4020

Jin-Zhong Xu1
Jian-Jun Miao2
Hong Lin1
Tao Ding1
Zhen-Yun Zhao1
Bin Wu1
Chong-Yu Shen1
Yuan Jiang1
1

National Bee-Product Reference

Laboratory, Jiangsu Entry-Exit
Inspection and Quarantine
Bureau, Nanjing, P. R. China
2
Nantong Environmental
Monitoring Center, Nantong,
P. R. China

Received June 19, 2009

Revised September 16, 2009
Accepted September 22, 2009

J. Sep. Sci. 2009, 32, 40204024

Research Article

Determination of amitraz and 2,4dimethylaniline residues in honey by using

LC with UV detection and MS/MS
A method for the determination and confirmation of amitraz and its degradation product
2,4-dimethylaniline (2,4-DMA) in honey is reported. Determination of the two compounds
was based on HPLC with UV detection and MS/MS (LC-MS/MS) after a liquidliquid
extraction with hexane and isopropyl alcohol. Chromatographic separation was achieved
by using a C18 column with a gradient mobile phase consisting of 0.02 M ammonium
acetate and ACN. Recoveries for fortified honey ranged from 83.4 to 103.4% for amitraz
and from 89.2 to 104.7% for 2,4-DMA with RSD values lower than 11.6% for HPLC and
LC-MS/MS methods. LOD was 6 mg/kg for amitraz and 8 mg/kg for 2,4-DMA, while LOQ
was 20 mg/kg for amitraz and 25 mg/kg for 2,4-DMA in HPLC method. LOD was 1 mg/kg
for amitraz and 2 mg/kg for 2,4-DMA, while LOQ was 5 mg/kg for amitraz and 10 mg/kg for
2,4-DMA in LC-MS/MS method.
Keywords: Amitraz / 2,4-Dimethylaniline / Honey / HPLC / LC-MS/MS
DOI 10.1002/jssc.200900437

1 Introduction
Amitraz (N-methylbis(2,4-xylyliminomethyl)amine) is a
triazapentadiene acaricide. It is widely applied on beehives
to control the beehive parasite Varroa jacobsoni destructor
which endangers beekeeping all over the world. However, it
also implies a risk of polluting honey and other honeybee
products at the same time. Maximum residual limit in
honey was set as 0.01 mg/kg in Germany and Italy and
0.2 mg/kg for European Union [1, 2]. Several methods have
been reported to determine amitraz residue in honey [36].
The amitraz molecule is very unstable and can hydrolyze
through a series of intermediate compounds to form
another environmentally stable toxic compound 2,4dimethylaniline (2,4-DMA) [3, 7]. The formulas are shown
in Fig. 1. So it is significant to develop a method to
determine amitraz and 2,4-DMA residues in honeybee
products synchronously.
Traditionally, amitraz was determined by GC with
ECD or MS detector [3, 4, 813], which required hydrolysis
of amitraz to 2,4-DMA and derivatization with heptafluorobutyric acid anhydride. These steps make the method
time consuming and troublesome. HPLC has also been
used to determine amitraz in honey or beeswax [5, 6, 14].
However, most of the articles do not include 2,4-DMA

Correspondence: Dr. Jin-Zhong Xu, National Bee-Product Reference Laboratory, Jiangsu Entry-Exit Inspection and Quarantine
Bureau, 99 Zhonghua Road, Nanjing 210001, P. R. China
E-mail: xujz@jsciq.gov.cn
Fax: 186-25-52345176

Abbreviation: 2,4-DMA, 2,4-dimethylaniline

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

in their analytes. GC-MS and LC-MS have been used

to determine amitraz in ground waters, soil, and
pear samples [1517]. But no articles were found using
LC-MS/MS for the determination of amitraz in honey
samples.
Amitraz in honeybee products is commonly extracted
with organic solvent like hexane [5] or by SPE [6] on polar
cartridges after diluting honey samples with water of
different pH values. Solid phase microextraction [12] has
been recommended for the determination of amitraz in
beeswax.
This paper describes a simple method for the simultaneous determination and confirmation of amitraz and 2,4DMA in honey. Determination procedure involves only a
liquidliquid extraction and direct analysis by HPLC with
UV detector and by LC-ESIMS/MS after the same extraction procedure. Several factors that affect the extraction
efficiency were studied.

2 Materials and methods

2.1 Chemicals and reagents
Amitraz and 2,4-DMA standards were purchased from
Dr. Ehrenstorfer (Augsburg, Germany). The stock standard
solutions of the two compounds were prepared in ACN
(amitraz is not stable in methanol) at a concentration of
1 mg/mL and were stored at 41C in the dark [7, 17]. They are
stable in 1 month. The solutions were diluted to required
concentrations with 1/1 ACN/water before use.
ACN and hexane of HPLC grade were purchased from
Sigma-Aldrich (Steinheim, Germany), sodium hydroxide
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Liquid Chromatography

J. Sep. Sci. 2009, 32, 40204024

CH3

CH3

4021

Table 1. Ion transitions and corresponding collision energies

CH3

used for amitraz and 2,4-DMA quantifications

N CH N CH N

CH3

H3C

Analyte

Ion transitions
(m/z)

Collision energy
(eV)

Quantification
ion

Amitraz

294.2-163
294.2-253
122.0-77.1
122.0-105.1
122.0-107.1

12
10
22
16
10

163

2,4-DMA
CH 3

107.1

NH 2

H 3C
Figure 1. Structures of amitraz and 2,4-dimethylaniline.

and iso-propyl alcohol of analytical grade were purchased

from Nanjing Chemical Reagent No. 1 Factory (Jiangsu,
China), and ammonium acetate of HPLC grade was
purchased from Tedia (OH, USA). The water used in all
experiments was purified on a Milli-Q system from Millipore (Bedford, MA, USA).

3 min, B 5 90%; 9 min, B 5 90%; 9.1 min, B 5 10%; 11 min,

B 5 10%. The flow rate was set at 250 mL/min, and the
injection volume at 25 mL. The MS detector was operated in
the positive ion mode. The capillary voltage was set at
4.8 kV, and the capillary temperature at 3301C. Nitrogen was
used as sheath gas and auxiliary gas, with gas pressure at
30 arb and 5 arb, respectively. Argon was used as collision
gas and its pressure was 1.5 mTorr. The collision energies
were separately optimized for the selected ion transitions of
both amitraz and 2,4-DMA (Table 1).
2.2.3 Extraction procedure

2.2 Apparatus
2.2.1 HPLC system
An Agilent 1100 LC system, consisting of a G1311A
quaternary pump, a G1322A degasser, a G1313A autosampler and a G1314A variable wavelength detector, was used
for HPLC analysis. The separation was performed on a
Cosmosil packed column, 5C18-MS-II (5 mm, 150  4.6 mm
id), with A: 0.02 M ammonium acetate and B: ACN as the
mobile phases. The gradient conditions were set as follows:
0 min, B 5 30%; 8.5 min, B 5 30%; 10 min, B 5 72%;
25 min, B 5 72%; 26 min, B 5 30%; 30 min, B 5 30%. The
flow rate was 1.0 mL/min, the injection volume was 40 mL,
and the column temperature was 301C. The wavelength of
the variable wavelength detector was set at 238 nm at
starting to detect 2,4-DMA, and changed to 289 nm after
10 min to detect amitraz. All of the data collections and
calculations were performed using Agilent Chemstation
revision A.10.02.
2.2.2 LC-ESI-MS/MS
The LC-ESI-MS/MS system consisted of a Surveyor LC
quaternary pump and an autosampler, coupled to a TSQ
Quantum triple stage quadrupole mass spectrometer
(Thermo Electron, San Jose, CA, USA). The chromatographic separation was performed on a Waters SunFire C18
column (3.5 mm, 100  2.1 mm id). A: 0.02 M ammonium
acetate and B: methanol were used as mobile phases. The
gradient conditions were set as follows: 0 min, B 5 10%;
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

For honey, a sample amount of 5 g was placed in

a centrifuge tube. Five milliliters of 0.1 M sodium hydroxide, 10 mL of hexane and 3 mL of isopropyl alcohol were
added and shaken on a vortex shaker (Taicang, Jiangsu,
China) for 1 min. The organic phase was separated by
centrifugation at 2000 rpm for 5 min and collected. The
operation was repeated once. The two organic portions were
combined and the solvent was evaporated to dryness in
chi, Plawil, Switzerland) under
a rotary evaporator (Bu
vacuum at 401C. The residues were first redissolved
with 500 mL of ACN and then with 500 mL of water; the
two portions were mixed together and filtered through
a 0.45 mm organic membrane filter prior to chromatographic analysis.
2.2.4 Calibration curve standard working solutions
Working solutions were prepared by adding blank sample at
five concentration levels: 25, 50, 100, 200, 500 and 1000 ng/
mL according to the extraction procedure (2.2.3). It is the
same as 5, 10, 20, 40, 100 and 200 mg/kg amitraz and 2,4DMA in sample. They are prepared just before use.

3 Results and discussion

3.1 Liquidliquid extraction
Ether, hexane, dichloromethane and ethyl acetate were
assayed to extract amitraz and 2,4-DMA from honey. The
results showed that dichloromethane and ethyl acetate were
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4022

J. Sep. Sci. 2009, 32, 40204024

J.-Z. Xu et al.

80

2,4-DMA
amitraz

70

Recovery (%)

60
50
40
30
20
10
0
4

10

12

pH

Figure 2. Effects of different pH on the extraction of amitraz and

2,4-DMA (blank samples spiked at 40 mg/kg).

not proper extraction solvents, obtaining very low recoveries

(o30%). Further experiments showed that, compared to
ether, hexane was better in extraction of amitraz and 2,4DMA from the matrix, resulting in higher recoveries and
lower RSDs. Thus hexane was chosen as the extraction
solvent.
The amitraz was easy to decompound in acid solution
and 2,4-DMA was alkaline compound. The effects of
different pH on the extraction of both amitraz and 2,4-DMA
were studied. The results are shown in Fig. 2. It can be
observed that, when the pH value of the solution decreased
to 4.0, the recovery of 2,4-DMA was rather low (o10%).
As the pH value increased, the recovery of 2,4-DMA
increased greatly. However, the recovery of amitraz was
not greatly affected by pH values. When pH value was
near 10.0, the recovery was relatively higher. So in the
later experiments, the pH value of the extraction solution
was fixed at about 10.5 by adding 5 mL of 0.1 M sodium
hydroxide into 5 g of honey sample. During the extraction
process, the amitraz was stable, and the hydrolysis
was below 10%. The result was the same with the
reference [7].
The addition of isopropyl alcohol can increase the
recoveries of both amitraz and 2,4-DMA a lot. If no addition
of isopropyl alcohol, the recovery of amitraz would decrease
to 41.9%, while that of 2,4-DMA to 26.6% (n 5 5). Isopropyl
alcohol can dissolve both in aqueous solution and in hexane;
it can decrease the surface energy of water, thus increase the
recoveries of the two compounds. (Isopropyl alcohol is an
often used cosurfactant, it helps dissolving organic
compound in water, thus increasing the recoveries of the
two compounds.)
As 2,4-DMA is volatile [3], heating temperature and time
will undoubtedly influence its recovery, so the strict control
of heating temperature and time is very important to obtain
relatively high and stable recovery of 2,4-DMA. However,
the decrease of heating temperature will prolong heating
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

time when evaporating. Different temperatures in the

evaporation step were tested. Results showed that when the
temperature of water bath was set at 401C and the corresponding evaporation time was about 5 min, best recoveries
of 2,4-DMA were obtained (about 6070%).
After rotary evaporation, several solvents including
methanol/water (3:7), ACN, ACN/water (8:2, 6:4, 3:7) and
water were tested to dissolve the residues. The
results showed that among these solvents, only with ACN,
good recovery of amitraz was obtained, as long as water
was included, the recovery of amitraz decreased
sharply, from 80 to 5.5%. The recovery of 2,4-DMA was just
opposite, obtaining good recovery with water or the mixture
of water and ACN mentioned above (460%). If only with
ACN, the recovery was much lower (o25.3%). As a
compromise, two steps were taken to dissolve both of the
two compounds, first with 500 mL of ACN, then with 500 mL
of water. The two portions of solution were filtered through
a 0.45 mm organic membrane filter prior to chromatographic analysis.

3.2 Quantification by HPLC

2,4-DMA is weakly retained in a reversed-phase column,
while the retention of amitraz is much stronger. So a
gradient is needed to obtain proper retention time for the
two compounds. The proportion of ACN was 30% at the
first 8.5 min to elute 2,4-DMA, then the proportion of ACN
rose to 72% at 10 min and was kept until 25 min for the
elution of amitraz. The increase of ACN in the mobile phase
can reduce the retention time of amitraz, but there was an
interference peak near the peak of amitraz. To separate the
two peaks, the proportion of ACN decreased, and different
C8 or C18 columns were tested (C18 columns included
Kromasil, Cosmosil, Lichrospher 100, Thermo BDS
HYPERSIL, 150 mm  4.6 mm id or 250 mm  4.6 mm id,
5 mm, C8 column was Agilent Eclipse XDB, 250 mm  4.6
mm id). The results showed that using Cosmosil C18
column and the gradient program introduced in Section
2.2.1, the interference peak could be well separated from
amitraz peak.
Results from the validation of amitraz and 2,4-DMA are
provided in Table 2. Calibration curves were constructed
from peak areas versus compounds concentrations.
Good linearity was observed at the range of 20 ng/mL
to 2 mg/mL for amitraz and 2,4-DMA. The linearity
equation was Y 5 76.7408X10.4682 for amitraz and
Y 5 77.5578X 0.5197 for 2,4-DMA with correlation coefficient R2 5 0.996 at least. LOD determined for the method
was 6 mg/kg for amitraz and 8 mg/kg for 2,4-DMA, giving a
response with a signal-to-noise ratio (S/N) of 3 and LOQ was
20 mg/kg for amitraz and 25 mg/kg for 2,4-DMA, giving an
S/N ratio of 10. In honey samples, recoveries ranged from
83.4 to 97.1%. RSD values were good, being lower than
10.4%. Chromatograms of honey samples free from residues and spiked samples are shown in Fig. 3.
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Liquid Chromatography

J. Sep. Sci. 2009, 32, 40204024

1 6 3 .1 0

Method

LC-MS/MS
Amitraz
HPLC

LC-MS/MS
2,4-DMA
HPLC

Concentration
(mg/kg)

10
20
50
20
50
100
10
20
50
40
100
200

Mean
recovery
(%)

Precision
RSD (%)

97.5
103.4
95.6
83.4
92.6
88.7
104.7
96.3
94.8
97.1
89.2
93.0

10.6
11.6
6.8
3.4
5.8
6.0
8.5
6.3
7.4
6.3
10.4
5.2

100
90
80
70
60
50
40
30
20
10
0
120

2 9 4 .0 1
2 5 3 .0 9

140

160

180

200
220
m /z

240

260

280

300

107.08

Relative Abundance

Compounds

Relative Abundance

Table 2. Mean recoveries and precisions of amitraz and 2,4DMA from honey at different fortification levels

4023

n 5 5.

100
90
80
70
60
50
40
30
20
10
0

77.09

105.05

79.10

103.06

122.06

70

75

80

85

90

95

100

105

110

115

120

125

130

m/z

Figure 4. MS/MS full scan spectra of amitraz and 2,4-DMA.

A
mAU
8
6
4
2
0
-2
-4

7.5

10

12.5

15

17.5

20

22.5

25

27.5

min

25

27.5

min

B
mAU
8
6
4
2
0
-2
-4

2,4-DMA

amitraz

7.5

10

12.5

15

17.5

20

22.5

Figure 3. Chromatograms of 2,4-DMA and amitraz with UV

detection at 238 nm and 289 nm, respectively. (A) Blank honey
sample, (B) blank sample spiked with 100 mg/kg of amitraz and
200 mg/kg of 2,4-DMA.

3.3 Quantification and confirmation by

LC-ESI-MS/MS
Using positive ion ESI, triple quadrupole mass spectrometer
parameters were optimized with amitraz and 2,4-DMA
standards. MS/MS-full scan spectra of amitraz and 2,4DMA are given in Fig. 4. Selected reaction monitoring
(SRM) was used to measure the transitions from the
precurser ions to product ions. Figure 5 shows typical
selected reaction monitoring chromatograms for the confirmation of amitraz and 2,4-DMA in honey samples.
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Compared to UV detection, the LC run time with MS

detection was much shorter (from 30 min with UV detector
to 11 min with MS detector). This is attributed to the
selectivity and specificity of MS spectrometry detection. No
interference peak was observed near the peaks of amitraz
and 2,4-DMA.
For a residue to be positively confirmed, the retention
time had to match that of a standard to within 5%. In
addition, the ion transitions monitored had to be present at
a signal-to-noise of greater than 10 and the relative abundances of the integrated peaks for each transition had to
match that observed in a matrix standard by710% (i.e. if the
relative abundance of an ion transition is 40% in the standard, the relative abundance had to be between 3050% in
the sample) [18]. Using these criteria, amitraz and
2,4-DMA were confirmed by analysis of control honey
fortified at three different levels: 10, 20, 50 mg/kg for amitraz
and 2,4-DMA. Recoveries and RSD results are shown in
Table 2. All the criteria could be well met, indicating this
confirmation method can be used in confirming amitraz
and 2,4-DMA residues in honey.
Linearity scope was from 5 ng/mL to 200 ng/mL for
amitraz and 2,4-DMA. The linearity equation was
Y 5 216890X 61987.6 for amitraz and Y 5 16767.8X1
13241.3 for 2,4-DMA with correlation coefficient R2 5 0.99
at least. LOD determined for the method was 1 mg/kg for
amitraz and 2 mg/kg for 2,4-DMA, giving a response with a
signal-to-noise ratio (S/N) of 3 and LOQ was 10 mg/kg for
amitraz and 2,4-DMA, giving an S/N ratio of 10 in spiked
sample. In honey samples, recoveries ranged from 94.8 to
104.7%. RSD values were lower than 11.6%. Chromatograms of honey samples free from residues and spiked
samples are shown in Fig. 5.
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4024

J. Sep. Sci. 2009, 32, 40204024

J.-Z. Xu et al.

A
1.41 2.17

Relative Abundance

100
90
80
70
60
50
40
30
20
10
0

0.98

2.85
2.92

0.69

6.24

3.95 4.08
4.77

6.43

6.09
5.28

10.94
10.61

6.57
6.74
6.98 7.18

10.57

The authors have declared no conflict of interest.

8.78 9.45 9.57

5 References

8.94

NL: 3.21E4
m/z= 162.90-163.10 F: +
c sid=-10.00 SRM ms2
294.20@cid-10.00
[162.99-253.00] MS
BLANK-LL-PH11

[1] Rial-Otero, R., Gaspar, E. M., Moura, I., Capelo, J. L.,

Talanta 2007, 71, 503514.

8.75
8.67

7.25 7.29
3.09
0.55

1.67

2.78

8.54
9.25
9.52 10.40

3.19 4.50
6.11 6.34
5.21 5.88

10

11

Time (min)

Relative Abundance

RT:0.00 - 11.01
RT: 5.19

100
90
80
70
60
50
40
30
20
10
0

[2] Council regulation No. 2377/90/EEC of 26 June 1990 (OJ

L 224 August 18, 1990, p. 1) laying down a community
procedure for the establishment of maximum residue
limits of veterinary medicinal products in foodstuffs of
animal origin.
[3] Jimenez, J. J., Nozal, M. J., Bernal, J. L., Santos, M.,
Mayorga, A. L., Anal. Bioanal. Chem. 2002, 374,
300304.

Relative Abundance

The authors gratefully acknowledge the project (No.

200810099) for financial support of General Administration of
Quality Supervision, Inspection and Quarantine of the PRC.

8.85

100
90
80
70
60
50
40
30
20
10
0

Relative Abundance

2.06

0.89

Compared to traditional determination method by GC,

this method is much simpler in pretreatment, requiring no
hydrolysis and derivatization step.

NL: 8.00E3
m/z= 107.00-107.20 F: +
c SRM ms2
122.00@cid-10.00
[77.12-107.10] MS
BLANK-LL-PH11

RT:0.00 - 11.01

NL: 4.97E5
m/z= 107.00-107.20 F: + c
SRM ms2
122.00@cid-10.00
[77.12-107.10] MS
Genesis 40ppb-LL-11

[4] Jimenez, J. J., Bernal, J. L., Nozal, M. J., Alonso, C.,

Anal. Chim. Acta 2004, 524, 271278.
[5] Martel, A., Zeggane, S., J. Chromatogr. A 2002, 954,
173180.
[6] Korta, E., Bakkali, A., Berrueta, L. A., Gallo, B., Vicente,
F., J. Chromatogr. A 2001, 930, 2129.
[7] Corta, E., Bakkali, A., Berrueta, L. A., Gallo, B., Vicente,
F., Talanta 1999, 48, 189199.
RT: 7.66

100
90
80
70
60
50
40
30
20
10
0

[8] Lodesani, M., Pellacani, A., Bergomi, S., Carpana, E.,

Rabitti, T., Lasagni, P., Apidologie 1992, 23, 257.

NL: 2.33E7
m/z= 162.90-163.10 F: + c
sid=-10.00 SRM ms2
294.20@cid-10.00
[162.99-253.00] MS
Genesis 40ppb-LL-11

[9] Fernandez Muino, M. A., Sancho, M. T., Muniategui, S.,

Huidobro, J. F., Simal Lozano, J., J. Food Prot. 1995, 58,
449.
[10] Korta, E., Bakkali, A., Berrueta, L. A., Gallo, B., Vicente,
F., Bogdanov, S., Anal. Chim. Acta 2003, 475, 97103.

10

11

Time (min)

Figure 5. LC-MS/MS chromatograms of (A) extract of blank

honey sample, (B) honey sample spiked with 40 mg/kg of amitraz
and 2,4-DMA standards. The upper peak is that of amitraz and
the lower peak is that of 2,4-DMA.

[11] Bernal, J. L., Nozal, M. J., Jimenez, J. J., J. Chromatogr.

A 1997, 765, 109114.
[12] Lenicek, J., Sekyra, M., Novotna, A. R., Vasova, E.,
Titera, D., Vesely, V., Anal. Chim. Acta 2006, 571, 4044.
[13] Xue, X. F., Zhao, J., Qiu, J., Zhou, Z.Q., Modern Scientific Instruments 2005, 1, 6567.
[14] Zhao, Z., Wu, B., Shen, C., Chen, H., Ding, T., Huang, J.,
Liu, Y., Apiculture. China 2005, 56, 46.
[15] Vega, A. B., Frenich, A. G., Vidal, J. L. M., Anal. Chim.
Acta 2005, 538, 117127.

4 Concluding remarks
A method has been developed for the simultaneous
determination of amitraz and its degradation product 2,4DMA in honey by HPLC and LC-MS/MS methods. Hexane
is proper solvent for the extraction of the two compounds.
The addition of isopropyl alcohol can increase recoveries of
amitraz and 2,4-DMA.

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[16] Fernandez-Alba, A. R., Aguera, A., Contreras, M.,

Penuela, G., Ferrer, I., Barceloo, D., J. Chromatogr. A
1998, 823, 3547.
[17] Tokman, N., Soler, C., Farre, M., Pico, Y., Barcelo, D., J.
Chromatogr. A 2009, 1216, 31383146.
[18] Andersen, W. C., Roybal, J. E., Gonzales, S. A., Turnipseed, S. B., Pfenning, A. P., Kuck, L. R., Anal. Chim. Acta
2005, 529, 145150.

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