Stability-indicating liquid chromatographic method development and validation for quantification of trifarotene in pure and topical drug product

Background Trifarotene is effective for treating acne and other skin issues. To ensure its quality and meet regulatory standards, a reverse phase liquid chromatography (RP-LC) stability-indicating method was developed and validated for its quantification in pure and topical dosage forms. An isocratic elution chromatographic method was employed, using an octadecylsilyl silica gel-packed column (150 mm × 4.6 mm, 3 µm particle size). The mobile phase was a mixture of phosphate buffer and acetonitrile (40:60 v/v). Chromatographic conditions included a flow rate of 0.5 mL/min, column temperature of 40 °C, detection at 265 nm, and injection volume of 20 µL. Results The developed analytical method reports the retention time of trifarotene 11.2 min, higher theoretical plate count, asymmetric peak, and good resolution between the peaks of trifarotene, phenoxyethanol, and environmentally generated impurities. Conclusion The analytical method has been found to be linear, accurate, robust, specific, and selective for impurities produced during forced degradation studies. The proposed analytical method can be utilized for routine pharmaceutical analysis of trifarotene to judge its quality and safety.


Background
A chronic inflammatory skin disease called acne vulgaris damages the pilosebaceous cells.As a result, papules, pustules, nodules, comedones (blackheads and whiteheads), and sometimes cysts appear [1].Acne treatment focuses on reducing sebum production, preventing follicle blockages, controlling bacterial colonization, reducing inflammation, and using topical medicines [2].
Trifarotene (Fig. 1), chemically (4-[3-(3-tert-butyl-4-pyrrolidin-1-ylphenyl)-4-(2-hydroxyethoxy)phenyl] benzoic acid) is a fourth-generation topical retinoid that specifically acts on the retinoic acid gamma receptor, which is mainly involved in the regulation of keratinocyte differentiation and the reduction of inflammation.Trifarotene is available in cream form 0.005% w/w and has been shown to be effective in treating acne on both the face and trunk [3,4].Trifarotene was approved on October 4, 2019, by the FDA in the United States under the name Aklief cream [5].According to the literature review, only one LC-MS method has been reported for the determination of trifarotene from plasma samples [6].This study effort only focuses on clinical studies and does not provide further details.Additionally, this trace analysis method cannot be used in routine pharmaceutical analysis that involves excipients.Moreover, there is no official guide for the analysis of trifarotene in any Pharmacopeia.Since trifarotene is in a low-dose form, a sensitive and selective method is needed to separate and measure it from other potential excipients.Therefore, this study aimed to develop an analytical method to measure trifarotene in topical dosage form using liquid chromatography by following the ICH guidelines [7].

Materials and chemicals
Horizon Healthcare, Pakistan graciously provided the trifarotene drug substance (purity: 99.6%, manufactured by Kaifeng Pharmaceutical, China) and excipients.We utilized water and acetonitrile of HPLC-grade manufactured by Honeywell.Aklief ® cream was purchased online.

Instrumentation and chromatographic conditions
The Shimadzu LC-10AD HPLC system included a thermostat oven, multi-channel photodiode array detector, vacuum degasser, coolant auto-sampler, and quaternary pump was used for the whole study.The system was connected to a computer running Lab solution software, which was used to control the equipment and estimate the results and responses.The system was operated isocratically at 40 °C column temperature and 0.5 mL/min running mobile phase consisted of 10 mM potassium dihydrogen phosphate (adjusted to pH 3.0 with 20% v/v solution of orthophosphoric acid) mixed with acetonitrile in 40:60 v/v ratio.The injection volume was 20 µL, 50% v/v solution of acetonitrile in distilled water used for needle rinsing.The photodiode array detector was set to scan wavelengths from 190 to 800 nm to identify the maximum absorbance.

Preparation of standard and calibration curve solutions
All solutions were made using acetonitrile and filtered through 0.2 µm filters.A stock solution with 100 µg/ mL of known concentration was prepared by mixing an appropriate amount of trifarotene in a flask with acetonitrile.A standard solution of 5 µg/mL was then made by diluting a portion of the stock solution.The calibration curve covered a range of concentrations from 2 to 10 µg/ mL.

Preparation of test and placebo solutions
The test solution was prepared by transferring 2 g of cream (equivalent to 0.1 mg nominal amount of trifarotene) into a 50 mL beaker, adding 10 mL of acetonitrile, and sonicating in an ultra-sonicator bath at 20 °C for 15 min to disperse all materials.Then, it was transferred into a 20 mL volumetric flask, and diluted to volume via rinsing a beaker with portions of acetonitrile.The placebo solution was prepared by transferring 10 mg of each excipient, (allantoin, copolymer of acrylamide and sodium acryloyldimethyltaurate, isohexadecane, cyclomethicone, ethanol, medium-chain triglycerides, phenoxyethanol, and propylene glycol) in 20 mL flask, mixed, and diluted with acetonitrile.Separate excipient solutions were also made for identification.

Forced degradation studies
The forced degradation studies were employed to investigate the intrinsic stability of trifarotene.A 100 µg/mL stock solution was prepared in diluent, which was then diluted to prepare a control standard solution of 5 µg/ mL concentration.The oxidative sample solution was prepared by mixing 5 mL of the stock solution with 5 mL of 3% hydrogen peroxide solution, diluted to 100 mL and kept at room temperature for 24 h.For acidic degradation, the sample solution was prepared by mixing 5 mL of standard stock solution with 5 mL of 0.1 M hydrochloric acid solution and left in dark for 24 h, then neutralized by adding 5 mL of 0.1 M sodium hydroxide solution and diluted to 100 mL.Similarly, for alkaline degradation, the sample solution was prepared by adding 5 mL of 0.1 M Sodium hydroxide solution, then neutralized and diluted.The thermal-humidity and photolytic environmental conditions were applied by keeping one sample solution of 5 µg/mL concentration at 60 °C and 75% relative humidity in climatic chamber for 24 h, and one sample solution was exposed to 2000 lx light by using xenon lamp for 24 h, respectively.Thermal, Thermal-humidity, and photolytic stability studies were also conducted on formulated cream samples at a nominal concentration of 5 µg/mL.For thermal studies, samples were placed in a thermostat oven at 80 °C for 3 h.Thermal-humidity and photolytic conditions were applied as previously described.

Analytical method development and optimization
The first step in developing this method was to determine the wavelength that minimizes baseline noise and provides sufficient signal strength, as well as the optimum system suitability.The ultraviolet-visible absorption spectrum provided the maximum absorbance of trifarotene at 265 nm (Fig. 2).In order to achieve better separation of trifarotene and excipients along with analyte better peak shape, various reversed-phase columns with different particle sizes, carbon contents, and lengths were tried.But the maximum efficiency and separation were achieved at Ultisil XB-C18 (150 mm × 4.6 mm, 3 µm).The injection volume higher than 50 µL cause increase in tailing factor.So, for the better peak asymmetry, 20 µL injection volume was chosen.To enhance the separation and improve the performance, we reduced the flow rate gradually from 1 to 0.5 mL/min and increased the column temperature from 30 to 40 °C.The details of the system suitability results provided in Table 1 indicated that all the experimentally measured system suitability parameters comply with the standard acceptance criteria.

Analytical method validation Specificity and forced degradation studies
By injecting blank, placebo, test, standard, and degradation solutions, the specificity of the method was examined and the impact of excipients and degradation products on the trifarotene retention time was assessed.The chromatograms in Fig. 3 revealed that, there was no interference of phenoxyethanol and degradation products observed in the retention time of trifarotene.The forced degradation studies results provided in Table 2.

Linearity, LOQ and LOD
The linearity was assessed by injecting three replicates of each of nine dilutions ranging from 40 to 200% (2-10 µg/  mL) of the target concentration.The calibration curve was a straight line rising concerning each concentration demonstrating slope (b = 133,207), intercept (a = − 248), correlation coefficient (r 2 = 0.9998), and standard deviation of intercept (SDI = 13,876).The limit of quantification and detection was calculated by dividing the value of the standard deviation of intercept by the value of slope and multiplying the resulting value with a factor (10 for LOQ and 3.3 for LOD).The quantitation concentration limit was recorded 1.04 µg/mL, and the detection concentration limit was 0.34 µg/mL.

Stability of solutions
The stability of the solutions was assessed by storing the standard and test solutions for one day at room temperature (25 °C and 60% relative humidity) and in a refrigerator (5 °C ± 3 °C).The % assay was measured at 6, 12, 18, and 24 h.After 24 h, the room temperature assays were 98.51% for the standard solution and 97.39% for the test solution, while the refrigerated assays were 100.27% for the standard solution and 99.85% for the test solution, compared to a freshly prepared standard solution.

Discussion
Since only one LC-MS analytical method was indicated from the literature and this method only focus on clinical studies [6].Hence, the goal of this study was to develop and validate an analytical method for determining trifarotene in low-dose topical dosage form.During the development of the analytical method, the spectrum of trifarotene showed maximum absorbance at two wavelengths: 265 and 295 nm.The 265 nm wavelength was chosen as the optimal wavelength to detect degradation impurities.The method was specific and selective, showing no interference from blank and potential excipient (phenoxyethanol).Forced degradation tests revealed trifarotene's instability in oxidative, alkaline, and thermal-humidity environments, but greater stability in acidic and photolytic conditions.Maximum impurities were generated in oxidative and alkaline environments, with all impurities successfully separated, demonstrating good selectivity.In formulated cream samples, the most significant degradation occurred under thermal and thermal-humidity conditions.The method followed Beer's law calibration curve providing the correlation coefficient > 0.990, with recovery rates within 98-102% and an overall % RSD of less than 2%, indicating accuracy and precision.Robustness tests showed that increased flow rate, column temperature, and acetonitrile ratio decreased retention time and resolution, while wavelength variation affected peak area.% RSD for recovery from these changes was less than 2%, indicating minimal impact on % recovery.The solutions were stable in the refrigerator, with slight degradation observed at room temperature.

Fig. 2
Fig. 2 UV-visible spectrum of Trifarotene; b Peak purity plot; c Peak profile plot

Fig. 3
Fig. 3 Chromatograms of trifarotene: a standard; b sample; c blank; d placebo; e oxidative degradation; f base degradation; g acidic degradation; h thermal & humidity degradation; i photolytic degradation; j formulated samples-Thermal degradation; k formulated samples-thermal-humidity degradation; l formulated samples-photolytic degradation

Table 1
System suitability results*Mean of six measurements ± standard deviation

Table 3
Recovery studies results*Mean of three measurements, SD; standard deviation, RSD; Relative standard deviation

Table 4
Robustness results *Resolution between trifarotene and phenoxyethanol peak