Malachite green MG and its hydrolysed metabolite Leucomalachite green LMG are organic compounds which have powerful anti-fungal properties and are used against Saprolegnia fungus either
Trang 1Malachite green (MG) and its hydrolysed metabolite Leucomalachite green (LMG) are organic compounds which have powerful anti-fungal properties
and are used against Saprolegnia
(fungus) either when present on fish or
as a prophylactic treatment to protect fish-eggs from infection In addition its antiparistic properties have caused its
use in treatments against Gyrodactylus (skin flukes), Dactylogyrus (gill flukes), Ichthyobodo (Costia), Trichodina, Chilodonella and Ichthyophthirius
(white spot)
MG and LMG are not listed as a veterinary medicines under Annexes
I, II or III of Council Regulation 2377/90
In recent years, there has been increasing concern of its usage due the harmful effects on humans
Administration of MG in aquaculture is therefore prohibited However, the low cost and efficacy of MG ensures its widespread use has continued illegally
We report here a highly sensitive surveillance method for detection and quantitation of both MG and the metabolite LMG in commercial aquaculture products by LC-MS/MS using an API 3200TM triple quadrupole mass spectrometer
Analysis of Malachite Green and Leucomalachite Green in Aquaculture Samples by LC/MS/MS
API 3200TM LC/MS/MS system
APPLICATION NOTE Malachite Green
Malachite Green MW = 329.4
Leucomalachite Green MW = 330.4
Trang 2Analytical Conditions
Column: RP-C18 Phenomenex Synergi
4μ Hydro-RP 80A (50 x 2mm)
Injection Volume 10μL
Mobile Phase
A= Water + 2 mM ammonium acetate
+ 0.1% formic acid
B= acetonitrile + 2 mM ammonium
acetate + 0.1% formic acid
Time (min) Flow Rate %A %B
Figure 1a MS/MS Spectra of Leucomalachite Green
Figure 1b MS/MS Spectra of Malachite Green
Analysis Conditions
Sample Preparation
Weigh 2 g of sample
Add 200 μL Std solution
(for spiked sample)
Add 200 μL IS solution 20 ng/mL
(for all samples)
Add 600 μL water
(800 μL if unknown sample)
Add 2 mL hydroxylamine HCL 5 g/L
Homogenise and let stand for 10 mins
Centrifuge at 14000 g for 5 min at
4˚ C, filterthrough 0.45μm disc
Reconstitute in 200 μL of 50:50
Mobile phase A and Mobile phase B
Inject 10 μL on HPLC column
Phenomenex Synergi 4μ Hydro-RP
80A (50 x 2.0mm)
MS/MS in MRM mode; positive ESI;
on API 3200™ LC/M/MS system
Mass Spectrometer API 3200TM LC/MS/MS System Turbo VTM TIS source
Positive Ion Mode Resolution was set for Q1 & Q3 at unit (peak width of 0.7 amu at half height) Malachite Green MRM pairs 329.2 / 313.2
329.2 / 208.2
DP 110 CE 47v Leucomalachite Green MRM pairs 331.2 / 239.2
331.2 / 165.0
DP 86 CE 77v
Trang 3Figure 2 MRM Chromatograms for Malachite Green (RT 1.0 min, top right) and
Leucomalachite Green (RT 2.7 min, bottom right) spiked into a real sample at
0.01ppb, with the corresponding blank response on the left.
Figure 3 MRM Chromatograms of Malachite Green spiked at 0.005ppb (RT 1.0
min, top right) and Leucomalachite Green (RT 2.7 min, bottom right) with the
corresponding blank response on the left.
Figure 4a Standard curves of MG over 4 orders of magnitude
Working range of the assay 0.005-50ppb Regression = 0.9999
Figure 4b Standard curves of LMG over 4 orders of magnitude
Working range of the assay 0.005-50ppb Regression = 0.9997
Linearity
Trang 4Headquarters
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For Research Use Only Not for use in diagnostic procedures.
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Applied Biosystems is a registered trademark and AB (Design) and Applera are trademarks of Applera Corporation or its subsidiaries in the U.S and/or certain other countries MDS and SCIEX are registered trademarks of MDS Inc API 3200 and Turbo V are registered trademarks of Applied Biosystems/MDS SCIEX All other trademarks are the sole property of their respective owners.
© Copyright 2006, Applera Corporation and MDS Inc All rights reserved Information subject to change without notice.
Printed in the USA 08/2006 Publication 114AP52-02
STATISTICS
Conclusion
This application note outlines the use
of an API 3200TM triple quadrupole mass
spectrometer for the rapid and sensitive
LC-MS/MS analysis of Malachite Green
and Leucomalachite green with a lower
limit of quantitation (LOQ) at 5 ppt The
robustness of the protocol is highlighted
by the linearity of the method over 4
orders of magnitude The excellent
performance of the API 3200TM system
platform provides quality results in
residue testing at an affordable price