INTRODUCTION:

In the year 2019, U.S. FDA approved trifarotene, a fourth-generation topical retinoid, to treat acne vulgaris under the trade name Aklief¹. Trifarotene works well to treat acne on face and trunk and is sold as a cream with a 0.005% w/w concentration². A common skin ailment, acne vulgaris, affects about 80% of young adults between the ages of 11 and 30.³ It leads to disfigurement, permanent scarring, and can significantly harm psychological development, resulting in social phobias, withdrawal from society, and clinical depression.^4,5

Triferotene is a type of topical retinoid designed to target retinoic acid receptor (RAR) specifically γ. Triferotene is efficacious even at low doses because of its great selectivity for RAR-γ, most prevalent isoform of RARs in skin. Triferotene, like other topical retinoids, decreases proliferation and encourages keratinocyte differentiation, which helps lessen hyperkeratinization.^6–8 Triferotene's selectivity for RAR-γ sets it apart from existing first generation topical retinoid which includes tretinoin, isotretinoin, and third-generation topical retinoids which includes adapalene, all of which are nonspecific and target both RARβ and RARγ.⁹ There is only one LC-MS technique available for detecting trifarotene in plasma samples, according to a comprehensive analysis of the analytical literature.¹⁰ Pharmacopoeia does not have any formal guidelines for trifarotene analysis. A sensitive and selective approach is required to test it accurately among potential excipients because of its low-dose administration. In accordance with ICH recommendations, the goal of this work was to create a liquid chromatography method for measuring trifarotene in topical dose forms.¹¹ Given this gap, an in-depth exploration of structurally related drugs was conducted to guide the development of a robust and reliable analytical method.^12-15 HPTLC is widely adopted due to its high sample throughput, minimal sample preparation, and low operational cost. It allows simultaneous analysis of multiple samples using small volumes of mobile phase, making it more time- and cost-efficient than HPLC¹⁶. Thus, this study was designed to conduct a validated simple HPTLC method to determine Triferotene in a cream formulation.

Figure 1: Structure of Trifarotene ^6-8

MATERIALS AND METHODS:

Materials:

TLC Silica gel F254 glass plates (20 cm × 10 cm, 250 µm thick; Sigma-Aldrich) were coated with the sample in the form of bands using a 100 µL CAMAG micro syringe and a CAMAG Linomat 5 sample applicator (CAMAG, Muttenz, Switzerland). 20 milliliters of mobile phase, that is consists of Chloroform: Ethyl acetate: Ethanol: Formic acid at a ratio (7.5:2:0.5:0.1 V/V/V/V), were maintained for 40 minutes in a CAMAG twin trough glass chamber (20 cm × 10 cm) until it reached saturation, keeping the lid closed. CAMAG TLC Plate Heater was used to activate the TLC plates for 20 minutes. Utilizing a nitrogen aspirator, the sample was applied in narrow bands. The chromatogram was developed up to an 80 mm distance in a linear increasing direction. Plates were scanned after development, by the help of CAMAG TLC Scanner 4 along with a 262 nm-wavelength deuterium light. CAMAG visionCATS program, version 3.0, had been utilized for peak evaluation. Samples and standards were weighed using a Sartorius Analytical Balance.

Chemicals and Reagents:

Pharmaceutical-grade trifarotene was acquired from the Central Drug Testing Laboratory (CDTL), located in Mumbai. Trifarotene cream (Aklief, 50 µg/g) was sourced from Galderma Laboratories. AR (Analytical Reagent) grade Ethanol (Hayman UK), Ethyl acetate, Formic acid, along with Chloroform, were from Rankem. We purchased TLC Silica gel 60 F254 glass plates from Sigma-Aldrich in India.

EXPERIMENTAL:

RP-HPTLC Method Development:

Chromatographic conditions:

Prior to chromatography, HPTLC Silica gel 60 plates were activated for 10 minutes at 110 °C¹⁷. To saturate the compartment, twenty millilitres mobile phase were added to covered development tank. For forty minutes, the assembly was kept to come to room temperature. Samples were spotted as narrow bands, each 8mm long. X and Y application locations were maintained at 8 and 20 mm, respectively, to prevent edge effects. Using a CAMAG twin trough glass chamber saturated with a mobile phase consisting of 7.5:2:0.5:0.1 v/v/v/v formic acid, ethanol, ethyl acetate, and chloroform, the chromatogram run was maintained at 80 mm for 40 minutes. This made it possible for the chromatogram to develop linearly and ascending. After development, an air dryer in a well-ventilated location was used to dry the HPTLC plates in a stream of air¹⁸. Using CAMAG TLC Scanner 4 in the reflectance–absorbance mode at 262 nm and a deuterium lamp as a radiation source, the separated components were subjected to spectro-densitometric analysis. The speed of the scan was 100 nm/s. Using vision CATS (CAMAG) software for data processing and peak area measurement, the evaluation was performed by linearly regressing the peak area response against the dose^19,20.

Determination of wavelength of maximum absorbance¹⁹

A UV spectrum was obtained by scanning the trifarotene solution between the ranges of near UV-visible region, i.e. 200-400 nm. A λ_max(maximum absorbance) of 262 nm was achieved and hence selected for Trifarotene analysis in cream dose form.

Preparation of Standard Solutions:

Ethanol served as a diluent in the preparation of a standard solution of100 µg/mL of trifarotene, after that, it was further diluted to 5µg/mL.

Preparation of sample solution:

A 10 mL volumetric flask containing 1.0 g of cream (0.05 mg trifarotene) was carefully weighed. The mixture was sonicated for fifteen minutes after the diluent was added. After adding diluent to raise the volume to the necessary amount, the final concentration was 5 µg/mL.

Method Validation:

System suitability:

This test evaluates the effectiveness of the HPTLC system. A blank preparation (single injection) and a standard preparation (six replicates) of trifarotene were analyzed, and the chromatograms were recorded to assess parameters like Rf Value.

Specificity:

For identification purposes, specificity, refers to the ability to differentiate between closely related compounds or to compare results with a known reference sample or impurity²¹. To ensure accurate analysis, it is recommended to inject a blank, which will help identify any peaks that may appear in the chromatogram. This process enhances the reliability of the results.

Accuracy:

The recovery experiments have been carried out at three distinct levels: 110 %, 120 %, and 130%. To perform the recovery tests, a predetermined amount of standard solution was added to the concentration of the pre-analyzed sample at 3 distinct levels. The average recovery was computed and documented after three tests were taken at each level²².

Linearity and Concentration Range²⁰:

Linearity of trifarotene had been carried out by appropriately diluting the standard stock solution within a 2–10 µg/ml concentration range were prepared with a concentration of 50% to 150%, and all were filled in a vial.

Robustness:

The method's robustness was assessed by adjusting certain parameters. This involved changing the saturation time by ±5 minutes, enabling the solvent front to move a distance within a range of ±5 mm, modifying the mobile phase volume by ±5 ml, and increasing ratio of some mobile phase components by ± 0.2 ml.

Precision:

Repeatability and intermediate precision investigations, which comprised intraday and interday precisions²¹, were used to confirm the accuracy of the suggested approach. A 5µg/ml trifarotene standard solution was applied six times to calculate the repeatability percentage RSD. Trifarotene standard solutions “containing 2.5, 5, and 7.5µg/ml were” applied three times on same day to plate in order to measure intraday precision. Analysing the 2.5, 5, and 7.5 µg/ml trifarotene standard solutions three times on the plate on three separate days allowed for the determination of interday precision¹⁶.

Sensitivity:

Sensitivity expressed as the limit of detection (LOD), the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated as an exact value under the experimental condition as well as limit of quantification (LOQ), which is the lowest amount of analyte that can be detected and quantified with suitable precision, accuracy and reproducibility²³. The LOD and LOQ was calculated based on the standard deviation of regression lines and slope of the calibration curve using the below equation:

LOD=3.3×σ/S, LOQ=10×σ/S

Where σ is the standard deviation of the regression line and S is the slope of the calibration curve. The results are summarized in table VI.

RESULTS AND DISCUSSION:

Analytical Method Validation:

According to ICH Q2 (R1) requirements²⁴, developed HPTLC method had been validated by examining factors, which is “including specificity, linearity, precision, accuracy, and robustness”.

Specificity:

It is “the capability of an analytical method for accurately calculate target analyte among” other sample elements. At the trifarotene retention time, no co-eluting peaks were seen. This validates the method's specificity and shows that the analyte's peak was pure. The findings are displayed in Figures 2 and 3.

Figure 2: Chromatogram of standard

Figure 3: Chromatogram of sample

System Suitability:

Six duplicates of the trifarotene standard solution were applied to the HPTLC system under ideal chromatographic conditions in order to assess system applicability. It was determined that the mean Rf value was 0.5517 with a %RSD of 1.3%, and the mean peak area was 0.00446 with a %RSD of 1.00%. The precision, reproducibility, and general appropriateness of the approach for the quantitative analysis of trifarotene were confirmed by the fact that all values were within acceptable bounds.

Linearity:

Trifarotene standard calibration curve was developed at concentrations between 0.5 and 3.5 µg/ml. To guarantee accuracy and repeatability, each concentration level was examined in triplicates using same chromatographic setup. Plotting the average trifarotene peak area ratio against the corresponding concentration data was done for each concentration in order to produce the calibration curve. As shown in Figure 4, the resulting graph showed a linear response, validating the method's suitability for statistical analysis within the given range.

Figure 1: Calibration curve of trifarotene at 262 nm

Precision:

Both “intra-day and inter-day investigations “were conducted at different concentration levels, 2.5, 5, and 7.5 µg/mL, to assess the repeatability” of the established approach. Specifically, the percentage RSD values of the intra-day precision were 0.853%, 0.487%, and 0.590%. As evidence of the method's dependability and reproducibility, the percentage RSD values for inter-day precision were” 0.775%, 0.457%, and 0.492%, respectively (Table III).

Accuracy:

Standard addition had been utilized to evaluate the method's accuracy at three different levels: 110%, 120%, and 130%²⁵. At these concentrations, 99.49%, 100.44%, and 99.47% of the trifarotene was recovered. Accuracy and dependability of the approach were verified by the entire mean recovery, which was 99.83% (Table IV).

Sensitivity:

Lowest concentration of the analyte that can be reliably detected or quantified were estimated²⁶. The lower LOD and LOQ values for trifarotene indicate that the developed method is sensitive. The RF value and % RSD of peak areas for trifarotene were found to be within the specified limits (Table 1).

Robustness:

By altering certain parameters, such as the solvent front distance, saturation period, and mobile phase composition, the method's robustness was assessed²⁴. Results showed minimal impact from these changes, with the mobile phase composition yielding an average assay value of 99.3%, saturation time resulting in 99.29%, and solvent front distance giving an average of 99.20%. These constant numbers attest to the method's robustness to small, intentional changes in parameters (Table 5).

Table 1: Regression analysis data

Parameters	HPTLC method
Linearity range	2 – 10 ng/band
Regression equation	y = 0.001x - 0.0007
Detection wavelength (nm)	262nm
Correlation coefficient (R²)	0.9992
Limit of detection (ng/band)	0.147ng/band
Limit of quantification (ng/band)	0.447ng/band

Table 2: Linearity data

Concentration (ng/band)	Peak area
2	0.00126
3	0.00214
4	0.00310
5	0.00422
6	0.00520
7	0.00615
10	0.00891

Table 3: Precision data

Conc. ng/band	Intraday Precision		Interday Precision


	Area ± SD	%RSD	Area ± SD	%RSD
2.5	0.00217 ±0.00001851	0.853	0.00218± 0.00001689	0.775
5	0.00455 ±0.00002216	0.487	0.00455± 0.0000208	0.457
7.5	0.00666 ±0.00003929	0.59	0.00665± 0.00003272	0.492

Table 4: Accuracy data

% Level	Amount Found (%)	% Recovery	Mean % recovery	SD of % recovery	% RSD of % recovery
100	99.93%	99.93	99.95	0.026457513	0.0264
100	99.94%	99.94
100	99.98%	99.98
110	109.30%	99.3	99.49	0.165227116	0.164
110	109.57%	99.57
110	109.60%	99.6
120	120.10%	100.1	100.44	0.314324673	0.315
120	120.72%	100.72
120	120.50%	100.5
130	129.40%	99.4	99.47	0.060827625	0.060
130	129.51%	99.51
130	129.50%	99.5

Table 5: Robustness studies

Parameters	Change in Parameter	% Assay	Mean %	SD	%RSD
Mobile phase composition-Chloroform: Ethyl acetate: Ethanol: Formic acid 7.5:2:0.5:0.1 (v/v/v/v)	74.8:20:5:1	99.04	99.3	0.367	0.37%
	75:20:5:1	99.14
	75.2:20:5:1	99.72
Saturation time	35min	99.71	99.29	0.365	0.37%
	40min	99.14
	45min	99.03
Distance travelled by the solvent	75mm	98.76	99.2	0.478	0.48%
	80mm	99.14
	85mm	99.71

Table 6: Analysis of trifarotene in cream dosage form

Drug	Brand name	Amount claimed	Amount found	%Amount estimated	SD	%RSD
Triferotene	Aklief	50µg/gm	49.54 µg/gm	99.09%	0.253	0.255

CONCLUSION:

The developed HPTLC method for trifarotene analysis demonstrated excellent simplicity, reliability, and precision. Comprehensive validation, as per ICH guidelines, confirmed its linearity, accuracy, recovery, robustness, and assay performance—all within acceptable limits. These results affirm the method’s suitability for routine quality control in the pharmaceutical industry.

LIST OF ABBREVIATIONS:

“ICH: International Council for Harmonization;

AR: Analytical reagent;

%RSD: Percentage Relative Standard Deviation;

NMT: Not More Than;

HPTLC: High-Performance Thin Liquid Chromatography;

UV-VIS: Ultraviolet-visible spectrophotometry;

SD: Standard deviation;

LOD: Limit of Detection;

LOQ: Limit of Quantification;”

AVAILABILITY OF DATA:

The supporting data of this study are available from the corresponding author. It will be provided as and when requested.

CONFLICT OF INTEREST:

None.

ACKNOWLEDGEMENTS:

The authors are grateful to the Central Drugs Testing Laboratory (CDTL), Mumbai, for providing the necessary facilities and permissions to carry out the HPTLC method development and validation work for the estimation of Trifarotene in topical cream formulations.

The contributions of each author are gratefully acknowledged:

Dr. Pratibha Daroi conceptualized and designed the research project.

Ms. Supriya Takalkar was responsible for the experimental execution and data collection.

Mr. Bushan Somawane provided technical supervision and overall guidance throughout the study.

Dr. Vijaykumar Munipalli contributed to the smooth conduct of the project by offering essential resources and logistical support.

The authors also express their appreciation to all staff members of CDTL, Mumbai, for their cooperation during the study.

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Received on 14.07.2025 Revised on 29.11.2025

Accepted on 15.02.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3251-3256.

DOI: 10.52711/0974-360X.2026.00463

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