Simultaneous determination of 17 sulfonamide residues in porcine meat, kidney and liver by solid-phase extraction and liquid chromatography–tandem mass spectrometry

Abstract

A precise and reliable method, using normal phase cartridge clean-up and liquid chromatography coupled with tandem mass spectrometry has been developed for measuring the residues of 17 sulfonamides in porcine tissues included in meat, liver and kidney. The parameters of mass spectrometer were optimized by continuous direct injection through a syringe pump. The composition and additives for mobile phases were also evaluated and the optimum one was methanol–water containing 0.1% formic acid. A liquid–liquid extraction and Sep-Pak silica clean-up procedure was developed for sample preparation. The method was validated and found to be satisfactorily linear, selective and robust. Recoveries ranged from 52 to 120% for all compounds at three different spiking levels and the limit of detections (LODs) ranged from 0.01 to 1.0 ng/g depending on various compounds.

Full text

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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
aInstitute of Nutrition and Food Hygiene, Beijing Center for Disease Prevention and Control, Beijing 100013, China
bNational Institute of Nutrition and Food Safety, Chinese Center for Disease Prevention and Control, Beijing 100050, China
cCollege of Environmental Sciences, Peking University, Beijing 100085, China
dDepartment of Applied Chemistry, Northeastern University, Shenyang 110004, China
Received 14 January 2005; received in revised form 29 April 2005; accepted 3 May 2005
Available online 20 June 2005
Abstract
A precise and reliable method, using normal phase cartridge clean-up and liquid chromatography coupled with tandem mass spectrometry
has been developed for measuring the residues of 17 sulfonamides in porcine tissues included in meat, liver and kidney. The parameters of
mass spectrometer were optimized by continuous direct injection through a syringe pump. The composition and additives for mobile phases
were also evaluated and the optimum one was methanol–water containing 0.1% formic acid. A liquid–liquid extraction and Sep-Pak silica
clean-upprocedurewasdevelopedforsamplepreparation.Themethodwasvalidatedandfoundtobesatisfactorilylinear,selectiveandrobust.
Recoveries ranged from 52 to 120% for all compounds at three different spiking levels and the limit of detections (LODs) ranged from 0.01
to 1.0ng/g depending on various compounds.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Sulfonamides; LC–MS/MS; Porcine tissues
1. Introduction
Sulfonamides (Fig. 1) are a group of synthetic antibiotics
that have played an important role as effective chemother-
apeutics in bacterial and protozoan infections in veterinary
medicinepractice.Theyhavebeenwidelyusedinanimalfeed
asgrowthpromoterstopreventandtreataseriesofdiseasesin
animals’feeding,suchasinfectiousdiseasesofdigestiveand
respiratory tracts. As a consequence of their extensive usage,
considerable attention has been paid to the potential human
health risk due to their carcinogenic potency and possible
antibiotic resistance [1–4]. To safeguard human health, the
∗Corresponding author. Tel.: +86 10 64212461x503;
fax: +86 10 64214405.
E-mail address: shaob@bjcdc.org (B. Shao).
European Union (EU) and other countries including China
have established safe maximum residue limits (MRLs) at the
total level of 100?g/kg in food of animal origin [5,6].
Pork is the main kind of meat consumed in China because
of its high nutrition, inexpensiveness and availability. It is
estimated that the average consumption in 2005 will be
54.4kg/person/year[7],andporkwillbethemaincontributor
for exposure to sulfonamides if not well controlled. There-
fore,itisnecessarytodevelopasensitiveandreliablemethod
to monitor the sulfonamide residue level in tissues. Many
methods such as GC–MS, GC, LC and LC–MS have been
developed to analyze sulfonamide residue in food. GC [8]
andLC[9–11]lacksensitivityandspecificityandcanonlybe
usedforscreeningofhighlevelsofresidues.GC–MS[12,13]
methods require derivation because of the high polarity and
low volatility of sulfonamides.
0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2005.05.007

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B. Shao et al. / Analytica Chimica Acta 546 (2005) 174–181
175
Fig. 1. Sulfonamides structure, name, CAS number and pKa1value.
Liquid chromatography coupled with electrospray ion-
ization mass spectrometry (LC–ESI-MS) has proven to be
a promising technique for residual analysis with its high
selectivity, specificity and sensitivity for the high polarity
of sulfonamides [13–23]. At present, only five LC–ESI-MS
methods have been reported to detect the residues of sulfon-
amides in meat [14,16,19,21,23]. However, when using the
highlysensitiveandspecificLC–MS,thesamplepurification
was ignored, which led to high limits of detection (LODs),
over 1?g/kg. This value was also obtained using LC with
fluorescence detection [24].
A clean sample is an important factor closely associated
with 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 and
determinedtheLODcomparedwiththatusingLC–MS.Hela
and Tarbin [13,18] used ion-exchange SPE to purify sample
extracts of egg and animal tissues. However, the fat in tissues

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B. Shao et al. / Analytica Chimica Acta 546 (2005) 174–181
is mainly responsible for interference. After liquid–liquid
extraction and solid-phase extraction (SPE) using reversed
phase and ion-exchange sorbents, only part of the fat is
removed.
To overcome these problems, in this study, a silica nor-
mal phase SPE method combined with LC–ESI-MS/MS was
applied to detect the wide range polarity of 17 sulfonamides
in pork, and porcine liver and kidney. Comprehensive sam-
ple preparation and mobile phase composition and additives
optimization 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 residue
grade, 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-
niumformatewereallanalyticalgrade(Beijing,China).Ultra
pure water was made by the Milli-Q ultrapure system (Mil-
lipore, Bedford, MA, USA). Sulfonamide standards were
obtained from Sigma (St Louis, MO, USA). Drug names,
structures and pKaare shown in Fig. 1. Internal standard,
13C6-sulfamethazine (13C6-SMA, 90%) was obtained from
Cambridge Isotope Laboratories (50 Frontage Road, MA,
USA). All standards were stored at −20◦C. Sep-Pak silica,
amino-propyl and cyan-propyl solid-phase extraction car-
tridges containing 500mg materials (3ml) were purchased
from Waters (Milford, MA, USA). To avoid cross contami-
nation, all the glassware was heated for 4h at 400◦C prior to
use. In addition, procedural blanks were conducted for each
batch of samples to ensure minimal contamination.
Stock solutions were prepared for all standard substances
at 1000mg/l in acetonitrile. Spiking and calibration mixtures
at various concentration levels were obtained by combining
aliquots of stock solutions and internal standard with mobile
phase and storing at 4◦C. The concentration of internal stan-
dardinallthecalibrationmixturesandfinalsamplesolutions
was 50?g/l.
2.2. Sample preparation
Ten grams of minced porcine tissues was placed into a
50ml polypropylene tube and spiked with 50ng13C6-SMA.
Ten grams baked anhydrous sodium sulfate and 15ml ace-
tonitrilewasthenadded.Thesamplewerehomogenizedwith
a refiner for about 1min, and then centrifuged at 8000rpm
for 10min at 0◦C. The supernatant was decanted into a sep-
aratory funnel. The residue was extracted two times with
15ml acetonitrile. The extracts were combined. Then 20ml
n-hexane was added into acetonitrile extracts, and each sam-
ple was mixed and separated. The upper layer was discarded
(n-hexane) and 5ml 1-propanol was added. The solvent was
evaporated to near dryness by a rotary evaporator at 30◦C.
The residue was dissolved by ultrasonication for 30s with
0.4ml chloroform and 3.6ml n-hexane. The solution then
was passed through the Sep-Pak Silica solid-phase extrac-
tion cartridge preconditioned by 6ml n-hexane without any
pressure.Fivemillilitersn-hexanewasusedtowashtheinter-
ference. The analytes were eluted sequentially with 6ml
methanol–acetone (v/v, 1:1) and 6ml acetone. The eluents
were dried under a gentle nitrogen stream, and reconstituted
with 1ml mobile phase for LC–MS/MS analysis.
2.3. LC–MS/MS analysis
Identification and quantification of analytes were carried
out 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
(150mm×2.1mm I.D., 3.5?m, Waters, USA) was used for
LC separation. The temperature of the column oven was
28◦C, the flow rate was 0.2ml/min, and the injection vol-
ume was 10?l. Methanol and water were used as mobile
phase containing 0.1% formic acid. The methanol was lin-
early increased from 3 to 15% in 13min, then increased to
40%in9minandheldfor3min,itwasthenincreasedto80%
in 5min, then increased to 100% in 1min and held for 5min,
and finally it was brought back to 3% and held for 20min
until the next injection.
The mass spectrometer was operated in electrospray pos-
itive ion mode. The capillary voltage was held at 3.5kV. The
cone voltage was 78V. The multiplier voltage was 650V.
The nebulizing, desolvation and cone gas were supplied with
UHP nitrogen. The nebulizing gas was adjusted to the max-
imum, and the flow of desolvation gas and cone gas were
set to 450 and 0l/h, respectively. The source temperature and
desolvation gas temperature were held at 100 and 300◦C
respectively. The radio frenquency (RF) lens 1 and RF lens
2 were set as 40 and 0.5V. The ion energy 1 and ion energy
1 were both 0.5V. The entrance and exit voltage were zero.
The collision gradient was 1.9. During tandem mass spec-
trometric analysis, UHP argon was used as collision gas and
the pressure of collision chamber was held 3.1×10−3mbar.
The multiple reaction monitoring transition conditions used
for quantitation of various sulfonamides are listed in Table 1.
3. Results and discussion
3.1. Optimizing LC–MS/MS condition
To achieve the maximum sensitivity, the mass spectrome-
try parameters including ionization mode, the capillary volt-
age, cone voltage, source temperature and desolvation gas
temperature, desolvation gas and cone gas flow, the pres-
sure of the collision chamber, and the collision energy were

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177
Table 1
Sulfonamide 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
SIM
SDZ
SPD
STZ
SMR
SDMD
SME
SMT
SMP
SCP
SMX
SIX
SMO
SDM
SQX
SNT
13C6-STZ
aDL: detection limit (three times of the signal-to-noise ratio).
3.49
12.23
13.19
15.53
15.78
17.34
20.74
21.21
21.33
21.71
22.42
23.49
24.66
24.66
28.10
29.17
32.35
20.67
173.2
279.3
251.3
250.3
256.4
265.3
279.2
281.3
271.2
281.3
285.2
254.3
268.2
268.3
311.4
301.2
336.4
284.5
156.0
124.1
156.1
156.0
156.1
156.0
186.0
156.0
156.0
156.0
156.0
156.0
156.0
156.0
156.1
156.0
156.0
185.8
132.0
186.1
108.1
184.2
108.1
172.1
124.1
126.1
108.0
126.1
207.1
108.1
113.1
113.1
108.1
107.9
294.3
123.9
9 0.7
0.2
0.3
0.2
0.3
0.1
0.5
0.3
0.1
0.1
0.3
0.1
0.2
0.2
0.1
0.1
0.2
156.0
92.1
18
15
15
19
15
20
15
14
15
12
13
15
16
19
15
10
19
92.1
110.1
156.0
108.0
92.0
108.1
92.1
108.1
108.1
92.1
198.1
161.2
first optimized by direct flow infusion of each standard. The
results indicated that the positive mode was more favorable
than the negative ion mode. The characteristic ion and col-
lision energy for each compound are listed in Table 1. All
the compounds except isotopic internal standard gave a char-
acteristic ion at m/z 156.0, although the abundance may be
different depending on the variable compounds.
As for the sulfonamides with a wide pKarange investi-
gated in our study, the properties of mobile phases such as
pH and additives can affect the LC resolution. In addition,
considering the characteristics of electrospray ionization, the
mobile phase composition and additive may significantly
affect the response. Therefore, the additive and the mobile
phase composition were comprehensively investigated. In
previous paper [22], the mobile phase parameter, concen-
tration of formic acid, pH of the aqueous solution and ratio
of the aqueous solution and the organic solvent were opti-
mized using methanol–acetonitrile 0.05M formic acid as
mobile phase only to achieve a satisfactory separation of
10 sulfanamides. For the mobile phase composition in this
study, the response using methanol–water as mobile phase
was significantly higher than that using acetonitrile–water.
In addition, the organic phase content can significantly affect
thesensitivity.Amongthe17sulfonamides,thesensitivityof
the sulfamilamide (SA) was far lower than others, as its elu-
tionatthetimeof3.49mincontainedabout7%ofmethanolin
the mobile phase composition. However, if the mobile phase
composition started with a high content methanol, the SA
would elute at a dead volume without retention due to its
high polarity.
Among these additives such as formic acid, acetic acid,
trifluoroacetic acid and ammonium acetate, different con-
centration levels were also optimized. Taking formic acid
as an example, the results suggest that the resolution and
sensitivityofthemixturestandardincreasedwiththeincreas-
ing of the formic acid concentration from 0.05% (pH=2.98)
to 0.1% (pH=2.28); however, the resolution and sensitivity
decreased when the formic acid concentration increased to
0.15%(pH=1.91).Thechangeofresolutioncanbeattributed
to 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 higher
than 0.1% may suppress the ionization. Acetic acid, trifluo-
roacetic acid and ammonium acetate were also optimized as
above, 0.2% of acetic acid (pH=3.43), 0.1% trifluoroacetic
acid(pH=2.28)and5mmol/lammoniumacetate(pH=6.16)
were the optimal composition and additives. In comparison
with the response using formic acid, 0.1% formic acid was
the optimum additive.
3.2. Sample clean-up
In addition to the instrument parameters, mobile phase
composition and additives, the clean-up procedure is an
important factor that has an influence on sensitivity. For
foods of animal origin, the fat is a critical factor, as it can
decrease the sensitivity and shorten the lifetime of a column.
Liquid–liquidextractionusingn-hexanecanonlyremovepart
of the fat. Therefore, normal phase SPE procedures are often
used to purify the bio-samples and enhance the method’s
sensitivity. However, only one method for sulfonamides has
reported the further clean up using normal phases SPE after
liquid–liquid extraction from tissue [25]. The Sep-Pak silica,
amino-propyl and cyan-propyl SPE were used to purify the
crude sample extracts at 100?g/kg fortification to evaluate
efficiency of clean up. The results (not listed in this paper)
indicated that the Sep-Pak silica was satisfactory using 6ml
acetone–methanol (1:1) and 6ml acetone as eluent.

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B. Shao et al. / Analytica Chimica Acta 546 (2005) 174–181
Fig. 2. The chromatograms of spiking samples containing 10?g/kg of each compound.

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B. Shao et al. / Analytica Chimica Acta 546 (2005) 174–181
179
Fig. 2. (Continued).
3.3. Method performance
The chromatograms of spiking samples containing
10?g/kg of each compound are shown in Fig. 2. Because of
their similar structures and the limited commercial availabil-
ity of stable isotope compounds, only one internal standard
wasused.Thecalibrationcurvesfordetectionoftheanalytes
were 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) against
the analytes concentration ranging from 1.0 to 200?g/l
containing 50?g/l internal standard, i.e. 10.0–2000pg with
10?l injection. The linearity obtained for all analytes were
good with correlation coefficients higher than 0.999. The
instrumental detection limits (IDL) with 10?l injection
rangedfrom0.1to0.7pgdependingondifferentcompounds,
Table 2
Recoveries, RSD and LOD of spiked porcine meat tissue
Compound Spiked level 1?g/kgSpiked level 5?g/kg Spiked level 10?g/kg LOD (ng/g)
Meat LiverKidney MeatLiver KidneyMeat Liver Kidney Meat Liver Kidney
SA
SIM
SDZ
SPD
STZ
SMR
SDMD
SME
SMT
SMP
SCP
SMX
SIX
SMO
SDM
SQX
SNT
Values in parentheses are % relative standard deviations for n=5.
65.2 (11.6)
69.8 (8.1)
98.2 (11.5)
105.3 (4.7)
68.3 (14.0)
102.3 (11.3)
112.1 (5.2)
65.6 (7.2)
65.2 (5.2)
89.1 (6.8)
90.7 (14.3)
111.9 (14.4)
90.8 (8.4)
87.7 (13.3)
78.9 (18.6)
59.9 (7.0)
53.5 (7.1)
69.6 (5.8)
66.2 (7.3)
78.0 (6.4)
88.0 (3.6)
70.2 (10.9)
85.1 (9.1)
105.4 (8.2)
56.7 (13.0)
83.0 (10.5)
86.0 (5.5)
89.9 (10.9)
70.9 (5.5)
95.2 (9.7)
95.1 (14.2)
84.6 (15.9)
67.3 (6.5)
52.33 (12.3)
74.3 (3.1)
58.1 (8.3)
67.9 (5.0)
88.9 (2.7)
85.4 (7.1)
87.7 (10.4) 114.0 (6.7)
114.9 (6.2)
55.5 (4.6)
84.1 (14.7)
83.3 (2.9)
91.1 (13.0)
78.2 (6.6)
107.1 (12.0)
111.2 (8.6)
84.6 (14.3)
64.2 (15.5)
53.0 (16.2)
75.8 (8.9)
67.8 (10.1)
95.1 (10.7)
91.4 (7.6)
64.7 (2.7)
72.8 (10.2)
64.4 (8.5)
71.8 (9.8)
89.4 (4.2)
88.7 (14.5)
89.3 (7.1)
111.7 (5.4)
67.4 (8.2)
97.8 (7.4)
84.9 (9.1)
94.0 (12.9)
78.1 (8.4)
89.67 (6.7)
82.3 (14.4)
60.4 (8.5)
61.5 (5.7)
78.8 (10.8)
76.4 (10.5)
93.9 (5.1)
112.3 (4.8)
72.8 (9.4)
87.1 (9.8)
78.8 (4.2)
79.7 (6.3)
66.7 (16.3)
79.8 (12.1)
86.7 (11.8)
73.0 (14.8)
57.6 (8.8)
52.4 (15.8)
90.9 (9.4)
69.0 (13.6)
102.0 (8.9)
89.4 (13.1)
85.0 (19.8)
108.3 (5.6)
111.0 (8.4)
81.3 (17.4)
117.4 (3.5)
112.2 (2.9)
94.9 (10.8) 101.8 (7.2)
91.7 (10.8)
85.2 (10.4) 104.4 (8.1)
64.5 (9.7)
97.1 (13.5)
64.5 (4.4)
62.7 (14.8)
74.5 (13.6)
72.7 (6.1)
75.9 (8.9)
82.6 (8.9)
90.3 (15.3)
97.2 (8.8)
119.2 (6.2)
65.4 (11.0)
105.9 (10.8) 110.1 (7.9)
90.0 (5.4)
68.8 (14.8) 0.3
68.5 (5.7)
60.9 (9.9)
70.7 (7.0)
76.7 (8.1)
100.5 (8.8)
114.1 (4.1)
68.3 (5.4)
0.50.4
0.02 0.02 0.04
0.02 0.08 0.33
0.02 0.02 0.06
0.01 0.06 0.15
0.03 0.03 0.07
0.05 0.04 0.14
0.01 0.05 0.12
0.05 0.08 0.11
0.05 0.06 0.08
108.0 (2.9)
71.4 (11.3)
97.1 (6.7)
95.9 (6.6)
83.3 (10.3)
97.2 (5.1)
94.7 (6.0)
60.8 (14.2) 101.4 (9.6)
83.8 (8.5)
60.8 (4.1)
52.5 (6.2)
82.5 (8.6)
95.3 (11.6) 0.03 0.03 0.11
75.1 (5.7)0.02 0.01 0.05
89.1 (12.1) 0.02 0.02 0.08
84.4 (10.9) 0.02 0.02 0.06
75.7 (9.5) 0.02 0.02 0.05
56.6 (9.6) 0.05 0.08 0.20
53.1 (8.9) 0.10 0.17 1.00
79.3 (9.6)
94.9 (16.4)
89.8 (14.2)
62.6 (8.5)
58.5 (14.5)
95.2 (16.4)
62.5 (9.0)
54.2 (14.1)

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B. Shao et al. / Analytica Chimica Acta 546 (2005) 174–181
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 at
each level were evaluated at three levels by spiking 10.0,
50.0 and 100.0ng of each standard analyte to 10.0g tissues.
The results are listed in Table 2. The average recoveries of
each compound are ranged from 52 to 120%. Generally, the
recoveriesinkidneywerehigherthanthoseinmeat,followed
bythoseinliver.SDMDiswidelyusedandcouldbefoundin
most of the sample; therefore, the recoveries were all higher
than 100 for all sample matrices. This may be the influence
of the background sample, although the background value
has been subtracted. In addition, according to the suggestion
of AOAC on the analysis of residues [28], these values are
acceptable. The reproducibilities of this method represented
by the percent of the relative standard deviation (RSD) at
each fortification level are also summarized in Table 2 and
showed that the method precision was within 20%, which is
satisfactory. The within-day and between-day reproducibil-
ities were evaluated by injecting a standard mixture five
times at three different levels each day for five consecutives
days. The within-day reproducibilities ranged from 1.9 to
3.8% and between-day reproducibilities ranged from 3.5
to 8.2%. The limits of detection for sulfonamides in pork,
liver and kidney tissues, defined as the concentration which
yield an S/N equal to three, are described in Table 2. The
LODs 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 meat
samples available from local market. Among the 15 samples,
SMEwasfoundin10sampleswithconcentrationlevelsrang-
ing from 0.01 to 15.43?g/kg, and SDMD ranging from 0.08
to7.34?g/kginnineofthe15samples.SDZ,SMXandSQX
werealsofoundonlyinonesample,withaconcentrationlevel
lowerthan0.4?g/kg.SAwasfoundintwoofthe15samples,
withaconcentration3.47and0.98?g/kg,respectively.Fig.3
shows a typical chromatogram of one sample available from
the 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/MS
method has been developed and optimized to measure
the residues of 17 sulfonamides in porcine tissues. The
methanol–watercontaining0.1%formicacidasmobilephase
showed good LC resolution and MS sensitivity. The LODs
were2–4magnitudeslowerthantheMRLsofdefinedbyEU.

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181
Acknowledgement
The authors wish to acknowledge the Beijing Natural
Science Foundation for financial support under grant num-
ber 7041004 and the Ministry of Science and Technology
(2001BA804A18-02, 2001BA804A55).
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