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Talanta 76 (2008) 1006–1014

Contents lists available at ScienceDirect

Talanta

journa l homepage: www.e lsev ier .com/ locate / ta lanta

se of experimental design and effective mobility calculations to develop aethod for the determination of antimicrobials by capillary electrophoresis

onica Cecılia Vargas Mamania, Jaime Amaya-Farfanb, Felix Guillermo Reyes Reyesc,ose Alberto Fracassi da Silvaa, Susanne Ratha,∗

Institute of Chemistry, Department of Analytical Chemistry, University of Campinas (UNICAMP), P.O. Box 6154,3084–971 Campinas, SP, BrazilDepartment of Food & Nutrition, University of Campinas (UNICAMP), P.O. Box 6121,3083–862 Campinas, SP, BrazilDepartment of Food Science, University of Campinas (UNICAMP), P.O. Box 6121,3083–862 Campinas, SP, Brazil

r t i c l e i n f o

rticle history:eceived 24 April 2008ccepted 25 April 2008vailable online 13 May 2008

a b s t r a c t

A capillary zone electrophoresis (CZE) method for the determination of chloramphenicol (CLP),danofloxacin (DANO), ciprofloxacin (CIPRO), enrofloxacin (ENRO), sulfamethazine (SMZ), sulfaquinoxa-line (SQX) and sulfamethoxazole (SMX) is described. For the development, the effective mobilities wereestimated and a central composite design was performed. The method was in-house validated for CLP,

eywords:hloramphenicolluoroquinolonesulfonamidesapillary zone electrophoresis

CIPRO, ENRO and SMX determination in pharmaceuticals. In comparison with the HPLC method recom-mended by the United States Pharmacopoeia, this CZE method exhibited the same performance, with theadvantage that seven different antimicrobials in pharmaceutical formulations could be simultaneouslydetermined.

© 2008 Elsevier B.V. All rights reserved.

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xperimental designffective mobilities

. Introduction

Sulfonamides (sulfamethoxazole (SMX), sulfamethazine (SMZ),ulfaquinoxaline (SQX)) and fluoroquinolones (enrofloxacinENRO), danofloxacin (DANO) and ciprofloxacin (CIPRO)) arentimicrobials widely used to treat a variety of infections in bothuman and veterinary medicine [1–4]. Chloramphenicol (CLP) is aroad-spectrum antibiotic not approved for use in animals for foodroduction in several countries, including the European Union,he USA and Brazil [5]. The use of this drug in humans is reservedor certain patients with serious infections, such as meningitis,yphus, and typhoid fever, who cannot take safer alternativesecause of resistance or allergies [2]. The chemical structures ofhese antimicrobials are shown in Fig. 1.

In recent years, methods have been published using capillary

one electrophoresis (CZE) for the determination of fluoro-uinolones [6–9], chloramphenicol [10–11] and sulfonamides12–17]. Several approaches have also been followed for theetermination of CLP, CIPRO and SMX by the United States Phar-

∗ Corresponding author. Tel.: +55 19 35213084; fax: +55 19 35213023.E-mail address: raths@iqm.unicamp.br (S. Rath).

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039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2008.04.062

acopoeia (USP) using high performance liquid chromatographyHPLC) for quality control of these antimicrobials [18]. However,he physicochemical properties of CLP, the fluoroquinolones andhe sulfonamides, their ionic natures, multiple ionization sites andifferent water solubilities, make these compounds more suitableor electrophoretic analysis. CZE continues to grow rapidly as annalytical technique for a wide range of applications, includinghe analysis of pharmaceuticals. CZE is also advantageous becauseower volumes of fluids and samples are employed, in comparison

ith HPLC [19].The official monographs of the United States Pharmacopoeia

escribe different HPLC methods for CLP, CIPRO and SMX. The sta-ionary phases employed for CLP, CIPRO and SMX determinationre octadecylsilane columns, whereas the mobile phases comprisemixture of water, methanol and glacial acetic acid for CLP; phos-horic acid, adjusted to pH 3 with triethylamine plus acetonitrileor CIPRO, and water, acetonitrile and triethylamine at pH 6.0 for,MX.

Nevertheless, several separation methods for the determinationf antimicrobials are presented in the literature most of them focus-ng on the quantitation of antimicrobials of the same group. Onlyfew papers describe the simultaneous determination of antimi-

robials of different groups, among which, �-lactam antibiotics,

M.C.V. Mamani et al. / Talanta 76 (2008) 1006–1014 1007

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Fig. 1. Structure of fluoroquinolon

etracyclines and chloramphenicol in milk [20] and streptomycinnd oxytetracycline in agricultural antimicrobials [21]. A reviewbout other applications has been written by Garcia-Ruiz andarina [22].There is an advantage in the quality control to use the same

ethod for the determination of several kinds of compounds, with-ut the need to change solvents, analytical columns and procedures.

Due to the different physicochemical properties of fluoro-uinolones, sulfonamides and chloramphenicol, the optimization

f separation with CZE is complex and difficult, because a highumber of parameters (temperature, pH, voltage, buffer com-osition) will affect the separation. Rather than following theonventional monovariate approach used to adjust the parameters,hus involving a large number of independent analyses, one could

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lfonamides and chloramphenicol.

dvantageously replace the procedure by statistically designedxperimental protocols, in which several factors are simultane-usly varied. The use of an experimental design can be extremelyeneficial in developing capillary electrophoretic methods.

This paper describes the development of a simple and compre-ensive CZE method for the simultaneous determination of CLP,ANO, CIPRO, ENRO, SMZ, SQX and SMX in pharmaceutical for-ulations, using effective mobility calculation and experimental

lanning. The method was validated for CLP, CIPRO, ENRO and

MX and its performance was compared with that of the officialPLC methods described in the United States Pharmacopoeia. Since

here is no Pharmacopoeia method available for ENRO, this fluoro-uinolone was treated in a similar manner as CIPRO for validationurposes.

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aeing over 1 day, under the same conditions, using solutions of eachanalyte at a single concentration level, 50 �g mL−1. The inter-assayprecision was determined for two concentration levels, 50 and100 �g mL−1, and the analyses were performed for 5 days.

Table 1Nominal values corresponding to −1, 0, +1, −1.41 and 1.41 in the first experimental

008 M.C.V. Mamani et al. / T

. Experimental

.1. Instrumental and operating conditions

The capillary electrophoresis was performed on a Hewlettackard 3D Capillary Electrophoresis system (Waldbronn, Ger-any), equipped with a DAD detector. CLP and fluoroquinolonesere detected at 270 nm and sulfonamides at 203 nm. Data were

ollected using the HP 3D Chemstation software from Hewlett-ackard. The separations were carried out in fused silica capillaries50 �m I.D.), with effective (l) and total (L) lengths of 60.0 and8.3 cm, respectively. Injection was done hydrodynamically for 20 st 20 mbar. Measurements of pH were made with a DM-20 pH-eter from Digimed (Sao Paulo, Brazil), using a combined glass

lectrode. When necessary, the electrolyte pH was adjusted using.1 mol L−1 NaOH or 0.1 mol L−1 phosphoric acid before making upo volume.

The high performance liquid chromatographic analyses wereerformed using a Waters chromatographic system (Milford, USA),omposed of a binary pumping system, model 1525, a diode arrayetector (DAD) model 2996 and a Rheodyne 7725 injector, with aample loop of 50 �L.

Chromatographic separation was achieved using a LiChrosphere18 column (125 mm × 4.6 mm, 5 �m) with a C18 guard-column4.0 mm × 4.0 mm, 5 �m), purchased from Merck (Darmstadt, Ger-

any). The mobile phase for CLP consisted of a mixture of water,ethanol, and glacial acetic acid (55:45:0.1, v/v/v). The mobile

hase for CIPRO was a mixture of 0.025 mol L−1 phosphoric acid,reviously adjusted with triethylamine to pH 3, and acetonitrile87:13, v/v). For SMZ, the mobile phase was obtained by mixing400 mL of water, 400 mL of acetonitrile and 2 mL of triethylaminend bringing the volume to 2000 mL with water. Remaining HPLConditions were as described in the United States Pharmacopoeia18].

.2. Standards and reagents

Standards of SMZ, SQX and SMX were purchased from SigmaSt. Louis, USA), while CLP was from ICN Biomedical Inc. (Cleveland,SA). CIPRO and ENRO were from Fluka Biochemicals (Steinheim,witzerland) and danofloxacin mesylate, from Pfizer (Guarulhos,razil).

Analytical grade disodium hydrogenphosphate, sodium tetrab-rate, sodium hydroxide, sodium carbonate, phosphoric acid,odium ethylenediaminetetraacetate (EDTA) and phosphoric acidere purchased from Merck (Darmstadt, Germany). Methanol andimethylformamide, HPLC-grade solvents, were purchased fromedia (Fairfield, USA).

Throughout the study, water was obtained from a Milli-Q sys-em from Millipore (Bedford, USA). Before analysis, all the solutionsere filtered through 0.22 �m nylon filters from Millipore (Sao

aulo, Brazil).

.3. Samples

The commercial samples of pharmaceutical formulations likephthalmic solutions containing CLP, tablets containing CIPRO,njections containing ENRO and tablets and oral suspensionsontaining SMX from different batches were purchased at localrugstores in Campinas, SP, Brazil.

.4. Standard solutions

Standard stock solutions of CLP, DANO, CIPRO, ENRO, SMZ, SQXnd SMX were prepared by dilution of appropriate volumes of the

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76 (2008) 1006–1014

tandards in methanol to a final concentration of 2 mg mL−1. Theseolutions were stored at −18 ◦C for up to 1 month. Working solu-ions in the concentration range of 50–250 �g mL−1 were preparedaily by dilution of the standard stock solutions in water.

.5. CZE procedures

Before daily use, the capillary was sequentially washed withater (10 min), 1 mol L−1 NaOH (2 min), 0.1 mol L−1 NaOH (3 min)

nd background electrolyte (5 min). After each determination, theapillary was sequentially washed with water (2 min), 1 mol L−1

aOH (1 min), 0.1 mol L−1 NaOH (1 min) and background electrolyte2 min).

The separation conditions for the determination of theeven antimicrobials were 60 mmol L−1 sodium phosphate plus0 mmol L−1 sodium tetraborate, pH 8.5; voltage, 24 kV and tem-erature, 26 ◦C.

.6. HPLC procedure

The HPLC determinations of CLP, CIPRO, ENRO and SMZ in theamples followed the official monograph of the United States Phar-acopoeia [18], without modification.

.7. Experimental design

Preliminary analytical trials were carried out to identify theignificant parameters. The results of this analysis showed the tem-erature and voltage to be significant. Therefore, these parametersere used to plan a subsequent higher order 22 design, with central

omposite. The variables evaluated were temperature (19–27 ◦C)nd voltage (16–24 kV) (Table 1). All statistical calculations wereeveloped with the software Statistic Statsoft, Inc., v. 5.5 (Tulsa,SA).

.8. Method validation

The method was in-house validated using the following perfor-ance criteria: linearity and linear range, sensitivity, selectivity,

ntra-assay and inter-assay precision, detectability, accuracy anduggedness. The linearity, linear range, sensitivity and detectabilityere established through the analytical curve obtained by triplicate

nalysis of CLP, DANO, CIPRO, ENRO, SMZ, SQX, SMX at five concen-ration levels 50, 100, 150, 200 and 250 �g mL−1. The sensitivity ishe slope of the analytical curve.

The detectability for each antimicrobial was calculated accord-ng to Miller and Miller [23], using the following expression:etectability = k sy/x/m, where k = 3, sy/x is the standard deviation ofhe residuals and m, the slope of the analytical curve.

The intra-assay precision of the method, expressed as the rel-tive standard deviation of peak area measurements (n = 5), wasvaluated through the results obtained with the method operat-

esign

ariables −1.41 −1 0 +1 1.41

—Temperature (◦C) 19 20 23 26 27—Voltage (kV) 16 17 20 23 24

M.C.V. Mamani et al. / Talanta

Table 2Nominal values corresponding to −1, 0, +1, −1.68 and 1.68 in the second experimen-tal design

Variables −1.68 −1 0 +1 1.68

ABC

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In Eq. (1), �j is the absolute electrophoretic mobility and ˛j is thedistribution function of species j. The distribution functions are

—pH 8.3 8.4 8.5 8.6 8.7—Phosphate concentration (mmol L−1) 58 59 60 61 62—Temperature (◦C) 24.3 25 26 27 27.7

The selectivity of the method was evaluated by exposing CLP,ANO, CIPRO, ENRO, SMZ, SQX, SMX, at the concentration of0 �g mL−1, to the following stress conditions for 1 h: 0.1 mol L−1

Cl, 0.1 mol L−1 NaOH, 3% (v/v) H2O2 or temperature (55 ◦C). Theolutions were analyzed considering the resolution between thenalytes and the possible presence of other substances formeduring the experiment and the analytical signal before and afterhe exposure of the analyte to the stress conditions, expressed asecovery.

The accuracy of the method was evaluated through analyses ofamples of pharmaceutical formulations containing the CLP, CIPRO,NRO, SMX by the proposed CZE method, using the procedureescribed below, and by HPLC according to the method described

n the United States Pharmacopoeia [18].The susceptibility of the developed analytical method to changes

as tested in order to evaluate the ruggedness of the method. Forhis purpose a 23 experimental design was employed (Table 2),here the following variables were tested: pH (8.3–8.7), phosphate

oncentration (58–62 mmol L−1) and temperature (24.3–27.7 ◦C).

.9. Sample analysis

The content of three samples of each commercial formulationrom the same batch containing either: CLP, CIPRO, ENRO or SMX,as used. In the case of CLP, an ophthalmic solution was used. An

ccurately measured volume, equivalent to about 150 �g mL−1 wasransferred, diluted with water and mixed.

In the case of CIPRO, tablets were analyzed. An accuratelyeighed quantity of the freshly mixed powder, equivalent to about

00 mg of ciprofloxacin hydrochloride, was transferred to a 50 mLolumetric flask, added with 2 mL of glacial acetic acid and 25 mL

f water. The mixture was sonicated for about 10 min, allowed toool, diluted to volume with water and filtered through a 0.22 �membrane filter. A volume of 150 �L of this solution was diluted to

0 mL with water in a volumetric flask (150 �g mL−1).

ig. 2. Simulated electrophoretic mobilities of sulfonamides, chloramphenicol anduoroquinolones as a function of pH in the range 0.0–14.0 in CZE. Electrolyte:0 mmol L−1 sodium phosphate plus 20 mmol L−1 sodium tetraborate, 24 kV, 26 ◦C.

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76 (2008) 1006–1014 1009

In the case of ENRO, injections for animal use were analyzed.liquots of 300 �L were transferred to a 100 mL volumetric flask

150 �g mL−1) and diluted with water.The last antibacterial, SMX, was determined in two pharmaceu-

ical formulations: tablets and an oral suspension. For tablets, anccurately weighed quantity of the freshly mixed powder (about00 mg SMX) was transferred to a 50 mL volumetric flask and 15 mLf methanol were added, followed by 25 mL of water. The mixtureas sonicated for about 10 min, allowed to cool to room tempera-

ure, diluted to volume with water and filtered through a 0.22 �membrane filter. A volume of 750 �L of this solution was diluted to

0 mL with water in a volumetric flask (150 �g mL−1). Oral suspen-ions were diluted as in the procedure for the tablets and processedn an identical manner. All samples were analyzed in quintuplicate.

The analyses by CZE were carried out as described in Sec-ion 2.5 and quantitation was accomplished through an externalnalytical curve with five concentration levels in the range of0–250 �g mL−1. The samples were also analyzed by HPLC usinghe methods described in the United States Pharmacopoeia [18].

. Results and discussion

.1. Eletrophoretic mobilities

The composition of the background electrolyte has great influ-nce on its separation ability for the antimicrobials under study.rincipally, the pH of the electrolyte influences the effective mobil-ties of the analytes. In order to assess the best pH-value of theackground electrolyte for the separation of all antimicrobialsnder study, the effective mobilities (�ef) for each compound werealculated using Eq. (1) [24] and a plot of �ef versus pH was con-tructed. This characterization of the electrophoretic behavior isuitable to indicate the pH regions where the solute mobilities differnd separation of the analytes would be possible.

ef =n∑

�j˛j (1)

elated to the pH and to the acid–base equilibrium constants.

ig. 3. Response surface for CLP, DANO, CIPRO, ENRO, SMZ, SQX and SMX showinghe number of separated compounds as a function of significant separation param-ters using temperature and voltage, obtained using a 22 factorial design with theentral composite design.

1010 M.C.V. Mamani et al. / Talanta 76 (2008) 1006–1014

Fig. 4. A typical electropherogram of 100 �g mL−1 of (1) CLP, (2) DANO, (3) CIPRO,(4) ENRO, (5) SMZ, (6) SQX and (7) SMX (tm1 7.2 min, tm2 7.4 min, tm3 7.7 min, tm4

8.3 min, tm5 9.8 min, tm6 10.7 min, and tm7 11.9 min, respectively). Capillary: uncoatedfused silica; background electrolyte, a solution of 60 mmol L−1 sodium phosphatep2s

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lus 20 mmol L−1 sodium tetraborate, pH 8.5; temperature, 26 ◦C; applied voltage,4 kV; detection wavelength, 270 nm for CLP and fluoroquinolones and 203 nm forulfonamides.

For the fluoroquinolones (FQ) two acid–base equilibria muste considered. The first one involves the cation form H2FQ+ andhe neutral HFQ (Ka1), while the second equilibrium involves theeutral HFQ and anionic FQ− forms (Ka2).

For the sulfonamides and chloramphenicol, just one acid–basequilibrium between the neutral form and the unprotonated anionas considered. Using Eq. (1), the pKa values and the eletrophoreticobilities [10,25–27], it was possible to calculate the �ef for each

ntimicrobial as a function of pH, using the software Microsoftxcel, Microsoft Corporation (USA). Fig. 2 shows the calculatedffective mobilities in which the effects of ionic strength were notaken into account. The results indicate that the separation of allhe antimicrobials can be performed in the pH range of 7.5–9, withll the analytes having a negative charge.

Consequently, the CZE method was developed using the high pHackground electrolyte, normal polarity, and cathodic EOF. Underhese conditions, the antimicrobials migrate against the EOF andhat having the smallest �ef would reach the detector first. Thus,he predicted migration order becomes: CLP, DANO, CIPRO, ENRO,MZ, SQX, and SMX.

.2. CZE method development

In view of reports on the influence of the electrolyte on selectiv-ty [15], it was deemed mandatory first to compare the performancef different types of background electrolytes for the separation of

he antimicrobials. Different electrolytes at pH 8.5 were evaluated:odium phosphate, Tris, sodium carbonate and sodium tetrabo-ate. The electrolytes were prepared in the concentration range of0–140 mmol L−1 with uniform steps of 40 mmol L−1.

s

Se

able 3uantitative features for CLP, CIPRO, ENRO, SMZ and SMX

arameter CLP CIPRO

inear range (�g mL−1) 50–250 50–25ensitivity (aU �g mL−1) (P < 0.05) 0.0083 ± 0.0001 0.00inearity (r) 0.999 0.99ntercept (P < 0.05) 0.02 ± 0.04 −0.02ntra-assay precision, 50 �g mL−1, n = 5 (R.S.D.%) 1.48 2.11nter-assay precision, 50 �g mL−1, n = 5 (R.S.D.%) 1.31 2.29nter-assay precision, 100 �g mL−1, n = 5 (R.S.D.%) 1.60 1.01etectability (�g mL−1) 10 12

.S.D.: relative standard deviation.

tandard solutions (50 �g mL−1) to temperature (55 ◦C), 0.1 mol L−1 HCl, 0.1 mol L−1

aOH or 3% (v/v) H2O2.

Considering the resolution of the analytes and the analysis time,he best conditions for the simultaneous separation of CLP, flu-roquinolones and sulfonamides were achieved with the sodiumhosphate plus sodium tetraborate electrolyte (40–100 mmol L−1).ubsequently, it was necessary to optimize the electrolyteoncentration, pH, temperature and voltage. An initial set ofrials was made using a monovariate approach, as was doneith the electrolyte concentration. The optimum concentrationas 60 mmol L−1. Concentrations below and above 60 mmol L−1

esulted in increased migration times and in higher currents,espectively. The effect of the pH was evaluated in the inter-al 6.8–10.2, permitting the best separation for all compoundst 8.5, confirming the pH-value previously established throughhe evaluation of the effective eletrophoretic mobilities. The opti-

um temperature and voltage conditions were selected throughhe response surfaces obtained by a 22 chemometric experimen-al design (11 tests). This procedure offers an efficient route foretermining the best resolution from a selected number of con-itions. Moreover, by using experimental design it is possible tovaluate the interaction of factors that have a significant effect onhe separation using a small number of experiments.

The experimental design was thus constructed by the use of aull 22 experimental design, including three central and four axialoints. The conditions are presented in Table 1.

Fig. 3 depicts the response surface, showing that the separa-ion of the seven antimicrobials (CLP, DANO, CIPRO, ENRO, SMZ,QX and SMX) under study could be achieved, for example, at4 kV and 26 ◦C, when using 60 mmol L−1 sodium phosphate plus0 mmol L−1 sodium tetraborate, pH 8.5 as background electrolyte.

Since the analytes migrate toward the detector pulled by thelectroosmotic flow (EOF), shorter analysis times can be obtainedy raising the pH of the background electrolyte, but incompleteeparations were obtained at pH values higher than 8.5.

A typical electropherogram of CLP, DANO, CIPRO, ENRO, SMZ,QX and SMX at the optimized conditions established through thexperimental design is shown in Fig. 4.

/DANO ENRO/DANO SMZ/SQX SMX/SQX

0 50–250 50–250 50–25070 ± 0.0001 0.0070 ± 0.0001 0.0050 ± 0.0001 0.0050 ± 0.00018 0.998 0.999 0.997± −0.04 −0.02 ± −0.05 −0.07 ± 0.02 −0.02 ± −0.04

1.39 1.19 1.402.13 1.01 1.691.50 2.52 1.98

15 10 17

alanta 76 (2008) 1006–1014 1011

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Table 4CLP content per mL of solution, determined in commercial ophthalmic samples byCZE and HPLC

Sample 1 Sample 2 Sample 3

CZE HPLC CZE HPLC CZE HPLC

Average content (mg mL−1) 4.48 3.60 4.43 3.59 4.23 3.64s (mg mL−1) 0.05 0.02 0.06 0.03 0.07 0.08R

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.3. Method validation

The results of the CZE method in-house validation for theetermination of CLP, CIPRO, ENRO, SMZ and SMX are summa-ized in Table 3. The accuracy was evaluated by comparing theesults obtained from the analysis of pharmaceutical formula-ions by the proposed CZE method with those obtained using theecommended HPLC method described in the United States Phar-acopoeia [18].The linearity, linear range and sensitivity were obtained from

nalytical curves using external standard ization for CLP and annternal standard (DANO) for CIPRO and ENRO, and SQX as internaltandard for SMZ and SMX, at five concentration levels for eachntimicrobial under study, with triplicate analyses. The linearityas tested using a pure error lack of fit test with simple regression,hich was not significant at the 5% level.

The selectivity of the method indicates the ability to accuratelyeasure the analyte response in the presence of potentially inter-

ering sample components or degradation products. In this study,he selectivity was evaluated by exposing the analyte to stress con-itions, such as acid (0.1 mol L−1 HCl), base (0.1 mol L−1 NaOH) andn oxidant (3%, v/v H2O2). The solutions were analyzed consid-ring the resolution between the analyte and other substancesormed during the experiment, and the analytical signal beforend after exposure of the analyte to the stress conditions. Theesults (Fig. 5) showed that the stabilities of the antimicrobials tohe three stressing conditions depend on the chemical structure.LP is the least stable compound to the alkaline conditions andLP degraded almost completely in 0.1 mol L−1 NaOH, while CIPRO,NRO, SMZ, SMX and TC were stable to alkaline, acid, tempera-ure, and peroxide stress. SQX was sensitive to the acid treatment.ANO was less stable under peroxide stress. The degradation prod-cts of CLP formed under the stressing conditions migrated inhorter times (approx. 6 min) than the other compounds. Antimi-robials like DANO, ENRO and SMX generated degradation productst 6–7 min. DANO had another degradation product evident at.9 min, but since no other compound appears at or about this

ime, the selectivity of the method can be confirmed. Co-migrationf any degradation products with the authentic standards did notccur as evidenced by the perfect overlay of the absorption spec-ra of the pure standards with those of the peaks obtained afteregradation.

doa(r

able 5IPRO and ENRO content per tablet and per mL of solution, determined in commercial sam

Sample CIPRO Sampl

CZE HPLC CZE

verage content (mg/tablet)a or (mg mL−1)b 501 450 47(mg/tablet)a or (mg mL−1)b 1.2 0.2 0.9.S.D. 2.5 0.04 1.8

= 5; s, estimate of the standard deviation (mg mL−1); R.S.D., relative standard deviation.a Units for CIPRO.b Units for ENRO.

able 6MX content per tablet and per mL of solution, determined in commercial samples by CZ

Sample 1t

CZE HPLC

verage content (mg/tablet)a or (mg mL−1)b 411 409(mg/tablet)a or (mg mL−1)b 7 4.7.S.D. 1.72 1.14

= 5; s, estimate of the standard deviation (mg mL−1); R.S.D., relative standard deviation.a Units for tablet.b Units for oral suspension.

.S.D. (%) 1.1 0.58 1.6 0.93 2.4 2.2

= 5; s, estimate of the standard deviation (mg mL−1); R.S.D., relative standard devi-tion.

The precision of the method for CLP, CIPRO, ENRO, SMZ and SMXas evaluated using the results obtained over 1 day of operationnder the same conditions (intra-assay precision) and for 5 daysinter-assay precision). The intra- and inter-assay precisions wereetermined at 100% of the target concentration from 5 determina-ions. The results were expressed as relative standard deviationsR.S.D.) and were lower than 2.1 and 2.3% for intra- and inter-assay,espectively (Table 3). Considering that regulatory agencies [18]ecommend that the precision should be lower than 2%, the valuesbtained by the CZE method are acceptable.

The established detectability represents the lowest concentra-ion of an analyte in sample solutions to be introduced in the CEquipment that can be detected. The detection and the quantita-ion limits of the method are not presented, due to the fact that thective compound is the major constituent of the formulation andhis parameter is not required for method validation for the qual-ty control of pharmaceutical products. Furthermore, these limits

ould depend on sample dilution before analysis.The accuracy of the method was assessed for CLP, CIPRO, ENRO

nd SMX by analyzing commercial formulations by the CZE methodnd the recommended United States Pharmacopoeia methods. Theean values obtained using the proposed method and the referenceethod (Tables 4–6), for all the samples, do not differ significantly

P = 0.05).Finally, the ruggedness of the method was assessed by intro-

ucing small changes to the procedure and examining the effect

n the results (analytical signal). For this purpose, the temper-ture (26.0 ± 0.8 ◦C), pH (8.5 ± 0.2) and electrolyte concentration60 ± 2 mmol L−1) were changed simultaneously using a 23 facto-ial design (17 tests). The response graphs can be seen in Figs. 6

ples by CZE and HPLC

e ENRO 1 Sample ENRO 2 Sample ENRO 3

HPLC CZE HPLC CZE HPLC

52 48 52 49 520.6 0.9 0.2 0.4 0.41.24 1.8 0.40 0.78 0.84

E and HPLC

Sample 2t Sample 3s

CZE HPLC CZE HPLC

435 382 39 367.6 7 0.7 0.651.75 1.79 1.67 1.8

1012 M.C.V. Mamani et al. / Talanta 76 (2008) 1006–1014

F 23 exv

(lbeib

simportant that the range of each variable that produces acceptable

F(c

ig. 6. Contour plots used to evaluate the ruggedness of the method selected by thes. electrolyte concentration).

CLP), 7 (DANO, CIPRO, ENRO) and 8 (SMZ, SMX, and SQX). The ana-ytical signal of CLP, DANO, CIPRO and ENRO were less influenced

y small variations in temperature in relation to the other param-ters, while SMZ and SQX were less affected by small variationsn the electrolyte concentration. In turn, SMX was most affectedy small variations in temperature, and pH. Considering that the

raas

ig. 7. Contour plots used to evaluate the ruggedness of the method selected by the 23 exppH vs. electrolyte concentration); (C) CIPRO (temperature vs. electrolyte concentration);oncentration); (F) ENRO (pH vs. electrolyte concentration).

perimental design. (A) CLP (temperature vs. electrolyte concentration); (B) CLP (pH

ame method should be applicable to all seven antimicrobials, it is

esults be incorporated in the analytical procedure in order to guar-ntee consistency of the results. The results obtained in this studyllowed establishing the following parameters: 60 ± 1 mmol L−1

odium phosphate, pH 8.5 ± 0.1 and 26.0 ± 0.5 ◦C.

erimental design. (A) DANO (temperature vs. electrolyte concentration); (B) DANO(D) CIPRO (pH vs. electrolyte concentration); (E) ENRO (temperature vs. electrolyte

M.C.V. Mamani et al. / Talanta 76 (2008) 1006–1014 1013

F e 23 e( tion);c

3

dCtotC(Snpasa

4

dtod

iac

oaan

weavdctf

ig. 8. Contour plots used to evaluate the ruggedness of the method selected by thpH vs. electrolyte concentration); (C) SMX (temperature vs. electrolyte concentraoncentration); (F) SMZ (pH vs. electrolyte concentration).

.4. Analysis of commercial samples

After validation, the CZE method was applied to assay threeifferent commercial samples labeled to contain 4 mg mL−1 ofLP, 500 mg of CIPRO, 50 mg mL−1 of ENRO, 400 mg of SMX inablet form and 40 mg mL−1 of SMX in oral suspension. Threef the samples were analyzed by HPLC. The average concen-rations determined in the samples were 4.2–4.5 mg mL−1 ofLP (Table 4), 501 mg/tablet of CIPRO, 47–49 mg mL−1 of ENROTable 5), 411–435 mg for SMX tablets and 39 mg mL−1 for theMX oral suspension. Considering that the active ingredienteeds to be in the range of 90–110% for nearly all the com-ounds and between 93 and 107% for the SMX tablets (Table 6),ll of the samples analyzed were in accordance with theirpecifications and also agreed with the results of the HPLCssay.

. Conclusions

Most of the methods for the determination of antimicrobialsescribed in Pharmacopoeias recommend analysis by HPLC. Never-heless, these methods require the use of relatively large volumesf HPLC-grade solvents. Recently, many CZE methods have beeneveloped. However, in spite of their advantages, such as analyt-

A

CC

xperimental design. (A) SQX (temperature vs. electrolyte concentration); (B) SQX(D) SMX (pH vs. electrolyte concentration; (E) SMZ (temperature vs. electrolyte

cal efficiency and cost, most of them continue to be considereds alternative methods in the quality control of pharmaceuti-als.

By combining the calculation of effective mobilities and the usef experimental design it was possible to arrive at optimal sep-ration conditions for the simultaneous determination of sevenntimicrobials of rather different nature, from a relatively smallumber of experiments.

The results obtained in this work confirm that the CZE method,hen properly optimized and validated, fulfills all the pre-

stablished requirements based on international regulations and isdequate to be used in the quality control of pharmaceuticals. Thisalidated electrophoretic method could be advantageous because itoes not require the usual frequent changes of operating analyticalonditions, besides reducing the cost of mobile phases and savingime of analysis in research and quality control of pharmaceuticalormulations.

cknowledgements

The authors gratefully acknowledge the financial support fromNPq and FAPESP (01/14072-6, 04/07312-9) and thank Professor.H. Collins for language assistance.

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eferences

[1] A. Anadon, M.R. Martinez-Larranaga, Livestock Prod. Sci. 59 (1999) 183.[2] L.S. Goodman, A.G. Gilman (Eds.), The Pharmacological Basis of Therapeutics,

9th ed., McGraw-Hill, New York, 1996, pp. 1131–1135.[3] M.D. Marzuela, M.C. Moreno-Bondi, J. Chromatogr. A 1034 (2004) 25.[4] J.A. Van Rhijn, J.J.P. Lasaroms, B.J.A. Berendsen, U.A.Th. Brinkman, J. Chromatogr.

A 960 (2002) 121.[5] E. Gikas, P. Kormall, D. Tsipi, A. Tsarbopoulos, J. Agric. Food Chem. 52 (2004)

1025.[6] P.H. Schmitt-Kopplin, J. Burhenne, D. Freitag, M. Spiteller, A. Kettrup, J. Chro-

matogr. A 837 (1999) 253.[7] K. Michalska, G. Pajchel, S. Tyski, J. Chromatogr. A 1051 (2004) 267.[8] D. Barron, J. Jimenez-Lozano, J. Barbosa, J. Chromatogr. B 759 (2001) 73.

[9] C. Lin, Y. Deng, W. Liao, S. Sun, W. Lin, C. Chen, J. Chromatogr. A 1051 (2004)

283.10] M.R. Gomez, R.A. Olsina, L.D. Martinez, M.F. Silva, Talanta 61 (2003) 233.11] A. Wang, L. Zhang, Y. Fang, Anal. Chim. Acta 394 (1999) 309.12] C. Lin, C. Chang, W. Lin, J. Chromatogr. A 768 (1997) 105.13] M.E.P. Hows, D. Perrett, J. Kay, J. Chromatogr. A 768 (1997) 97.

[

[[[

76 (2008) 1006–1014

14] L.Y. Fan, H.L. Chen, X.G. Chen, Z. De Hu, J. Sep. Sci. 26 (2003) 1376.15] M.S. Fuh, S. Chu, Anal. Chim. Acta 499 (2003) 215.16] S. Lamba, K.S. Sunil, A. Asthana, M. Shelke, Anal. Chim. Acta 552 (2005) 110.17] B. Santos, A. Lista, B.M. Simonet, A. Rios, M. Valcarcel, Electrophoresis 26 (2005)

1567.18] The United States Pharmacopoeia, The National Formulary, 28th ed., United

States Pharmacopoeia Convention, Rockville, 2005, pp. 1886–1887.19] A. Weston, P.R. Brown (Eds.), LC and CZE Principles and Practice, Academic Press,

San Diego, 1997, p. 279.20] S.M. Santos, M. Henriques, A.C. Duarte, V.I. Esteves, Talanta 71 (2007) 731.21] P.P. Maia, J.A. Farfan, S. Rath, F.G.R. Reyes, J. Pharm. Biomed. Anal. 43 (2007)

450.22] C. Garcıa-Ruiz, M.L. Marina, Electrophoresis 27 (2006) 266.23] J.C. Miller, J.N. Miller (Eds.), Statistics for Analytical Chemistry, 3rd ed., Ellis

Horwood, New York, 1993, p. 233.24] R. Kuhn, S. Hoffstetter-Kuhn, Capillary Electrophoresis: Principles and Practice,

Springer-Verlag, Berlin, 1993, pp. 74–80.25] C. Lin, W. Lin, Y.C.S. Wang, J. Chromatogr. A 792 (1997) 37.26] M.F.M. Tavares, V.L. Guffin, J. Chromatogr. A 686 (1994) 129.27] D. Barron, J. Jimenez-Lozano, J. Barbosa, J. Chromatogr. A 919 (2001) 395.