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Food Chemistry 196 (2016) 1144–1149

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Short communication

Development of an immunoaffinity chromatography and HPLC-UV


method for determination of 16 sulfonamides in feed
Ho Jin Kim a,b, Min Hee Jeong a, Hye Jin Park a, Won Chan Kim b, Jang Eok Kim b,⇑
a
National Agricultural Products Quality Management Service, Kimchun 740-871, Republic of Korea
b
School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: A novel and simple method for detecting 16 sulfonamides (SAs) in animal feed using high performance
Received 13 April 2015 liquid chromatography equipped with a photo-diode array detector (HPLC/PDA) and immunoaffinity
Received in revised form 4 September 2015 chromatography was developed. The chromatographic peaks of the 16 SAs were successfully identified
Accepted 5 October 2015
by comparing their retention times and UV spectra with reference standards. Method validation was per-
Available online 9 October 2015
formed with linearity, sensitivity, selectivity, accuracy and precision. The limits of detection (LODs) for
the instrument used to study sulfonamides ranged from 14.1 to 45.0 lg/kg, and the limits of quantifica-
Keywords:
tion (LOQs) ranged from 46.9 to 150.0 lg/kg. Average recoveries of the 16 SAs ranged from 78.2% to
Immunoaffinity chromatography
Sulfonamide
105.2%. Method replication resulted in intraday and interday peak area variation of <5.5%. The developed
Animal feed method was specific and reliable and is suited for the routine analysis of SAs in animal feed.
Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction need to be developed to determine the sulfonamides in animal


feed.
Many different types of antibiotics exist, but among these types, Although many analytical methods have been developed for
sulfonamides (SAs) are the most commonly used chemotherapeu- identifying and quantifying sulfonamide residues in animal tissues,
tics in veterinary practices because they are relatively inexpensive an acceptable routine method is still needed that is simple, reliable,
wide-spectrum antimicrobial agents (Biswas, Rao, Kondaiah, inexpensive, fast, less hazardous to operators and the environment,
Anjaneyulu, & Malik, 2007). SAs have treated various diseases in and capable of determining residues at concentrations lower than
farmed animals, and account for a high proportion of total world- the MRL. The existing methods include high performance liquid
wide antibiotic use (Kowalski, Plenis, Oledzka, & Konieczna, 2011). chromatography (HPLC) (Sandra, Danijela, Dragana, Horvat, &
All SAs contain a common structure, an unsubstituted amine on a Macan, 2006), LC coupled with mass spectrometry (MS) (Martos
benzene ring and a sulfonamide group para to the amine (Fig. 1). As et al., 2010; Silvia, Jesús, & Damià, 2008), gas chromatography
public awareness of food safety and quality issues grow, maximum (GC) (Naziha, Amel, & Jean, 2005), and capillary electrophoresis
residue limits (MRL) for antibiotics including SAs have been estab- (CE) (Sushma, Sunil, Amit, & Manjusha, 2005). Among these meth-
lished by law in many countries to protect consumers against pos- ods, HPLC with ultraviolet detection is currently the most widely
sible adverse health effects from antibiotic residues in foods. used method. Despite the use of selective detection techniques
According to European Union (EU) regulations, the MRL for the such as MS, sample preparation is a major challenge for the analyt-
total SAs in edible animal tissues is 100 lg/kg (Council ical procedures used in determining the chemical residues in ani-
Regulation (EEC) No. 37/2010 of 22 December, 2009), while the mal feed. Compared with other sample treatment methods,
Codex Alimentarious Commission (CAC) set the limits at 25 lg/kg which include liquid–liquid extraction (Bohm, Stachel, & Gowik,
and 100 lg/kg for SAs in cattle milk and products of animal origin 2009), solid–liquid extraction (Muhammad, Ping, & Yi, 2009) and
(muscle, fat, kidney, and liver), respectively (CAC 32th session). solid-phase extraction (Aurore, Cédric, Terence, Lucien, & Torsten,
Therefore, to meet the legislative requirements, analytical methods 2010; Jesús, Silvia, & Damià, 2010), immunoaffinity chromatogra-
phy (IAC) (Bingyu et al., 2008; Cun et al., 2008) provides better
sample cleanup and higher selectivity. Furthermore, IAC can sim-
⇑ Corresponding author at: School of Applied Biosciences, College of Agriculture plify the sample treatment process and a need a smaller volume
and Life Sciences, Kyungpook National University, Daehakro 80, Daegu 702-701, of toxic solvent compared with other sample treatment tech-
Republic of Korea. niques. IAC could increase the selectivity of sulfonamides by easily
E-mail address: jekim@knu.ac.kr (J.E. Kim).

http://dx.doi.org/10.1016/j.foodchem.2015.10.014
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
H.J. Kim et al. / Food Chemistry 196 (2016) 1144–1149 1145

Fig. 1. Chemical structures of 16 sulfonamides antibiotics analyzed in this study.

eliminating fat, protein, carbohydrate, and salt present in the MA, USA). The analytical grade of potassium chloride, sodium chlo-
assorted animal feed because of its enzyme-linked antibody which ride, KH2PO4, Na2HPO412H2O, and Tween-20 were purchased
is selectively coupled to sulfonamides. from Sigma–Aldrich (St. Louis, USA). All other organic chemicals
To the best of our knowledge, this study is the first demonstrat- and organic solvents were reagent grade or higher.
ing IAC/HPLC-UV procedures that have been developed and vali-
dated for the simultaneous determination of 16 sulfonamides in 2.3. Standard preparation
animal feed. Because antibiotic use has been linked to a wide range
of human health hazards, SAs levels in animal feed must be con- The standard stock solutions of the 16 SAs were prepared in
trolled and monitored to guarantee the safety of animal products methanol. The SAs concentrations ranged from 1.0 to 10 mg/ml.
such as meat, milk and eggs which are highly consumed in would The appropriate volumes of each stock solution were mixed
wide. together. Then, the mixture was serially diluted in water/methanol
(1/1) to prepare the working standard solutions. All standard solu-
tions were stored in a refrigerator.
2. Materials and methods
2.4. Sample preparation
2.1. Samples
The samples were prepared for extraction by IAC. Five grams of
Two methods for obtaining samples were used when preparing
homogenized sample were placed into a centrifuge tube and then
the animal feed samples. The first method was purchasing animal
mixed with 20 ml of the extraction solvent (ultrapure water/etha-
feed from local markets. The second method directly acquired ani-
nol, 20:80 v/v). The mixture was homogenized for 3 min, and son-
mal feed from local farm. The collected samples were composed of
icated (S450H; HUCOM system, Suwon, Korea) for 30 min at room
four different animal feed (the ratio of the first method to the sec-
temperature. It was then centrifuged at 3000 rpm for 5 min. The
ond method for obtaining samples were 37:10 for bovine, 25:10 for
supernatant (5 ml) was diluted to 50 ml with ultrapure water
pork, 32:10 for chicken, and 33:10 for duck). These samples were
and filtered (Whatman No. 4). Twenty milliliters of the filtrate
obtained from markets or farms in five different cities. The four
was passed through the immunoaffinity columns for sulfonamides
types of dried animal feeds were pulverized into a fine powder
(IAC-SULF). Ten milliliters of phosphate buffered solution (PBS)
using a pulverizer (HMF-100; HANIL, Seoul, Korea). All samples
containing 0.1% (v/v) Tween-20 was passed through the IAC-
were stored under refrigeration.
SULF, and washed with 10 ml of water, and the column effluent
was discarded. The sample was eluted form the IAC-SULF by pass-
2.2. Chemicals and reagents ing 2 ml of methanol, and all eluted samples were collected in a
new test tube. The sample eluate was evaporated under a gentle
Sulfacetamide (SA), sulfadiazine (SDZ), sulfathiazole (ST), sul- stream of nitrogen at 50 °C, and the residue was dissolved in
fapyridine (SPD), sulfamerazine (SMR), sulfamethizole (SMTZ), sul- 500 ll of solution (water:methanol, (9:1, [v/v])) and filtered
famethazine (SM2), sulfachloropyridazine (SCP), sulfamethoxazole through a 0.22 lm disposable filter prior to chemical analysis. All
(SMZ), sulfamonomethoxine (SMM), sulfisoxazole (SIZ), sul- measurements were performed in triplicate.
fadimethoxine (SDM), and sulfaquinoxaline (SQX) were purchased
from Sigma–Aldrich (St. Louis USA). Sulfamethoxydiazine (SMD), 2.5. HPLC analysis
sulfisomidine (SIM2), sulfachloropyrazine (SPZ) and IAC purchased
were from Clover Technology (CA, USA). HPLC-grade methanol and The HPLC analysis was performed on a Shiseido Nanospace SI-2
ethanol were purchased from Merck (Darmstade, Germany), and system (Shiseido, Tokyo, Japan) equipped with a binary solvent
acetic acid from Kanto (Tokyo, Japan). Water was purified using a delivery pump, an auto sampler, and a photodiode array detector
Milli-Q Rios/Elix water purification system (Millipore, Bedford, (PDA) and controlled by the EZchrom elite software. A reversed
1146 H.J. Kim et al. / Food Chemistry 196 (2016) 1144–1149

Fig. 2. HPLC chromatograms of a mixture of 16 SAs detected at 270 nm. (1) SA, (2) SIM2, (3) SDZ, (4) ST, (5) SPD, (6) SMR, (7) SMD, (8) SMTZ, (9) SM2, (10) SCP, (11) SMZ, (12)
SMM, (13) SIZ, (14) SPZ, (15) SDM, (16) SQX.

Table 1
Validation parameters of the developed RP-HPLC/UV method with IAC-SULF.

Peak# Compound RTa (min) r2b LODc LOQd Rse Asymm.f Precisions (% RSDg) Recovery (%)h
(lg/kg) (lg/kg)
Intercept Slope Intra-day (n = 6) Inter-day (n = 9)
1 SA 8.87 11160 2386 0.9997 19.6 65.2 – 0.88 2.4 3.4 95.0
2 SIM2 13.43 12757 3232 0.9995 23.7 78.9 26.22 1.07 2.8 3.6 103.5
3 SDZ 14.71 10521 2499 0.9998 26.5 88.2 6.44 1.13 4.3 3.2 102.1
4 ST 15.69 9852 2710 0.9999 25.0 83.3 4.61 1.06 1.7 2.3 90.2
5 SPD 16.61 10849 2840 0.9999 45.0 150.0 4.08 1.10 5.5 4.0 78.2
6 SMR 18.31 11783 2541 0.9995 34.6 115.4 8.15 0.85 2.2 3.3 105.5
7 SMD 21.07 11232 3619 0.9992 37.5 125.0 10.86 1.14 3.2 2.4 95.3
8 SMTZ 21.65 10731 3132 0.9999 20.5 68.2 1.48 0.97 1.1 1.8 87.0
9 SM2 23.08 11015 3309 0.9997 34.6 115.4 5.09 0.93 2.2 1.2 105.2
10 SCP 24.52 9617 2749 0.9996 30.0 99.0 3.47 0.97 1.1 0.8 101.5
11 SMZ 25.94 10555 703 0.9991 14.1 46.9 3.80 0.92 1.2 1.9 80.2
12 SMM 26.63 10061 4820 0.9993 15.3 51.1 2.31 0.88 2.0 2.3 104.2
13 SIZ 27.57 11089 4084 0.9997 18.8 62.5 1.28 0.83 1.9 2.1 98.4
14 SPZ 36.10 9625 5508 0.9992 26.5 88.2 17.50 0.88 1.8 2.5 90.4
15 SDM 43.34 10914 3713 0.9997 16.1 53.6 15.43 0.90 2.6 1.6 84.5
16 SQX 45.54 10434 5644 0.9990 25.0 83.3 6.74 0.91 1.4 1.9 90.4
a
Retention time (min).
b
Coefficients of correlation.
c
Limit of detection, the lowest analyte concentration that produces a response detectable above the noise level of the system.
d
Limit of quantification, the lowest level of analyte that can be accurately and precisely measured.
e
Resolution.
f
Asymmetry.
g
Relative standard deviation, expressed as %.
h
Average of recoveries at two spike levels (high and low).

the following gradient elution: 0 min (7% B), 0–5 min (7–7% B),
Table 2
Incidence and concentration of SAs in animal feeds. 5–10 min (7–10% B), 10–20 min (10–15% B), 20–22 min (15–21%
B), 22–32 min (21–32% B), 32–50 min (32–40% B) and
Sample Analyzed Detected Range of total SAs Kind of
50–50.1 min (40–7% B). The sample injection volume was 3 ll,
category samples sample (lg/kg) SAs
and the flow-rate was set at 0.4 ml/min. The peaks were detected
Bovine 47 1 368 SMZ
at 270 nm throughout the analysis. Before use, the mobile phase
150 SM2
Pork 35 1 180 SMZ was filtered through 0.45 mm membrane filters (Millipore, Milford,
155 SM2 MA, USA) and degassed under a vacuum.
Chicken 42 1 161 SMZ
Duck 32 1 460 SMZ
2.6. Method validation

Method validation was performed according to the guidelines


phase Unison UK-C18 (150 mm  3.0 mm, 3 lm particle size) set by the International Conference on Harmonization (ICH,
(Tokyo, Japan) column was used for all separations at a column 2005) and the International Union of Pure and Applied Chemistry
temperature of 30 °C. The mobile phase was composed of buffer (IUPAC, 2002). The method was validated for linearity, sensitivity,
A (0.1% acetic acid in PBS) and B (0.1% acetic acid in methanol) with selectivity, accuracy, and precision.
H.J. Kim et al. / Food Chemistry 196 (2016) 1144–1149 1147

Fig. 3. HPLC chromatograms obtained from animal feeds: (a) bovine, (b) pork, (c) chicken, (d) duck.
1148 H.J. Kim et al. / Food Chemistry 196 (2016) 1144–1149

3. Results and discussion in 4 different kinds of animal feeds; bovine, pork, chicken and duck,
and SMZ was found in all of them. It is considered that SMZ was
3.1. Method validation detected because it is one of the most widely used sulfonamide
antibiotics (Drillia et al., 2005). Also, in bovine and pork, not only
The developed RP-HPLC/UV method using IAC-SULF was vali- SMZ but SM2 was detected. The amount was ranged from 150 to
dated to verify its performance was compatible with the required 460 lg/kg, and the detailed results are shown in Table 2. Chro-
performance for routine SAs analysis in animal feeds. Several per- matograms from feeds containing SAs are shown in Fig. 3. The
formance characteristics were used including selectivity, linearity, results showed SAs in the feeds collected from local farms but
sensitivity, accuracy, and precision, to validate the measurement not in those feeds purchased directly from markets, it appears that
method. local farms are mixing SAs in the animal feeds. Therefore, the indis-
The selectivity was determined by the absence of interference criminate SAs use as additives in animal feeds must be stopped by
in the same chromatographic window, which was examined using government regulations, as well as by MRL standards for the
the SAs solution and blank. No interfering peaks were observed in proper amount of SAs. We believe that the above developed anal-
the blank chromatograms at the quantification wavelength ysis method for SAs could solve these problems.
(270 nm). As shown in Fig. 2 and Table 1, the chromatograms of A simple qualitative and quantitative method for the simultane-
the SAs indicate that the separation of all 16 compounds was suc- ous determination of 16 SAs from animal feed using RP-HPLD/UV-
cessfully achieved in less than 46 min, with good resolution and IAC-SULF was successfully developed and validated. The proposed
symmetric peaks. Resolution and asymmetry values were in the method provided appropriate accuracy and precision, and success-
range of 1.45–15.27, 0.83–1.14. The peak identification results of fully analyzed different animal feeds. These satisfactory results
the 16 studied compounds showed satisfactory selectivity on the demonstrated that these HPLC method also provides a good alter-
HPLC system. native for routine analyses because of its simplicity, specificity and
The linearity was assessed by building external calibration sensitivity, which allow its use in several potential applications as
curves for each compound using a working solution containing a reliable, quality evaluation method for animal feeds.
the SAs. Calibration curves were obtained by plotting the analyte
peak area versus its concentration for seven different concentra-
tions. Each concentration of the mixed standard solution was
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