Research Article Fast HPLC-MS/MS Method for …downloads.hindawi.com/journals/jamc/2015/959675.pdf · Fast HPLC-MS/MS Method for Determining Penicillin Antibiotics in Infant Formulas

Research ArticleFast HPLC-MSMS Method for DeterminingPenicillin Antibiotics in Infant Formulas Using MolecularlyImprinted Solid-Phase Extraction

Moacutenica Diacuteaz-Bao Rociacuteo Barreiro Joseacute Manuel MirandaAlberto Cepeda and Patricia Regal

Department of Analytical Chemistry University of Santiago de Compostela 27002 Lugo Spain

Correspondence should be addressed to Patricia Regal patriciaregalusces

Received 2 December 2014 Revised 13 January 2015 Accepted 27 January 2015

Academic Editor Adam Voelkel

Copyright copy 2015 Monica Dıaz-Bao et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The dairy cattle may suffer from different infections relatively often but the inflammation of the mammary gland is very importantto the farmer These infections are frequently treated with penicillin antimicrobial drugs However their use may result in thepresence of residues in animal products such as milk powder andor infant formulas and it represents a potential risk forconsumers To monitor this the EU has defined safe maximum residue limits (MRLs) through Commission Regulation (EU)number 372010 Although LC-MS is a trustful option for confirmation and quantification of antibiotics the analysis of realsamples with complex matrices frequently implies previous clean-up steps In this work precipitation polymerization has beenused and different molecularly imprinted polymer (MIP) sorbents were tested and optimized for the fast and simultaneous solid-phase extraction (MISPE) of eight common penicillins (ampicillin amoxicillin oxacillin penicillin G penicillin V cloxacillindicloxacillin and nafcillin) The extracts were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MSMS) and the applicability of these polymers as sorbents for the extraction of penicillins at MRL levels in milk powder (infantformulas) was proved The limits of detection and quantification were below the legal tolerances except for LOQ for oxacillin andcloxacillin

1 Introduction

The term antibiotic refers to a very diverse range of chemicalsubstances that possess antibacterial activity They can beeither broad spectrum or narrow spectrum [1] 120573-Lactamantibiotics (BLAs) constitute one of the most widely usedantimicrobial drugs in veterinarymedicine especially to treatand prevent bacterial infections (respiratory urinary andmammary gland or skin infections) of dairy cattleThis groupof antibiotics can be classified into several groups accordingto their structural characteristics penicillins cephalosporinsand more recently carbapenems Their unique structuralfeature is the presence of the four-membered BLAs (2-azetidinone) ring [2]

Penicillins are antimicrobial agents used against variousmicroorganisms and exert their activity inhibiting the synthe-sis of the peptidoglycan layer of bacterial cell walls [1] These

molecules derive their activity from the 6-aminopenicillanicacid nucleus which is effective against mainly Gram-positivebacteria (Figure 1) Amoxicillin ampicillin penicillin Gpenicillin V cloxacillin dicloxacillin oxacillin and nafcillinare registered drugs for the treatment of food-producinganimals [3] In dairy cattle in addition to digestive andrespiratory diseases the inflammation of themammary glandis very problematic for the farmer Mastitis although ananimal welfare problem is a big economic problem becauseit increases the somatic cell count a milk quality indicator[4] These infections are frequently treated with penicillinsand they are also used for prevention on a regular basis Theincorrect use of these antibiotics may result in the presenceof residues in edible tissues or derived products such as milk[5 6] It is well known that the improper usemay have adverseeffects on consumer health including bacterial resistance tothese drugs in humans and also problems in dairy industry

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2015 Article ID 959675 8 pageshttpdxdoiorg1011552015959675

2 Journal of Analytical Methods in Chemistry

N

O

H

N

S

O

O

OH

R CH3

CH3

Figure 1 General structure of penicillins (R = lateral amino chain)

Table 1MRLs established byCommissionRegulation (EU) number372010 for penicillins residues in milk

Analyte MRL (120583gsdotkgminus1)Oxacillin 30Cloxacillin 30Dicloxacillin 30Nafcillin 30Penicillin G 4Penicillin V 4Ampicillin 4Amoxicillin 4

[7] To avoid health risks for the consumer the EUhas definedsafe maximum residue limits (MRL) for penicillins throughCommission Regulation (EU) number 372010 The limitsimplemented in milk for penicillin antibiotics are shown inTable 1

The determination of these compounds at their lowMRLrequires sensitive analytical methods to comply with currentlegislation BLAs have poor stability in standard solutions andsome specific compounds such as amoxicillin suffer degrada-tion in solutions in one week of even fewer days [8] For thisreason fast and straightforward methods of determinationare generally preferred [9] High performance liquid chro-matography (HPLC) has been the most frequent techniquefor analyzing penicillins and other BLAs in differentmatricesusually coupled with mass spectrometry (MS) [1 3 7 10 11]Usually this technique implies previous clean-up steps usingcommon solid-phase extraction (SPE) proceduresThe clean-up of the samples plays indeed a key role in determining thedetection capability of the instrumental techniques becauseof its ability to reduce matrix interferences [12] The maindrawback of SPE techniques is the lack of selectivity ofthe sorbents Molecularly imprinted polymers (MIP) aresynthetic materials with recognition sites that specificallybind target molecules in mixtures with other compoundsMIP sorbents which imitate natural recognition are capableof meeting the demands of SPE and they may be reusedseveral times with optimum recoveries [13] The possibleapplication of MIP as selective sorbents for the solid-phaseextraction of some components of BLA group has beenreported in recent years [12 14ndash16] During the developmentof MIP-based methods it is interesting to develop polymersthat allow the simultaneous detection of various analytes

The aim of the present work was testing the suitability ofvarious MIP synthesized using different templates and cross-linker monomers for the simultaneous determination ofeight penicillins (ampicillin amoxicillin oxacillin penicillinG penicillin V cloxacillin dicloxacillin and nafcillin) Thepolymer was used as sorbent for the extraction of theseantimicrobial drugs from milk and then followed by LC-MSMS analysis The effectiveness of these polymers wasproved in spiked infant formulas at the level of interest(MRLs)

2 Materials and Methods

21 Chemicals and Reagents Ampicillin (AMP) amoxicillin(AMX) oxacillin (OXA) penicillin G (PEN G) penicillinV (PEN V) cloxacillin (CLOX) dicloxacillin (DICLOX)nafcillin (NAFC) and piperacillin (PIPE) were obtainedfrom Sigma-Aldrich (Madrid Spain) The chemicals usedfor the polymers synthesis were methacrylic acid (MAA)divinylbenzene 80 (DVB-80) ethylene glycol dimethacry-late (EGDMA) trimethylolpropane trimethacrylate (TRIM)and the initiator 221015840-azobis-(2-methylbutyronitrile) (AIMN)from Sigma-Aldrich MAA EGDMA and TRIM were freedfrom stabilizers by distillation under reduced pressure andAIMN was recrystallized from methanol prior to use Addi-tionally DVB was freed from stabilizers by passing through asmall column packed with neutral alumina (Aldrich) HPLCgrade solvents were supplied by Merck (Madrid Spain)

22 Preparation of Polymers The polymers were prepared byprecipitation polymerization OXA AMX and NAFC wereused as template molecules and MAA was used as functionalmonomer As cross-linkers DVB EGDMA and TRIM weretested including different solvents in the polymerizationmixture All the polymers were prepared by mixing thetemplate functional monomer and cross-linking monomerusing a ratio of 1 6 20 under dilution conditions (4wt) in the porogen solvent and adding AIMN (2 wtrelative tomonomers) as initiatorThe different combinationsare shown in Table 2 The polymerization mixtures weresimultaneously introduced into a temperature controllableincubator equipped with a low-profile roller at 24 rpm and60∘C for 24 hours The polymer particles were separated andcleaned by vacuumfiltration through a nylonmembrane filterof 045 120583m of pore diameter using 50mL of acetonitrile and50mL of methanol Then the imprint molecule was removedby Soxhlet extraction for 8 h using a methanolacetic acidmixture (1 1) In each case nonimprinted polymers (NIP)were prepared in the same way but without the addition oftemplate

23 LC-MSMS Analysis The obtained recoveries using dif-ferent polymers were calculated using an HPLC-MSMSmethod Separation was performed in a 1100 series HPLCsystem from Agilent Technologies (Minnesota USA) ASynergi 4 120583m MAX-RP 80A (100 times 2mm) column fromPhenomenex (Torrance CA USA) was used The mobilephase was acetonitrile (A) mixed on a gradient mode with

Journal of Analytical Methods in Chemistry 3

Table 2 Composition of different MIP synthesized for the simultaneous extraction of penicillin drugs

Template Functional monomer Cross-linkers Porogens Initiator Polymerization problems

Oxacillin DVB ACNTOL AIMN Not dissolvedEGDMA MeOH AIMN

Amoxicillin DVB ACNTOL AIMN Not dissolvedEGDMA MeOH AIMN

Nafcillin

MAA DVB ACNTOL AIMNTRIM ACN AIMNTRIM MeOH AIMN

EGDMA ACN AIMNEGDMA MeOH AIMN

MAA methacrylic acid EGDMA ethylene glycol dimethacrylate DVB divinylbenzene AIMN 221015840-azobis-(2-methylbutyronitrile) TRIM trimethylol-propane trimethacrylate MeOH methanol ACN acetonitrile TOL toluene

Table 3 MRM transitions of each analyte and collision energy (CE)for mass spectrometry detection and chromatographic retentiontime

Compound Mw Precursor ion Fragment ion CE (volts)lowast RT

Amoxicillin 3654 366 349 13 21114 25

Oxacillin 4014 402 144 33 145186 21

Cloxacillin 4359 436 178 31 149220 21

Penicillin G 3344 335 217 17 129202 31

Penicillin V 3504 351 229 19 137257 17

Ampicillin 3494 350 106 21 64160 9

Dicloxacillin 4703 470 212 33 157254 23

Nafcillin 4144 415 199 17 152171 51

lowastCE collision energy in volts

02 aqueous formic acid (B) at a flow rate of 300 120583Lminminus1After the first 2 minutes with very aqueous mobile phaseat 90 (B) binary gradient mixing was initiated as follows(B) 90 to 0 for 16min and 0 to 90 again for 3minat this point the gradient was kept isocratic for 4min Theobtained chromatographic retention times (RT) are includedin Table 3 AQ-Trap 2000mass spectrometer with ESI Sourcefrom AB SCIEX (Toronto Canada) was used working inpositive mode For quantification the most intense multi-ple reaction monitoring (MRM) transition was monitoredalong with a second transition for qualitative confirmation(Table 3)

24 Optimization of Molecularly Imprinted Solid-PhaseExtraction (MISPE) Molecularly imprinted and nonim-printed polymers (005 g) were placed in empty SPE glasscartridges The cartridges were coupled to an SPE manifold

and several experiments were carried out loading 1120583g of eachanalyte in 1mL of different loading solvents (acetonitrileand toluene) and using 6mL of various washing solutions(acetonitrile methanol and toluene with different ofacetonitrile) In parallel the same experiments were carriedout on NIP cartridges in order to prove the existence oftemplate-specific imprinted sites into the MIP The obtainedelution (3mL of methanol 1 acetic acid) was evaporatedunder nitrogen stream and redissolved in 1mL of mobilephase for HPLC-MSMS analysis Recovery values werecalculated using a standard calibration curve

25 MISPE of Infant Formulas After MISPE optimizationinfant formulas (03 g) spiked at the level of interest (MRLTable 1) with a mixture of the selected penicillins in ace-tonitrile were extracted using the selected polymer (NAFC-MAA-EGDMA-ACN-AIMN) and the optimized loading-washing-elution conditions Thus the MISPE protocol wasacetonitrile-acetonitrile-methanol 1 acetic acid Sampleswere diluted with 1mL of acetonitrile vortexed for 1minand centrifuged at 5000 g for 10min and the supernatant wasdirectly loaded into the selected MIP cartridge The columnwas washed with 6 times 1mL acetonitrile and the elution wasperformedwith 3times 1mL of elution solvent acidifiedmethanol(1 acetic acid) After washing and eluting the extract wasdried under a nitrogen stream at 30∘C and redissolved in100 120583L of mobile phase B Thirty microliters of extract wereimmediately injected into the chromatographic system andassayed with the developed HPLC-MSMS method

3 Results and Discussion

31 Preparation of Polymers and MISPE Optimization Theusedpolymerization techniquewas precipitation polymeriza-tion which yields an increased rebinding capacity and morehomogeneous distribution binding sites with microsphericalshapes of uniform sizes In this study oxacillin amoxicillinand nafcillin were selected as representative structures ofthe penicillin group and possible adequate templates tosynthesize MIP sorbents that would be effective in extract-ing the selected analytes These templates were combined


Table 4Maximum recoveries achieved for the different penicillin-basedMIP sorbents duringMISPE optimization using standard solutions

MIPa Recovery ()AMX OXA CLOX PEN G PEN V AMP DICLOX NAFC

OXA-MAA-EGDMA-MeOHb 45 60 28 77 34 50 25 36AMX-MAA-EGDMA-MeOHb 15 10 10 6 7 10 7 8NAFC-MAA-DVB-ACNTOLc 27 31 27 22 35 45 22 50NAFC-MAA-TRIM-ACNc 25 65 66 54 62 45 58 56NAFC-MAA-TRIM-MeOHc 22 82 61 54 65 38 57 51NAFC-MAA-EGDMA-ACNc 58 88 82 56 70 65 63 77NAFC-MAA-EGDMA-MeOHc 20 58 43 42 56 40 51 49aPolymerization mixture that is template-functional monomer-cross-linking monomer-porogen (initiator was always AIMN) bcOptimized extractionconditions for maximum recoveries elution always with methanol 1 acetic acid bloading toluene washing toluene 10 cloading and washing acetonitrile

Table 5Mean recoveries (ten samples) precision limit of detection (LOD) and limit of quantification (LOQ) obtained for the determinationof penicillin drugs in milk powder (infant formula) using NAFC-MAA-EGDMA-ACN polymer as MISPE sorbent in combination withHPLC-MSMS

Parametera AnalyteAMX OXA CLOX PEN G PEN V AMP DICLOX NAFC

Recovery () 60 84 91 60 60 66 62 74CVr () 92 121 36 55 139 103 14 81CVR () 107 192 193 99 160 129 24 111LOD (120583g kgminus1) 09 236 155 07 12 11 14 74LOQ (120583g kgminus1) 31 784 516 24 39 36 48 246aCVr repeatability greater intraday CV CVR within-lab reproducibility greater interday CV

with MAA and DVB EGDMA andor TRIM in differ-ent porogen solutions After polymerization the preparedMISPE cartridges were tested using standard solutions ofthe selected penicillins The eligibility criterion in terms ofrecovery requirements to select the best polymer was set ata minimum of 50 recovery percentage for all the selectedanalytes Table 4 shows themaximum achieved recoveries foreach polymer obtained during the optimization experiments(different loading and washing steps elution was always withmethanol 1 acetic acid)

In general recoveries with OXA- andor AMX-basedMIP were lower than those with the NAFC-based polymersThese polymers provided good retention of penicillins in pre-liminary experiments using acetonitrile as loading solventThe acetonitrile was evaporated after the sorption step (pass-through the cartridges) and redissolved in mobile phase forHPLC analysis proving that approximately 90 of mostpenicillins were retained in the MIP However the analyteswould elute during the washing steps (using acetonitrileor methanol) obtaining no more than 30 of recoveryduring elution For this reason additional experimentswere performed with toluene in both loading and washingsteps and also using toluene with different percentagesof acetonitrile (5 10 and 20) as washing solution Eventhen the elution was not homogeneous for all penicillinsgetting good recoveries in some cases (OXA and PEN Gwith OXA-based polymer) but not for the whole group

of antimicrobials NAFC was a more adequate template toobtain polymers and its combination with MAA EGDMAand acetonitrile as porogen solvent permitted to obtain MIPsorbent for the simultaneous extraction of all the selectedanalytes (Table 4) A more homogeneous recovery for allpenicillins was preferred according to the low MRL of theseanalytes in milk Additionally this polymer provided thehigher differences (gt30) between MIP and NIP cartridgesAlso some polymerization problems were found when usingOXA andor AMX as templates in combination with DVB inacetonitriletoluene porogen mainly due to the low solubilityof these drugs in nonpolar solvents (the mixture ACNTOLpresents the lower polarity of all the tested porogens) In thiscase no polymerizationwas achieved because it was not pos-sible to dissolve the template in the polymerization mixturewhich is a key factor in MIP synthesis When using DVB ascross-linker the combination of acetonitrile and toluene isrequired to obtain polymers with good performance andwiththe production of monodisperse imprinted-polymer beadswith well-developed permanent pore structures [13]

Summing up NAFC-MAA-EGDMA-ACN was selectedas SPE sorbent because it provided the higher reten-tion capacity for the eight penicillins studied (ampicillinamoxicillin oxacillin penicillin G penicillin V cloxa-cillin dicloxacillin and nafcillin) enabling the simultaneousextraction of these drugs at the level of interest in milkTherefore this polymerwas selected for further investigations


22 (B leche) of data08212013 peniswiff (turbo spray)XIC of +MRM (18 pairs) 40201440 amu from sample

Inte

nsity

(cps

)

1800

1600

1400

1200

1000

800

600

400

200

0

1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24

121213471418

1454

Max

Max Max Max

Max Max

Max Max

Max

Inte

nsity

(cps

)

Inte

nsity

(cps

)125

100

Inte

nsity

(cps

)

10

0

20

25

Inte

nsity

(cps

)

20

0

40

50

Inte

nsity

(cps

)10

Inte

nsity

(cps

)

0

500

1000

1500

1813

0

30

20

38

Inte

nsity

(cps

)

10

0

30

20

38

50

00

Inte

nsity

(cps

) 125

100

50

00

Time (min)10 12 14 16 18 20

Time (min)10 12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)10

20

40

60

0 Inte

nsity

(cps

)

20

40

60

0

12 14 16 18 20

Time (min)10 12 14 16 18 20

PEN G

AMP

NAFCPEN V

PIPE (IS)

DICLOX AMX

OXA CLOX1025

1204

1352 18111946

Max

XIC of +MRM (18 pairs) 4020144 XIC of +MRM (18 pairs) 4360178

XIC of +MRM (18 pairs) 3501106

Time (min)2 4 6 8 10

Time (min)5 10 15 20



Time (min)10 12 14 16 18 20


Time (min)10 12 14 16 18 20


1181

1212

1418

1454

1643 1813 2089 1400

1487

1599 1688

18901949

633

656

1058

128 842 1502

1566

1854

1959

2094

110

219245

449474

656

788

819921

1140

1107

1263 1482

15791768

1813

1372

1446 1581

1918 1270

625 cps

625 cps 625 cps

875 cps500 cps250 cps

375 cps 125 cps 18125 cps

250 cps


2 0 1

3 1 2

0 1

Figure 2 Chromatogram of a blank infant formula (internal standard piperacillin)

and validationwith real samples After optimizing theMISPEconditions an adequate and detectable amount of PIPE wasadded to the samples to act as internal standard

32 MISPE Application to Infant Formulas and AnalyticalPerformance The developed procedure with NAFC-basedMIP was applied for the determination of penicillin residuesin infant formulas as representative milk powder samplesAcetonitrile as loading and washing solvent provided cleanextracts with satisfactory recoveries for all penicillins in realsamples (ge60) as it is shown in Table 5 In 2010 Yin etal hinted the suitability of nafcillin as template using this

compound as pseudo-template to develop a MISPE protocolfor selective screening of other four penicillins in water amuch less complex aqueous matrix [14] In this study thetemplate was also determined and no measurable bleedingof nafcillin was detected in the blank samples Apart fromnafcillin seven different penicillin drugs were extracted withthis polymeric sorbent and determined by LC-MSMS Thematrix was selected based on the importance that residues ofantimicrobials in milk may have for vulnerable populationas babies and children Powdered infant formulas weresampled as they were liquid formulas because there is noMRL specifically established for powdered formulas Thusa sample of 03 g of powder formula was analyzed as it


1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24

XIC of +MRM (18 pairs) 40201440 amu from sample14 (leite po LD) of data08212013 peniswiff (turbo spray)

Max

6000

5000

4000

3000

2000

1000

0

1426

1454

PEN G

AMP

NAFC PEN V PIPE (IS)

DICLOX AMX

OXACLOX

Max Max Max

Max Max Max

Max Max Max

Inte

nsity

(cps

)In

tens

ity (c

ps)

200

400

Inte

nsity

(cps

)

0

1000

2000

2525

Inte

nsity

(cps

)0

2000

4000

Inte

nsity

(cps

)

0

2000

4000

6000

Inte

nsity

(cps

)

0

1000

2000

3000

Inte

nsity

(cps

)

400

800

600

1000

Inte

nsity

(cps

)

500

1000

1500

Inte

nsity

(cps

)

500

0

1000

1963

1500600

800

0

Inte

nsity

(cps

)

200

400

600

0


Time (min)10 12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18


Time (min)12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)2 4 6 8 10

Time (min)12 14 16 18 20

1296

1492 1426

14541492

6271566 212

15151365 1268

18375 cps

18375 cps

25250 cps

19625 cps

60000 cps

30500 cps11250 cps44125 cps

8875 cps

6000 cps

1 4 8

9 0 6

9 9 3

Figure 3 Chromatogram of an infant formula spiked at the MRL levels for ampicillin amoxicillin oxacillin penicillin G penicillin Vcloxacillin dicloxacillin and nafcillin (internal standard piperacillin)

is the amount necessary to obtain 2mL of liquid formulaaccording to manufacturerrsquos directions The sample size wasalso selected based on the instrumental detection limits

The analytical MISPE procedure was fast as it onlyincluded a simple protein precipitation with acetonitrilefollowed by the direct loading of the extract into the NAFC-MAA-EGDMA-ACN polymeric SPE cartridge Protein pre-cipitation was necessary to avoid the interference of macro-molecules present in milk with the active imprinting sites[16]The total clean-up time was always less than 20 minutesUsually the determination of penicillins in milk is includedin multiresidue and multiclass methods implying variouspurification steps such as defatting centrifugation dilution

and less selective SPE protocols [1 3 7 10 11] Thus thepresent experiments were considered successful as the goalof the study was to develop a fast reusable affordable andsimple method for the analysis of these drugs in milk Theobtained recoveries were similar to or lower than thoseobtained by Ghidini et al who used only sample (acidic)precipitation centrifugation and filtration However theyused a higher initial sample amount and injected a highervolume of the final extract [9]

The selected polymer was chosen basing on the previousrecovery experiments and aiming at a reasonable recovery(compromise between matrix effects and low MRLs) forall the analytes The recovery came out at between 60 and


91 similar values to those recently obtained for someof the compounds by Giovannoli et al who used silicabeads as support for imprinting and applied the method tomilk samples [12] These authors prepared silica-imprintedbeads using 6-aminopenicillanic acid as mimic templateThe imprinted beads were used to extract penicillin V naf-cillin oxacillin cloxacillin and dicloxacillin in milk at MRLlevels and the extracts were analyzed using electrokineticchromatography The possible use of magnetic molecularlyimprinted polymers (MMIP) to selectively separate some 120573-lactam antibiotics from milk has been reported [15] usingalso MAA as functional monomer and EGDMA as cross-linking agent However in that previous study the selectedtemplate molecule was penicillin V and the magnetic MIPsorbent was applied to determine only PEN V AMX andOXA in milk samples The recoveries and detection limitswere similar to those obtained in this study using a lesscomplicated approach

As an example of successful clean-up Figure 2 showsa chromatogram of a blank sample and Figure 3 shows achromatogramobtained during the analysis of a spiked infantformula containing 4 120583g Lminus1 of AMX PEN G PEN V andAMP and 30 120583g Lminus1 of OXA CLOX DICLOX and NAFC(MRLs) No interfering peaks could be observed in thechromatogram The obtained recoveries limit of detection(LOD) and limit of quantitation (LOQ) for each antibioticare summarized in Table 5 It is clear that the detection limitsachieved by the developed procedure are low enough to allowthe analysis of penicillins in milkmilk powder samples atreal concentration levels except for the LOQs for OXA andCLOX Respectively LOD and LOQ values were expressedas the concentration of the target analyte that produced achromatographic signal response three and ten times higherthan the chromatographic baseline or background noisesurrounding that peak The linearity of the method wasevaluated by preparing blank and spiked samples (at MRLand 15 2 and 3 times the MRL concentration) Calibrationcurveswere constructed by plotting the concentration againstarea ratio (analyte peak areainternal standard peak area)Linearity coefficients ranged from 095 to 099 Precision wasexpressed as intra- and interday precision (CV) from fiverepeated experiments performed at two times during thesame day (ten injections) and on three consecutive workingdays (fifteen injections) Precision is concentration depen-dent and acceptable precision at the lower concentrationswas set at 20 (Table 5) If highly contaminated samplesneed to be analyzed the recommendation is diluting thesamples previous MISPE andor preparing MISPE cartridgeswithmore polymeric content Otherwise the imprinting siteswould be saturated and the analytical signal would reach amaximum or even decrease

4 Conclusions

Various imprinted polymers have been prepared to obtain aMIP-based material with proper characteristics to be usedas selective MISPE sorbent for the fast and simultane-ous extraction of eight important penicillins in milkmilkpowder Amongst the different combinations tested only

the MIP prepared in acetonitrile using nafcillin as templatemolecule and EGDMA as cross-linker monomer showed areasonable recovery for all the selected analytesThe latest factproves that testing various assayspolymeric combinations ispreferable when designing MIP for solid-phase extractionespecially in the case of a group of structurally relatedcompounds

From the observed data it may be concluded that thisNAFC-based MIP sorbent is suitable for the extraction ofpenicillin drugs from milk powder Molecularly imprintingtechnologies provide an alternative solution for SPE allowinga simple and straightforward clean-up strategy With thedeveloped MISPE protocol enough recovery was achievedfor eight common penicillin antibiotics at their low levelof interest (MRL) The combination of this fast extractionapproach with LC-MSMS determination resulted in anaffordable analyticalmethodwith good performance in termsof linearity and precision

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This research was supported by the Project EM 2012153 fromConsellerıa de Cultura Educacion e Ordenacion Universi-taria Xunta de Galicia

References

[1] E N Evaggelopoulou and V F Samanidou ldquoDevelopmentand validation of an HPLC method for the determinationof six penicillin and three amphenicol antibiotics in giltheadseabream (Sparus Aurata) tissue according to the EuropeanUnion Decision 2002657ECrdquo Food Chemistry vol 136 no 3-4 pp 1322ndash1329 2013

[2] F J Lara M del Olmo-Iruela C Cruces-Blanco C Quesada-Molina and A M Garcıa-Campana ldquoAdvances in the determi-nation of 120573-lactam antibiotics by liquid chromatographyrdquo TrACTrends in Analytical Chemistry vol 38 pp 52ndash66 2012

[3] B J A Berendsen H W Gerritsen R S Wegh et al ldquoCompre-hensive analysis of szlig-lactam antibiotics including penicillinscephalosporins and carbapenems in poultry muscle usingliquid chromatography coupled to tandem mass spectrometryRapidDetection in Food and FeedrdquoAnalytical andBioanalyticalChemistry vol 405 no 24 pp 7859ndash7874 2013

[4] J E Hillerton and E A Berry ldquoTreating mastitis in the cowmdashatradition or an archaismrdquo Journal of Applied Microbiology vol98 no 6 pp 1250ndash1255 2005

[5] M Khaskheli R S Malik M A Arain A H Soomro and HH Arain ldquoDetection of 120573-lactam antibiotic residues in marketmilkrdquo Pakistan Journal of Nutrition vol 7 no 5 pp 682ndash6852008

[6] M Roca L Villegas M L Kortabitarte R L Althaus and MP Molina ldquoEffect of heat treatments on stability of 120573-lactams inmilkrdquo Journal of Dairy Science vol 94 no 3 pp 1155ndash1164 2011

[7] M Becker E Zittlau and M Petz ldquoResidue analysis of 15penicillins and cephalosporins in bovine muscle kidney and


milk by liquid chromatography-tandem mass spectrometryrdquoAnalytica Chimica Acta vol 520 no 1-2 pp 19ndash32 2004

[8] L Jank R B Hoff P C Tarouco F Barreto and TM Pizzolatoldquo120573-lactam antibiotics residues analysis in bovine milk by LC-ESI-MSMS a simple and fast liquid-liquid extractionmethodrdquoFood Additives and Contaminants Part A Chemistry AnalysisControl Exposure and Risk Assessment vol 29 no 4 pp 497ndash507 2012

[9] S Ghidini E Zanardi G Varisco and R Chizzolini ldquoResiduesof 120573-lactam antibiotics in bovine milk confirmatory analysisby liquid chromatography tandem mass spectrometry aftermicrobial assay screeningrdquo Food Additives ampContaminants vol20 no 6 pp 528ndash534 2003

[10] A Junza N Dorival-Garcıa A Zafra-Gomez et al ldquoMulticlassmethod for the determination of quinolones and 120573-lactams inraw cow milk using dispersive liquid-liquid microextractionand ultra high performance liquid chromatographyndashtandemmass spectrometryrdquo Journal of Chromatography A vol 1356 pp10ndash22 2014

[11] C P Rezende M P Almeida R B Brito C K Nonaka andM O Leite ldquoOptimisation and validation of a quantitativeand confirmatory LC-MS method for multi-residue analyses of120573-lactam and tetracycline antibiotics in bovine musclerdquo FoodAdditives amp Contaminants Part A vol 29 no 4 pp 541ndash5492012

[12] C Giovannoli L Anfossi F Biagioli C Passini and C Bag-giani ldquoSolid phase extraction of penicillins from milk by usingsacrificial silica beads as a support for a molecular imprintrdquoMicrochimica Acta vol 180 no 15-16 pp 1371ndash1377 2013

[13] P Regal M Dıaz-Bao R Barreiro A Cepeda and C FenteldquoApplication of molecularly imprinted polymers in food anal-ysis clean-up and chromatographic improvementsrdquo CentralEuropean Journal of Chemistry vol 10 no 3 pp 766ndash784 2012

[14] J Yin ZMengM Du C LiuM Song andHWang ldquoPseudo-template molecularly imprinted polymer for selective screeningof trace 120573-lactam antibiotics in river and tap waterrdquo Journal ofChromatography A vol 1217 no 33 pp 5420ndash5426 2010

[15] X Zhang L Chen Y Xu et al ldquoDetermination of 120573-lactamantibiotics in milk based on magnetic molecularly imprintedpolymer extraction coupled with liquid chromatography-tandem mass spectrometryrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol878 no 32 pp 3421ndash3426 2010

[16] C Quesada-Molina B Claude A M Garcıa-Campana M delOlmo-Iruela and P Morin ldquoConvenient solid phase extractionof cephalosporins in milk using a molecularly imprinted poly-merrdquo Food Chemistry vol 135 no 2 pp 775ndash779 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


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Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


N

O

H

N

S

O

O

OH

R CH3

CH3

Figure 1 General structure of penicillins (R = lateral amino chain)

Table 1MRLs established byCommissionRegulation (EU) number372010 for penicillins residues in milk

Analyte MRL (120583gsdotkgminus1)Oxacillin 30Cloxacillin 30Dicloxacillin 30Nafcillin 30Penicillin G 4Penicillin V 4Ampicillin 4Amoxicillin 4

[7] To avoid health risks for the consumer the EUhas definedsafe maximum residue limits (MRL) for penicillins throughCommission Regulation (EU) number 372010 The limitsimplemented in milk for penicillin antibiotics are shown inTable 1

The determination of these compounds at their lowMRLrequires sensitive analytical methods to comply with currentlegislation BLAs have poor stability in standard solutions andsome specific compounds such as amoxicillin suffer degrada-tion in solutions in one week of even fewer days [8] For thisreason fast and straightforward methods of determinationare generally preferred [9] High performance liquid chro-matography (HPLC) has been the most frequent techniquefor analyzing penicillins and other BLAs in differentmatricesusually coupled with mass spectrometry (MS) [1 3 7 10 11]Usually this technique implies previous clean-up steps usingcommon solid-phase extraction (SPE) proceduresThe clean-up of the samples plays indeed a key role in determining thedetection capability of the instrumental techniques becauseof its ability to reduce matrix interferences [12] The maindrawback of SPE techniques is the lack of selectivity ofthe sorbents Molecularly imprinted polymers (MIP) aresynthetic materials with recognition sites that specificallybind target molecules in mixtures with other compoundsMIP sorbents which imitate natural recognition are capableof meeting the demands of SPE and they may be reusedseveral times with optimum recoveries [13] The possibleapplication of MIP as selective sorbents for the solid-phaseextraction of some components of BLA group has beenreported in recent years [12 14ndash16] During the developmentof MIP-based methods it is interesting to develop polymersthat allow the simultaneous detection of various analytes

The aim of the present work was testing the suitability ofvarious MIP synthesized using different templates and cross-linker monomers for the simultaneous determination ofeight penicillins (ampicillin amoxicillin oxacillin penicillinG penicillin V cloxacillin dicloxacillin and nafcillin) Thepolymer was used as sorbent for the extraction of theseantimicrobial drugs from milk and then followed by LC-MSMS analysis The effectiveness of these polymers wasproved in spiked infant formulas at the level of interest(MRLs)

2 Materials and Methods

21 Chemicals and Reagents Ampicillin (AMP) amoxicillin(AMX) oxacillin (OXA) penicillin G (PEN G) penicillinV (PEN V) cloxacillin (CLOX) dicloxacillin (DICLOX)nafcillin (NAFC) and piperacillin (PIPE) were obtainedfrom Sigma-Aldrich (Madrid Spain) The chemicals usedfor the polymers synthesis were methacrylic acid (MAA)divinylbenzene 80 (DVB-80) ethylene glycol dimethacry-late (EGDMA) trimethylolpropane trimethacrylate (TRIM)and the initiator 221015840-azobis-(2-methylbutyronitrile) (AIMN)from Sigma-Aldrich MAA EGDMA and TRIM were freedfrom stabilizers by distillation under reduced pressure andAIMN was recrystallized from methanol prior to use Addi-tionally DVB was freed from stabilizers by passing through asmall column packed with neutral alumina (Aldrich) HPLCgrade solvents were supplied by Merck (Madrid Spain)

22 Preparation of Polymers The polymers were prepared byprecipitation polymerization OXA AMX and NAFC wereused as template molecules and MAA was used as functionalmonomer As cross-linkers DVB EGDMA and TRIM weretested including different solvents in the polymerizationmixture All the polymers were prepared by mixing thetemplate functional monomer and cross-linking monomerusing a ratio of 1 6 20 under dilution conditions (4wt) in the porogen solvent and adding AIMN (2 wtrelative tomonomers) as initiatorThe different combinationsare shown in Table 2 The polymerization mixtures weresimultaneously introduced into a temperature controllableincubator equipped with a low-profile roller at 24 rpm and60∘C for 24 hours The polymer particles were separated andcleaned by vacuumfiltration through a nylonmembrane filterof 045 120583m of pore diameter using 50mL of acetonitrile and50mL of methanol Then the imprint molecule was removedby Soxhlet extraction for 8 h using a methanolacetic acidmixture (1 1) In each case nonimprinted polymers (NIP)were prepared in the same way but without the addition oftemplate

23 LC-MSMS Analysis The obtained recoveries using dif-ferent polymers were calculated using an HPLC-MSMSmethod Separation was performed in a 1100 series HPLCsystem from Agilent Technologies (Minnesota USA) ASynergi 4 120583m MAX-RP 80A (100 times 2mm) column fromPhenomenex (Torrance CA USA) was used The mobilephase was acetonitrile (A) mixed on a gradient mode with






Nafcillin






Amoxicillin 3654 366 349 13 21114 25

Oxacillin 4014 402 144 33 145186 21

Cloxacillin 4359 436 178 31 149220 21

Penicillin G 3344 335 217 17 129202 31

Penicillin V 3504 351 229 19 137257 17

Ampicillin 3494 350 106 21 64160 9

Dicloxacillin 4703 470 212 33 157254 23

Nafcillin 4144 415 199 17 152171 51





















Inte

nsity

(cps

)

1800

1600

1400

1200

1000

800

600

400

200

0

1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24

121213471418

1454

Max

Max Max Max

Max Max

Max Max

Max

Inte

nsity

(cps

)

Inte

nsity

(cps

)125

100

Inte

nsity

(cps

)

10

0

20

25

Inte

nsity

(cps

)

20

0

40

50

Inte

nsity

(cps

)10

Inte

nsity

(cps

)

0

500

1000

1500

1813

0

30

20

38

Inte

nsity

(cps

)

10

0

30

20

38

50

00

Inte

nsity

(cps

) 125

100

50

00

Time (min)10 12 14 16 18 20

Time (min)10 12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)10

20

40

60

0 Inte

nsity

(cps

)

20

40

60

0

12 14 16 18 20

Time (min)10 12 14 16 18 20

PEN G

AMP

NAFCPEN V

PIPE (IS)

DICLOX AMX

OXA CLOX1025

1204

1352 18111946

Max



Time (min)2 4 6 8 10

Time (min)5 10 15 20



Time (min)10 12 14 16 18 20


Time (min)10 12 14 16 18 20


1181

1212

1418

1454

1643 1813 2089 1400

1487

1599 1688

18901949

633

656

1058

128 842 1502

1566

1854

1959

2094

110

219245

449474

656

788

819921

1140

1107

1263 1482

15791768

1813

1372

1446 1581

1918 1270

625 cps

625 cps 625 cps


375 cps 125 cps 18125 cps

250 cps


2 0 1

3 1 2

0 1






1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24


Max

6000

5000

4000

3000

2000

1000

0

1426

1454

PEN G

AMP


DICLOX AMX

OXACLOX

Max Max Max

Max Max Max

Max Max Max

Inte

nsity

(cps

)In

tens

ity (c

ps)

200

400

Inte

nsity

(cps

)

0

1000

2000

2525

Inte

nsity

(cps

)0

2000

4000

Inte

nsity

(cps

)

0

2000

4000

6000

Inte

nsity

(cps

)

0

1000

2000

3000

Inte

nsity

(cps

)

400

800

600

1000

Inte

nsity

(cps

)

500

1000

1500

Inte

nsity

(cps

)

500

0

1000

1963

1500600

800

0

Inte

nsity

(cps

)

200

400

600

0


Time (min)10 12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18


Time (min)12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)2 4 6 8 10

Time (min)12 14 16 18 20

1296

1492 1426

14541492

6271566 212

15151365 1268

18375 cps

18375 cps

25250 cps

19625 cps

60000 cps

30500 cps11250 cps44125 cps

8875 cps

6000 cps

1 4 8

9 0 6

9 9 3









4 Conclusions






Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






Nafcillin






Amoxicillin 3654 366 349 13 21114 25

Oxacillin 4014 402 144 33 145186 21

Cloxacillin 4359 436 178 31 149220 21

Penicillin G 3344 335 217 17 129202 31

Penicillin V 3504 351 229 19 137257 17

Ampicillin 3494 350 106 21 64160 9

Dicloxacillin 4703 470 212 33 157254 23

Nafcillin 4144 415 199 17 152171 51





















Inte

nsity

(cps

)

1800

1600

1400

1200

1000

800

600

400

200

0

1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24

121213471418

1454

Max

Max Max Max

Max Max

Max Max

Max

Inte

nsity

(cps

)

Inte

nsity

(cps

)125

100

Inte

nsity

(cps

)

10

0

20

25

Inte

nsity

(cps

)

20

0

40

50

Inte

nsity

(cps

)10

Inte

nsity

(cps

)

0

500

1000

1500

1813

0

30

20

38

Inte

nsity

(cps

)

10

0

30

20

38

50

00

Inte

nsity

(cps

) 125

100

50

00

Time (min)10 12 14 16 18 20

Time (min)10 12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)10

20

40

60

0 Inte

nsity

(cps

)

20

40

60

0

12 14 16 18 20

Time (min)10 12 14 16 18 20

PEN G

AMP

NAFCPEN V

PIPE (IS)

DICLOX AMX

OXA CLOX1025

1204

1352 18111946

Max



Time (min)2 4 6 8 10

Time (min)5 10 15 20



Time (min)10 12 14 16 18 20


Time (min)10 12 14 16 18 20


1181

1212

1418

1454

1643 1813 2089 1400

1487

1599 1688

18901949

633

656

1058

128 842 1502

1566

1854

1959

2094

110

219245

449474

656

788

819921

1140

1107

1263 1482

15791768

1813

1372

1446 1581

1918 1270

625 cps

625 cps 625 cps


375 cps 125 cps 18125 cps

250 cps


2 0 1

3 1 2

0 1






1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24


Max

6000

5000

4000

3000

2000

1000

0

1426

1454

PEN G

AMP


DICLOX AMX

OXACLOX

Max Max Max

Max Max Max

Max Max Max

Inte

nsity

(cps

)In

tens

ity (c

ps)

200

400

Inte

nsity

(cps

)

0

1000

2000

2525

Inte

nsity

(cps

)0

2000

4000

Inte

nsity

(cps

)

0

2000

4000

6000

Inte

nsity

(cps

)

0

1000

2000

3000

Inte

nsity

(cps

)

400

800

600

1000

Inte

nsity

(cps

)

500

1000

1500

Inte

nsity

(cps

)

500

0

1000

1963

1500600

800

0

Inte

nsity

(cps

)

200

400

600

0


Time (min)10 12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18


Time (min)12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)2 4 6 8 10

Time (min)12 14 16 18 20

1296

1492 1426

14541492

6271566 212

15151365 1268

18375 cps

18375 cps

25250 cps

19625 cps

60000 cps

30500 cps11250 cps44125 cps

8875 cps

6000 cps

1 4 8

9 0 6

9 9 3









4 Conclusions






Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of














Inte

nsity

(cps

)

1800

1600

1400

1200

1000

800

600

400

200

0

1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24

121213471418

1454

Max

Max Max Max

Max Max

Max Max

Max

Inte

nsity

(cps

)

Inte

nsity

(cps

)125

100

Inte

nsity

(cps

)

10

0

20

25

Inte

nsity

(cps

)

20

0

40

50

Inte

nsity

(cps

)10

Inte

nsity

(cps

)

0

500

1000

1500

1813

0

30

20

38

Inte

nsity

(cps

)

10

0

30

20

38

50

00

Inte

nsity

(cps

) 125

100

50

00

Time (min)10 12 14 16 18 20

Time (min)10 12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)10

20

40

60

0 Inte

nsity

(cps

)

20

40

60

0

12 14 16 18 20

Time (min)10 12 14 16 18 20

PEN G

AMP

NAFCPEN V

PIPE (IS)

DICLOX AMX

OXA CLOX1025

1204

1352 18111946

Max



Time (min)2 4 6 8 10

Time (min)5 10 15 20



Time (min)10 12 14 16 18 20


Time (min)10 12 14 16 18 20


1181

1212

1418

1454

1643 1813 2089 1400

1487

1599 1688

18901949

633

656

1058

128 842 1502

1566

1854

1959

2094

110

219245

449474

656

788

819921

1140

1107

1263 1482

15791768

1813

1372

1446 1581

1918 1270

625 cps

625 cps 625 cps


375 cps 125 cps 18125 cps

250 cps


2 0 1

3 1 2

0 1






1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24


Max

6000

5000

4000

3000

2000

1000

0

1426

1454

PEN G

AMP


DICLOX AMX

OXACLOX

Max Max Max

Max Max Max

Max Max Max

Inte

nsity

(cps

)In

tens

ity (c

ps)

200

400

Inte

nsity

(cps

)

0

1000

2000

2525

Inte

nsity

(cps

)0

2000

4000

Inte

nsity

(cps

)

0

2000

4000

6000

Inte

nsity

(cps

)

0

1000

2000

3000

Inte

nsity

(cps

)

400

800

600

1000

Inte

nsity

(cps

)

500

1000

1500

Inte

nsity

(cps

)

500

0

1000

1963

1500600

800

0

Inte

nsity

(cps

)

200

400

600

0


Time (min)10 12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18


Time (min)12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)2 4 6 8 10

Time (min)12 14 16 18 20

1296

1492 1426

14541492

6271566 212

15151365 1268

18375 cps

18375 cps

25250 cps

19625 cps

60000 cps

30500 cps11250 cps44125 cps

8875 cps

6000 cps

1 4 8

9 0 6

9 9 3









4 Conclusions






Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Inte

nsity

(cps

)

1800

1600

1400

1200

1000

800

600

400

200

0

1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24

121213471418

1454

Max

Max Max Max

Max Max

Max Max

Max

Inte

nsity

(cps

)

Inte

nsity

(cps

)125

100

Inte

nsity

(cps

)

10

0

20

25

Inte

nsity

(cps

)

20

0

40

50

Inte

nsity

(cps

)10

Inte

nsity

(cps

)

0

500

1000

1500

1813

0

30

20

38

Inte

nsity

(cps

)

10

0

30

20

38

50

00

Inte

nsity

(cps

) 125

100

50

00

Time (min)10 12 14 16 18 20

Time (min)10 12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)10

20

40

60

0 Inte

nsity

(cps

)

20

40

60

0

12 14 16 18 20

Time (min)10 12 14 16 18 20

PEN G

AMP

NAFCPEN V

PIPE (IS)

DICLOX AMX

OXA CLOX1025

1204

1352 18111946

Max



Time (min)2 4 6 8 10

Time (min)5 10 15 20



Time (min)10 12 14 16 18 20


Time (min)10 12 14 16 18 20


1181

1212

1418

1454

1643 1813 2089 1400

1487

1599 1688

18901949

633

656

1058

128 842 1502

1566

1854

1959

2094

110

219245

449474

656

788

819921

1140

1107

1263 1482

15791768

1813

1372

1446 1581

1918 1270

625 cps

625 cps 625 cps


375 cps 125 cps 18125 cps

250 cps


2 0 1

3 1 2

0 1






1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24


Max

6000

5000

4000

3000

2000

1000

0

1426

1454

PEN G

AMP


DICLOX AMX

OXACLOX

Max Max Max

Max Max Max

Max Max Max

Inte

nsity

(cps

)In

tens

ity (c

ps)

200

400

Inte

nsity

(cps

)

0

1000

2000

2525

Inte

nsity

(cps

)0

2000

4000

Inte

nsity

(cps

)

0

2000

4000

6000

Inte

nsity

(cps

)

0

1000

2000

3000

Inte

nsity

(cps

)

400

800

600

1000

Inte

nsity

(cps

)

500

1000

1500

Inte

nsity

(cps

)

500

0

1000

1963

1500600

800

0

Inte

nsity

(cps

)

200

400

600

0


Time (min)10 12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18


Time (min)12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)2 4 6 8 10

Time (min)12 14 16 18 20

1296

1492 1426

14541492

6271566 212

15151365 1268

18375 cps

18375 cps

25250 cps

19625 cps

60000 cps

30500 cps11250 cps44125 cps

8875 cps

6000 cps

1 4 8

9 0 6

9 9 3









4 Conclusions






Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


1 2 3 4 5 6 7 8 9 10

Time (min)11 12 13 14 15 16 17 18 19 20 21 22 23 24


Max

6000

5000

4000

3000

2000

1000

0

1426

1454

PEN G

AMP


DICLOX AMX

OXACLOX

Max Max Max

Max Max Max

Max Max Max

Inte

nsity

(cps

)In

tens

ity (c

ps)

200

400

Inte

nsity

(cps

)

0

1000

2000

2525

Inte

nsity

(cps

)0

2000

4000

Inte

nsity

(cps

)

0

2000

4000

6000

Inte

nsity

(cps

)

0

1000

2000

3000

Inte

nsity

(cps

)

400

800

600

1000

Inte

nsity

(cps

)

500

1000

1500

Inte

nsity

(cps

)

500

0

1000

1963

1500600

800

0

Inte

nsity

(cps

)

200

400

600

0


Time (min)10 12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18

Time (min)10 12 14 16 18 20



Time (min)12 14 16 18


Time (min)12 14 16 18 20

Time (min)2 4 6 8 10

Time (min)2 4 6 8 10

Time (min)12 14 16 18 20

1296

1492 1426

14541492

6271566 212

15151365 1268

18375 cps

18375 cps

25250 cps

19625 cps

60000 cps

30500 cps11250 cps44125 cps

8875 cps

6000 cps

1 4 8

9 0 6

9 9 3









4 Conclusions






Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




4 Conclusions






Acknowledgment


References




























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Fast HPLC-MS/MS Method for …downloads.hindawi.com/journals/jamc/2015/959675.pdf · Fast HPLC-MS/MS Method for Determining Penicillin Antibiotics in Infant Formulas