Beruflich Dokumente
Kultur Dokumente
Food Safety
Authors
Feng Liang, Peibin Hu, and Ping Li
Agilent Technologies
Beijing, China
Abstract
This application note demonstrates a complete method to
rapidly and precisely determine residue levels of malachite green and leucomalachite green in fish with the new
Agilent 6410 LC/MS triple quadrupole system. Using positive mode electrospray ionization (ESI+) and multiple
reaction monitoring (MRM), qualification and quantification were accomplished without the traditional tedious
PbO2 oxidation process. The LC/MS/MS methods LOQ is
0.01 g/Kg, which easily meets the import requirement of
2 g/Kg set by Japan and the EU.
Introduction
Experimental
Acetonitrile
Reagents
MG
LMG
Acetic acid
Ammonium acetate
Sigma-Aldrich,
CAS 569-64-2, USA
Dr. Ehreastorfer's lab,
D-86199, 99% pure,
Augsburg, Germany
CAS 75-05-8; Burdick &
Jackson; Morristown,
New Jersey, USA
Merck, Germany
CAS 631-61-8, Acros
Organics, Morris Plains,
New Jersey, USA
H
C
N
+
Cl
Malachite green
Figure 1.
Leucomalachite green
Calibration Solutions
Instrumentation
A stock standard solution of MG and LMG in acetonitrile was prepared at 100 g/mL and stored at
%18 oC, avoiding light. The stock solution was
diluted in 50:50 acetonitrile:water to make the calibration solutions+10, 50, 100, 500, 1000, 5000, and
10,000 fg/L.
LC
Column
Column temp.
Mobile phase
Sample Preparation
To 5 g tilapia tissue was added 1 mL (0.25 mg/mL)
hydroxylamine, 2 mL 1 M toluene sulfonic acid,
2 mL of 0.1 M ammonium acetate buffer (pH 4.5),
and 40 mL acetonitrile. The mixture was then
homogenized for 2 min. The supernatant was
decanted, and to the precipitate was added 20 mL
acetonitrile. This was filtered and added to the
supernatant. To the combined acetonitrile
extracts, 35 mL water and 30 mL methylene chloride were added. The solution was shaken and the
methylene chloride layer collected. A second
extract of 20 mL methylene chloride was made,
and this layer added to the first extract. The methylene chloride was taken to dryness with a gentle
stream of nitrogen and the extract reconstituted in
100 L of acetonitrile
Column flow
Gradient
Injection vol.
1100 LC
C18, 2.1 x 150 mm, 5 m
40 oC
A % 10 mmol/L ammonium acetate
(adjust to pH 4.5 with acetic acid)
B % acetonitrile
0.3 mL/min
Time
%B
0
30
1
50
2
95
8
95
8.01
30
13
30
10 L
MS
Table 1.
Time
Compound
Precursor
Product
Dwell
(ms)
Fragmentor
(V)
Collision
Energy (V)
MG
329.3
329.3
313.3
208.2
40
40
100
100
40
40
LMG
331.3
331.3
316.3
239.2
40
40
100
100
30
30
(a) 20 V, (b) 30 V, and (c) 40 V. Due to their structural differences, the voltage required for optimum
fragmentation of each compound is different. For
MG, the optimum fragmentation was observed at
40 V. The ion m/z 313 was due to the neutral loss
of methane. The ion at m/z 208 was due to the neutral loss of N,N-dimethylaniline. For LMG, the optimum fragmentation was observed at 30 V. The ion
at m/z 316 was due to the loss of a methyl radical.
The ion at m/z 239 resulted from a subsequent loss
of a benzene radical or, more likely, the rearrangement and neutral loss of toluene.
70 V
3
1
x107
5
90 V
3
1
x107
100 V
3
1
0
10
11
12
Figure 2.
EICs of malachite green and leucomalachite green at fragmentor values of 70 V, 90 V, and 100 V.
329.3
x105
+ Product Ion (5.499-5.633 min, 17 scans) (329.3, 331.4 **) optimizing MS2_FRG100_CE20_2.d
1.2
1.0
0.8
0.6
0.4
0.2
193.1 208.3
0.0
236.9
268.4
284.3
313.4
331.8
x105 + Product Ion (8.349-8.466 min, 15 scans) (331.4, 329.3 **) optimizing MS2_FRG100_CE20_2.d
239.8
2.0
1.6
316.7
1.2
0.8
0.4
120.8 134.5
0.0
40
60
80
100
120
140
165.6
160
195.8
180
209.8
272.8
286.6 301.8
223.9
200
220
240
260
280
300
320
340
329.3
+ Product Ion (5.457-5.591 min, 17 scans) (331.4, 329.3 **) optimizing MS2_FRG100_CE30_3.d
6
5
4
313.4
3
2
208.2
1
134.3
165.1
192.8
217.4
237.2 251.4
270.3 285.3298.9
+ Product Ion (8.349-8.457 min, 14 scans) (331.4, 329.3 **) optimizing MS2_FRG100_CE30_3.d
239.8
x105
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
315.8
134.5
40
60
80
100
120
140
209.8 223.9
165.8 180.6 194.7
160
180
200
220
240
Abundance vs. mass-to-charge (m/z)
260
272.7 286.8
301.9
280
300
331.8
320
340
x104
313.3
+ Product Ion (5.474-5.591 min, 15 scans) (331.4, 329.3 **) optimizing MS2_FRG100_CE40_4.d
4.0
3.0
208.2
2.0
1.0
165.3
0.0
192.1
117.9 134.1
91.5
221.0
329.4
284.2
241.4
270.3
299.4
+ Product Ion (8.340-8.499 min, 20 scans) (329.3, 331.4 **) optimizing MS2_FRG100_CE40_4.d
x105
239.8
2.5
2.0
1.5
1.0
194.7
0.5
0.0
40
60
80
165.8 180.7
120.6 134.5
91.6
100
120
140
315.8
208.7 223.8
160
180
200
220
240
Abundance vs. mass-to-charge (m/z)
260
280
300
320
340
2.4
y = 23363.3374 * - 1766.9951
R 2 = 0.99946103
2.2
2.0
1.8
Response
1.6
1.4
1.2
1.0
R 2 = 0.9995
0.8
0.6
0.4
0.2
0.0
0
5
6
7
Concentration (ng/mL)
10
11
12
Figure 4a. Calibration curve of malachite green, linear range: 10 ppt to 10 ppb.
x10 6
Leucomalachite green - 7 Levels, 7 Levels Used, 14 Points, 14 Points Used, 0 QCs
1.0
y = 93199.4712 * - 7543.3588
R 2 = 0.99942595
0.9
0.8
Response
0.7
0.6
0.5
R 2 = 0.9994
0.4
0.3
0.2
0.1
0.0
0
5
6
7
Concentration (ng/mL)
10
11
12
Figure 4b. Calibration curve of leucomalachite green, linear range: 10 ppt to 10 ppb.
8.433
12
x101
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
0
5
6
7
Abundance vs. acquisition time (min)
10
11
12
10
11
12
12
1.4
1.2
1.0
0.8
5.440
0.6
0.4
0.2
0.0
0
5
6
7
Abundance vs. acquisition time (min)
8.315
x102
12
3.2
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
0
5
6
7
Abundance vs. acquisition time (min)
10
11
12
Figure 6. MRM result of talapia extract spiked with 100-ppt MG and LMG.
Conclusions
This application note demonstrates a complete
method to rapidly and precisely determine residue
levels of malachite green and leuco-malachite
green in fish. Using positive mode electrospray
ionization (ESI+) and multiple reaction monitoring
(MRM) technique, the LC/MS/MS method shows
detection limit of 10 ppt, which easily meets the
import requirement set by Japan or EU.
References
1. S. Srivastava, R. Sinha, and D. Roy, Toxicological effects of malachite green. Aquatic Toxicology 2004, 66, (3), 319%329.
2. The Rapid Alert System for Food and Feed
(RASFF) Annual Report 2005. 2005, 29.
3. C. A. Hajee and N. Haagsma, Simultaneous
determination of malachite green and its
metabolite leucomalachite green in eel plasma
using post-column oxidation. Journal of Chromatography B Biomed Appl. 1995, 669, (2),
219%227.
Acknowledgement
The authors would like to thank Dr. Yanqin Liu for
providing the standard solutions and sample
extracts.
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.
Information, descriptions, and specifications in this publication are subject to change
without notice.
Agilent Technologies, Inc. 2006
Printed in the USA
October 25, 2006
5989-5807EN