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Journal of

Research in Pharmacy

Research Article

www.jrespharm.com

Characterization with new developed UPLC method and stability study. J Res Pharm. 2019; 23(3): 426-440.

Ξ 2019 Marmara Uniǀersity Press

ISSN: 2630-6344

https://doi.org/10.12991/jrp.2019.150 426
Cefaclor monohydrate loaded microemulsion formulation for topical application: Characterization with new developed UPLC method and stability study $B $OSHU g=7U5. 1 * , Umay Merve *U9(1 2

1 Department of Pharmaceutical Technology, Faculty of Pharmacy, Anadolu University, (VNLüHOLU, Turkey.

2 DepartPHQP RI 3OMUPMŃHXPLŃMO 7HŃOQRORJ\ )MŃXOP\ RI 3OMUPMŃ\ TXNXURYM University, Adana, Turkey.

* Corresponding Author. E-mail: aaozturk@anadolu.edu.tr (A.A.g.); Tel. +90-222-335 05 80/3731. Received: 01 November 2018 / Revised: 26 November 2018 / Accepted: 27 November 2018

ABSTRACT: The purpose of this study was to formulate Cefaclor monohydrate (CEF) loaded microemulsion

formulations with the help of pseudo-ternary phase diagrams for topical application. Additionally, in this study also a

new ultra-performance liquid chromatography (UPLC) method was developed for the determination of CEF, which

was not previously entered into the literature. The droplet size, polydispersity index, pH, rheology, drug content, FT-

IR, dissolution study and release kinetic study have been used in the characterization of microemulsion. The UPLC

method developed was validated for linearity, specificity, precision, sensitivity, accuracy, range and robustness.

Linearity was determined to be at a concentration range of 5-DD źJBPI-1. The method developed was decided to be

precise due to RSD values of <2%. Recovery of the method was satisfactory owing to <2%RSD value. The drug content

was found to be in the range of 99.54-100.01% in stability study, indicating the uniformity of the high drug content. The

release of CEF from microemulsion showed conformity with the zero-order kinetics. The droplet size of the formulations

were measured ranged in 170.6-174.4 nm. The droplet size distribution of the formulations were observed range in

0.154-0.150. The results showed that nano-sized and monodisperse formulations were prepared. The storage stability

of CEF loaded optimum microemulsion was followed to ICH Q1(R2) at 251C/60%5% relative humidity up to six-

months. As a result of the stability study, microemulsion was found to be physically and chemically stable. According

to the results, microemulsion formulation prepared have longer release times than the release of pure CEF.

KEYWORDS: Cefaclor monohydrate; UPLC; microemulsion; stability study.

1. INTRODUCTION

Cephalosporins are a group of bactericidal semi-synthetic beta-lactam antibiotic drug active ingredients

that containing four generations of compounds grouped by pharmacokinetic / pharmacodynamic and

microbiological properties. This group is widely used worldwide in the treatment of human and veterinary

GLVHMVHV L1@B 7OH JURXS RI ŃHSOMORVSRULQ·V ROLŃO MUH OHVV MOOHUJHQLŃ POMQ SHQLŃLOOLQ·V MQG OHVV VHQVLPLYH PR NHPM-

lactamases, are considered broad-spectrum antibiotics used against both gram-negative and positive bacterial

strains and effectively break in microbial growth [1,2]. Cephalosporins can be administered orally and

parenterally. They show rapid distribution in biological systems and have half-lives ranging from 0.25 to 9

hours [2]. Cefaclor monohydrate (CEF) belongs to semisynthetic cephalosporin antibiotic group for oral

administration. The chemical name and molecular formula of CEF is 3-Chloro-7-d-(2-phenylglycinamido)-3-

cephem-4-carboxylic acid monohydrate and C15H14ClN3O4S, respectively. Figure 1 shown chemical structure

of CEF. N S O NH O NH2 Cl OOH HH . H2O

Figure 1. Chemical structure of CEF.

úG úG

Cefaclor monohydrate microemulsion formulation

Journal of Research in Pharmacy

Research Article

https://doi.org/10.12991/jrp.2019.150

J Res Pharm 2019; 23(3): 426-440

427

0HMQ VHUXP OHYHOV RI 7 13 MQG 23 źJBPI-1 averaged over 30 to 60 minutes after oral doses of 0.25 g, 0.5

g and 1 g on an empty stomach, respectively. A large part of the drug is thrown from the body in first 2 hours

after oral administration of CEF. In normal subjects without an antimicrobial disease the serum half-life varies

between 0.6 and 0.9 hours. The plasma half-life of this molecule is 2.3 to 2.8 hours in the literature [4].

Ultra-performance liquid chromatography (UPLC) system operates with sub-2 micron

chromatographic particles at pressures in the of 6000-15000 psi range. The reduction in particle size to below

2 micron provide improved chromatographic resolution and more optimal responses compared to

conventional High performance liquid chromatography (HPLC) with larger particles. UPLC also provides a

better and wider range of linear speed, velocities, faster analysis time and better chromatographic resolution.

High chromatographic resolution, resulting in increased signal to noise ratio and narrow peak width

compared to conventional HPLC, is useful not only for drug formulations, but also to allow for the

identification of a large number of metabolites at the physiological level [5]. As a result, it can be said that

UPLC offers important advantages over traditional reversed phase HPLC (RP-HPLC), with two quartets peak

capacity, almost ten times faster in speed and three to five times greater sensitivity compared to the

conventional 3.5-micron stationary phase [6]. As noted earlier, it is in the literature that UPLC results in 20%

more detectable components compared to HPLC for separation of human serum metabolites [7]. CEF is

officially in the European Pharmacopoeia (EP-2014), the United States Pharmacopoeia (USP-NF 33-2015) and

the British Pharmacopoeia (BP-2017). The HPLC methods developed for CEF are available in these

pharmacopoeias [4]. While many HPLC methods for CEF have been introduced into the literature; a simple,

precise, specific and highly sensitive & accurate UPLC method is not yet available [4, 8-10].

Microemulsions (MEs) are drug delivery systems that have recently attracted attention in drug research

and development studies. MEs are high thermodynamically and kinetically stable, optical transparent, low

viscosity and isotropic system comprising a water phase, an oil phase and a surfactant and usually together

with a co-surfactant. According to the preparation method, ME systems are separated into oil in water (O/W),

water in oil (W/O) and bicontinuous ME. In the literature, many studies have been conducted to demonstrate

the improved bioavailability of drugs when using ME. Various ME systems with surfactants and oils have the

advantages of a large surface area required for transport of drugs in the gastrointestinal tract and topical way

for low free energy and absorption. As a result, MEs have been proposed to positively influence drug

absorption in a variety of ways, including protecting the drug from oxidative and enzymatic degradation and

enhancing membrane permeability and lymphatic transport, and have also been suggested to prolong drug

release in oral use [11, 12]. Another important issue of ME is topical application. The small droplet size of ME

provides a OMUJH VXUIMŃH MUHM MQG XQLIRUP GLVPULNXPLRQ RQ POH VNLQ ÀOP IRUPMPLRQ SHUIHŃP RŃŃOXVLYHQHVV

aesthetic qualities and skin feel. MEs may increase the penetration of the drug active substance into the skin

by a number of mechanisms. They provide high dissolution capacity for both hydrophilic and lipophilic drug

active substance, so increasing the loading capacity and dosing of the formulation. MEs provide good surface

contact with the surface of the stratum corneum, coupled with large surface area and good skin contact,

obstructive nature. The oil and surfactant in the microemulsion formulation have a direct penetration

enhancing effect on the lipid structure of the stratum corneum [13]. In this study, CEF loaded topical microemulsion formulations were prepared and characterized for

droplet size, polydispersity index, pH, rheology, FT-IR, drug content, dissolution study and release kinetics

study with DDSolver software program. A new UPLC method, which was not previously reported in the

literature, has been developed and validated. This method was used for the determination of CEF in the new

formulation and dissolution study. In the last part of the study, a 6-month stability study was performed on

the selected optimum microemulsion formulation.

2. RESULTS AND DISCUSSION

2.1. Method optimization

The optimization of chromatographic separation for analysis of CEF, have been started by testing some

parameters such as particle size of stationary phase, column length, temperature, flow rate and the

composition of the mobile phase. For this purpose, firstly, two different size C18 columns (2.1 x 50 mm and 2.1

[ 100 PP ROLŃO OMV 1B8 —P SMUPLŃOH VL]H RHUH PHVPHG RLPO GLIIHUHQP ŃRPSRVLPLRQV RI RMPHU MQG PHPOMQRO MV

mobile phase. Since, suitable retention time was obtained with short column, the study was continued with

2.1 x 50 mm C18 column. Because the peak symmetry was not smooth, buffer solution was added to the mobile

phase mixture. After this step, different buffer systems (acetate buffer, disodium hydrogen phosphate buffer,

potassium dihydrogen phosphate buffer) were added to the mobile phase mixture. In terms of peak

Cefaclor monohydrate microemulsion formulation

Journal of Research in Pharmacy

Research Article

https://doi.org/10.12991/jrp.2019.150

J Res Pharm 2019; 23(3): 426-440

428

morphology and retention time, it was decided that the most appropriate buffer was acetate buffer. Thus, after

selecting acetate buffer, different concentrations (0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 M) were tested, and 0.1 M was

selected as the optimum buffer concentration. After all this steps, mobile phase composition was determined

as methanol: water: 0.1 M acetate buffer (40:50:10, v/v/v). At this stage, flow rates of 0.1 to 0.5 mL.min-1 were

tried to be observed, with careful consideration of peak morphology and retention time. When the flow rate

was reduced, expansion at the peak base was observed. When the flow rate was increased, the active substance

peak was observed at 0.6 minutes and coincided with the mobile phase peak. These conditions were overcome

with a flow rate of 0.25 mL.min-1 B HQ PHUPV RI UHPHQPLRQ PLPH 2DƒF 30ƒF 3DƒF MQG 40ƒF RHUH PHVPHG MQG 40ƒF

was found to be the most suitable colon temperature. When operating the instrument under these conditions,

ŃOURPMPRJUMPV RHUH H[MPLQHG MP GLIIHUHQP MNVRUNMQŃH·V MQG POH RMYHOHQJPO MP ROLŃO POH PM[LPXP

absorbance was observed was chosen as 265 nm. The best chromatographic separation occurred on C18 (2.1x50

PP 1B8 —P RLPO M PRNile phase consisting methanol: water: 0.1 M acetate buffer at a flow rate of 0.25 mL

minï1 and wavelength at 265 nm. Table 1 shows the UPLC methodology applied for selected method.

Table 1. Summary conditions of the UPLC method.

Device Agilent Technology 1290 Infinity

Column Zorbax Eclipse Plus C18 (2.1x50 mm, 1.8 —P Mobile phase 40:50:10 (v/v/v) methanol: water: 0.1 M acetate buffer

Oven temperature 40ƒF

Flow rate 0.25 mL.min-1

úQÓHŃPLRQ YROXPH 0.5 —I

Wavelength 265 nm

Retention time 1.6 min

2.2. Method validation

Method validation studies for CEF were carried out according to the literature and International

Conference on Harmonization (ICH) guideline Q2(R1) [14,15,16]. Linearity of CEF for the method used was

found to be 5-DD —JBPI-1 while regression equation was determined to be y=1.5931x-1.6058 by plotting

concentration (x) versus normalized peak area ratio (y). Determination coefficient (R2) of 0.9999 was highly

significant. Linearity test results are shown in Table 2 and regression curve is presented in Figure 2. Range is

the interval between the upper and lower concentration of active agent that have been indicated to be

determined with precision, accuracy and linearity using the method as written. The accuracy and precision of

the method are within the acceptable range [15]. In this study the range was observed linearly to the highest

ŃRQŃHQPUMPLRQ 220 źJBPI-1, R2:0.9999).

Table 2. Series and area values prepared for linearity and range study. CONC* (—JBPI-1)

Area/Rt**

SET 1 SET 2 SET 3 Mean SD SE

5.00 9.95 10.37 8.71 9.68 0.86 0.50

15.00 26.13 25.77 25.52 25.81 0.31 0.18

25.00 40.39 42.83 40.38 41.20 1.41 0.81

35.00 56.21 58.65 56.06 56.98 1.45 0.84

45.00 72.49 75.67 70.90 73.02 2.43 1.40

55.00 90.74 91.99 86.40 89.71 2.94 1.69

82.50 134.63 135.74 132.82 134.40 1.47 1.21

96.25 156.72 160.91 159.64 159.09 2.15 1.47

110.00 177.88 179.15 180.91 179.31 1.52 1.23

220.00 358.12 359.14 360.36 359.21 1.12 1.06

*CONC: Concentration, **Area/Rt: Normalized peak area ratio.

Cefaclor monohydrate microemulsion formulation

Journal of Research in Pharmacy

Research Article

https://doi.org/10.12991/jrp.2019.150

J Res Pharm 2019; 23(3): 426-440

429
Figure 2. Regression profile of CEF (*Area/Rt: Normalized peak area ratio).

The ability to detect and measure is important performance characteristics of each measurement

process. A representative feature of any analytical method developed; it can be defined as the smallest

concentration that can be detected or quantified with a certain degree of precision [17]. In general, a limit of

detection (LOD) is detected as the lowest concentration in a sample under the conditions specified in the test,

but is not considered to be quantifiable. Limit of quantitation (LOQ) is the lowest concentration of an analyte

in a test and can be determined with acceptable precision and accuracy under the specified test conditions.

Detection and quantification limits are the two principal components of method validation [15]. In this study,

I2G MQG I24 RHUH ŃMOŃXOMPHG N\ OLQHMU UHJUHVVLRQ MQG IRXQG MV 0BD82 —JBPI-1 MQG 1B76D —JBPI-1 respectively.

Results of intermediate precision and repeatability tests on different concentrations are given in Table

3. RSD values for both intermediate precision and repeatability were <2 %. Therefore, the method developed

for CEF was found to be precise according to the suggestions in ICH Q2(R1) guidelines [16]. As shown in Table 3 perfect recoveries of CEF at various concentrations were obtained between 99.699

- 100.015 % and also RSD values for all concentration were <2 %. Table 3 indicates good accuracy of the UPLC

method developed in this study. Results were obtained for area response and retention time, RSD % was calculated and examined for

robustness. RSD % for retention time for six different conditions were between 0.18 and 0.64 % (Table 3), which

LV RHOO LQVLGH POH SURSRVHG MŃŃHSPMQŃH NMVLV RI "D B 56G IRU MUHM UHVSRQVH RMV IURP 0B1D PR 0B87 Rhich

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consistent in front of the wavelength, temperature and flow rate. The selectivity of the analytical method developed is very important for pharmaceutical technology.

There are many excipients in the formulations developed and the peaks of these materials are not in conflict

with the active substance in the chromatogram. Furthermore, in the characterization method of formulations

such as in vitro dissolution study, they should not disturb the peak of the active substance in the medium

used. It is noted that the substances in the medium used do not coincide with the chromatogram of the active

substance. Characteristic UPLC chromatogram of CEF is given at Figure 3. It can be seen that chromatogram

recorded for the combination of non-functioning components exposed no peaks at retention time of 1.6

minutes (Figure 3).

2.3. Preparation of microemulsion formulation

The substances to be used in the microemulsion formulation were selected carefully. Isopropyl

myristate (IPM) is often used as an oil phase [18,19]. In addition to previous reports also confirmed that IPM

was an excellent enhancer for transdermal delivery [20,21]. Appropriate excipient selection and safety

evaluation especially of the co-surfactants is crucial in the formulation of microemulsions. Generally non-

ionic surfactants are chosen because of their good cutaneous tolerance, lower irritation potential and toxicity.

There is wide use of nonionic surfactants in topical microemulsion formulations as solubilizing agents. Span

80 and Tween 80 were preferred as surfactants in system. Another important parameter in the formulation of

Cefaclor monohydrate microemulsion formulation

Journal of Research in Pharmacy

Research Article

https://doi.org/10.12991/jrp.2019.150

J Res Pharm 2019; 23(3): 426-440

430

microemulsion system is the choice of co-surfactants. In permeability studies, Propylene glycol was found to

enhance penetration. Due to this property, it has been used as a cosurfactant in the formulation [18, 22].

Table 3. Precision, accuracy and robustness study results.quotesdbs_dbs41.pdfusesText_41

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