Determination of Malachite Green in Fish by LC-MS/MSHubert P.O.
Tang & Dr. Jacky Y.Y. Choi Chemists
Food & Environmental Hygiene C Section Government
Laboratory
14 September 2005
MG & LMGMG & LMG
Part BPart B –– Instrumental AnalysisInstrumental Analysis ––
ResultsResults –– Quality AssuranceQuality Assurance
Discussion Discussion
Malachite Malachite / / ‘mælkat –– a decorative green stonea
decorative green stone ––
–– ““chch”” / / tt∫∫ ; e.g. Channel / ; e.g. Channel / tt∫∫æænlnl
–– Chaos / Chaos / kkee asas
IntroductionIntroduction
Malachite greenMalachite green ((MGMG)) – direct dyeing silk, wool,
leather, etc – Biological stain – Acid base indicator (Green at pH
2;
colorless at pH 14) – Effective fungicide & parasiticide in
fish
IntroductionIntroduction
+
IntroductionIntroduction
CH3
IntroductionIntroduction
Toxicity of MGToxicity of MG –– May cause liver tumors in miceMay
cause liver tumors in mice –– Carcinogenicity for MG in humans
Carcinogenicity for MG in humans
still unknownstill unknown –– Not yet evaluated by JECFA or IARC
Not yet evaluated by JECFA or IARC
IntroductionIntroduction
Literatures
IntroductionIntroduction
Update on the CFIA monitoring activities for Update on the CFIA
monitoring activities for malachite green malachite green
http://www.inspection.gc.ca/english/corpaffr/newcom/2005/2005070http://www.inspection.gc.ca/english/corpaffr/newcom/2005/2005070
6e.shtml6e.shtml
–– National Toxicity Program (2005). TRNational Toxicity Program
(2005). TR--527 527 Toxicity and carcinogenesis studies of
malachite Toxicity and carcinogenesis studies of malachite green
chloride and green chloride and leucomalachiteleucomalachite green
in F344/N green in F344/N rats and B6C3F1 mice (Feed studies) rats
and B6C3F1 mice (Feed studies)
http://http://ntp.niehs.nih/gov/index.cfm?objectidntp.niehs.nih/gov/index.cfm?objectid=070B74C5=070B74C5--C780C780--E272E272--
6721383F7A1113BC6721383F7A1113BC
–– Reduced form of MGReduced form of MG –– Persistent metabolite in
tissuePersistent metabolite in tissue – C23H26N2
– Mol wt 330
RegulationsRegulations –– Codex not yet established food safety
standards Codex not yet established food safety standards
for MG in foodfor MG in food
–– Not allowed in US, EU (including UK), Canada, Not allowed in US,
EU (including UK), Canada, Japan, Thailand, Singapore as veterinary
drug on Japan, Thailand, Singapore as veterinary drug on food
animals, aquaculture or fish for human food animals, aquaculture or
fish for human consumptionconsumption
–– Recently included in the Harmful Substances in Recently included
in the Harmful Substances in Food Regulation in Hong KongFood
Regulation in Hong Kong
IntroductionIntroduction
RegulationsRegulations –– ““Zero toleranceZero tolerance”” in Hong
Kongin Hong Kong –– Minimum Required Performance Minimum Required
Performance
Limit (MRPL) for residue of MG and Limit (MRPL) for residue of MG
and LMG of LMG of 2 micrograms/kg2 micrograms/kg (Commission
Decision 2004/25/EC)(Commission Decision 2004/25/EC) Designed to
Designed to harmoniseharmonise the analytical capabilities of the
analytical capabilities of EU member statesEU member states
IntroductionIntroduction
Methods published in literaturesMethods published in literatures ––
Sample extractionSample extraction
by acidic buffer and by acidic buffer and acetonitrileacetonitrile
presence of antioxidants or enzyme presence of antioxidants or
enzyme inhibitorinhibitor may contain ionmay contain ion--pairing
reagentspairing reagents
IntroductionIntroduction
Methods published in literaturesMethods published in literatures ––
SPE CleanSPE Clean--upup
Cationic exchange SPE (SCX or Cationic exchange SPE (SCX or
PRS)PRS) Alumina (Neutral)Alumina (Neutral)
IntroductionIntroduction
LC (major)LC (major) –– C18, CN, phenylC18, CN, phenyl--hexylhexyl
columnscolumns
GC (minor)GC (minor) –– for LMG only for LMG only
IntroductionIntroduction
Methods published in literaturesMethods published in literatures ––
DetectionDetection
LC post column oxidation and LC post column oxidation and visible
detection (visible detection (620nm620nm)) Visible for MG (Visible
for MG (620nm620nm) or ) or fluorescence for LMG (fluorescence for
LMG (Ex=265nm Ex=265nm and and EmEm=360nm=360nm)) APCIAPCI-- or
ESIor ESI-- MS/MSMS/MS
Sample PreparationSample Preparation
Sample ExtractionSample Extraction
2mL NH4OAc buffer2mL NH4OAc buffer (pH4.5)(pH4.5)
5mL 5mL AcetonitrileAcetonitrile
Vortex for 2 minutesVortex for 2 minutes
Hydroxylamine
N CH3
AntiAnti--oxidantsoxidants –– Hydroxylamine
hydrochlorideHydroxylamine hydrochloride –– L(+)L(+)--ascorbic
acidascorbic acid
Sample Preparation Sample Preparation (cont(cont’’d)d)
Centrifuge at Centrifuge at high speedhigh speed
1. 2mL 1. 2mL McIlvaineMcIlvaine bufferbuffer (pH 6)(pH 6)
2. 2mL saturated 2. 2mL saturated NaClNaCl 3. Dichloromethane3.
Dichloromethane
Vortex for 1 minutesVortex for 1 minutes
Fast LCFast LC--MS/MSMS/MS QuantitationQuantitation &&
confirmationconfirmation
Sample Preparation Sample Preparation (cont(cont’’d)d)
Centrifuge and Centrifuge and collect lower organic collect lower
organic
layerlayer
under Nunder N22 at 40at 40--5050ooCC
Reconstitute withReconstitute with 5:95 acetic 5:95 acetic
acid:MeCNacid:MeCN
CationCation ExchangeExchange
Solid Phase ExtractionSolid Phase Extraction
SPE cartridgeSPE cartridge –– Merck SCX (500mg, 3mL)Merck SCX
(500mg, 3mL) –– Ion exchange capacity : 0.75meq/gIon exchange
capacity : 0.75meq/g –– Others:Others:
e.g. e.g. BakerbondBakerbond SCX (500mg, 3mL); SCX (500mg, 3mL);
Strata SCX (500mg, 3mL); Oasis MCX Strata SCX (500mg, 3mL); Oasis
MCX (60mg, 3mL), etc.(60mg, 3mL), etc.
Solid Phase ExtractionSolid Phase Extraction
O
Si
O
Four major stepsFour major steps –– ConditioningConditioning ––
Sample loadingSample loading –– WashingWashing ––
ElutingEluting
Solid Phase ExtractionSolid Phase Extraction
MeCN 5:95 HOAc:MeCN
SorbentSorbent MassMass – Mass of solute equivalent to 5%
sorbent
mass i.e. 5mg of TOTAL solute mass per 100mg sorbent
Loading/ Eluting flow rate – ↓flow rate ↑time for
sorbent-analyte
interaction (slow kinetics)
Loading/ Eluting flow rateLoading/ Eluting flow rate – ↓flow rate
↑time for sorbent-analyte
interaction (slow kinetics) ~ 1~ 1 mLmL/min or 1 drop per
second/min or 1 drop per second
Solid Phase ExtractionSolid Phase Extraction
Wash volume – 5 to 10 sorbent bed volumes – usu. 125µL per 100mg of
sorbent
DryDry – Critical when elution solvents not miscible
with wash solvent – No overdrying ⇒↓ analyte recovery
ElutionElution – 4-8 times sorbent bed volumes
MG & LMGMG & LMG
Analysis of Malachite Greenin Fish Samples
MG & LMG
