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A multidimensional high performance liquid chromatography method coupled with amperometric detection using a boron-doped diamond electrode for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk

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A multidimensional high performance liquid chromatography method coupled with amperometric detection using a boron-doped diamond electrode for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk
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  Analytica Chimica Acta 654 (2009) 127–132 Contents lists available at ScienceDirect AnalyticaChimicaActa  journal homepage: www.elsevier.com/locate/aca A multidimensional high performance liquid chromatography methodcoupled with amperometric detection using a boron-doped diamondelectrode for the simultaneous determination of sulfamethoxazole andtrimethoprim in bovine milk Leonardo S. Andrade, Marcela C. de Moraes, Romeu C. Rocha-Filho,Orlando Fatibello-Filho, Quezia B. Cass ∗ Departamento de Química, Universidade Federal de São Carlos, C.P. 676, 13560-970 São Carlos - SP, Brazil a r t i c l e i n f o  Article history: Received 15 July 2009Received in revised form18 September 2009Accepted 23 September 2009 Available online 27 September 2009 Keywords: Multidimensional HPLCRestricted-access media (RAM)Bovine milkSulfonamides determinationElectrochemical detectionBoron-doped diamond electrode a b s t r a c t The development and validation of a multidimensional HPLC method using an on-line clean-up columncoupledwithamperometricdetectionemployingaboron-dopeddiamond(BDD)electrodeforthesimul-taneousdeterminationofsulfamethoxazole(SMX)andtrimethoprim(TMP)inbovinemilkarepresented.Aliquotsofpre-preparedskim-milksamplesweredirectlyinjectedintoaRAMoctyl-BSAcolumninorderto remove proteins that otherwise would interfere with milk analysis. After exclusion of the milk pro-teins, SMX and TMP were transferred to the analytical column (an octyl column) and the separation of the compounds from one another and from other endogenous milk components was achieved. SMX andTMP were detected amperometrically at 1.25V  vs.  Ag/AgCl (3.0molL  − 1 KCl). Results with good linearityin the concentration ranges 50–800 and 25–400  gL  − 1 for SMX and TMP, respectively, were obtainedand no fouling of the BDD electrode was observed within the experimental period of several hours. Theintra- and inter-assay coefficients of variation were less than 10% for both drugs and the obtained  LOD values for SMX and TMP were 25.0 and 15.0  gL  − 1 , respectively. © 2009 Elsevier B.V. All rights reserved. 1. Introduction A series of synthetic bacteriostatic compounds is used in dairyfarming for the treatment of diseases evolving from bacterialinfections, such as mastitis, pneumocystis pneumonia, chronicbronchitis, meningococcal meningitis, acute otitis, and toxoplas-mosis [1]. Sulfonamides have been used for several decades in the treatment of bacterial infection. They competitively inhibit thebacterial enzyme dihydropholate synthetase [2,3]. In veterinary medicine, sulfonamides are commonly used to treat or preventlivestock diseases, such as respiratory and gastrointestinal tractinfections [4]. The corresponding pharmaceutical products usually consist of a sulfonamide mixed with another drug that increasesitsantibacterialactivity(ina5:1ratiosulfonamide:potentiator),asfor example the sulfamethoxazole (SMX) and trimethoprim (TMP)mixture,whichiscommonlyknownasSMX–TMP(fortheirchemi-calstructures,seedePaulaetal.[5]).However,theuseofSMX–TMP mayleadtoresiduesinmilk,whichsubsequentlymayinducealler-gicreactionsinhumans.Inaddition,theycangiverisetoanincrease ∗ Corresponding author. Tel.: +55 16 3351 8087; fax: +55 16 3351 8350. E-mail address:  quezia@pesquisador.cnpq.br (Q.B. Cass). in the resistance of pathogenic bacteria to them, which may resultinhealthproblems[1].Toprotectconsumer’shealth,themaximum residue limit ( MRL ) adopted by the US Food and Drug Administra-tion (FDA) for sulfonamides in milk is 100  gkg − 1 [6]. In the sameway, the European Medicines Agency (EMEA) has set the  MRL  of trimethoprim in meat and milk as 50  gkg − 1 [7]. Therefore, thesearchforanalyticalmethodsthatcandetecttheseindividualsub-stances at low concentration levels is quite relevant.Commonly, the monitoring of sulfonamides in milk is carriedout using microbial methods, but the use of instrumental meth-ods of analysis based on LC with UV or fluorescence becomesnecessary when more conclusive results are required [2]. How- ever, problems involving the direct injection of milk samples intochromatographic phases can limit their use for practical purposes,since proteins present in this type of biofluid promptly clog thechromatographiccolumn,leadingtoasignificantlossofchromato-graphicefficiency[8].Aimingtoavoidanypretreatmentbeforethe chromatographicanalysis,theinterestinhavingon-linetechniquesforthehandlingofuntreatedbiologicalsampleshasbeenincreased.Themainadvantageofon-linetechniquesistheirnon-requirementof further handling of the samples; thus they are highly suitable tobecome fully automated techniques that can be used on site [9].Inthissense,HPLCmethodsincorporatingrestricted-accessmedia 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.09.035  128  L.S. Andrade et al. / Analytica Chimica Acta 654 (2009) 127–132 (RAM)columnscoupledon-linewithanalyticalcolumnshavebeenreported for analyte determination in several biological fluids bydirect injection [4,5,10–13].A significant number of analytical HPLC procedures aredescribed in the literature for the analysis of sulfonamides infood biological matrices, which include milk [1,2,13–19], egg [5,15,18–23], meat [15,24–28], and shrimp [29]. However, among these procedures there are only a few reports on the analysis of sulfonamideorthecombinationSMXandTMPusingRAMcolumns[30]. For example, Pereira and Cass [13] investigated the simulta- neous determination of SMX and TMP in cow’s milk (after beingsubjected to SMX–TMP treatment) employing an HPLC methodwithanon-linesampleclean-upusingaRAMcolumn.Themethodwas successfully applied to the measurement of residue levels of SMXandTMP,whereastheRAMoctyl-bovineserumalbumin(BSA)column showed an excellent lifetime. de Paula et al. [5] developed andvalidatedamethodforthesimultaneousdeterminationofSMXand TMP in whole-egg samples, also employing an HPLC methodwithanon-linesampleclean-upusingaRAMcolumn.Theobtainedresults showed that the method was suitable for monitoring SMXand TMP residues in eggs. Kishida [23] proposed a RAM-HPLCmethod for the simultaneous determination of sulfamonomethox-ine, sulfadimethoxine, and their N4-acetyl metabolites in eggsusing a green chemistry approach. The method was consideredsimple, rapid, and highly reproducible and reliable.Most of the above reviewed reports used UV–vis detection fortheir analyses and, as far as we could verify, there are only fewreports about these applications using electrochemical detection.In some instances, the electrochemical detection of sulfonamideswascarriedout,butusingclassicalelectrochemicaltechniquessuchasamperometry[19,27,28],square-wavevoltammetry[31,32],and differential pulse voltammetry (DPV) [33–35]. The use of elec- trochemical detectors in HPLC is attractive because they can behighly selective and sensitive. Furthermore, the electrolyte ionicstrength, buffer type, and pH can be modified in order to maxi-mize the sensitivity of these detectors. On the other hand, theymay present some disadvantages, as for example the tendencyto be deactivated/fouled by the adsorption of oxidation/reductionproducts and by changes of the electrode surface also caused byoxidation/reduction. In other words, from time to time they mayneed cleaning (often mechanical). However, when boron-dopeddiamondelectrodes(BDD)wereused,inseveralinstancesnodeac-tivation has been found during the electrochemical detection of different compounds, including sulfonamides [28,33,36,37]. This is one reason why BDD electrodes seem so promising for elec-troanalytical purposes. Besides, the wide potential window (up to3V) presented by BDD electrodes in aqueous solutions, as well astheir very low and stable voltammetric background current, long-termresponsestability,andlowsensitivitytodissolvedoxygenareimportant characteristics that allow the detection of many elec-troactive species that otherwise would be masked by the waterdecomposition reactions [38,39].Thus,thepurposeofthispaperistodescribetheresultsobtainedin the development and validation of a multidimensional HPLCmethod using an on-line sample clean-up column and employingamperometric detection at a BDD electrode for the simultaneousdetermination of SMX and TMP in bovine milk. 2. Experimental  2.1. Reagents The solutions were prepared using deionized water (Milli-pore, Brazil). The following reagents were of analytical grade:BSA (Sigma, USA; fraction V powder, minimum 98%); glutaralde-hyde, potassium dihydrogen phosphate, and sodium borohydride Fig. 1.  Schematic representation of the electrochemical detector used in themultidimensional-HPLC-flow measurements: (a) solution inlet; (b) solution outlet;(c)stainless-steeltube(cathode);(d)referenceelectrode;(e)silicon-rubberO-ring;(f) BDD electrode; (g) screw (electrical contact for the BDD electrode). (Merck, Darmstadt, Germany). SMX (99.85%) and TMP (98.88%)were generously supplied by Laboratório Teuto-Brasileiro (Anápo-lis, Brazil). Acetonitrile (ACN), 2-propanol, and methanol were of HPLC grade (J.T. Baker, USA). All solutions were filtered using a47-mm diameter, 0.45-  m Nylon membrane from Millipore.  2.2. Equipment, electrode, and apparatus TheHPLCsystemconsistedoftwoShimadzuLC-10ATVPpumps(Kyoto, Japan), with one of the pumps having a FCV-10AL valve forselecting solvent, a SIL 10AVP auto-injector, a DGU-14A degasser,a CTO-10 column oven, an SPD-6AV UV–vis detector (at 240 and269nm), and an SCL 10AVP interface. An HPLC 7000 Nitronic EAsix-port valve (Sulpelco, St. Louis, USA) was used for automatedcolumn-switching. Data acquisition was controlled with the Shi-madzu Class-VP Software. The quantification of SMX and TMP inmilk samples was carried out using an electrochemical detectorthat was developed in our laboratory and consisted of two acrylicblocks, as shown in Fig. 1. In one of them, three holes were drilled. The first hole (a) allows the introduction of the solution into thedetector, the second one (b) the exit of the solution, and the thirdone the insertion of a miniaturized Ag/AgCl (3.0molL  − 1 KCl) ref-erence electrode (d) [40], which was used in all measurements. A stainless-steel tube (c), used as counter electrode, was embeddedin the solution exit hole (b). A silicone rubber O-ring (e) and theBDD electrode (f) were fixed between the two acrylic blocks, leav-ing a 0.64-cm 2 exposed area. The electrical contact for the BDDelectrode was made through a stainless-steel screw (g) at the goldplated bottom of the silicon wafer.The BDD film (8000ppm) used as electrode (1.2cm × 1.2cm)waspreparedattheCentreSuissedeElectroniqueetdeMicrotech-  L.S. Andrade et al. / Analytica Chimica Acta 654 (2009) 127–132  129 nique SA (CSEM), Neuchatêl, Switzerland, as described elsewhere[41]. Prior to use, the BDD electrode surface was cleaned with 2-propanolandrinsedwithultra-purewater.Then,inordertoattaina hydrophilic (oxygen-terminated) surface [39], the BDD elec- trode was anodically pre-treated in a 0.5molL  − 1 H 2 SO 4  solutionby applying 0.5Acm − 2 , during 60s. The amperometric measure-ments were carried out using an Autolab PGSTAT-30 (Ecochemie)potentiostat/galvanostat controlled with the GPES 4.0 software.Hydrodynamicvoltammograms(HDVs)wereobtainedforeachcompound before the amperometric determinations and the elec-trode potential applied in these analyses was selected taking intoaccount the limiting current range in the HDVs.  2.3. Columns The analytical (150mm × 4.6mm i.d.) and the RAM-BSA(100mm × 4.6mm i.d.) columns were packed with Luna ® octylsilica (10  m, 120Å; Phenomenex, USA) by the ascending slurrymethod at 7500psi, using methanol for both the preparation andpacking of the slurry (50mL). After, the columns were conditionedwith methanol at a flow rate of 1.0mLmin − 1 for 12h [5,13].The BSA immobilization was done  in situ , based on the protocolproposedbyMenezesandFelix[42],asdescribedelsewhere[5,13]. WhentheRAM-BSAcolumnwasnotinuse,itwaskeptinwaterandstored at 4 ◦ C.  2.4. Solutions 2.4.1. Buffer solutions KH 2 PO 4  was used to prepare the 0.01molL  − 1 phosphate buffersolutions (pH 5.0 and 6.0), whose pH was adjusted with a1.0molL  − 1 sodium hydroxide solution, as required.  2.4.2. Standard solutions Firstly, methanolic stock solutions (1.0gL  − 1 ) of SMX and TMPwere prepared. From these, standard methanolic solutions wereprepared: 5.00, 10.0, 20.0, 30.0, 40.0, 60.0, and 80mgL  − 1 , for SMX;2.50,5.00,10.0,20.0,30.0,and40.0mgL  − 1 ,forTMP.Theconcentra-tionsoftheworkingsolutionsfortheconstructionofthecalibrationcurves were: 50, 100, 200, 400, 600, and 800  gL  − 1 , for SMX;25.0, 50.0, 100, 200, 300, and 400  gL  − 1 , for TMP. Three standardsolutions for the quality control (QC) samples of each drug wereindividually prepared: 80, 300, and 700  gL  − 1 , for SMX; 40, 150,and 350  gL  − 1 , for TMP.  2.4.3. Spiked samples Cow’s skim milk was used in the development and validationof the method. The preparation of the spiked milk samples wascarried out by transferring 10.0-  L aliquots of each appropriateworking standard solution into culture tubes and evaporating thesolventunderanitrogenstream.Allmilksampleswerecentrifuged(10,000rpm for 15min, at 20 ◦ C), resulting in an upper and verythin fatty layer, a middle aqueous layer, and a small cell pellet atthe lower layer in the centrifuge tube. Middle layers (1.000mL)were used to prepare the samples by reconstituting the dried ana-lytes.Thesolutionsweremixedbyvortexagitationfor30s.Aliquotsof 250  L were transferred to auto-sampler vials and 200  L wasinjected into the column-switching HPLC system.  2.5. Method validation The method validation was carried out according to interna-tionally accepted criteria [43] and took the following parameters intoaccount:linearity,selectivity,accuracyandprecision,recovery,limit of quantification ( LOQ  ), and limit of detection ( LOD ).  2.5.1. Linearity and selectivity The linearity of the SMX and TMP responses was assessedthrough calibration standards in triplicate. The SMX and TMP cal-ibration curves were constructed by plotting the chromatographicpeak area (charge,  Q  ) against the concentration of each drug.Aiming to assess the interference of endogenous compounds, theselectivity was verified by analyzing drug-free milk samples andSMX–TMP-spiked milk samples.  2.5.2. Precision, accuracy, and recovery The inter- and intra-day variability of the method was deter-minedbyanalyzingreplicatesofthreeQCsamples.Fivesamplesof eachconcentrationwerepreparedinmilkinthreenon-consecutivedays. The extraction recoveries of each drug were estimated usingspiked milk samples prepared in the same concentrations of thethree QC samples used for the determination of the intermediateprecision.  2.5.3. Limits of quantification and detection The acceptance criteria for  LOQ   were that the precision and theaccuracyforthreeextractedsamplespresentedlessthan20%vari-ability.The LOD wasmeasuredtakingintoaccountasignal-to-noiseratio of three. Both  LOQ   and  LOD  were measured by preparing sev-eral diluted solutions of SMX–TMP-spiked milk samples.  2.6. Column-switching procedure Fig. 2 illustrates the column-switching system used, which wasalternated between positions 1 and 2 and controlled through thetimedeventsusingtheClass-VPSoftware(thetime-sequenceusedis listed in Table 1). Initially, the column-switching system was set to position 1 and the milk sample injected into the RAM column.The six-port valve remained in this position for 11.70min whilethe macromolecules were discharged into the waste. At the sametime,theanalyticalcolumnwasconditionedwiththemobilephasedelivered by pump 2. Then, the valve was changed to position 2,redirecting the flow from the waste to the analytical column; thetransfer of the effluent fraction containing the analytes occurredbetween times 11.70 and 13.70min. Then, the valve was switchedback to position 1 for cleaning and conditioning the RAM column,while the compounds SMX and TMP were analyzed on the octylanalytical column. A flow-rate of 1.0mLmin − 1 was used and theUV–vis and electrochemical (BDD electrode) detections were car-ried out sequentially at 265nm and 1.25V  vs.  Ag/AgCl (3.0molL  − 1 KCl), respectively. The total analysis time for each measurementwas 32min. 3. Results and discussion The optimum potential for the amperometric detection of SMXandTMPwasinferredfromHDVs,whichwereobtainedusingonlythe analytical column and with the analytes reconstituted in themobile phase. Fig. 3A shows the HDVs obtained at a BDD electrode for 200-  L injections of 2.0mgL  − 1 SMX or TMP in 0.05molL  − 1 phosphatebuffersolution(pH5):ACN(82:18,v/v)atseveralpoten-tialsfrom1.00Vto1.30V vs. Ag/AgCl(3.0molL  − 1 KCl).TakingtheseHDVs into account as well as the need for a good compromisebetween the analytical-signal magnitude and the time requiredto stabilize the background current ( ∼ 20nA, after  ∼ 40min), theBDD electrode potential for the SMX/TMP determination was cho-senas1.25V vs. Ag/AgCl(3.0molL  − 1 KCl).AtypicalchromatogramrecordedatthiselectrodepotentialispresentedinFig.3B.Asitcan be observed from the SMX and TMP peak responses, a very goodchromatographic separation was attained under these conditions.Inordertoattainaselectiveseparationoftheanalytesfromtheendogenous milk compounds, typical chromatograms of drug-free  130  L.S. Andrade et al. / Analytica Chimica Acta 654 (2009) 127–132 Fig. 2.  Schematic diagram for the column-switching HPLC system. (A), (B), (C), and (D): mobile phases; W: waste. and SMX/TMP-spiked milk exclusion profiles were obtained usingthe RAM-BSA column (Fig. 4A). For this, only the UV–vis detector wasused.Asitcanbeobservedintheobtainedchromatograms,theproteinchromatographicbandsreturnedtothebaselinein ∼ 5min(corresponding to the necessary time for the exclusion of the milkproteins);after ∼ 12min,bothSMXandTMPco-elutedasanarrowband (highlighted as a “window” in the figure insert). The “win-dow” time is defined as the exact time needed for transferringSMXandTMPfromtheRAMtotheanalyticalcolumn,i.e.,thetimeperiod when the valve was set to position 2 (see Fig. 2). After the exclusionofthemilkproteinsandtransferenceoftheanalytes,thelatter were analyzed on the C 8  column using a 0.05molL  − 1 phos-phate buffer solution (pH 5):ACN (82:18, v/v). As it can be seen inFig.4B,thechromatogram(amperogram)obtainedfromtheanalyt-icalcolumnbyusingtheBDDelectrochemicaldetectorpresentedagood selectivity, without interference of endogenous compounds.This result clearly shows that the RAM-BSA column provides anadequate clean-up of the milk samples.The analytical curves obtained from the amperograms for SMXand TMP presented a good linearity in the investigated con-centration ranges (50–800 and 25–400  gL  − 1 , respectively). Thefollowing regression equations and correlation coefficients wereobtained: Q/ nC = 11 . 6 + 1 . 09[ c/ (  gL  − 1 )] for  SMX   ( r   = 0 . 9995) Q/ nC =− 1 . 62 + 1 . 65[ c/ (  gL  − 1 )] for  TMP   ( r   = 0 . 9996)The precision was expressed by the coefficient of variation (CV%)of the triplicates and presented values lower than 10%. Based on asignal-to-noise ratio of 3:1, the  LOD  values attained for SMX andTMP in the milk samples were 25.0 and 15.0  gL  − 1 , respectively.The  LOQ   values were 50 and 25  gL  − 1 for SMX and TMP, respec-tively. Therefore, the obtained  LOD  and  LOQ   values attest that themethod here reported is quite suitable for the monitoring of SMXand TMP residues in milk, since these values are lower than therespective MRL valuesformilk[6,7].Ontheotherhand,itshouldbe noticed that the BDD electrode did not present any fouling withinthe experimental period of several hours.The intra- and inter-day precision and accuracy of the methodwere determined by analyzing five replicates of three quality-control samples on three non-consecutive days. The precisionswereexpressedascoefficientsofvariation(CV%);theaccuracywasevaluated by back-calculation and expressed as the percent devia-tionbetweentheamountfoundandtheamountaddedatthethreeconcentrations examined. The obtained results, which led to satis-factory values of intra- and inter-assay precision and accuracy, areshown in Table 2. All values are within the acceptance criteria for determinations in biological fluids [44].These results are comparable to those obtained by Pereira andCass [13], using an UV–vis detector. The  LOD  values obtained bythem for SMX and TMP in bovine milk were 15.0 and 25.0  gL  − 1 ,respectively.Thus,theresultobtainedforTMPusingtheBDDdetec-tor (15.0  gL  − 1 ) is better than the one obtained using the UV–visdetector,whiletheoneforSMXusingtheelectrochemicaldetector(25.0  gL  − 1 )isworse.Itshouldbeemphasizedthatthechromato-graphic conditions here used for the electrochemical detectionwere the same optimized for the UV–vis detector [13]; only the buffer concentration was changed (increased by five times) whenthe electrochemical detection was used, in order to increase theionic strength.The main purpose of this work was to investigate the viabilityof the BDD electrochemical detector for SMX and TMP determina-tion in bovine milk. The obtained results clearly indicate that theBDD electrode performance is quite promising, especially becausethe chromatographic conditions used were the ones optimized foran UV–vis detector [13]. Recently [33], we found that the magni- tude of the peak current obtained for the SMX and TMP oxidation  Table 1 Time events for the column-switching procedure and mobile phases used.Time (min) Pump Event Valve position0.00–5.00 Pump 1 (eluent A) Exclusion of milk proteins by RAM column 1Pump 2 (eluent D) Conditioning of the analytical column 15.01–13.70 Pump 1 (eluent B) Elution of retained analytes from RAM 111.70–13.70 Pump 1 (eluent A) Transference of analytes to the analytical column 213.71–32.00 Pump 2 (eluent D) SMX and TMP analysis 113.70–22.00 Pump 1 (eluent C) Washing of the RAM column 122.01–32.00 Pump 1 (eluent A) Conditioning of the RAM column 1Pump 1 - eluents: (A) 0.05molL  − 1 KH 2 PO 4  (pH 6.0):ACN (95:5, v/v); (B) 0.05molL  − 1 KH 2 PO 4  (pH 6.0):ACN (83:17, v/v); (C) ACN:H 2 O:isopropanol (75:15:10, v/v/v). Flowrate: 1.0mLmin − 1 . Pump 2 - eluent (D): 0.05molL  − 1 KH 2 PO 4  (pH 5.0):ACN (82:18, v/v). Flow rate: 1.0mLmin − 1 .  L.S. Andrade et al. / Analytica Chimica Acta 654 (2009) 127–132  131 Fig. 3.  (A) Hydrodynamic voltammograms obtained for SMX (full line) and TMP(dashed line). (B) Chromatogram obtained at 1.25V  vs.  Ag/AgCl (3.0molL  − 1 KCl).[SMX]=[TMP]=2.0mgL  − 1 . Chromatographic conditions: 0.05molL  − 1 KH 2 PO 4  (pH5.0):ACN (82:18, v/v). (by DPV) increases significantly as the pH decreases from 7 to 2.ThechromatographicconditionsusedinthedeterminationofSMXand TMP in milk are limited by its physical properties, i.e., the pHrangewhereitsprecipitationoccurs.Astheisoelectricpointofmilkprotein occurs at pH 4.6, the on-line milk deproteinization by theRAM-BSA column was carried out at pH 6. Nevertheless, it shouldbenoticedthatitispossibletoworkinchromatographicconditionsat pH values lower than 4.6, even as low as 2.0 (when working inextreme conditions). In this case, a maximization of the SMX andTMPanalytical-signalsensibilitiescouldbeachievedusingtheBDDelectrochemical detector.Finally, it should be highlighted that very few 2D-LC methodswith electrochemical detection have been developed [30] and, as far as we could verify, the method here described is the first bydirect injection of milk samples containing SMX–TMP and usingelectrochemical detection. It should also be noted that analyses of several samples might be carried out without the need for elec-trode cleaning while using a low-cost “homemade” detector withan excellent sensitivity. Moreover, the quality of the performanceof the detector and the lifetime and stability of both columns weremaintained even after injection of over 20.6mL of milk samples. Fig. 4.  Typical chromatograms: (A) drug-free milk (dashed line) and SMX/TMP-spiked milk (full line) exclusion profiles showing the window time used;(B) drug-free milk (dashed line) and SMX/TMP-spiked milk (full line).[SMX]=[TMP]=2.0mgL  − 1 . Chromatographic conditions: (A) RAM octyl-BSA;mobile phase, 0.05molL  − 1 KH 2 PO 4  (pH 6.0):ACN (95:5, v/v);   =265nm; (B)analytical column (octyl-Luna); mobile phase, 0.05molL  − 1 KH 2 PO 4  (pH 5.0):ACN(82:18, v/v);  E  =1.25V  vs.  Ag/AgCl (3.0molL  − 1 KCl).  Table 2 Accuracy, intra- and inter-day variability, and recovery for the assay of SMX andTMP in bovine milk.Sample (  gL  − 1 ) Accuracy (%) a 1st day 2nd day 3rd day RecoverySMX80 97  ±  6 98  ±  1 104  ±  7 100  ±  5300 99  ±  4 105  ±  4 101  ±  3 102  ±  4700 96  ±  4 97  ±  1 100  ±  4 98  ±  5TMP40 98  ±  8 100  ±  5 104  ±  3 101  ±  5150 101  ±  7 108  ±  2 106  ±  6 105  ±  5350 102  ±  4 108  ±  5 107  ±  5 105  ±  5 a n =5. 4. Conclusions HDVs obtained at a BDD electrode allowed concluding that itspolarization at 1.25V  vs.  Ag/AgCl (3.0molL  − 1 KCl) was suitable forSMX–TMP determination in bovine milk, with a good compromisebetween the analytical-signal magnitude and the time requiredto stabilize the background. Additionally, no fouling of the BDDelectrode was observed within the experimental period of several
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