Vol. 14 | No.

2 |1435-1440| April - June | 2021

ISSN: 0974-1496 | e-ISSN: 0976-0083 | CODEN: RJCABP
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DETERMINATION OF TRICLOSAN IN INDONESIAN

HOUSEHOLD, PERSONAL CARE, AND COSMETIC
PRODUCTS BY ISOCRATIC REVERSED-PHASE HIGH-
PERFORMANCE LIQUID CHROMATOGRAPHY
N. Rusdiana1,2, , M.S. Wibowo1 and R.E. Kartasasmita1
1
Department of Pharmacochemistry, School of Pharmacy, Bandung Institue of Technology, Jl.
Ganesha 10 Bandung 40132, West Java Indonesia
2
College of Pharmacy Muhammadiyah Tangerang, Jl Syech Nawawi 13 Tangerang 15720,
Banten Indonesia

Corresponding Author: [email protected]

ABSTRACT
Triclosan (5 - chloro - 2 - (2,4 - dichlorophenoxy) phenol) is a biocide compound commonly added to the various
household, personal care, and cosmetic products. This research aims to determine triclosan content in those products
by a chromatographic method. Product samples containing were analyzed using HPLC equipped with C18 column
UV detection at 280 nm and mobile phase using a mixture of acetonitrile - double distilled water (75:25). All samples
were confirmed to contain triclosan at levels not more than permissible level by the regulation in Indonesia.
Keywords: HPLC, Household, Personal Care, Cosmetic, Triclosan.
RASĀYAN J. Chem., Vol. 14, No.2, 2021

INTRODUCTION
Triclosan (1) has been used for more than 40 years as a preservative in cosmetics 1–3 and almost in all
personal care 4–6 and household products.7–9 Triclosan is a very strong microbial inhibitor, which can be
used to control dental infections caused by bacteria.10 The chemical name of triclosan is 5 - chloro - 2 - (2,4
- dichlorophenoxy) phenol, while its molecular formula is C12H7Cl3O2 and its molecular weight is 289.5 g
per mol. This compound is a white crystalline powder, which easily dissolves in alkaline solutions and
organic solvents, soluble in acetone, ethanol, methanol, and slightly soluble in water. Several
chromatographic methods have been reported for the determination of triclosan levels in various samples.
Especially HPLC methods have been designed that allow the determination of the effectiveness of
separation and quantification of samples.11–16 In our experiment, a reverse phase HPLC method with an
isocratic gradient system has been prepared for the determination of triclosan in the household, personal
care, and cosmetic products.

Fig.-1: Chemical Structures of Triclosan (1)

EXPERIMENTAL
Instrumentation
The sample analysis products were performed on a Waters e2695 series HPLC system (Millford, USA)
equipped with UV-Vis detector and Agilent ZORBAX Eclipse Plus C-18 column (5 m, 4,6 x 150 mm)
(USA). The signal output was monitored and integrated using Waters Empower 3 software.

Rasayan J. Chem., 14(2), 1435-1440(2021)

http://dx.doi.org/10.31788/ RJC.2021.1425861 This work is licensed under a CC BY 4.0 license.
 Vol. 14 | No. 2 |1435-1440| April - June | 2021

Chemicals and Solvents

Triclosan was purchased from Sigma Aldrich (purity 99.9 percent), while acetonitrile and methanol (Merck)
double distilled water were purchased from IPHA Laboratories (Bandung, Indonesia).

Mobile Phase Preparation

A 500 mL mobile phase was prepared to consist of a mixture of 375 mL acetonitrile and 125 mL of double-
distilled water.

Diluent Preparation
The mobile phase was used as diluent.

Standard Preparation: (For Triclosan 1000 g / mL)

25 mg triclosan was accurately weighed and transferred into a 25 mL measuring flask and dissolved with
the mobile phase. To enhance the solubility, the mixture was ultrasonicated until triclosan was completely
dissolved.

Sample Preparation
A sample of products containing triclosan was chosen to represent solid, semi-solid, and liquid preparations.
0.5 g of solid sample was accurately weighed in a 15 mL plastic centrifuge tube, added with 7.5 mL of
acetonitrile gradually, mechanically agitated for 3 minutes, ultrasonicated for 5 minutes, and then
centrifuged for 10 minutes. The supernatant was quantitatively pipetted.
0.5 g of semi-solid samples were accurately weighed in a 10 mL volumetric flask, added with 3 mL of
methanol, ultrasonicated for 10 minutes, and filled with methanol to mark (solution 1), and then shaken.
2.0 mL of solution 1 was pipetted into a 10 mL volumetric flask, diluted with methanol to mark, and then
shaken.
250 L of the liquid sample was pipetted into a 10 mL volumetric flask, added by mL of acetonitrile and
double distilled water (75:25) gradually. Afterward, it was ultrasonicated for 10 minutes, diluted with
mobile phase to mark, and then shaken.
All samples were filtered using a 0.45 µm membrane filter. The resulting filtrate was then used as the
solution to be tested.

Chromatographic Conditions
The column oven for ZORBAX Eclipse plus 5 μm (4.6 x 150 mm) Columns was set at 30 °C. The mobile
phase was controlled at a flow rate of 0.6 mL per min. The sample injection volume was 10 μL, the detector
was set at 280 nm and the chromatography runtime was 10 minutes.

Analysis of Triclosan in the Household, Personal Care, and Cosmetic Products

Identification of triclosan in the samples was conducted by comparison of triclosan peak retention time in
the chromatogram of standard solution with those of sample solutions. If the retention times of the two
peaks in these chromatograms were not significantly different, the corresponding peak in the chromatogram
of the sample was identified as triclosan. The determination of triclosan in the samples was conducted by
the external calibration method. To determine the compliance of triclosan levels in the sample, the results
were compared with the maximum use level according to regulation in Indonesia (0.3-2%).
RESULTS AND DISCUSSION
Method Development
To develop a suitable and robust method for the determination of triclosan, an HPLC system equipped with
a ZORBAX Eclipse plus 5 μm (4.6 x 150 mm) column controlled at 30 °C and UV detection at 280 nm was
used. Different mobile phases were tested to achieve the best chromatographic performance. A mixture of
acetonitrile and double distilled water with a ratio 75:25 v / v and a flow rate of 0.6 mL/minute gave the
best and consistent chromatogram. The following chromatogram (Fig.-2) shows repetitive peaks of the
standard of triclosan which were consistently eluted at 4.434 ± 0.01 minutes.
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Fig-2: HPLC Chromatogram of Triclosan Standard

The results of system suitability of criteria for triclosan revealed that all parameters fulfilled the
requirements. Triclosan was eluted and gave nearly a symmetric peak (low tailing factor), a high number
of theoretical plates, and gut injection repeatability. The overall system suitability criteria result Table-1.
Table-1: System Suitability of Criteria for Triclosan Standard (n=9)
Parameter Triclosan Acceptable limit17
tR (SD) in minutes 4.434 (0.01) 
Tailing factor 1.112 2
Number of theoretical plates 710098  2000
Injection repeatability (% RSD) 0.40 1
Linearity
Figure-3 shows the calibration curve of triclosan in the concentration range of 10 - 60 ppm with a linear
regression equation of y = 19410x + 39153 and r2 value of 0.9997. This result indicates that there is a linear
relationship between the measurement responses and the concentration of triclosan. In addition, the linearity
was tested by comparing the relative procedural standard deviation of linear regression (linear VX0) with
that of quadratic regression quadratic (quadratic VX0). Since linear VX0 is less than quadratic VX0, the linear
regression is better than quadratic regression, which is proof of linearity.18 The VX0 calculation results are
presented in Table-2.

14,00,000
12,00,000 y = 19410x + 39153
10,00,000 R² = 0.9997
8,00,000
Area

6,00,000
4,00,000
2,00,000 Concentration (μg/ml)
-
0 10 20 30 40 50 60 70

Fig.-3: Linear Calibration Curve of Triclosan

Table-2: Linear and Quadratic VX0 Calculation Results
No C (mg/L), Xi AUC, Yi Xi2 Xi3 Xi4 Yi2 XiYi Xi2Yi
1 10 238473 100 1000 10000 56589530552 2384733 23847333
2 20 418786 400 8000 160000 175381992708 8375727 167514533
3 30 624562 900 27000 810000 390077691844 18736860 562105800
4 40 820131 1600 64000 2560000 672614310954 32805227 1312209067
5 50 1019780 2500 125000 6250000 1039951248400 50989000 2549450000
6 60 1201551 3600 216000 12960000 1443725589625 72093080 4325584788
 210 4323284 9100 441000 22750000 3778620373082 185384626 8940711522
y̅ 720547.2
Linear VX0 (%) 0.863
Quadratic VX0 (%) 1.210

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Precision
For precision determination, a sample matrix was spiked with triclosan standard solution at 30 ppm.
Repetitive determination of triclosan in that spiked sample gave RSD value of 0.56% (N = 9). According
to AOAC17, RSD value for repetitive determination in the range 10-100 ppm and 100-1000 ppm should be
not more than 4 - 6% and 3% - 4%, respectively. In addition, the calculation of Horrat value gave a result
0.19%, while the acceptance value is ≤ 2%17. These results highlighted that the analytical method used has
good precision. The result of the precision determination is depicted in Table-3.
Table-3: The Result of Precision Determination of Triclosan (n=9)
Sample No. Obtained Concentration
(Spiked concentration 30 ppm) (μg/mL)
1 29.62
2 29.76
3 30.09
4 30.03
5 30.03
6 29.93
7 30.06
8 30.10
9 30.03
x 30.00
SD 0.2
RSD (%) 0.56
RSD Horwitz 3.38
HORRAT 0.19
Accuracy
The accuracy was determined from the % of recovery determination of spiked triclosan standard solution
to the sample matrix at the concentration level of 80%, 100% and 120% of the predicted levels of analyte
in the sample. The results of these determinations were in the range of 97% - 104%, while the acceptable
% of recovery of AOAC17 for the concentration range of 10-100 ppm is 80-115%. Thus, the % of recovery
obtained can be accepted based on the AOAC criteria, and hence the method shows good accuracy. The
results of accuracy determination were presented in Table-4.
Limit of Detection (LOD) and Limit of Quantification (LOQ)
The LOD and LOQ values of triclosan were determined using statistical calculation method17 from the
calibration curve equation. Table-5 shows the results of LOD and LOQ calculations.
Table-4: Accuracy in the Assay Determination of Triclosan
Spike Level Spiked (ppm) Obtained (ppm) % of Recovery Mean % Recovery
27.3 22.97 74
Of 80% 29 22.97 10 97%
29.7 22.97 115
31.5 23.33 86
100% 32.1 23.33 92 94%
33.4 23.33 106
37.7 22.27 106
120% 35.5 22.27 91 104%
38.7 22.27 113

Table-5: LOD and LOQ of Triclosan

Sample No. Concentration (ppm) yr yi yr-yi (yr-yi)2
1. 10.02 238473.33 233834.52 4638.80 21518552.64
2. 20.05 418786.33 428516.04 -9729.71 94667338.41
3. 30.08 624562.00 623197.57 1364.42 1861667.04
4. 40.11 820130.66 817879.09 2251.57 5069579.17
5. 50.14 1019780.00 1012560.61 7219.38 52119476.46

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6. 60.17 1201551.33 1207242.14 -5690.81 32385336.66

∑(yr-yi)2 207621950.4
1
Sy/x 5882.49
(Sy/x)/b 0.30 ppm
LOD 1.00 ppm
LOQ 3.03 ppm
This method has the LOD and LOQ values of 1.00 and 3.03 ppm, respectively. Since the expected
concentration of triclosan in the samples will be higher than LOQ values, this method will be able to
determine triclosan in the samples with good precision and accuracy.
Identification and Determination of Triclosan in the Samples
The identification of triclosan in the samples was conducted based on the presence of peaks in the
chromatograms eluted at the retention times not significantly different from those of triclosan standard. In
addition, in the case of doubt, a co-chromatography procedure was carried out, i.e., triclosan standard
solution was spiked into the sample solution. The chromatograms revealed that the retention times of
triclosan were in the range of 4.09 - 4.36 with an average of 4.155 minutes, while the retention of standard
triclosan was averagely 4.434 minutes. The slight difference in the retention time is still acceptable due to
possible matrix influences in the sample solutions. Commonly, the influences of the matrix could lead to
column enrichment and result in earlier retention time. Fig.-4 shows the overlay chromatograms of several
sample solutions.

Fig.-4. Overlay Chromatograms of Sample Solutions

Triclosan was detected in several samples which were further classified into two categories by the
regulation, i.e., rinse-off products and non-rinse-off products. Hand sanitizer, feminine hygiene, antiseptic
soap, detergent, mouthwash, and laundry freshener were included in the rinse-off products. The triclosan
levels in these six products were in the range of 0.024 – 0.321%. Furthermore, deodorant, face mask, and
face powder were included in non-rinse-off products, in which three product samples were identified to
contain triclosan in the range of 0.024 – 0.070%. All the determination results (Table-6) and revealed that
the triclosan level in those products was not more than the permitted level according to regulation in
Indonesia (2% for rinse-off products and 0.3% for non-rinse-off products).
Table-6: Analytical Results of Household, Personal Care, and Cosmetic Products by IRP-HPLC UV
No. Samples %Found  SD (N=5) Maximum Level19 (%)
Solid-base Semisolid-base Liquid-base
Rinse-off Products
1. Hand sanitizer 0.3210.002
2. Laundry freshener 0.0290.002
3. Detergent 0.0240.000
2
4. Feminine hygiene 0.0830.000
5. Mouthwash 0.1250.006
6. Body wash liquid soap 0.2080.001
Non-Rinse-off Products
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1. Face masks 0.070.000

2. Deodorant 0.0400.000 0.3
3. Face powder 0.0240.000
CONCLUSION
An HPLC method equipped with C18 columns controlled at 30 C, UV detection system at 280 nm and the
mobile phase has been developed and was successfully applied for determination of triclosan in several
household, personal care, and cosmetic products samples. The triclosan levels in those products were in the
range of 0.024 – 0.321% which were not more than the maximum permitted level according to regulation
in Indonesia.
ACKNOWLEDGEMENT
The author would like to thank BUDI DN No. 20161141041010 and LPDP Indonesia for their financial
support.
REFERENCES
1. J. D. Lee, J. Y. Lee, S. J. Kwack, C. Y. Shin, H. J. Jang, H. Y. Kim, M. K. Kim, D. W. Seo, B. M. Lee
and K. B. Kim, Toxicological Research. 35(2), 137(2019), DOI:10.5487/TR.2019.35.2.137
2. T. Liu, and D. Wu, International Journal of Cosmetic Science, 34, 489(2012), DOI:10.1111/j.1468-
2494.2012.00742.x
3. N. Halla, I. P. Fernandes, S. A. Heleno, P. Costa, Z. B. Otmani, K. Boucherit, A. E. Rodrigues, I. C. F.
R. Ferreira, and M. F. barreiro, Molecules, 23(1571), 1(2020), DOI:10.3390/molecules23071571
4. E. Manova, N. van Goetz, C. Keller, M. Siegrist, and K. Hungerbühler, International Journal of
Environmental Research and Public Health, 10, 2778(2013), DOI:10.3390/ijerph10072778
5. H. Shaaban, and W. Alhajri, Journal of Environmental and Public Health, 2020, Article ID 8434508
(2020), DOI:10.1155/2020/8434508
6. J. W. H Biesterbos, T. Dudzina, C. J. E. Delmaar, M. I. Bakker, F. G. M. Russel, N. van Goetz, P. T. J.
Scheepers, and N. Roeleveld, Food and Chemical Toxicology, 55, 8(2013),
DOI:10.1016/j.fct.2012.11.014
7. A. B. Dann, and A. Hontela, Journal of Applied Toxicology, 31, 285(2011), DOI: 10.1002/jat.1660
8. D. Mackay, and L. Barnthouse, Integrated Environmental Assessment and Management, 6, 390(2010).
DOI:10.1002/ieam.73
9. X. May, D. H. Bennett, B. Ritz, D. L. Cassady, and K. Lee, and I. H. Picciotto, Food and Chemical
Toxicology. 48, 3109(2010), DOI: 10.1016/j.fct.2010.08.004
10. S. Subramonian, S. Chandran, M. Murugan, and T. Murugan, Rasayan Journal of Chemistry, 9,
335(2016).
11. N. Aminu, S. Y. Chan, N. H. Khan, and S. M. Toh, Acta Chromatographica, 30, 219(2018),
DOI:10.1556/1326.2017.00286
12. R. K. Bhogadi, A. Satyanarayana, N. Someswara Rao, S. Arutla, and A. Malleswarareddy, Rasayan
Journal of Chemistry, 7, 380(2014).
13. S. Shiyan, S. Shiyan, R. Martien, and A. K. Nugroho, Rasayan Journal of Chemistry, 12(3),
1098(2019), DOI: 10.31788/RJC.2019.1235276
14. F. I. Nuryana, M. A. Chozin, and D. Guntoro, Rasayan Journal of Chemistry, 12(1), 360(2019),
DOI:10.31788/RJC.2019.1215024
15. V. Sathiyanarayanan, H. Venkatasubramanian, and D. Easwaramoorthy, Rasayan Journal of
Chemistry, 12, 2141(2019), DOI: 10.31788/RJC.2019.1245419
16. G. Ramanaiah, D. Ramachandran, G. Srinivas, J. Gowardhane, P. Rao, V. Srilakshmi, American
Journal of Analytical Chemistrys, 3, 378(2012), DOI: 10.4236/ajac.2012.35050
17. Association of Official Analytical Chemist (AOAC), Official Methods of Analysis of the Association of
Official Analytical Chemist International, 18th ed, Washington DC (2011)
18. W. Gottwald, Statistik fuer Anwender, Auflage, Wiley-VCH, Weinheim, 1, s. 89 - 146 (2000)
19. ASEAN Member countries, ASEAN Definition of Cosmetics and Illustrative List By Category of
Cosmetic Products, 138 (2019).
[RJC-5861/2020]
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