Rapid determination of sulfonamides in milk using micellar electrokinetic chromatography with fluorescence detection

Analytica Chimica Acta 552 (2005) 110–115

Rapid determination of sulfonamides in milk using micellarelectrokinetic chromatography with fluorescence detection

Sushma Lamba, Sunil Kumar Sanghi∗, Amit Asthana, Manjusha ShelkeRegional Research Laboratory, CSIR, Microfluidics and MEMS, Near Habibganj Naka, Hoshangabad Road, Bhopal, MP 462026, India

Received 8 April 2005; received in revised form 18 May 2005; accepted 19 May 2005Available online 3 October 2005

Abstract

A new method was developed for the determination of sulfonamides in milk by using micellar electrokinetic chromatography coupledwith fluorescence detection. Separation of fluorescamine-derivatized sulfonamides was accomplished by using a buffer 13.32 mM disodiumhydrogen phosphate, 6.67 mM potassium dihydrogen phosphate and 40 mM sodium dodecyl sulphate at pH 7.5 in addition to positivepower supply at 21 kV at 25◦C. Detection was performed using UG-11 excitation filter and 495 nm emission filters. The proposed capillaryelectrophoresis method allows the separation of five sulfonamides within 7 min with a limit of detection of 1.59–7.68 nmol/L for all the

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sulfonamides considered for present study. A simple sample preparation method with fairly good recoveries 85–114% is also pcurrent paper. Inter-day and intra-day validation of the separation method shows fairly good results. Robustness of the method hastudied.© 2005 Elsevier B.V. All rights reserved.

Keywords: Sulfonamides; Fluorescamine; Micellar electrokinetic chromatography; Milk

1. Introduction

Sulfonamide group of drugs is often used in veterinarypractice for therapeutic and prophylactic purposes[1]. Theyare also used in the treatment of human infections, but toa lesser extent[2]. Improper administration of these antibi-otics can leave residues in edible animal products like meat,milk, egg and fish[3–5]. One of the drugs, sulfamethazine issuspected to be carcinogenic and produce thyroid tumors inrodent[6] and others are known to cause allergic reactionsin human. Owing to their potential impact on human health,the European Union has adopted a maximum residue level(MRL) of 100�g/kg in edible animal tissue and 10�g/L inmilk [7]. Therefore there is a need for the development of

Abbreviations: MEKC, micellar electrokinetic chromatography; CE,capillary electrophoresis; MS, mass spectrometry; MS/MS, tandem massspectrometry; FT-IR, Fourier transform infrared; S.D., standard deviation;R.S.D., relative standard deviation

∗ Corresponding author. Tel.: +91 755 2489402; fax: +91 755 2488323.E-mail address: [email protected] (S.K. Sanghi).

sensitive and selective method for monitoring their reslevel in edible animal products.

There are several analytical methods for the determtion of sulfonamides, among them high performance liqchromatography with ultra-violet detection (HPLC–UV)the most widely applied[3,8]. HPLC with photodiode arradetection has also been applied for the determinatiosulfonamides in milk[9,10]; but the matrix influence fromtissue sample reduces the selectivity of the HPLC-UV detion. Therefore either a good matrix cleanup procedurequired[11] or a very selective detector is needed.chromatography coupled with mass spectrometry (GC–methods are relatively sensitive and selective[12,13], butroutine residue analysis by these methods are not feabecause of many purification steps required prior toanalysis of thermally labile and non-volatile sulfonamidHowever, several methods involving mass spectrometryfor detection such as liquid chromatography mass strometry (LC–MS)[14–16], LC tandem mass spectrome(LC–MS/MS) [17], gas chromatography mass spectrotry, HPLC atmospheric pressure chemical ionization m

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.

S. Lamba et al. / Analytica Chimica Acta 552 (2005) 110–115 111

spectrometry (APCI-MS)[18], packed column supercriticalfluid chromatography (pSFC) APCI-MS[19], have been suc-cessfully used for the determination of sulfonamides. Packedcolumn supercritical fluid chromatography interfaced to FT-IR spectrometry has also been applied to determine eightsulfonamides[20], but FT-IR detection is non-specific forsuch compounds. Capillary zone electrophoresis interfacedwith nano-electrospray MS/MS/MS has been successfullyapplied for the detection of sulfonamides in milk. However,the separation suffers from interferences from salt and fatfrom milk [21].

Fluorescence spectroscopic methods coupled with LChave been widely applied to the analytical problems, requir-ing highly sensitive detection. The sensitivity and selectivityof fluorometry after pre- and post-column derivatization haveencouraged their widespread use in the analysis of sulfon-amides. Post-column derivatization with fluorescamine hasbeen applied for the HPLC determination of sulfonamides inhuman saliva[22] and in salmon[23,24]. Another approachis the pre-column derivatization of sulfonamides with fluo-rescamine[25–27]and OPA[28]. HPLC in combination withfluorescence detection have an excellent limit of detection butat the cost of using toxic solvents for separation and prolongedanalysis time. Capillary electrophoresis has been proven tobe a highly efficient and rapid analytical technique for var-ious applications[29–32]. CE has inherent advantage overH velys con-s thed ne onC t-r es hro-m DS)a

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Stock solutions of sulfonamides (10−2 M) were pre-pared by dissolving appropriate amounts of the sulfon-amides in 3 mL of 3 M HCl, and then made up to 10 mLwith distilled water. An intermediate composite standardsolution and working standard solution were prepared bytaking an aliquot of the stock solution and diluting themixture with distilled water. All solutions were storedin dark at ∼4◦C and were stable for at least 1 month.Fluorescamine{4-phenylspiro[furan-(3H),1-phthalan]-3,3′-dione} was obtained from fluka (Buchs, Switzerland) asderivatizing reagent. Fluorescamine (10 mM solution in ace-tone) was prepared daily and refrigerated when not in use. Allother pure analytical quality chemicals were obtained fromstandard suppliers and used as received.

Acetate buffer (pH 5) used for reaction was prepared bymixing 5.01 mM acetic acid and 9.99 mM sodium acetate.The buffer used for the separation of sulfonamides was of pH7.5 and was prepared by mixing 13.32 mM disodium hydro-gen phosphate, 6.67 mM potassium dihydrogen phosphateand 40 mM sodium dodecyl sulphate (SDS).

2.2. Instrumentation and separation conditions

A Prince-C 255 instrument with programmable injectorand high voltage source (Prince Technologies, The Nether-l arriedo ion2 m-e icesL gthw doww romt cmc ami-c res-c tru-m curyl h aS asa tedl ticalc was8

nal-y wasu

2

singw ithd llaryw fol-l in,w for

PLC or GC such as highly efficient separations in relatihort time, small sample volumes and almost negligibleumption of organic solvents. CE is not very common foretermination of sulfonamides. Some work has been doE in this area, using UV detection[33–35]and amperome

ic detection[36]. Several others[37–39]have examined theparation of sulfonamides by micellar electrokinetic catography (MEKC) using sodium dodecyl sulphate (Ss a micellar phase.

In the present work, the applicability of capillary elrophoresis (CE) method for the analysis of residualonamides in milk has been evaluated. Present workith pre-column derivatization of sulfonamides with flu

escamine followed by MEKC coupled with sensitive flescence detection.

. Materials and methods

.1. Chemicals and solutions

Sulfanilamide, sulfathiazole, sulfamethoxazole, suuanidine, sulfadiazine and sodium acetate were obt

rom Sigma (St. Louis, MO, USA). Glacial acetic acid, ddium hydrogen phosphate and potassium dihydrogenhate were obtained from E. Merck (India). Sodium dodulphate (SDS) was used as surfactant and was obtrom BDH (India). Methanol (HPLC grade), hydrochic acid (AR), acetone (HPLC grade) was from RanbIndia).

ands) was used for the experiments. Separation was cut at 21 kV applied voltage (during reaction optimizat0 kV was applied). Fused silica capillary with internal diater of 75�m was purchased from Composite Metal Servtd. (Worcestershire, UK). A capillary of 53.2 cm total lenas used as separation column. A 1 cm detection winas created by burning off the coating at 30.2 cm f

he capillary inlet. For the study of reaction pH, a 54.4apillary was used. Samples were introduced hydrodynally, by applying pressure of 40 mbar for 12 s. For fluoence detection, an ARGOS 250 B instrument (Flux Insents, Switzerland) equipped with a 75 W xenon–mer

amp was used. The excitation light was filtered througchott glass UG-11 filter and a 495 nm cut off filter wpplied for the limited light. For the decoupling of emit

ight glycerol was applied between the capillary and opone. The voltage used for PMT (photo multiplier tube)00 V.

For data processing, DAx 7.1 Data Acquisition and asis Software (Prince Technologies, The Netherlands)sed.

.3. Electrophoretic method

Before first use, a new capillary was charged by rinith 0.1 M NaOH for 40 min, followed by a 15 min rinse weionized water. At the beginning of each day the capias regenerated by rinsing with methanol for 10 min,

owed by water for 5 min, 1 M hydrochloric acid for 10 mater for 5 min, 0.1 M sodium hydroxide for 20 min, water

112 S. Lamba et al. / Analytica Chimica Acta 552 (2005) 110–115

5 min and background electrolyte (BGE) for 20 min. Beforeeach run the capillary was first rinsed with 0.1 M NaOH for2 min. At the end of each day the capillary was flushed withdeionized water for 10 min followed by drying with air for2 min.

2.4. Sample preparation

The buffalo’s milk used in this experiment was purchasedfrom the local market. For the extraction of sulfonamidefrom milk, 100�L of 0.09 M hydrochloric acid was addedto 1 mL portion of the sample and diluted to 10 mL withmethanol. After shaking for 1 min the sample was allowed tostand for 15 min and the supernatant was derivatized for finalanalysis.

2.5. Derivatization procedure

To 1 mL of sample/working standard in acetate buffer (pH5), 40�L of fluorescamine solution (10 mM) was added andstirred vigorously for 1 min. The sample was allowed to standat room temperature for 20 min before injection.

3. Result and discussion

3

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s solu-t asesb owerp p ofs ferc M),w

3ctor,

w ey Mfl ationc ,c toa ts int enceq ine[ rbidw wasa gentc e alsod reas-i rela-t slowsd rom

Fig. 1. Effect of pH on fluorescence yield. Capillary: 54.4 cm (30.2 cm effec-tive length)× 75�m I.D. BGE: 13.32 mM disodium hydrogen phosphate,6.67 mM potassium dihydrogen phosphate (pH 7.5) and 40 mM sodiumdodecyl sulphate (SDS). Applied voltage: 21.4 kV. Peaks: 1, sulfanilamide;2, sulfaguanidine; 3, sulfadiazine; 4, sulfamethoxazole; 5, sulfathiazole.

several minutes to several hours, as also observed by Steinet al.[41], compared to milliseconds in aqueous medium.

Under optimized reaction condition the reaction com-pletes in 20 min and does not further increase with reactiontime (Fig. 1).

3.2. Optimization of separation conditions

3.2.1. Effect of pHThe changes in pH were found to affect the migration

behavior of sulfonamides (Fig. 2). At pH 7.5 (13.32 mMdisodium hydrogen phosphate and 6.67 mM potassium dihy-drogen phosphate) the effective mobilities of sulfadiazine,sulfamethoxazole, sulfathiazole changes effectively, howeverbaseline separation of the pair sulfanilamide and sulfaguani-dine was not observed. Hence at pH 7.5 sodium dodecylsulphate (SDS) was added for micellar electrokinetic sep-aration of the sulfonamides.

F een7

.1. Optimization of the derivatization conditions

.1.1. Effect of pH on reactionIt was found that maximum yield was obtained when

elected sulfonamides were derivatized in an aqueousion of pH between 3 and 5. At higher pH the yield decreecause of probable hydrolysis of fluorescamine and at lH the yield decreases due to protonation of amino grouulfonamides[40]. The best combination of pH and bufoncentration was found in acetate buffer pH 5 (15 mhich was selected for further studies.

.1.2. Effect of amount of fluorescamine and co-solventThe concentration of fluorescamine is another fa

hich affect the reaction yield[40]. The fluorescencield was optimized by using different amount of 10 muorescamine while keeping the sulfonamide concentronstant. It appeared that 40�L of 100 mM fluorescamineorresponding to 0.4 mM concentration is sufficientchieve the maximum yield. At higher reagent amoun

he reaction mixture, the yield decreases due to fluorescuenching by one of the hydrolysis product of fluorescam

40]. It was observed that reaction mixture becomes tuhen higher concentration of reagent (>0.5 mM)dded in the reaction mixture. It was found that the reaoncentration needed to achieve maximum fluorescencepended upon the amount of organic co-solvent. On inc

ng the percentage of acetone on the reaction mixture aive decrease in fluorescence intensity was observed. Itown the reaction of sulfonamides with fluorescamine f

ig. 2. Effective mobility of sulfonamides obtained at varied pH (betw.0 and 9.0). Operating conditions and peak numbering as inFig. 1.

S. Lamba et al. / Analytica Chimica Acta 552 (2005) 110–115 113

3.2.2. Effect of SDS concentrationThe pH studies make it clear that a more powerful sep-

aration technique like MEKC is needed to separate the fivesulfonamides. In current studies SDS the most common sur-factant used in MEKC has been employed for the separationof sulfonamides. Background electrolyte (BGE) system con-taining 20 mM phosphate buffer (pH 7.5) at different SDSconcentration up to 50 mmol/L was used to study the effectof SDS concentration on resolution. The results obtainedare shown inFig. 3, where effective mobilities were plottedagainst SDS concentration. An increase in migration timeand resolution of the five fluorescamine-derivatized sulfon-amides was observed when the SDS concentration in the BGEincreased. The results indicate markable change in reten-tion behavior of sulfathiazole and sulfaguanidine. Retentionof both the compound increases with increase in SDS con-centration. This is in accordance with the typical partitionbehavior of the compounds. Baseline resolution of all thefluorescamine-derivatized drugs was obtained at SDS con-centrations≥40 mmol/L. The migration and resolution offluorescamine derivatives of sulfonamides are significantlyaffected by the SDS concentration. Sodium dodecyl sulphate(40 mM) at pH 7.5 was found to be optimum for the separa-tion of all the five fluorescamine-derivatized sulfonamides.Further increase in SDS concentration increases separationtime as well as current. Separation of all five fluorescamine-dn e of2

3tion

o n of4 om-p tion.I onsb

F bili-t peakn

Fig. 4. Separation of a standard mixture of five derivatized sulfonamidesby MEKC. Applied voltage: 21 kV. Other operating conditions are as forFig. 1. Sample concentration: 10−6 mol/L of each compound. Peaks: *,electro-osmotic flow; 1, sulfanilamide; 2, sulfaguanidine; 3, sulfadiazine;4, sulfamethoxazole; 5, sulfathiazole.

3.3. Validation of the method

3.3.1. Recovery studiesThe extraction recovery was determined by comparing

the corrected peak areas of sulfonamides extracted fromspiked milk samples with that of the unextracted stan-dards containing the same amount of sulfonamides. For thedetermination of sulfonamides in milk three spiked sam-ples at concentrations of 5, 10 and 15�mol/L were used.The three spiking standards were prepared by transferringmilk (1 mL) into three different 10 mL calibrated flasks,then adding the spiked solution to the flasks and dilut-ing with methanol to 10 mL to give the desired concen-trations[19]. For the sample preparation and derivatizationsame procedure is applied as described in Sections2.4 and2.5. The results are shown inTable 1. The average recov-ery ranged from 85 to 114% in the concentration range of5–15�mol/L. The reproducibility of the extraction proce-dure is determined by three replicates at fortification level10�mol/L. Each replicate represents the mean of threevalues. The relative standard deviation of recoveries wereless than 4% for the sulfonamides, except for sulfathiazole(9.75%).

3.3.2. Precisionnedtan-ilityandsured-pro-ageboth.38)

erivatized sulfonamides is depicted inFig. 4. The plateumber under the optimum conditions was in the rang2 000–105 000.

.2.3. Effect of injection timeEffect of increase in the volume of the sample injec

n resolution was also studied. Hydrodynamic injectio0 mbar for 12 s (81.07 nL) was found to be the best cromise between good limits of detection and resolu

ncrease in injection volume lead to better limit of detectiut at the cost of resolution.

ig. 3. Effect of SDS concentration of the BGE on the effective moies of the fluorescamine derivatives. Other operating conditions andumbering as inFig. 1.

The precision of the analytical method is determiby comparing the effective mobilities of sulfonamide sdards.Table 2represents inter- and intra-day repeatabin terms of percentage R.S.D. in effective mobilitiescorrected peak area. Inter-day repeatability was meawithin 15 days. As indicated inTable 3 excellent intraday (%R.S.D. < 0.83) and inter-day (%R.S.D. < 1.77) reducibility for effective mobilities were achieved. PercentR.S.D. for corrected peak area was also fairly good forintra-day (%R.S.D. < 4.74) and inter-day (%R.S.D. < 4except for sulfathiazole.

114 S. Lamba et al. / Analytica Chimica Acta 552 (2005) 110–115

Table 1Recovery at different spiking level

Sulfonamides Recovery (%)

5�mol/L 10�mol/L 15�mol/L

Average± S.D. %R.S.D. Average± S.D. %R.S.D. Average± S.D. %R.S.D.

Sulfanilamide 87.33± 3.45 3.96 98.80± 1.10 1.12 101.15± 2.51 2.49Sulfaguanidine 85.12± 2.29 2.69 96.69± 1.60 1.65 97.40± 4.60 4.73Sulfadiazine 114.63± 4.62 4.03 101.22± 1.12 1.11 98.90± 2.45 2.48Sulfamethoxazole 100.28± 4.22 4.20 101.87± 0.75 0.74 100.84± 0.52 0.52Sulfathiazole 94.34± 9.62 10.20 105.61± 4.91 4.66 108.40± 3.13 2.89

Table 2Precision of analytical method

Sulfonamides %R.S.D. correctedpeak area

%R.S.D. effectivemobility

Intra-dayb Inter-dayc Intra-day Inter-day

Sulfanilamide 1.30 1.87 0.83 1.65Sulfaguanidine 2.54 1.92 0.75 1.28Sulfadiazine 1.30 1.87 0.69 1.77Sulfamethoxazole 1.05 4.36 0.72 1.74Sulfathiazole 4.74 12.17 0.68 1.30

a Sulfadiazine was taken as internal standard.b n = 6.c n = 6 and each value is average of three replicates.

3.3.3. Calibration curve and sensitivityCalibration curves were plotted between the concentration

range 50–1000 nmol/L and were found to be linear over thisrange. The correlation coefficients,r, of the standard curves,concentration limit of detection (LOD) and limits of quanti-tation (LOQ) for five sulfonamides under the selected condi-tions are summarized inTable 3. Limit of detection is definedas concentration, which gives a signal to noise ratio of 3:1.The detection limits is between the range 1.59–7.68 nmol/L,which was far below the maximum residue level for milk. TheLOQ is defined as average of background plus 10 standarddeviations.

The electropherogram of the blank milk sample (Fig. 5B)shows a small peak having same effective mobility as thoseof sulfaguanidine in the spiked milk sample (Fig. 5A), whichdenotes to 2.02�mol/L sulfaguanidine in the milk. Sulfadi-azine was used as internal standard.

Fig. 5. The electopherogram of milk samples (A) spiked with five sul-fonamides at 10�mol/L. (B) Blank milk sample with internal standardsulfadiazine at 10�mol/L. Peaks: *, electro-osmotic flow; 1, sulfanilamide;2, sulfaguanidine; **, unknown; 3, sulfadiazine; 4, sulfamethoxazole; 5, sul-fathiazole. Other operating conditions as inFig. 4.

3.3.4. RobustnessRobustness relates to the capacity of the method to remain

unaffected by small but deliberate variations introduced in

Table 3Calibration curve and detection limits

Sulfonamides y = a + bxa LOD (nmol/L) LOQ (nmol/L)

a ± S.D. Sab b ± S.D. Sb

c r2

Sulfanilamide 1.18× 10−5 ± 1.18× 10−5 1.12× 10−5 4827.65± 4827.65 21.78 0.9999 1.59 5.3Sulfaguanidine −7.6× 10−5 ± 7.55× 10−5 1.98× 10−5 3935.59± 3935.59 38.43 0.9996 2.36 7.88Sulfadiazine 3.17× 10−6 ± 3.17× 10−5 1.3× 10−5 3449.12± 3449.12 25.18 0.9997 3.29 10.97S −5 −5 −6 350.35± 350.35 9.69 0.9997 7.68 25.59S 340.82± 1340.82 50.84 0.9942 7.04 23.47

ope.

ulfamethoxazole 1.54× 10 ± 1.54× 10 5× 10 1ulfathiazole 4.47× 10−5 ± 4.47× 10−5 2.62× 10−5 1a x: concentration (mol/L);y: peak area/migration time;a: intercept;b: slb Standard error in intercept.c Standard error in slope.

S. Lamba et al. / Analytica Chimica Acta 552 (2005) 110–115 115

Table 4Robustness of the method

Parameter changed Parameter studied

Effectivemobilities(%R.S.D.)

Correctedpeak area(%R.S.D.)

NaOH rinsing (1.5, 2.0, 2.5 min) 0.72–1.0 –Buffer rinsing (1.5, 2.0, 2.5 min) 1.0–9.6 –Injection time (11.4, 12.0, 12.6 s) – 3.6–14.2SDS concentration (36, 40, 44 mM) 1.7–2.9 −Phosphate concentration (18, 20, 22 mM) 5.5–6.2 −Separation temperature (23, 25, 27◦C) 0.8–1.6 −

method parameters. The most relevant factors to investigateare the electrolyte composition, injected volume, separationtemperature and rinse time etc. These factors are variedaround the value set in the method to reflect the changeslikely to arise in different test environment. The results ofrobustness study are summarized inTable 4indicating thatthe method is fairly robust under different test conditions.

4. Conclusion

An MEKC method has been developed for the determina-tion of sulfonamides in milk after pre-column derivatizationwith fluorescamine. The method has been validated and theresults showed acceptable performances. The LOD and LOQfor all sulfonamides were adequate for practical analysis inmilk sample. The simplified extraction procedure, includingextraction with methanol, enables quantitative determinationof five of the most used sulfonamides at concentration farbelow maximum residue level (10�g/L) The advantage ofMEKC for sulfonamides analysis is the elimination of theneed for expensive organic solvents and column. Analysis byCE is also simple, rapid and robust.

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