Recent advances in the analysis of antibiotics by capillary electrophoresis

Ca men Ga cía-Ruiz Ma ía Luisa Ma ina Depa amen o de Química Analí ica, Facul ad de Química, Uni e sidad de Alcalá, Alcalá de Hena es, Mad id, Spain Recei ed June 5, 2005 Re ised July 28, 2005 Accep ed July 29, 2005 Re iew Recen ad ances in he analysis o an ibio ics by capilla y elec opho esis In his e iew, he main aspec s ela ed o he sepa a ion o di e en g oups o an ibi- o ics by CE as well as he di e en applica ions epo ed in he li e a u e om he beginning 2003 ill May 2005 will be p o ided o he eade s. Fi s ly, he expe imen al condi ions employed o achie e he analysis o an ibio ics by CE a e gi en. Then, he main applica ions pe o med in he pha maceu ical, clinical, ood, and en i onmen al ields ha e been e iewed making emphasis on sample p epa a ion equi emen s needed in each case. Finally, he main conclusions and u u e p ospec s in his ield a e p esen ed. Keywo ds: An ibio ics / Capilla y elec opho esis / Re iew DOI 10.1002/elps.200500430 1 In oduc ion The e m “an ibio ic” e e s o a e y di e se ange o chemical subs ances (p oduced om bac e ia o ungi (na u al way) o in a semisyn he ic o syn he ic way) ha possess an ibac e ial ac i i y, ha is, ha kill o inhibi he g ow h o mic oo ganisms. They a e used in human and animal medicine o p e en and ea diseases (Eu opean Medicines Agency, h p://www.emea.eu.in /; checked on May 2005) [1]. Al hough HPLC is mainly used o he analysis o an ibi- o ics by sepa a ion echniques, CE is being inc easingly employed due o i s a o able cha ac e is ics (high e i- ciency, la ge lexibili y, and low consump ion o samples and eagen s). In addi ion, CE is being used in ou ine analysis because i allows ob aining app op ia e analy i- cal cha ac e is ics and good quan i a i e esul s. The analysis o an ibio ics by CE is mainly included in wo di - e en wo king modes: (i) CZE whe e a sepa a ion bu e wi hou o wi h addi i es is used o he sepa a ion o ionic o ionogenic an ibio ics based on hei di e en elec o- pho e ic mobili ies, and (ii) MEKC whe e a micella sys em (su ac an a a concen a ion highe han i s CMC) is added o he sepa a ion bu e o pe o m he sepa a ion o neu al and/o ionic o ionogenic an ibio ics based on he gene a ion o a pseudos a iona y phase in which analy e pa i ion akes place. Al hough much less used, CEC and nonaqueous CE (NACE) ha e also been used o he analysis o an ibio ics [2]. In o de o p o ide o he eade s an upda ed iew o he sepa a ion condi ions as well as he di e en applica ions epo ed in he analysis o an ibio ics using CE as sepa a ion echnique, his e iew co e s he li e a u e dealing on he analysis o an ibio ics by CE om he beginning o 2003 ill May 2005. Li e a u e published be o e his da e on his subjec has al eady been e iewed by Flu e [2–5]. The expe imen al condi ions employed o achie e he analysis o an ibio ics by CE a e i s p e- sen ed in his a icle. Sec ion 3 o his wo k desc ibes he main applica ions pe o med in he pha maceu ical, clin- ical, ood, and en i onmen al ields making emphasis on sample p epa a ion equi emen s needed in each case. Finally, he main conclusions and u u e p ospec s in his ield a e p esen ed. 2 Analysis o an ibio ics by CE Table 1 summa izes he an ibio ics analyzed du ing he las 2.5 yea s using CE as he sepa a ion echnique. They ha e been classi ied in di e en an ibio ic g oups indica - ing hei molecula o mula, o mula weigh , CAS numbe (numbe assigned by he Chemical Abs ac Se ice (CAS) o iden i y a speci ic chemical compound), and s uc u e. In addi ion o he CE sepa a ion condi ions, he Co espondence: P o esso D . Ma ía Luisa Ma ina, Depa amen o de Química Analí ica, Facul ad de Química, Uni e sidad de Alcalá, C a. Mad id-Ba celona Km. 33.600, E-2887 Alcalá de Hena es, Mad id, Spain E-mail: [email p o ec ed] Fax: 134-91-885-4971 Abb e ia ions: LLE, liquid–liquid ex ac ion; OPA, 1,2-ph halic di- ca boxaldehyde; SLE, solid–liquid ex ac ion 266 Elec opho esis 2006, 27, 266–282 ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com Elec opho esis 2006, 27, 266–282 CE and CEC 267 Table 1. G oup, subs ance, molecula o mula, o mula weigh , CAS numbe , s uc u e, CE sepa a ion condi ions, de ec- ion sys em, and LODs o he an ibio ics included in his e iew G oup o an ibio ic Subs ance Molecula o mula ( o mula weigh )a) CAS numbe b) S uc u e CE sepa a ion condi ions De ec ion (LOD) Re e - ence â-Lac am an i- bio ics Ampicillin C16H19N3O4S (349.40) 69-53-4 40 mM phospha e- bo a e (pH 7.5) 175 mM SDS (capilla y 50 cm (57 cm)650 mm ID; 23–30 kV; 257C) UV-200 nm (0.1%) [6] 40 mM bo a e (pH 8.5) 1100 mM SDS (capilla y 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.2 mg/mL) [7] 20 mM bo a e (pH 8) 160 mM SDS (capilla y 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 bo a e (pH 8.5) 1100 mM SDS (capilla y 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.15 mg/mL) [7] 20 mM bo a e (pH 8) 160 mM SDS (capilla y 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 bo a e (pH 8.5) 1100 mM SDS (capilla y 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.3 mg/mL) [7] 10 g/L phospha e- bo a e (pH 8.7) 114.4 g/L SDS (capilla y 52 cm (60 cm)675 mm ID; 18 kV; 257C) UV-214 nm (1 mg/mL) [9] 80 mM bo a e (pH 8.0) (capilla y 60 cm675 mm ID; 15 kV; 357C) UV-185 nm (3.5 mg/mL) [10] 20 mM bo a e (pH 8) 160 mM SDS (capilla y 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.005 mg/mL) [8] ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com 268 C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2006, 27, 266–282 Table 1. Con inued G oup o an ibio ic Subs ance Molecula o mula ( o mula weigh )a) CAS numbe b) S uc u e CE sepa a ion condi ions De ec ion (LOD) Re e - ence Amoxicillin C16H19N3O5S (365.40) 26787- 78-0 5–25 mM phospha e- bo a e (capilla y 47–77 cm6 75–100 mm ID; 25 kV; 257C) UV-200 nm (0.015 mg/mL) [11] 40 mM bo a e (pH 8.5) 1100 mM SDS (capilla y 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.3 mg/mL) [7] 20 mM bo a e (pH 8) 160 mM SDS (capilla y 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) 7240- 38-2 40 mM bo a e (pH 8.5) 1100 mM SDS (capilla y 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.2 mg/mL) [7] 20 mM bo a e (pH 8) 160 mM SDS (capilla y 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 bo a e (pH 8.5) 1100 mM SDS (capilla y 50 cm (57 cm)675 mm ID; 10 kV; 207C) UV-210 nm (0.2 mg/mL) [7] 20 mM bo a e (pH 8) 160 mM SDS (capilla y 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.005 mg/mL) [8] Diclo- xacillin C19H16Cl2N3 O5SNa? H2O (492.31) 13412- 64-1 20 mM bo a e (pH 8) 160 mM SDS (capilla y 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.005 mg/mL) [8] Na cillin – – 20 mM bo a e (pH 8) 160 mM SDS (capilla y 55.5 cm (64 cm)675 mm ID; 15 kV; 257C) UV-210 nm (0.015 mg/mL) [8] ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com Elec opho esis 2006, 27, 266–282 CE and CEC 269 Table 1. Con inued G oup o an ibio ic Subs ance Molecula o mula ( o mula weigh )a) CAS numbe b) S uc u e CE sepa a ion condi ions De ec ion (LOD) Re e - ence Tica cillin C15H14N2 Na2O6S2 (428.39) 4697-14-7 20 mM phospha e- bo a e (pH 8.66) 11.44% SDS (capilla y 60 cm675 mm ID; 18 kV; 257C) UV-214 nm (1.5 mg/mL) [12] Cephalexin C16H17N3 O4S?xH2O (347.39) 15686- 71-2 20 mM bo a e (pH 9.23) 120 mM SDS 11% B ij 35 (capilla y 28 cm (50 cm)675 mm ID; 15 kV; 307C) UV-210 nm (2 mg/mL) [13] Ce azolin C14H13N8 NaO4S3 (476.49) 27164- 46-1 25 mM phospha e (pH 6.8) (capilla y 40 cm (48.5 cm)650 mm ID; 25 kV; 257C) UV-270 nm [14] Aminogly- coside an i- bio ics Kanamycin B C18H37N5O10 (581.59) 29701- 07-3 R1= –H, R2= –NH2,R 3= –OH, R4= –OH, R5= –CH2NH2,R 6= –H, R7= –H, R8= –CH2OH 30 mM bo a e (pH 10.0) 116% me hanol (capilla y 31.5 cm (40 cm)650 mm ID; 15 kV; 207C) UV-335 nm (OPA de i a- i e; ,0.6 mg/ mL) [17] Amikacin – 149022- 22-0 R1= –COCH(CH3)2CH2NH2,R 2= –OH, R3= –OH, R4= –OH, R5= –CH2NH2, R6= –H, R7= –H, R8= –CH2OH 100 mM bo a e (pH 10.0) 120 mM deoxy- chola e 115 mM b- CD (capilla y 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA de i a i e) [15] ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com 270 C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2006, 27, 266–282 Table 1. Con inued G oup o an ibio ic Subs ance Molecula o mula ( o mula weigh )a) CAS numbe b) S uc u e CE sepa a ion condi ions De ec ion (LOD) Re e - ence Tob amycin C18H37N5O9 (467.51) 32986- 56-4 R1= –H, R2= –NH2,R 3= –H, R4= –OH, R5= –CH2NH2,R 6= –H, R7= –H, R8= –CH2OH 100 mM bo a e (pH 10.0) 120 mM deoxy- chola e 115 mM b- CD (capilla y 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA de i a i e) [15] Gen amicin (C1,C 1a, C2a,C 2) – 1405- 41-0 R1= –H, R2= –NH2,R 3= –H, R4= –H, R5= –CH(NH2)CH3,R 6= –CH3,R 7= –CH3,R 8=–H 100 mM bo a e (pH 10.0) 120 mM deoxy- chola e 115 mM b- CD (capilla y 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA de i a i e) [15, 16] 1 mM ci a e (pH 3.5) 10.2 mM CTAB (capilla y 53 cm650 mm ID; 15 kV; 257C) Po en ial g adien de ec ion (,9mg/mL) [18] 60 mM CHES (pH 9.5) 131.6% me hanol (capilla y 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) 53179- 09-2 R1= –H, R2= –NH2,R 3= –H, R4= –H, R5= –CH2NH2,R 6= –CH3,R 7= –CH3, R8=–H 100 mM bo a e (pH 10.0) 120 mM deoxy- chola e 115 mM b- CD (capilla y 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA de i a i e) [15, 16] Ne ilmicin C21H41N5 O7?2.5H2 O4S (720.78) 56391- 57-2 R1=–C 2H5,R 2= –NH2,R 3= –H, R4= –H, R5= –CH2NH2,R 6= –CH3, R7= –CH3,R 8=–H 100 mM bo a e (pH 10.0) 120 mM deoxy- chola e 115 mM b- CD (capilla y 24.5 cm650 mm ID; 12 kV; 257C) UV-340 nm (OPA de i a i e) [15, 16] ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com Elec opho esis 2006, 27, 266–282 CE and CEC 271 Table 1. Con inued G oup o an ibio ic Subs ance Molecula o mula ( o mula weigh )a) CAS numbe b) S uc u e CE sepa a ion condi ions De ec ion (LOD) Re e - ence Dihyd o- s ep o- mycin C21H41N7O12? 3/2H2SO4 (730.71) 5490- 27-7 R = –CH2OH 80 mM bo a e (pH 8.0) (capilla y 60 cm675 mm ID; 15 kV; 357C) UV-185 nm (15 mg/mL) [10] S ep omy- cin 2C21H39 N7O12? (H2SO4)3 (1457.38) 3810- 74-0 R = –CHO 20 mM phospha e- bo a e (capilla y 47 cm6100 mm ID; 25 kV; 257C) UV-200 nm (0.04 mg/mL) [11] Glyco- pep ide an i- bio ics a-A opa cin (R = –H) b-A o- pa cin (R = –Cl) –– 20 mM bo a e (pH 9.2) 175 mM SDS (capilla y 50 cm (57 cm)675 mm ID; 15 kV; 307C) UV-200 nm (,0.01 mg/mL) [19] Ris o- ce in A Ris o- ce in B –– Ris obiose[ O -a-L- hamnopy anosyl- (1–6)- O -b-D-glucopy anosyl] a he han is o e ose[ O -a-D-a abino u anosyl- (1–2)- O -a-D-mannopy anosyl-(1–2)- O -a-L- hamnopy anosyl-(1–6)- O - b-D-glucopy anosyl] is a ached o ing II in Ris oce in B Vancomycin C66H75Cl2 N9O24 HCl?xH2O (1485.71) 123409- 00-7 ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com 272 C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2006, 27, 266–282 Table 1. Con inued G oup o an ibio ic Subs ance Molecula o mula ( o mula weigh )a) CAS numbe b) S uc u e CE sepa a ion condi ions De ec ion (LOD) Re e - ence Sul onamide an i- bio ics Sul ame- hazine C12H14N4 O2S (278.33) 57-68-1 35 mM phospha e (pH 6.5) (capilla y 72 cm (80.5 cm)650 mm ID; 25 kV; 257C) UV-205 nm (,0.005 mg/mL) [20] Sul ame- azine C11H12N4O2S (264.30) 127-79-7 Sul adiazine C10H10N4O2S (250.28) 68-35-9 Sul adime- hoxine C12H14N4O4S (310.33) 122-11-2 Sul amono- me hoxine C11H12N4O3S (280.30) 1220-83-3 Sul a- phenazole C15H14N4O2S (314.36) 526-08-9 Sul a- quinoaline –– Sul isoxa- zole C11H13N3O3S (267.30) 127-69-5 ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com Elec opho esis 2006, 27, 266–282 CE and CEC 273 Table 1. Con inued G oup o an ibio ic Subs ance Molecula o mula ( o mula weigh )a) CAS numbe b) S uc u e CE sepa a ion condi ions De ec ion (LOD) Re e - ence Te acycline an i- bio ics Te a- cycline C22H24N2 O8?xH2O (444.43) 60-54-8 R1= –H, R2= –CH3,R 3= –OH, R4= –H 50 mM ci ic acid (pH 2.5) (capilla y 37 cm675 mm ID; 10 kV; 257C) UV-260 nm (,0.002 mg/mL) [21] Oxy e a- cycline C22H24N2 O9?2H2O (496.46) 6153- 64-6 R1= –H, R2= –CH3,R 3= –OH, R4= –OH Doxy- cycline C22H24N2O8 HCl 1/2 (H2O) 1/2 (C2H6O) (512.94) 24390- 14-5 R1= –H, R2= –CH3,R 3= –H, R4= –OH Fluo oquino- lone an i- bio ics O loxacin C18H20FN3O4 (361.37) 82419- 36-1 R1= –CH3,R 2= –CH(CH3)CH2O– = R350 mM phospha e (pH 2.8) 140 mg/mL me hyl b-CD (capilla y 30 cm (37 cm)650 mm ID; 20 kV; 257C) UV-280 nm (0.003 mg/mL o each enan- iome ) [22] En o- loxacin C19H22FN3O3 (359.39) 93106- 60-6 R1= –CH2CH3,R 2=,R 3= –H 25 mM phospha e- bo a e (capilla y 77 cm675 mm ID; 25 kV; 257C) UV-280 nm (0.005– 0.003 mg/mL) [11] Cip o- loxacin C17H18FN3O3 (331.34) 85721- 33-1 R1= –H, R2=,R 3=–H No loxacin C16H18FN3O3 (319.33) 70458- 96-7 R1= –H, R2= –CH2CH3,R 3=–H O he an i- bio ics ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com 274 C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2006, 27, 266–282 Table 1. Con inued G oup o an ibio ic Subs ance Molecula o mula ( o mula weigh )a) CAS numbe b) S uc u e CE sepa a ion condi ions De ec ion (LOD) Re e - ence Flo phenicol C12H14Cl2 FNO4S (358.21) 73231- 34-2 R1= –SO2CH3,R 2= –COCHCl2,R 3= –F 25 mM phospha e-bo- a e (capilla y 57 cm675 mm ID; 25 kV; 257C) UV-200 nm (0.015/ 0.008 mg/ mL) [11] 50 mM bo a e (pH 9.0) 125 mM SDS (capilla y 52.5 cm (60 cm)675 mm ID; 15 kV; 207C) UV-214 nm [23] Thiam- phenicol C12H15Cl2 NO5S (356.22) 15318- 45-3 R1= –SO2CH3,R 2= –H, R3= –OH Chlo am- phenicol Cl2CHCON HCH(CH2 OH)CH (OH)C6H4 NO2 (323.13) 56-75-7 R1= –NO2,R 2= –COCHCl2,R 3= –OH 50 mM bo a e (pH 9.0) 125 mM SDS (capilla y 52.5 cm (60 cm)675 mm ID; 15 kV; 207C) UV-214 nm [23] Fos omycin C3H5O4PNa2 (182.02) 26016- 99-9 25 mM benzoic acid 10.5 mM CTAB (pH 6.95 o 8.05) (capilla y 56 cm (64.5 cm)650 mm ID; -25 kV; 257C) Indi ec UV- 254 nm (1 mg/mL) [24] B ij 35: lau yl polyoxye hylene e he . a) In o ma ion ob ained om (Sigma-Ald ich, h p://www.sigmaald ich.com; checked on May 2005 ). b) CAS numbe : he numbe assigned by he Chemical Abs ac Se ice (CAS) o iden i y speci ic chemical compounds. A chemical may ha e mo e han one CAS numbe . No all chemicals ha e an assigned CAS numbe (Cali o nia pes icide in o ma ion po al e ms and de ini ions, h p://calpip.cdp .ca.go /c docs/calpip/p od/in odocs/glossa y.c m; checked on May 2005). de ec ion condi ions employed and he LODs de e mined o he di e en an ibio ics included in his e iew a e indica ed in he able. The esul s ob ained in he analysis o an ibio ics by CE a e p esen ed as ollows, including i s he mos widely analyzed by his echnique. 2.1 â-Lac am an ibio ics b-Lac am an ibio ics can be classi ied in o se e al g oups acco ding o hei s uc u al cha ac e is ics, bu hei unique s uc u al ea u e is he p esence o he ou - membe ed b-lac am (2-aze idinone) ing. They include penicillins and cephalospo ins (also aminocillins, ca bapenems, and monobac ams) (b-lac am an ibio ics, h p://www.cic.kl e.hu/,gunda /be alaca.h m; checked on May 2005). Penicillins included in his e iew, ampi- cillin, penicillin V, penicillin G, amoxicillin, oxacillin, clox- acillin, dicloxacillin, na cillin, and ica cillin, ha e been sepa a ed using bo a e o phospha e-bo a e bu e s a basic pH usually wi h SDS micelles [6–12]. As example, Fig. 1 shows he sepa a ion o a mix u e o eigh o hese penicillins in bo a e bu e a pH 8 wi h SDS micelles in abou 20 min a e an on-column sample p econcen a- ion by s acking [8]. In addi ion, he use o UV-de ec ion a low wa eleng hs ( om 185 o 214 nm) enabled o ob ain LODs anging om 0.005 o 1.5 mg/mL (see Table 1). The cephalospo ins cephalexin and ce azolin ha e also been sepa a ed by CE. Cephalexin has been analyzed using bo a e bu e a pH 9.23 in combina ion wi h an anionic ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com Elec opho esis 2006, 27, 266–282 CE and CEC 281 www.epa.go /ne lesd1/chemis y/ppcp/images/iom-2003. pd ; checked on May 2005). The mos p e alen an ibio ics ound in he en i onmen ha e been mac olide, luo - oquinolone, and sul onamide g oups [27]. Al hough e a- cyclines o penicillins ha e been ound only in some cases and gene ally a low concen a ions, he wo appli- ca ions ound in he li e a u e in he pe iod o ime e iewed in his wo k a e ocused on he de e mina ion o hese an ibio ics in wa e samples. The sepa a ion and de e mina ion o penicillins in a m wa e samples we e pe o med by CE using bo a e bu e a pH 8.5 wi h SDS micelles and UV-de ec ion a 210 nm. In his s udy, a e y simply sample ea men was pe - o med since he wa e samples we e only il e ed be o e in oduc ion in he CE equipmen . Unde hese condi ions LODs o abou 0.2 mg/mL we e achie ed al hough, as he au ho s indica ed, hey would be imp o ed i a solid- phase concen a ion s ep is included in o he sample p epa a ion p ocess [7]. Th ee e acyclines ( e acycline, oxy e acycline, and doxycycline) we e de e mined in g oundwa e and su - ace wa e samples a e hei p econcen a ion on a STRATA-X solid-phase minicolumn inse ed in a low mani old coupled online o CE. Figu e 7 shows he elec- ophe og am co esponding o a wa e sample spiked wi h a 5 ng/mL concen a ion o each e acycline de i a- i e. This CE me hod enabled he de ec ion up o 2 ng/mL o e acyclines in wa e samples [21]. Figu e 7. Analysis by CE o a wa e sample spiked wi h 5 ng/mL o each analy e. Elec opho e ic condi ions: used-silica capilla y, ld= 37 cm and 75 mm ID; sepa a ion bu e , 50 mM ci ic acid (pH 2.5); sepa a ion empe a- u e, 257C; applied ol age, 10 kV; sample injec ion 20 psi o 10 s. UV-de ec ion a 260 nm. Peak iden i ica ion: a, e acycline; b, oxy e acycline; c, doxycycline. Re- p in ed om [21], wi h pe mission. 4 Conclusions and u u e p ospec s The sepa a ion o mix u es o an ibio ics has been suc- cess ully pe o med by CE, mainly using MEKC. The lack o sensi i i y o some classes o an ibio ics, such as aminoglycoside an ibio ics, has been o e come de i a- izing hem o o m UV-abso bing de i a i es o UV- abso bing bo a e complexes o using al e na i e de ec- ion sys ems. Al hough de ec ion by di ec UV-abso p ion o an ibio ics o hei de i a i es was used in mos o he pape s included in his e iew, he use o al e na i e de ec ion sys ems such as MS o elec ochemical de ec- ion o sol e sensi i i y p oblems o he analysis o some an ibio ics cons i u e a u u e p ospec in his ield. Al hough he sample p epa a ion equi ed o he de e - mina ion o an ibio ics in pha maceu ical p epa a ions has usually been simple, a mo e elabo a ed sample ea men be o e he injec ion in he CE sys em is usually needed o he analysis o an ibio ics in complex ma ices, i.e., in biological, ood, and en i onmen al samples. In his sense, o line sample ea men by SPE, LLE, and SLE has been pe o med. Howe e , he online coupling o sample ea men sys ems o CE seems o be e y p omising because i enables he au oma iza- ion o he analy ical p ocess, one in e es ing example being included in his e iew [21]. In addi ion, hese sample ea men p ocedu es can concen a e an ibio ic samples p io o he injec ion in he CE sys em. I is also impo an o ema k he in e es o he use o on-column sample p econcen a ion echniques based on elec o- pho e ic p inciples, such as s acking p econcen a ion, which has been achie ed e y ecen ly o a g oup o penicillins [8]. In ac , he achie emen o low de ec ion limi s is impo an o he analysis o biological, ood, and en i onmen al samples. Thus, he maximum esi- due le els o an ibio ics in oods a e in he mg/mL le el [26] and he concen a ions o an ibio ics in en i on- men al samples may ange om he mg/mL o he pg/ mL le els. The de elopmen o new applica ions o an ibio ics, especially in he en i onmen al ield whe e hei de e mi- na ion a low concen a ion le els has an inc easing in e es , is also a u u e p ospec in he analysis o an ibi- o ics by CE. Finally, ano he end in analy ical chemis y ha also a ec s he analysis o an ibio ics by CE is he ans e o CE me hods o minia u ized sys ems such as mic ochips. As an illus a i e example, Fig. 8 shows he sepa a ion o wo penicillin an ibio ics in seconds when hei de ec ion a he mic omola concen a ion was pe o med by ampe ome ic de ec ion [28]. ©2006 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com 282 C. Ga cía-Ruiz and M. L. Ma ina Elec opho esis 2006, 27, 266–282 Figu e 8. Sepa a ion o 13 mM ampicillin (AMP) and 12 mM penicillin G (PEN) by CE and pulsed ampe ome ic de ec ion. CE chip wi h channels o 50 mm wid h, 50 mm deep and sample loop 580 mm long. Sepa a ion bu e , 10 mM bo a e (pH 9.45); applied ol age, 1.7 kV; sample injec ion o 10 s. Pulsed ampe ome ic de ec ion using a po en ial o 0.5 V. Rep in ed om [28], wi h pe mission. M. L. Ma ina hanks he Minis y o Science and Technol- ogy (Spain) o he esea ch p ojec BQU2003-03638. C. Ga cía-Ruiz also hanks his Minis y o he Ramón y Cajal p og am (RYC-2003-001). 5 Re e ences [1] He nández, M., Bo ull, F., Calull, M., T ends Anal. Chem. 2003, 22, 416–427. [2] Flu e , C. L., Elec opho esis 2003, 24, 4116–4127. [3] Flu e , C. L., Elec opho esis 2001, 22, 4249–4261. [4] Flu e , C. L., Elec opho esis 1999, 20, 3269–3279. [5] Flu e , C. L., Elec opho esis 1997, 18, 2427–2437. [6] Dolez ˇalo á, M., Kun eo á, B., Jobánek, R., J. Sep. Sci. 2004, 27, 560–568. [7] Nozal, L., A ce, L., Ríos, A., Valcá cel, M., Anal. Chim. Ac a 2004, 523, 21–28. [8] Puig, P., Bo ull, F., Calull, M., Aguila , C., Elec opho esis 2005, 26, 954–961. [9] Pajchel, G., Michalska, K., Tyski, S., J. Ch oma og . 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