Recent advances in the analysis of antibiotics by capillary electrophoresis

Carmen García-Ruiz María Luisa Marina Departamento de Química Analítica, Facultad de Química, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain Received June 5, 2005 Revised July 28, 2005 Accepted July 29, 2005 Review Recent advances in the analysis of antibiotics by capillary electrophoresis In this review, the main aspects related to the separation of different groups of antibiotics by CE as well as the different applications reported in the literature from the beginning 2003 till May 2005 will be provided to the readers. Firstly, the experimental conditions employed to achieve the analysis of antibiotics by CE are given. Then, the main applications performed in the pharmaceutical, clinical, food, and environmental fields have been reviewed making emphasis on sample preparation requirements needed in each case. Finally, the main conclusions and future prospects in this field are presented. Keywords: Antibiotics / Capillary electrophoresis / Review DOI 10.1002/elps.200500430 1 Introduction The term “antibiotic” refers to a very diverse range of chemical substances (produced from bacteria or fungi (natural way) or in a semisynthetic or synthetic way) that possess antibacterial activity, that is, that kill or inhibit the growth of microorganisms. They are used in human and animal medicine to prevent and treat diseases (European Medicines Agency, http://www.emea.eu.int/; checked on May 2005) [1]. Although HPLC is mainly used for the analysis of antibiotics by separation techniques, CE is being increasingly employed due to its favorable characteristics (high efficiency, large flexibility, and low consumption of samples and reagents). In addition, CE is being used in routine analysis because it allows obtaining appropriate analytical characteristics and good quantitative results. The analysis of antibiotics by CE is mainly included in two different working modes: (i) CZE where a separation buffer without or with additives is used for the separation of ionic or ionogenic antibiotics based on their different electrophoretic mobilities, and (ii) MEKC where a micellar system (surfactant at a concentration higher than its CMC) is added to the separation buffer to perform the separation of neutral and/or ionic or ionogenic antibiotics based on the generation of a pseudostationary phase in which analyte partition takes place. Although much less used, CEC and nonaqueous CE (NACE) have also been used for the analysis of antibiotics [2]. In order to provide to the readers an updated view of the separation conditions as well as the different applications reported in the analysis of antibiotics using CE as separation technique, this review covers the literature dealing on the analysis of antibiotics by CE from the beginning of 2003 till May 2005. Literature published before this date on this subject has already been reviewed by Flurer [2–5]. The experimental conditions employed to achieve the analysis of antibiotics by CE are first presented in this article. Section 3 of this work describes the main applications performed in the pharmaceutical, clinical, food, and environmental fields making emphasis on sample preparation requirements needed in each case. Finally, the main conclusions and future prospects in this field are presented. 2 Analysis of antibiotics by CE Table 1 summarizes the antibiotics analyzed during the last 2.5 years using CE as the separation technique. They have been classified in different antibiotic groups indicating their molecular formula, formula weight, CAS number (number assigned by the Chemical Abstract Service (CAS) to identify a specific chemical compound), and structure. In addition to the CE separation conditions, the Correspondence: Professor Dr. María Luisa Marina, Departamento de Química Analítica, Facultad de Química, Universidad de Alcalá, Ctra. Madrid-Barcelona Km. 33.600, E-2887 Alcalá de Henares, Madrid, Spain E-mail: [email protected] Fax: 134-91-885-4971 Abbreviations: LLE, liquid–liquid extraction; OPA, 1,2-phthalic dicarboxaldehyde; SLE, solid–liquid extraction 266 Electrophoresis 2006, 27, 266–282 ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2006, 27, 266–282 CE and CEC 267 Table 1. Group, substance, molecular formula, formula weight, CAS number, structure, CE separation conditions, detection system, and LODs of the antibiotics included in this review Group of antibiotic Substance Molecular formula (formula weight)a) CAS numberb) Structure CE separation conditions Detection (LOD) Reference â-Lactam antibiotics Ampicillin C16H19N3O4S (349.40) 69-53-4 40 mM phosphateborate (pH 7.5) 175 mM SDS (capillary 50 cm (57 cm)650 mm ID; 23–30 kV; 257C) UV-200 nm (0.1%) [6] 40 mM borate (pH 8.5) 1100 mM SDS (capillary 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.2 mg/mL) [7] 20 mM borate (pH 8) 160 mM SDS (capillary 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.015 mg/mL) [8] Penicillin V C16H17N2O5SK (388.48) 132-98-9 40 mM borate (pH 8.5) 1100 mM SDS (capillary 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.15 mg/mL) [7] 20 mM borate (pH 8) 160 mM SDS (capillary 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.005 mg/mL) [8] Penicillin G C16H17KN2O4S (372.48) 113-98-4 40 mM borate (pH 8.5) 1100 mM SDS (capillary 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.3 mg/mL) [7] 10 g/L phosphateborate (pH 8.7) 114.4 g/L SDS (capillary 52 cm (60 cm)675 mm ID; 18 kV; 257C) UV-214 nm (1 mg/mL) [9] 80 mM borate (pH 8.0) (capillary 60 cm675 mm ID; 15 kV; 357C) UV-185 nm (3.5 mg/mL) [10] 20 mM borate (pH 8) 160 mM SDS (capillary 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.005 mg/mL) [8] ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com 268 C. García-Ruiz and M. L. Marina Electrophoresis 2006, 27, 266–282 Table 1. Continued Group of antibiotic Substance Molecular formula (formula weight)a) CAS numberb) Structure CE separation conditions Detection (LOD) Reference Amoxicillin C16H19N3O5S (365.40) 2678778-0 5–25 mM phosphateborate (capillary 47–77 cm6 75–100 mm ID; 25 kV; 257C) UV-200 nm (0.015 mg/mL) [11] 40 mM borate (pH 8.5) 1100 mM SDS (capillary 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.3 mg/mL) [7] 20 mM borate (pH 8) 160 mM SDS (capillary 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.025 mg/mL) [8] Oxacillin C19H18N3 NaO5S? H2O (441.43) 724038-2 40 mM borate (pH 8.5) 1100 mM SDS (capillary 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.2 mg/mL) [7] 20 mM borate (pH 8) 160 mM SDS (capillary 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.005 mg/mL) [8] Cloxacillin C19H17ClN3 NaO5S (457.86) 642-78-4 40 mM borate (pH 8.5) 1100 mM SDS (capillary 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.2 mg/mL) [7] 20 mM borate (pH 8) 160 mM SDS (capillary 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.005 mg/mL) [8] Dicloxacillin C19H16Cl2N3 O5SNa? H2O (492.31) 1341264-1 20 mM borate (pH 8) 160 mM SDS (capillary 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.005 mg/mL) [8] Nafcillin – – 20 mM borate (pH 8) 160 mM SDS (capillary 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.015 mg/mL) [8] ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2006, 27, 266–282 CE and CEC 269 Table 1. Continued Group of antibiotic Substance Molecular formula (formula weight)a) CAS numberb) Structure CE separation conditions Detection (LOD) Reference Ticarcillin C15H14N2 Na2O6S2 (428.39) 4697-14-7 20 mM phosphateborate (pH 8.66) 11.44% SDS (capillary 60 cm675 mm ID; 18 kV; 257C) UV-214 nm (1.5 mg/mL) [12] Cephalexin C16H17N3 O4S?xH2O (347.39) 1568671-2 20 mM borate (pH 9.23) 120 mM SDS 11% Brij 35 (capillary 28 cm (50 cm)675 mm ID; 15 kV; 307C) UV-210 nm (2 mg/mL) [13] Cefazolin C14H13N8 NaO4S3 (476.49) 2716446-1 25 mM phosphate (pH 6.8) (capillary 40 cm (48.5 cm)650 mm ID; 25 kV; 257C) UV-270 nm [14] Aminoglycoside antibiotics Kanamycin B C18H37N5O10 (581.59) 2970107-3 R1= –H, R2= –NH2,R 3= –OH, R4= –OH, R5= –CH2NH2,R 6= –H, R7= –H, R8= –CH2OH 30 mM borate (pH 10.0) 116% methanol (capillary 31.5 cm (40 cm)650 mm ID; 15 kV; 207C) UV-335 nm (OPA derivative; ,0.6 mg/ mL) [17] Amikacin – 14902222-0 R1= –COCH(CH3)2CH2NH2,R 2= –OH, R3= –OH, R4= –OH, R5= –CH2NH2, R6= –H, R7= –H, R8= –CH2OH 100 mM borate (pH 10.0) 120 mM deoxycholate 115 mM bCD (capillary 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA derivative) [15] ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com 270 C. García-Ruiz and M. L. Marina Electrophoresis 2006, 27, 266–282 Table 1. Continued Group of antibiotic Substance Molecular formula (formula weight)a) CAS numberb) Structure CE separation conditions Detection (LOD) Reference Tobramycin C18H37N5O9 (467.51) 3298656-4 R1= –H, R2= –NH2,R 3= –H, R4= –OH, R5= –CH2NH2,R 6= –H, R7= –H, R8= –CH2OH 100 mM borate (pH 10.0) 120 mM deoxycholate 115 mM bCD (capillary 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA derivative) [15] Gentamicin (C1,C 1a, C2a,C 2) – 140541-0 R1= –H, R2= –NH2,R 3= –H, R4= –H, R5= –CH(NH2)CH3,R 6= –CH3,R 7= –CH3,R 8=–H 100 mM borate (pH 10.0) 120 mM deoxycholate 115 mM bCD (capillary 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA derivative) [15, 16] 1 mM citrate (pH 3.5) 10.2 mM CTAB (capillary 53 cm650 mm ID; 15 kV; 257C) Potential gradient detection (,9mg/mL) [18] 60 mM CHES (pH 9.5) 131.6% methanol (capillary 40 cm (50.2 cm)675 mm ID; 23 kV; 207C) UV-230 nm (,0.0001 mg/L) [25] Sisomicin 2C19H37 N5O7? 5H2O4S (1385.45) 5317909-2 R1= –H, R2= –NH2,R 3= –H, R4= –H, R5= –CH2NH2,R 6= –CH3,R 7= –CH3, R8=–H 100 mM borate (pH 10.0) 120 mM deoxycholate 115 mM bCD (capillary 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA derivative) [15, 16] Netilmicin C21H41N5 O7?2.5H2 O4S (720.78) 5639157-2 R1=–C 2H5,R 2= –NH2,R 3= –H, R4= –H, R5= –CH2NH2,R 6= –CH3, R7= –CH3,R 8=–H 100 mM borate (pH 10.0) 120 mM deoxycholate 115 mM bCD (capillary 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA derivative) [15, 16] ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2006, 27, 266–282 CE and CEC 271 Table 1. Continued Group of antibiotic Substance Molecular formula (formula weight)a) CAS numberb) Structure CE separation conditions Detection (LOD) Reference Dihydrostreptomycin C21H41N7O12? 3/2H2SO4 (730.71) 549027-7 R = –CH2OH 80 mM borate (pH 8.0) (capillary 60 cm675 mm ID; 15 kV; 357C) UV-185 nm (15 mg/mL) [10] Streptomycin 2C21H39 N7O12? (H2SO4)3 (1457.38) 381074-0 R = –CHO 20 mM phosphateborate (capillary 47 cm6100 mm ID; 25 kV; 257C) UV-200 nm (0.04 mg/mL) [11] Glycopeptide antibiotics a-Avoparcin (R = –H) b-Avoparcin (R = –Cl) –– 20 mM borate (pH 9.2) 175 mM SDS (capillary 50 cm (57 cm)675 mm ID; 15 kV; 307C) UV-200 nm (,0.01 mg/mL) [19] Ristocetin A Ristocetin B –– Ristobiose[ O -a-L-rhamnopyranosyl- (1–6)- O -b-D-glucopyranosyl] rather than ristotetrose[ O -a-D-arabinofuranosyl- (1–2)- O -a-D-mannopyranosyl-(1–2)- O -a-L-rhamnopyranosyl-(1–6)- O - b-D-glucopyranosyl] is attached to ring II in Ristocetin B Vancomycin C66H75Cl2 N9O24 HCl?xH2O (1485.71) 12340900-7 ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com 272 C. García-Ruiz and M. L. Marina Electrophoresis 2006, 27, 266–282 Table 1. Continued Group of antibiotic Substance Molecular formula (formula weight)a) CAS numberb) Structure CE separation conditions Detection (LOD) Reference Sulfonamide antibiotics Sulfamethazine C12H14N4 O2S (278.33) 57-68-1 35 mM phosphate (pH 6.5) (capillary 72 cm (80.5 cm)650 mm ID; 25 kV; 257C) UV-205 nm (,0.005 mg/mL) [20] Sulfamerazine C11H12N4O2S (264.30) 127-79-7 Sulfadiazine C10H10N4O2S (250.28) 68-35-9 Sulfadimethoxine C12H14N4O4S (310.33) 122-11-2 Sulfamonomethoxine C11H12N4O3S (280.30) 1220-83-3 Sulfaphenazole C15H14N4O2S (314.36) 526-08-9 Sulfaquinoaline –– Sulfisoxazole C11H13N3O3S (267.30) 127-69-5 ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2006, 27, 266–282 CE and CEC 273 Table 1. Continued Group of antibiotic Substance Molecular formula (formula weight)a) CAS numberb) Structure CE separation conditions Detection (LOD) Reference Tetracycline antibiotics Tetracycline C22H24N2 O8?xH2O (444.43) 60-54-8 R1= –H, R2= –CH3,R 3= –OH, R4= –H 50 mM citric acid (pH 2.5) (capillary 37 cm675 mm ID; 10 kV; 257C) UV-260 nm (,0.002 mg/mL) [21] Oxytetracycline C22H24N2 O9?2H2O (496.46) 615364-6 R1= –H, R2= –CH3,R 3= –OH, R4= –OH Doxycycline C22H24N2O8 HCl 1/2 (H2O) 1/2 (C2H6O) (512.94) 2439014-5 R1= –H, R2= –CH3,R 3= –H, R4= –OH Fluoroquinolone antibiotics Ofloxacin C18H20FN3O4 (361.37) 8241936-1 R1= –CH3,R 2= –CH(CH3)CH2O– = R350 mM phosphate (pH 2.8) 140 mg/mL methyl b-CD (capillary 30 cm (37 cm)650 mm ID; 20 kV; 257C) UV-280 nm (0.003 mg/mL of each enantiomer) [22] Enrofloxacin C19H22FN3O3 (359.39) 9310660-6 R1= –CH2CH3,R 2=,R 3= –H 25 mM phosphateborate (capillary 77 cm675 mm ID; 25 kV; 257C) UV-280 nm (0.005– 0.003 mg/mL) [11] Ciprofloxacin C17H18FN3O3 (331.34) 8572133-1 R1= –H, R2=,R 3=–H Norfloxacin C16H18FN3O3 (319.33) 7045896-7 R1= –H, R2= –CH2CH3,R 3=–H Other antibiotics ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com 274 C. García-Ruiz and M. L. Marina Electrophoresis 2006, 27, 266–282 Table 1. Continued Group of antibiotic Substance Molecular formula (formula weight)a) CAS numberb) Structure CE separation conditions Detection (LOD) Reference Florphenicol C12H14Cl2 FNO4S (358.21) 7323134-2 R1= –SO2CH3,R 2= –COCHCl2,R 3= –F 25 mM phosphate-borate (capillary 57 cm675 mm ID; 25 kV; 257C) UV-200 nm (0.015/ 0.008 mg/ mL) [11] 50 mM borate (pH 9.0) 125 mM SDS (capillary 52.5 cm (60 cm)675 mm ID; 15 kV; 207C) UV-214 nm [23] Thiamphenicol C12H15Cl2 NO5S (356.22) 1531845-3 R1= –SO2CH3,R 2= –H, R3= –OH Chloramphenicol Cl2CHCON HCH(CH2 OH)CH (OH)C6H4 NO2 (323.13) 56-75-7 R1= –NO2,R 2= –COCHCl2,R 3= –OH 50 mM borate (pH 9.0) 125 mM SDS (capillary 52.5 cm (60 cm)675 mm ID; 15 kV; 207C) UV-214 nm [23] Fosfomycin C3H5O4PNa2 (182.02) 2601699-9 25 mM benzoic acid 10.5 mM CTAB (pH 6.95 or 8.05) (capillary 56 cm (64.5 cm)650 mm ID; -25 kV; 257C) Indirect UV254 nm (1 mg/mL) [24] Brij 35: lauryl polyoxyethylene ether. a) Information obtained from (Sigma-Aldrich, http://www.sigmaaldrich.com; checked on May 2005 ). b) CAS number: the number assigned by the Chemical Abstract Service (CAS) to identify specific chemical compounds. A chemical may have more than one CAS number. Not all chemicals have an assigned CAS number (California pesticide information portal terms and definitions, http://calpip.cdpr.ca.gov/cfdocs/calpip/prod/infodocs/glossary.cfm; checked on May 2005). detection conditions employed and the LODs determined for the different antibiotics included in this review are indicated in the table. The results obtained in the analysis of antibiotics by CE are presented as follows, including first the most widely analyzed by this technique. 2.1 â-Lactam antibiotics b-Lactam antibiotics can be classified into several groups according to their structural characteristics, but their unique structural feature is the presence of the fourmembered b-lactam (2-azetidinone) ring. They include penicillins and cephalosporins (also aminocillins, carbapenems, and monobactams) (b-lactam antibiotics, http://www.cic.klte.hu/,gundat/betalaca.htm; checked on May 2005). Penicillins included in this review, ampicillin, penicillin V, penicillin G, amoxicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, and ticarcillin, have been separated using borate or phosphate-borate buffers at basic pH usually with SDS micelles [6–12]. As example, Fig. 1 shows the separation of a mixture of eight of these penicillins in borate buffer at pH 8 with SDS micelles in about 20 min after an on-column sample preconcentration by stacking [8]. In addition, the use of UV-detection at low wavelengths (from 185 to 214 nm) enabled to obtain LODs ranging from 0.005 to 1.5 mg/mL (see Table 1). The cephalosporins cephalexin and cefazolin have also been separated by CE. Cephalexin has been analyzed using borate buffer at pH 9.23 in combination with an anionic ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2006, 27, 266–282 CE and CEC 281 www.epa.gov/nerlesd1/chemistry/ppcp/images/iom-2003. pdf; checked on May 2005). The most prevalent antibiotics found in the environment have been macrolide, fluoroquinolone, and sulfonamide groups [27]. Although tetracyclines or penicillins have been found only in some cases and generally at low concentrations, the two applications found in the literature in the period of time reviewed in this work are focused on the determination of these antibiotics in water samples. The separation and determination of penicillins in farm water samples were performed by CE using borate buffer at pH 8.5 with SDS micelles and UV-detection at 210 nm. In this study, a very simply sample treatment was performed since the water samples were only filtered before introduction in the CE equipment. Under these conditions LODs of about 0.2 mg/mL were achieved although, as the authors indicated, they would be improved if a solidphase concentration step is included into the sample preparation process [7]. Three tetracyclines (tetracycline, oxytetracycline, and doxycycline) were determined in groundwater and surface water samples after their preconcentration on a STRATA-X solid-phase minicolumn inserted in a flow manifold coupled online to CE. Figure 7 shows the electropherogram corresponding to a water sample spiked with a 5 ng/mL concentration of each tetracycline derivative. This CE method enabled the detection up to 2 ng/mL of tetracyclines in water samples [21]. Figure 7. Analysis by CE of a water sample spiked with 5 ng/mL of each analyte. Electrophoretic conditions: fused-silica capillary, ld= 37 cm and 75 mm ID; separation buffer, 50 mM citric acid (pH 2.5); separation temperature, 257C; applied voltage, 10 kV; sample injection 20 psi for 10 s. UV-detection at 260 nm. Peak identification: a, tetracycline; b, oxytetracycline; c, doxycycline. Reprinted from [21], with permission. 4 Conclusions and future prospects The separation of mixtures of antibiotics has been successfully performed by CE, mainly using MEKC. The lack of sensitivity for some classes of antibiotics, such as aminoglycoside antibiotics, has been overcome derivatizing them to form UV-absorbing derivatives or UVabsorbing borate complexes or using alternative detection systems. Although detection by direct UV-absorption of antibiotics or their derivatives was used in most of the papers included in this review, the use of alternative detection systems such as MS or electrochemical detection to solve sensitivity problems for the analysis of some antibiotics constitute a future prospect in this field. Although the sample preparation required for the determination of antibiotics in pharmaceutical preparations has usually been simple, a more elaborated sample treatment before the injection in the CE system is usually needed for the analysis of antibiotics in complex matrices, i.e., in biological, food, and environmental samples. In this sense, offline sample treatment by SPE, LLE, and SLE has been performed. However, the online coupling of sample treatment systems to CE seems to be very promising because it enables the automatization of the analytical process, one interesting example being included in this review [21]. In addition, these sample treatment procedures can concentrate antibiotic samples prior to the injection in the CE system. It is also important to remark the interest of the use of on-column sample preconcentration techniques based on electrophoretic principles, such as stacking preconcentration, which has been achieved very recently for a group of penicillins [8]. In fact, the achievement of low detection limits is important for the analysis of biological, food, and environmental samples. Thus, the maximum residue levels of antibiotics in foods are in the mg/mL level [26] and the concentrations of antibiotics in environmental samples may range from the mg/mL to the pg/ mL levels. The development of new applications for antibiotics, especially in the environmental field where their determination at low concentration levels has an increasing interest, is also a future prospect in the analysis of antibiotics by CE. Finally, another trend in analytical chemistry that also affects the analysis of antibiotics by CE is the transfer of CE methods to miniaturized systems such as microchips. As an illustrative example, Fig. 8 shows the separation of two penicillin antibiotics in seconds when their detection at the micromolar concentration was performed by amperometric detection [28]. ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com 282 C. García-Ruiz and M. L. Marina Electrophoresis 2006, 27, 266–282 Figure 8. Separation of 13 mM ampicillin (AMP) and 12 mM penicillin G (PEN) by CE and pulsed amperometric detection. CE chip with channels of 50 mm width, 50 mm deep and sample loop 580 mm long. Separation buffer, 10 mM borate (pH 9.45); applied voltage, 1.7 kV; sample injection for 10 s. Pulsed amperometric detection using a potential of 0.5 V. Reprinted from [28], with permission. M. L. Marina thanks the Ministry of Science and Technology (Spain) for the research project BQU2003-03638. C. García-Ruiz also thanks this Ministry for the Ramón y Cajal program (RYC-2003-001). 5 References [1] Hernández, M., Borrull, F., Calull, M., Trends Anal. Chem. 2003, 22, 416–427. [2] Flurer, C. L., Electrophoresis 2003, 24, 4116–4127. [3] Flurer, C. L., Electrophoresis 2001, 22, 4249–4261. [4] Flurer, C. L., Electrophoresis 1999, 20, 3269–3279. [5] Flurer, C. L., Electrophoresis 1997, 18, 2427–2437. [6] Dolez ˇalová, M., Kunteová, B., Jobánek, R., J. Sep. Sci. 2004, 27, 560–568. [7] Nozal, L., Arce, L., Ríos, A., Valcárcel, M., Anal. Chim. Acta 2004, 523, 21–28. [8] Puig, P., Borrull, F., Calull, M., Aguilar, C., Electrophoresis 2005, 26, 954–961. [9] Pajchel, G., Michalska, K., Tyski, S., J. Chromatogr. A 2004, 1032, 265–272. [10] Michalska, K., Pajchel, G., Tyski, S., J. Chromatogr. B 2004, 800, 203–209. [11] Kowalski, P., Oledzka, I., Lamparczyk, H., J. Pharm. Biomed. Anal. 2003, 32, 937–947. [12] Pajchel, G., Tyski, S., J. Pharm. Biomed. Anal. 2003, 32, 59– 69. [13] Steppe, M., Prado, M. S. A., Tavares, M. F. M., Pinto, T. J. A., Kedor-Hackmann, E. R. M., Santoro, M. I. R. M., J. AOAC Int. 2003, 86, 707–713. [14] Klekner, A., Ga’spa’r, A., Kardos, S., Szabó, J., Cse’csei, G., J. Neurosurg. Anesthesiol. 2003, 15, 249–254. [15] Wienen, F., Holzgrabe, U., Electrophoresis 2003, 24, 2948– 2957. [16] Deubner, R., Holzgrabe, U., J. Pharm. Biomed. Anal. 2004, 35, 459–467. [17] Kaale, E., Van Schepdael, A., Roets, E., Hoogmartens, J., Electrophoresis 2003, 24, 1119–1125. [18] Yuan, L. L., Wei, H. P., Li, S. F. Y., Electrophoresis 2005, 26, 196–201. [19] Lucas, C., Foley, J. P., Ahuja, E. S., Biomed. Chromatogr. 2003, 17, 172–181. [20] Fuh, M.-R. S., Chu, S.-Y., Anal. Chim. Acta 2003, 499, 215– 221. [21] Nozal, L., Arce, L., Simonet, B. M., Ríos, A., Valcárcel, M., Anal. Chim. Acta 2004, 517, 89–94. [22] Awadallah, B., Schmidt, P. C., Wahl, M. A., J. Chromatogr. A 2003, 988, 135–143. [23] Hillaert, S., Van den Bossche, W., J. Pharm. Biomed. Anal. 2004, 36, 437–440. [24] Petsch, M., Mayer-Helm, B. X., Sauermann, R., Joukhadar, C., Kenndler, E., Electrophoresis 2004, 25, 2292–2298. [25] Kaale, E., Long, Y. H., Fonge, H. A., Govaerts, C., Desmet, K., Van Schepdael, A., Hoogmartens, J., Electrophoresis 2005, 26, 640–647. [26] EEC Establishment of maximum residue levels of veterinary medical products in foodstuffs of animal origin, Council Regulation No. 2377/90 of EEC. [27] Beausse, J., Trends Anal. Chem. 2004, 23, 753–761. [28] García, C. D., Henry, C. S., Anal. Chem. 2003, 75, 4778– 4783. ©2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com