APPLICATION NOTE Malachite Green Analysis of Malachite Green and Leucomalachite Green in Aquaculture Samples by LC/MS/MS API 3200 LC/MS/MS system TM Introduction 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 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) 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 3200 triple quadrupole mass spectrometer 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 Malachite Green MW = 329.4 TM Leucomalachite Green MW = 330.4 Mass Spectrometer API 3200 LC/MS/MS System Turbo V TIS source Positive Ion Mode Resolution was set for Q1 & Q3 at unit (peak width of 0.7 amu at half height) Analysis Conditions Sample Preparation TM TM Weigh 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) Malachite Green MRM pairs 329.2 / 313.2 329.2 / 208.2 DP 110 CE 47v Add mL hydroxylamine HCL g/L Homogenise and let stand for 10 mins Leucomalachite Green MRM pairs 331.2 / 239.2 331.2 / 165.0 DP 86 CE 77v Centrifuge at 14000 g for 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 Analytical Conditions Column: RP-C18 Phenomenex Synergi 4μ Hydro-RP 80A (50 x 2mm) Figure 1a MS/MS Spectra of Leucomalachite Green Injection Volume 10μL Mobile Phase A= Water + mM ammonium acetate + 0.1% formic acid B= acetonitrile + mM ammonium acetate + 0.1% formic acid Time (min) 0.0 4.0 6.0 6.1 8.0 Flow Rate 0.2 mL/min 0.2 mL/min 0.2 mL/min 0.2 mL/min 0.2 mL/min %A 95 5 95 95 %B 95 95 5 Figure 1b MS/MS Spectra of Malachite Green Linearity Figure 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 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 orders of magnitude Working range of the assay 0.005-50ppb Regression = 0.9999 Figure 4b Standard curves of LMG over orders of magnitude Working range of the assay 0.005-50ppb Regression = 0.9997 STATISTICS Concentration (ppt) MG % CV A MG % CV A LMG % CV A LMG % CV A 9.8 98.3 3.0 103.0 N/A N/A N/A N/A 10 4.9 102.2 4.6 92.9 8.9 103.7 N/A N/A 50 1.6 100.9 2.4 104.4 4.0 87.3 6.6 100.8 100 4.0 99.5 4.3 101.7 1.6 88.7 6.9 99.6 500 1.0 99.0 0.9 101.9 0.7 98.0 3.2 97.5 1000 1.4 100.7 1.3 104.8 1.1 96.9 1.5 92.6 5000 1.4 100.2 1.3 101.9 0.4 107.8 0.7 102.1 10000 1.3 98.7 0.9 103.0 0.5 109.9 0.8 105.7 50000 N/A N/A 0.5 86.4 0.2 105.8 0.7 102.4 100000 N/A N/A N/A N/A 0.1 101.8 0.7 99.3 Conclusion This application note outlines the use of an API 3200 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 ppt The robustness of the protocol is highlighted by the linearity of the method over orders of magnitude The excellent performance of the API 3200 system platform provides quality results in residue testing at an affordable price TM TM For Research Use Only Not for use in diagnostic procedures Applera Corporation is committed to providing the world’s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Applied Biosystems/MDS SCIEX is a joint venture between Applera Corporation and MDS Inc 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 Headquarters 850 Lincoln Centre Drive | Foster City, CA 94404 USA Phone 650.638.5800 | Toll Free 800.345.5224 www.appliedbiosystems.com International Sales For our office locations please call the division headquarters or refer to our Web site at www.appliedbiosystems.com/about/offices.cfm