The Qualitative, Quantitative and
Confirmatory Determination of Controlled
(and Prohibited) Drugs and Pesticides in
Food Using LC-MS/MS
Bryn Shurmer, M.Sc.
Andrew Harris, M.Sc.
Perry Martos, Ph.D., CIH
Trace Organics and Pesticide Section
Laboratory Services Division
University of Guelph
Guelph, Ontario, Canada
Overview: Tandem MS and Food Safety
Laboratory Services at the University of Guelph
-Agonists in Beef
Malachite Green in Fish
Require sub ppb detection limits using LC-MS/MS. For
small molecule analyses this ensures data are irrefutable –
a consistent theme with international approach to calling
“positives” for legal concerns.
Conclusions
Laboratory Services
Number of sections

Microbiology, Food Chemistry, Microscopy, Dairy
Testing….
Trace Organics and Pesticides

Regulatory – ISO 17025




Food - pesticide and drug residues
PMRA (SCC Accredited - GLP)
OECD guidelines
Unique analyses for on-campus researchers
General Parameters
Drug Residues (more than 15 different drug
classes)


Food (muscle, liver, fish tissue, etc.)
Thyroid, diaphragm
Pesticide Residues (over 260 compounds)

Food (fruits, vegetables, muscle)
New Pesticides and Drugs - almost all analyses
require LC-MS/MS
LC-MS/MS Equipment
Sciex 4000
Sciex 4000 QTrap
Waters – Quattro Ultima
SCIEX 4000 QTrap
Waters Quattro Ultima
Analysis of Clenbuterol
With LC-MS/MS
Clenbuterol
OH
Cl
HN
CH3
H2N
Cl
H3C
CH3
Formula:
C12 H18 Cl2 N2O
Monoisotopic mass
276.0796
Consequences
Growth promoter in livestock
Used in humans as a performance enhancing drug
Test for clenbuterol in retina and confirm in liver
(pharmakodynamic studies proved retina was best
target organ to test for residues – about 1 g sample
size)
Farmer’s product is held and if tested positive,
then destroyed, but…there are very serious human
health concerns to contend with…
Health Consequences
Analysis
Instrument Conditions
Positive Electrospray Ionization on Waters Quattro Ultima
TIC of 3 MRM Transitions
277>203
277>168
277>132
Blank
retina
BA Feb 25 2004 11 Sm (SG, 2x5)
5: MRM of 3 Channels ES+
TIC
5.55e3
100
11.85
10.47
Clenbuterol
%
0
10.25
10.50
10.75
11.00
11.25
11.50
11.75
12.00
12.25
12.50
12.75
Time
13.00
2- Fortified blank retina (10ng/g)
BA Feb 25 2004 12 Sm (SG, 2x5)
100
5: MRM of 3 Channels ES+
TIC
1.27e4
11.72
Clenbuterol
%
12.74
0
10.25
10.50
10.75
11.00
11.25
11.50
11.75
12.00
12.25
12.50
12.75
Time
13.00
Test retina sample X
BA Feb 25 2004 17 Sm (SG, 2x5)
100
5: MRM of 3 Channels ES+
TIC
4.80e4
11.73
Clenbuterol
%
0
10.25
10.50
10.75
11.00
11.25
11.50
11.75
12.00
12.25
12.50
12.75
Time
13.00
Compound name: Clenbuterol
Correlation coefficient: r = 0.990989, r^2 = 0.982058
Calibration curve: 135.44 * x + 11.9519
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None
Clenbuterol Calibration by MRM
(2 to 20 ng/g)
19.4
Residual
-27.4
ppb
2930
Response
0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
ppb
20.0
Analysis of
Malachite Green
With LC-MS/MS
Scope of the Problem
http://www.cnn.com/2004/TECH/science/09/27/environment.salmon.reut/
Used as anti-microbial, anti-fungal, and antiparasitic treatment for fish
Scope of the Problem
http://www.cnn.com/2004/TECH/science/09/27/environment.salmon.reut/
“SANTIAGO, Chile (Reuters) -- A cargo of
farmed Chilean salmon tainted with a potentially
harmful toxin and rejected for import in
Europe has returned to Chile and could be reexported to a country with less stringent laws,
consumer and environmental groups warned.”
This is the latest in a series of tainted shipments that
has Chile's $1.15 billion-a-year salmon industry on
the defensive. Fish and fish products are one of
Chile's top exports.
Health authorities in Holland rejected a container
bound for France, which was packed with 20 tons of
frozen salmon in August after detecting the fungicide
malachite green, which is used to control molds that
kill the eggs of farmed fish.
The use of malachite green, which can cause
cancer, is prohibited in many parts of the world.
Chile banned it in 2002.
Earlier this year Holland rejected a different 180tonne shipment of salmon for the same reason and
environmental group Ecoceanos says part of that
shipment was later sold in Estonia.
"The food has to be destroyed, because it is
contaminated and cannot be consumed," said Juan
Carlos Cardenas, a veterinarian and director of
Ecoceanos.
Chilean government health officials in southern
Chile, where the container now sits, confirmed
Holland's findings and outlawed the sale of the
salmon in Chile. However, they said it was up to the
company to decide whether to sell it elsewhere or
have it destroyed.
"We have control of the container and the company must
decide if it re-exports it to a country without these limitations,"
said health ministry official Yuri Carvajal in a statement on
Friday.
Officials at a salmon producers association contacted by
Reuters were not immediately available for comment.
The government has not identified the companies that
produced the fish.
Chile exported $820 million worth of salmon and trout to
world markets in the first half of 2004, mainly to the
United States and Japan.
Chilean salmon farmers said in January their fish were among
the safest to eat and contained low levels of potentially
harmful chemicals -- such as polychlorinated biphenyls, or
PCBs, dioxins and others -- compared to their North American
counterparts.
Malachite Green (MG)
Ph
C
Me 2 + N
NMe 2
·
Cl -
Formula:
C23 H25 N2
Monoisotopic weight
329.2018
Leucomalachite Green (LMG)
Ph
CH
Me 2 N
NMe 2
Formula:
C23 H26 N2
Monoisotopic mass
330.2096 amu
Analysis
Analysis
Target Analyte Identification





Analyte extraction and concentration
Retention time (tR)
Hits with MRM, at least three transitions, TIC of the
transitions
A full spectrum would be more definitive, but is not
possible for sub ppb concentrations using conventional
LC-MS/MS
Sciex 4000 Q can yield full spectral detail at sub ppb
concentrations (data to follow)
Instrument Conditions
Positive Electrospray Ionization on Sciex API4000QTrap
TIC of 4 MRM transitions for each compound
Leucomalachite Green:
331>316
331>239
331>223
331>194
Malachite Green:
329>313
329>284
329>241
329>208
Traditional Approaches to Analyze Malachite Green
1995 – GC-MS
1995 - LC-Post-column reaction with Pb(IV)O (5ppb)
1997 – LC-Post-column electrochemical oxidation
(25ppb)
1997 - HPLC using electrochemistry with UV-VIS diode
array and fluorescence detection
1998 - HPLC-visible and ESP-LC-MS
1998 - Isotope dilution LC-APCI/MS
2003 - Ion-trap LC-MS “…very low level (<1µg/kg)
quantitation of drug residues using this instrument will
require additional investigation, and may be more readily
achieved using an alternate platform, such as a triple
quadrupole instrument”
2004 - LC-MS/MS
Process – Optimization, Chromatography
Infusion, 15 µL/min of 10 to 100 ppb
 MS scan
 Product Ion Scan
 EPI
 MS/MS/MS
Chromatography
Optimization (Infusion)
MG – 15 µL/min of 100 ppb (+ve ESI, MG+) (scan)
Ph
C
Me 2 + N
NM e 2
·
Cl -
LMG – 15 µL/min of 100 ppb (+ve ESI, [LMG+H]+) (scan)
Ph
CH
Me 2 N
NM e 2
LMG - Products of [M+H]+ 331
LMG – EPI (331>(scan))
LMG - MS/MS/MS (331>239>scan)
Chromatography
Standard (10 µL of 50 ppb mix)
TIC: from Sample 1 (MG and LMG 50ppb each) of Oct 6 2004 tests.wiff (Turbo Spray)
Max. 1.8e6 cps.
6.21
1.8e6
1.7e6
1.6e6
1.5e6
LMG
1.4e6
1.3e6
6.71
1.2e6
In te n s ity , c p s
1.1e6
MG
1.0e6
9.0e5
8.0e5
7.0e5
6.0e5
5.0e5
4.0e5
3.0e5
2.0e5
1.0e5
0.0
0.17 0.69 0.99
1.24
1
1.90 2.19
2
2.41
3.11 3.21
3
3.63 4.12 4.39 4.72
4
Time, min
4.98
5
5.41
5.91
6
7.12 7.26
7
7.69
8
Precision:
0.5 ppb and 25 ppb standards, by TIC of several MRM
transitions
% RSD (0.5 ppb) % RSD (25 ppb)
LMG
5.3
4.3
MG
9.6
5.0
MG Calibration by MRM (0.4 to 25 ppb)
Aqua Oct 07 2004.rdb (MG): "Quadratic" Regression ("No" weighting): y = -9.23 x^2 + 2.39e+003 x + 42.2 (r = 0.9996)
5.6e4
5.4e4
5.2e4
5.0e4
4.8e4
4.6e4
4.4e4
4.2e4
4.0e4
3.8e4
3.6e4
A re a , c o u n ts
3.4e4
3.2e4
3.0e4
2.8e4
2.6e4
2.4e4
2.2e4
2.0e4
1.8e4
1.6e4
1.4e4
1.2e4
1.0e4
8000.0
6000.0
4000.0
2000.0
0.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Concentration, ppb
15
16
17
18
19
20
21
22
23
24
25
MG Calibration (0.4 to 25 ppb) (EPI)
LMG Calibration by MRM (0.4 to 25 ppb)
October 15 2004 Aqua.rdb (LMG): "Quadratic" Regression ("No" weighting): y = -31.4 x^2 + 8.13e+004 x + -1.39e+004 (r = 1.0000)
2.0e6
2.0e6
1.9e6
1.8e6
1.7e6
1.6e6
1.5e6
1.4e6
1.3e6
A re a , c o u n ts
1.2e6
1.1e6
1.0e6
9.0e5
8.0e5
7.0e5
6.0e5
5.0e5
4.0e5
3.0e5
2.0e5
1.0e5
0.0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Concentration, ng/mL
15
16
17
18
19
20
21
22
23
24
25
LMG Calibration (0.4 to 25 ppb) (EPI)
TIC: from Sample 2 (2.0 ppb std) of Oct 08 2004 MRM.wiff (Turbo Spray)
Max. 8.7e4 cps.
6.11
8.5e4
8.0e4
7.5e4
7.0e4
MRM of Standard
(10 µL of 2.0 ppb)
6.5e4
6.0e4
Intensity, cps
5.5e4
5.0e4
LMG
4.5e4
4.0e4
3.5e4
MG
3.0e4
2.5e4
6.77
2.0e4
1.5e4
5.89
1.0e4
5000.0
0.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
Time, min
5.6
5.8
6.0
6.2
6.4
6.6
6.8
TIC: from Sample 8 (5x LOQ matrix) of Oct 08 2004 MRM.wiff (Turbo Spray)
7.0
7.2
7.4
Max. 8.7e4 cps.
6.11
8.5e4
LMG
8.0e4
7.5e4
7.0e4
6.5e4
MRM of Spike
(10 µL of 2.6 ppb in-vial)
6.0e4
Intensity, cps
5.5e4
5.0e4
4.5e4
4.0e4
MG
3.5e4
3.0e4
2.5e4
2.0e4
6.75
1.5e4
5.87
1.0e4
6.31
5000.0
0.0
6.47
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
Time, min
5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
TIC: from Sample 6 (Matrix blank) of Oct 08 2004 MRM.wiff (Turbo Spray)
Max. 1.2e4 cps.
8.5e4
8.0e4
7.5e4
7.0e4
6.5e4
MRM of Matrix Blank
6.0e4
Intensity, cps
5.5e4
5.0e4
4.5e4
4.0e4
3.5e4
3.0e4
2.5e4
2.0e4
1.5e4
5.88
1.0e4
5000.0
4.45
0.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.60
4.6
4.94
4.8
5.05
5.0
6.14
5.42
5.65
5.2
5.4
Time, min
5.74
5.6
5.8
6.0
6.31
6.2
6.51 6.58
6.4
6.6
6.8
TIC: from Sample 9 (04-0616648) of Oct 08 2004 MRM.wiff (Turbo Spray)
7.0
7.2
7.4
Max. 1.2e4 cps.
8.5e4
8.0e4
7.5e4
7.0e4
6.5e4
6.0e4
Intensity, cps
5.5e4
5.0e4
MRM of Negative Sample
4.5e4
4.0e4
3.5e4
3.0e4
2.5e4
2.0e4
1.5e4
5.87
1.0e4
5000.0
0.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
Time, min
5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
TIC: from Sample 10 (04-0616681) of Oct 08 2004 MRM.wiff (Turbo Spray)
Max. 2.1e6 cps.
8.5e4
8.0e4
7.5e4
7.0e4
6.5e4
6.0e4
Intensity, cps
5.5e4
5.0e4
MRM of Incurred Positive Sample
4.5e4
4.0e4
3.5e4
3.0e4
2.5e4
2.0e4
1.5e4
1.0e4
5000.0
0.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
Time, min
5.6
5.8
6.0
6.2
6.4
6.6
6.8
TIC: from Sample 2 (2.0 ppb std) of Oct 08 2004 MRM.wiff (Turbo Spray)
7.0
7.2
7.4
Max. 8.7e4 cps.
6.11
8.5e4
8.0e4
7.5e4
7.0e4
MRM of Standard
(10 µL of 2.0 ppb)
6.5e4
6.0e4
Intensity, cps
5.5e4
5.0e4
4.5e4
4.0e4
3.5e4
3.0e4
2.5e4
6.77
2.0e4
1.5e4
5.89
1.0e4
5000.0
0.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
Time, min
5.6
5.8
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
Questionable Positive (EPI) (~400 ppt)
For Malachite Green
Questionable Positive (by MRM, ion ratios):
(there is zero tolerance for malachite green)
“…freezing in fish tissue can oxidize LMG back to parent compound…”
25 ppb Mixed Standard (EPI)
LMG
MG
0.4 ppb Mixed Standard (EPI)
LMG
MG
Matrix Blank (EPI)
LMG
MG
The Questionable Sample (EPI)
LMG
MG
The Questionable Sample (EPI used to prove MG)
Sample
0.4 ppb
Matrix Blank
The Questionable Sample (Product Ion Experiment)
MG
LMG
Conclusions
LC-MS/MS has proven to be an invaluable
tool for the analysis and confirmation of
prohibited chemicals used in food
In those cases where the compound(s) is not
registered for use in Canada, the LOD is often
used as a benchmark for MRL (any comments
on this?)
Thank you…