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Development and validation (according to the 2002/657/EC regulation) of
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a method to quantify sulfonamides in porcine liver by fast partition at very low

temperature and LC-MS/MS
Renata Pereira Lopes,a Daniella Vasconcellos Augusti,b Leonardo Francisco de Souza,b Flavio Alves Santos,b
Josefa Abucater Lima,b Eug^enia Azevedo Vargasb and Rodinei Augusti*a
Received 28th September 2010, Accepted 2nd December 2010
DOI: 10.1039/c0ay00587h

A novel multi-residue method for the quantification of 15 sulfonamides in porcine liver is described. It
involves the application of a liquid–liquid extraction with fast partition at very low temperature (LLE-
FPVLT) procedure followed by HPLC-MS/MS (high performance liquid chromatography coupled to
tandem mass spectrometry) analysis. By this innovative method, acetonitrile is added to a minced
porcine liver sample and the resulting suspension centrifuged and immersed in a container with liquid
nitrogen for 15 s. The acetonitrile phase, which remains liquid under these conditions, is isolated,
evaporated to dryness, recomposed with formic acid 0.1% v/v, and injected into the liquid
chromatograph. The whole analytical procedure was validated according to the European Commission
Decision 2002/657/EC and acceptable values (except for sulfanilamide) were obtained for the following
parameters: linearity (0.97 < R2 < 0.99), decision limit (107.70 mg kg1 < CCa < 128.65 mg kg1),
detection capability (115.40 mg kg1 < CCb < 157.29 mg kg1), limit of detection (5.58 mg kg1 < LOD <
16.75 mg kg1), limit of quantification (18.41 mg kg1 < LOQ < 55.26 mg kg1), accuracy (recovery rates),
precision (repeatability, intermediate precision, and measurement uncertainty tests), selectivity, and
robustness. Recoveries higher than 70%, at three concentration levels (0.5, 1.0 and 1.5 of the maximum
residue limit, MRL), were attained for the majority of the sulfonamides. These very promising results
point to the inclusion of the present methodology into the National Residue Control Plan scope of the
Ministry of Agriculture, Livestock and Food Supply of Brazil.

Introduction kg. These limits demand, therefore, the development of analyt-

Sulfonamides (Fig. 1), derivatives of sulfanilamide (p-amino-
benzene sulfonic acid), have been widely used as veterinary drugs
for prophylactic and therapeutic purposes. About 30 sulfon-
amides have been employed so far, some of them with good
antibiotic and growth-promoter effects. However, several classes
of bacteria have become resistant to many drugs due to the
misuse of sulfonamides. This practice can also signify a potential
risk to human health since many diseases may emerge, such as
thyroid cancer and anaphylactic reactions.1
To limit human exposure, many organizations, such as the
Codex Alimentarius,2 European Union (EU)3 and US Food and
Drug Administration (FDA),4 have established maximum
residue limits (MRLs) for these drugs in animal products. For
instance, the European Union defined that the residue content of
all the sulfonamides in meat should not be higher than 100 mg per
ical methods capable to detect and quantify, with speed and
sensitivity, drug residues in complex matrixes, such as milk and
muscle tissues.5 Enzyme linked immune sorbent assay (ELISA),
although being a non-specific method, has been largely used.6
Other analytical methods have been used, such as thin-layer
chromatography (TLC),7 high-performance liquid chromatog-
raphy (HPLC),8,9 high performance liquid chromatography
coupled with tandem mass spectrometry (HPLC-MS/MS),10–12
and capillary electrophoresis-mass spectrometry.13 Hence,
whereas ELISA is the most typical method for a fast screening
and batch analysis, HPLC-MS/MS has been widely used due to
its high sensitivity and broad linear range and to its capability to
supply accurate and precise quantifications.14–17

a
Departamento de Quımica, Universidade Federal de Minas Gerais, Belo
Horizonte/MG, Brazil 31270-901. E-mail: augusti@ufmg.br
b
Laborat
orio Nacional Agropecu
ario (LANAGRO), Pedro Leopoldo/MG,
Brazil Fig. 1 General structure of the sulfonamides.

606 | Anal. Methods, 2011, 3, 606–613 This journal is ª The Royal Society of Chemistry 2011

The detection of antibiotic residues at low levels in complex
samples, such as animal tissues, requires highly efficient sample
preparation steps. For instance, methods for the analysis of
sulfonamides in biological matrixes usually include the use of
extraction procedures, such as liquid–liquid (LLE) and solid
phase (SPE), to promote both the sample cleanup and analytes
pre-concentration, respectively. However, this two-stage proce-
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dure presents some remarkable disadvantages such as the high
costs (due to the large amounts of solvent required and the
infeasibility of cartridges reuse), long time of execution (due to
the saturation of the adsorbent sites that results in a low flow of
solvent through the cartridge), and poor reproducibility.
Aiming at surpassing such disadvantageous aspects, an alter-
native LLE procedure was recently introduced. Basically, the
method consists of adding small portions of acetonitrile on a given
sample, followed by homogenization (usually by using a grinder
apparatus), centrifugation (which allows the precipitation of solid
components), and filtration. The isolated liquid phase (comprised
mostly by water and acetonitrile) is then placed in a refrigerator at
20  C for ca. 12 hours. Under these conditions water is frozen
slowly whereas acetonitrile remains liquid. The acetonitrile phase
containing the analytes that preferentially migrate to it is then
promptly isolated and subsequently analyzed. This methodology,
known as liquid–liquid extraction with cleanup by precipitation at
low temperature (LLE-PLT), is simple and efficient, uses reduced
amounts of organic solvent, requires few manipulation steps, and
demands no extra purification stages. It has been successfully
applied to the analysis of organophosphorous insecticides in olive
oil18 as well as for the detection of organic residues in diverse
matrixes, such as milk and water.19,20
The primary intention of this work is therefore to develop and
validate (according to the European Commission Decision 2002/
657/EC)3 a multi-residue method for the quantification of 15
sulfonamides in porcine liver via HPLC-MS/MS analyses. A
novel high-throughput methodology for the sample treatment,
called LLE-FPVLT (liquid–liquid extraction with fast partition
at very low temperature), is described herein and its advanta-
geous features, such as speed and reliability, demonstrated.
Finally, it is also our intention to apply such innovative proce-
dure to control and monitor drug residues within the scope of the
National Residue Control Plan of the Ministry of Agriculture,
Livestock and Food Supply of Brazil.21
Experimental
Reagents and solutions
All reagents were of analytical grade. HPLC-grade acetonitrile and
methanol were supplied by Merck (Darmstad, Germany). Formic
acid was purchased from Fluka (Ronkonkoma, NY, USA).
Ultrapure water was generated by a Millipore Milli-Q system
(Milford, MA, USA). Purified samples were filtered through
PTFE Millipore (Milford, MA, USA) filters (13 mm; 0.45 mm).
All the standards were of high purity grade (>99.0%). The
following standards were purchased from Riedel de Ha€en
(Seelze, Germany): sulfacetamide, sulfadimethoxine, sulfame-
thoxypyridazine, and sulfadoxine. On the other hand, sulfa-
chloropyridazine, sulfamethazine, sulfaguanidine,
sulfamerazine, sulfamethizole, sulfanilamide, sulfaquinoxaline,
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sulfisoxazole, sulfadiazine, sulfathiazole, sulfapyridine (used as
an internal standard), tilmicosin, clindamycin, erythromycin,
tylosine, and lincomycin (prototype macrolides) were supplied by
Sigma-Aldrich (St Louis, MO, USA). Finally, sulfamethoxazole
(99.0%) was furnished by Globe Quımica (Cosm opolis, SP,
Brazil). Each individual stock solution was prepared at 1.00 g L1
in methanol and stored at 20  2  C in a refrigerator. The
working solutions were prepared by appropriate dilutions of the
stock solutions with Milli-Q water.
Chromatographic conditions
The HPLC-MS/MS system consisted of an Alliance 2795
(Micromass, Manchester, UK) chromatographer and a Quattro
Premier XE (Micromass, Manchester, UK) triple quadrupole
mass spectrometer. The separation of the sulfonamides was
accomplished at 30  C with a Zorbax Eclipse XDB C-18
(dimensions: 4.6  150 mm; particle size: 5 mm) column (Agilent,
Santa Clara, CA, USA). The flow rate and injection volume were
0.3 mL min1 and 50 mL, respectively. The mobile phases used
were water/acetonitrile (95 : 5)/formic acid 0.1% (A) and water/
acetonitrile (5 : 95)/formic acid 0.1% (B). The gradient elution
program was as follows: A (90%)–B (10%) (5 min); A (80%)–B
(20%) (4 min); A (45%)–B (55%) (0.5 min); A (20%)–B (80%) (4.5
min); and A (10%)–B (90%) (3 min). The total chromatography
run time was 17 minutes.
Mass spectrometric conditions
Electrospray ionization was performed in the positive ion mode
for all the analytes. The mass spectrometer was operated under
the following conditions: capillary voltage 2.8 kV; source
temperature 120  C; desolvation temperature 300  C; cone gas
flow 46 L h1; and desolvation gas flow 697 L h1. In order to
determine the m/z transitions, solutions of each analyte at 10 mg
L1 in methanol–water (1 : 1 v/v) containing 0.01% (v/v) of for-
mic acid were directly infused, via a micro syringe (Hamilton,
Reno, NV) at a flow rate of 10 mL min1, into the mass spec-
trometer ion source. A summary of the precursor and product
ions, collision energy, and dwell time achieved for each analyte is
presented in Table 1.
LLE-FPVLT (liquid–liquid extraction with fast partition at very
low temperature) procedure
The sulfonamides were extracted from the spiked porcine liver
samples using the LLE-FPVLT (liquid–liquid extraction with
fast partition at very low temperature) procedure. Briefly, the
procedure was as follows: after being cut into small pieces, 2.00 
0.01 g of the porcine liver was weighed and spiked with proper
amounts of the sulfonamides and sulfapyridine (used as an
internal standard at 100 mg kg1) solutions. This mixture was
transferred to a 50 mL polypropylene centrifuge tube and 4.0 mL
of acetonitrile were added. The resulting suspension was then
homogenized (by using an Ultra Turrax grinder apparatus) for 1
min. After centrifugation at 4000 rpm for 10 min at 10  1  C, the
tube was immersed for 15 seconds in a container with liquid
nitrogen (the total time elapsed between the addition of aceto-
nitrile and freezing was approximately 15 minutes). The aqueous
phase was frozen whereas the acetonitrile layer, that remained
Anal. Methods, 2011, 3, 606–613 | 607
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Table 1 Multiple reaction monitoring optimized conditions achieved for each sulfonamide

Sulfonamide Retention time/min Transitions (m/z) Cone voltage/V Collision energy/eV Dwell time/s

Sulfaguanidine 3.77 215.0 > 156.0 21 15 0.025

215.0 > 108.0 21 25 0.025
Sulfanilamide 4.41 173.0 > 156.0 10 16 0.050
173.0 > 108.0 10 11 0.050
Sulfacetamide 5.53 215.0 > 156.0 15 20 0.025
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215.0 > 108.0 15 20 0.025

Sulfadiazine 7.01 251.1 > 156.1 20 25 0.050
251.1 > 92.0 20 25 0.050
Sulfathiazole 7.27 255.8 > 107.9 25 14 0.010
255.8 > 155.8 26 14 0.010
Sulfapyridine (internal standard) 7.68 249.9 > 155.8 25 18 0.010
249.9 > 184.0 25 18 0.010
Sulfamerazine 8.42 265.1 > 156.0 25 18 0.010
265.1 > 92.0 25 18 0.010
Sulfamethazine 9.54 279.0 > 155.8 27 18 0.010
279.0 > 185.9 27 16 0.010
Sulfamethizole 9.71 271.0 > 156.0 21 24 0.025
271.0 > 92.0 21 24 0.025
Sulfachloropyridazine 11.05 284.9 > 91.9 26 27 0.025
284.9 > 155.8 26 15 0.025
Sulfamethoxypyridazine 11.05 281.0 > 125.9 25 20 0.050
281.0 > 155.8 25 17 0.050
Sulfadoxine 11.28 311.1 > 156.1 20 25 0.010
311.1 > 92.0 20 25 0.010
Sulfamethoxazole 11.45 254.0 > 156.0 20 25 0.010
254.0 > 92.0 20 25 0.010
Sulfisoxazole 11.69 267.8 > 156.0 20 25 0.010
267.8 > 113.2 20 25 0.010
Sulfaquinoxaline 12.15 301.0 > 91.9 27 27 0.010
301.0 > 107.9 27 27 0.010
301.0 > 155.8 27 18 0.010
Sulfadimethoxine 12.21 311.0 > 155.8 30 20 0.010
311.0 > 218.0 30 20 0.010
311.0 > 245.0 30 20 0.010

liquid under these conditions, was collected and evaporated to

dryness at 40  1  C under an air stream. In sequence, 1.0 mL of
formic acid 0.1% was added to the residue and the solution
injected into the HPLC system. A schematic representation of
such extraction procedure is displayed in Fig. 2.

Method validation
Method validation was performed following the directives from
European Commission Decision 2002/657/EC,3 ISO 11843-
2:2000,22 and ISO/TS/2148:2004.23 The following parameters
were assessed for each sulfonamide: linearity, matrix effects, limit
of detection (LOD), limit of quantification (LOQ), decision limit
(CCa), detection capability (CCb), accuracy (recovery), preci-
sion (repeatability, intermediate precision, and measurement
uncertainty), selectivity, and robustness.

Calibration curves: linearity and matrix effects

Two calibration curves, standard (in solvent) and matrix-
matched standard (MMS), were built at the following levels (3
replicates per level for three different days): 0, 50, 75, 100, 125,
and 150 mg kg1 of liver, which correspond to 0  MRL, 0.50 
MRL, 0.75  MRL, 1.00  MRL, 1.25  MRL, and 1.50 
MRL, respectively. For the MMS calibration curves, the porcine
liver extracts were spiked with proper amounts of the solutions of Fig. 2 Schematic representation of the LLE-FPVLT extraction proce-
sulfonamides and sulfapyridine (internal standard at 100 mg dure.

608 | Anal. Methods, 2011, 3, 606–613 This journal is ª The Royal Society of Chemistry 2011

kg1). The data were treated by using the Waters QuanLynx
module of the MassLynx software. The calibration curves were
built by plotting the response (sulfonamide/sulfapyridine peak
area ratio) versus the sulfonamide concentration. A simple
regression using the least square method was applied. The line-
arity was appraised by the coefficient of determination (R2),
which was calculated as an average of the values obtained for the
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three different days. To verify matrix effects, the average
responses at each level (n ¼ 3) of the standard and matrix-
matched standard calibration curves were statistically compared
using the Snedecor F and Student’s t tests24 at a significance level
of 95%.
Limit of detection (LOD) and limit of quantification (LOQ)
The limit of detection (LOD) was calculated according to the
equation: LOD ¼ 3  RSD  C, where C is the lowest
concentration (usually 0.5  MRL) that furnished a relative
standard deviation (RSD) smaller than 20% (n ¼ 9). Analo-
gously, the limit of quantification was calculated as: LOQ ¼ 10 
RSD  C.25
Decision limit (CCa) and detection capability (CCb)
The CCa and CCb parameters were calculated using the cali-
bration curve procedure according to the guidelines from ISO
11843-2:200022 and normative as described by the European
Commission Decision 2002/657/EC.3
Accuracy and precision
The accuracy was determined via the attainment, by two analysts
for three different days, of the recovery rates from samples
spiked with the sulfonamides at three distinct levels: 50, 100, and
150 mg kg1 (n ¼ 6 replicates per level). Recoveries were calcu-
lated by comparing the concentrations of the extracted sulfon-
amides with those from the MMS (matrix-matched standard)
calibration curves. These data were also used to determine the
precision of the method via the evaluation of the following
parameters: repeatability, intermediate precision, and measure-
ment uncertainty (MU). Repeatability and intermediate preci-
sion, expressed as a relative standard deviation (RSD), were
evaluated from the responses obtained at the three levels (50, 100,
and 150 mg kg1; n ¼ 6 replicates per level) for three different days
by one (n ¼ 18 per level) or two (n ¼ 36 per level) analysts,
respectively. Measurement uncertainty (MU) was accessed
according to the guidelines from ISO/TS 21748:2004.23
Selectivity
The porcine liver samples were spiked with some prototype
macrolides (another class of drugs usually employed to treat
farm animals), i.e. tilmicosin, clindamycin, erythromycin, tylo-
sine, and lincomycin, to evaluate the selectivity of the method.
Hence, batches of porcine liver samples containing the sulfon-
amides at three levels (50, 100, and 150 mg kg1) were prepared in
triplicate (n ¼ 3). To these samples the following concentrations
of the macrolides were added: 1000 (tilmicosin), 200 (clindamy-
cin), 200 (erythromycin), 1500 (tylosine), and 1500 (lincomycin)
mg kg1. A second set of samples, containing the sulfonamides
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but without the macrolides, was prepared as a control group. All
the samples were submitted to the whole LLE-FPVLT extraction
procedure and analyzed as previously described.
Robustness
The robustness of the method was evaluated by changing three
parameters, typical of the extraction procedure. A two-level
factorial design was then applied and the factors considered
were: (1) acetonitrile trademark: Merck (Darmstad, Germany) or
Tedia (Fairfield, OH); (2) freezing method applied on the porcine
liver suspensions: liquid nitrogen for 15 s or refrigeration in
a freezer for 12 h; (3) temperature used for the organic layer
evaporation: 40 or 50  C. Then, a total of 24 assays were executed
(23 ¼ 8 experiments carried out in triplicate). The data manipu-
lation was performed by using the Statistica software.
Results and discussion
The extracted-ion chromatograms for the 15 sulfonamides are
shown in Fig. 3 (note the presence of sharp peaks related to each
sulfonamide). To obtain this chromatogram, a matrix-matched
standard (MMS) solution with all the sulfonamides was prepared
(at a concentration of 100 mg kg1 each) while selecting a specific
m/z transition for each analyte (Table 1).
With the data from the standard and MMS calibration curves
an unequivocal detection of matrix effects could be achieved.
Hence, sulfaguanidine was the only analyte that exhibited
a remarkable matrix effect, as the slope of both calibration
curves (standard and MMS) showed to be statistically distinct
(plots not shown). Despite of this result, MMS calibration curves
were built for all the 15 sulfonamides and then used in the
subsequent studies.
Almost all the analytes exhibited a linear behavior over the
concentration range evaluated (0–150 mg kg1), as verified by the
good linear determination coefficients (0.97 < R2 < 0.99, Table
2). The exception was sulfanilamide, for which a calibration
curve with a smaller R2 (0.94) was obtained (this anomalous
result is not fully understood and therefore a reasonable expla-
nation for it was not postulated herein). The results from Table 2
also indicated that the method presented good detection capa-
bility because the LOD and LOQ obtained are below the MRL
established (100 mg kg1) for all the sulfonamides.
The decision limit (CCa) is defined as the limit above which the
sample contains the analyte with a probability error of a. In the
case of substances with established MRL, such as sulfonamides,
a ¼ 5%. The detection capability (CCb) is the smallest content of
the substance that may be detected, identified, and quantified in
a sample, with a statistical certainty of 1  b.23 The values
obtained for CCa and CCb3 are indicated in Table 2. These
values, close enough to the MRL for the sulfonamides (100 mg
kg1) with a possible exception of sulfanilamide, thus corrobo-
rate the good method performance.
The results for accuracy, estimated from the recovery data, are
illustrated in Fig. 4. All the sulfonamides showed results
consistent with the acceptable range specified by the Codex Ali-
mentarius (>70%).2 Moreover, the good performance of the
present methodology could be verified by comparison with
similar reports described in the literature. For example, in two
Anal. Methods, 2011, 3, 606–613 | 609
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Fig. 3 Extracted-ion chromatograms resulting from the application of the LLE-FPVLT procedure on porcine liver sample spiked with the 15
sulfonamides.

earlier studies the authors made use of LLE1 and SPE (silica as repeatability and intermediate precision (Table 3) also showed to
a stationary phase)10 to analyze sulfonamides in bovine meat and be consistent with the Codex Alimentarius normative,2 except for
observed recovery rates similar to those described herein. The sulfadiazine, sulfaguanidine, sulfamethoxazole, sulfathiazole

610 | Anal. Methods, 2011, 3, 606–613 This journal is ª The Royal Society of Chemistry 2011
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Table 2 Results (R2, LOD, LOQ, CCa, CCb) obtained from the application of the modified LLE-PLT method on the spiked porcine liver samples for
the analysis of 15 sulfonamides (n ¼ 36)

Analyte R2 LOD/mg kg1 LOQ/mg kg1 CCa/mg kg1 CCb/mg kg1

Sulfaguanidine 0.98 7.27 24.00 128.65 157.29

Sulfanilamide 0.94 23.54 77.68 136.89 173.77
Sulfacetamide 0.99 8.19 27.03 107.70 115.40
Sulfadiazine 0.97 10.57 34.89 122.87 145.73
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Sulfathiazole 0.99 11.28 37.21 113.78 127.55

Sulfamerazine 0.99 7.30 24.10 110.93 121.86
Sulfamethazine 0.99 7.03 23.18 109.45 118.89
Sulfamethizole 0.99 5.59 18.44 110.74 121.48
Sulfachloropyridazine 0.99 10.84 35.77 109.70 119.39
Sulfamethoxypyridazine 0.99 6.35 20.94 108.32 116.63
Sulfadoxine 0.98 10.61 35.01 118.61 137.22
Sulfamethoxazole 0.98 16.75 55.26 123.26 146.53
Sulfisoxazole 0.99 12.58 41.52 112.96 125.92
Sulfaquinoxaline 0.98 5.58 18.41 115.95 131.89
Sulfadimethoxine 0.98 10.79 35.59 116.06 132.12

(repeatability) and sulfaguanidine, sulfamethoxazole (interme-
diate precision) that presented slightly out-of-range results.
The estimation of the measurement uncertainties (MU) was
carried out following an empirical ‘‘top down’’ and ‘‘bottom up’’
model, in which the uncertainties from sample preparation,
standard dilution, as well as chromatography and mass spec-
trometry detection variability were included.23 The values
obtained, expressed as a relative standard deviation (RSD) for
each concentration level (50, 100 and 150 mg kg1), are exposed in
Table 4. The highest values were obtained for sulfisoxazole and
sulfadimethoxine (>26% and >25%, respectively) whereas for the
other sulfonamides much smaller measurement uncertainties
were achieved (<16%).
The results related to the determination of the method
robustness demonstrated that whereas the acetonitrile trademark
(Merck or Tedia) and the evaporation temperature (40 or 50  C)
presented no significant effects (i.e. the recovery rates were
identical within the error range) for all the sulfonamides, the
freezing method (in liquid nitrogen for 15 s or in a refrigerator at
20  C for 12 h) was shown to be of major relevance. Hence,
a fortunate result could be verified herein: the use of liquid
nitrogen favored the attainment of higher recovery rates. These
remarks could be better visualized in Pareto charts built from the
recovery rates obtained for all the sulfonamides (a Pareto chart
indicates the magnitude and the significance of each variable as
well as double and triple interactions among them and contains
a reference line so that any effect that goes beyond this line is
potentially important).26 Hence, a careful inspection on the
Pareto chart for sulfachloropyridazine displayed in Fig. 5
(similar charts were obtained for all the other sulfonamides,
although not shown herein) clearly supports these alleged effects.
Finally, to assess the selectivity of the method the whole
extraction procedure was performed by using porcine livers
spiked with the sulfonamides and some macrolides (clindamycin,
erythromycin, tylosine, and lincomycin, chosen as prototype
interfering compounds). The presence of such substances,
however, was not able to produce a measurable effect (within
a 95% confidence level) on the recovery rates for the sulfon-
amides. Hence, one can conclude that the present methodology
has a high selectivity for the sulfonamides.

Fig. 4 Recoveries for the sulfonamides as obtained by applying the LLE-FPVLT extraction procedure (n ¼ 36) on spiked porcine liver samples.

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Table 3 Repeatability and intermediate precision, given as a relative standard deviation (RSD), calculated for the sulfonamides by one or two analysts,
respectively

Repeatability Intermediate precision

RSD (%) (n ¼ 18) RSD (%) (n ¼ 36)

1 1 1
Analyte 50 mg kg 100 mg kg 150 mg kg 50 mg kg1 100 mg kg1 150 mg kg1
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Sulfaguanidine 19.7 20.5 26.1 28.0 23.3 21.7

Sulfanilamide 15.7 15.2 14.3 17.1 16.0 12.8
Sulfacetamide 11.1 14.9 13.4 12.0 14.1 14.3
Sulfadiazine 15.6 19.2 22.4 20.0 17.3 18.4
Sulfathiazole 15.6 19.2 22.8 18.1 15.0 12.9
Sulfamerazine 8.9 10.8 10.5 9.0 9.6 9.4
Sulfamethazine 11.1 13.4 11.8 11.0 13.3 11.7
Sulfamethizole 11.4 5.1 8.3 20.8 16.3 17.2
Sulfachloropyridazine 7.7 3.2 5.4 7.8 5.7 7.8
Sulfamethoxypyridazine 8.1 5.4 3.5 9.2 7.7 5.6
Sulfadoxine 7.9 5.3 7.5 8.7 6.2 8.9
Sulfamethoxazole 25.1 25.2 27.3 19.5 19.8 22.2
Sulfisoxazole 19.1 16.0 11.7 17.3 16.3 17.1
Sulfaquinoxaline 15.7 6.8 10.7 15.2 10.1 13.3
Sulfadimethoxine 12.9 5.8 5.6 12.0 6.5 8.1

Table 4 Measurement uncertainties (MU) calculated for the 15 sulfonamides at three different levels (50, 100 and 150 mg kg1) (n ¼ 36)

Estimation of the measurement uncertainty (MU)

Analyte 50 mg kg1 100 mg kg1 150 mg kg1

Sulfaguanidine 11.1 11.5 12.0

Sulfanilamide 12.4 12.6 13.9
Sulfacetamide 10.0 10.9 12.1
Sulfadiazine 11.0 12.0 14.0
Sulfathiazole 11.9 11.9 12.4
Sulfamerazine 11.9 12.6 14.2
Sulfamethazine 10.3 10.4 11.1
Sulfamethizole 12.3 13.5 16.2
Sulfachloropyridazine 11.3 11.4 11.9
Sulfamethoxypyridazine 13.2 14.0 14.4
Sulfadoxine 10.8 11.2 11.7
Sulfamethoxazole 9.7 9.9 9.9
Sulfisoxazole 26.2 26.6 27.4
Sulfaquinoxaline 13.7 14.3 14.9
Sulfadimethoxine 25.3 26.1 27.2

Fig. 5 The Pareto chart built from the recovery rates achieved upon the
extraction of sulfachloropyridazine spiked on porcine liver samples.
Conclusion
A whole analytical procedure, including an innovative extrac-
tion/cleanup methodology (called herein as liquid–liquid
extraction with fast partition at very low temperature, LLE-
FPVLT) and analyses by HPLC-MS/MS, was successfully
employed to quantify sulfonamides in porcine liver samples. The
LLE-FPVLT procedure was demonstrated to be fast, simple,
efficient, and of very easy execution. Furthermore, it must be
emphasized that such methodology makes use of reduced
amounts of acetonitrile, requires few manipulation steps, and
demands no additional purification stages. The whole analytical
procedure was validated according to the European Commission
Decision 2002/657/EC.3 Although some parameters showed to be
slightly unsuitable for a couple of sulfonamides (linearity:
sulfanilamide; accuracy: sulfamethoxazole; repeatability: sulfa-
diazine, sulfaguanidine, sulfamethoxazole, sulfathiazole; inter-
mediate precision: sulfaguanidine and sulfamethoxazole;
measurement uncertainty: sulfadimethoxime, sulfamethoxazole),

612 | Anal. Methods, 2011, 3, 606–613 This journal is ª The Royal Society of Chemistry 2011

the quite similar recoveries and the reasonably low coefficients of
variation achieved at the three levels (0.5  MRL, 1.0  MRL,
and 1.5  MRL) for all the sulfonamides ensure its usefulness.
Furthermore, the results reported herein point toward the
application of such sample treatment methodology, in combi-
nation with HPLC-MS/MS, to multi-residue and multi-classes
analyses, which are underway in our laboratory.
Published on 27 January 2011. Downloaded by Universidade Federal de Ouro Preto on 22/07/2016 04:28:24.
Acknowledgements
The authors acknowledge financial support and fellowships from
the Conselho Nacional de Desenvolvimento Cientıfico e Tec-
ogico (CNPq), Fundação de Amparo a Pesquisa do Estado de
nol
Minas Gerais (FAPEMIG), and Ministerio da Agricultura,
Pecuaria e Abastecimento (MAPA)—Brazil.
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