1. INTRODUCTION:

Triclosan (TCS) is an antimicrobial compound found in various medicated cosmetic products like toothpaste, soaps, detergents, mouthwashes, handwashes, facewashes, and surgical cleaning solutions. Triclosan is a chlorinated phenoxy phenol derivative^1,2.

At high concentrations, triclosan acts as a bactericide by attacking bacterial membranes and cytoplasm. On the other hand, triclosan seems to be bacteriostatic at lower concentrations by preventing bacteria from producing fatty acids. Triclosan attaches to the bacterial enzyme enoyl-acyl carrier protein reductase (ENR). Nicotinamide adenine dinucleotide (NAD) and this triclosan-ENR combination combine to form a stable ternary complex called triclosan-ENR-NAD, which is unable to take part in the production of bacterial fatty acids. Due to this, it causes weakening of the cell membrane and cell death. Since humans lack the ENR enzyme, they are unaffected^2,3.

Despite its widespread usage, triclosan raises several health concerns. Hence, it is considered harmful. There are concerns that the triclosan used in many different applications could cause or promote bacterial resistance to antibiotics. Antibiotic resistance is a more serious issue, which has resulted in treatment failures for infectious diseases in both humans and animals⁴. An increased risk of food sensitivities, allergies, and asthma may result from an elevated amount of TCS. Furthermore, it has been discovered that TCS suppresses natural killer cell immunity by inhibiting its lytic function⁵. Triclosan may be an endocrine disruptor as well as affecting the body's thyroid hormone metabolism. TCS is classified as an endocrine-disrupting chemical (EDC) in humans as well as other species. Exposure to the ingredient is of particular concern to women due to its ability to pass through the placenta and into breast milk^1,5. Concerns exist over triclosan and its association with dioxin, a highly carcinogenic substance that can lead to significant health issues such as liver toxicity and immune system weakness. Research on triclosan's connection to cancer shows mixed results. According to some research, prolonged exposure to the chemical raises your risk of developing several types of cancer. However, more research has indicated that it may be utilized as a cancer treatment, namely for prostate cancer^1,3,5,6. Triclosan affects the reproductive function. TCS may reduce testosterone levels in men by blocking the production of testicular steroid hormones or by interacting with hormone receptors. Moreover, TCS bioaccumulation in reproductive organs has a direct impact on sperm production and semen quality and may cause cancer in those tissues. TCS exposure in females also affects the next generation, in addition to affecting reproductive hormone levels and ovarian function impairment^5,6.

Triclosan's potential effects on public health are being examined because the US Geological Survey has listed it as a "contaminant of emerging concern (CEC)" because of potential health risks⁷. In 2016, the FDA banned TCS from soap products. However, it is still allowed in handwash, toothpaste, and mouthwash. Beginning in January 2017, the European Union (EU) prohibited TCS from producing any biocidal products for personal hygiene. TCS is a common ingredient in toothpaste because it prevents gingivitis. TCS is still used in hand sanitizer products in the healthcare sector, where it is thought to be advantageous because hand sanitizers increase hand-washing compliance⁵. Triclosan use in cosmetics is limited to a maximum concentration of 0.3% in Europe, Canada, USA, India, and Australia, whereas in Japan maximum concentration allowed is 0.1% ^8,9.

Triclosan has adverse effects, hence cosmetic items shouldn't contain more of it than is permitted by the regulatory guidelines. Because of this, triclosan estimation is essential for evaluating whether or not triclosan is present at its limit. The quantity of triclosan in commercial products can be estimated using a variety of methodologies. The colorimetry method of triclosan estimation is an easy, cost-effective, and reliable method. Colorimetry is effective for both qualitative and quantitative estimation as it is based on the color reaction. The colored end product of colorimetry can be personally observed, allowing for qualitative estimation, or it can be detected using a spectrophotometer, allowing for quantitative estimation. Hence, the colorimetry technique is selected for qualitative and quantitative evaluation of triclosan in medicated cosmetic products. Many literatures are accessible for the determination of triclosan by the colorimetry method employing diazotization reaction^10-16.

Colorimetry is a frequently employed analytical technique that utilizes color estimation to determine the quantity of a colored chemical in a sample solution at visible light wavelengths. The intensity of the sample solution’s color determines the concentration of the colored compound. A radiation can pass through the sample holder that contains the analyte in the solution. The absorption of the radiation is proportional to the analyte's concentration. The sample's color is either an inherent property of the solution or can be changed by adding the right chemicals. It is possible to calculate the test sample's concentration by comparing its absorption to standards¹⁷. The colorimetry estimation method applies to many pharmaceuticals and related compounds ^18-26.

The colorimetric test of phenolic compounds can be carried out using the Folin–Ciocalteu reagent (FCR), sometimes referred to as Folin's phenol reagent or Folin–Denis reagent. It is composed of phosphomolybdic acid in phosphoric acid and sodium tungstate^27,28. The FCR reagent functions as the oxidizing agent that can take an electron from the electron donor in an electron-transfer reaction, which is the basis of the FCR method. The FCR reagent in an alkaline solution is reduced by the phenolic component, changing its color from yellow to blue. The reducing activity of the phenolic compounds is directly correlated with the amount of color shift that happens at the end of the reaction²⁹.

The following is the colorimetry reaction for triclosan estimation by FCR reagent in alkaline conditions. As per this reaction, the triclosan gets oxidized and FCR gets reduced to acquire a blue color product in an alkaline condition.

2. MATERIAL:

Instruments and Apparatus: UV-Visible Spectrophotometer: Shimadzu – UV 1900I, Digital Balance: Aczel -CY612, Sonicator: Lab junction digital ultrasonic cleaner, Beaker, Pipette, Volumetry flask: Borosil

Reagents: Triclosan: Ottokemi (batch no:0037), Folin–Ciocalteu reagent (FCR): Molychem (MCM-5372), Sodium Hydroxide: Finar AR (batch no:895970330IV), Water: type II water

Formulation: ToothPaste: Sample 1 and 2 [Triclosan-0.3%W/W], Soap: Sample 3 and 4 [Triclosan-0.5%W/W], Hand Wash: Sample 5 and 6, Facewash: Sample 7 and 8

3. METHOD:

3.1 Preparation of reagent solution:

3.1.1 Preparation of 0.5N NaOH: 2gm of NaOH Standard was dissolved in 100ml distilled water to make 0.5N NaOH solution.

3.1.2 Preparation of standard stock of triclosan(10000µg/ml): 1gm triclosan standard was dissolved in 100ml 0.5N NaOH to make 10000µg/ml solution.

3.1.3 Preparation of working standard of triclosan(1000µg/ml): 1ml standard stock of triclosan was diluted in 10ml with distilled(deionized) water to make 1000µg/ml solution.

3.1.4 Preparation of FCR dilute solution: 2.0ml FCR was diluted in 10ml distilled water.

3.2 Optimization of method: The experimental conditions for the colorimetry reaction were methodically developed by assessing how various reagents, their concentrations, and the quantity or volume of reagents affected the method's sensitivity.

3.2.1 Optimization of reagents: The effect of different reagents and the sequence of addition of reagents on the colorimetry reaction for triclosan estimation was observed and recorded.

3.2.2 Optimization of concentration of reagent: The effect of concentrations of sodium hydroxide on the colorimetry reaction for triclosan estimation was investigated over the range of 0.1N-1N. The effect of the concentration of FCR was also studied from concentrated to diluted (FCR in ml: Water in ml = 0.5:10, 2:10, 5:10).

3.2.3 Optimization of the amount or volume of reagents: Using an experimental technique known as full factorial design, the volume or amount of sodium hydroxide and FCR for the colorimetry reaction were optimized. The purpose of the experiment was to determine each reagent's function as well as the impact of their interactions on the colorimetry reaction. JMP (Version 17.2.0) was the program utilized to apply the strategy.

3.2.4 Optimization of resting time: The reaction mixture of triclosan, sodium hydroxide, and FCR was kept aside for 0, 5, 15, 30, and 45 minutes for the optimization of the time required to complete the colorimetry reaction.

3.2.5 Optimized Method: A 10ml volumetric flask was filled with 0.1 ml of triclosan standard stock solution, to which first 3 ml of 0.5N NaOH and then 3 ml of FCR diluted solution were added. The mixture was shaken thoroughly, and the volume was adjusted to 10 ml with distilled water to obtain 100 μg/ml triclosan solution. The mixture solution was left for 15 minutes to finish the reaction. The absorbance was measured in a UV-visible spectrophotometer in the 400–1000 nm wavelength range. 725nm was selected as the wavelength maxima for taking further readings.

3.3 Validation of method: According to ICH recommendations Q2R1, the validation parameters such as linearity, accuracy, precision, limit of detection, limit of quantitation, and robustness were examined ³⁰.

3.3.1 Linearity: 0.2, 0.4, 0.6, 0.8, and 1 ml were collected from the 1000 μg/ml triclosan working standard solution in a 10-milliliter volumetric flask. To this, 3 ml of 0.5N NaOH and 3 ml of Diluted FCR were added, and the volume was adjusted to 10 ml using distilled water to achieve a concentration range of 20–100μg/ml. At 725 nm, absorbance was measured. Plotting absorbance against concentrations facilitated the construction of calibration curves, and the regression equations were derived.

3.3.2 Precision: The repeatability was verified (n=6), by repeatedly measuring the absorbance of triclosan (60μg/ml) standard solutions and reporting the results. The intra-day and inter-day precisions (n=3) were determined by assessing the corresponding responses on the same day and other days, respectively, for three different triclosan concentrations (40, 60, and 80 μg/ml). Relative standard deviation was used to report the results.

3.3.3 Accuracy: The standard addition method was used to assess the accuracy parameter. A pre-quantified sample solution of triclosan (60μg/ml) was mixed with a known quantity of standard triclosan solutions (0, 40, 60, and 80μg/ml). At a chosen wavelength, the absorbance of triclosan was measured, and the recovery percentage was computed.

3.3.4 LOD, LOQ: The formulas LOD = 3.3 × N/S and LOQ = 10 × N/S were used to determine the limits of detection (LOD) and quantification (LOQ). Here, S is the calibration curve's slope and N is the standard deviation of the y-intercept.

3.3.5 Robustness: The experimental parameters that were slightly altered, like the solvent proportions, duration, and detecting wavelength to study the robustness. Changes were observed to have an impact on the regression coefficient, linearity, absorbance, and λmax. To determine the method's robustness, % RSD was calculated.

3.4 Analysis of formulations: The extraction method for the sample preparation was optimized by performing different trials(n=10). As per the optimized method, about 2 gm of triclosan sample products were weighed, and to this, 100 ml 0.5N NaOH solution was added. The ultrasonic cleaner was used to extract the samples for five minutes. The extracted materials were put into a centrifugal tube and rotated at 3000 rpm for 10 minutes. Whatman filter paper was used to filter the liquid supernatant. To the 10ml of filtrate, 3ml 0.5N NaOH and then 3ml FCR dilute solution were added. Shook well. To this, 4 ml of distilled water was added to make the dilution. Keep the mixture solution aside for 15 minutes to complete the reaction. Take the absorbance in a UV-visible spectrophotometer for the evaluation of triclosan in sample products.

4. RESULT AND DISCUSSION:

4.1 Optimization of method:

4.1.1 Optimization of reagents: The different reagents were studied for optimization of the colorimetry reaction. To make an alkaline condition, sodium carbonate was used in place of sodium hydroxide, but sodium carbonate gave precipitation with FCR. Therefore, sodium hydroxide was selected to make an alkaline condition. Methanol was used to dissolve triclosan, but it gave a turbid solution after adding FCR. So, sodium hydroxide was chosen to dissolve triclosan. In the colorimetry reaction, FCR was added before sodium hydroxide. This sequence of additions gave precipitation. To correct this, sodium hydroxide was added before the FCR.

4.1.2 Optimization of concentration of reagent: The lower concentration of sodium hydroxide created a solubility issue, and the higher concentration of sodium hydroxide gave precipitation. So, 0.5N NaOH was selected for the colorimetry reaction. The concentrated FCR gave precipitation, and the more diluted FCR gave less intense color in the colorimetry reaction for triclosan estimation. So, 2.0ml FCR diluted in 10ml water was chosen for the reaction.

4.1.3 Optimization of the amount or volume of reagents: A full 5² factorial design was used to investigate the effects of FCR and sodium hydroxide volume on the creation of a colorful product. Table 1 shows the experimental matrix with the results obtained. The triclosan concentration in each experiment was 100µg/ml.

Table 1: Experiment matrix with the results for the optimization of the amount of reagents for colorimetry estimation of triclosan

Sr. No	Concentration of 0.5 N NaOH in ml	Concentration of Dilute FCR in ml	Absorbance at 725nm	Observation
1	1	1	0.278
2	2	2	0.390
3	3	3	0.529
4	4	4	0.523
5	5	5	0.520
6	1	2	-	Precipitation
7	1	3	-	Precipitation
8	1	4	-	Precipitation
9	1	5	-	Precipitation
10	2	1	0.141
11	2	3	-	Precipitation
12	2	4	-	Precipitation
13	2	5	-	Precipitation
14	3	1	0.158
15	3	2	0.212
16	3	4	-	Precipitation
17	3	5	-	Precipitation
18	4	1	0.162
19	4	2	0.229
20	4	3	0.299
21	4	5	-	Precipitation
22	5	1	0.178
23	5	2	0.290
24	5	3	0.383
25	5	4	0.447

Figure 1 displays the Pareto graph for the estimated effects of the elements being studied and their interactions. The graph's horizontal bars show how important each of the parameters under investigation is in relation to the colorimetric reaction. The length of each bar on the chart represents the value of the estimated effect. A vertical line on the graph represents a statistically significant value. If the bar crosses this line, it indicates the significant effect of the factor. Based on this graph, the most significant effects were NaOH concentrations and the interaction between NaOH and FCR. The estimated effect values of all factors are presented in Table 2.

Figure 1: The Pareto graph for the estimated effects of factors on colorimetry reaction.

Table 2: The estimated effect values of all factors.

Term	Estimate	Std Error	t Ratio	Prob> [t]
Intercept	0.18956	0.029472	6.43	<.0001
NaOH(1,5)	0.15048	0.04168	3.61	0.0016
FCR(1,5)	-0.0378	0.04168	-0.91	0.3747
NaOH*FCR	0.13744	0.058944	2.33	0.0298

To validate the value of the derived mathematical model, the analysis of variance (ANOVA) of the experiment design was carried out. Table 3 presents the findings of the ANOVA study.

Table 3: Result of ANOVA study

Source	DF	Sum of Squares	Mean Square	F Ratio
Model	3	0.41897434	0.139658	6.4315
Error	21	0.45601182	0.021715	Prob > F
C. Total	24	0.87498616		0.0029

Using the response surface methodology, the optimization procedure was developed. It was feasible to examine how the elements chosen concurrently affected the relevant response (absorbance measurement) using the response surface. The relationship between dependent variables is displayed on the response surface. The optimal absorbance responses are shown in Figure 2. For this investigation, the ideal conditions were expected to yield a maximum absorbance value of 0.529 (±0.0256) with 3 ml of 0.5N NaOH and 3 ml of diluted FCR.

Figure 2: Three-dimensional plot of the optimized surface response of absorbance measurements at different NaOH and FCR concentrations.

4.1.4 Optimization of resting time:

The reaction mixture should be allowed to rest for 15 minutes, as indicated by Table 4, to avoid variations in absorbance and wavelength maxima.

Table 4: Optimization of resting time for reaction mixture

Time (min)	Absorbance at 725nm	Wavelength maxima
0	0.481	741nm
5	0.497	732nm
15	0.509	725nm
30	0.500	725nm
45	0.501	725nm

4.2 Validation of method:

4.2.1 Linearity: At 725 nm, the triclosan linearity (n=5) was found to be between 20 to 100µg/ml, with mean absorbances ranging from 0.241+0.12 to 0.732+0.21. The regression equation was determined to be y=0.0062x + 0.1187 with an R² value of 0.9985. The calibration curve of triclosan and overly spectra of triclosan are shown in Figures 3 and 4, respectively.

Figure 3: Calibration curve of triclosan standard

Figure 4: Overly spectra of triclosan standard

4.2.2 Precision: The %RSD for repeatability(n=6) was found to be 1.2%. For intra-day (n=3) precision, the %RSD was determined to be between 0.16 and 0.35 percent, while for inter-day (n=3) precision, it was between 0.28 and 0.40 percent. This value indicates the method is precise. Accuracy: The percentage recovery (n=3) for triclosan by the standard addition method (40,60,80µg/ml) was found in the range of 99.25-100.16%. This value represents the accuracy of the method. The results showed that the LOD was 1.55µg/ml and the LOQ was 4.71µg/ml. Robustness: By deviating the parameters such as solvent proportions (NaOH + FCR), time, and wavelength of detection, the percentage RSD for robustness studies was 0.29-0.67%, or less than 2%. The method's robustness is shown by this value.

4.3 Analysis of formulations:

The formulations analyzed and their assay results are listed in Table 5.

5. CONCLUSION:

A new, easy, and effective colorimetry method was created using FCR as a chromogenic reagent for the estimation of triclosan in various medicated cosmetic products. The experimental condition was optimized using a 5² full factorial experimental design with response surface methodology in terms of the concentration and volume of reagents. The impacts of the factors under study and their interactions were evaluated by the Pareto graph. To validate the value of the derived mathematical model, the analysis of variance (ANOVA) of the experiment design was carried out. The developed technique was validated as per ICH recommendations, and all validation parameters were determined to be within the specified limits. The suggested approach was shown to be cost-effective, innovative, straightforward, sensitive, accurate, reproducible, and precise; it can be applied to both qualitative and quantitative triclosan analysis in various medicinal cosmetic goods that contain triclosan.

6. ABBREVIATION:

TCS: Triclosan

FCR: Folin–Ciocalteu reagent

NaOH: Sodium Hydroxide

ICH: International Council for Harmonisation

ANOVA: Analysis of variance

7. ACKNOWLEDGMENTS:

The authors would like to thankful to Mr. Chirag Sharma, Dravya Analytical Solution LLP, Ahmedabad, India, for providing valuable contributions to perform this research work.

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Received on 18.05.2024 Revised on 07.12.2024

Accepted on 11.04.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5814-5820.

DOI: 10.52711/0974-360X.2025.00838

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Formulation	Label Claim	Assay % of label claim + RSD (n=3)	Mean Assay concentration
ToothPaste: Sample 1	Triclosan-0.3%W/W	92.21% + 0.54
ToothPaste: Sample 2	Triclosan-0.3%W/W	96.70% + 0.39
Soap: Sample 3	Triclosan-0.5%W/W	90.75% + 0.42
Soap: Sample 4	Triclosan-0.5%W/W	91.16% + 0.66
Hand Wash: Sample 5	-	-	2855microgram/gm
Hand Wash: Sample 6	-	-	2589microgram/gm
Facewash: Sample 7	-	-	3735microgram/gm
Facewash: Sample 8	-	-	4322microgram/gm