28

Electrophoresis 2014, 35, 28–49

Elena Dom´ınguez-Vega1∗ 2∗ ´ ´ Virginia Perez-Fern andez Antonio Luis Crego2 ´ Mar´ıa Angeles Garc´ıa2 Mar´ıa Luisa Marina2

Review

1 Department

of Biomolecular Analysis, Faculty of Sciences, Utrecht University, Utrecht, The Netherlands 2 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Biology, Environmental Sciences and ´ Chemistry, University of Alcala, Alcala´ de Henares (Madrid), Spain

Antibiotics are a class of therapeutic molecules widely employed in both human and veterinary medicine. This article reviews the most recent advances in the analysis of antibiotics by CE in pharmaceutical, environmental, food, and biomedical fields. Emphasis is placed on the strategies to increase sensitivity as diverse off-line, in-line, and on-line preconcentration approaches and the use of different detection systems. The use of CE in the microchip format for the analysis of antibiotics is also reviewed in this article. Moreover, since the use of antibiotics as chiral selectors in CE has grown in the last years, a new section devoted to this aspect has been included. This review constitutes an update of previous published reviews and covers the literature published from June 2011 until June 2013.

Received July 26, 2013 Revised October 6, 2013 Accepted October 6, 2013

Keywords: Antibiotics / Analysis / Chiral selectors / CE

Recent advances in CE analysis of antibiotics and its use as chiral selectors

1 Introduction Antibiotics represent a group of drugs widely employed both in human and veterinary medicine to prevent outbreak diseases due to bacterial infections [1]. In order to show the great variety of antibiotics available nowadays, Table 1 includes the most employed antibiotic families and representative examples belonging to each group. Since the 1940s, antibiotics have played a critical role in protecting public health, but unfortunately their inappropriate use is threatening their efficacy. Thus, the use of antibiotics in the food industry (they are employed as growth promoting agents [2]) is responsible for drug-resistant bacteria that reach the population through their diet. Likewise, the use of antibiotics in lactating breeding animals may leave residues of these compounds in milk, creating resistant

Correspondence: Dr. Mar´ıa Luisa Marina, Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Biology, Environmental Sciences and Chemistry, Uni´ Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcala´ versity of Alcala, de Henares (Madrid), Spain E-mail: [email protected] Fax: +34-918854971

Abbreviations: ASE, accelerated solvent extraction; [BMIM]BF4 , 1-butyl-3-methylimidazolium hexafluoroborate; [BMIM]PF6 , 1-butyl-3-methylimidazolium tetrafluorophosphate; C12 MIM, 1-docdecyl-3-methylimidazolium; CSP, chiral stationary phase; CTAB, cetyltrimethylammonium bromide; DLLME, dispersive liquid–liquid microextraction; DOX, doxorubicin; DSPE, dispersive solid-phase extraction; L-His, L-Histidine; LVSS, large-volume sample stacking; MES, 2-(Nmorpholino)ethanosulfonic acid; MRL, maximum residue limit; MSPD, matrix solid-phase dispersion; MSPE, magnetic solid-phase extraction; NACE, nonaqueous capillary electrophoresis; o-MWCNT, oxidized multi-walled carbon nanotube; poly(DVB-OMA), polydivinylbenzene-n-octyl methacrylate  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

DOI 10.1002/elps.201300347

strains, allergic hypersensitivity, or failures in the fermentation process of dairy products [3]. In this sense, and considering that in recent years food safety problems have become a frequent recurring phenomenon, consumer protection has increased drastically in the European Union around antibiotic residues. As antibiotics are continuously released to the environment as a result of manufacturing processes, household discharges, or excretion by urine from humans and animals, the chemical pollutants released to the environment include antibiotics used in both, human and veterinary medicine [4], causing the appearance of resistant bacterial strains. All these facts have prompted the development of multiple analytical methods for antibiotic determination in many different matrices. CE has proven efficacy in analyzing antibiotics mainly due to its versatility, wide variety of detection systems, low consumption of samples and reagents and multiple separation modes that facilitates the separation of a wide range of antibiotics. CE’s main limitation is its limited sensitivity when coupled to ultraviolet (UV) detection systems, although this detection system is still the most employed in the separation and determination of samples antibiotic drugs by CE due to its simplicity and low cost. However, there are many other detection systems that have been employed in the analysis of antibiotics by CE: laser-induced fluorescence, electrochemiluminiscence, electrochemical detection, conductivity detection, or MS, among others permitting better sensitivity. Finally, CE in microchip format has also attracted attention in the last years; the two new works published in the period of time covered in this review by using chip-based microfluidic systems for the analysis of antibiotics are a good case in point A wide variety of samples analyzed by CE for the determination of antibiotics exists. In addition to pharmaceutical ∗

These authors have contributed equally to this work.

Colour Online: See the article online to view Figs. 1, 3, and 5 in colour. www.electrophoresis-journal.com

Example

Amikacin

Vancomycin

Family

Aminoglycosides

Glycopeptides

␤-Lactams

Family

Structure

Structure

Table 1. Main families of antibiotics and representative examples belonging to each group

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Lincosamides

Ansamycins

Family

Lincomycin

Geldanamycin

Example

Structure

Electrophoresis 2014, 35, 28–49

CE and CEC 29

www.electrophoresis-journal.com

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Furazolidone

Bacitracin

Nitrofurans

Polypeptides

Sulfadiazine

Daptomycin

Lipopeptides

Structure

Tetracyclines

Quinolones

Oxazolidonones

Macrolides

Family

Doxycicline

Ciprofloxacin

Linezolid

Erythromycin

Example

Structure

E. Dom´ınguez-Vega et al.

Sulfonamides

Example

Family

Table 1. Continued

30 Electrophoresis 2014, 35, 28–49

www.electrophoresis-journal.com

Electrophoresis 2014, 35, 28–49

formulations, milk, meat, fish, or eggs as food samples, waters as environmental samples, and urine as biological fluids, have been analyzed. The analysis of antibiotic residues in these kind of samples in most cases requires a pretreatment step of the sample primarily through SPE [5–11], liquid– liquid extraction [12, 13], and solid–liquid extraction [14]. Besides these common techniques, accelerated solvent extraction (ASE) [15], matrix solid-phase dispersion (MSPD) [16], magnetic solid-phase extraction (MSPE) [17], dispersive liquid–liquid microextraction (DLLME) [18], dispersive solid– phase extraction (DSPE) [19] and the extraction with oxidized multi-walled carbon nanotubes (o-MWCNTs) have also been utilized [19]. Additionally, the interest for the enantiomeric separation of all type of chiral compounds in pharmaceutical, clinical, environmental and food analysis has increased. In this regard, CE has proven to be a powerful separation technique for chiral analysis. For this purpose, the use of chiral selectors to achieve the enantioseparation is necessary because enantiomers of a chiral compound have identical electrophoretic mobility. These chiral selectors can be used as additives dissolved in the BGE or linked to a chiral stationary phase (CSP). A great number of chiral selectors are available for their use in CE. Although in general terms cyclodextrins are the most popular, different types of antibiotics play also an important role in this area [20]. This review covers all the literature concerning the separation and determination of antibiotics by CE from June 2011 to June 2013 as a continuation of previous reviews concerning this field [21–28]. All CE modes are considered and the most significant applications of the developed methodologies are described. The works included in this review are classified based on the detection system utilized. According to this criterion, the review is divided in four main sections: (i) CE with UV detection, (ii) CE with other spectroscopic detection systems, (iii) CE with conductivity detection, and (iv) CE coupled to MS detection. Finally, a section regarding CE in microchip format and a new section covering recent applications based on the use of antibiotics as chiral selectors in CE are also included.

2 CE with UV detection In the separation of antibiotics by CE, UV-Vis absorbance detection is the first option considered due to its usefulness for a large number of compounds, its simplicity, low cost, and the possibility of using all separation modes in CE [29]. Moreover, it is a nondestructive detection system providing not only quantitative data but also qualitative information [29]. However, there is an important limitation in the sensitivity of UV detectors. In UV detection, the sensitivity obtained is proportional to the optical path length that in CE depends on the inner diameter of the capillary. Considering that the capillaries employed range from 25 to 75 ␮m of inner diameter, the LODs achieved are quite limited. However, several options exist to improve sensitivity in CE including path length  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CE and CEC

31

expansion using bubble capillaries or z-shaped cells, and the application of on-line sample preconcentration techniques. In general, most of the works developed by CE-UV for the determination of antibiotics are focused on three types of matrices (waters, urine, and milk), which consequently require new sample treatment methodologies and preconcentration procedures to enable the detection of trace levels. Regarding separation, CE enables the use of a wide variety of separation modes, for the analysis of different types of compounds. In the field of antibiotic analysis, CZE (see Table 2), is mostly employed coupled with UV detection although EKC with MEKC, ionic liquids, or MEEK have also been employed as it can be observed in Table 3. CZE is based on the free mobility of the analytes in an aqueous solution under an electric field and consequently, it can only be used for charged or ionizable analytes. In the period covered in this review, 12 new works have appeared in which CZE is used for the separation of ␤-lactams [4, 30, 31], fluoroquinolones [5, 15, 17, 19, 30–32], sulfonamides [15, 18, 33], and tetracyclines [16]. ␤-Lactam antibiotics are widely used to treat bacterial infections of various organs and include a large group of antibiotics such as cephalosporins or penicillins. Because of their extensive use they can readily be found in aquatic environments but their determination can be labor-intensive due to their low concentrations [4, 18, 19]. Three different works exist regarding the separation and determination of ␤-lactams (all as cephalosporins) by CZE-UV in the period reviewed [4,30,31]. The analysis of five cephalosporins (cephoperazone, cephalexin, cefazolin, ceftiofur, and cephadroxil) in environmental waters was achieved by CZE-UV upon the extraction of analytes by SPE with Oasis HLB cartridges [4]. As explained above, the main problem in the analysis of environmental waters is the low concentrations of analytes. For this reason, the use of preconcentration techniques such as in-capillary preconcentrations is essential in this field. In this work, a large-volume sample stacking (LVSS) preconcentration procedure was applied in which the sample was diluted in a low conductivity solvent (water:ACN 6:4 v/v) and injected into the capillary until the whole capillary was filled with the sample. A negative voltage was then applied in order to eliminate the matrix from the capillary and finally the voltage was stopped and reversed in order to separate the stacked compounds. The use of a 70 mM ammonium acetate buffer (pH 7.0) and a voltage of 22.5 kV enabled the baseline separation of the five cephalosporines in less than 8 min with recoveries of the whole method from 84 to 112% for all compounds except for cephalexin in groundwater (only a 14% of recovery was obtained). The simultaneous determination of ␤-lactams and fluoroquinolones was performed by Sun et al. [30, 31]. The combination of these two types of antibiotics has favorable synergistic action for ␤-lactams, preventing clinical isolates that can cause urinary tract infections. Two cephalosporins (cefazolin, cefminox) and one fluoroquinolone (gatifloxacin) were simultaneously analyzed in urine samples of patients after intravenous administration [30]. As antibiotics are www.electrophoresis-journal.com

Cefazolin Ceftiofur Cephalexin Cephapirin Cephoperazone

Cefazolin Cefminox Gatifloxacin

Ceftriaxone Levofloxacin

Ciprofloxacin Danofloxacin Difloxacin Enrofloxacin Flumequine Levofloxacin Lomefloxacin Marbofloxacin Moxifloxacin Oxolinic acid Perfloxacin Ciprofloxacin Enrofloxacin

␤-Lactams

␤-Lactams and Fluoroquinolones

␤-Lactams and Fluoroquinolones

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Fluoroquinolones

CZE

CZE

CZE

CZE

CZE

CE mode

268 nm

250 nm oxolinic acid and flumequine 280 nm the rest of antibiotics

254 nm

272 nm

250 nm (cephoperazone, cephazolin and cephalexin) 270 nm (cephapirin) 292 nm (ceftiofur)

Detection wavelength

BGE: 50 mM phosphate buffer (pH 8.4) Capillary: 50 cm × 75 ␮m Voltage: 25 kV Temperature: 25⬚C Injection: 0.5 psi × 5 s

BGE: 70 mM acetate buffer (pH 7.0) Capillary: 40 cm × 50 ␮m Voltage: 22.5 kV Temperature: 25⬚C Injection: 1 bar × 1 min (LVSS) BGE: 25 mM borate buffer (pH 9.2) Capillary: 40 cm × 75 ␮m Voltage: 18 kV Temperature: 25⬚C Injection: 50 mbar × 4 s BGE: 25 mM tetraborate buffer (pH 9.2) Capillary: 40 cm × 75 ␮m Voltage: 20 kV Temperature: 25⬚C Injection: 50 mbar × 4 s BGE: 65 mM phosphate buffer (pH 8.5) Capillary: 60 cm × 75 ␮m Voltage: 15 kV Temperature: 25⬚C Injection: 20 psi × 3 s (LVSS)

CE conditions

SPE

DSPE with o-MWCNTs

Waste, mineral and tap waters

Milk

Protein precipitation with ACN and dilution of the supernatant

Protein precipitation with ACN and dilution of the supernatant

SPE

Sample treatment

Human urine

Human urine

Environmental waters

Sample

[30]

[31]

[19]

5.9–15.0 ␮g/mL

2.5–7.5 ␮g/mL

7.42–16.70 ␮g/L

[5]

[4]

0.1–0.3 ␮g/L

0.02–0.03 mg/kg

Ref.

LOD

E. Dom´ınguez-Vega et al.

Fluoroquinolones

Analyte

Antibiotic Family

Table 2. Separation and determination of antibiotics by CE with UV detection

32 Electrophoresis 2014, 35, 28–49

www.electrophoresis-journal.com

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Tetracyclines

Sulfonamides

Sulfonamides

Doxycycline Oxytetracycline Tetracycline

CZE

268 nm

254 nm

254 nm

CZE

CZE

272 nm

CZE

Ciprofloxacin Enrofloxacin Lomefloxacin Ofloxacin Sulfadiazine Sulfadimethoxine Sulfamerazine Sulfadiazine Sulfadimidin Sulfadoxin Sulfamerazine Sulfapyridine Sulfamethazine

Fluoroquinolones and Sulfonamides

260 nm

260 nm

Detection wavelength

CZE

CZE

Ciprofloxacin Danofloxacin Difloxacin Enrofloxacin Flumequine Marbofloxacin Oxolinic acid Ciprofloxacin and other drugs

Fluoroquinolones

Fluoroquinolones

CE mode

Analyte

Antibiotic Family

Table 2. Continued

BGE: 20 mM phosphate buffer (pH 8.5) with 10% ACN Capillary: 40 cm × 75 ␮m Voltage: 25 kV Temperature: 25⬚C BGE: phosphate buffer (pH 7.4) Voltage: 25 kV Temperature: 25⬚C Injection: 0.8 psi × 60 s BGE: 30 mM phosphate buffer (pH 11.5) with 1 mM EDTA Capillary: 50 cm × 75 ␮m Voltage: 25 kV Temperature: 25⬚C Injection: 30 mbar × 3 s

BGE: 50 mM tetraborate buffer (pH 9.0) Capillary: 50 cm × 75 ␮m Voltage: 30 kV Temperature: 25⬚C Injection: 0.5 psi × 4 s BGE: 25 mM borate buffer (pH 8.6) Capillary: 40 cm × 75 ␮m Voltage: 20 kV Temperature: 25⬚C Injection: 50 mbar × 5 s

BGE: 40 mM phosphate buffer (pH 8.1) with 1 mM EDTA Capillary: 41.7 cm × 50 ␮m Voltage: 15 kV Temperature: 25⬚C Injection: 0.5 psi × 5 s

CE conditions

MSPD

Filtration and direct injection

HSA-antibiotic incubated solutions

Milk

DLLME

ASE

Dilution in water

MSPE

Sample treatment

Lake, pond and tap water

Fish

Pharmaceutical formulation and human urine

Milk

Sample

[32]

[15]

[18]

1 ␮g/mL

0.14–0.29 ␮g/mL

0.04–0.57 ␮g/mL

0.074–0.081 ␮g/mL

[16]

[33]

[17]

9–12 ␮g/L

-

Ref.

LOD

Electrophoresis 2014, 35, 28–49

CE and CEC 33

www.electrophoresis-journal.com

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Biapenem

Amoxicillin Ampicillin Nafcillin Oxacillin Penicillin G Penicillin V

Ciprofloxacin Norfloxacin Ofloxacin

Ciprofloxacin Enoxacin Enrofloxacin Gatifloxacin Norfloxacin Ofloxacin Pazufloxacin Tosufloxacin Gatifloxacin Lomefloxacin Pazufloxacin Rufloxacin

Dimetridazole Ipronidazole Metronidazole Ornidazole Ronidazole Ternidazole and three metabolites

␤-Lactams

␤-Lactams

Fluoroquinolones

Fluoroquinolones

Nitroimidazoles and their metabolites MEKC

320 nm

250 nm

280 nm

MEKC with IL

EKC with IL

280 nm

200 nm

200 nm

Detection wavelength

MEKC

MEEKC

MEKC

CE mode

BGE: 20 mM borate –10 mM phosphate buffer (pH 8.6) with 0.3 mM C12 MIM-OH ionic liquid Capillary: 40 cm × 75 ␮m Voltage: 12 kV Injection: siphoned by 30 cm for 20 s BGE: 20 mM phosphate buffer (pH 6.5) with 150 mM SDS Capillary: 56 cm × 50 ␮m Voltage: 25 kV Temperature: 20⬚C Injection: 50 mbar × 15 s (sweeping)

BGE: 22.5 mM formic acid (pH 4.3) with 150 mM SDS Capillary: 50 cm × 50 ␮m Voltage: –22 kV Temperature: 25⬚C Injection: 3.4 kPa × 10 s (sweeping) BGE: 70 mM acetate buffer (pH 8.0):1-butanol:SDS 80:15:5 v:v:v Capillary: 40 cm × 50 ␮m Voltage: –29 kV Temperature: 37.5⬚C Injection: 50 mbar × 60 s (NSM) BGE: 25 mM borate buffer (pH 9.3) with 5% MeOH and 100 mM SDS Capillary: 40 cm × 50 ␮m Voltage: 20 kV Temperature: 20⬚C Injection: 50 mbar × 5 s BGE: 10 mM borate buffer (pH 7.1) with 1.7% SDS and 1.5% [BMIM]PF6 Capillary: 52 cm × 50 ␮m Voltage: 18 kV Temperature: 25⬚C Injection: siphoned for 5 s

CE conditions

River waters

SPE

Direct injection

Direct injection

Standards

Standards

Direct injection

SPE

Direct injection

Sample treatment

Standards

Porcine organs

Pharmaceutical formulation

Sample

0.469–1.105 ␮g/L

0.10–0.14 mg/L



[6]

[37]

[36]

[35]

[7]

0.06–0.11 ␮g/mL

2.07–2.49 mg/L

[34]

Ref.

0.5 ␮g/mL

LOD

E. Dom´ınguez-Vega et al.

NSM, normal stacking mode.

Fluoroquinolones

Analyte

Antibiotic Family

Table 3. Separation and determination of antibiotics by EKC with UV detection

34 Electrophoresis 2014, 35, 28–49

www.electrophoresis-journal.com

Electrophoresis 2014, 35, 28–49

metabolized and excreted by urine, the analysis of this matrix is also of great interest [30, 31]. The samples were previously treated with ACN to precipitate the proteins and directly diluted and injected in the CE system. A 25 mM borate buffer (pH 9.2) was used and the separation of the three antibiotics was achieved under optimal conditions in less than 15 min with recoveries above 93%. The same authors carried out the simultaneous separation of ceftriaxone (cephalosporin) and levofloxacin (fluoroquinolone) for their determination in human urine in the same experimental conditions as before [31]. Under the optimized conditions, the two drugs could effectively be separated in approximately 6 min with LODs below 7.5 ␮g/mL. Fluoroquinolones are synthetic antibacterial compounds used in humans and food-producing animals for the treatment of various bacterial infections. Some of them are specifically used in veterinary practice while others are only designed for humans [38]. Their use in both cases is increasing and as a result, they can be easily found in environmental waters and the milk produced by the animals treated with these antibiotics. Herrera-Herrera et al. [19] developed a new method using CZE-UV for the determination of 11 fluoroquinolones (moxifloxacin, lomefloxacin, danofloxacin, ciprofloxacin, levofloxacin, marbofloxacin, enrofloxacim, difloxacin, perfloxacin, oxolinic acid, and flumequine) in different water samples after DSPE. The main novelty of this work is the use of o-MWCNTs as stationary phase for the preconcentration step only used once before for the extraction of fluoroquinolones from human plasma [39]. These materials have an adequate sorption capacity for the extraction of both organic and inorganic compounds [40]. Once again, the low concentrations of analytes in the aqueous samples requires not only the selective extraction and preconcentration by DSPE but also the use of an in-capillary preconcentration technique as LVSS in order to improve the sensitivity of the CE method. The separation was achieved using a 65 mM phosphate buffer (pH 8.5) and the sample diluted with water was injected at 20 psi during 30 s. The matrix of the sample was eliminated by applying a negative polarity and when the current became 95–99% of the value obtained with the BGE, the voltage was turned off, polarity reversed, and the separation achieved. Under optimal conditions, the separation of the 11 fluoroquinolones was achieved in 18 min with recoveries above 75% and the LVSS preconcentration method allowed to obtain LODs at the ␮g/L level. Two CZE methodologies for the determination of fluoroquinolones in milk have recently been developed [5, 17]. The use of antibiotics in lactating breeding animals can lead to residues in milk that must be controlled in order to meet applicable regulatory requirements, to protect consumers and the environment. In this sense, the Commission of the European Community [41] and the United States Food and Drug Administration (US FDA) [42] have established a maximum residue limit (MRL) for enrofloxacin and its main metabolite ciprofloxacin of 0.1 mg/kg in milk. Pi˜ neiro et al. [5] developed a new CZE-UV method with phosphate buffer as separation media for the determination of enrofloxacin and ciprofloxacin  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CE and CEC

35

in milk samples extracted by SPE with Oasis HLB cartridges. The recoveries obtained were higher than 89% and the LODs achieved were low enough to determine the compounds at the MRL cited before. For danofloxacin, ciprofloxacin, marbofloxacin, enrofloxacin, difloxacin, oxolinic acid, and flumequine the Commission of the European Union has adopted MRLs of 30 ␮g/kg for danofloxacin, 50 ␮g/kg for flumequine, 75 ␮g/kg for marbofloxacin, and 100 ␮g/kg for enrofloxacin and ciprofloxacin; oxilinic acid and difloxacin have however been banned for milk-producing animals [41]. Ibarra et al. [17] developed a CZE method for the determination of the above-mentioned antibiotics in milk samples using a 40 mM phosphate buffer (pH 8.1) as separation media and accomplishing the separation in less than 8.5 min. The milk samples containing the analytes were extracted by MSPE with magnetite particles covered with octylphenyl silica adsorbents. This technique is based on the dispersion of a magnetic adsorbent in the liquid sample and after their adsorption in the surface of the magnetic particles, the analytes are eluted in an appropriate solvent and injected in the CE system. Upon the optimization of the variables involved in the sample treatment procedure such as composition of the magnetic support, sample pH and amount of magnetic adsorbent, the quantitative extraction of the seven fluoroquinolones with recoveries from 74 to 98% in real milk samples was achieved. All milk samples analyzed were positive to the presence of the fluoroquinolones researched. Solangi et al. [32] developed a rapid and simple methodology for the simultaneous separation of ciprofloxacin, a fluoroquinolone antibiotic, from other coadministered drugs (paracetamol and diclofenac) by CZE-UV. Separation was achieved in only 6.5 min with a 50 mM sodium tetraborate buffer (pH 9.0) reaching LODs of 1 ␮g/mL. The method was successfully applied to the analysis of these drugs in commercial pharmaceutical formulations and urine samples from patients treated with this mixture of drugs. The results were compared to those obtained by the HPLC method of the pharmacopeia and good agreement was found. Sun et al. [15] developed a methodology for the simultaneous separation and determination of fluoroquinolone and sulfonamide antibiotics by CZE-UV. In this work, the analysis of four fluroquinolones (ciprofloxacin, enrofloxacin, lomefloxacin, and ofloxacin) and three sulfonamides (sulfadiazine, sulfamerazine, and sulfadimethoxine) in several fish samples was accomplished. The method consisted in using a 25 mM borate buffer (pH 8.6) that allowed the separation of the seven antibiotics in less than 7.5 min. The samples analyzed were extracted by ASE with ACN providing recoveries above 83% for all compounds in shrimp, eel, and tuna samples. This methodology, combining CZE with ASE, provides a rapid and simple extraction procedure as compared to conventional extraction techniques, enabling satisfactory recoveries, and the effective separation of the fluoroquinolones and sulfonamides studied. Sulfonamides, which are derivatives of sulfanilamide, are widely used for the treatment of infections in the digestive and respiratory tracts. Besides the work on the simultaneous www.electrophoresis-journal.com

36

E. Dom´ınguez-Vega et al.

Figure 1. (A) Scheme of the DLLME procedure employed for the extraction of the sulfonamide antibiotics. (B) Typical electropherograms of sulfonamide antibiotic standards at 10 ␮g/mL (i) before and (ii) after DLLME. Separation conditions: BGE: 20 mM sodium dihydrogen phosphate (pH 8.5) – 10% ACN; capillary: 40 cm × 75 ␮m; voltage: 25 kV; temperature: 25⬚C, detection wavelength: 254 nm. SPD: Sulfapyridine, SDM: Sulfadimidin, SDX: Sulfadoxin, SMR: Sulfamerazine, SDZ: Sulfadiazine. Reprinted with permission from [18].

separation of fluoroquinolones and sulfonamides described above, two additional works concerning the separation and determination of sulfonamides by CZE with UV-Vis detection [18, 33] are also noteworthy. The simultaneous separation of sulfadiazine, sulfapyridine, sulfadimidin, sulfadoxin, and sulfamerazin was applied to the analysis of lake, pond, and tap waters [18]. As these compounds are frequently administered to humans and animals their appearance in environmental samples such as natural waters is not surprising. DLLME is a novel miniaturized sample treatment technique that employs low amounts of organic solvents compared to traditional extraction techniques. In this technique, the extraction solvent is injected by a syringe into the sample; the particles of extracting solvent then interact with the analyte in an efficient way enabling its extraction as shown in Fig. 1A. DLLME with DMSO as disperser solvent and chlorobenzene as extraction solvent have been used for the extraction of the above-mentioned antibiotics in different waters. This work represents the first attempt in the coupling of DLLME with CE-UV for the analysis of sulfonamide antibiotics in waters. The separation was accomplished by CZE-UV with a sodium dihydrogen phosphate buffer with 10% ACN enabling the separation of the five analytes in less than 5 min. Fig. 1B shows the electropherograms obtained after and before the DLLME process. As it  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2014, 35, 28–49

can be observed, when employing DLLME, the antibiotics sulfadoxin and sulfamerazin were slightly worse separated than when directly injecting the standards, but sensitivity was considerably increased thus permitting their determination in real samples. With DLLME, the LODs obtained for real water samples were below 0.57 ␮g/mL for all the antibiotics involved and the recoveries achieved were above 54% for the compounds with the lowest recoveries for sulfadimidin and the highest ones for sulfamerazin. Additionally, the interactions between the sulfamethazine antibiotic and HSA were studied [33]. HSA is a primary protein in blood plasma that provides about 80% of the osmotic pressure in blood [43] and it does have important functions in the transport and disposition of various endogenous and exogenous chemicals (i.e. drugs). As the presence of this antibiotic could affect the normal physiological functions of HSA, the study of the interaction between HSA and sulfamethazine is paramount [44]. Accordingly, a CE-frontal analysis method is proposed in this work [33]. The experiments were carried out incubating 25 ␮M HSA with different concentrations of sulfamethazine for 15 min and then the supernatant was injected in the CE system. The difference between the initial concentration of antibiotic and the free concentration of antibiotic in the supernatant provided the amount of sulfamethazine bounded to HSA. Finally, the interactions between HSAsulfamethazine were spectroscopically characterized and the binding sites were identified by fluorescence and circular dichroism. In the last work performed by CZE-UV in the period reviewed in this article, a new methodology for the separation of three tetracycline antibiotics was proposed [16]. Tetracyclines are a kind of broad-spectrum antibiotics produced by Actinomycetes. They can be natural or semy-synthetic and are commonly applied to food-producing animals due to their broad spectrum. As they are widely employed in a variety of food-producing animals, they are usually found in meat, eggs, and milk. The European Union stipulates that the total content of tetracycline antibiotics shall not exceed 100 ␮g/kg in milk and consequently, control of this kind of matrices is required. Mu et al. [16] reported a CZE method for the simultaneous determination of tetracycline, oxytetracycline, and doxycycline in milk samples. The effect of the electrolyte composition, pH, temperature, and applied voltage was researched and the optimal conditions consisted of 30 mM sodium hydrogen phosphate buffer (pH 11.5) with 1 mM EDTA, 25 kV and 25⬚C. Regarding the analysis of real samples, a previous extraction step was optimized. The selected sample preparation technique was MSPD enabling recoveries above 95.4% for the three tetracyclines. EKC is a CE separation mode combining electrophoretic and chromatographic principles in order to achieve the separation of neutral and charged compounds and to increase selectivity. Thus, this separation mode based on the use of a pseudostationary phase in the BGE, is a good choice to improve the resolution of highly related compounds such as antibiotics. The interaction of the analyte with the pseudostationary phase results in a separation not only based on the www.electrophoresis-journal.com

Electrophoresis 2014, 35, 28–49

electrophoretic behavior but also on the affinity for pseudostationary phase. Although there is a variety of surfactants that can be employed as micellar pseudostationary phases in MEKC, the anionic surfactant SDS is undoubtedly the most popular. In fact, SDS was the surfactant employed in all the works included in this review concerning the analysis of antibiotics by MEKC-UV (see Table 3). However, SDS micelles in MEKC can sometimes show insufficient selectivity to separate highly hydrophobic compounds. Many alternative approaches have recently been developed to improve the resolution obtained in MEKC separations, but the use of other additives in the BGE together with micelles has proved to be the most effective [45–47]. Ionic liquids are a good option as additives in the separation media. They are materials with melting points at or close to room temperature, with unique physicochemical properties of broad liquid range, low vapor pressure, and high thermal stability compared to conventional organic solvents [48, 49]. Consequently, ionic liquids are used in CE as additives in the BGE to increase resolution. Additionally, MEEKC is another EKC mode which offers the possibility of highly effective separations of both charged and neutral solutes covering a wide range of solubilities [50]. In this CE mode, a micro-emulsion in the BGE is used as the pseudostationary phase to separate solutes based on their hydrophobicities and their electrophoretic mobilities. Micro-emulsions are composed of water-immiscible organic solvent (oil), water, and a surfactant. When the surface tension between the oil and water is reduced with the aid of a surfactant, stable, dispersed, and surfactant coated droplets are formed [51]. These droplets have proven promising separation media in EKC. Table 3 groups the antibiotic families separated by EKC, ␤-Lactams [7, 34], fluoroquinolones [35–37], and nitroimidazoles [6]. Carbapenem antibiotics are a type of ␤-lactam antibiotics widely used for many bacterial infections, such as Escherichia coli and Klebsiella pneumoniae. However, their activity is limited because some bacteria have become resistant to this type of antibiotics. For this reason, the research and study of new carbapenem antibiotics is becoming more extensive. Biapenen is a new parenteral carbapenem antibiotic approved for use in 2002 in Japan. Michalska et al. [34] reported a new method using MEKC-UV for the separation of biapenem from its related substances or impurities using a sweeping preconcentration method. The optimum separation was achieved with a 22.5 mM formic acid (pH 4.3) BGE with 150 mM SDS added to the electrolyte as sweeping agent. The evaluated MEKC method was applied to the analysis of a medicinal product containing biapenem for intravenous drip infusion and enabled to detect amounts higher than 0.5 ␮g/mL of the active compound. Huang et al. [7] described for the first time the use of a normal stacking mode, which is a in-capillary preconcentration technique, coupled to MEEKC system for the separation of six ␤-lactams (oxacillin, penicillin V, penicillin G, nafcillin, ampicillin, and amoxicillin) and their determination in porcine organs after SPE with C18 cartridges. Optimiza C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CE and CEC

37

Figure 2. Electropherogram of eight fluoroquinolones in (A) 10 mM sodium borate buffer (pH 7.1) – 1.7% SDS and 1.5% [BMIM]PF6 , and (B) 10 mM sodium borate buffer (pH 7.1) – 1.7% SDS. Other separation conditions: capillary: 52.0 cm × 50 ␮m; voltage: 18 kV; temperature: 25⬚C, detection wavelength: 280 nm. 1: Ofloxacin, 2: Norfloxacin, 3: Gatifloxacin, 4: Ciprofloxacin, 5: Enoxacin, 6: Pazufloxacin, 7: Tosufloxacin, 8: Enrofloxacin. Reprinted with permission from [36].

tion of the type and concentration of the oil phase (1-butanol) and the conditions of normal stacking mode step allowed the separation of the six penicillin antibiotics in less than 10 min and with a sensitivity increase about 12-fold compared with conventional injection. Finally, the proposed method was successfully applied in the analysis of several food samples spiked with the analytes, detecting concentrations above 0.11 ␮g/mL. EKC-UV has also been applied to the separation of fluoroquinolone antibiotics [35–37]. The separation of three fluoroquinolones (ciprofloxacin, norfloxacin, and ofloxacin) was carried out by MEKC with SDS in the BGE [35]. These three antibiotics are in general the most frequent fluoroquinolone derivatives and, although they are not therapeutically employed simultaneously, complex mixtures of them can be found in the environment. This method allowed the simultaneous determination of three analytes in a simple by adding 100 mM SDS and a 5% of methanol to a buffer solution of 25 mM sodium tetraborate (pH 9.3). Separation was performed in approximately 8.5 min with LODs lower than 2.49 mg/L. Chen et al. employed the ionic liquid 1-butyl3-methylimidazolium hexafluorophosphate ([BMIM]PF6 ) in combination with SDS in a 10 mM sodium borate buffer (pH 7.1) for the simultaneous separation of enoxacin, tosufloxacin, ofloxacin, norfloxacin, gatifloxacin, pazufloxacin, enrofloxacin, and ciprofloxacin [36]. The effect of several parameters on the separation selectivity such as pH, concentration of BGE, concentration of ionic liquid, and surfactant, etc., allowed fixing the best experimental conditions for achieving the complete resolution. Figure 2 shows the electropherograms obtained for the eight fluoroquinolones studied both with and without the addition of the ionic liquid to www.electrophoresis-journal.com

38

E. Dom´ınguez-Vega et al.

the BGE. As it can be observed, when only SDS was added to the separation media (Fig. 2B) only six peaks were observed because some of the analytes coeluted in a single peak. However, the addition of [BMIM]PF6 allowed to achieve the complete baseline separation of all the antibiotics (Fig. 2A). The conditions chosen as optimal were a BGE consisting of 1.7% SDS and 1.5% [BMIM]PF6 dissolved in a 10 mM sodium borate buffer (pH 7.1) applying a separation voltage of 18 kV. Under these conditions, the complete separation of the eight analytes was achieved in less than 15 min. Finally, in the case of fluoroquinolones that are anphoteric, a micellar phase in the BGE is not required because these charged compounds can interact in a selective way with ionic liquids in an EKC mode. The interactions between ionic liquidcation and the analytes with opposite charge include electrostatic interactions, hydrophobic interactions and hydrogen bonding [52–54]. Thus, Liu et al. studied the influence of 1-dodecyl-3-methyl-imidazolium (C12 MIM) basic ionic liquid on the separation of gatifloxacin, lomefloxacin, rufloxacin, and pazufloxacin antibiotics by EKC-UV [37]. The presence of 0.3 mM C12 MIM-OH in a 20 mM tetraborate and 10 mM phosphate buffer significantly improved the resolution of fluoroquinolones without detectable loss in the UV detection sensitivity. Under optimized conditions, the four fluoroquinolones were separated in 14 min with LODs between 0.10 and 0.14 mg/L. Nitroimidazoles are a family of antibiotics characterized by the presence of an imidazole cycle with an NO2 group. They possess antibacterial and antiprotozoal properties and can be employed mainly against anaerobic bacteria and anaerobic protozoans. These compounds have also been used as growth promoters, but their use for this purpose has been banned since 2006 [55]. Regarding 5-nitroimidazoles, (NO2 group at position 5 of the ring) they have been forbidden as veterinary drugs due to the adverse health effects reported [56, 57]. For this reason, their determination in animal products and environmental samples, especially waters, is clearly necessary. Metronidazole, dimetridazole, ronidazole, ipronidazole, ornidazole, and ternidazole are antibiotics belonging to 5-nitroimidazoles commonly employed. Hern´andez-Mesa et al. [6] developed a method for these six antibiotics and their main metabolites by MEKC with SDS as surfactant in the BGE. Separation was carried out in less than 21 min with a 20 mM phosphate buffer (pH 6.5) with 150 mM SDS. In order to improve sensitivity, an extended light path capillary and an on-line preconcentration approach based on sweeping, with the sample prepared in the BGE without the micelles, were employed enabling LODs lower than 1.1 ␮g/L. The method was applied to the analysis of these antibiotics in river waters extracted by SPE with Oasis HLB cartridges.

3 CE with other spectroscopic detection systems LIF detection offers high sensitivity and additional selectivity compared to UV-Vis absorption detection, which makes it one  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2014, 35, 28–49

of the most interesting detector systems for CE. LIF detection with argon-ion laser is still one of the most used and most sensitive detection techniques in CE. However, the use of LED, solid-state laser, or deep-UV lasers as excitation sources has increased [58]. Table 4 groups the works performed by CE-LIF in the field of antibiotic analysis in the reviewed period of time. Two out of the three works using LIF system, used argon-ion laser [11, 13] and the other solid-state laser [59]. Antracyclines are a group of antibiotic popularly employed as anticancer drugs possessing intrinsic fluorescence, making LIF the ideal detection system for this kind of compounds. Doxorubicin (DOX) is a clinically important anthracycline presenting anticancer effects, offering therapeutic effectiveness against a variety of human tumors, such as breast cancer, ovarian cancers, or leukemia [62]. However, a number of undesirable secondary effects such as cardiac and liver toxicity and drug resistance may be accompanied with DOX treatment. As a result, although DOX is currently prescribed in most cancer chemotherapy regimens, it still remains the focus of modern research. Two sensitive methods have been developed using CE-LIF for the determination of DOX in cancer cells [13, 59]. In the first one, the determination of DOX in single human leukemia cells was performed by MEKC-LIF [59]. Human leukemia cells were treated with DOX and the DOX uptake was analyzed in single cells in order to avoid problems derived from cell heterogeneity and to obtain more comprehensive information about DOX uptake. The procedure of injection of the single cell is represented in Fig. 3A. Thus, a single cell was selected by microscope and rapidly injected via a pulse of vacuum. Once the single cell was introduced into the capillary, the capillary tip was moved into the cell lysis solution for 5 min and this solution was injected applying a new pulse of vacuum. Then, the capillary inlet was reinserted into the CE buffer vial containing 10 mM sodium borate with 10 mM SDS at pH 9.3 and voltage was applied, allowing the separation of the lysated cell. Figure 3B shows the electropherogram obtained for a lysated cell after DOX uptake. The presence of DOX derived from the treated cell was observed. In addition, it is important to highlight the excellent sensitivity obtained with this system (LOD 56 zmol). Because SDS eliminates DOX fluorescence quenching and sample loss is avoided when in-capillary single-cell lysis is performed, this method directly detects the real DOX uptake of single K562 cells and, consequently, presented accurate information on both cell-to-cell heterogeneity in DOX uptake and the patterns of DOX uptake in K562 cells as functions of drug concentration and exposure time. In the second work, a MEKC-LIF method for the determination of DOX in subcellular fractions of DOX treated cells was reported [13]. This method is a modification of a previous method reported by Anderson et al. [63] and consisted in the use of 10 mM sodium borate buffer with 100 mM SDS at pH 9.3 offering efficient and reproducible separations for the analysis of DOX. The LOD obtained with the proposed methodology was 11 nM, being suitable for the determination of DOX in chinese hamster ovary cells (CHO-K1). Subsequently, the method was employed for the evaluation of the intercellular accumulation www.electrophoresis-journal.com

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Doxorubicin

Acetylspiramycin Azithromycin Roxithromycin Tilmicosin

Tetracycline

Anthtracyclines

Macrolides

Tetracyclines

Chlorotetracycline Doxycycline Oxytetracycline Tetracycline

Doxorubicin

Anthtracyclines

Tetracyclines

Analyte

Antibiotic Family

NACE

CZE

EKC

MEKC

MEKC

CE mode

LIF ␭exc: 325 nm ␭em: 514 nm

ECL ECL cell: 5 mM [Ru(bpy)3 2+] 50 mM phosphate (pH 8.0); detection potential: 1.25 V

ECL ECL cell: 5 mM [Ru(bpy)3 2+] 50 mM phosphate (pH 8.0); detection potential: 1.2 V

LIF ␭exc: 488 nm ␭em: 514 nm

LIF ␭exc: 473 nm ␭em: 514 nm

Detection and conditions

BGE: 10 mM borate buffer (pH 9.3) with 10 mM SDS Capillary: 45 cm × 30 ␮m Voltage: 20 kV Temperature: room temperature Injection: injected by vacuum and assisted with an inverted microscope BGE: 10 mM borate buffer (pH 9.3) with 100 mM SDS Capillary: 52 cm × 150 ␮m Voltage: 25 kV Temperature: 25⬚C Injection: siphoned by 17.5 cm ×5s BGE: 15 mM phosphate buffer (pH 7.5) containing 0.5% v/v BMIMBF4 ionic liquid Capillary: 50 cm × 50 ␮m Voltage: 25 kV Temperature: room temperature Injection: 10 kV × 10 s BGE: 10 mM phosphate buffer (pH 9.0) Capillary: 50 cm × 75 ␮m Voltage: 12 kV Temperature: room temperature Injection: 12kV × 10 s BGE: 500 mM acetate buffer in n-methylformamide Capillary: 20 cm × 100 ␮m Voltage: 25 kV Temperature: 25⬚C Injection: 10 kV × 300 s

CE conditions

Table 4. Separation and determination of antibiotics by CE with other spectroscopic detection methods

Urine, plasma, feed, and milk

Fish

Urine Tablets Pig fodder Egg samples

Cancer cells

Cancer cells

Sample

SPE

Protein precipitation with TCA and extraction in Na3 PO4

Urine: dilution and filtration Tablets: extraction with MeOH Pig fodder: extraction with MeOH:water (5/1, v/v) Egg samples: deproteinization by heating (90⬚C) and dilution

Cell lysis and LLE

In-capillary cell lysis

Sample treatment

[61]

1.8 ng/mL

[11]

[60]

1.3 – 70 nM

1.3 -13.3 ng/mL

[13]

[59]

Ref.

11 nM

56 zmol

LOD

Electrophoresis 2014, 35, 28–49

CE and CEC 39

www.electrophoresis-journal.com

40

E. Dom´ınguez-Vega et al.

Electrophoresis 2014, 35, 28–49

Figure 3. (A) Schematic representation of the homemade CELIF system employed for singlecell analysis. (B) DOX uptake of a single K562 cell exposed to 1 ␮M DOX for 3 h. Separation conditions: BGE: 10 mM borate buffer (pH 9.3) – 10 mM SDS; capillary: 45 cm × 30 ␮m; voltage: 20 kV. Reprinted with permission from [59].

of three subcellular fractions of DOX treated cells using both free and liposomal carrier form. Results obtained from this study indicated that the higher quantities of DOX were present in the nuclear fraction and provide direct evidence of the enhanced efficiency of liposomal carriers in delivering DOX into the nucleus. Amino groups in the structure of antibiotics favor using ECL detection, and hence this detection system has been widely used in the determination of antibiotics [21–23]. ECL detection is a kind of chemiluminescence where chemical luminescence emission is generated in the process of substance oxidation and reduction at an electrode surface. This detection system has many advantages such as high sensitivity, simplicity, low cost instrumentation, low background noise, and wide linear range. In recent years, ECL based on tris(2,2’bipyridyl) ruthenium (II) (Ru(bpy)3 2+ ) has gained considerable attention due to high luminescence efficiency, stability in aqueous media and its ability to react with primary, secondary and tertiary amines [64]. As shown in Table 4, in the period of time covered in this review, Ru(bpy)3 2+ was employed in ECL detection for the determination by CE of Macrolides [60] and tetracyclines  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[61]. Macrolides are extensively used as veterinary medicines and feed additives, but their excessive use is producing superbugs resistant to antibiotics, as admitted by Food authorities that recommend reducing their use on farms and have established MRLs of macrolides in foods. Liu et al. [60] developed a methodology for the determination of azithromycin, tilmicosin, acetylspiramycin, and roxithromycin in human urine, tablets, pig fodder, and egg samples by EKC-ECL. A remarkable detail of this work is the use of an ionic liquid in the BGE to enhance the separation. In this work, the use of 0.5% of 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF4 ) in 15 mM phosphate buffer at pH 7.5 significantly improved the separation of the four macrolides with a separation time of around 7 min. The LODs obtained (between 1.3 and 70 nM) together with the efficient separation obtained in CE shows the good rapport of CE with ECL in terms of the efficiency and sensitivity needed to determine traces of antibiotics in food and biological samples. Additionally, the determination of tetracycline in crucian carp muscle using CZE-ELC was carried out by Deng et al. [61]. Tetracycline has commonly been used for the prevention and treatment of bacterial fish diseases in the last two decades because of its broad www.electrophoresis-journal.com

Electrophoresis 2014, 35, 28–49

antibacterial spectrum and cost-effectiveness, and. consequently, the MRL of tetracyclines in fish are regulated. After optimization of different electrophoretic parameters, the use of a 10 mM phosphate buffer and a separation voltage of 12 kV allowed the separation of tetracycline from the impurities in less than 7 min with good sensitivity (LOD 1.8 ng/mL). This method was successfully applied for tetracycline pharmacokinetics in crucian carp and to determine residue levels in edible muscles after a simple deproteinization of the sample. This study showed that tetracycline was still present in fish muscles in concentrations above the MRLs after 96 h of the antibiotic administration. Finally, four tetracylines (tetracycline, chlorotetracycline, oxytetracycline, and doxycycline) were determined in milk and biological samples using a nonaqueous capillary electrophoresis (NACE) method with LIF detection [11]. As mentioned before, tetracyclines are one of the most employed antibiotics in the food industry and consequently are subject to regulation. In order to reach the sensitivity needed, the combination of LIF detection and field amplified sample stacking in-capillary preconcentration method with electromigration injection was used. This on-line preconcentration step allowed improving sensitivity although resolution was lost. In order to eliminate this overlapping, 100 mM SDS in 500 mM magnesium acetate tetrahydrate in nmethylformamide was used for its ability to resolve tetracycline antibiotics. The LODs in this work ranged from 1.3 to 13.3 nM. Urine, plasma, feed, and milk samples were analyzed using the developed method after SPE extraction and precipitation of the remaining proteins. This proved its potential for the routine analysis of tetracycline residues in milk and biological samples.

4 CE with conductivity detection The capacitively coupled contactless conductivity detector (C4 D) has progressed in the last years and the number of applications of CE-C4 D has considerably increased in the field of antibiotic analysis [22,23] as it can be observed in Table 5. C4 D presents advantages as the non-necessity of light absorption by the analytes, easy handling, reduced background noise (in comparison with normal conductivity detection) and robustness. It can also be an interesting alternative for determination of antibiotics with lack of chromophore groups precluding the need of tedious and time-consuming derivatizations. However, this detection system does not always provide high sensitivity [69]. CE-C4 D has been used for the determination of different aminoglycosides [65–68], and sulfamethoxazole and trimethoprim [12] in the reviewed period. The lack of absorption properties of aminoglycosides makes CE in combination with C4 D a simple and good alternative for pharmaceutical analysis where extremely low LODs are not needed. The determination of kanamycin and related substances in pharmaceutical formulations was achieved using CZE-C4 D by el-Attug et al. [65]. Kanamycin is an aminoglycoside used  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CE and CEC

41

for the treatment of serious infections by susceptible strains. This antibiotic is produced by fermentation and its production can generate closely related substances such as paromamine. Moreover, degradation of kanamycin can rise for the formation of several compounds. In order to achieve the separation of all these compounds, a BGE consisting on a mixture of 2-(N-morpholino)ethanosulfonic acid (MES) and L-Histidine (L-His), also called “good buffer” for its low background conductivity was chosen. Cetyltrimethylammonium bromide (CTAB) was added in concentrations below the critical micellar concentration in order to improve the resolution, allowing the baseline separation of Kanamycin from six related substances in less than 6 min. This methodology also allowed the simultaneous determination of sulfate ions from kanamycin sulfate being a suitable alternative for biological assay for kanamycin, and titration for sulfate indicated in the pharmacopeia. The same research group reported a similar methodology for the determination of the purity of tobramycin, another aminoglycoside, in pharmaceutical products [66]. In this case, kanamycin b, nebramine, and neamine are the three known impurities of tobramycin. For the separation of these four compounds the MES-L-His buffer with CTAB in concentrations below the critical micellar concentration was again employed. Good selectivity and LOD in the range of mg/L was obtained for this aminoglycoside. Finally, the method was successfully applied for the determination of the purity of tobramycin in commercial pharmaceutical products after a careful validation. The same strategy was applied for the simultaneous determination of amikacin and six impurities [67]. Amikacin is an aminoglycoside used in the treatment of Gram-negative infections caused by bacteria resistant to other aminoglycosides as it has fewer target sites for enzymatic attack. Because of the structure of its precursor kanamycin, a number of similar impurities can be formed including positional isomers during the synthesis process. As a result of its good characteristics, the system MES/L-His/CTAB was employed once again as BGE for the separation of this aminoglycoside. The method was simple, fast (less than 6 min), and robust and the sensitivity obtained was good enough to determine amikacin and its impurities in commercial samples without the need of tedious derivatization steps. Amikacin was also determined using CZE-C4 D in bronchial epithelial lining fluid [68]. Amikacin is often used in neonates for the treatment of some gram-negative infections as some kinds of pulmonary infections. As its bactericidal efficacy is related to reach therapeutic levels at the infection site, the determination of amikacin in epithelial lining fluid of newborns after treatment was studied. Because the amount of urea is constant in epithelial lining fluid and in serum, it is often used as an endogenous marker to correct the dilution produced for the bronchoalveolar lavage employed for the epithelial lining fluid collection. Consequently, the simultaneous determination of amikacin and urea was carried out in this work. Urea was determined by measuring NH4 + after enzymatic conversion of urea with urease enzyme resulting from its comigration with sodium ions present in biological www.electrophoresis-journal.com

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Tobramycin and three impurities

Amikacin and six impurities

Amikacin and diverse ions

Aminoglycosides

Aminoglycosides

Aminoglycosides

Sulfamethoxazole Trimethoprim

Kanamycin and related substances

Aminoglycosides

CZE

CZE

CZE

CZE

CZE

CE mode BGE: 40 mM MES (pH 6.35) with L-His and 0.6% CTAB Capillary: 41 cm × 75 ␮m Voltage: –30 kV Temperature: 25⬚C Injection: 3.45 kPa × 5s BGE: 25 mM MES (pH 6.4) with L-His and 0.3 mM CTAB Capillary: 41 cm × 75 ␮m Voltage: –30 kV Temperature: 25⬚C Injection: 3.45 kPa × 5s BGE: 20 mM MES (pH 6.6) with L-His and 0.3% CTAB Capillary: 48 cm × 75 ␮m Voltage: –30 kV Temperature: 25⬚C Injection: 0.5 psi × 5 s BGE: 30 mM malic acid (pH 4.1) with L-arginine and 10 mM 18-crow-6 Capillary: 41 cm × 75 ␮m Voltage: 30 kV Temperature: 25⬚C Injection: 5.5 kPa × 4 s BGE: 10mM phosphate buffer (pH 7.1) Capillary: 10/39 cm × 50␮m Voltage: 30 kV Temperature: 25⬚C Injection: 5 kPa × 5 s

C4 D Excitation: 1200 kHz; Amplitude: 100 V

C4 D Excitation: 1200 kHz; Amplitude: 100 V

C4 D Excitation: 1200 kHz; Amplitude: 100 V

C4 D Excitation: 700 kHz; Amplitude: 100 V

C4 D Excitation: 1200 kHz; Amplitude: 100 V

CE conditions

Detection and conditions

Pharmaceutical formulations

Bronchial epithelial lining fluid

Pharmaceutical formulations

Pharmaceutical formulations

Pharmaceutical formulations

Sample

Extraction with MeOH and dilution in water

Centrifugation and treatment with urease

Dilution

Dilution

Dilution

Sample treatment

1.1–3.3 ␮M

0.92 mg/L

0.5 mg/L

0.4 mg/L

0.7 mg/L

LOD

[12]

[68]

[67]

[66]

[65]

Ref.

E. Dom´ınguez-Vega et al.

Sulfonamides Trimethoprim

Analyte

Antibiotic Family

Table 5. Separation and determination of antibiotics by CE with conductivity detection

42 Electrophoresis 2014, 35, 28–49

www.electrophoresis-journal.com

CE and CEC

Electrophoresis 2014, 35, 28–49

samples. 18-crown-6 ether was used as additive in the BGE to allow the separation of amikacin and NH4 + from other ions (potassium, sodium, lithium, magnesium, and calcium) present in biological fluids. Although the separation was really promising and the analysis time was really short (< 3 min) the sensitivity obtained with this method was insufficient to determine the low concentration of amikacin in bronchial epithelial lining fluid and unfortunately an alternative and more sensitive method by LC with pulsed electrochemical detection was finally employed for the clinical and pharmacokinetic studies of amikacin [70]. Finally, an interesting method was reported employing two C4 D detectors for the determination of one sulfonamide (sulfamethoxazole) and trimethoprim [12]. This combination of antibiotics is widely utilized both in human and veterinary medicine for their synergetic action on the folic acid metabolism. In this case, 10 mM lithium phosphate buffer at pH 7.1 was selected because of its high EOF that is necessary to determine sulfamethoxazole in anionic form and its low conductivity provided good signal to noise ratio. It has to be mentioned that two C4 D detectors were employed for the determination of antibiotics, one at 10 cm from the inlet of the capillary and the second one at 39 cm. Separation was possible even in the first detector (separation time

Recent advances in CE analysis of antibiotics and its use as chiral selectors.

Antibiotics are a class of therapeutic molecules widely employed in both human and veterinary medicine. This article reviews the most recent advances ...
1MB Sizes 0 Downloads 0 Views