404 Chiang Mai J. Sci.

2018; 45(1)

Chiang Mai J. Sci. 2018; 45(1) : 404-412

http://epg.science.cmu.ac.th/ejournal/
Contributed Paper

Development and Validation of Atomic Absorption

Spectroscopic Method for Analysis of ZnO in Blemish
Balm Cream
Sophi Damayanti* [a], Tuan Norhafiza Binti Tuan Mat [a], Pimpichaya Tiyasuwan [b],
Ondrej Bolcarovic [c], Wipawadee Yoo-in [b], Andhika B. Mahardhika [a] and
Supat Jiranusornkul* [b]
[a] Department of Pharmacochemistry, School of Pharmacy, Bandung Institute of Technology,
Bandung 40132, Indonesia.
[b] Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200,
Thailand.
[c] Department of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno 61242,
Czech Republic.
* Author for correspondence; e-mail: supat.jira@cmu.ac.th; sophi.damayanti@fa.itb.ac.id

Received: 23 April 2017

Accepted: 8 August 2017

ABSTRACT
Blemish balm cream is one of the recently famous cosmetics that are used by women
in order to protect skin from UV radiation and give coverage at the same time. Zinc oxide
acts as an active sunscreen agent in BB Cream by refracting and absorbing UV rays to prevent
skin cancer and damage of other organs. However, ZnO has the ability to penetrate skin
and enter the bloodstream and may cause skin side effects such as photosensitization.
Therefore the maximum concentration of ZnO in sunscreen that stated by FDA is only up to
25 percent. The purposes of the present study are to develop and validate an analytical method
for determination of ZnO in cosmetic preparations by using Atomic Absorption Spectroscopy
(AAS) and to apply the method to the samples from various market origins. Recommended
parameters of Zn metal measurement were Hollow Cathode Lamp (operating current 7.0
mA), wavelength at 213.9 nm, air-acetylene flame, and slit width of 1.0 nm. Linearity of Zn
standard solution showed a good correlation with linear equation was y = 0.054x + 0.0571,
regression coefficient (R2) was 0.9993 and VX0 was 1.59. Limit of Detection was determined
in a value 0.314 μg/mL whereas Limit of Quantification was 0.952 μg/mL. The Coefficient
Variant of precision test was 0.99% and 1.09% for intraday and interday respectively.
Accuracy was shown in terms of percentage recovery that lies in a range of 100.85-102.56%.
Dry and wet digestion method was done on Standard Reference Material and placebo cream.
% CV for intra- and inter-day precision for dry digestion were 1.86% and 2.33%, respectively,
whereas those of wet digestion were 1.18% and 1.39%, respectively. The percentage recoveries
for dry and wet digestion were 95.51-98.72% and 99.57-101.03% respectively. Statistical
analysis using paired t-test showed that both methods differ significantly. Hence eleven samples
of marketed products were digested using wet digestion method. Results of total percentage
Chiang Mai J. Sci. 2018; 45(1)
of ZnO in the samples were in the range of 0.48-6.55%. This AAS method fulfills validation
criteria and could be developed to determine ZnO in the samples.
Keywords: sunscreen, cosmetics, Zn oxide analysis, validation
1. INTRODUCTION
BB cream or blemish balm cream
was first for mulated in Germany by a
dermatologist to protect skin after laser
treatment. It’s later being further developed
by Korean cosmetics companies. Although
it has been marketed in the worldwide under
various beauty brands, BB cream is majorly
used by Asian populations. BB cream, so
called ‘magic cream’, is able to provide
all-in-one facial cosmetics like moisturizing,
foundation, replace serum action, and as well
as effective UV protection.
Zinc oxide (ZnO) is one of the active
sunscreen agents that commonly used in BB
Cream and other sunscreen products. As an
active sunscreen agent, ZnO works by
scattering, reflecting and absorbing UV
radiation from sunlight [1]. Years ago, ZnO
left a white residue on the skin but nowadays
it was developed by reducing its size with
nanotechnology. Nano-sized particles make
ZnO hard to see on the skin so it seems like
there is no white stain left after applying
the cream. According to World Health
Organization (WHO), prolonged exposure
towards sunlight can be a cause of dermal
damage. Moreover, sunscreen application
is able to give protection and can prevent
from it [2-3].
However, Therapeutic Goods
Administration (TGA)from Australian
Government reported that ZnO itself
has photocatalytic activity in which that it is
able to yield Reactive Oxygen Species (ROS)
[4-5]. In addition, zinc oxide in form of
nanoparticles are able to penetrate into the
skin thus it may cause damage to skin due to
405
its photocatalytic [6-7]. Food and Drug
Administration (FDA) stated that the used of
ZnO as sunscreen should be not more than
25% [8]. Therefore there is need to develop
an efficient and accurate analytical method
of quantification ZnO in cosmetics.
Analysis of Zn can be done by using
spectrophotometric method described by
Benamor and co-workers [9]. Principle of
this method is based on color formation
reaction between Zn2+ and xylenol orange
in constant pH 5.5 environment. Rahula
conducted a study that compares method
of analysis of ZnO content in sunscreen
using atomic absorption spectroscopy (AAS)
and spectroscopy where the result showed
that data obtained by AAS are very close to
the actual ZnO content in commercial samples
[10]. On the other hand, spectrophotometric
method is more problematic with a greater
tendency to provide values higher than actual.
Other than spectrophotometry, various
analytical techniques that can be used to
analyze Zn are AAS, inductively coupled
plasma and neutron activation analysis [11].
AAS is a sensitive analytical method to
provide quantitative information of elements
in part per million (ppm) with the lowest
detection limit for Zn is 1.0 part per billion
(ppb). Therefore method development from
AAS would be feasible enough to analyze
metal as an active substance in a sample.
However, AAS requires sample pre-treatment
prior to analysis in which that the digestion
of matrix carrying substances of interest
should be done [12].
The aim of this study were to develop
406
and validate the method of analysis of ZnO
in BB cosmetic preparation by using AAS and
to apply the method to the samples from
various market origins.
2. MATERIAL AND METHODS
2.1 Chemical and Reagents
Zinc chloride standard solution
1000 μ g/mL was obtained from Merck
Chemical with SRM Certificates and AAS
Grade, Hydrochloric acid (HCl) 37% and
Ethanol 96% was from Merck Chemical
with ACS Grade, Cetyl alcohol, Liquid
paraffin, Span 80, Tween 80 is pharmaceutical
grade from Brataco Chemicals, BB cream
product samples was obtained from different
markets in Indonesia, Thailand and Czech
Republic.
2.2 Placebo Cream Preparation
Placebo cream preparation was prepared
by using formulation as shown in Table 1.
The cream was prepared by mixing the oil
and water phase components in two different
evaporating dishes. Both of the phases were
heated using water bath until temperature of
70 C. Further, oil and water phase was then
poured into a mortar and being triturated
until the cream was formed. About 2 g of
prepared cream was then taken and spiked
with standard zinc solution at 4, 5 and
6 μg/mL for accuracy and precision test
purposes. The cream was treated using both
of dry and wet digestion similar to sample
treatment.
2.3 Validation Method of AAS
2.3.1 Linearity
Standard solutions with 2, 4, 6, 8, and
10 μg/mL were prepared from 1000 μg/mL
stock standard solution. The absorbance
was measured for each of concentration.
Linearity was assessed from the calibration
curve absorbance of the standard solution
Chiang Mai J. Sci. 2018; 45(1)
as a function of the concentration. It was
evaluated by coefficient relation (R2) and
coefficient regression function (VX0).
2.3.2 Limit of detection (LOD) and limit
of quantification (LOQ)
LOD and LOQ were calculated based
on data from regression line of the calibration
curve. LOD is equalled to three point three
times of standard deviation of the calculated
concentration. LOQ is calculated ten times.
2.3.3 Precision
Repeatabilities of intraday and interday
were done by measuring absorbance in
three consecutive days and in a day with
3 different time intervals. Absorbance was
obtained then calculated for percentage
coefficient variation (%CV). Precision test
was done using six replicates of 5 μg/mL
standard solution and six replicates of Zinc
SRM that has been spiked with 50 μg/mL
of standard solution. Then, they treated
using both dry and wet digestion method
prior to analysis.
2.3.4 Accuracy
Accuracy was determined by percentage
recovery of the analyzed material based on
regression equation. It was done on standard
solution at concentration of 4, 5, and
6 μg/mL and placebo cream that has been
spiked with standard solution of 40, 50, and
60 μ g/mL in order to represent 80, 100,
and 120% concentration of Zinc SRM
(spiked-sample). Three replicates were done
for each of concentration. For spiked-samples,
both methods of digestion which are dry and
wet digestion were done prior to analysis.
2.3.6 Dry and wet digestion
The sample was placed onto hotplate by
swirling a few times. The samples were taken
from hot plate after 20-25 minutes of heating
Chiang Mai J. Sci. 2018; 45(1)
at 250 °C and they were allowed to cool at
room temperature and being filtered.
Another 5 mL HCl 37% was added into the
filtrate then heating process was furthered
for another 10-15 minutes. After being filtered,
the mixture was again heated prior to addition
of 5 mL of deionised water and 5 mL of
HCl 37%. The end filtrate obtained was then
marked up to calibrated mark of 100 mL
volumetric flask.
2.3.7 Optimization of AAS
The parameters of AAS were adjusted
to meet the specific measurement of Zinc
metal. Type of gas used as a flame source
was air-acetylene with flow rate of 4 l/minute,
Table 1. Formulation for 100 g of placebo cream.
Ingredient Amount (%)
Cetyl alcohol 15
Liq. paraffin 20
Span 80 0.8
Tween 80 4.2
Ethanol 96% 12
Deionized water 48
3. RESULT AND DISCUSSION
Absorbance of standard solution series
in a range of 2-10 μg/mL was measured by
using AAS to check for its linearity where the
absorbance of the standard itself depicts
the function of concentration in this analysis.
From data collected, a calibration curve was
plotted from standard mean absorbance data
against the standard solution concentration.
Evaluation of linearity is observed from R2
where it should be nearest to 1 and by using
VX0 that it should be below than 2 [13-16].
1 shows the result of linearity test.
Measurement of linearity test was done using
Zinc SRM solution with 3 replicates for each
407
selected the signal at the hollow cathode
lamp with operating current at 7.0 mA,
1.0 nm slit width, the specific wavelength
for Zinc determination that is 213.9 nm and
1ml/minute flow rate of sample aspirated.
2.3.8 Sampling
Samples of BB cream products
containing ZnO that originated from
different manufacturers was collected.
The samples were digested using both dry
and digestion method in which that after the
desired substance obtained, dilution with
deionised water was done to get absorbance
reading lies within the linear range using
Varian AAS Spectra.
Amount (g) Uses
15 Thickening agent
20 Oil phase
0.8 Oil phase surfactant
4.2 Water phase surfactant
12 Preservative
48 Solvent
of concentration. Linear equation obtained
was y = 0.054x + 0.0571 with R2 and VX0
are 0.9993 and 1.59 respectively.
Figure 1. The linearity of Zn determination
from series of standard solution by atomic
absorption spectrometry.
408 Chiang Mai J. Sci. 2018; 45(1)

Both the LOD and LOQ were calculated
from the calibration curve. The result of
LOD was 0.314 μ g/mL and LOQ was
0.952 μg/mL.
Precision the test was conducted using
both Zn standard solution and using
placebo which has been spiked with Zinc
standard solution. The chosen concentration
was the concentration that lies within the
linearity range which is 5 μ g/mL of Zn
standard solution was used for this purpose.
A total of six replicates was analyzed during
each of testing period of both intraday and
interday. Result shown in Table 2 shows
that intraday and interday precision of Zinc
SRM were both having %CV below than 2%.
Meanwhile, the result for precision test using
spiked placebo with 50 μg/mL and has been
treated with both wet and dry digestion is
shown in Table 3 below and was interpreted
as %CV. Generally, requirement for %CV
should be less than 2% [17].

Table 2. Precision test for Zn standard solution at 5 μg/mL concentration.

Precision test Period Standard solutiona %CV b

Intraday Morning 5.14 ± 1.13 0.99
Afternoon 5.22 ± 0.60
Evening 5.17 ± 0.61
Interday Day 1 5.13 ± 1.22 1.09
Day 2 5.21 ± 0.76
Day 3 5.17 ± 0.77

Notes: a Average of concentration (μg/mL) ± % CV of each period; (n = 6).

b
Average of %CV for intraday and interday respectively.

Table 3. Precision test for SRM spiked with Zn standard solution to be in 5 μg/mL
concentration.
Precision test Period SRM with %CV b SRM with %CV
Dry digestiona 1.86 Wet digestion 1.88
Intraday Morning 4.90 ± 1.90 5.10 ± 0.78
Afternoon 4.85 ± 1.42 5.10 ± 1.56
Evening 4.86 ± 2.33 2.33 5.14 ± 1.13 1.39
Interday Day 1 4.90 ± 0.81 5.09 ± 1.14
Day 2 4.80 ± 2.19 5.12 ± 1.26
Day 3 4.95 ± 2.72 5.14 ± 1.75
Notes: a Average of concentration (μg/mL) ± %CV of each period; (n = 6)
b
Average of %CV for intraday and interday respectively.

The critical point of Zn analysis is during
the sample preparation as the right approach
to sample preparation is very important in
obtaining correct results. Digestion method
of the matrix to be analyzed by AAS can be
wet digestion and dry digestion which is
also known as dry ashing. Wet digestion can
be further divided to conventional heating,
microwave-assisted heating and UV-assisted
digestion [18]. Oliveira mentioned that for
mineral oxides it is better to use dry digestion
[19]. However the choice of method
Chiang Mai J. Sci. 2018; 45(1) 409

also depends on the sample itself whether it
is a solid sample, liquid, or slurry form. For
cosmetics that are commonly found as creams
and lotion that the consistency is very slurry
instead of dry digestion, it is better to be
digested by wet digestion [20]. Idera and
co-workers suggested that nitric/peroxide
and aqua regia were the most suitable for Zn
extraction with 98% recovery from fish
tissues whereas our wet digestion gave a
better recovery with greater than 99% [21].
From the result obtained, it shows that
%CV of dry digestion for interday precision
did not meet the requirement with value of
2.33%. Meanwhile, intraday and interday
precision result for wet digestion met the
requirement of %CV which is below than 2.
Accuracy test is an analysis to measure the
suitability of the system to recover the
substance content after analysis where
concentration of 4, 5, and 6 μg/mL of Zn
standard solution is used as representative
of 80, 100, and 120 % of actual amount
of substance. Analysis was done using
3 replicates of each of concentration.
Results for this test were expressed in terms
of percentage recovery with the requirement
for recovery of 80-110 % [22]. The accuracy
test result for standard solution is shown
in Table 4.

Table 4. Percentage recovery data for result of accuracy test on Zn standard solution.
Certified concentration (μg/mL) Standard solutiona %Recoveryb
4.00 4.10 ± 0.09 102.56 ± 2.22
5.00 5.18 ± 0.04 103.59 ± 0.89
6.00 6.05 ± 0.09 100.85 ± 1.48
Notes: a Average of concentration (μg/mL) ± %CV; (n = 3)
b
Average of % Recovery ± %CV

Similar to precision test, accuracy test also
conducted on Placebo spiked with Zn SRM
solution and it was treated with both dry
ashing and wet digestion method of sample
preparation. The result is shown in Table 5
and it can be observed that recovery of Zn
for dry digestion and wet digestion method
met the percentage recovery requirement.
Range of percentage recovery for dry
digestion was 95.51-98.72 % meanwhile for
wet digestion percentage recovery was in
between the range of 99.57-101.03 %.

Table 5. Percentage recovery data for result of accuracy test on SRM treated with dry and
wet digestion.

Certified concentration SRM with %Recoveryb SRM with %Recovery

(μg/mL) Dry digestiona Wet digestion
4.00 3.82 ± 0.09 95.51 ± 2.22 4.00 ± 0.08 100.00 ± 1.92
5.00 4.82 ± 0.04 96.41 ± 0.89 5.05 ± 0.04 101.03 ± 0.89
6.00 5.92 ± 0.08 98.72 ± 1.28 5.97 ± 0.04 99.57 ± 0.74
Notes: a Average of concentration (μg/mL) ± %CV; (n = 3)
b
Average of %Recovery ± %CV
410 Chiang Mai J. Sci. 2018; 45(1)

The main objective of this study is to
develop the method for quantitative ZnO
analysis in cosmetic products. Two methods
of digestion were compared which are wet
digestion method and dry digestion method.
Although both methods showed a good
recovery that meet percentage recovery
requirement, statistical analysis was used to
confirm that if the method shows differences.
From the result obtained in accuracy and
precision test statistical analysis by using paired
t-test method was perfor med where
calculation is displayed briefly in the Table 6.
Result showed that tvalue is higher than the tcritical
(p<0.05) which indicates that there is
significantly different between wet digestion
and dry ashing method. In other words, wet
digestion method is more effective compared
to dry ashing method. Similar to study
of Soylak (2006) reported that sample
preparation for determination of trace metals
content in instant soup powder by using AAS.
It showed the effectiveness of wet digestion
over dry ashing [23].

Table 6. Comparison of dry and wet digestion method by paired t-test statistical analysis.
Mean Standard Standard t-value Degree of t-critical
difference deviation error of d freedom
( d )a (Sd) ( Sd ) (n-1)
√n

Precisionb 0.232 0.115 0.027 8.593 17 2.11

Precisionc 0.303 0.104 0.024 12.404 17 2.11
Accuracyd 0.171 0.092 0.031 5.581 8 2.31

Notes:a Difference between Zn concentrations from wet digestion with dry digestion.
b
Precision intraday; tvalue > tcritical, method significantly different ; (n = 18).
c
Precision interday; tvalue > tcritical, method significantly different ; (n = 18).
d
tvalue > tcritical, method significantly different ; (n = 9).

Furthermore, there is study of was subjected to wet digestion as sample

development and validation of atomic
absorption spectrometry for the determination
of zinc by Rohman, et al. The study used wet
digestion method but different in details,
the result showed that developed method
was accurate for quantification of zinc in
cream cosmetics as same as this study [24].
When compare the result of this study and
the result from Rohman’s study, this study
showed the better result in linearity, precision,
and accuracy.
Since the best method verified was wet
digestion, Samples of BB cream products
preparation method prior to analysis.
Samples were collected from different origin
and the results of percentage ZnO content is
shown in Table 7. ZnO as an active sunscreen
agent is commonly found in those cosmetics
that claimed to use mineral based ingredients.
Further studies, the need of characterization
of the ZnO in samples may be investigated
by scanning electron microscopy (SEM),
X-ray diffraction (XRD) and Atomic
Absorption Spectroscopy (AAS) to get more
information from different metrics [25-26].
Chiang Mai J. Sci. 2018; 45(1)
Table 7. Total percentage of ZnO content in the sample of BB cream products.
Sample Origin
1 Indonesia
2 Indonesia
3 Indonesia
4 Germany
5 Czech Republic
6 Korea
7 Korea
8 Singapore
9 Thailand
10 Thailand
11 Thailand
Notes: Average of %ZnO content ± SD of each samples analyzed; (n = 3),
N.D. = not detectable
4. CONCLUSION
Method of analysis of ZnO was
developed and validated by using two
different methods of sample preparation,
comparing wet digestion method to dry
digestion method. In general, the method
showed good linearity by the parameter of
R2 and VX0 was 0.9993 and 1.59, respectively.
LOD and LOQ were 0.314 and 0.952
μg/mL. Statistical analysis of precision and
accuracy data for placebo cream (p<0.05)
results in t value was higher than the t critical
which showing that both methods are
significantly different. Therefore, the analysis
of ZnO in BB cream product samples was
prepared by using wet digestion method.
From the analysis, the total ZnO concentration
in BB cream products was determined in
the range of 0.48-6.55 %.
ACKNOWLEDGEMENTS
The author would like to thank to all the
contributors.
411
Concentration of total ZnO (%)
0.82 ± 0.03
1.01 ± 0.02
3.70 ± 0.11
N.D.
N.D.
5.49 ± 0.15
6.55 ± 0.06
4.15 ± 0.06
4.80 ± 0.25
0.48 ± 0.05
0.84 ± 0.13
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