Dexamethasone | Target Protein Ligan Signal

Determination of dexamethasone acetate in CETETH 20-based in liquid crystalline systems using HPLC

1Department of Drugs and Medicines, School of Pharmaceutical Sciences, S~ao Paulo State University (UNESP), Araraquara, SP, Brazil 2Laboratório de Controle de Qualidade,
Faculdade de Farmácia, Universidade Federal de Goiás—UFG, Goiânia, Goiás, Brazil

Correspondence
Ana Carolina Kogawa, Laboratório de Controle de Qualidade, Faculdade de Farmácia, Universidade Federal de Goiás, Rua 240, s/n, Setor Leste Universitário, CEP 74605-170, Goiânia, Goiás, Brazil.
Email: [email protected]; [email protected]
Abstract
Dexamethasone acetate (DEX), a potent anti-inflammatory, is used primarily in the treatment of inflammatory and autoimmune diseases. It was incorporated in CETETH 20 (polyoxyethylene 20 cetyl alcohol)–based liquid crystalline systems to enhance the purpose of the drug. Concomitant with the pharmaceutical technology per- formed, a HPLC method was developed and validated for the quantification of dexa- methasone acetate in CETETH 20-based liquid crystalline systems for the evaluation of the drug in the new matrix. The method was performed using a C18 column with acetonitrile:methanol:water (35:35:30, v/v/v) as the mobile phase at a flow rate of 0.8 mL min-1 at 239 nm. The method was linear in the range of 1–25 μg mL-1; the limit of quantification and limit of detection were 0.05 and 0.16 μg mL-1, respec- tively; the accuracy of the method was 99.92% (relative standard deviation < 1%), and it presented intra-day and inter-day precision with deviations less than 1%. In this context, the method was successfully used to determine the incorporation effi- ciency of DEX in CETETH 20-based liquid crystalline systems and can be easily used by pharmaceutical companies and laboratories around the world.

K E Y W O R D S
anti-inflammatory, CETETH 20, dexamethasone acetate, HPLC, liquid crystalline systems

1| INTRODUCTION

Corticosteroids are steroid molecules synthesized and secreted by the adrenal cortex. The main steroids are mineralocorticoid and glucocor- ticoid. Mineralocorticoids affect electrolyte balance, and their main endogenous hormone is aldosterone. Cortisol is the primary glucocor- ticoid, acting on cardiovascular function in regulating metabolism, growth, and immunity. Synthetic corticosteroids are largely bound to albumin and thus may be in free form; they are available to act on the target cell, being mainly represented by hydrocortisone, corticoste- rone, and dexamethasone (Katzung, 2006; Rang, Dale, & Ritter, 2001) The use of dexamethasone acetate (DEX, Figure 1) is recommended in cutaneous therapy because of its anti-inflammatory and immunosup- pressive effects and is being incorporated in creams, lotions,

ointments, and microemulsions (Bhardwaj & Burgess, 2010; Brunton, Lazo, & Parker, 2006).
The nanostructured systems such as liquid crystals have been studied by many researchers; these systems have the ability to increase the responsiveness of the drug, minimize side effects, and incorporate different types of drugs in relation to its solubility. These pharmaceutical systems can be administered by different routes such as nasal, oral, intramuscular, and cutaneous (Calixto et al., 2018; Calixto, Victorelli, Dovigo, & Chorilli, 2018; Chorilli et al., 2011; Fonseca-Santos, Gremi~ao, & Chorilli, 2017; Formariz, Urban, Silva Júnior, Gremi~ao, & Oliveira, 2005; Rodero et al., 2018; Victorelli, Calixto, Ramos, Bauab, & Chorilli, 2018).
The liquid crystals are formed by water, oil, and surfactant (or a co-surfactant). They generate isotropic or anisotropic mixtures and

https://doi.org/10.1002/bmc.5054

330 UV–Vis PDA spectrophotometric detector (set at 239 nm), and a Rheodine VS 7125 injection valve, was used for the analysis of DEX. A reversed-phase C18 column (250 × 4.6 mm, 5 μm, Phenomenex, Allcrom, S~ao Paulo, Brazil) was used.

FIGURE 1 Chemical structure of dexamethasone
2.3 | Chromatographic conditions
are classified into two types: lyotropic (formation of systems with the

addition of solvents) or thermotropic (formation of systems is depen- dent on temperature). These systems can increase the solubility of drugs, achieving better efficacy, reducing side effects, and prolonging drug release (Formariz, Urban, Silva Júnior, Gremi~ao, & Oliveira, 2005; Guo, Wang, Cao, Lee, & Zhai, 2010; Oyafuso et al., 2017; Urban, Mainardes, & Gremi~ao, 2009). Polyoxyethylene 20 cetyl alcohol (CETETH 20) is an ethoxylated cetyl alcohol surfactant with no poly- oxypropylene units. It is a nonionic block copolymer pertaining to Brij family, which is pharmaceutically accepted and has been extensively studied as drug delivery systems (Wang, Thakur, Fan, &
Michniak, 2005).
Capillary electrophoresis, thin-layer chromatography-densitome- try, and flow-injection chemiluminescence methods have been used to determine DEX in pharmaceutical dosage forms (Guo, Chen, Yang, &
Hou, 2004; Krzek, Maslanka, & Lipner, 2005; Wu & Lv, 2007). HPLC is the method of choice for many pharmaceutical analyses, including topical formulations, which have a complex composition. The litera- ture shows methods for evaluating DEX in matrices such as creams (Garcia, Breier, Steppe, Schapoval, & Oppe, 2003), ointments (Zivanovic, Zecevic, Markovic, Petrovic, & Ivanovic, 2005), and micro- emulsions (Urban, Mainardes, & Gremi~ao, 2009) but none in liquid crystalline systems.
Therefore, this work aims at developing and validating a method using HPLC for the quantitative evaluation of DEX in liquid crystalline systems, showing compliance with selectivity, linearity, precision, and accuracy.

2| EXPERIMENTAL

2.1| Materials

Polyoxyethylene 20 cetil ether—Brij 58, CETETH 20 (Sigma-Aldrich, Steinheim, Germany), PEG-12-dimethicone (Dow Corning, Midland, MI, USA), PEG-DM (Dow Corning, Midland, MI, USA), DEX (Purifarma, Anápolis, Brazil), and HPLC-grade methanol and acetonitrile (J. T. Baker, Center Valley, PA, USA) were used. Purified water from a
The mobile phase comprised of methanol:acetonitrile:water (35:35:30, v/v/v). The mobile phase was filtered through a 0.45-μm membrane filter and degassed using an ultrasonic device. The analysis used an isocratic system with a flow rate of 0.8 mL min-1 and a wave- length of 239 nm at 25ti C. The injection volume was 50 μL.

2.4| Preparation of standard and sample preparation

A standard stock solution of 100 μg mL-1 of DEX was prepared in methanol. Then, seven dilutions (1, 2.5, 5, 10, 15, 20, and 25 μg mL-1) were prepared using methanol as the diluent. Similarly, another seven dilutions were obtained at concentrations of 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, and 2.5 μg mL-1 to determine the limit of detection (LOD) and limit of quantification (LOQ). The solutions were filtered using a 0.22-μm-pore-size filter (Millipore, Bedford, MA, USA) before being injected into the system.

2.5| Method validation

The validation of the method followed the parameters of ANVISA (2017) and ICH (2005) with respect to selectivity, linearity, precision, and accuracy.
Selectivity was evaluated for analyzing the responses of the stan- dard and sample chromatograms, investigating the possible interfer- ence of the matrix components.
Linearity was determined by the regression line from the peak area and concentration for seven standard solutions (1, 2.5, 5, 10, 15, 20, and 25 μg mL-1) prepared in methanol. Each concentration was prepared in triplicate and on three different days.
LOD and LOQ were determined, using Equations 1 and 2, from a specific calibration curve obtained with another seven solutions (0.1, 0.25, 0.5, 1.0, 1.5, 2.0, and 2.5 μg mL-1), which were closest to the LOQ.

Milli-Q plus purification system (Millipore, Billerica, MA, USA) was used.
LOD = 3:3σ=S
ð1Þ

2.2| Instrumentation

A Varian HPLC system (Varian, Palo Alto, CA USA), consisting of a ProStar/Dynamax 210/215 solvent delivery module, ProStar
LOQ = 10σ=S ð2Þ

σ is the standard deviation of the response and S is the slope of the calibration curve.
Precision was analyzed at two levels, repeatability or intra-day pre- cision and intermediate precision, using three different DEX standard

solutions (1, 10, and 25 μg mL-1). At the repeatability level, the analyses were performed on the same day with the same conditions and by the same analyst. At the intermediate precision, the analyses were made on different days with the same conditions and by different analysts. The results were reported as relative standard deviations (RSD %).
Accuracy was proved using the recovery test. The amounts of standard DEX solution were added to the formulation matrix to obtain the solutions of three concentrations (1, 10, and 25 μg mL-1). The experimentally determined concentration and the theoretical concen- tration were related.

2.6| Method applicability

2.6.1| Preparation of the liquid crystalline systems

The mixtures of CETETH 20 and PEG-DM were used in weight ratios from 1:9 to 9:1 to obtain the ternary phase diagrams.
Purified water was slowly added (1.0 g) by constant agitation using a bath at 60ti C to melt the surfactant. The change from translu- cent dispersions (TDs) to semisolid transparent systems (SSTSs) and isotropic transparent liquid systems (ITLSs) or phase separations (PSs) was achieved at 25ti C by adding 0.5 mL of water. The samples, after each transition, were maintained at the same temperature (25ti C) for 24 h to complete the system equilibration, and then the percentages of the three components were calculated. This is important to obtain the points, which define the boundaries between the ternary phase

2.6.2| Incorporation of DEX into the formulations

An excess of DEX (100 mg g-1) was incorporated into the formulations developed. The formulations were placed in centrifugal tubes, and DEX was added to the tubes and shaken using a rotating shaker for 48 h at 25ti C. After shaking, the tubes were centrifuged at 360g for 20 min with the aim of separating the undissolved DEX and maintaining the supernatant that contains DEX solubilized at saturation. Then, an ali- quot of the supernatant was pipetted and diluted in methanol, filtered using a 0.22-μm-pore filter, and analyzed using HPLC.

3| RESULTS AND DISCUSSION

3.1| Method development

The mobile phase consisting of methanol and purified water in vari- able proportions was tested. The combination of these solvents in the proportion of 65:35 (v/v) presented a peak with an irregular shape after 10 min. Therefore, acetonitrile was included, and after some var- iations, a regular and symmetric peak was observed using acetonitrile, methanol, and purified water in the proportion of 35:35:30 (v/v/v) in about 10 min. The most adequate flow was 0.8 mL min-1 (Figure 2).

TABLE 1 Centesimal composition of liquid crystalline formulations
Composition (%, w/w)

diagram regions.
The formulations, named F1, F2, F3, F4, F5, and F6 (Figure 3),
Components
F1 F2 F3 F4 F5 F6

Water 60 50 40 50 40 30
were chosen for incorporation of DEX. Their compositions are pro-

vided in Table 1, and they were prepared by weighting each compo- nent, which were mixed using a bath at 60ti C. The samples were stored in vials after cooling.

FIGURE 3 Ternary phase diagram of CETETH 20, PEG-DM, and water. F1–F6 points chosen for incorporation of dexamethasone acetate (DEX). TD, translucent dispersion; SSTS, semisolid transparent systems; ITLS, isotropic transparent liquid system; PS, phase separation
PEG-DM 30 30 30 10 20 30
CETETH 20 10 20 30 40 40 40 Note. CETETH 20, polyoxyethylene 20 cetyl alcohol.

the correlation coefficient (r = 0.9996) were obtained by the method of least squares. The r-value near 1 indicates linearity in the proposed range.

3.2.3| LOD and LOQ

The LOD and LOQ were 0.05 and 0.16 μg mL-1, respectively. They were calculated using the standard deviation and slope data (Equations 1 and 2) from a specific analytical curve (0.2–2.0 μg mL-1). The results prove that it is possible to accurately and safely quantify DEX in this interval using the conditions proposed in the method.

3.2.4| Precision

The precision results, repeatability and intermediate precision, show an RSD less than 1% (Table 2) at all levels analyzed (1, 10, and 25 μg mL-1), which indicates the proximity of the values and confirms the precision of the method.

3.2.5| Accuracy

The average recovery of DEX from the formulation matrix was 99.92% (Table 3), close to 100% and between the specification of 98 and 102% recommended by the AOAC (2002) and by Horwitz,
FIGURE 2 Chromatogram of (a) dexamethasone acetate (DEX)

standard solution at a concentration of 25 μg mL-1, (b) DEX sample in liquid crystalline formulation, and (c) supernatant from liquid crystalline formulations
TABLE 2 Precision results for the different levels of DEX in the standard solutions

Standard solution (μg mL-1)a
Measured concentration ± SD (μg mL-1)
RSD
(%)

Analysis repeatability (n = 3)
3.2| Method validation 1 0.99 ± 0.006 0.58

3.2.1| Selectivity
10
25
10.00 ± 0.04 25.00 ± 0.04
0.44
0.21

Intermediate precision (n = 3)

For this study, a blank liquid crystalline formulation—F1 (without DEX) was prepared, and the supernatant, after centrifugation and dilution with methanol, was analyzed using HPLC. The chromato- gram of the sample (Figure 2) shows the DEX peak at ti 10 min, which is consistent with the chromatogram of the standard
Day 1 1
10
25

0.99 ± 0.005 10.00 ± 0.01 24.96 ± 0.06

0.58
0.12
0.29

Day 2
(Figure 2). No other signals around the retention time of DEX

(ti10 min) were observed in the chromatogram for the supernatant from the blank liquid crystalline formulation—F1 (Figure 2), which indicates no interference in the quantitative determination of DEX using the formulation components.
1 0.99 ± 0.005 0.58
10 10.03 ± 0.015 0.12
25 24.02 ± 0.05 0.26 Day 3

1 0.98 ± 0.006 0.59

3.2.2| Linearity
10
25
10.02 ± 0.015 25.03 ± 0.03
0.15
0.13

Note. DEX, dexamethasone acetate; RSD, relative standard deviation; SD,

Linearity was evaluated using seven concentrations ranging from 1 to 25 μg mL-1, and the regression equation (y = 0.0021x + 0.0007) and
standard deviation. an = 3.

TABLE 3 Accuracy results for the concentrations of DEX recovered from the formulation matrix
DEX concentration added (μg mL-1)a DEX concentration recovered (μg mL-1)a Recovery (%) RSD (%)
1 0.99 99.66 0.58
10 10.02 100.23 0.23
25 24.97 99.88 0.21

Note. DEX, dexamethasone acetate; RSD, relative standard deviation. an = 3.

Kamps, and Boyer (1980) for pharmaceutical analysis. Therefore, the results indicate the proximity of the values to a true value, which confirms the accuracy of the method.

3.3| Method applicability

The proposed analytical method was performed to evaluate the incor- poration of DEX into the formulations. The indirect method was cho- sen, in which the supernatant containing the free DEX was analyzed using HPLC. The selectivity test demonstrated that no interfering or unusual peaks were observed in the chromatograms during the drug quantification.
Liquid crystalline systems were obtained by phase diagram, which is a tool that provides an overview to study, design, and explain how liquid crystals are self-assembled (Oliveira, Scarpa, Correa, Cera, &
Formariz, 2004). The ternary phase diagrams constructed for the oil phase, PEG-DM, and water are shown in Figure 3. The systems were classified as ITLS when they were liquid and exhibited transparency. The systems were classified as SSTS when they were semisolid and transparent. When the systems were opaque, independent of the vis- cosity, they were classified as TD. Both ITLS and SSTS systems were spontaneously formed, showing thermodynamic stability. The incor- poration of DEX into the formulations was measured, which is pro- vided in Table 4.
DEX has poor solubility in water, and it is classified as a lipophilic compound and has a partition coefficient (octanol/water) of 2.91 (Xuan, Xu, Li, Gao, & Li, 2015). The formulations were able to solubi- lize DEX varying from 1.28 to 5.30 mg mL-1, and the solubilization was dependent on surfactant and oil ratios. The high ratios of CETETH-20 and PMG in formulations were able to solubilize the high amounts of DEX, the use of these vehicles could define the potency of topical preparations, and its selection is crucial for product perfor- mance. Furthermore, oil/water partition coefficients of the vehicles
lead to the different solubilities and could be related to the amount of drug permeating through the skin (Xiong, Li, Su, Liu, & Wang, 2011).

4| CONCLUSION

The HPLC method using the RP C18 column and acetonitrile:metha- nol:water (35:35:30, v/v/v) as the mobile phase with a flow rate of 0.8 mL min-1 at 239 nm was suitable for the determination of DEX in CETETH 20-based liquid crystalline systems. It can also be considered for the evaluation of the DEX release profile. The method is feasible and precise, and the protocol of extraction is not costly and does not require any additional step of purification or solid-phase extraction. The analytical method can be adapted for other pharmaceutical prod- ucts containing DEX and addresses the applicability to other laborato- ries with scope in the academic or industrial field.

ACKNOWLEDGMENTS
The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundaç~ao de Amparo à Pesquisa do Estado de S~ao Paulo, and Programa de Apoio ao Desenvolvimento Científico da Universidade Estadual Paulista for financial support.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the authors on reasonable request.

ORCID
Márcia Helena Oyafuso https://orcid.org/0000-0003-3556-5274
Bruno Fonseca-Santos https://orcid.org/0000-0002-4259-0008
Ana Carolina Kogawa https://orcid.org/0000-0003-2834-6532
Flávia Chiva Carvalho https://orcid.org/0000-0001-7586-539X

TABLE 4 Values of incorporated DEX (mg mL-1) in the formulations
DEX (mg mL-1) incorporated into the formulation

F1 F2 F3 F4 F5

F6
Maria Palmira Daflon Gremi ~ao https://orcid.org/0000-0001-6950- 7852
Marlus Chorilli https://orcid.org/0000-0002-6698-0545

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RSD (%) 1.10 1.67 0.92 4.71 3.18 3.76 Note. DEX, dexamethasone acetate; RSD, relative standard deviation.
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How to cite this article: Oyafuso MH, Fonseca-Santos B, Kogawa AC, Carvalho FC, Gremi~ao MPD, Chorilli M. Determination of dexamethasone acetate in CETETH 20- based in liquid crystalline systems using HPLC. Biomedical Chromatography. 2020;e5054. https://doi.org/10.1002/
bmc.5054