International Journal of Cosmetic Science, 2012, 1–6 doi: 10.1111/j.1468-2494.2012.00742.

High-performance liquid chromatographic determination of

triclosan and triclocarban in cosmetic products

T. Liu* and D. Wu†

*China National Cosmetics Quality Supervision & Inspection Center, Beijing 100094, China and †Key Laboratory of Biomedical Information
Engineering of Education Ministry, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China

Received 9 April 2012, Accepted 10 July 2012

Keywords: cosmetic product, high–performance liquid chromatographic, triclocarban, triclosan, usage

Synopsis are antimicrobial compounds commonly added because of their

sanitizing properties to a wide range of household and personal care
A high–performance liquid chromatographic (HPLC) method for the
products, including shampoos, soaps, creams, mouthwash and
determination of triclosan and triclocarban in cosmetic products
toothpaste [1]. These compounds, which are known endocrine
was developed. Triclosan and triclocarban quantities in 168 of
disruptors, can transform into more toxic and persistent species
cosmetics were investigated and statistical analyzed with this
such as chlorinated phenols, polychlorinated biphenyl ethers,
method. The optimal condition are as follows: An Agilent SB-C8
polychlorinated dibenzodioxins, as well as mono- and dichlori-
analytical column (250 4.6 mm, 4.5 m) was utilized, and mixed
nated anilines. The absorption and chronic systemic circulation
buffer solution of methanol and 0.01 mol L1 phosphate (pH 3.0)
of TCS in humans has been recently observed, and several arti-
(72 : 28, V/V) were used for isocratic elution at a total flow rate of
cles on the possible side effects of this compound and/or its
1.0 mL min1. It is found the calibration curves had a good linear
metabolites have been published over the past years [2–5]. TCS
regression with UV detection (280 nm) within test range of
exposure is considered by the United States Environmental Pro-
0–110 g mL1 with the correlation coefficients of 0.999 in all cases.
tection Agency in the aggregate risk assessment [6]. TCS is
This method is simple, selective, convenient, and reproducible for
reportedly one of the most frequently detected organic pollutants
the determination of triclosan and triclocarban in commercial
in the aquatic environment and municipal sludge, and TCC is a
cosmetic products.
co-contaminant with TCS in both wastewater and surface water
[7]. Therefore, there is concern about their addition to cosmetic
Résumé products, the risk they pose to consumer safety and the poten-
tial impacts of their release to the environment. Because of
Un procédé de chromatographique liquide à haute performance
public health concerns, the maximum allowable concentrations
(CLHP) pour la détermination de triclosan et de triclocarban a été
of TCC and TCS in cosmetic products are regulated by the
développé pour une application dans le domaine des produits cos-
appropriate licensing authorities in most countries. In China,
métiques. Les quantités de triclosan et de triclocarban dans 168
the allowed concentrations of TCS (0.3%) and TCC (0.2%) as
échantillons ont été déterminées par cette méthode et analysées
preservatives in cosmetics are specified by the Hygienic Standard
statistiquement. Les conditions optimales sont: Colonne Agilent
of Cosmetics. In Europe, the maximum allowable concentration
SB-C8 analytique (250 4.6 mm, 4.5 m), solution tampon de
according to the EU Cosmetics Directive 76/768/EEC is 0.3%,
méthanol et de 0.01 mol L 1 phosphate (pH 3.0) (72:28,V/V)
but the applications of TCS and TCC in cosmetics have recently
pour élution isocratique, débit 1.0 mL min 1. Les courbes de cali-
become more limited [8].
bration montrent une bonne régression linéaire avec la détection
The present study was performed because we were unable to
UV à 280nm dans la gamme 0 – 110 g ml 1, avec un coefficient
find published data on the usage of TCS and TCC in cosmetic
de corrélation de 0.999 dans tous les cas. La méthode est simple,
products. We were keen on establishing a methodology for
sélective, pratique et reproductible, et elle est adaptée pour la
measuring TCS and TCC, as well as obtaining information on
détermination de triclosan et du triclocarban dans les produits
their use in cosmetics. This article presents a reliable quantita-
cosmétiques commercialisés.
tive analytical method for determining TCS and TCC in a broad
range of cosmetic products using high-performance liquid chro-
Introduction matography (HPLC). Data on their addition to various cosmetic
products are also provided. Compared with the available analyt-
Triclosan (5-chloro-2-[2.4-dichloro-phenoxy]-phenol) (TCS) and ical methods for the determination of these two compounds [9–
triclocarban (N-(4-chlorophenyl)-N-(3,4-dichlorophenyl)urea) (TCC) 22], the proposed method is rapid, simple, selective and suitable
for quality control, as well as for the routine analysis of vari-
ous cosmetic products (e.g. body shampoo, bar soap, creams,
Correspondence: D. Wu, Key Laboratory of Biomedical Information
Engineering of Education Ministry, School of Life Science and Technol- mouthwash, toothpaste, etc.) in the market. Information on
ogy, Xi’an Jiaotong University, Xi’an 710049, China. Tel.: 86-29- TCS and TCC contents and labelling in the past 2 years is
82663941; e-mail: daochengwu@gmail.com provided.

Ó 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie 1

Determination of triclosan and triclocarban
Experimental
Chemicals and reagents
Methanol was purchased from the Beijing Chemical Reagents Com-
pany (Beijing, China), filtered through a 0.45-lm membrane and
sonicated before use as the mobile phase. TCS was purchased from
Alfa Aesar. TCC was purchased from the Tokyo Chemical Industry
Co., Ltd. (Tokyo, Japan). All other reagents including sodium dihydro-
gen phosphate monohydrate and phosphoric acid were of analytical
grade. Water of Milli-Q (Millipore, Bedford, MD, U.S.A.) quality was
used. Cosmetics were obtained from local markets in Beijing (China).
Apparatus
An Agilent series 1200LC comprising a degasser, a quaternary
pump, a thermostated column compartment, an autosample and a
diode array detection (DAD) was used. Data collection and
treatment were performed using Agilent Chemstation.
High–performance liquid chromatographic was performed using
Agilent EclipseXDB-C8 or Agilent ZORBAX SB-C8 analytical col-
umn (250 mm 9 4.6 mm, 5 lm). The column temperature was
set at 35°C. The flow of the mobile phase was set at 1.0 mL min 1.
The detection wavelength of DAD was 280 nm, and 10 lL of
sample extract or calibration standard solution was analysed by
isocratic elution using mixed methanol and 0.01 mol L 1 phosphate
buffer solution (pH 3.0; 72:28, V/V) as HPLC mobile phase.
Calibration standard solutions of TCS and TCC
Preparation of stock standard solution
Separate stock solutions of 1 mg mL 1 TCS and 1 mg mL 1 TCC
were prepared by dissolving an appropriate amount of each sub-
stance in methanol.
Preparation of calibration standard solutions
The working standard solutions were prepared by mixing individ-
ual standard solutions and diluting to the appropriate concentra-
tion. Portions (10 mL) of the TCS and TCC stock solutions (2.3.1)
were pipetted separately into a 100-mL volumetric flask, which
was filled up to the mark with methanol. Appropriate amounts of
mixed standard solution (0, 0.5, 0.1, 0.2, 0.5, 1.0, 5.0 and
10.0 mL) were pipetted to 10-ml volumetric flasks and diluted to
the mark with methanol.
Calibration curves of standard solutions
An aliquot of each standard (2.3.2) was injected. The peak areas
were measured and those of the analytes (y) were plotted against the
respective concentrations (microgram per millilitre) of TCS and TCC
(x). Least square linear regression analysis was used to determine the
slope, y-intercept and correlation coefficients of the standard plots.
Sample preparation
Based on experimental results, different sample preparation proce-
dures were summarized as the followings.
Preparation for body shampoo, creams, mouthwash and detergent
products
Approximately 0.5 g (reported to 0.0001 g) of sample was placed in
a 25-mL screw-capped test tube, to which 25 mL of methanol was
Ó 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie
2 International Journal of Cosmetic Science, 1–6
T. Liu and D. Wu
added. After adequate mixing and sonication for 30 min to extract
TCS and TCC, the mixture was filtered through a 0.45-lm mem-
brane.
Preparation for bar soap products
Using a stainless grater or knife, a sufficient amount of grated soap
was obtained from the interior of a product bar and ground into
pieces. Approximately 0.5 g (reported to 0.0001 g) of bar soap
pieces was placed in a 25-mL screw-capped test tube, to which
5 mL of acetone was added. The mixture was stirred well, and
20 mL of methanol was added to the sample. After adequate mix-
ing and sonication for 30 min to extract TCS and TCC, the mixture
was filtered through a 0.45-lm membrane.
Preparation of toothpaste products
Approximately 0.5 g (reported to 0.0001 g) of toothpaste was
placed in a 25-mL screw-capped test tube, to which 5 mL of H2O
was added. The mixture was stirred well for proper dissolution.
After adding 20 mL of methanol, adequate mixing and sonication
for 30 min to extract TCS and TCC, the mixture was filtered
through a 0.45-lm membrane.
Assay validation
The validation of the proposed liquid chromatographic method
included assessments of selectivity, precision, linearity, accuracy,
range, limit of detection (LOD) and limit of quantification (LOQ).
To demonstrate selectivity, chromatograms of TCS and TCC
standard solutions were compared with the standard mixture and
sample chromatograms. The measurement methods for instrumen-
tal precision and repeatability were commonly suitable for the
determination of precision. Repeatability was checked via six
consecutive injections of the sample solution. The quantity and
relative standard deviation (RSD) were calculated.
Linearity was examined by preparing solutions of working
standard mixtures at five concentration levels depending on the
target analyte concentration. Calibration curves were constructed
by plotting peak areas vs. solute concentrations.
The accuracy was determined as the recovery of known amount
of analyte spiked into a placebo. The spiked samples were prepared
at levels over a range that covered the expected analyte content.
The resultant samples were also prepared and analysed as
described in Sample preparation, and the quantity of each solute
was subsequently obtained from the corresponding calibration
curves. The percentage difference between the amounts determined
and spiked (recovery) was considered as a measure of accuracy.
Range criteria were defined based on the linearity and accuracy of
data.
The LOQ was calculated as the analyte concentration that
yielded a signal-to-noise ratio of 10, and this concentration was
experimentally confirmed.
The LOD was the lowest analyte concentration in a sample that
resulted in a peak three times the height corresponding to noise.
This concentration was experimentally confirmed.
Results and discussion
An HPLC method for the determination of TCS and TCC contents
in cosmetic products was developed. The experimental condi-
tions were investigated, and the proposed method was also vali-
dated.
Determination of triclosan and triclocarban
Figure 1 UV spectra of triclosan (5-chloro-2-[2.4-dichloro-phenoxy]-phenol) (TCS) and triclocarban (N-(4-chlorophenyl)-N-(3,4-dichlorophenyl)urea) (TCC).
Chromatography
The chromatographic condition for the separation and determina-
tion of TCS and TCC was optimized during the developmental
phase while investigating the influences of different parameters on
separation, including analytical wavelength, column type and
composition of mobile phase.
Analytical wavelength
Figure 1 illustrates the UV spectra of TCS and TCC at different
wavelengths from 200 to 400 nm. TCC had a maximum
absorbance at 205 and 264 nm, whereas TCS had a maximum
absorbance at 202, 235 and 281 nm. The selected optimum
absorbance to obtain the best sensitivity was 280 nm.
Mobile phase
The optimal conditions of HPLC for determining TCS and TCC
were carried out under isocratic conditions. Various mobile phase
systems with different compositions, for example, methanol and
0.01 mol L 1 phosphate buffer solution (pH 3.0), as well as
water and methanol were investigated. The ratio of methanol to
water or methanol to buffer for the mobile phase was adjusted to
optimize the retention time and resolution of TCS and TCC (i.e. a
Figure 2 High–performance liquid chromatographic (HPLC) chromatogram of calibration standard solutions; mobile phase: methanol and 0.01 mol L
phate buffer solution (72 : 28, V/V).
Ó 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie
International Journal of Cosmetic Science, 1–6
T. Liu and D. Wu
higher amount of buffer and water can increase the retention
time and resolution). TCS and TCC showed worse resolution and
cannot be completely separated using Agilent Eclipse XDB-C18
and Agilent ZORBAX SB-C18 columns, which hampered accurate
determination. Therefore, Agilent ZORBAX SB-C8 and Agilent
Eclipse XDB-C8 columns were selected as analytical columns.
There was no significant difference between the peak shape and
column efficiency of the Agilent ZORBAX SB-C8 and Eclipse XDB-
C8 columns when water and methanol (25 : 75, V/V) were used
as effluent to separate TCS and TCC. When mixed methanol and
0.01 mol L 1 phosphate buffer solution (pH 3.0) were used as
the mobile phase, the ZORBAX SB-C8 column provided better
chromatographic performance such as efficient symmetrical peaks.
As it was showed in Figs 2 and 3, analysis time of Fig. 2 condi-
tion (mixed methanol and 0.01 mol L 1 phosphate buffer solu-
tion (pH 3.0) were used as the mobile phase) is little longer than
Fig. 3 condition (water and methanol (25 : 75, V/V) were used
as the mobile phase). It is clear the retention time of TCC in
Figs 2 and 3 only has about 3 min difference, and retention time
of TCS in Figs 2 and 3 only has about 4 min difference. To get
better sample separation, the mixture of methanol and
0.01 mol L 1 phosphate buffer solution (72 : 28, V/V) was used
as the HPLC mobile phase, and the ZORBAX SB-C8 column was
1
phos-
3
Determination of triclosan and triclocarban T. Liu and D. Wu

Figure 3 High–performance liquid chromatographic (HPLC) chromatogram of calibration standard solutions; mobile phase: water and methanol (25 : 75,
V/V).

Figure 4 High–performance liquid chromatographic (HPLC) chromatogram of spiked cosmetic sample; mobile phase: methanol and 0.01 mol L 1
phosphate
buffer solution (72 : 28, V/V).

selected as the analytical column. The bases of these choices
were the optimum peak profiles, resolution, good retention times
and elution of the compounds within 30 min, as well as the
avoidance of interference of coexisting ingredients (e.g. some per-
fume ingredients) in the TCS and TCC assay. The optimum flow
rate was 1.0 mL min 1 because it provided good resolution, high
sensitivity and proper analysis time, among others. Figures 2 and
3 demonstrate the HPLC chromatograms of the calibrated stan-
dard solutions. Figures 4 and 5 are the HPLC chromatograms of
the spiked cosmetic sample and toothpaste sample that contained
TCS, respectively.
Calibration curves, linearity and range
Linearity was determined for TCS and TCC using working
standard mixture solutions at seven concentrations. The correla-
tion coefficient requirement R2 > 0.995 is considered acceptable
[23] and was achieved for both compounds. The calibration
curves for both compounds showed good linearity within the
examined concentration range. The statistical relationships, such
as regression plot, correlation coefficient, test ranges and other
details of the calibration curves of each solute, are summarized
in Table I.
Accuracy and precision
The accuracy of an analytical procedure expresses the closeness of
agreement of the obtained value with the value accepted either as
the conventional true value or as an accepted reference value [24].
The accuracy and precision of the proposed method were evaluated
by analysing samples from the same cosmetic sample (n = 6). The
accuracy was determined by spiking TCS and TCC in the placebo
preparation. For the proposed assay method, body shampoo, bar
soap, creams and toothpaste placebos were spiked at three

Ó 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie

4 International Journal of Cosmetic Science, 1–6
Determination of triclosan and triclocarban T. Liu and D. Wu

Figure 5 High–performance liquid chromatographic (HPLC) chromatogram of toothpaste sample containing triclosan (5-chloro-2-[2.4-dichloro-phenoxy]-phe-
nol) (TCS); mobile phase: methanol and 0.01 mol L 1 phosphate buffer solution (72 : 28, V/V).

Table I The statistical relationships of the calibration curves for triclosan and triclocarban measurement

Validation parameters

Repeatability Accuracy

Range
Preservatives Linearity Regression plot R2 RSD (%) (lg ml 1) Recovery (%) RSD (%) LOQ (lg g 1) LOD (lg g 1)

TCS y = 9.2584x – 10.001 0.9998 0.073 0–110 99.5 0.59 50 15

TCC y = 29.807x – 18.269 0.9999 0.099 0–110 100.1 0.96 17 5

LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation; TCC, triclocarban (N-(4-chlorophenyl)-N-(3,4-dichlorophenyl)urea); TCS, triclosan
(5-chloro-2-[2.4-dichloro-phenoxy]-phenol).

concentration levels for TCS and TCC and then analysed according to respectively. The LOQ and LOD results for TCS and TCC are
the proposed method. The recovery was calculated, and the accuracy presented in Table I.
results are shown in Table I. The excellent recoveries of the standard
addition amounts suggest the accuracy of the proposed method. Table II Usage of TCS and TCC in cosmetics
The precision of an analytical procedure expresses the close-
ness of agreement among series of measurements obtained from
Sample Concentration of
multiple sampling of the same homogeneous sample under pre-
Sample type Number with TCS TCS (%)
scribed conditions. The repeatability, as one of the characteristics
of precision, of the proposed analytical method was determined
by six injections of the experimental sample. The results listed in Cleansing product
Table I for TCS and TCC met the proposed requirement Shampoo 40 2 0.02–0.05
RSD  2% [25]. Body wash 19 3 0.07–0.25
Bar soap 5 0 /
Reproducibility was determined by recovery experiments. The Facial cleanser 13 2 0.05–0.10
average recoveries of TCS and TCC were >99.5%. The precision Hair conditioner 2 0 /
calculated as RSD was <0.96%. Skin beauty/care product
Anti-acne facial cream 7 2 0.04–0.29
Body lotion (moisturizer, 52 2 0.14–0.21
LOD and LOQ cream)
Toothpaste 30 5 0.05–0.29
The sensitivity of the assay was determined in terms of the LOD
and LOQ. These values were estimated for each of the examined
compounds. The LOQ and LOD were calculated as the TCS and/or TCC, triclocarban (N-(4-chlorophenyl)-N-(3,4-dichlorophenyl)urea); TCS, Triclo-
TCC concentrations that yielded signal-to-noise ratios of 10 and 3, san (5-chloro-2-[2.4-dichloro-phenoxy]-phenol).

Ó 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie

International Journal of Cosmetic Science, 1–6 5
Determination of triclosan and triclocarban T. Liu and D. Wu

soap. Compared with other cosmetics, TCS was more easily

Selectivity
The selectivity of the procedure was evaluated by analysing 168
different commercially available cosmetic products. The chromato-
grams clearly indicated that there was no interference from the
formulation excipients.
Usage of TCS and TCC in cosmetics
The proposed method was suitable for TCS and TCC assay
because the validation confirmed that the method was selective,
linear, accurate and reproducible. A total of 168 different com-
mercially available cosmetic products were assayed using the
proposed procedure, and the usage of TCS and TCC in the 168
different cosmetics was also investigated. The assay results indi-
cated that the proposed analytical method was adequate for
determining TCS and TCC in commercial cosmetics.
Table II shows that TCC was not found in all 168 products,
whereas TCS was detected in 16 cosmetics, accounting for about
10% of the total products. TCS was found in the following cleans-
ing products: two of 40 (5%) shampoos, three of 19 (16%) body
washes and 2 of 13 (15%) facial cleansers. The concentration of
TCS ranged from 0.02% to 0.25%. TCS was found in the following
skin beauty/care products: two of 7 (30%) anti-acne facial creams
and two of 52 (4%) body lotions (moisturizer, cream). Among the
30 toothpaste samples, 17% contained TCS, which ranged from
0.05% to 0.29%. TCS was also found in hair conditioner and bar
detected in anti-acne facial cream, and high concentrations of TCS
were most frequently measured in this product.
Conclusions
A reversed-phase (RP) high-performance liquid chromatographic
procedure was developed to determine TCS and TCC contents in
commercial cosmetic products. The method was also validated in
terms of LOD, LOQ, repeatability, reproducibility and accuracy.
The optimum conditions and analytical characteristics for the
RP-HPLC determination of TCS and TCC exhibited good resolu-
tion, short analysis time and rather high sensitivity. The usage
of TCS and TCC in 168 cosmetics was investigated and statisti-
cally analysed. Information on the TCS and TCC contents, as
well as labelling, was provided. These data can be used as
benchmarks for new developments.
Acknowledgements
This study was supported in part by a grant of Shaanxi Province
Science and Technology and Innovation Project (no. 2011KTCL03-
07) and the Open Research Fund of the State Key Laboratory of
Precision Spectroscopy, East China Normal University. The authors
express their sincere gratitude to Ms. Ruiyun He (China National
Cosmetics Quality Supervision & Inspection Center) for providing
helpful advices to this study.

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