TSKgel® TECHNICAL INFORMATION SHEET
No. 122
Analysis of Malachite Green and Leucomalachite Green by LC/MS
Malachite Green is used as a synthetic antimicrobial for treating water mold disease in aquarium fish, but has been
identified as carcinogenic, and use in farm fish, etc., for human consumption is banned in Japan. In the Food Sanitation
Law in Japan, the maximum residue limit has been specified as “not detected.” In other countries this agent is widely
used in farmed salmon and eel, and reports have described detection in farmed eel imported into Japan from China.
The LC/MS procedures used to analyze Malachite Green and its metabolite, Leucomalachite Green, are described as
analytical methods in regulations announced by the Ministry of Health, Labour and Welfare. Below is an example of
simultaneous analysis of Malachite Green and Leucomalachite Green based on these regulations. As each of these is a
basic substance containing a dimethylamino group, the decision was made to use a solvent with an acid composition.
The calibration curves created each displayed good linearity in a concentration range of 0.05-50ppb.
Figure 1. Structural formula of Malachite Green and Leucomalachite Green
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
Malachite Green
Leucomalachite Green
Table: Analysis conditions
Column:
Mobile phase:
Gradient:
Flow rate:
Temperature:
TSKgel ODS-100V, 3µm, 2.0mm ID × 15cm
A:10mmol/L ammonium formate , pH 3.75
B: acetonitrile
0min (10%B) → 10 min (100 %B) → 15 min (100 %B)
0.2mL/min
40°C
Injection vol.:
5µL
Instrument:
QTRAP (Applied Biosystems)
Ion Source:
Polarity:
ESI
®
Positive
Figure 2. Chromatograms of Malachite Green and Leucomalachite Green
8.0E+04
5.0ppb each
Leucomalachite Green
Malachite Green
Intensity, cps
Intensity (cps)
Intensity,
cps
6.0E+04
(m/z; 331/239)
(m/z; 329/313)
4.0E+04
2.0E+04
0.0E+00
0
2
4
6
8
10
12
14
Time Time,
(minutes)
min
8.0E+02
Leucomalachite Green
0.05ppb each
(m/z; 331/239)
Malachite Green
(m/z; 329/313)
5.0ppb each
Intensity cps
(cps)
Intensity,
Intensity,
cps
6.0E+02
4.0E+02
2.0E+02
0.0E+00
0
2
4
6
8
10
12
14
Time Time,
(minutes)
min
Figure 3. Malachite Green and Leucomalachite Green calibration curves
R2 = 0.9999
2.0E+06
R2 = 0.9998
2.0E+06
Leucomalachite Green
Peak area (counts)
Peak area (counts)
Peak area (counts)
Peak area (counts)
Malachite Green
1.5E+06
1.0E+06
5.0E+05
1.5E+06
1.0E+06
5.0E+05
0.0E+00
0.0E+00
0
10
20
30
40
0
50
10
20
30
40
Concentration
Concentration (ppb)
(ppb)
Concentration
Concentration(ppb)
(ppb)
2
50