advances in the determination of β-lactam antibiotics by liquid chromatography

Trends Trends in Analytical Chemistry, Vol. 38, 2012

Advances in the determinationof b-lactam antibiotics by liquidchromatographyFrancisco J. Lara, Monsalud del Olmo-Iruela, Carmen Cruces-Blanco,

Carolina Quesada-Molina, Ana M. Garcıa-Campana

b-lactam antibiotics have been the most widely used antimicrobial drugs for more than 80 years and still constitute the most

important group of antibiotics. Their extensive use in veterinary medicine practices as growth promoters, and chemotherapeutic

and/or prophylactic agents causes numerous residues in foodstuffs which present a serious health hazard. For this reason,

sensitive and specific methods for the quantification of these compounds in numerous matrices have been published.

This article reviews the current status of the application of liquid-chromatography-based analytical methods in the analysis of

b-lactam antibiotics. These methods are classified according to the different detection systems used: UV-visible spectropho-

tometry, mass spectrometry and other detection techniques such as fluorescence or chemiluminescence. We include applications

in different fields (e.g., food, environmental, clinical and pharmaceutical).

ª 2012 Elsevier Ltd. All rights reserved.

Keywords: Antibiotic; b-lactam; Cephalosporin; Clinical analysis; Environmental analysis; Food analysis; High-performance liquid chromatography

(HPLC); Mass spectrometry (MS); Penicillin; Pharmaceutical analysis

Francisco J. Lara,

Monsalud del Olmo-Iruela,

Carmen Cruces-Blanco,

Carolina Quesada-Molina,

Ana M. Garcıa-Campana*

Department of Analytical

Chemistry, Faculty of Sciences,

University of Granada,

18071 Granada, Spain

*Corresponding author.

Fax: +34 958249510;

E-mail: [email protected]

52

1. Introduction

The term antibiotic refers to a very diverserange of chemical substances producedfrom bacteria or fungi in a natural, semi-synthetic or synthetic way that possessantibacterial activity, by killing or inhib-iting the growth of microorganisms. Theyare used in human and animal medicineto prevent and treat diseases. Among thedifferent types, b-lactam antibiotics have along history in the treatment of infectiousdiseases, though their use was and con-tinues to be confounded by the develop-ment of resistance in target organisms. Fornearly six decades, penicillins have beenwidely used to treat bacterial infections.However, the development of multidrugresistance has reduced the effectiveness ofb-lactams and antimicrobial drugs. Thistype of antibiotics can be classified intoseveral groups according to their struc-tural characteristics, but their uniquestructural feature is the presence of thefour-membered b-lactam (2-azetidinone)ring. They include: penicillins, cephalo-

0165-9936/$ - see front matter ª 2012 Elsevier Ltd. All rights

sporins, and more recently, carbapenems(Fig. 1).

Penicillins work against bacterial infec-tions, inhibiting the formation of the cellwall in susceptible bacteria. The basicstructure of penicillins is a thiazolidizinering connected to a b-lactam ring, towhich a side chain is attached. The ceph-alosporin ring structure is derived from7-aminocephalosporinic acid (7-ACA)while the penicillins are derived from 6-aminopenicillanic acid (6-APA). Carba-penems are compounds fairly recentlyadded to b-lactams, and are used againstserious infectious diseases or methicillin-resistant Staphylococcus aureus.

In the past few years, public concernabout the utilization of antibiotics in food-producing animals and their misuse inhumans has increased due to the transfer ofantibiotic-resistant bacteria to man. In thissense, the control of antibiotic residues inedible animal tissues is mandatory. TheEuropean Union (EU) Directive (96/23/CE)[1] established the control measures andthe alert plans to be applied for the detec-tion of special substances and their

reserved. doi:http://dx.doi.org/10.1016/j.trac.2012.03.020

mailto:[email protected]

http://dx.doi.org/10.1016/j.trac.2012.03.020

(A)

N

SCH3

CH3

COOH

HHN

O

R

O

(B)

N

S

O R1

COOH

HHN

O

R2

(C)

N

H

O

R1

COOH

R3

R2

Figure 1. b-lactam chemical structures: (A) penicillins, (B) cephalosporins, (C) carbapenems.

Trends in Analytical Chemistry, Vol. 38, 2012 Trends

residues, potentially toxic for the consumer, in live ani-mals or products of animal origin used in human feeding.Also, maximum residue limits (MRLs) of antibiotics infoodstuffs of animal origin [e.g., multiple animal tissues(e.g., muscle, liver, kidney, fat), milk or eggs] were re-cently revised by the Commission Regulation (EU) No 37/2010 for safe consumption [2]. Technical guidelines andperformance characteristics (e.g., detection level, selec-tivity and specificity for residue control in the frameworkof Directive 96/23/EC) are described in CommissionDecision 2002/657/EC [3], including additional require-ments for confirmatory methods by introducing the con-cept of identification points (IPs) in order to achieveunambiguous identification of the legislated residues thatare monitored.

However, personal-care products and pharmaceuticalsubstances, including antibiotics, have attracted fast-growing interest as emerging pollutants, and recentstudies have shown that a multitude of drugs are presentin aquatic systems. It is important to consider that thelarge amount of antibiotics consumed by humans and

animals, which are continually introduced into theenvironment make them potential pollutants that arecome from a variety of sources, including dischargesfrom domestic wastewater-treatment plants (WWTPs)and pharmaceutical companies, run-off from animal-feeding operations, infiltration from aquaculture activi-ties or from compost made of animal manure containingantibiotics [4,5]. However, these antimicrobials have notbeen included in the list of priority and hazardous sub-stances in the Water Framework Directive of theEuropean Union [6].

The low concentration levels and the complexity ofnumerous matrices [e.g., groundwater and surface-watersamples, foods of animal origin, biological fluids (e.g.,blood and plasma from human or animal origin)] make itnecessary to use highly sensitive, selective methods fordetermination of b-lactam antibiotics. High-performanceliquid chromatography (HPLC) has been widely used forthe analysis of antibiotics in food and environmentalsamples, mainly in combination with mass spectrometry(MS), as has been clearly stated in some general reviews.

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These reviews included b-lactams [5,7,8] and specificallyabout analytical methodologies for these compounds[9,10].

The present review gives an overview of existinganalytical methods using LC. We consider differentdetection techniques and sample treatment, as well asapplications in different fields (e.g., clinical, pharma-ceutical, food and environmental analysis).

2. Analysis of b-lactam antibiotics by HPLC with UVdetection

2.1. Food analysisUV detection by using diode-array detection (DAD) is avery popular technique coupled with HPLC but it hassome limitations in determining b-lactams (e.g., lowsensitivity due to the lack of chromophores). Moreover,HPLC-UV is not completely reliable for confirmation, noteven using DAD. That is why HPLC-MS is the preferredtechnique for identification and quantitation of b-lac-tams in foodstuffs.

In monitoring residues of b-lactam antibiotics in foodderived from animals, considering the low MRLs estab-lished by international legislation, it is necessary to applypreconcentration steps in sample treatment to reachlimits of detection (LODs) close to or below the MRLs.Table 1 shows a summary of the proposed methods foranalysis of these compounds in different matrixes usingHPLC-UV detection.

Because b-lactam antibiotics are usually employed intreating mastitis in cows, milk is the most frequentlyanalyzed sample [11], but, once the chromatographicseparation has been developed, sample treatments can beadjusted and optimized for different types of sample. Forexample, a capillary HPLC method has been proposed forthe simultaneous determination of 10 b-lactams in foodsof animal origin (e.g., milk or chicken muscle) [12].LODs for chicken muscle and milk samples were belowthe legislated MRLs, established as 25–300 lg/kg forchicken muscle and 4–200 lg/L for milk. CapillaryHPLC shows several advantages compared to analyticalHPLC, such as better resolution, lower LOD and lowersolvent consumption, being more environmentallyfriendly than conventional HPLC. Fig. 2 shows the sep-aration of 10 b-lactams in 15 min using this technique.

Recently, ultrasound-assisted matrix-solid-phase dis-persion (MSPD) was applied to isolate eight cephalospo-rins from milk [13]. Extraction yield using this strategywith Nexus polymeric sorbent was higher than otherapplied solid-phase extraction (SPE) procedures for thesame matrix. High recoveries can also be obtained whenPENG, OXA and CLOX are extracted from beef and milkusing ion-paired extraction (IPE) with tetrabutylammo-nium bromide as ion-pairing agent and binary water–acetonitrile as extractant [14].

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The evolution of column technology has led to sub-2-lm particle sizes being used for ultra-high performancechromatography (UHPLC). Compared to LC, UHPLCimproves efficiency, resolution and sensitivity, and sig-nificantly reduces sample-analysis time and mobile-phase solvent consumption. This superior performancerequires expensive pumps capable of operating at pres-sures up to 1200 bar.

An interesting option not needing new instrumenta-tion is the use of analytical columns with core-shelltechnology. These columns are packed with 2.6 lmcore-shell particles that consist of a solid core (1.9 lm)and a porous shell. As a result, shorter diffusion pathsare obtained, allowing high efficiency even at high flowrates [15,16]. They are available with different chemis-tries: phenyl-hexyl, C18, C8, pentafluorophenyl (PFP)and HILIC. This technology has been used to determinepenicillins and amphenicols in milk with sample treat-ment based on MSPD and QuEChERS (Quick, Easy,Cheap, Effective, Rugged, and Safe) [17].

2.2. Environmental analysisIn water analysis, considering the extremely low level ofresidues, the large volumes used in the preconcentrationsteps, usually by SPE, increase total analysis time. In thissense, the selection of the sorbents and the volume ofsample to be processed are crucial. For example, differenttypes of wastewaters [industrial, influent and effluentsewage-treatment plant (STP)] have been analyzed forsimultaneous determination of different b-lactam anti-biotics [18]. Two SPE cartridges were compared forsample clean-up and preconcentration:(1) a reversed-phase silica-based cartridge (Bond Elut

C18, Varian Inc.); and,(2) a strong polymeric mixed mode anion exchanger

(Oasis MAX, Waters).The matrix components in industrial and urban

wastewater samples reduced the preconcentration effi-ciency in both sorbents, especially for the Bond Elut C18.

Capillary HPLC also demonstrated its usefulness in theanalysis of different samples of river and well waters[12]. In this case, 250 mL of water were used in thepreconcentration step, using Oasis HLB cartridges.

Molecularly-imprinted polymer (MIP) applied to SPE(MISPE) can also be an interesting option to achieveselective extraction of the target compound when thecommonly applied sorbents lack selectivity. A MISPEprocedure has been developed for the selective precon-centration of different b-lactam antibiotics in environ-mental waters, by using a MIP imprinted with penicillinG [19]. Various parameters affecting the extraction effi-ciency of the polymer have been evaluated to achieve theselective preconcentration of the antibiotics from aque-ous samples and to reduce non-specific interactions. Thisresulted in a MISPE-HPLC method for the direct extrac-tion of the analytes from the sample matrix with a

Table 1. Determination of b-lactams by HPLC-UV Detection

Sample Analyte Sample treatment Chromatographic/detectioncharacteristics

Recovery (%) LODs Ref.

Milk andveterinaryformulation

CTX, CLX Protein precipitation. NexusSPE. Elution with ACN. Ev.

C18

MeOH/acetate buffer (pH 4)k = 265 nm

84–121 0.1 lg/L CTX0.3 lg/L CLX

[11]

Milk, chickenmuscle, waters

AMOX, AMPI, PEN G, PENV, PIPE, NAFCI, OXA,CLOX, DICLOX,CLAV.ACID

Ex with ACN. SPE withOasis HLB and Alumina.Elution with H2O

Luna C18 (150 · 0.5 mm,5 lm)Solvent A: water/0.01% TFASolvent B: ACN/0.01% TFAgradientk = 220 nm

82.9–98.2 0.04–0.06 lg/L waters0.80–1.40 lg/L foods

[12]

Milk CDX, CCL, CLX, CTX, CFZ,CXM, CPZ, CTF

UA-MSPD with Nexussorbent. Elution withMeOH, Ev

Chromolith RP-18e(100 · 4.6 mm,5 lm)Solvent A: HCOOH 0.1%Solvent B: MeOH/ACN (75:25)gradientk = 265 nm except for CFZ and CXM275 nm

93.8–115.3 103–112.3 lg/Kg [13]

Beef, milk PENG, OXA, CLOX Ion-paired extraction withTBABr and water-ACN

Xbridge C18 (250 · 4.6 mm,5 lm)Solvent A: 5 mM phosphate (pH 6.6)Solvent B: ACNIsocratic (75% A, 25% B)k = 226, 240 nm

72.3–98.9% 1–2 ng/mL [14]

Milk AMOX, AMPI, OXA, CLOX,DICLOX

UA-MSDP – QuEChERS Core shell Kinetex C18(150 · 4.6 mm, 2.6 lm)Solvent A: 0.05 M CH3COONH4

Solvent B: ACNGradientk = 226, 240 nm

70–120% 6–12 lg/Kg [17]

Wastewater PENG, AMOX, AMPI,PENV, OXA, CLOX,DICLOX, NAFCI

Anion-exchange SPE C18

H2O/TFA-ACN/TFA, gradientk = 220 nm

a46–91 industriala28–91 influent STPa39–114 effluent STP

b2.9–25.6 lg/L industrialb2.5–12.4 lg/L influent STPb2.2–12.7 lg/L effluent STP

[18]

Environmentalwater samples

PENG, PENV, AMPI,AMOX, NAFCI, OXA,CLOX, DICLOX

MISPE (template PENG) C18

H2O TFA-ACN TFAk = 220 nm

93–100 tap water90–100 river water

c0.38–0.98 lg/L Milli Qc0.9–2.9 lg/L tap waterc1.3–5.8 lg/L river water

[19]

River waterHuman urine

CLXCLX, AMOX

MISPE (template CLX)Urine: pH = 3Water: tandem SPE withHLB

C18 (250 · 4.6 mm,5 lm)ACN 1% acetic acidk = 252 nm

>50 river water78 CLX and 60 AMOX

nr [20]

Pharmaceuticals,human bloodserum, urine

CLX, CDX, CCL, CTX Serum: Add ACN, V, C, Ev,SPE with Diol Bond ElutUrine: D and F

C18

Acetate bf (pH 4.0)-MeOHk = 265 nm

76.3–112.0 0.01 ng/lL CDX and CLX0.005 ng/lL CTX and CCL

[21]

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Table 1 (continued)

Sample Analyte Sample treatment Chromatographic/detectioncharacteristics


Serum, bronchialsecretions

CAZ, MPM HPLC-ISP: C8 or C18

cartridge and in-line filter.Serum: DISputum: Add NaH2PO3 bf(pH 5.0), H, C

C18

ACN-NaH2PO3 bf (pH 5.0)k = 258, 296 nm

Sputum:56.7–73.4 MPM49.3–56.9 CAZSerum:105.2–89.4 MPM109.0–95.8 CAZ

<0.5 lg/mL [22]

Pharmaceuticalformulation

AMOX, AMPI, CLX nr FIA-Monolithic C18

MeOH-acetic/acetate (pH 6.2)k = 250 nm

101.4 AMOX nr [23]

Human urine Ciprofloxacin, CLOX,ibuprofen

D C18

Formic/formate (pH 3)-ACN-MeOHk = 220, 280 nm

92–110 0.41 lg/mL CLOX [24]

Rabbit serum,tissue cage fluid(TCF)

TIPC, clavulanate Add ACN, CAdd DCM, C

C18 IPACN-phosphate bf pH 4.1 (TBAS),gradientk = 218, 254 nm

76.81 lg/mL serum79.93 lg/mL TCF

1 lg/mL (LOQ) [25]

Human plasma DORI, ERTA, IMI, MPM Add MES bf pH 6Add ACN, C, EvAdd MES bf pH 6

Core shell Kinetex PFP(100 · 4.6 mm,2.6 lm)Solvent A: MethanolSolvent B: 0.1 M phosphate (pH = 7)Gradientk = 295 nm

87.9–99.7 0.50 mg/L (LOQ) [26]

AMOX, Amoxicillin; AMPI, Ampicillin; CAZ, Ceftazidime; CCL, Cefaclor; CDN, Cephradine; CDX, Cefadroxil; CEP, Cephapirin; CFM, Cefepime; CFPM, Cefpirome; CFX, Cefoxitin; CFZ,Cefazolin; CPZ, Cefoperazone CLAV.ACID, Clavulanic acid; CLOX, Cloxacillin; CLX, Cephalexim; CMNX, Cefminox; CMZ, Cefmetazole; CTF, Ceftiofur; CTX, Cefotaxime; CXM, CefuroximeDICLOX, Dicloxacillin; DORI, Doripenem; ERTA, Ertapenem; IMI, Imipenem; MPM, Meropenem; NAFCI, Nafcillin; OXA, Oxacillin; PENG, Penicillin G; PENV, Penicillin V; PIPE, Piperacillin;PFP, Pentafluorophenyl; TIPC, Ticarcillin; bf, buffer; C, Centrifugation; Co, Concentration; D, Dilution; DI, Direct injection; Ev, Evaporation; Ex, Extraction; F, Filtration; H, Homogenization; He,Heat; HTAB, Hexadecyltrimethylammonium bromide; IP, Ion pair; ISP, Integrated sample preparation; ISPR, Isopropanol; MIP, Molecularly imprinted polymer; UA-MSPD, Ultrasound-assistedmatrix solid-phase dispersion; nr, not reported; S8, Octanesulfonate; S12, Dodecanesulfonate; STP, Sewage-treatment plant; TBABr, Tetrabutylammonium; TBAS, Tetrabutylammonium hydrogensulfate; TEA, Tetraethylammonium; TFA, Trifluoroacetic acid; TrEA, Triethylamine; ulC, Ultracentrifugation; V, VortexaSamples spiked with all the antibiotics at 25 lg/L and 75 lg/L.bProcessing 250 mL of wastewater samples.cPercolation of 50 mL of water.

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Figure 2. Chromatograms of (A) cow raw milk and (B) chicken muscle spiked with 5 lg/L or lg/kg, respectively, of each b-lactam; (C) river watersample; and, (D) well-water sample spiked with 0.7 lg/L of each b-lactam. (1) Clavulanic acid, (2) amoxicillin, (3) ampicillin, (4) piperacillin, (5)penicillin G, (6) penicillin V, (7) oxacillin, (8) cloxacillin, (9) nafcillin and (10) dicloxacillin. (I.S.) p-amino benzoic acid 50 lg/L (Reprinted from[12] with permission).


selective wash using just 10% (v/v) organic solvent. Onthe basis of UV detection only, the method showed goodrecoveries and precision for both tap and river water,suggesting that this MIP procedure can be applied to thedirect preconcentration of b-lactam antibiotics in envi-ronmental waters.

Also, a cephalexin MIP was synthetized for the deter-mination of this compound in river waters [20]. In orderto increase the sample volume for a higher sensitivity, atandem SPE system incorporating Oasis HLB sorbent wasimplemented, making possible the determination ofcephalexin with recoveries greater than 50%.

2.3. Clinical and pharmaceutical analysisThe application of HPLC with UV detection to the anal-ysis of b-lactams in clinical and pharmaceutical matriceshas been used extensively [21]. It included monitoringthe concentrations of b-lactam antibiotics and theirprecursors to optimize their production.

Some progress in sample treatment includes the use ofdirect injection of samples without any previous sampletreatment as in a MISPE procedure to determine amox-icillin and cephalexin in urine, only adjusting the urine

sample to acidic pH [20] or the analysis of serumsamples without any pretreatment, by integrating theextraction column and the separation column in thesame set-up [22]. After the matrix passed the extractioncolumn, the retained analyte was quantitatively trans-ferred to the analytical column where separation byisocratic HPLC was performed.

Other advances are focused on getting simpler designsand very low-cost equipment. In that sense, multi-syringechromatography (MSC) involving a multi-syringe mod-ule, solenoid valves and a chromatographic column hasproved to be a satisfactory, cheap alternative to HPLC forthe analysis of three b-lactam antibiotics (amoxicillin,ampillicin and cephalexin) present in a generic formula-tion of amoxicillin [23]. Fig. 3 shows the set-up used forthis determination.

Selectivity is a key point in these methods becauseseveral active principles are usually co-administered. AC18 analytical column with isocratic elution was used todetermine cloxacillin together with ciprofloxacin andibuprofen in human urine without interferences [24].

Sometimes, gradient elution must be used to achieve acomplete separation (e.g., to carry out pharmacokinetic


Figure 3. Multi-syringe chromatography (MSC) set-up used to determine amoxicillin, ampicillin and cephalexin. MS, Multi-syringe burette; M,Manometer; V1–V3, Solenoid valves; MP, Mobile phase; S, Sample; W, Waste; RP-18, Monolithic column; DA, Diode-array detector; U, Unusedsyringe (Reprinted from [23] with permission).


studies involving the carboxypenicillin ticarcillin and theb-lactamase inhibitor clavulanate in rabbit serum andtissue-cage fluid samples) [25].

Columns based on core-shell technology have alsobeen used in pharmacokinetic studies of carbapenem.Retention times were significantly decreased and authorsreferred to this technique as UHPLC even though theydid not use sophisticated ultra-high-pressure pumps[26].

3. Analysis of b-lactam antibiotics by HPLC withMS detection

3.1. Food analysisFood samples (e.g., milk) are typically screened forb-lactam antibiotics by non-specific methods based onenzymatic reactions or microbial growth inhibition.These methods are fast and sensitive but they cannotidentify individual compounds and they yield false posi-tives.

MS is the preferred detection technique to confirm thepresence of b-lactams in foodstuffs. In spite of the highselectivity of this detection technique, previous chro-matographic separation is necessary in order to avoidion suppression of co-eluting compounds. Most of theproposed methods are also able to quantify, althoughearly designs of mass spectrometers had some difficulties


[27,28]. Both trap and triple-quadrupole analyzers caneasily achieve the minimum number of IPs required bythe EU for unambiguous b-lactam identification in thistype of sample. At least two fragments must be detected,the most intense signal being used for quantification andthe second one for identification. The ratio between theintensities of both fragments has to be within the per-mitted tolerance for identification to be successful.

b-lactams were first determined by HPLC-MS usingthermospray but currently electrospray ionization (ESI)is the preferred interface between both techniques due tothe ease of b-lactam ionization. Although b-lactamshave a carboxylic group, positive ionization is normallyused, due to the higher sensitivity. In some cases, betterresults have been reported in negative mode, avoidingmatrix interferences that significantly weaken the signal[29]. Collision-induced fragmentation in positive-ionmode leads to cleavage of the b-lactam ring. Therefore,two fragments are usually detected:(1) the class-specific fragment m/z 160; and,(2) the compound-specific daughter ions

[M + H � 159]+.The most abundant ion to be fragmented can be the

protonated molecular ion, the sodium adduct [30] oradducts with methanol ([M+CH3OH+H]+) [31], but it isimportant to realize that related metabolites can also befound, especially in case of ceftiofur [32] or amoxicillin[33].

Figure 4. Mass-spectral fragmentation pathways of (a) cephalosporin antibiotics and (b) penicillin antibiotics (Reprinted from [58] with permis-sion).


On one hand, methanol should be avoided in themobile phase, due to the potential for analyte degrada-tion [34]. But, on the other hand, it can provide betterchromatographic peaks. Usually, if the chromatographicrun is fast enough, degradation effects are not significant[35]. HPLC-UV methods cannot be applied directly to MSif they use non-volatile compounds in the mobile phase,such as buffers or ion-pair reagents. Problems can alsobe anticipated if non-volatile compounds are used aseluting solvents in SPE. The separation capacity of LCcan be improved tremendously with the new sub-2-lmchromatography columns. This improvement allows thedevelopment of multi-residue methods to determine in ashort analysis time several analytes from different fami-lies likely to be found in a given sample. This is the caseof b-lactams, which can be found together with otherantibiotics [36–39].

Milk is one of the samples analyzed most often for themonitoring of b-lactams, and is usually extracted usingacetonitrile. Acidic solutions can also be used, butattention must be paid to stability of some of them {e.g.,cephapirin [29] and amoxicillin at extreme pHs [40]}.

b-lactam antibiotics exhibit significant binding tomatrix components, especially proteins, but enzymaticdegradation of proteins can lead to antibiotic degrada-tion as well. The b-lactams detected most in milk arepenicillin G and amoxicillin [29,41].

Once acetonitrile has been evaporated, samples arereconstituted in a suitable solvent and SPE can be ap-plied. On-line SPE has also been proposed for fullyautomated analysis of b-lactams, and satisfactory resultswere achieved although relative standard deviationswere sometimes above 30% [35].

Newer strategies for sample treatment include mag-netic MIP extraction, which is an interesting optionwhen we are interested in class-specific analysis ratherthan a multi-residue method [42]. For solid samples

(e.g., meat, kidney or liver), sample treatments that haveincluded:(1) liquid membrane extraction, achieving LODs of

1 ng/kg for penicillin G and penicillin V [43];(2) pressurized liquid extraction (PLE) to determine

cloxacillin, cefalexin and dicloxacillin in meat withLODs below the MRLs [44]; and,

(3) dispersive SPE [34,45], which is an alternative toclassic cartridge-based SPE, simplifying and speed-ing up the sample preparation.

However, b-lactam residues were not detected in eggsof laying hens orally dosed with these antibiotics [28].

3.2. Environmental analysisGenerally, drugs are absorbed by the organism after in-take and are metabolized. However, a significant amountof the original substance will leave the organismunmetabolized via urine or feces. WWTPs are unable toeliminate these pharmaceutical residues completely andeventually they will contaminate the aquatic environ-ment.

Intact b-lactams do not occur frequently in the envi-ronment due to the poor stability of the b-lactam ring.Analytical methods, especially sample treatments, mustprovide very low LODs (ng/L). Although SPE is still thesample treatment used most for liquid samples [46],some variations have been applied to improve sensitivity.These modifications include the use of two different SPEcartridges plugged together [47] or a derivatization stepto form penicilloyl methyl esters. This reaction takesplace ‘‘in situ’’ during the SPE solvent evaporation andcan increase sensitivity of dicloxacillin by a factor of 9[48].

In solid samples (e.g., soil), PLE has been successfullyapplied using a methanol:water mixture (80:20, v/v) asextracting solvent [47]. Analytical methods for envi-ronmental samples are usually multi-residue methods



and are optimized for a wide variety of antibiotic familiesincluding b-lactams and many other pharmaceuticals,so they have to find a compromise among all theinstrumental parameters involved [49,50].

3.3. Clinical and pharmaceutical analysisSome applications of MS in pharmaceutical analysis in-clude the determination of b-lactam contamination innon-b-lactam pharmaceuticals. Cross-contamination is acritical issue because it may cause unexpected, adversereactions [51]. Another field of application is therapeuticdrug monitoring (TDM). The purpose of TDM is to providea rational dosing of the drug that depends on the needs ofeach individual, based on pharmacokinetic parameters.UHPLC has been applied in TDM to determine six b-lac-tams in neonate plasma [52], achieving an analysis timeof approximately 3.5 min per sample. Also, b-lactamshave been determined in plasma [53] and serum [54]samples by SPE using Oasis HLB cartridges with goodrecoveries (>75%) and LODs in the ng/mL range.

Degradation of b-lactams is quite likely, due to theinstability of the four-member ring, so analytical meth-ods are needed to check the purity. MS is essential toidentify degradation products but, unfortunately, non-volatile buffers and ion-pairing agents incompatible withMS have to be used to achieve a complete chromato-graphic separation.

An interesting alternative to make HPLC conditionscompatible with MS is based on multi-stage LC. Briefly, asmall volume eluted from the first column is diverted to asecond column. The mobile phase of the first columncontains all the additives required for the separation, andthe mobile phase of the second column is fully MScompatible. Using this approach, it was possible todetermine the degradation products of a newly-synthe-sized b-lactam antibiotic [55]. In this case, it was nec-essary to add a third column to avoid the contaminationof the ion-pairing reagent with the second column.

Coupling two columns in-line has also been used foron-line purification of plasma samples. The first column,used as the on-line extraction column, contains bigparticles (30 lm) and high flow rates are applied (4 mL/min). The second column is the analytical column, whereseparation takes place. Satisfactory precision was ob-tained and the total analysis time was just 1.6 min [56].

b-lactams often decompose at high temperatures inatmospheric pressure chemical ionization (APCI) inter-faces. If softer conditions are applied for the ionization,some b-lactams are not detected unless an ionization-accelerating solvent is used. Bromoform has been re-ported as a satisfactory ionization-accelerating solvent,being able to form adducts with these labile b-lactams, soimproving sensitivity and making it easy to identify[M+Br]� because of the stable-isotope-abundance ratioof 1:1 [57]. Nevertheless, ESI is the preferred ionization


mode and fragmentation patterns for b-lactams are wellknown (Fig. 4) [58].

Table 2 includes the methods for the analysis of thesecompounds in different fields of application by using LCwith MS detection.

4. Analysis of b-lactam antibiotics by otherdetection techniques

4.1. Chemiluminescenceb-lactams as a group possess poor chromophores, espe-cially those lacking an aromatic ring, so other detectiontechniques have been tested [e.g., chemiluminescence(CL), fluorescence and electrochemical].

CL has been the used most due to its inherent sensi-tivity and selectivity. b-lactams can participate in CLreactions in two ways:(1) enhanced CL emission; or,(2) being derivatized with a suitable CL reagent.

In the first group (1), it was found that dicloxacillinand clavulanic acid exhibit the highest CL enhancementwhen luminol is oxidized with H2O2 [59]. It appears thata strained b-lactam ring is essential for this particular CLenhancement. Attention must be paid to mobile-phasecomposition as there are buffers (e.g., borate) and or-ganic solvents (e.g., acetonitrile) not supporting CLemission.

In the second group (2), it is possible to use derivati-zation reagents [e.g., 4-(2�-cyanoisoindolyl)phenylisoth-iocynate (CIPIC)]. CIPIC reacts with the primary aminogroup of the drug to form a CIPIC conjugate, whichemits CL when oxidized with H2O2. Cefaclor has beendetermined using this strategy in human serum andbetter sensitivity was obtained than with fluorescenceand MS detection [60,61]. Borate and acetonitrile sup-port this CL emission.

4.2. FluorescenceFluorescence detectors have also been tested for thedetermination of b-lactams. Lower LODs are expectedbut, because of the lack of a fluorophore in thesecompounds, derivatization is usually required. This is atedious process and the efficiency might not be optimalwhen b-lactam concentration is low. CIPIC derivate usedfor CL detection could also be used for fluorescence, butbetter sensitivity was achieved with CL [61].

A labeling reaction involving trichloroacetic acid(TCA) and formaldehyde at 100�C for 30 min was usedto determine amoxicillin [62]. Previously, the analytewas extracted from catfish and salmon tissues withphosphate buffer (pH 4.5), proteins were precipitatedwith TCA and b-lactams were recovered with SPE (C18).LODs were 0.5 lg/kg and 1.2 lg/kg for catfish andsalmon, respectively.

Table 2. Determination of b-lactams by HPLC-MS detection

Sample Analyte Sample treatment Chromatographic/detection characteristics


Natural andwastewater

AMOX, AMPI, OXA, CLOX,CEP

SPE with Oasis HLB, Ev,add mobile phase

C18-LC/MS/MS with ESI0.1% formic acid-ACN-MeOH gradient

>75% exceptAMOX<40%

8–10 ng/L surface H2O13–18 ng/L influent8–15 ng/L effluent

[46]

Beef kidney tissue DECEP, AMOX, CEP,DCCD, AMPI, CFZ, PENG,OXA, CLOX, NAFCI,DICLOX

H, Ex H2O-ACN, V, C, EvACN, SPE with C18, Ev

C18-LC/MS/MS with ESI0.1% formic acid in water-0.1% formic acid in MeOH,gradient

nr (DECEP: 10–50; AMOX:50–100; CEP: 10; DCCD:500; AMPI:10; CFZ:10;PENG:10; OXA:10; CLOX:10; NAFCI:10;DICLOX:100–500) ng/g

[27]

Eggs Sulfonamides, tetracyclines,fluoroquinolones and b-lactams (AMOX, CEP,AMPI, PENG, CLOX)

Bl, Add sodium succinatebf, H, C, SPE with OasisHLB

Phenyl-bonded silicacolumnLC/MS/MS with ESI0.1% formic acid-ACN,gradient

AMOX CEP <25%; AMPI,PENG, CLOX: 30–50%

Screening limits approx.50 ppb

[28]

Bovine milk AMPI, AMOX, PENG,PENV, CLOX, CEP, CTF

Add ACN, V, C, Ev, addphosphate bf, V, SPEcleanup with Oasis HLB

C18 LC-IT-MS/MSH2O-1% acetic ac/MeOH-1%acetic ac, gradient

115–85a (0.2 AMPI, 0.4 CTF, 0.8CEP, 1AMOX and PENG, 2CLOX and PENV)c ng/mL

[30]

Raw bovine milk DCFA C, add extraction solution(0.4% DTE in borate bf pH9), V, Dr withiodacetamide, V, pHadjusted to 3.5, C, SPE withOasis HLB, Ev, C

Phenylether LC-ESI MS/MS0.005% Formic acid-MeOH, gradient

87.0–95.2% 96.1 lg/Kg [32]

Pig kidney, liver,muscle and fat.

AMOX and relatedmetabolites

Add phosphate bf, V, C, addTCA, SPE with C18, elutewith ACN, Ev, R

RP-LC-PI-ESI-MS/MSH2O-9.6 mM PFPA/ACN-H2O(1:1 v/v) + 9.6 mM PFPA,gradient

39.2–104.0a 0.2–12.0 ng/g [33]

Milk 7 b-lactam, 12 macrolide, 2liconsamide, morantel,orbifloxacin

Add ACN, C, Ev, add ACN(50%), F

UPLC HSS T3 column(100 mm · 2.1 mm, 1.8 lm)Solvent A: 0.05% FA inwaterSolvent B: ACNGradient

140.3–50.5 0.1–2.5 ng/mL [49]

Bovine milk PENG, AMPI, OXA, AMOX,DICLOX, CLX, CEP

Add 10% acetic ac, V, C, F C18 LC-PI-ESI-MSH2O-0.1% Formic ac/ACN-0.1%Formic ac, gradient

(PENG: 52, AMPI: 76, OXA:43.5, AMOX: 52, DICLOX:28.5, CLX: 82, CEP: 78.5)b

PENG,CLX, CEP:1 lg/L,AMPI:2 lg/L, OXA:5 lg/L,AMOX:<1 lg/L,DICLOX:4 lg/L

[41]

Milk AMOX, OXA, PENV Magnetic molecularlyimprinted polymer

C18-LC-MS/MS0.1% formic acid aqueoussolution/methanol (40:60,v/v)

71.6–90.7% 1.6–2.8 ng/mL [42]

(continued on next page)

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Table 2 (continued)

Sample Analyte Sample treatment Chromatographic/detection characteristics


Cow milk, bovinekidney and livertissues

AMPI, CLOX, PENV, PENG SLM n-undecane:di-n-hexylether

C18 LC-PI-ESI-MSAcetic ac-MeOH-H2O

34–66 milka

47–85 kidney, liveraPENG, PENV:1 ng/Kg in kidney, liver0.7 lg/L in milkAMPI:1.4 lg/Kg in kidney, liver1.7 lg/L in milk

[43]

Bovine kidney tissue DECEP, AMOX, DCCD,AMPI, CFZ, PENG, OXA,CLOX, NAFCI, DICLOX

H, Ex H2O-ACN, dispersiveSPE cleanup with C18, C,Ev, F

C18-LC/MS/MSA: 0.1% formic acid; B:50%ACN-50% MeOH

70% except DCCD 58% nr [45]

Non-b-lactampharmaceuticals

CMZ, CPDXPR Di, S, C, F RP-LC-PI-ESI-MSH2O-ACN-Formic acid

96.7–102.2 CMZ88.9–94.2 CPDXPR

0.002 mg/Kg [51]

Human plasma CXM Add 0.2 mM NH4Ac (pH 5),V, C, SPE with Oasis HLB,Ev, R in mobile phase

RP-LC-NI-ESI-MS/MSACN:0.2 mM NH4Ac:glacial acetic acid(70:30:0.020, v/v/v)

>76.4% LOQ: 81.0 ng/mL [53]

Human serum AMPI, CFZ, CFM, CMZ,CTX, DORI, MPM, PIPE

Add 10 mM ammoniumformate, V, SPE with OasisHLB, Ev, R in 10 mMammonium formate andmethanol (95:5)

C18-LC/MS/MS with ESIA: 10 mM aqueousammonium formatecontaining 0.1% formicacid.B: methanol containing0.1% formic acid

Relative recoveries: 80.2–98.6%Absolute recoveries:83.7%–104.9%

CFP: 0.05 lg/mL, DORI:0.10 lg/mL, MPM: 0.05 lg/mL, CMZ: 0.01 lg/mL, CTX:0.05 lg/mL, AMPI: 0.01 lg/mL, CFZ: 0.05 lg/mL, PIPE:0.001 lg/mL

[54]

Human plasma A b-lactam candidate(comp I) and its open-ringtransformation product(comp II)

Add equal vol. IS in 0.5 MNH4Ac (pH 4), V, C

High Flow C18-LC-ESI-MS/MSOasis HLB for online ExA: 1 mM formic acid inwater;B: 1 mM formic acid inMeOH; C:1 mM formicacid in MeOH-H2O

70% comp I95% comp II

0.980 ng/mLd [56]

No sample CTX, CPZ, CMX, CPM,CTRX, CPIZ, CMNX, CZX,CFX, CMZ, CTM, CXM,LOMX, AMPI, PENG,CBPC, SBPC, PIPE, ASPC,AMOX, TIPC

Identification by FIAC18 LC-APCI-MSA: ACN-1% acetic acid, B:ACN-Bromoform asionization accelerationsolvent (100:1)

nr nr [57]

Human blood CCL Dr Py/CIPIC at 80�CC, F

C18-LC/APCI/MS40%ACN-10% 0.1 M TrEA

nr 10 nm/50 lL [61]

Bovine milk AMOX, AMPI, CLOX, OXA,PENG

C, Ex 100 mM phosphate bfpH 9.2, liq-liq Ex withhexane, SPE with C18, volreduction, F

C18 LC-ESI MS/MSA: 0.1% formic acid B: 65%ACN, 35% H2O, 0.1%formic acid, gradient

Stability studies in milk andmilk extracts at 4, �20 and�76�

nr [67]

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Fish AMOX, AMPI, CEP, PENG,OXA, CLOX, DICLOX

Ex ACN-hexane, C, Ev, addH2O-ACN

Phenyl-LC-ESI-MS/MSA: 0.1% formic acid with10 lMNaOH in waterB: ACN

AMOX: 10%AMPI, PENG <50%CEP, OXA, CLOX, DICLOX:>50%

0.1 mg/Kg AMOX, AMPI,CEP, OXA, CLOX, DICLOX1 mg/Kg PENG

[68]

Bovine muscle AMOX Ex H2O, C, Add ACN, C,Add dichloromethane

C18-LC-MS/MSA: ACN; B: H2O + 0.005%FA;C: H2O.

86% nr [69]

ERTA Dried blood spots Ex with 30/70 (v/v) water/methanol

Agilent Zorbax Eclipse PlusC18, (50 mm · 1.8 lm,2.1 mm)Solvent A: 0.1% FA in waterSolvent B: 0.1% FA in ACNGradient

94–155% 0.2 mg/L [70]

AMOX, Amoxicillin; AMPI, Ampicillin; ASPC, Aspoxicillin; CBPC, Carbenicillin; CCL, Cefaclor; CEP, Cephapirin; CFM, Cefepime; CFPM, Cefpirome; CFX, Cefoxitin; CFZ, Cefazolin; CLOX,Cloxacillin; CLX, Cephalexim; CMNX, Cefminox; CMX, Cefmenoxime; CMZ, Cefmetazole; CPDXPR, Cefpodoxime proxetil; CPIZ, Cefpimizole; CPL, Cephalin; CPM, Cefpiramide; CPZ,Cefoperazone; CQM, Cefquinome; CTF, Ceftiofur; CTM, Cefotiam; CTRX, Ceftriazone; CTX, Cefotaxime; CXM, Cefuroxime; CZX, Ceftizoxime; DCCD, Desfuroylceftiofur cysteine disulfide;DCFA, Desfuroylceftiofur free acid; DECEP, Deacetylcephapirin; DICLOX, Dicloxacillin; DORI, Doripenem; ERTA, ertapenem; LOMX, Latamoxef; MPM, Meropenem; NAFCI, Nafcillin; OXA,Oxacillin; PENG, Penicillin G; PENV, Penicillin V; PIPE, Piperacillin; SBPC, Sulbenicillin; TIPC, Ticarcillin; APCI, Atmospheric pressure chemical ionization; bf, buffer; Bl, blend; C, Centri-fugation; CID, Collision-induced dissociation; CIPIC, 4-(2-cyanoisoindolyl) phenylisothiocyanate; CTAB, Cetyltrimethylammonium bromide; Di, Dissolve; Dr, Derivatization; DTE, Dithio-erythritol; ESI, Electrospray ionization; Ev, Evaporation; Ex, Extraction; F, Filtered; FA, Formic acid; H, Homogenize; IT, Ion trap; NI, Negative ionization ; nr, not reported; PFPA,Pentafluoropropionic acid; PI, Positive ionization; PPC, Perfusive-particle chromatography; Py, Pyridine; R, reconstitution; RP, Reverse phase; S, Sonicate; SLM, Supported liquid membrane;TCA, Trichloroacetic acid; TrEA, Triethylamine; ulC, Ultracentrifugation; V, Vortex.a Recovery range covers all the analytes and concentrations.b Average recovery.c For a nominal sample size of 5 mL.d Corresponding to limits of quantification.

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Table 3. Determination of b-lactams by HPLC with other detection techniques

Sample Analyte Sample treatment Determination technique Recovery (%) LODs Ref.

No sample PENG, PENV,DICLOX

C18 LC-CL35% MeOH-0.01 M NaH2PO4

PDr: 10�4 M Luminol in 0.001 MNaOH, 0.03% H2O2 in 0.001 MNaOH

nr nr [59]

Human blood CCL Dr Py/CIPIC at 80�CC, F

C18 LC-FL34% ACN-10% 0.1 M TrEACL detectionH2O2, borate bf (pH 9.5), ACN

94–106 10 pmol/25 lLpmol/25 lL

[61][60]

Catfish andsalmontissues

AMOX Ex phosphate bf (pH 4.5)C18 SPEDr TCA-formaldehyde at 100�C

C18 LC-FL80% ammonium formate(0.05 M, pH 5.8) – 20% ACN.

67.0–82.0 0.5 lg/kg catfish0.8 lg/kg salmon

[62]

Bovine milk AMOX, AMPI Add 0.01 MKH2PO4 + Na2WO4 + H2SO4

C18 SPEDr salicylaldehyde

ODS-3 LC-FLACN:0.02 M KH2PO4 bf, pH 5.5Gradient

81.6–87.0 1.1 ng/mL AMOX1.0 ng/mL AMPI

[63]

Humanserum andplasma

AMOX Add HClO4 + sodium acetateDr fluorescamine

C18 LC-FL0.02 M methanesulfonic acid–ACN (92:8,v/v)

100.2–106.8% 0.1 lg/mL [64]

Human urine AMOX,amoxicilloicacid

Method 1: Ex MeOH + ACNMethod 2: SPE with Bond Elut

C18 LC-FL0.1% Formic acid: ACNgradient

Method 1: 55–91.4Method 2: 46.6–98.0

Method 1: 0.24 lg/mLAMOX, 0.24 lg/mLamoxicilloic acidMethod 2: 0.02 lg/mLAMOX, 0.04 lg/mLamoxicilloic acid

[65]

Raw milk AMPI, CEP C, solidification fat layer, addACN, F, Ev, C18 SPE, F

C8 LC-IPADACN-acetate bf (pH 3.75)-water

67–80a 5 lg/L [66]

AMOX, Amoxicillin; AMPI, Ampicillin; CCL, Cefaclor; CDN, Cephradine; CDX, Cefadroxil; CEP, Cephapirin; CLX, Cephalexim; DICLOX, Dicloxacillin; PEN, Penicillin; PENG, Penicillin G;PENV, Penicillin V; bf, buffer; C, centrifugation; CL, chemiluminescence; CIPIC, 4-(2-cyanoisoindolyl) phenylisothiocyanate; Dr, derivatization; Ev, evaporation; Ex, extraction; F, filtration; FL,fluorescence; IPAD, integrated pulsed amperometric detection; MFS, micro-flow system; nr, no reported; PDr, Post-column derivatization; Py, pyridine; TCA, Trichloroacetic acid; TrEA, Tri-ethylamine; V, Vortexa Recovery range covers all the analytes and concentrations.

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Precolumn derivatization with salicylaldehyde avoidssome co-eluting HPLC peaks in milk samples and is alsoa highly sensitive method to determine amoxicillin andampicillin [63].

Post-column derivatization is also possible using fluo-rescamine. 0.1 lg/mL was the LOD for amoxicillin inhuman serum with this approach [64].

It is also possible to get good sensitivity for amoxicillinand ampicillin without derivatization, exciting at235 nm (kem = 310 nm) but a preconcentration stepusing SPE is required [65].

4.3. Electrochemical detectionSulfur-containing b-lactams can be determined usingelectrochemical methods [e.g., pulsed electrochemicaldetection (PED)]. Sulfur compounds are preadsorbed tothe oxide-free noble metal (gold) surface by a non-bonded electron pair from the sulfur group (sulfur cannotbe fully oxidized). The adsorbed sulfur moiety is thenoxidized concurrently with the gold surface. A detectorsignal results from analyte oxidation and gold-oxideformation. Unfortunately, formation of the gold oxide isassociated with unstable baselines and large backgroundsignals. While in PED, current is measured after a pulseand a short delay, to allow charging current to decay, inintegrated pulsed amperometric detection (IPAD), cur-rent is integrated continuously during a cycle where theelectrode is oxidized and then reduced back to the ori-ginal state. The advantage of IPAD is that the contri-bution of surface-oxide formation from the detectorsignal is removed and the effect on the baseline is greatlyminimized. IPAD has been used to determine cephapirinand ampicillin in milk samples and no derivatization wasrequired. Better sensitivity and selectivity was achievedthan obtained using UV detection [66].

Table 3 describes of the methods for the analysis ofb-lactam antibiotics by HPLC using different detectiontechniques.

5. Conclusions

This review summarizes the different methodologiesproposed for monitoring b-lactam antibiotics by LC indifferent fields of application (e.g., food, environmental,clinical and pharmaceutical analysis). This techniqueoffers the best option for robust quantification in multi-residue analysis of these compounds. Although UV-Visdetection has been the detection technique applied mostin LC, MS appears to be the most powerful detectionsystem, mainly in the control of b-lactam antibiotics infood safety or environmental pollution, due to the pos-sibility of unambiguous identification when applyingtandem MS, by achieving the identification points re-quired in legislation.

Other detection systems have been developed less fre-quently, and are simpler and cheaper. Consideringsample preparation as one of the most critical steps inthe analytical procedure and taking into account theneed to minimize preparation, extraction and clean up inthe analysis of b-lactam antibiotics in the differentsamples, we included description of the different strate-gies carried out depending on the matrix.

We await important advances in on-line sampletreatments, the use of new column technologies and thedevelopment of greener alternatives.

AcknowledgementsProject P08-AGR-4268 (Proyecto de Excelencia, Juntade Andalucıa) supported this work.

References

[1] European Commission, Council Directive (EEC) 96/23/EC, Off. J.

Eur. Commun. L125 (1996) 10.

[2] Commission Regulation (EU) No 37/2010 of 22 December 2009

on pharmacologically active substances and their classification

regarding maximum residue limits in foodstuffs of animal origin,

Off. J. Eur. Commun. L15 (2010) 1.

[3] European Commission, Commission Decision (EEC) 2002/657/EC,


[4] M.A. Soliman, J.A. Pedersen, I.H. Suffet, J. Chromatogr., A 1029

(2004) 223.

[5] M.S. Dıaz-Cruz, M.J. Lopez De Alda, D. Barcelo, Trends Anal.

Chem. 22 (2003) 340.

[6] European Commission, Commission Decision (EEC) 2000/60/EC,


[7] C. Blasco, Y. Pico, C.M. Torres, Trends Anal. Chem. 26 (2007)

895.

[8] A.A.M. Stolker, T. Zuidema, M.W.F. Nielen, M.W.F. Nielen, Trends

Anal. Chem. 26 (2007) 967.

[9] S.R. El-Shaboury, G.A. Saleh, F.A. Mohamed, A.H. Rageh, J.

Pharm. Biomed. Anal. 45 (2007) 1.

[10] L. Kantiani, M. Farre, D. Barcelo, Trends Anal. Chem. 28 (2009)

729.

[11] V.F. Samanidou, E.D. Tsochatzis, I.N. Papadoyannis, Michrochim.

Acta 160 (2008) 471.

[12] M.I. Bailon-Perez, A.M. Garcıa-Campana, M. del Olmo-Iruela, L.

Gamiz-Gracia, C. Cruces-Blanco, J. Chromatogr., A 1216 (2009)

8355.

[13] E.G. Karageorgou, V.F. Samanidou, J. Sep. Sci. 33 (2010) 2862.

[14] C. Kukusamude, R. Burakham, O. Chailapakul, S. Srijaranai,

Talanta 92 (2012) 38.

[15] F. Gritti, G. Guiochon, J. Chromatogr., A 1217 (2010) 1604.

[16] V.F. Samanidou, E.G. Karageorgou, Drug Test. Anal. 3 (2011)

234.

[17] E.G. Karageorgou, V.F. Samanidou, J. Sep. Sci. 34 (2011) 1893.

[18] E. Benito-Pena, A.I. Partal-Rodera, M.E. Leon-Gonzalez, M.C.

Moreno-Bondi, Anal. Chim. Acta 556 (2006) 415.

[19] J.L. Urraca, M.C. Moreno-Bondi, A.J. Hall, B. Sellergren, Anal.

Chem. 79 (2007) 695.

[20] A. Beltran, N. Fontanals, R.M. Marce, P.A.G. Cormack, F. Borrull,

J. Sep. Sci. 32 (2009) 3319.

[21] V.F. Samanidou, E.A. Hapeshi, I.N. Papadoyannis, J. Chromatogr.,

B 788 (2003) 147.



[22] M. Ehrlich, F.D. Daschner, K. Kummerer, J. Chromatogr., B 751

(2001) 357.

[23] H.M. Gonzalez-San Miguel, J.M. Alpızar-Lorenzo, V. Cerda, Anal.

Bioanal. Chem. 387 (2007) 663.

[24] A. Espinosa-Mansilla, A. Munoz de la Pena, D. Gonzalez Gomez, F.

Canada-Canada, J. Sep. Sci. 29 (2006) 1969.

[25] C. Li, Q. Geng, D.P. Nicolau, C.H. Nightingale, J. Chromatogr., B

794 (2003) 227.

[26] E. Dailly, R. Bouquie, G. Deslandes, P. Jolliet, R. Le Floch, J.

Chromatogr., B 879 (2011) 1137.

[27] C.K. Fagerquist, A.R. Lightfield, Rapid Commun. Mass Spectrom.

17 (2003) 660.

[28] D.N. Heller, C.B. Nochetto, N.G. Rummel, M.H. Thomas, J. Agric.

Food Chem. 54 (2006) 5267.

[29] M. Becker, E. Zittlau, M. Petz, Anal. Chim. Acta 520 (2004) 19.

[30] D.M. Holstege, B. Puschner, G. Whitehead, F.D. Galey, J. Agric.

Food Chem. 50 (2002) 406.

[31] S. Grujic, T. Vasiljevic, M. Lausevic, T. Ast, Rapid Commun. Mass

Spectrom. 22 (2008) 67.

[32] M. Becker, E. Zittlau, M. Petz, Eur. Food Res. Technol. 217 (2003)

449.

[33] S. De Baere, M. Cherlet, K. Baert, P. De Backer, Anal. Chem. 74

(2002) 1393.

[34] K. Mastovska, A.R. Lightfield, J. Chromatogr., A 1202 (2008)

118.

[35] L. Kantiani, M. Farre, M. Sibum, C. Postigo, M.L. De Alda, D.

Barcelo, Anal. Chem. 81 (2009) 4285.

[36] A. Junza, R. Amatya, D. Barron, J. Barbosa, J. Chromatogr., B 879

(2011) 2601.

[37] D. Ortelli, E. Cognard, P. Jan, P. Edder, J. Chromatogr., B 877

(2009) 2363.

[38] A. Stolker, P. Rutgers, E. Oosterink, J. Lasaroms, R. Peters, J. van

Rhijn, M. Nielen, Anal. Bioanal. Chem. 391 (2008) 2309.

[39] Y. Tang, H. Lu, H. Lin, Y. Shih, D. Hwang, J. Chromatogr., B.

881–882 (2012) 12.

[40] A. Menelaou, A.A. Somogyi, M.L. Barclay, F. Bochner, J.

Chromatogr., B 731 (1999) 261.

[41] S. Ghidini, E. Zanardi, G. Varisco, R. Chizzolini, Food Addit.

Contam. 20 (2003) 528.

[42] X. Zhang, L. Chen, Y. Xu, H. Wang, Q. Zeng, Q. Zhao, N. Ren, L.

Ding, J. Chromatogr., B 878 (2010) 3421.

[43] T.A.M. Msagati, M.M. Nindi, Food Chem. 100 (2007) 836.

[44] V. Carretero, C. Blasco, Y. Pico, J. Chromatogr., A. 1209 (2008)

162.

[45] C.K. Fagerquist, A.R. Lightfield, S.J. Lehotay, Anal. Chem. 77

(2005) 1473.


[46] J.M. Cha, S. Yang, K.H. Carlson, J. Chromatogr., A 1115 (2006)

46.

[47] T. Christian, R.J. Schneider, H.A. Farber, D. Skutlarek, M.T.

Meyer, H.E. Goldbach, Acta Hydrochim. Hydrobiol. 31 (2003) 36.

[48] F. Bruno, R. Curini, A.D. Corcia, M. Nazzari, R. Samperi, Rapid

Commun. Mass Spectrom. 15 (2001) 1391.

[49] M.S. Dıaz-Cruz, D. Barcelo, Anal. Bioanal. Chem. 386 (2006) 973.

[50] S. Grujic, T. Vasiljevic, M. Lausevic, J. Chromatogr., A 1216

(2009) 4989.

[51] N. Fukutsu, Y. Sakamaki, T. Kawasaki, K. Saito, H. Nakazawa,

Chem. Pharm. Bull. 54 (2006) 1469.

[52] M.J. Ahsman, E.D. Wildschut, D. Tibboel, R.A. Mathot, Antimic-

rob. Agents Chemother. 53 (2009) 75.

[53] P. Partani, S. Gurule, A. Khuroo, T. Monif, S. Bhardwaj, J.

Chromatogr., B 878 (2010) 428.

[54] T. Ohmori, A. Suzuki, T. Niwa, H. Ushikoshi, K. Shirai, S. Yoshida,

S. Ogura, Y. Itoh, J. Chromatogr., B 879 (2011) 1038.

[55] L. Rovatti, M. Garzotti, A. Casazza, M. Hamdan, J. Chromatogr., A

819 (1998) 133.

[56] M. Jemal, Z. Ouyang, Y.Q. Xia, M.L. Powell, Rapid Commun. Mass

Spectrom. 13 (1999) 1462.

[57] S. Horimoto, T. Mayumi, K. Aoe, N. Nishimura, T. Sato, J. Pharm.

Biomed. Anal. 30 (2002) 1093.

[58] X.M. Chong, C.Q. Hu, Chromatographia 68 (2008) 759.

[59] J.H. Miyawa, S.G. Schulman, J.H. Perrin, Biomed. Chromatogr. 11

(1997) 224.

[60] M. Kai, H. Kinoshita, K. Ohta, S. Hara, M.K. Lee, J. Lu, J. Pharm.

Biomed. Anal. 30 (2003) 1765.

[61] M. Kai, H. Kinoshita, M. Morizono, Talanta 60 (2003) 325.

[62] C.Y.W. Ang, W. Luo, E.B. Hansen Jr., J.P. Freeman, H.C.

Thompson Jr., J. AOAC Int. 79 (1996) 389.

[63] W. Luo, E.B. Hansen Jr., C.Y.W. Ang, J. Deck, J.P. Freeman, H.C.

Thompson Jr., J. Agric. Food Chem. 45 (1997) 1264.

[64] H.J. Mascher, C. Kikuta, J. Chromatogr., A 812 (1998) 221.

[65] R. Fernandez-Torres, M.O. Consentino, M.A.B. Lopez, M.C. Moc-

hon, Talanta 81 (2010) 871.

[66] C.O. Dasenbrock, W.R. LaCourse, Anal. Chem. 70 (1998) 2415.

[67] S. Riediker, A. Rytz, R.H. Stadler, J. Chromatogr., A 1054 (2004)

359.

[68] S. Smith, C. Gieseker, R. Reimschuessel, C. Decker, M.C. Carson, J.

Chromatogr., A 1216 (2009) 8224.

[69] B. Lugoboni, T. Gazzotti, E. Zironi, A. Barbarossa, G. Pagliuca, J.

Chromatogr., B 879 (2011) 1980.

[70] G. la Marca, E. Giocaliere, F. Villanelli, S. Malvagia, S. Funghini,

D. Ombrone, L. Filippi, M. De Gaudio, M. De Martino, L. Galli, J.

Pharm. Biomed. Anal. 61 (2012) 108.

advances in the determination of β-lactam antibiotics by liquid chromatography

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