Validation of a chromatographic method for α-phellandrene quantification in skin permeation studies

Abstract

Alpha-phellandrene (α-PHE), a cyclic monoterpene abundant in volatile oils, has anti-inflammatory and healing properties. A cutaneous formulation using this bioactive could provide benefits in wound healing. However, a simple method that quantifies α-PHE in the skin is still required. Thus, the study aims to develop and validate a high-performance liquid chromatographic (HPLC-UV) method to quantify α-PHE extracted from the skin. The method uses a reversed-phase C₁₈ column (15 cm × 4.6

mm, 5 μm) and a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) flowing at 0.5 mL/min. The phase concentration (A/B %) along the 40-min run time varied as described: 15/85 (0.01-10 min), 0/100 (10.1-20 min), and 15/85 (20.1-40 min). α-PHE was detected at 263 nm. The method was selective against skin and formulation interferents, linear (r = 0.9995) in a concentration range of 0.5-15.0 μg/mL, precise with an overall variation coefficient lower than 5.5%, accurately achieving recovery from the skin within 92-102%, and sensitive for the proposed application (detection limit = 0.05 µg/mL, quantification limit = 0.14 μg/mL). Finally, the validated method was tested in a skin penetration study, attesting to its success in developing topical formulations containing α-PHE.

Keywords:
Essential oil; Alpha phellandrene; Validation; Analytical method; HPLC-UV; Topical application.

INTRODUCTION

The considerable extension of a tropical forest in the Amazon region reveals its potential for bioeconomic exploration (Barbosa et al., 2023). Medicinal plants are abundant reservoirs of bioactive substances with therapeutic potential. Among these compounds, essential oils represent complex combinations of volatile natural products from aromatic plants produced as secondary metabolites (Salas-oropeza et al., 2021; Siqueira et al., 2016). Such volatile compounds have presented several pharmacological applications and are considered potential sources for developing new drugs (Mohammed et al., 2024; Oliveira et al., 2018).

Protium heptaphyllum (Aubl.), also known as breu or white pitch oil, is a tree belonging to the Burseraceae family (Mendes et al., 2019; Piva et al., 2019; Silva et al., 2016; Vieira et al., 2014). It produces an amorphous and aromatic resin utilized in folk medicine for its analgesic and anti-inflammatory properties. This resin, abundant in essential oils, has alpha-phellandrene (α-PHE) as one of its predominant constituents (Bandeira et al., 2001; Mendes et al., 2019; Silva et al., 2016).

Phellandrenes are cyclic monoterpenes (Figure 1) consisting of α-PHE and beta-phellandrene (Cheng et al., 2017; Thangaleela et al., 2022). α-PHE (5-isopropyl- 2-methyl-1,3-cyclohexadiene) is a colorless to slightly yellow liquid with a mobile consistency and a peppery, woody, and herbaceous aroma. It is a cyclic monoterpene with both double bonds being endocyclic, distinguishing it from beta-phellandrene, which has one exocyclic bond (Radice et al., 2022).

FIGURE 1
Chemical structure of phellandrenes.

It has been discovered that α-PHE inhibits leukocyte rolling and adhesion, significantly reduces the production of the pro-inflammatory cytokines IL-6 and TNF-α, and exhibits antinociceptive effects (Radice et al., 2022; Scherer et al., 2019; Siqueira et al., 2016). Although nonsteroidal anti-inflammatory drugs and corticosteroids are among the most commonly prescribed medications worldwide for various acute and chronic pains and inflammatory conditions (Siqueira et al., 2016), they are associated with side effects, including gastrointestinal and cardiovascular complications, nephrotoxicity, idiosyncratic drug toxicity, and hepatotoxicity (Bindu, Mazumder, Bandyopadhyay, 2020; Chen et al., 2021). Thus, considering those anti-inflammatory and analgesic properties, the alternative use of α-PHE and extracts containing this biomarker could replace treatment with anti-inflammatory drugs.

The use of α-PHE as an active agent for topical purposes is still relatively underexplored. Moreover, the chromatographic methods for this biomarker in literature do not consider skin interference (Çelik et al., 2020; Coronel et al., 2006; Dugo et al., 2005; Kbaydet et al., 2024; Liu et al., 2024; Piva et al., 2019; Ramos et al., 2000; Siani et al., 1999; Singh et al., 2000; Verzera et al., 1998). The cutaneous application of this bioactive molecule implies the existence of a reliable analytical methodology capable of quantifying α-PHE in the presence of skin layers.

Thus, the objective of the research outlined herein was to validate a chromatographic method for quantifying α-PHE amidst skin interferences, facilitating the development of a cutaneous drug delivery system incorporating this natural compound.

MATERIAL AND METHODS

Material

The α-PHE pattern (>88%) used in this study was commercially acquired from Sigma-Aldrich^® (Steinhiem, Germany), and the essential oil from the plant white Pitch oil (Protium heptaphyllum) was provided by the Federal University of Pará (UFPA). HPLC-grade ethanol and acetonitrile were acquired from Dinâmica-Química contemporânea LTDA (São Paulo, Brazil). Span^® 80 and Kolliphor EL^® were obtained from Sigma-Aldrich (Steinhiem, Germany), and Labrasol^® was obtained from Gattefossé (Saint- Priest, France). All preparations and analyses were performed with ultrapure water (Millipore, Illkirch Graffenstaden, France).

The skin used in the permeation and recovery experiments was from porcine ears obtained from a local slaughterhouse (Via Carnes, Formosa, Brazil).

Preparation of skin extract

Porcine skin samples were cut into 2 cm² circles and then into small pieces. They were then placed in closed glass flasks with 5 mL of ethanol for α-PHE extraction. The samples were kept under magnetic stirring (300 rpm) for 24 h at room temperature. The solvent was then filtered through a 0.45 μm membrane filter.

Preparation of stock solutions

The stock solution (20.645 μg/mL) was prepared by diluting 13.8 μL of α-PHE (density 0.85 g/mL at 25 °C) in 25 mL of ethanol. Next, 500 μL of this solution was transferred to a 10 mL flask, supplemented with ethanol, and stored in a refrigerator (2° - 8 °C) for later use in the validation of the method. Three independent stock solutions were obtained. Analytical curves in ethanol were prepared from these solutions, varying the concentration from 0.5 - 15.0 μg/mL.

Scan

The maximum absorption wavelength of α-PHE was initially selected based on spectrophotometric scans of an α-PHE ethanolic solution at 100 μg/mL using UV spectrophotometric equipment (Lambda Bio Model, PerkinElmer, UK).

Apparatus and Chromatographic Conditions

The quantitative analysis of α-PHE was performed with high-performance liquid chromatography (HPLC) using a Shimadzu LC 20-AD equipment, equipped with a DAD detector (SPD-20A), a pump (LC-20D),a degasser (DGU-20A3), an automatic injector (model 9SIL-20AD), an oven (model CTO-20AS) and a computer equipped with the Shimadzu LC analysis software. The C₁₈Discovery HPLC Supelco Sigma-Aldrich reversed-phase column (15 cm × 4.6 mm, 5 μm) was used as a stationary phase. The mobile phase consisted of a mixture of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). A gradient elute system was used, varying phase concentration (A/B %) along the 40 min of run time as described: 15/85 (0.01-10 min), 0/100 (10.1-20 min), and 15/85 (20.1-40 min). A 0.5 mL/min flow rate and an injection volume of 50 μL were used. The column furnace was set to 45 °C. The detection was performed at 263 nm, representing the maximum absorption wavelength in the UV spectrum for α-PHE.

Validation of the Analytical Method

The validation of the proposed chromatographic method followed the guidelines of the International Conference of Harmonization (ICH, 2023). Different chromatographic conditions were tested, such as mobile phase composition, flow rate, and injection volume, to obtain α-PHE chromatographic peaks with acceptable analytical performance, considering the parameters of selectivity, linearity, detection, and quantification limits (LD and LQ, respectively), precision, accuracy, and robustness, according to guidelines.

Selectivity

The standard solution of α-PHE (5.0 μg/mL) was evaluated in the absence (blank solution) and in the presence of skin and of the topical formulations (microemulsions, prepared according to the described below). Assays were prepared from six independent sources of each matrix; the skin matrix was prepared as described above. The results were examined by comparing retention time and peak area. Moreover, Protium heptaphyllum extract was evaluated in the absence of α-PHE. All statistical calculations were performed using the GraphPad Prism 8.0 software (Boston, MA, USA).

Linearity

Six dilutions (0.5, 1.0, 3.0, 5.0, 10.0, and 15.0 μg/ mL), in triplicate of each concentration, were prepared from the stock solution with a concentration of 20.645 μg/mL, using ethanol as a diluent. The statistical analysis of the data was obtained using the linear regression method, angular coefficient significance, and proportionality tests using Student’s t-test (p = 0.05). Response factors were calculated considering the ratio between peak area and analyte concentration (Angelo et al., 2016b; Cardoso et al., 2023; Pereira et al., 2018).

Limit of detection and quantification

The minimum concentrations of α-PHE to be detected and quantified (LD and LQ, respectively) were calculated based on the standard deviation (σ) and slope (S) of the three independent calibration curves according to the following Equations (1 and 2):

(1) $$LD=\frac{(3,3x\sigma )}{S}$$

(2) $$LQ=\frac{(10x\sigma )}{S}$$

Precision

Precision was analyzed in repeatability and intermediateprecisionstudies.Thesampleswere prepared at three different calibration curve concentrations (0.5, 5.0, and 15.0 μg/mL) and analyzed in triplicate for each sample for the repeatability tests (intraassay). Samples at three different concentrations (0.5, 5.0, and 15.0 μg/mL) were analyzed in triplicate for each concentration in HPLC on different days by two different analysts to analyze intermediate precision (inter-assay). The precision results were expressed as a coefficient of variation (CV), calculated according to Equation (3), using the standard deviation of the mean (σ) and the mean concentration:

(3) $$CV(\mathrm{\%})=\left(\frac{\sigma }{\text{Mean concentration}}\right)\times 100$$

Accuracy

Accuracy was determined asthepercentage of α-PHE recovered from the skin. First, the skin was cut into small pieces. Then, different known volumes of the α-PHE ethanol solution were added to the cut skin to obtain the concentrations of 0.5 μg/mL, 5.0 μg/mL, and 15.0 μg/mL (n = 3 for each concentration) considering the final volume (5 mL), the samples were left in the fume hood until the solvent completely evaporated at room temperature. After, 5 mL of ethanol was added to each flask, and the samples were left under constant agitation for 24 h for α-PHE extraction. Finally, the samples were filtered in a 0.45 μm membrane and analyzed by the HPLC method described above. The recovery of α-PHE was determined according to the amount extracted from the skin relative to the amount added.

Accuracy was expressed by the ratio of the experimentally determined mean concentration to the corresponding theoretical concentration, according to Equation (4):

(4) $$\text{Recovery}(\mathrm{\%})=\left(\frac{\text{Measured concentration}}{\text{Theoretical concentration}}\right)\times 100$$

Robustness

Deliberated variations in the parameters of the method, i.e., oven temperature (OT), flow rate (FR), and mobile phase (W), were evaluated according to a factorial design to evaluate the robustness of the method (Angelo et al., 2016a; Quintão et al., 2022). The variation of the levels concerning the developed method considering the three parameters (OT = 45 °C; FR = 0.5 mL/min; and W = 15/85% (Water/Acetonitrile) (v/v)) was ± 1 °C; ± 0.05 mL/min; and ± 1%, respectively. All assays were performed in triplicate.

For each response (e.g., peak area, retention time, and tailing factor), a prediction equation was calculated using the stepwise analysis of multiple regressions. The model was validated by the analysis of variance (ANOVA) with a significance level of 0.05. All statistical analyses were calculated using the Action Stat software.

Application of the developed method in skin penetration studies

Preparation of the microemulsions

As a proof-of-concept, the α-PHE was incorporated into microemulsions. Two microemulsions were developed, each consisting of 31.58% oil phase, 21.05% surfactant (Labrasol^®: Span^® at a 4:1 w/w ratio), and 47.37% aqueous phase (ultrapure water). The oil phase of the first formulation (ME1) comprised 1.5% α-PHE and 30.08% Kolliphor EL, while the second formulation (ME2) contained 5.75% White Pitch oil (composed by 24.3% of α-PHE) and 25.83% Kolliphor EL. The concentration of α-PHE was chosen based on the maximum amount that could be solubilized within the formulations.

The droplet hydrodynamic diameter, polydispersity index (PDI), and zeta potential of ME1 and ME2 were assessed using Zetasizer Nano ZS equipment (Malvern Instruments, Worcestershire, UK). The pH values of microemulsions were evaluated using a pH meter (DigiMed, model DM-22, São Paulo, Brazil), equipped with an electrode for semisolid formulations.

Preparation of penetration studies

The skin penetration of α-PHE was evaluated in vitro using Franz-type diffusion cells coupled to the Phoenix^® DB-6 system (Teledyne Hanson Inc., Chatsworth, CA, USA). The diffusional area of the cells was 16 mm in diameter. Skin fragments separated the donor and acceptor compartments of the diffusion cells. The donor compartment was filled with 500 µL ME1 or ME2, and the acceptor compartment was filled with 15 mL of PBS with 3.0 % (w/v) Tween 80^®. The cells were kept at 35 ± 2 °C, maintained by a water bath, and magnetically stirred for 12 h. After the treatment, the skin was removed from the diffusion cells and cleaned, with an absorbent paper and water, before extracting the bioactive compound. The validated chromatographic method determined the amount of α-PHE recovered from the skin.

RESULTS AND DISCUSSION

Method development

Figure 2 shows the UV/Vis absorbance spectrum of the α-PHE in ethanol at 100 μg/mL, in which the wavelength of 263 nm represented the maximum absorption for the drug.

FIGURE 2
Absorption spectrum in the UV/VIS region of α-PHE at 100 μg/mL. Scan range: 200 to 800 nm.

Some mobile phases were used to identify and quantify the α-PHE in the skin and the developed formulations and to verify the best analytical condition to separate the analyte of interest from possible interferers of such chemical and biological matrices.

Briefly, water, acetonitrile, and ethanol were tested as mobile phases, with or without additives (acid), in different proportions and forms of elution (isocratic and concentration gradient), as well as stationary phases with different characteristics (Table I). Initially, some attempts were made using the isocratic method. However, the observed peaks were very early and presented low resolution according to the parameters established by the FDA (1994) and ICH guideline Q2 (R2) (2023). Ethanol was used as a cleaning solution for the injection needle.

TABLE I
Conditions tested in the isocratic method

Trial	Mobile phase (v/v)	Flow rate (mL/min)	Temperatu-re (°C)	Retention time (min)	Tailing factor	Theoretical plates	Peak characteristics
1	A:B (60:40)	0.8	45	8.5	1.046	6434	Suitable peak
2	A:B (80:20)	0.8	45	6.3	0.856	7252	Shoulder peak
3	A:B (80:20)	0.5	45	10.1	0.860	6809	Suitable peak
4	A:B (85:15)	0.5	45	8.2	0.878	8900	Suitable peak

Notably, the execution time initially tested in the assays needed to be increased to separate the peaks. Therefore, the run was extended to 40 min to improve peak resolution and eliminate all lipophilic interferers during the run.

After the tests, the gradient method showed better efficacy and was chosen for the study. The mobile phase comprised a mixture of water/acetonitrile with formic acid, eluted following a gradient described above with a flow rate of 0.5 mL/min. The chromatogram obtained from the developed method is shown in Figure 3A, in which α-PHE appeared at 8.1 min. The analyte peak showed theoretical plates of 8800 (N>2000), and the reject factor was within the limit of T<2.0 (0.9).

FIGURE 3
Selectivity of the method in the quantification of α-PHE at 5.0 μg/mL with retention time of 8.10 min. Representative chromatograms of α-PHE and blank injections of pitch oil, microemulsion, and skin (A). Analysis of peak areas (B), and retention times (C) comparing the chromatograms of α-PHE in the presence of matrices. Statistical analysis by ANOVA, followed by Tukey’s multiple comparisons test; p>0.05. α-PHE - alpha-phellandrene; ME - microemulsion.

Selectivity

Each chromatogram of the isolated matrices (skin and formulation) was compared to that from the α-PHE (Figure 3A), and no interfering peaks were detected in its same retention time. The pitch oil extracted from Protium hephytalium showed the biomarker α-PHE with an estimated percentage of 24.3%.

In addition, peak areas and retention time of α-PHE in the presence of each interferent were analyzed to ensure that impurities and chemical or biological matrices did not interfere with drug quantification (Figures 3B and 3C). The results showed that the retention time of the drug peak was not altered in the presence of the biological matrix (skin) and the formulation components (p>0.05).

Still within the tolerance of the ICH guidelines (2023), the results indicate that the method developed is selective against the analyzed interferers.

Linearity

The linear regression calculation of the curve for α-PHE (Figure 4) resulted in the equation y = 145445x - 3462.6, where y represents the peak area and x is the drug concentration in μg/mL. This linear regression showed a correlation coefficientvalue of 0.9995, corresponding to the limits above the regulatory requirements established by guidelines of the International Conference on Harmonization (2023).

FIGURE 4
Analytical curve of α-PHE prepared with ethanol. Calibration curve equation: y = 145445x - 3462.6, r = 0.9995. The data are presented as the average of 3 replicates ± standard deviation.

Limits of detection and quantification

The calculated LD and LQ were 0.05 µg/mL and 0.14 µg/mL, respectively. These established low values are enough to reliably quantify α-PHE in skin permeation studies, as demonstrated in the next experiments.

Precision

The coefficient of variation values determined during the repeatability test employing three concentrations of α-PHE was lower than 2.5% (Table II). Likewise, in terms of intermediate precision (inter-day precision), the values ranged from 0.3% to 5.0% (Table II). These results suggest that the method maintains consistent responses despite operational variations and is, therefore, precise.

TABLE II
Results of HPLC-UV precision tests for determination of α-PHE in standard solutions

		Repeatability
Theoretical concentration (pg/mL)			Experimental concentration (pg/mL)	CV (%)
	1.0		1.13 ±0.01	1.2
	5.0		5.68 ±0.09	1.6
	15.0		15.36 ±0.39	2.5
		Intermediate precision
		Theoretical concentra-tion (pg/mL)	Experimental concentration (pg/mL)	CV (%)
		1.0	0.94 ±0.01	0.9
	Day 1	5.0	4.95 ±0.15	0.3
		15.0	15.40 ±0.60	3.9
Analyst 1
		1.0	1.03 ±0.04	3.7
	Day 2	5.0	4.83 ±0.16	3.4
		15.0	14.69 ±0.81	5.5
		1.0	0.79 ±0.01	0.8
	Day 1	5.0	4.13 ±0.14	3.3
		15.0	13.15 ±0.66	5.0
Analyst 2
		1.0	0.92 ± 0.05	5.0
	Day 2	5.0	4.95 ±0.18	3.6
		15.0	15.23 ±0.76	5.0

Accuracy

For bioanalytical assays, a limit range of 15% (85% to 115%) is allowed in the values obtained (ICH, 2019). The results presented in Table III show that all the recovery percentages for α-PHE in the skin were higher than 92%, with coefficients of variation lower than 5%. This high recoverability allows the elimination of a correction factor in permeation studies, which is accepted in the literature for values above 70% (Oliveira et al., 2020).

TABLE III
Results of HPLC-UV accuracy tests for determination of α-PHE in standard solutions. α-PHE: alpha-phellandrene

Bio-	α-PHE	α-PHE
logical	theoretical	experimental	Recovery (%)
Matrix	(μg/mL)	(μg/mL)
	1.0	0.92 ± 0.1	92.3 ± 0.1
Skin	5.0	4.96 ± 0.1	99.2 ± 0.2
	15.0	15.23 ± 0.7	101.6 ± 0.7

Robustness

The method’s robustness was studied by evaluating intentional modifications in OT, FR, and W parameters using a factorial design (Table IV).

TABLE IV
Effects of the analytical parameters on the area, retention time, and tailing factor of the developed method for quantification of α-PHE

Trial
		1 2	3 4	5	6 7	8
	OT	44 44	44 44	46	46 46	46
Inde-pendent	FR	0.45 0.45	0.55 0.55	0.45	0.45 0.55	0.55
variables	W	84 86	84 86	84	86 84	86
Effect
		Area	Retention time		Tailing factor
	OT	-9023.3	-0.1583	*	0.0071*
Inde-pendent	FR	-236041*	-1.66*		0.0054*
variables	W	-898.67	-0.6483	*	0.0299*

• OT (◦C): Oven temperature; FR (mL/min): flow rate; W (%): acetonitrile percentage in mobile phase.
• ^*
  p < 0.05.

The mobile phase flow significantly influenced the peak area (p<0.05). It was evident that as the flow increased, there was a marked decrease in peak area. Retention time was significantly affected by the three variables tested (p<0.05). Temperature and solvent flow rate changes were inversely proportional to retention time; thus, increasing these parameters resulted in shorter retention times. Moreover, mobile phase modifications also result in retention time alterations. In fact, the increase in the organic phase (acetonitrile) resulted in an expected decrease in retention time, considering the lipophilic characteristics of α-PHE. Finally, the three variables tested significantly modified the tailing factor response (p<0.05). By increasing temperature, flow rate, and mobile phase (acetonitrile), it was possibleto observe an increase in the tailing factor. However, according to FDA guidelines (1994), the tailing factor was within the limit of T<2.0 in all the trials conducted, ranging between 0.780 and 0.829.

The significant alterations in some parameters may compromise the chromatographic method’s performance; in this way, there is a necessity for strict control over OT, FR, and W during analyses. These variations arose from a broader initial range of values, which exceed recommended levels. This allowed the study to identify the most critical parameters that should be more precisely controlled to maintain the method’s precision. To ensure the method’s reliability for α-PHE quantification, proper preventive maintenance of the equipment is essential, and it will maintain accurate pump operation, thereby preventing fluctuations in temperature, flow rate, and mobile phase mixture. Nonetheless, further studies analyzing smaller variations in the analyzed parameters would be necessary to confirm method robustness.

Application of the developed method in skin penetration studies

The in vitro skin permeation studies demonstrated the applicability of the validated analytical method. For this purpose, microemulsions were formulated and tested. The droplets’ size of such microemulsions were monodispersed, with PDI values below 0.2 and droplet sizes ranging from 153 to 250 nm, a pH of 6.2, and surface charges between -8.44 and -10.5 mV, which is considered neutral. Permeation studies using ME1 and ME2 were carried out over 12 h, and the amount of α-PHE that penetrated the skin was measured posttreatment (Figure 5).

FIGURE 5
Recovered α-PHE from skin after 12h of treatment with ME1 and ME2. The data represent averages of 5 replicates ± standard deviation. ME1 - oil phase: 1.5% α-PHE and 30.08% Kolliphor EL; surfactant: 21.05% Labrasol^®: Span^® (4:1 w/w), and aqueous phase: 47.37% ultrapure water; ME2 - oil phase: 5.75% White Pitch oil and 25.83% Kolliphor EL, surfactant: 21.05% Labrasol^®: Span^® (4:1 w/w), and aqueous phase: 47.37% ultrapure water. Statistical analysis by t-Tests, (^*) p<0.05.

No quantifiable amounts of α-PHE were found in the receptor medium of the diffusion cells, indicating that this bioactive did not permeate through the skin. The amount of α-PHE that penetrated the skin was statistically different (p<0.05) between the applied formulations, as ME2 accumulated 1.8-fold more α-PHE into the skin than the ME1 (53.1 ± 23.9 versus 29.4 ± 3.8 µg/cm²), which were effectively distinguished using the developed method. The higher coefficient of variation observed for the active penetration values for ME2, despite not having statistically affected its comparison with ME1, possibly comes from changes in the biological material used, or inherent errors in the processing of the skin during the extraction process.

A previous study using α-PHE loaded in ethosomal suspension and ethosomal gel reported skin deposition levels of the bioactive at 26.8 ± 0.3 and 48.8 ± 1.5 µg/ cm² after 24 h, respectively (Soba et al., 2021), whereas our results demonstrated similar deposition outcomes in just 12 h. Moreover, previous studies indicates that plant extracts can positively influence drug behavior in topical applications, sometimes enhancing skin permeation. This effect may stem from compounds in the extract that interact with skin layers, acting as permeation enhancers for the active ingredient (Back et al., 2018). In this way, the presence of the white pitch oil on ME2 might have influenced the mechanism by which the active ingredient’s permeation is promoted, resulting in a measurable impact on its efficacy. Furthermore, the analytical method demonstrated sufficient sensitivity and precision to detect and differentiate these variations.

Hence, these results could adequately confirm that the proposed analytical methodology can effectively quantify α-PHE in the skin, supporting the development of new formulations of this bioactive for topical application.

CONCLUSION

This work validated a selective chromatographic method for α-PHE quantification in the skin to evaluate topical delivery of pharmaceutical systems. The method could selectively quantify α-PHE from any interference, whether from the skin or components of a topical formulation. The method proved linearity over a wide concentration range and demonstrated accuracy, precision, and low LD and LQ during all analyses.

• ACKNOWLEDGMENTS
  The authors thank the Brazilian funding agencies FAP-DF and CNPq for supporting this research, as well as CAPES for providing the scholarship for D. S. Negreiros and C. O. Cardoso. The authors also thank “Frigorífico Via Carnes” for providing porcine ear skin.

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Edited by

• Associated Editor:
  Anibal de Freitas Santos Junior

Publication Dates

• Publication in this collection
  12 Jan 2026
• Date of issue
  2025

History

• Received
  30 Sept 2024
• Accepted
  09 Dec 2024