Simultanea 17 Sulfonamida in Porcine

Analytica Chimica Acta 546 (2005) 174–181

Simultaneous determination of 17 sulfonamide residues in porcine meat,kidney and liver by solid-phase extraction and liquid

chromatography–tandem mass spectrometry

Bing Shaoa,∗, Dan Donga,d, Yongning Wub, Jianying Huc, Juan Menga,Xiaoming Tua, Shukun Xud

a Institute of Nutrition and Food Hygiene, Beijing Center for Disease Prevention and Control, Beijing 100013, Chinab National Institute of Nutrition and Food Safety, Chinese Center for Disease Prevention and Control, Beijing 100050, China

c College of Environmental Sciences, Peking University, Beijing 100085, Chinad Department of Applied Chemistry, Northeastern University, Shenyang 110004, China

Received 14 January 2005; received in revised form 29 April 2005; accepted 3 May 2005Available online 20 June 2005

Abstract

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A precise and reliable method, using normal phase cartridge clean-up and liquid chromatography coupled with tandem mass sas been developed for measuring the residues of 17 sulfonamides in porcine tissues included in meat, liver and kidney. The paass spectrometer were optimized by continuous direct injection through a syringe pump. The composition and additives for moere also evaluated and the optimum one was methanol–water containing 0.1% formic acid. A liquid–liquid extraction and Seplean-up procedure was developed for sample preparation. The method was validated and found to be satisfactorily linear, selectivecoveries ranged from 52 to 120% for all compounds at three different spiking levels and the limit of detections (LODs) ranged

o 1.0 ng/g depending on various compounds.2005 Elsevier B.V. All rights reserved.

eywords:Sulfonamides; LC–MS/MS; Porcine tissues

. Introduction

Sulfonamides (Fig. 1) are a group of synthetic antibioticshat have played an important role as effective chemother-peutics in bacterial and protozoan infections in veterinaryedicine practice. They have been widely used in animal feeds growth promoters to prevent and treat a series of diseases innimals’ feeding, such as infectious diseases of digestive andespiratory tracts. As a consequence of their extensive usage,onsiderable attention has been paid to the potential humanealth risk due to their carcinogenic potency and possiblentibiotic resistance[1–4]. To safeguard human health, the

∗ Corresponding author. Tel.: +86 10 64212461x503;ax: +86 10 64214405.E-mail address:[email protected] (B. Shao).

European Union (EU) and other countries including Chave established safe maximum residue limits (MRLs) atotal level of 100�g/kg in food of animal origin[5,6].

Pork is the main kind of meat consumed in China becof its high nutrition, inexpensiveness and availability. Iestimated that the average consumption in 2005 wi54.4 kg/person/year[7], and pork will be the main contributfor exposure to sulfonamides if not well controlled. Thefore, it is necessary to develop a sensitive and reliable meto monitor the sulfonamide residue level in tissues. Mmethods such as GC–MS, GC, LC and LC–MS havedeveloped to analyze sulfonamide residue in food. GC[8]and LC[9–11]lack sensitivity and specificity and can onlyused for screening of high levels of residues. GC–MS[12,13]methods require derivation because of the high polaritylow volatility of sulfonamides.

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.05.007

B. Shao et al. / Analytica Chimica Acta 546 (2005) 174–181 175

Fig. 1. Sulfonamides structure, name, CAS number and pKa1 value.

Liquid chromatography coupled with electrospray ion-ization mass spectrometry (LC–ESI-MS) has proven to bea promising technique for residual analysis with its highselectivity, specificity and sensitivity for the high polarityof sulfonamides[13–23]. At present, only five LC–ESI-MSmethods have been reported to detect the residues of sulfon-amides in meat[14,16,19,21,23]. However, when using thehighly sensitive and specific LC–MS, the sample purificationwas ignored, which led to high limits of detection (LODs),over 1�g/kg. This value was also obtained using LC withfluorescence detection[24].

A clean sample is an important factor closely associatedwith the sensitivity of an analytical method. The preva-lent clean-up procedures are liquid–liquid extraction (LLE)using hexane to remove fat[14,27]and reversed phase solid-phase extraction (SPE) used to concentrate the samples[9,17,18,26]. Fuh [25] used neutral Alumina and macrop-orous copolymer SPE column for clean-up as well as capil-lary electrophoresis to detect eight sulfonamides in meat anddetermined the LOD compared with that using LC–MS. Helaand Tarbin[13,18]used ion-exchange SPE to purify sampleextracts of egg and animal tissues. However, the fat in tissues

176 B. Shao et al. / Analytica Chimica Acta 546 (2005) 174–181

is mainly responsible for interference. After liquid–liquidextraction and solid-phase extraction (SPE) using reversedphase and ion-exchange sorbents, only part of the fat isremoved.

To overcome these problems, in this study, a silica nor-mal phase SPE method combined with LC–ESI-MS/MS wasapplied to detect the wide range polarity of 17 sulfonamidesin pork, and porcine liver and kidney. Comprehensive sam-ple preparation and mobile phase composition and additivesoptimization procedures with good resolution and high sen-sitivity are descried.

2. Experimental

2.1. Standards and reagents

Organic solvents such as methanol, acetonitrile, hex-ane, chloroform and 1-propanol were all pesticide residuegrade, commercially available from Scharlau Chemic S.A.(Barcelona, Spain). Formic acid (99%), acetic acid (99%)and trifluoroacetic acid (99%) were from Acros Organics(New Jersey, USA). Anhydrous sodium sulfate and ammo-nium formate were all analytical grade (Beijing, China). Ultrapure water was made by the Milli-Q ultrapure system (Mil-lipore, Bedford, MA, USA). Sulfonamide standards wereo es,s ,1 mC MA,U ,a car-t sedf i-nu eachb

ncesa resa ininga bilep n-d ionsw

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to a5T l ace-t witha rpmf sep-a with1 0 mln sam-

ple was mixed and separated. The upper layer was discarded(n-hexane) and 5 ml 1-propanol was added. The solvent wasevaporated to near dryness by a rotary evaporator at 30◦C.The residue was dissolved by ultrasonication for 30 s with0.4 ml chloroform and 3.6 mln-hexane. The solution thenwas passed through the Sep-Pak Silica solid-phase extrac-tion cartridge preconditioned by 6 mln-hexane without anypressure. Five millilitersn-hexane was used to wash the inter-ference. The analytes were eluted sequentially with 6 mlmethanol–acetone (v/v, 1:1) and 6 ml acetone. The eluentswere dried under a gentle nitrogen stream, and reconstitutedwith 1 ml mobile phase for LC–MS/MS analysis.

2.3. LC–MS/MS analysis

Identification and quantification of analytes were carriedout using an Alliance 2695 (Waters, USA) liquid chromatog-raphy equipped with a Quattro Ultima Pt (Micromass, UK)tandem mass spectrometer. The Xterra® MS C-8 column(150 mm× 2.1 mm I.D., 3.5�m, Waters, USA) was used forLC separation. The temperature of the column oven was28◦C, the flow rate was 0.2 ml/min, and the injection vol-ume was 10�l. Methanol and water were used as mobilephase containing 0.1% formic acid. The methanol was lin-early increased from 3 to 15% in 13 min, then increased to40% in 9 min and held for 3 min, it was then increased to 80%i in,a minu

y pos-i Thec 0 V.T withU ax-i weres e andd 00r lens2 ergy1 zero.T pec-t s andtT sedf

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me-t olt-a n gast pres-s ere

btained from Sigma (St Louis, MO, USA). Drug namtructures and pKa are shown inFig. 1. Internal standard3C6-sulfamethazine (13C6-SMA, 90%) was obtained froambridge Isotope Laboratories (50 Frontage Road,SA). All standards were stored at−20◦C. Sep-Pak silicamino-propyl and cyan-propyl solid-phase extraction

ridges containing 500 mg materials (3 ml) were purcharom Waters (Milford, MA, USA). To avoid cross contamation, all the glassware was heated for 4 h at 400◦C prior tose. In addition, procedural blanks were conducted foratch of samples to ensure minimal contamination.

Stock solutions were prepared for all standard substat 1000 mg/l in acetonitrile. Spiking and calibration mixtut various concentration levels were obtained by combliquots of stock solutions and internal standard with mohase and storing at 4◦C. The concentration of internal staard in all the calibration mixtures and final sample solutas 50�g/l.

.2. Sample preparation

Ten grams of minced porcine tissues was placed in0 ml polypropylene tube and spiked with 50 ng13C6-SMA.en grams baked anhydrous sodium sulfate and 15 monitrile was then added. The sample were homogenized

refiner for about 1 min, and then centrifuged at 8000or 10 min at 0◦C. The supernatant was decanted into aratory funnel. The residue was extracted two times5 ml acetonitrile. The extracts were combined. Then 2-hexane was added into acetonitrile extracts, and each

n 5min, then increased to 100% in 1 min and held for 5 mnd finally it was brought back to 3% and held for 20ntil the next injection.

The mass spectrometer was operated in electrosprative ion mode. The capillary voltage was held at 3.5 kV.one voltage was 78 V. The multiplier voltage was 65he nebulizing, desolvation and cone gas were suppliedHP nitrogen. The nebulizing gas was adjusted to the m

mum, and the flow of desolvation gas and cone gaset to 450 and 0 l/h, respectively. The source temperaturesolvation gas temperature were held at 100 and 3◦Cespectively. The radio frenquency (RF) lens 1 and RFwere set as 40 and 0.5 V. The ion energy 1 and ion enwere both 0.5 V. The entrance and exit voltage werehe collision gradient was 1.9. During tandem mass s

rometric analysis, UHP argon was used as collision gahe pressure of collision chamber was held 3.1× 10−3 mbar.he multiple reaction monitoring transition conditions u

or quantitation of various sulfonamides are listed inTable 1.

. Results and discussion

.1. Optimizing LC–MS/MS condition

To achieve the maximum sensitivity, the mass spectrory parameters including ionization mode, the capillary vge, cone voltage, source temperature and desolvatio

emperature, desolvation gas and cone gas flow, theure of the collision chamber, and the collision energy w


Table 1Sulfonamide data acquisition parameters (LC–MS/MS) and instrumental detection limit

Chemicals Retention time (min) MH+ (m/z) Daughter ion (m/z) Collision energy (eV) DLa (pg)

SA 3.49 173.2 156.0 132.0 9 0.7SIM 12.23 279.3 124.1 186.1 156.0 18 0.2SDZ 13.19 251.3 156.1 108.1 92.1 15 0.3SPD 15.53 250.3 156.0 184.2 15 0.2STZ 15.78 256.4 156.1 108.1 92.1 19 0.3SMR 17.34 265.3 156.0 172.1 110.1 15 0.1SDMD 20.74 279.2 186.0 124.1 156.0 20 0.5SME 21.21 281.3 156.0 126.1 108.0 15 0.3SMT 21.33 271.2 156.0 108.0 92.0 14 0.1SMP 21.71 281.3 156.0 126.1 108.1 15 0.1SCP 22.42 285.2 156.0 207.1 12 0.3SMX 23.49 254.3 156.0 108.1 92.1 13 0.1SIX 24.66 268.2 156.0 113.1 108.1 15 0.2SMO 24.66 268.3 156.0 113.1 108.1 16 0.2SDM 28.10 311.4 156.1 108.1 92.1 19 0.1SQX 29.17 301.2 156.0 107.9 15 0.1SNT 32.35 336.4 156.0 294.3 198.1 10 0.213C6-STZ 20.67 284.5 185.8 123.9 161.2 19

a DL: detection limit (three times of the signal-to-noise ratio).

first optimized by direct flow infusion of each standard. Theresults indicated that the positive mode was more favorablethan the negative ion mode. The characteristic ion and col-lision energy for each compound are listed inTable 1. Allthe compounds except isotopic internal standard gave a char-acteristic ion atm/z156.0, although the abundance may bedifferent depending on the variable compounds.

As for the sulfonamides with a wide pKa range investi-gated in our study, the properties of mobile phases such aspH and additives can affect the LC resolution. In addition,considering the characteristics of electrospray ionization, themobile phase composition and additive may significantlyaffect the response. Therefore, the additive and the mobilephase composition were comprehensively investigated. Inprevious paper[22], the mobile phase parameter, concen-tration of formic acid, pH of the aqueous solution and ratioof the aqueous solution and the organic solvent were opti-mized using methanol–acetonitrile 0.05 M formic acid asmobile phase only to achieve a satisfactory separation of10 sulfanamides. For the mobile phase composition in thisstudy, the response using methanol–water as mobile phasewas significantly higher than that using acetonitrile–water.In addition, the organic phase content can significantly affectthe sensitivity. Among the 17 sulfonamides, the sensitivity ofthe sulfamilamide (SA) was far lower than others, as its elu-tion at the time of 3.49 min contained about 7% of methanol int asec SAw itsh

cid,t con-c cida n and

sensitivity of the mixture standard increased with the increas-ing of the formic acid concentration from 0.05% (pH = 2.98)to 0.1% (pH = 2.28); however, the resolution and sensitivitydecreased when the formic acid concentration increased to0.15% (pH = 1.91). The change of resolution can be attributedto the change of polarity for the sulfonamides with differ-ent pKa, which induced to the change of the capacity fac-tor of each sulfonamide. However, the concentration higherthan 0.1% may suppress the ionization. Acetic acid, trifluo-roacetic acid and ammonium acetate were also optimized asabove, 0.2% of acetic acid (pH = 3.43), 0.1% trifluoroaceticacid (pH = 2.28) and 5 mmol/l ammonium acetate (pH = 6.16)were the optimal composition and additives. In comparisonwith the response using formic acid, 0.1% formic acid wasthe optimum additive.

3.2. Sample clean-up

In addition to the instrument parameters, mobile phasecomposition and additives, the clean-up procedure is animportant factor that has an influence on sensitivity. Forfoods of animal origin, the fat is a critical factor, as it candecrease the sensitivity and shorten the lifetime of a column.Liquid–liquid extraction usingn-hexane can only remove partof the fat. Therefore, normal phase SPE procedures are oftenused to purify the bio-samples and enhance the method’ss hasr afterl ,a thec ee per)i 6 mla

he mobile phase composition. However, if the mobile phomposition started with a high content methanol, theould elute at a dead volume without retention due toigh polarity.

Among these additives such as formic acid, acetic arifluoroacetic acid and ammonium acetate, differententration levels were also optimized. Taking formic as an example, the results suggest that the resolutio

ensitivity. However, only one method for sulfonamideseported the further clean up using normal phases SPEiquid–liquid extraction from tissue[25]. The Sep-Pak silicamino-propyl and cyan-propyl SPE were used to purifyrude sample extracts at 100�g/kg fortification to evaluatfficiency of clean up. The results (not listed in this pa

ndicated that the Sep-Pak silica was satisfactory usingcetone–methanol (1:1) and 6 ml acetone as eluent.


Fig. 2. The chromatograms of spiking samples containing 10�g/kg of each compound.


Fig. 2. (Continued).

3.3. Method performance

The chromatograms of spiking samples containing10�g/kg of each compound are shown inFig. 2. Because oftheir similar structures and the limited commercial availabil-ity of stable isotope compounds, only one internal standardwas used. The calibration curves for detection of the analyteswere obtained by performing a linear regression analysis on

standard solution using the ratio of the standard area to inter-nal standard area (each sulfonamide for13C6-SMA) againstthe analytes concentration ranging from 1.0 to 200�g/lcontaining 50�g/l internal standard, i.e. 10.0–2000 pg with10�l injection. The linearity obtained for all analytes weregood with correlation coefficients higher than 0.999. Theinstrumental detection limits (IDL) with 10�l injectionranged from 0.1 to 0.7 pg depending on different compounds,

Table 2Recoveries, RSD and LOD of spiked porcine meat tissue

Compound Spiked level 1�g/kg Spiked level 5�g/kg Spiked level 10�g/kg LOD (ng/g)

Meat Liver Kidney Meat Liver Kidney Meat Liver Kidney Meat Liver Kidney

SA 65.2 (11.6) 69.6 (5.8) 74.3 (3.1) 75.8 (8.9) 72.8 (10.2) 82.3 (14.4) 90.9 (9.4) 74.5 (13.6) 68.8 (14.8) 0.3 0.5 0.4SIM 69.8 (8.1) 66.2 (7.3) 58.1 (8.3) 67.8 (10.1) 64.4 (8.5) 60.4 (8.5) 69.0 (13.6) 72.7 (6.1) 68.5 (5.7) 0.02 0.02 0.04SDZ 98.2 (11.5) 78.0 (6.4) 67.9 (5.0) 95.1 (10.7) 71.8 (9.8) 61.5 (5.7) 102.0 (8.9) 75.9 (8.9) 60.9 (9.9) 0.02 0.08 0.33SPD 105.3 (4.7) 88.0 (3.6) 88.9 (2.7) 91.4 (7.6) 89.4 (4.2) 78.8 (10.8) 89.4 (13.1) 82.6 (8.9) 70.7 (7.0) 0.02 0.02 0.06STZ 68.3 (14.0) 70.2 (10.9) 85.4 (7.1) 64.7 (2.7) 88.7 (14.5) 76.4 (10.5) 85.0 (19.8) 90.3 (15.3) 76.7 (8.1) 0.01 0.06 0.15SMR 102.3 (11.3) 85.1 (9.1) 87.7 (10.4) 114.0 (6.7) 89.3 (7.1) 93.9 (5.1) 108.3 (5.6) 97.2 (8.8) 100.5 (8.8) 0.03 0.03 0.07SDMD 112.1 (5.2) 105.4 (8.2) 114.9 (6.2) 108.0 (2.9) 111.7 (5.4) 112.3 (4.8) 111.0 (8.4) 119.2 (6.2) 114.1 (4.1) 0.05 0.04 0.14SME 65.6 (7.2) 56.7 (13.0) 55.5 (4.6) 71.4 (11.3) 67.4 (8.2) 72.8 (9.4) 81.3 (17.4) 65.4 (11.0) 68.3 (5.4) 0.01 0.05 0.12SMT 65.2 (5.2) 83.0 (10.5) 84.1 (14.7) 97.1 (6.7) 97.8 (7.4) 87.1 (9.8) 117.4 (3.5) 105.9 (10.8) 110.1 (7.9) 0.05 0.08 0.11SMP 89.1 (6.8) 86.0 (5.5) 83.3 (2.9) 95.9 (6.6) 84.9 (9.1) 78.8 (4.2) 112.2 (2.9) 90.0 (5.4) 82.5 (8.6) 0.05 0.06 0.08SCP 90.7 (14.3) 89.9 (10.9) 91.1 (13.0) 83.3 (10.3) 94.0 (12.9) 79.7 (6.3) 94.9 (10.8) 101.8 (7.2) 95.3 (11.6) 0.03 0.03 0.11SMX 111.9 (14.4) 70.9 (5.5) 78.2 (6.6) 97.2 (5.1) 78.1 (8.4) 66.7 (16.3) 91.7 (10.8) 79.3 (9.6) 75.1 (5.7) 0.02 0.01 0.05SIX 90.8 (8.4) 95.2 (9.7) 107.1 (12.0) 94.7 (6.0) 89.67 (6.7) 79.8 (12.1) 85.2 (10.4) 104.4 (8.1) 89.1 (12.1) 0.02 0.02 0.08SMO 87.7 (13.3) 95.1 (14.2) 111.2 (8.6) 60.8 (14.2) 101.4 (9.6) 86.7 (11.8) 64.5 (9.7) 94.9 (16.4) 84.4 (10.9) 0.02 0.02 0.06S (16.4)S (9.0)S (14.1)

V

DM 78.9 (18.6) 84.6 (15.9) 84.6 (14.3) 83.8 (8.5) 95.2QX 59.9 (7.0) 67.3 (6.5) 64.2 (15.5) 60.8 (4.1) 62.5NT 53.5 (7.1) 52.33 (12.3) 53.0 (16.2) 52.5 (6.2) 54.2

alues in parentheses are % relative standard deviations forn= 5.

73.0 (14.8) 97.1 (13.5) 89.8 (14.2) 75.7 (9.5) 0.02 0.02 0.0557.6 (8.8) 64.5 (4.4) 62.6 (8.5) 56.6 (9.6) 0.05 0.08 0.2052.4 (15.8) 62.7 (14.8) 58.5 (14.5) 53.1 (8.9) 0.10 0.17 1.00


Fig. 3. The typical chromatogram of one sample available from local market.

respectively, which was estimated at a signal-to-noise (S/N)ratio of 3. The recoveries of analytes in five replicates ateach level were evaluated at three levels by spiking 10.0,50.0 and 100.0 ng of each standard analyte to 10.0 g tissues.The results are listed inTable 2. The average recoveries ofeach compound are ranged from 52 to 120%. Generally, therecoveries in kidney were higher than those in meat, followedby those in liver. SDMD is widely used and could be found inmost of the sample; therefore, the recoveries were all higherthan 100 for all sample matrices. This may be the influenceof the background sample, although the background valuehas been subtracted. In addition, according to the suggestionof AOAC on the analysis of residues[28], these values areacceptable. The reproducibilities of this method representedby the percent of the relative standard deviation (RSD) ateach fortification level are also summarized inTable 2andshowed that the method precision was within 20%, which issatisfactory. The within-day and between-day reproducibil-ities were evaluated by injecting a standard mixture fivetimes at three different levels each day for five consecutivesdays. The within-day reproducibilities ranged from 1.9 to3.8% and between-day reproducibilities ranged from 3.5to 8.2%. The limits of detection for sulfonamides in pork,liver and kidney tissues, defined as the concentration whichyield an S/N equal to three, are described inTable 2. TheLODs for all the compounds were lower than 1�g/kg, which

was much higher than those reported in previous papers[13–23].

4. Analysis of real samples

This method has been used to analyze 15 porcine meatsamples available from local market. Among the 15 samples,SME was found in 10 samples with concentration levels rang-ing from 0.01 to 15.43�g/kg, and SDMD ranging from 0.08to 7.34�g/kg in nine of the 15 samples. SDZ, SMX and SQXwere also found only in one sample, with a concentration levellower than 0.4�g/kg. SA was found in two of the 15 samples,with a concentration 3.47 and 0.98�g/kg, respectively.Fig. 3shows a typical chromatogram of one sample available fromthe local market with SA (0.98�g/kg), SDMD (0.81�g/kg)and SMT (0.03�g/kg) found in the sample.

5. Conclusions

In this study, a clean-up procedure and LC–MS/MSmethod has been developed and optimized to measurethe residues of 17 sulfonamides in porcine tissues. Themethanol–water containing 0.1% formic acid as mobile phaseshowed good LC resolution and MS sensitivity. The LODswere 2–4 magnitudes lower than the MRLs of defined by EU.


Acknowledgement

The authors wish to acknowledge the Beijing NaturalScience Foundation for financial support under grant num-ber 7041004 and the Ministry of Science and Technology(2001BA804A18-02, 2001BA804A55).

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Documents

Simultanea 17 Sulfonamida in Porcine