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European Journal of Applied Sciences – Vol. 11, No. 3
Publication Date: June 25, 2023
DOI:10.14738/aivp.113.14907
Peña-Velasco, G., Amador-González, E., Melgoza-Contreras, L. M., & Hernández-Baltazar, E. (2023). Assessment of Topical
Formulations Skin Permeation Using Raman Spectroscopy. European Journal of Applied Sciences, Vol - 11(3). 793-804.
Services for Science and Education – United Kingdom
Assessment of Topical Formulations Skin Permeation Using
Raman Spectroscopy
Peña-Velasco, Gabriela
Universidad Autó nóma del Estadó de Mórelós,
Facultad de Farmacia, Cuernavaca, Mórelós
Amador-González, Enrique
Universidad Naciónal Autó nóma de Me xicó,
Facultad de Quí mica, Me xicó
Melgoza-Contreras, Luz María
Universidad Autó nóma Metrópólitana,
Departamentó de Ciencias Bióló gicas, Me xicó
Hernández-Baltazar, Efrén
Universidad Autó nóma del Estadó de Mórelós,
Facultad de Farmacia, Cuernavaca, Mórelós
GRAPHICAL ABSTRACT
ABSTRACT
The use of new noninvasive analytical techniques and procedures for the
assessment of topical formulation skin permeation has been a challenge for
pharmaceutics sciences. In recent years, Raman spectroscopy has been limited to
the identification of components inside a sample or unknown substances. In this
work, a handheld Raman spectrometer was used in the follow-up of active
pharmaceutical ingredients (APIs) in topical formulations. Thus, in combination
with Franz cells and Tape stripping procedures, permeation flux and retained drug
amount in layers of skin were evaluated in hydrogels of lidocaine (LD) and
meloxicam (MX) with myristate isopropyl (IPM). The proposed method allows
lower analysis time, simple operation, collects direct measures of APIs without
preview sample treatment, and avoids the use of solvents in support of eco-friendly.
Diagrama Descripción generada automáticamente
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The obtained results demonstrate the effective use of IPM as a permeation
promotor agent increasing the permeation flux of 59.9 to 72.2 cm/min and 31.8 to
41.0 cm/min for LD and MX hydrogels, respectively. The developed analytical
method by Raman spectroscopy obtained determination coefficients of r2≤ 0.993,
an inter-day precision (repeatibility) of %RSD ≤ 5%, limits of detection (LODs) and
quantification (LOQs) from 0.05 and 0.07 mg/mL, respectively. Finally, the
advantages and limitations of this proposed quantification alternative were
compared with other analytical methods, which could suggest their potential
application and incorporation in standardized guidelines of skin permeation.
Keywords: Raman spectroscopy, hydrogels, skin permeation, lidocaine, meloxicam.
INTRODUCTION
In recent years, portable Raman spectrometers equipment has been further used in the
pharmacy industry for identifying APIs, in real-time monitoring processes, and indirect
quantification of powders [1–3]. In regard to bioanalysis, micro-spectroscopy and Confocal
Raman micro-spectroscopy (CRM) are techniques that dominant to a great number of research
[4–6]. Specifically for the permeation skin studies, until now, cells Franz diffusion with
subsequent analysis for high-resolution liquids chromatographic or ultraviolet spectrometry,
Tape Stripping coupled Confocal laser scanning microscopy (CLSM), Skin biopsy, and Suction
blister, have been the mainly used techniques for the assessment permeation flux of API
principles across the skin [7,8]. The majority with limitations such as ethical difficulties, high
cost, variations in skin types (synthetic, animal or human), low reproducibility, laborious and
high consumption time analysis, invasive processes [7,9]. Therefore, the importance of the use
of new techniques that facilitate the assessment of permeation fluxes of APIs in topical
formulations could make an important contribution to the field of pharmaceutic and
cosmetology sciences. The purpose of the present research was the study the application of
portable Raman spectroscopy combined with Franz cells and Tape stripping for assessment
and monitoring permeation flux of two hydrogel formulations. Lidocaine (LD) and meloxicam
hydrogels were used as models of topical pharmaceutical formulations, with IPM as an
enhancer permeation agent.
EXPERIMENTAL SECTION
Materials
In all the experiments high purity deionized water was used. Lidocaine hydrochloride
(C14H22N2O · HCl · H2O) and Meloxicam (C14H13N3O4S2) standards were high purity (≥ 98%). For
the preparation of hydrogels, sodium carboxymethyl cellulose, glycerin, and IPM were used as
excipients. All the reagents used were purchased from Sigma Aldrich. For the assessment
permeation flux of APIs, HAWP type 0.45μm Millipore nitrocellulose filter and vertical Franz
type diffusion cells were used. LD and MX standards stock solutions were prepared at 100 and
2 mg/mL, respectively. From them and using the corresponding dilution of individual stock
solutions were constructed the calibration curves for the quantification of target analytes and
calculated statical parameters. Table 1 shows the mainly properties of model drugs studied.
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Peña-Velasco, G., Amador-González, E., Melgoza-Contreras, L. M., & Hernández-Baltazar, E. (2023). Assessment of Topical Formulations Skin
Permeation Using Raman Spectroscopy. European Journal of Applied Sciences, Vol - 11(3). 793-804.
URL: http://dx.doi.org/10.14738/aivp.113.14907
Table 1 Properties of model drugs for hydrogel formulations.
Lidocaine Hydrochloride Meloxicam
Molecular weight (g/mol) 270.8 351.4
pka 7.7 4.08
LogP 2.44 3.54
Solubility (mg/mL at 25°C) 50 < 0.1
TPSA (Å2) 32.3 136
LogP = octanol-water partition coefficient, TPSA= Topological Polar Surface Area
Methods
Preparation of Hydrogels:
Two different formulations of LD and MX hydrogels were prepared and evaluated, respectively
(with and without IPM added in them). Table 2 shows the composition of each formulation.
Finally, the prepared hydrogels in an amber glass recipient at room temperature for 24 h before
use were stored. The fabricated hydrogels were labeled as LD and MX for lidocaine and
meloxicam hydrogel, respectively, the formulations with IPM added were identified as LD-IPM
and MX-IPM, respectively.
Table 2 Composition of prepared lidocaine and meloxicam hydrogels formulations.
LD Hydrogel LD-IPM
Hydrogel
MX
Hydrogel
MX-IPM
Hydrogel
Carboxymethylcellulose sodium (2%) * * * *
Glycerin (10%) * * * *
Meloxicam (0.3%) * *
Lidocaine hydrochloride (10%) * *
IPM (1%) * *
Water q.s. 100 mL 100 mL 100 mL 100 mL
Procedure for the Obtention of Skin Membrane:
Used the test No. 428: Skin absorption: in vitro method from the Guidelines for the Testing of
Chemicals [10], skin from human or animal sources can be used and it is essential that skin is
properly prepared [11]. Pig skin has been reported with similar features to human beings,
making it a suitable animal model for permeation tests [8]. Therefore, excised skin ear pig
dorsal surface was used, purchased from the slaughterhouse in Cuernavaca, Morelos, México.
Briefly, the pig skin membrane was prepared as previously reported [9], a first step of cleaning
with distilled water, for removing traces of dirt, blood, and/or others. The membrane skin was
extracted manually with a scalpel, separating the subcutaneous fatty layer from the skin
(extracting the dermis without subcutaneous tissue) leaving the stratum corneum intact. The
skin membrane obtained and conditioned with phosphate‐buffered saline (PBS pH 7.4) was
stóred at −20 °C until use.
Franz Diffusion Cell Permeation Studies:
Vertical Franz-type diffusion cells with an average area and volume of 3.31 cm2 and 24.84 cm3
were used. Therefore, skin pig circular samples of around 3.31 cm2 were placed between the
donor and receiver chamber of a Franz cell, with the stratum corneum facing the donor
chamber. The receptor compartment was filled with 25 ml of phosphate-buffered saline (pH
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7.4), magnetically stirred at 400 rpm, and at 32°C was kept. Later, 500 mg of the test hydrogel
formulation was applied to the skin surface in the donor cell and was sealed with parafilm to
avoid evaporation. The LD and MX permeation flux were monitored for 2 and 6 h, respectively,
taking samples (1 ml) at the desired time intervals and replacing the volume with the same
account of PBS. For comparative purposes, Franz diffusion cell study in synthetic membrane
(HAWP type 0.45μm Millipore nitrocellulose filter) was realized using the same conditions and
equipment previously described. The flux value (Jss) was obtained from the slope of the linear
regression analysis for each experiment. The apparent permeability coefficient (Papp) was
calculated as previous reports [12,13] according to Fick law diffusion, taking initial drug
concentration (C0) using the equation (1):
Papp = Jss⁄C0
Furthermore, the profile concentration by skin deepness was studied by the Tape stripping
technique. At the conclusion of the Franz diffusion cell permeation study, each skin sample was
removed from the donor chamber and the residual drug delivery formulation was carefully
removed. Thus, on the area that was in contact with the formulations, the technique of Tape
stripping was performed as described previously in the literature [13–15]. Fifteen tapes were
used for the remotion of the stratum corneum. Each tape strip was placed on a slide and directly
measured (three points of the surface) by portable Raman spectroscopy (Figure 1) allowing
analysis time ≤ 2 y 10 min fór Franz cells and Tape stripping, respectively. All experiments for
triplicate were carried out.
Fig. 1. Tape stripping in vitro procedure used in this work.
Spectroscopy Analysis Conditions:
The monitoring and quantification of target drugs LD and MX were carried out by Raman
spectroscopy using TruScan Ahura Scientific handheld equipment with laser excitation
wavelength light at 785 ± 0.5 nm in the Raman spectrum range 250 cm-1 to 2875 cm-1, previous
reports associated the use of this type of laser source with fluorescence less effect in biological
samples [4].
As a mentioned preview, for the permeation study, the aliquot was placed in a vial. For Tape
stripping, the strip was placed on a slide. In both cases, the samples without needing added
solvents or calibration steps were directly measured for triplicate.
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Peña-Velasco, G., Amador-González, E., Melgoza-Contreras, L. M., & Hernández-Baltazar, E. (2023). Assessment of Topical Formulations Skin
Permeation Using Raman Spectroscopy. European Journal of Applied Sciences, Vol - 11(3). 793-804.
URL: http://dx.doi.org/10.14738/aivp.113.14907
Statistical Analysis:
The analytical parameters obtained for the developed method were calculated and expressed
with corresponding standard deviations. Finally, permeation data were analyzed by T-student
test at a p-value of 0.05 of significance.
RESULTS AND DISCUSSION
In vitro Permeation Study of LD and MX Hydrogels
Pig skin samples and synthetic membrane (nitrocellulose filters) were used for these
experiments. Figure 2 a) y b) shows results for study in synthetic membrane. The values Papp
were highest for the prepared formulations with IPM added (Table 3), corroborating with
previous reports [16–18] about their activity as an effective permeation promotor. The Papp
calculated from the slope of the drug release curves were 59.9 and 72.2 cm/min for LD
hydrogels and for MX, Papp= 0.48 and 0.61 cm/min, in both cases with and without IPM,
respectively.
Regarding permeation skin experiments, it was obtained the under the same procedure as for
synthetic membranes. The obtained results (Figure 3 a) y b)) show effectively that an increase
in permeation flux was kept in prepared formulations with IPM for both drugs, obtaining 41.0
y 0.473 cm/min Papp values for LD and MX hydrogels, respectively, in comparison with 31.8 y
0.44cm/min for LD and MX hydrogels without IPM. It is important to mention due to despite
the diverse chemical features of LD and MX (mainly Log P, polarity, pka), the IPM allowed the
improvement in permeation through the skin due to having been related to a lower flux through
it keeping drugs in the skin [19]. Moreover, is interesting observe the difference between MX
and LD permeation through the skin, being less facilitated to the first them. Since, as can be seen
in Table 2, the apparent permeability coefficient had an insignificance increase (0.44 to 0.47
cm/min); which could be explained due to its lipophilic character (Log P = 3.43), favoring a
preference to be accumulated through the corneum stratus layers [20]. The effect of the use of
IPM is shown in Table 3, the accumulated account of lidocaine and meloxicam were higher in
all formulations with IPM added.
0 20 40 60 80 100 120
0
50
100
150
200
250
300
350 A)
Qacum(mg/cm
2)
Time (min)
LD-IPM Hydrogel LD Hydrogel
0 40 80 120 160 200 240 280 320 360
0
4
8
12
16 B)
Qacum(mg/cm
2)
Time (min)
MX-IPM Hydrogel MX Hydrogel
Fig. 2 In vitro profiles hydrogels permeation of a) LD-10% and b) MX-0.3% using a synthetic
membrane (nitrocellulose filters).
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Table 3 Parameters obtained from In vitro permeation study MX and LD (mean ± SD, n
= 3).
Synthetic Membrane Pig skin Membrane
Formulation Papp
(cm/min)
Qacum
(mg/cm2
)
Formulation Papp
(cm/min)
Qacum
(mg/cm2
)
LD Hydrogel 59.9 ± 0.13 310 ± 0.20 LD Hydrogel 31.8 ± 0.21 207 ± 0.71
LD-IPM
Hydrogel
72.2 ± 0.15 349 ± 0.11 LD-IPM
Hydrogel
41.0 ± 0.12 243 ± 0.32
MX Hydrogel 0.48 ± 0.09 14 ± 0.42 MX Hydrogel 0.44 ± 0.07 9.0 ± 0.37
MX-IPM
Hydrogel
0.61 ± 0.05 15 ± 0.34 MX-IPM
Hydrogel
0.47 ± 0.03 12.0 ± 0.19
0 20 40 60 80 100 120
0
50
100
150
200
250 A)
Qacum(mg/cm
2)
Time (min)
LD-IPM Hydrogel LD Hydrogel
0 40 80 120 160 200 240 280 320 360
0
4
8
12
16 B)
Qacum(mg/cm
2)
Time (min)
MX-IPM Hydrogel MX Hydrogel
Fig. 3 In vitro profiles hydrogels permeation of a) LD and b) MX using skin pig membrane.
Tape Stripping Test
Tape stripping (TS) is a simple, low-cost, viable, and minimally invasive technique for the
quantification of pharmaceuticals compounds through the skin, considering layers depth
where they could be found by chemometrics analysis [7,14]. In this work, this technique was
applied separately to the permeation in Franz cells study [21]. Figure 4 a) y b) shows the
obtained results for the Tape stripping test from the MX and LD hydrogels topically applied in
skin pig membranes. The ≥50% amóunt óf accumulated pharmaceutical cómpóunds was
retained in layers of skin for all formulations. As expected, the IPM added hydrogels
formulations show lower drug retention in the corneum stratus layers, corroborating the
obtained results in the Franz cells permeation study. The most interesting of this graphic is
specifically, in the case of MX, a pronounced difference in the retention of the drug in the skin
is observed in the formulation with isopropyl myristate (IPM) compared to the formulation
without it (Qacum with IPM =1.6393 mg vs Qacum without IPM = 2.3756mg), what could be seen
as an opposite effect to the results obtained in Franz cells permeation study, however, could
due to the short exposure time of the hydrogels formulations on skin membranes (2h for TS vs
6h Franz cells, respectively) moreover of lipophilic character of this formulation [22].
Moreover, as mentioned preview, the properties of model drugs are key factor, Table 1 shows
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Peña-Velasco, G., Amador-González, E., Melgoza-Contreras, L. M., & Hernández-Baltazar, E. (2023). Assessment of Topical Formulations Skin
Permeation Using Raman Spectroscopy. European Journal of Applied Sciences, Vol - 11(3). 793-804.
URL: http://dx.doi.org/10.14738/aivp.113.14907
clearly the hydrophilic character of Lidocaine (Kow = 2.44 and higher solubility in water) which
that facilitate their dispersion in the vehicle, moreover lower values of molecular weight (270
vs 351 g/mol for LD and MX, respectively) and topological polar surface area of 32.3 Å2 in
comparison to 136 Å2 of meloxicam, could explain the improve their pass across the layers
skin. Therefore, meloxicam to be an API lipofilic strong API(solubility in water ≤ 0.1) and higher
molecular weight have a highest trend to remain between layers skin, according to obtained
results of TS and Franz cells experiments. In addition, the use of hydrophilic formulation could
be relational with an improvement Tape stripping protocol due to not affecting the adhesion
impairment [22].
A)
0
10
20
30
40
50
60
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Accumulated amount of LD (%)
Ratio of LD with respect applied dosis (mg)
Strip number
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B)
Fig. 4 Tape stripping test results of hydrogels of a) Lidocaine and b) Meloxicam.
(The values are the amount of drug retained in SC and related to drug concentration detected
in each applied tape and analyzed by Raman spectroscopy).
For the LD hydrogels Tape stripping test, no greater difference was observed in the cumulative
amount obtained for both formulations Qacum with IPM = 41.71mg vs Qacum without IPM = 39.57
mg, previous reports associated a greater potentiating effect of IPM in LD formulations in
combination with other permeation promotors agents [23]. However, less amount of LD
retained in the first superficial layers of the skin is observed in the formulation without the
permeation promotor agent, indicating a lower depth of penetration of it, corroborating IPM
function [24]. The tape stripping study shows that 50% of drug models remained between
corneum stratum (SC) layers, being MX the API highest retained for their lipophilic properties
(Table 1).
Analytical Parameters for Drugs Quantification by Raman Spectroscopy
According to USP 43 - 1225, the validation procedure is key for insurance that the developed
quantification method the requirements for its intended analytical applications [25]. The
obtained analytical parameters are present in Table 4. Linearity, precision, recovery, limits of
detection (LOD), and quantification (LOQ) were calculated for Franz cells permeation
experiments.
The developed method was lineal with determination coefficients (r2 ) higher than 0.998, the
range lineal was 75 - 91 and 1 - 2 mg/mL for LD and MX, respectively. Precision (inter-day)
evaluated as reproducibility, lower than 5%, and recovery percentages in the range of 98 -
102% considering a method successful according to established in “Harmonized guidelines for
single laboratory validation of methods of analysis” [26]. To distinguish between these two
0
0.5
1
1.5
2
2.5
3
0
0.05
0.1
0.15
0.2
0.25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Ratio of MX with respect applied
dosis (mg)
Strip number
Accumulated amount of MX (%)
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Peña-Velasco, G., Amador-González, E., Melgoza-Contreras, L. M., & Hernández-Baltazar, E. (2023). Assessment of Topical Formulations Skin
Permeation Using Raman Spectroscopy. European Journal of Applied Sciences, Vol - 11(3). 793-804.
URL: http://dx.doi.org/10.14738/aivp.113.14907
applications of Raman spectroscopy, the method for the quantification of drugs in Tape
stripping experiments by measuring drugs concentration contained in hydrogel formulations
directly was developed. Table 4 shows indicate also analytical parameters calculated for Tape
stripping test. Coefficients (r2) higher than 0.993, the range lineal was 2 - 10 and 0.05 - 0.03
mg/mL for LD and MX, respectively. Precision (inter-day) evaluated as reproducibility, lower
than 5%, and recovery percentages in the range of 95 - 102%. It is important to note that under
current good validation practice regulations, users of the analytical methods described in the
USP-NF are not required to verify the accuracy and reliability of these methods, but merely to
verify their suitability under actual conditions of use [25]. Therefore, both quantification
methods were satisfactorily developed and applicated to the monitoring of LD and MX through
the skin, demonstrating the potential use of this spectroscopy technique as a viable, eco- friendly, and fast tool for the analysis of dissolved drugs or pharmaceuticals formulations.
Table 4 Analytical parameters for the quantification of MX and LD using portable
Raman spectroscopy (n = 3).
Type
experiment
Franz cells permeation study Tape stripping
LD MX LD MX
Regression
equation
12.622x – 28.714 795.43x + 8.7857 2464.3x – 48.714 62012x +683.9
r
2 0.9992 0.9998 0.9994 0.9936
Linear range
(mg/mL)
75 - 91 1-2 2 – 10 0.05 – 0.3
LOD
(mg/mL)
1.26 0.05 0.16 0.05
LOQ
(mg/mL)
9.99 0.17 0.49 0.07
Precision
(% RSD) *
1.15 1.53 4.9 4.4
Recovery
(%)
99.22 100.46 99.94 100.53
*Calculated at the lowest level of linear range.
Comparison with Conventional Quantification Methods
Interestingly, Raman spectroscopy was observed to allow monitoring of drug permeation in the
skin (according to USP 42) and provide results in accordance with those obtained by
quantification traditional methods. Table 5 shows that the use of this technique yields similar
results that studies carried out with techniques such as ultraviolet spectroscopy (UV), and high- resolution liquid chromatography (HPLC).
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European Journal of Applied Sciences (EJAS) Vol. 11, Issue 3, June-2023
Table 5 Comparison of the analytical techniques for quantification of drugs in the
permeation of topically formulations.
Formulation
Type
Drug Instrumental
Technique
Sample
Preparation
(Solvents
added)
Test Analysis
Time
(min)
Ref
Cream,
ointment.
Fludrocortisone
and Diclofenac
sodium
HPLC Yes:
Mobile phase
Extraction of
drug from
tape
Tape
stripping
39 [19]
Hydrogels Diclofenac HPLC Yes:
Mobile phase
Extraction of
drug from
tape
Tape
stripping
67 [2]
Hydrogels and
organogels
Meloxicam UV-Vis
Spectroscopy
Blanks used
(placebo gel)
Franz cells NR [10]
Hydrogels Meloxicam
and Lidocaine
Portable
Spectroscopy
Raman
None Tape
stripping
Franz cells
10
and
2
This work
NR= Not reported.
CONCLUSIONS
The present research confirmed the successful use of a portable Raman spectrometer for
monitoring the skin permeation of two hydrogel formulations. The use of advanced techniques
could represent an alternative for the development and analysis of new topical formulations.
Thus, the developed protocol designed in this work allowed the monitoring and quantification
of lidocaine and meloxicam, two active pharmaceutical ingredients with different polarity in
addition to IPM as an enhancer permeation inside of topical formulation. These experiments
confirmed that lidocaine showed a higher and facilitated permeation across the skin could
suggest their systemic action. Instead, meloxicam was higher retained (around 83%) between
the SC layers being a better candidate drug for local action. Also, the use of IPM increased the
permeation flux of both drugs, and their incorporation in hydrogels formulation was not
affected the topical formulation analysis by Raman spectroscopy. Finally, one of the more
significant findings to emerge from this study is an easy, fast, low-cost, and non-invasive
method with huge potential for monitoring skin permeation drugs by use of portable Raman
spectroscopy. Considerably more work will need to be done to determine its capability or
effectiveness in detecting APIs in topical formulations and incorporating them into guidelines
by the regulatory agencies.
ACKNOWLEDGMENTS
The authors thank the financial support from IIQUIAP FROM PROINNOVA 0154498. Peña- Velasco G. acknowledges to the CONACyT-México for the scholarship support CVU 588460.
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