CHEM. RES. CHINESE UNIVERSITIES 2012, 28(2), 195—199
Investigation of Breaking Efficiency of N-Glycosidic
Bond Between Sulfonamide and Honey
WANG Yan-hua1, LI Li1, CHEN Chia-hung2, CHENG Shuenn-ren3, LEE Mu-sheng2,5,
YU Pu-ping4 and CHANG-CHIEN Guo-ping2*
1. College of Biology and Enviornmental Engineering, Zhejiang Shuren University,
Hangzhou 310015, P. R. China;
2. Super Micro Mass Research & Technology Center, 3. Department of Business Administration,
4. Department of Mechanical Engineering, Cheng Shiu University, Kaohsiung 833, Taiwan, P. R. China;
5. Environmental Protection Bureau Kaohsiung City Government, Keelung 20143, Taiwan, P. R. China
Abstract Sulfonamide residue in honey existed in a form bonded to sugar via the N-glycosidic bond. It would result
in the possible underestimation of concentration of sulfonamide if it is not decomposed by chemical methods. However, in Mainland China and Taiwan(P. R. China), the regulation for sulfonamide residue analysis does not include
hydrolysis and has been applied to a very broad range of samples, for example, egg, milk, meat, seafood as well as
honey. This paper demonstrates the necessity of hydrolysis of it prior to extracting honey. The breaking efficiencies of
N-glycosidic bond were investigated by 2 mol/L hydrochloric acid, pure methanol and 0.5 mol/L hydrochloric acid in
methanol, respectively. It was found that acid plays the key role in breaking the N-glycosidic bond, and it was also
noticed that the dissolution of liberated sulfonamide in methanol could carry the reaction forward in favor of breaking
the N-glycosidic bond.
Keywords Sulfonamide; Honey; Liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESIMS/MS); N-Glycosidic bond
Article ID 1005-9040(2012)-02-195-05
1
Introduction
Sulfonamide is an antimicrobial agent widely used in
animal as a growth promoter as well as for therapeutic purpose.
Sulfathiazole was initially recommended to control the American Foulbrood, one of the most widespread and lethal diseases
affecting honey bee. Its use has been banned nowadays due to
its residue in honey lasting for months[1,2]. Nevertheless those
compounds with similar moiety have still been discovered in
several honey samples from many countries[3]. For this reason,
it is very important to analyze sulfonamide residue in honey to
assure that this natural product does not imply any risk to consumers.
Some countries in the European Union have established
their own tolerated levels for these antibiotics. For example,
Belgium and UK have set limits of 20 and 50 ng/g, respectively
for total sulfonamide in honey[4]. Switzerland has set a maximum residue level(MRL) of 50 ng/g for the sum of sulfonamide and analog[5]. However, in many other countries, no MRL
has been set in food, which means that those antibiotics, if
present, must be kept below the limit of quantitation(LOQ) of
the analytical method. The European Commission Decision
2002/657/EC further states that the method based only on
chromatographic analysis without the use of molecular
spectrometric detection is not suitable for confirmation. To our
knowledge, only liquid chromatography coupled to electrospray ionization tandem mass spectrometry(LC-ESI-MS/MS)
instrumentation could meet these criteria for sulfonamide detection. Many scholars have presented rapid confirmatory methods for monitoring sulfonamide residue in honey, milk, egg,
chicken muscle, aquatic product on the basis of different pretreatments coupled with LC-ESI-MS/MS[6―9]. Shao et al.[6]
reported a method for measuring the residue of 17 sulfonamide
compounds in porcine tissue via extracting sulfonamide from
acetonitrile followed by solid phase extraction(SPE) coupled
with LC-ESI-MS/MS. Sergi et al.[7] presented a LC-ESIMS/MS confirmatory procedure for monitoring 13 sulfonamide
compounds in animal tissue by matrix solid-phase dispersion
technique.
It is a fact that the regulation for sulfonamide residue
analysis has been used for a very broad range of samples in
many regions. For example, the sulfonamide residue in egg,
milk, meat, seafood and honey is detected via a similar pretreatment process in Mainland China. To our knowledge, matrixes from different foods have diverse chemical components.
In this instance of honey, there is a lot of sugar responsible for
the matrix effect on analyte[10,11]. Since 1990, it has been clear
that sulfonamide residue present in honey combines with
———————————
*Corresponding author. E-mail: [email protected]
Received May 26, 2011; accepted November 8, 2011.
Supported by the National Natural Science Foundation of China(No.20907046) and the Natural Science Foundation of Zhejiang Province of China(No.Y4100623).
196
CHEM. RES. CHINESE UNIVERSITIES
reducing-sugar via N-glycosidic bond. This sugar-bonded form
is difficult to detect[12]. Only when the sugar-bonded sulfonamide is decomposed by chemical method, could it be liberated
and become detectible. In Mainland China, the regulation of
detecting sulfonamide residue in honey includes the following
steps: the sample is extracted from acetonitrile, cleaned up by
hexane and detected by LC-ESI-MS/MS. The similar pretreatment process could also be found in the regulation of Taiwan,
P. R. China. It could be speculated that acetonitrile in the
process above mentioned is not able to extract the sulfonamide
residue bonded to sugar. Therefore the actual concentration of
sulfonaminde residue in honey is most likely underestimated.
However, some methods concerning acidic hydrolysis have
been developed to determine sulfonamide residue in honey in
recent years. Verzegnassi et al.[5] and Thompson et al.[13] respectively detected several sulfonamide compounds in honey
by acid hydrolysis to liberate sugar-bonded sulfonamide in the
pretreatment process. Sheridan et al.[8] used a multi-screening
approach to monitor 14 sulfonamide compounds and one chloramphenicol with the help of an acid hydrolysis to liberate the
sugar-bonded sulfonamide followed by SPE to remove potential interference. An absolute recovery of more than 60% has
been achieved. Bernal et al.[11] has recently detected 14 sulfonamide compounds in honey simultaneously by high performance liquid chromatography(HPLC) with the aid of fluorescence, in which pure methanol was chosen as extracting agent
for the following reasons: it could break the N-glycosidic bond
between sugar and the sulfonamide, further decrease the surface tension, avoiding the emulsion formation and also be capable of dissolving the sulfonamide.
Considering that in Mainland China and Taiwan(P. R.
China), the regulation for sulfonamide residue analysis in honey does not include hydrolysis, we adopted the method developed by Food and Drug Administration in Taiwan(P. R.
China) that encompasses liquid-liquid extraction(LLE) coupled
to LC-ESI-MS/MS, with the main purpose to confirm the necessity of hydrolysis prior to extracting honey. The detection
was performed 10 min and one night respectively after the sulfonamide standard solution spiked into honey. The first part of
the paper compares the recoveries of the two spiked batches.
The second part confirms the necessity of prior acidic hydrolysis to avoid possible underestimation of sulfonamide in honey.
The third part evaluates the efficiencies of different reagents in
breaking the N-glycosidic bond, namely, 2 mol/L HCl, pure
methanol, and 0.5 mol/L HCl in methanol.
All chemical analysis was carried out in the Super Micro
Mass Research and Technology Center at Cheng Shiu
University(Taiwan, P. R. China), whose laboratory was
accredited by the Food and Drug Administration in Taiwan, P.
R. China.
2
Materials and Method
2.1
Materials and Reagents
2.1.1
Materials
Five
sulfonamide
compounds,
sulfamerazine(SMZ),
Vol.28
sulfamethazine(STZ), sulfamonomethoxine(SDT), sulfaquinoxaline(SQX) and sulfadimethoxine(SDX) in honey were
detected. HPLC grade methanol, hexane, acetonitrile, formic
acid and standard compound were purchased from J. T. Baker
Co.(USA). Distilled-deionized water was generated in-house
on a Milli-Q-Plus water system. High-speed screw-top polypropylene centrifuge tubes were obtained through Fisher
Scientific(USA). Blank honey was obtained from Super Micro
Mass Research and Technology Center at Cheng Shiu University(Taiwan, P. R. China).
2.1.2 Standard Stock Solution
Each standard of 5 mg was put into a 50 mL volumetric
flask to obtain 100 mg/L standard stock solution by adding pure
methanol to the mark. This solution was used immediately in
preparation for the following working standard solution. The
excessive solution was stored in a dark glass bottle at –20 °C
and could be stable for at least 6 months. The solution should
be warmed up to room temperature before use.
2.1.3
Working Standard Solution
Each standard stock solution of 5 mL was pipetted into a
50 mL volumetric flask and brought to the mark with 20%
(volume fraction) methanol to obtain 10 mg/L working standard solution, which was later diluted by 20% methanol to
obtain 1 mg/L working standard solution. This solution was
stored in a dark glass bottle at 4 °C and could remain stable for
at least one month. The solution should be warmed up to room
temperature before use. Matrix-matched calibration curves
were all constructed in a calibration range of 10, 25, 50, 100
and 250 μg/L.
2.2
2.2.1
Sample Preparation
Groups of Samples
Three groups of samples were prepared, namely, groups A,
B and C. For each sample, 2 g of honey was weighed into a 50
mL disposable centrifuge tube. Three levels of concentrations
were spiked into each group.
Before the spiked samples were extracted, group A was
maintained for 10 min. While both groups B and C were kept
overnight to ensure complete bonding of sulfonamide in standard solution and sugar in honey. After one night, each sample
in group C was added with 5 mL of 2 mol/L HCl and left at
room temperature for 1 h. All spiked samples from groups A, B
and C were extracted via LLE. Meanwhile, a set of matrixmatched calibration curves were constructed following the
process of LLE(the samples used were immediately extracted
after being spiked).
To compare the efficiencies of different reagents, namely,
2 mol/L HCl, pure methanol, and 0.5 mol/L HCl in methanol in
breaking the N-glycosidic bond, three new groups, D, E and F
were prepared. Each group was spiked at three concentrations:
25, 100 and 250 μg/L, respectively. All the samples were maintained at room temperature overnight. Then samples in D, E
and F were respectively added with 5 mL of 2 mol/L HCl, 5 mL
of pure methanol and 5 mL of 0.5 mol/L HCl in methanol. After
maintained for 1 h at room temperature, all spiked samples
were extracted following the LLE procedure.
No.2
WANG Yan-hua et al.
For better comparison, we set groups D, E and F with
their corresponding groups D', E' and F'. The corresponding
groups were meant to offset the matrix effect from three
reagents. Honey of 2 g was first mixed with 5 mL of 2 mol/L
HCl in D', 5 mL of pure methanol in E' and 5 mL of 0.5 mol/L
HCl in methanol in F'. They were left at room temperature for 1
h. Samples were immediately extracted after a series of standard solution were spiked, which could prevent sulfonamide
from bonding with reducing-sugar.
In this paper, we defined the breaking efficiency as follows: to divide the peak area of the liberated sulfonamide in
groups D, E and F by the peak area of all free sulfonamide in
groups D', E' and F'.
2.2.2
2.3
of 10 μL was employed for all diluted honey solutions. The
HPLC flowage directly entered into the MS detector between
8.5 and 15.5 min by means of a cut-off valve. Analytes were
detected via electrospray ionization in the positive mode.
Nitrogen was used both as TurboIonSpray and curtain gas at
flow rates of 7.5 and 10 mL/min, respectively. The block
source temperature was maintained at 35 °C. The collision
energy voltages applied were summarized in Table 2. Data
processing was done with the aid of Analyst software.
Table 2
Analysis by LC-ESI-MS/MS
Analysis was performed on Agilent Technology 1200 Series coupled to an Agilent Technology 6410 triple-stage quadrupole mass spectrometer equipped with a TurboIon Spray
ionization source. The HPLC column was Supelco Discovery
HS-C18(2.1 mm×50 mm i.d., 3 μm). The mobile phase was
constituted by solvent A(0.2% formic acid in water, volume
fraction) and solvent B(0.2% formic acid in acetonitrile, volume fraction). The gradient elution program employed for the
separation and cleanup is given in Table 1. An injection volume
Table 1
Gradient elution program
Time/min
Flow rate/
(mL·min–1)
Volume ratio of
solvent A to solvent B
0
0.5
0.2
0.2
97:3
97:3
6
0.2
55:45
10
0.2
50:50
13
0.2
50:50
15
0.2
15:85
15.1
0.2
20
0.2
20.1
0.3
97:3
28
0.3
97:3
28.1
0.2
97:3
0:100
0:100
Transition reactions and conditions for the
measurement of sulfonamide
Compd.
Molecular
weight
SMZ
264.3
Parent ion
265
STZ
278.3
279.1
SDT
280.3
281.1
SQX
300.2
301.2
SDX
310.3
311.1
LLE Step
A mixture of 10 mL of acetonitrile/methanol(95:5, volume
ratio) was rotated for 1 min and left for 3 min. Anhydrous
magnesium sulfate of 2 g was added to eliminate trace of water,
the mixture was left for 3 min and centrifuged for 10 min at
4000 r/min. Then the supernatant was collected. The residue
was extracted from 10 mL of acetonitrile/methanol once more
via the same procedure. Supernatants from two extractions
were combined, to which 10 mL of hexane was added, the
mixture was left for 3 min and then kept still. The hexane layer
was discarded; the acetonitril/methanol layer was once again
extracted from 10 mL of hexane via the procedure above mentioned. The acetonitril/methanol layer was collected into a 50
mL dark glass bottle and evaporated to dryness under a stream
of nitrogen at 45 °C. The dry residue was reconstituted with 2.5
mL of 20%(volume fraction) methanol solution and mixed by
vortex for 5 min, and filtered through a 0.22 μm nylon filter
directly into an HPLC vial. All spiked samples mentioned
above were analyzed in triplicate.
197
3
m/z
Product ion
156
92
186.1
124
126
108.1
156
92
108.1
92
Collision
energy/eV
15
25
15
25
15
20
15
30
30
35
Results and Discussion
3.1 Method Validation of LLE Developed by FTA
in Taiwan, P. R. China
The linearity of matrix-matched calibration standard curve
reached a corresponding correlation coefficient(r2) of higher
than 0.995 in all the analytes. To assess accuracy and precision,
each analytical batch consisted of a set of calibration standard,
procedural blank spiked(blank), a series of spiked samples. It
was found that all the accuracies of QC samples were within
current recommended acceptance criteria, and the relative
standard deviations(RSD, n=3) for all spiked samples were
below 10%.
3.2
Binding of Sulfonamide and Honey
The mean recoveries of every analyte in groups A, B and
C were obtained via matrix-matched calibration curves, as
listed in Table 3. It was observed that there were significant
differences in recoveries of five sulfonamide compounds in
both groups A and B. Group A fell into a range of
53.01%―119.97%. Group B gave consistently low recoveries
of 13.15%―51.24%, especially that of SQX, which was lower
than the limit of detection at a level of 25 μg/L, implying a
remarkable decrease in the concentration of free sulfonamide
with an extension of maintaining time. Many scholars[12―17]
also noticed that the concentration of sulfonamide in honey
decreased over a period of time. Our present result further
shows that the spiked sulfonamide could bond to sugar easily in
honey at room temperature.
It was also shown that the change of recovery depended
on not only the structure of analyte, but also the spiked
concentration. In groups A and B, lower concentration samples
usually obtained lower recoveries. For example, the recovery in
198
CHEM. RES. CHINESE UNIVERSITIES
group B at 25 μg/L level could only be as high as 30%, while at
100 and 250 μg/L levels the recoveries were higher. It could be
possible that only a certain amount of spiked sulfonamide
Table 3
Analyte
SMZ
STZ
Vol.28
actually bonds to the limited reducing-sugar in honey. On the
other hand, the structure of sulfonamide affects the formation
of the N-glycosidic bond.
Average recoveries(%) for each analyte at three spiked levels for groups A, B and C
Group A
25 μg/L
81.93
70.53
Group B
100 μg/L
118.98
119.97
Group C
250 μg/L
118.98
96.49
25 μg/L
18.65
20.05
100 μg/L
39.71
25.88
250 μg/L
23.88
17.42
25 μg/L
94.57
116.71
100 μg/L
104.07
124.41
250 μg/L
96.82
122.28
138.48
SDT
73.50
116.26
94.48
13.15
32.56
27.59
147.70
140.92
SQX
53.01
117.30
116.22
ND*
48.48
47.38
192.70
231.09
268.45
SDX
78.92
116.48
119.38
30.16
30.15
51.24
294.16
302.44
308.22
* ND: not detected.
As the bond between the sulfonamide and the reducingsugar in honey could form easily at room temperature, it is
strongly recommended that the regulation in Mainland China
and Taiwan(P. R. China) for sulfonamide residue analysis in
honey should break the bond prior to LLE to avoid underestimate of the residue.
3.3
Effect of Acid Hydrolysis
We estimated the effect of acid hydrolysis of groups B and
C. First, 5 mL of 2 mol/L HCl was mixed with the spiked group
C, it was kept for 1 h at room temperature, followed by LLE.
The recovery of group C is also listed in Table 3. It is noticeable that some of the recoveries have gone over the acceptance
criteria. One reason could be that the same matrix-matched
calibration curve did not reflet perfectly for samples in group C,
due to their different left time before LLE. Another reason for
Table 4
Analyte
SMZ
STZ
25 μg/L
102.66
115.40
3.4 Comparison of the Breaking Efficiency by
Acid and Methanol in LLE
In our assignment, three reagents, 5 mL of 2 mol/L HCl,
5 mL of pure methanol and 5 mL of 0.5 mol/L HCl in methanol
were respectively investigated. The breaking efficiency was the
peak area of the liberated sulfonamide in groups D, E and F
divided by that in groups D′, E′ and F′, as listed in Table 4.
Breaking efficiency(%) of three reagents at three spiked levels
Group D
100 μg/L
95.66
97.24
Group E
250 μg/L
89.82
91.97
Group F
25 μg/L
2.27
15.65
100 μg/L
27.78
29.29
250 μg/L
21.24
22.05
25 μg/L
74.47
90.75
100 μg/L
82.64
74.15
250 μg/L
89.80
92.25
SDT
103.42
92.66
86.21
28.35
28.36
30.86
78.96
91.30
91.81
SQX
82.28
68.37
68.60
12.50
2.30
1.64
82.19
88.87
90.01
SDX
114.26
89.81
88.51
2.33
0.61
0.17
97.65
92.01
94.58
It was observed that the breaking efficiencies were
68.37%―114.26% for 2 mol/L HCl, 0.61%―30.86% for pure
methanol and 74.15%―97.65% for 0.5 mol/L HCl in methanol,
respectively. In general, the two reagents containing
HCl(groups D and F) obtained high breaking efficiency, whereas methanol hardly had the ability of breaking the
N-glycosidic bond in our experiment. Despite the methanol
included in group F, acid played the key role in breaking the
N-glycosidic bond. Our result is consistent with that of many
previous reports. In group F, we should always notice the solubility of free sulfonamide in methanol. Thermodynamically, the
rapid dissolution of sulfonamide in methanol could cause a
shift in chemical equilibrium, further promoting the liberation
of sulfonamide. So it would be possible that the mixture of
hydrochloric acid and methanol could increase the breaking
rate for sugar-bonded sulfonamide.
4
that could be the matrix effect resulted from HCl that remarkably enhanced the signal. Nevertheless, it is clearly indicated
that the acidic hydrolysis prior to LLE has drastically increased
the recoveries. There is no doubt that hydrochloric acid could
release the sugar-bound sulfonamide back to free form. The
extent of recovery increment was also highly dependent on
both the spiked level and the structure of analyte.
Conclusions
We adopted the method developed by Food and Drug Administration in Taiwan, P. R. China. The goal was to confirm
the necessity of breaking the N-glycosidic bond between sulfonamide residue and honey. Firstly, it was validated once more
that the spiked sulfonamide could easily bond with the
reducing-sugar in honey at room temperature. So it was
strongly recommended that in Mainland China and Taiwan, P.
R. China, the regulation for sulfonamide residue analysis in
honey should include hydrolysis prior to LLE to liberate sulfonamide to avoid its underestimation. It was observed that the
breaking efficiencies of reagents were 68.37%―114.26% for 2
mol/L HCl, 0.61%―30.86% for pure methanol and 74.15%―
97.65% for 0.5 mol/L HCl in methanol. It was also noticeable
that the dissolution of liberated sulfonamide in methanol could
carry the reaction forward in favor of breaking the N-glycosidic
bond. So the mixture of hydrochloric acid and methanol could
increase the breaking rate for sugar-bonded sulfonamide.
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