/
4.0/
7.6/PS
6c 6
/

/

/
1.0/
3.1/
6.0/
10.5/PS
6d 6
/

/

/
1.1/
2.9/
5.7/
10.4/PS
7a 4
/

/

/
1.1/
2.1/
4.7/
7.1/PS
7b 5
/

/

/
1.2/
2.5/
5.5/
8.2/PS
7c 7
/

/
1.0/
2.2/
4.9/
7.4/PS 11.1/PS
7d 6
/

/
1.5/
2.9/
5.5/
7.9/PS 12.4/PS
8a 4
/

/
1.0/
2.2/
3.9/
6.4/PS 12.2/PS
8b 5
/
1.0/
1.9/
4.2/
5.8/PS 7.6/PS 15.4/PS
8c 6
/
1.1/
2.6/
4.9/
6.6/PS 7.0/PS 17.1/PS
8d 6
/

/
1.2/
3.0/
4.8/
6.5/PS 15.4/PS

, no change.

of AA [65]. It has also been found to slow down the degradation of

AA in o/w cream formulations [3].
The creams containing CT appeared identical to the formulations
prepared without it (section Appearance of the Creams) and were
stored and analysed in a similar manner as described earlier. Maxi-
mum degradation of AA has been observed at pH 6 in all formula-
tions; therefore, the pH of the creams containing CT has also been
set at the same value for comparison purposes. The r of AA in the
presence of CT (15.5–36.3%) has been found to be better than
those prepared without it (9.8–30.5%). These results indicate that
the synergistic or inhibitory effect of CT is also present in oily
media which slows down the oxidation of AA. The degradation of
AA in the presence of CT also follows an apparent first-order kinet-
ics with the lowest rate found for formulation 6, that is,
1.19 9 102 day
1 and the highest for formulation 2, that is, Figure 4 Fourier transform infrared spectra of AA, CT and their physical
2.17 9 102 day
1 (Table III). The formulations showed a similar mixtures in 1 : 1, 1 : 2, 2 : 1 ratios along with chemical structures of (a)
order of effectiveness of emollients and humectants as observed in AA (b) CT.

© 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie 501
International Journal of Cosmetic Science, 36, 494–504
Stability of AA in water-in-oil creams M. A. Sheraz et al.

Study of interaction between AA and CT by FTIR spectrometry
FTIR spectrometry has been employed to observe any possible
interaction between AA and CT at the molecular level. This tech-
nique can be used for the detection of any possible interaction or
change from one polymorphic form to the other as a result of a
shift or change in the intensity of absorption band height or area
such as broadening of the peak [66, 67].
AA is a six-carbon keto-lactone that contains four OH groups, of
which two are attached to the lactone ring carbons (enolic OH
groups) and the remaining two are on the side chain C atoms
(Fig. 4). The ability of AA to donate hydrogen atoms (C3 and C4)
makes it a good reducing agent, and therefore, it acts as an antiox-
idant. A comprehensive structural and vibrational study of AA has
been reported by Singh et al. [68]. According to these authors, AA
exhibits four bands in the region 3200–3600 cm
1 for the O–H
stretching vibrations. The bands in the region 2850–3150 cm
1
correspond to the C–H/CH2 stretching modes. The other major
peaks of AA are observed at 1753 (C=O), 1652 (C=C), 1313 (C–H),
1111 (C–O, lactone ring), 1022 (C–O–H, lactone ring), 988 (CH2),
869–680 (C–C) cm
1. CT contains three carboxylic acids in its
structure and an OH group attached to the central carbon atom. It
exhibits absorption bands at 3420 cm
1 for the non-ionized OH
and 2630 cm
1 for the ionized hydroxyl group (Fig. 4). Similarly,
it gives a stretching band at 1730 cm
1 for C=O, 1625 and
1400 cm
1 for COO
vibration and stretching, respectively,
1200 cm
1 for C–O stretching or OH deformation and 940 cm
1
for out-of-plane OH bending (Fig. 4) [69, 70]. The FTIR spectra of
the physical mixtures of AA and CT in 1 : 1, 1 : 2 and 2 : 1 ratios
are shown in Fig. 4. In all the three ratios, a combination of both
compounds can be observed whereas no prominent changes like a
shift or broadening of the peak has been noted in any of the sam-
ples. This indicates that there is no prominent interaction between
the two compounds in physical mixtures and the decrease in rates
of degradation of AA (Table III) is due to the antioxidant activity of
CT that has protected AA from oxidation.
Conclusion
Degradation of AA has been found to be influenced by a number of
factors in the w/o cream formulations. The formulation ingredients,
that is, emollients and humectants, have been shown to play an
important role as they influence the viscosity of the creams and thus
affect the degradation of the vitamin. Castor oil and glycerine are
found to be the most viscous emollient and humectant, respectively,
and hence stabilized AA in cream formulations at pH 4. Inclusion of
a suitable stabilizer like CT in cream formulations is advantageous
for AA to protect it from chemical degradation during storage. All
these factors which affect the chemical stability of AA do not have
any significant effect on the physical stability of AA as creaming and
phase separation has been observed in all creams at certain time peri-
ods. These findings show that even if a drug is chemically stable it is
not necessary that the same formulation may also be stable physi-
cally. A careful approach and detailed study of the selection and
interaction of ingredients of high purity is necessary for the formula-
tion of a safe, effective and stable dosage form.
Acknowledgements
The study was supported and carried out at the Baqai Institute of
Pharmaceutical Sciences, Baqai Medical University, Karachi.

References
1. Ahmad, I., Sheraz, M.A., Ahmed, S., Shaikh,
R.H., Vaid, F.H.M., Khattak, S.U.R. and An-
sari, S.A. Photostability and interaction of
ascorbic acid in cream formulations. AAPS
PharmSciTech. 12, 917–923 (2011).
2. Ahmad, I., Sheraz, M.A., Ahmed, S., Bano,
R. and Vaid, F.H.M. Photochemical interac-
tion of ascorbic acid with riboflavin, nicotin-
amide and alpha-tocopherol in cream
formulations. Int. J. Cosmet. Sci. 34, 123–
131 (2012).
3. Ahmad, I., Sheraz, M.A., Ahmed, S., Shad,
Z. and Vaid, F.H.M. Photostabilization of
ascorbic acid with citric acid, tartaric acid
and boric acid in cream formulations. Int. J.
Cosmet. Sci. 34, 240–245 (2012).
4. Gallarate, M., Carlotti, M.E., Trotta, M.
and Bovo, S. On the stability of ascorbic
acid in emulsified systems for topical and
cosmetic use. Int. J. Pharm. 188, 233–241
(1999).
5. Placzek, M., Gaube, S., Kerkmann, U., Gil-
bertz, K.P., Herzinger, T., Haen, E. and
Przybilla, B. Ultraviolet B-induced DNA
damage in human epidermis is modified by
the antioxidants ascorbic acid and D-alpha-
tocopherol. J. Invest. Dermatol. 124, 304–
307 (2005).
6. Rowe, R.C., Sheskey, P.J. and Quinn, M.E.
Handbook of Pharmaceutical Excipients, 4th
edn, pp. 29–31, 43–46, 77–78, 126–127,
155–156, 181–183, 283–286, 378–
380,445–447, 592–594, 766–770. Phar-
maceutical Press, London, UK (2009).
7. Sheraz, M.A., Khan, M.F., Ahmed, S. and
Ahmad, I. Pharmacological effects of ascor-
bic acid. In: Vitamin C: Dietary Sources, Tech-
nology, Daily Requirements and Symptoms of
Deficiency (Guine, R.P.F., ed.), pp. 191–208.
Nova Science Publishers, Inc., New York
(2013).
8. Ball, G.F.M. Vitamins in Foods Analysis, Bio-
availability and Stability, pp. 289–305. CRC
Press, Boca Raton, FL (2006).
9. DeRitter, E. Vitamins in pharmaceutical for-
mulations. J. Pharm. Sci. 71, 1073–1096
(1982).
10. Johnston, C.S., Steinberg, F.M. and Rucker,
R.B. Ascorbic acid. In: Handbook of Vitamins,
4th edn (Zempleni, J., Rucker, R.B., McCor-
mick, D.B. and Suttie, J.W., eds.), pp. 489–
520. CRC Press, Boca Raton, FL (2007).
11. Li, M., Suzuki, E. and Kurata, T. Effects of
2,3-diketo-L-gulonic acid on the oxidation of
yolk lipoprotein. Biosci. Biotechnol. Biochem.
65, 599–604 (2001).
12. Sheraz, M.A., Ahmed, S., Ahmad, I., Qadeer,
K. and Vaid, F.H.M. Photodegradation and
photostabilization of ascorbic acid in phar-
maceutical preparations. Int. J. Chem. Anal.
Sci. 1, 68–70 (2010).
13. O’ Neil, M.J. (ed.). Monograph on ascorbic
acid (819). In: The Merck Index, 15th edn,
pp. 142–143. Royal Society of Chemistry,
Merck & Co., Inc., White House Station, NJ
(2013).
14. Vaid, F.H.M., Shaikh, R.H., Ansari, I.A. and
Ahmad, I. Spectral study of the photolysis of
aqueous thiamine hydrochloride and ascor-
bic acid solution in the presence and
absence of riboflavin. J. Chem. Soc. Pak. 27,
227–232 (2005).
15. Vaid, F.H.M., Shaikh, R.H., Ansari, I.A. and
Ahmad, I. Chromatographic study of the
photolysis of aqueous thiamine hydrochlo-
ride and ascorbic acid solutions in the pres-
ence and absence of riboflavin. J. Chem. Soc.
Pak. 28, 461–464 (2006).

502 © 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie
International Journal of Cosmetic Science, 36, 494–504
Stability of AA in water-in-oil creams M. A. Sheraz et al.

16. Pinnell, S.R., Yang, H., Omar, M. et al. Topi-
cal L-ascorbic acid: percutaneous absorption
studies. Dermatol. Surg. 27, 137–142
(2001).
17. Akhtar, N., Ahmad, M., Khan, H.M.S., Ak-
ram, J., Gulfishan, G., Mahmood, A. and
Uzair, M. Formulation and characterization
of a multiple emulsion containing 1%
l-ascorbic acid. Bull. Chem. Soc. Ethiop. 24,
1–10 (2010).
18. Akhtar, N., Zulfiqar, N., Gulfishan., Ahmed,
M., Khan, H.M.S. and Saeed, T. Effect of
L–ascorbic acid on the formulation and
characterization of a multiple emulsion from
paraffin oil. J. Chem. Soc. Pak. 32, 724–730
(2010).
19. Austria, R., Semenzato, A. and Bettero, A.
Stability of vitamin C derivatives in solution
and topical formulations. J. Pharm. Biomed.
Anal. 15, 795–801 (1997).
20. Branco, G.F., Rodrigues, M.I., Gioielli, L.A.
and Castro, I.A. Effect of the simultaneous
interaction among ascorbic acid, iron and
pH on the oxidative stability of oil-in-water
emulsions. J. Agric. Food Chem. 59, 12183–
12192 (2011).
21. Darr, D., Dunston, S., Faust, H. and Pinnell,
S. Effectiveness of antioxidants (vitamin C
and E) with and without sunscreens as topi-
cal photoprotectants. Acta Derm. Venereol.
76, 264–268 (1996).
22. Farahmand, S., Tajerzadeh, H. and Farboud,
E.S. Formulation and evaluation of a vita-
min C multiple emulsion. Pharm. Dev. Tech-
nol. 11, 255–261 (2006).
23. Khalid, N., Kobayashi, I., Neves, M.A., Uem-
ura, K. and Nakajima, M. Preparation and
characterization of water-in-oil emulsions
loaded with high concentration of L-ascorbic
acid. LWT. Food Sci. Technol. 51, 448–454
(2013).
24. Khalid, N., Kobayashi, I., Neves, M.A., Uem-
ura, K. and Nakajima, M. Preparation and
characterization of water-in-oil-in-water
emulsions containing a high concentration
of L-ascorbic acid. Biosci. Biotechnol. Bio-
chem. 77, 1171–1178 (2013).
25. Lee, J.S., Kim, J.W., Han, S.H. et al. The stabil-
ization of L-ascorbic acid in aqueous solution
and water-in-oil-inwater double emulsion by
controlling pH and electrolyte concentration.
J. Cosmet. Sci. 55, 1–12 (2004).
26. Maia, A.M., Baby, A.R., Pinto, C.A., Yasaka,
W.J., Suenaga, E., Kaneko, T.M. and Velas-
co, M.V. Influence of sodium metabisulfite
and glutathione on the stability of vitamin C
in O/W emulsion and extemporaneous
aqueous gel. Int. J. Pharm. 322, 130–135
(2006).
27. Pakpayat, N., Nielloud, F., Fortun
e, R., To-
urne-Peteilh, C., Villarreal, A., Grillo, I. and
Bataille, B. Formulation of ascorbic acid mi-
croemulsions with alkyl polyglycosides. Eur.
J. Pharm. Biopharm. 72, 444–452 (2009).
28. Raschke, T., Koop, U., D€ using, H.J. et al.
Topical activity of ascorbic acid: from in
vitro optimization to in vivo efficacy. Skin
Pharmacol. Physiol. 17, 200–206 (2004).
29. Rozman, B. and Gasperlin, M. Stability of
vitamins C and E in topical microemulsions
for combined antioxidant therapy. Drug
Deliv. 14, 235–245 (2007).
30. Rozman, B., Zvonar, A., Falson, F. and Gas-
perlin, M. Temperature-sensitive microemul-
sion gel: an effective topical delivery system
for simultaneous delivery of vitamins C and
E. AAPS PharmSciTech. 10, 54–61 (2009).
31. Rozman, B., Gasperlin, M., Tinois-Tesso-
neaud, E., Pirot, F. and Falson, F. Simulta-
neous absorption of vitamins C and E from
topical microemulsions using reconstructed
human epidermis as a skin model. Eur. J.
Pharm. Biopharm. 72, 69–75 (2009).
32. Zhang, L., Lerner, S., Rustrum, W.V. and
Hofmann, G.A. Electroporation-mediated
topical delivery of vitamin C for cosmetic
applications. Bioelectrochem. Bioenerg. 48,
453–461 (1999).
33. Elmore, A.R. Final report of the safety
assessment of L-ascorbic acid, calcium ascor-
bate, magnesium ascorbate, magnesium
ascorbyl phosphate, sodium ascorbate, and
sodium ascorbyl phosphate as used in cos-
metics. Int. J. Toxicol. 24, 51–111 (2005).
34. Kwakye, J.K. The use of stabilizers in the UV
assay of ascorbic acid. Talanta 51, 197–200
(2000).
35. Lin, F.H., Lin, J.Y., Gupta, R.D. et al. Ferulic
acid stabilizes a solution of vitamins C and E
and doubles its photoprotection of skin. J.
Invest. Dermatol. 125, 826–832 (2005).
36. Selimovic, A., Salkic, M. and Selimovic, A.
Direct spectrophotometric determination of
l-ascorbic acid in pharmaceutical prepara-
tions using sodium oxalate as a stabilizer.
Int. J. Basic Appl. Sci. 11, 106–109
(2011).
37. Tournas, J.A., Lin, F.H., Burch, J.A., Selim,
M.A., Monteiro-Riviere, N.A., Zielinski, J.E.
and Pinnell, S.R. Ubiquinone, idebenone,
and kinetin provide ineffective photoprotec-
tion to skin when compared to a topical
antioxidant combination of vitamins C and
E with ferulic acid. J. Invest. Dermatol. 126,
1185–1187 (2006).
38. Homann, P. and Gaffron, H. Photochemistry
and metal catalyses: studies in a flavin sen-
sitized oxidation of ascorbic acid. Photochem.
Photobiol. 3, 499–515 (1964).
39. Betageri, G. and Prabhu, S. Semisolid prepa-
rations. In: Encyclopedia of Pharmaceutical
Technology, 2nd edn (Swarbrick, J. and Boy-
lan, J.C., eds.), pp. 2436–2457. Marcel Dek-
ker, New York (2002).
40. Block, L.H. Pharmaceutical emulsions and
microemulsions. In: Pharmaceutical Dosage
Forms: Disperse Systems, Vol. 2, 2nd edn,
Chap. 2 (Lieberman, H.A., Rieger, M.M. and
Banker, G.S., eds.). Marcel Dekker, New
York (1996).
41. Flynn, G.L. Cutaneous and transdermal
delivery-processes and systems of delivery.
In: Modern Pharmaceutics, Chap. 8 (Banker,
G.S. and Rhodes, C.T., eds.), pp. 187–235.
Marcel Dekker, New York (2002).
42. Lu, G.W. and Flynn, G.L. Cutaneous and
transdermal delivery-processes and systems
of delivery. In: Modern Pharmaceutics Applica-
tions and Advances, Vol. 2, 5th edn, Chap. 3
(Florence, A.T. and Siepmann, J., eds.).
Informa Healthcare Inc., New York (2009).
43. Vimaladevi, M. Textbook of Cosmetics. Chap. 2
and 17. CBS Publishers, New Delhi (2005).
44. British Pharmacopoeia. Monograph on Ascor-
bic Acid. Electronic version. Her Majesty’s
Stationary Office, London, UK (2013).
45. Ganshirt, H. and Malzacher, A. Separation
of several vitamins of the B group and C by
chromatography. Naturwissenschaften 47,
279–280 (1960).
46. Bolliger, H.R. and Konig, A. Water-soluble
vitamins. In: Thin-layer Chromatography
(Stahl, E., ed.), pp. 304–306. Springer-Ver-
lag, Berlin, Germany (1969).
47. Saari, J.C., Baker, E.M. and Sauberlich, H.E.
Thin-layer chromatographic separation of
the oxidative degradation products of ascor-
bic acid. Anal. Biochem. 18, 173–177 (1967).
48. Zeng, W., Martinuzzi, F. and MacGregor, A.
Development and application of a novel UV
method for the analysis of ascorbic acid. J.
Pharm. Biomed. Anal. 36, 1107–1111
(2005).
49. Davies, M.B., Austin, J. and Partridge, D.A.
Vitamin C. Its Chemistry and Biochemistry,
pp. 57–65. The Royal Society of Chemistry,
Cambridge, UK (1991).
50. Moura, T., Gaudy, D., Jacob, M. and Cass-
anas, G. pH influence on the stability of
ascorbic acid spray-drying solutions. Pharm.
Acta Helv. 69, 77–80 (1994).
51. Meher, J.G., Yadav, N.P., Sahu, J.J. and Sin-
ha, P. Determination of required hydro-
philic–lipophilic balance of citronella oil and
development of stable cream formulation.
Drug Dev. Ind. Pharm. 39, 1540–1546
(2013).
52. Raghavendra, S.N. and Raghavarao,
K.S.M.S. Effect of different treatments for the
destabilization of coconut milk emulsion. J.
Food Eng. 97, 341–347 (2010).
53. Watanabe, Y., Suzuki, T., Nakanishi, H.,
Sakuragochi, A. and Adachi, S. Effect of

© 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie 503
International Journal of Cosmetic Science, 36, 494–504
Stability of AA in water-in-oil creams
ascorbic acid or acyl ascorbate on the sta-
bility of catechin in oil-in-water emulsion.
J. Am. Oil Chem. Soc. 89, 269–274
(2012).
54. Golubitskii, G.B., Budko, E.V., Basova, E.M.,
Kostarnoi, A.V. and Ivanov, V.M. Stability
of ascorbic acid in aqueous and aqueous–
organic solutions for quantitative determina-
tion. J. Anal. Chem. 62, 742–747 (2007).
55. Bisby, R.H., Morgan, C.G., Hamblett, I. and
Gorman, A.A. Quenching of singlet oxygen
by trolox C, ascorbate and amino acids:
effect of pH and temperature. J. Phys. Chem.
103, 7454–7459 (1999).
56. Blaug, S.M. and Hajratwala, B. Kinetics of
aerobic oxidation of ascorbic acid. J. Pharm.
Sci. 61, 556–562 (1972).
57. Fasman, G.D. (ed.). Handbook of Biochemistry
and Molecular Biology, 3rd edn, Vol. 1, pp.
122–130. CRC Press, Cleveland, OH (1976).
58. Hendrickson, R. (ed.). Topical drugs. In:
Remington: The Science and Practice of Phar-
macy, 21st edn, Vol. 2, pp. 1280–1281. Lip-
pincott Williams & Wilkins, Philadelphia,
PA (2005).
59. Srivastava, P. Excipients for semisolid prepa-
rations. In: Excipient Development for Pharma-
ceutical, Biotechnology, and Drug Delivery
Systems (Katdare, A. and Chaubal, M.V.,
eds.), p. 203. Informa Healthcare, New York
(2006).
504
60. Idson, B. and Lazarus, J. Semisolids. In: The
Theory and Practice of Industrial Pharmacy
(Lachman, L., Lieberman, H.A. and Kanig,
J.L., eds.), p. 544. Lea & Febiger, Philadel-
phia, PA (1987).
61. Billany, M. Suspensions and emulsions. In:
Pharmaceutics: The Design and Manufacture of
Medicines (Aulton, M.E., ed.), 3rd edn, pp.
383–405. Churchill Livingstone, London,
UK (2007).
62. Rungseevijitprapa, W., Siepmann, F., Siep-
mann, J. and Paeratakul, O. Disperse sys-
tems. In: Modern Pharmaceutics – Basic
Principles and Systems, Vol. 1, 5th edn (Flor-
ence, A.T. and Siepmann, J., eds.), pp. 357–
421. Informa Healthcare Inc., New York
(2009).
63. Schramm, L.L. Emulsions, Foams, and Suspen-
sions–Fundamentals and Applications. Wiley
VCH Verlag GmbH & Co-KGaA, Weinheim,
Germany (2005).
64. Higashi-Okai, K., Ishikawa, A., Yasumoto, S.
and Okai, Y. Potent antioxidant and radical-
scavenging activity of Chenpi–compensatory
and cooperative actions of ascorbic acid and
citric acid. J. UOEH 31, 311–324 (2009).
65. Doores, S. pH control agents and acidulants.
In: Food Additives (Branen, L.A., Davidson,
P.M., Salminen, S. and Thorngate, J.H.,
eds.), p. 637. Marcel Dekker, New York
(2002).
© 2014 Society of Cosmetic Scientists and the Soci
et
e Francßaise de Cosm
etologie
International Journal of Cosmetic Science, 36, 494–504
M. A. Sheraz et al.
66. Ahmed, S., Sheraz, M.A. and Rehman,
I.U. Studies on tolfenamic acid-chitosan
intermolecular interactions: effect of ph,
polymer concentration and molecular
weight. AAPS PharmSciTech. 14, 870–879
(2013).
67. Istanbullu, H., Ahmed, S., Sheraz, M.A. and
Rehman, I.U. Development and character-
ization of novel polyurethane films impreg-
nated with tolfenamic acid for therapeutic
applications. Biomed. Res. Int. 2013,
178973 (2013).
68. Singh, P., Singh, N.P. and Yadav, R.A.
Study of the optimized molecular structures
and vibrational characteristics of neutral L-
Ascorbic acid and its anion and cation using
density functional theory. J. Chem. Pharm.
Res. 2, 656–681 (2010).
69. Masoudpanah, S.M. and Seyyed Ebrahimi,
S.A. Fe/Sr ratio and calcination temperature
effects on processing of nanostructured
strontium hexaferrite thin films by a sol–gel
method. Res. Chem. Intermed. 37, 259–266
(2011).
70. Rocha, R.A. and Muccillo, E.N.S. Physical
and chemical properties of nanosized pow-
ders of gadolinia-doped ceria prepared by
the cation complexation technique. Mat.
Res. Bull. 38, 1979–1986 (2003).