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HUMAN HEALTH • ENVIRONMENTAL HEALTH
What’s really in our food?
June 20152015
2
10/1
Food Integrity & Safety Issues…
Peanut Butter
- Salmonella
>€45M Fake Butter -
Non Food Oils
£100M
Spices -
Dye
Enhanced
Juices-
Fungicides
Eggs-
Dioxins
$1B
loss
Wine -
Dilution >18
dead
6 dead,
>300,000
sick
Milk Powder
Melamine
Spinach -
Salmonella
$350M
loss
Recalled
>250,000
Frozen Berries
- Hep A $1.7mil
3
Food industry trends in context to science …
Consumer Trust & Brand Value
Regulatory Safety
Quality & Consistency
Authenticity & Origin
Deter, Prevent, Detect
Fraud
Product Innovation
Value Chain Safety
Supply Chain Integrity
Brand Innovation
Industry Initiatives
4
10/1
A Focus on …
• Food authenticity
◦ Ingredient substitution
◦ Economic adulteration
◦ Incorrect labelling
◦ A question of provenance
• Food Safety
◦ Levels of toxic elements
◦ Residuals
◦ Contamination
5
Economically motivated adulteration
• “… the deliberate adulteration of
food… to inflate profits by fraudulent means” (FDA)
• Examples:
◦ Dilution of valuable oils with
cheaper ones
◦ Substitution of cheaper juices for
pomegranate juice
• Likely to involve high
concentrations of the adulterant
• The tools used to test for EMA may
also be useful for screening for
other types of contamination or
substitution, and in industries other
than food
6
Real-Time Detection Using Direct Sampling TOF Mass Spectroscopy
• Development of technique
known as “field-free APCI”
• A stream of Nitrogen gas is
heated
• Ionized by a corona discharge
needle.
• The ionized nitrogen flows over
the target sample, generating
ions from it.
• Readily swap between direct
sampling source and LC
introduction
Whole Foods
Liquids
Powders
7
How AxION DSA works
8
148.07 155.14
163.05
164.06
168.01 175.08
180.08
182.04 189.05
191.08
197.92
198.09
199.09
202.08
208.11
209.10
210.11
212.11
219.11
221.91
222.11
223.10
226.08 226.12
227.12
228.21
239.13
240.09
243.11
249.13
255.15
0 160 170 180 190 200 210 220 230 240 250 260m/z
Carbamates analysis in orange juice…
Spectrum of juice indicating presence of carbamates
9
Adulteration of our everyday commodities
10
No chromatography, No Sample Preparation
LIQUID EXTRACT SAMPLED DIRECTLY ON MESH ANSWER IN <30 SECONDS
Real-time Detection for Critical Authenticity Issues
Vanilla widely used AND the 2nd most
expensive spice in the world
12,000 Tons Synthetic Vanillin produced but only 20 tons extracted
from the bean Synthetic $12/kg vs.
$2,000/kg For Natural
4-Hydroxybenzaldehyde
ABSENT in Synthetic
Vanillin
11
No chromatography, No Sample Preparation, results in 30 seconds
LIQUID MILK SAMPLED DIRECTLY ON MESH
Real-time Detection – Dietary Reconstruction for Authenticity
Hippuric acid identified as a grass
feed marker in literature.
Shop bought organic and non-organic milk
analysed directly
Current USDA certification demands
that cows are fed grass for AT LEAST 120
days per year
12
Cow ß-LG A
Buffalo ß-LG
Cow
ß-LG B
Little Sample Preparation, LC-TOF measurement
LIQUID MILK SAMPLED AFTER PROTEIN PRECIPITATION
Real-time Detection – Species ID and Intact Protein Profiling
Protein development also key area of
research TOF-MS very suitable for measuring INTACT
PROTEINS
Increased interest in non-cows milk for health and taste
benefits
Cow ß-LG A
Buffalo ß-LG
Buffalo: 50% Cows Milk Substitution
Buffalo: 5% Cows Milk Substitution
Sheep ß-LG-A
Cow ß-LG-B
Sheep ß-LG-B Cow ß-LG-A
Sheep: 50% Cows Milk Substitution
Other protein work: Soy
Proteins, Camel Milk
Powder and Human Milk
13
Little Sample Preparation, DSA-TOF measurement
MILK SAMPLED AFTER PROTEIN PRECIPITATION
Detecting Fraud – Melamine in Liquid Milk & Milk Powder
Continued threat in milk powder but also
liquid milk. Rapid screening tool in DSA technology.
Melamine is a nitrogen rich compound added to diluted milk to falsify
the test for protein content.
Detection Limit:
adult food of 2.5ppm
Infant food 1ppm
DSA method readily
detects down to 1ppm
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
32.3 32.4 32.5 32.6 32.7 32.8 32.9 33.0 33.1 33.2 33.3 33.4
EICm/z (127.069:127.077)
Amplitude
Time (min)
Cu
EIC of varying concentrations of
melamine spiked in milk powder
127.0724
0
50
100
150
200
250
300
350
400
126.5 127.0 127.5 128.0 128.5 129.0
Mass
Amplitud
e
m/z
Cur
Exact mass = 127.0727
ppm error = 2.3
Mass spectrum of melamine
spiked in milk powder (1 ppm)
50 ppm spike
5 ppm spike 1 ppm spike control
Calibration curve of melamine spiked in liquid milk y = 0.0013x - 0.011
R² = 0.9997
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 100 200 300 400 500 600Conc of melamine (ng/mL)
Are
a r
atio
14
No chromatography, No Sample Preparation
LIQUID MILK SAMPLED DIRECTLY ON MESH ANSWER IN <30 SECONDS
Deterring Fraud – Screening for Future Adulterants
List of 5 additional future adulterants to
melamine well documented:
Urea, Biuret, Cyanuric Acid, Dicyandiamdie,
Cyromaxin,
Common Adulterants Sodium Thiosulphate,
Hydrolyzed leather meal,
calcium hydroxide, salicylic
acid, gelatin
Future threats Cyanoguanidine,
Amidinourea,
Aminotriazole,
Isobutylidenodiurea,
caprolactam, biuret
15
10/1
Pomegranate Juice Adulteration
DSA-TOF No sample prep; results in seconds
Tartaric acid is only present in grape juice; so it can be used as a marker for the detection of pomegranate juice adulteration with grape juice.
Can easily identify as low as 1% grape juice mixed in pomegranate juice
Pomegranate juice No Tartaric acid
detected
1 3 3 . 0 1 4 1
1 4 9 . 0 0 8 8
1 9 1 . 0 2 0 2
0
1 0 0 0 0
2 0 0 0 0
3 0 0 0 0
4 0 0 0 0
5 0 0 0 0
6 0 0 0 0
7 0 0 0 0
8 0 0 0 0
9 0 0 0 0
0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0
M a s sR E T ( 0 2 . 2 9 1 3 3 )
Am
pli
tu
de
m / z
Pomegranate juice with 1% grape juice
1 3 3 . 0 1 4 8
1 4 9 . 0 1 0 5
1 9 1 . 0 2 0 1
0
2 5 0 0 0
5 0 0 0 0
7 5 0 0 0
1 0 0 0 0 0
1 2 5 0 0 0
0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0
M a s sR E T ( 0 2 . 0 4 7 9 1 )
Am
pli
tu
de
m / z
Tartaric Acid
[M-H]-
Mass Accuracy = 8.72 ppm
Pomegranate juice with 2% grape juice
Tartaric Acid
[M-H]-
Mass Accuracy = 2.68ppm
1 3 3 . 0 1 4 9
1 4 9 . 0 0 9 6
1 9 1 . 0 2 0 4
0
2 5 0 0 0
5 0 0 0 0
7 5 0 0 0
1 0 0 0 0 0
1 2 5 0 0 0
0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0
M a s sG E N ( 0 1 . 6 5 4 5 8 : 0 1 . 7 0 4 5 8 )
Am
pli
tu
de
m / z
Tartaric Acid
[M-H]-
Mass Accuracy = 2.68 ppm
Pomegranate juice with 5% grape juice
16
10/1
Fruit
Fungicide screening Organic Authenticity Mycotoxin analysis
DSA-TOF No sample prep; results in seconds
Surface analysis of fungicides on oranges with a swab test. Can easily analyze for pesticides and fungicides on food surfaces with DSA.
DSA-TOF No sample prep; results in seconds
LC-UV Quantitative analysis
Fungicides such as imazalil are banned in organic production and MRL’s are in place for conventional fruits. Rapid analysis of organic and non-organic citrus fruits can detect the presence of any fungicides.
EIC for imazalil in two organic and five conventional fruits
1% Grape juice in pomegranate juice
Patulin in apple juice
Patulin is a naturally occurring toxin that is not destroyed by the pasteurisation process. Due to its potential toxicity MRL have been set.
Thiabendazole
[M+H]+
0.5ppm
Imazalil
[M+H]+
2.36 ppm
17
No chromatography, Little Sample Preparation, results in 30 seconds
Olive oil diluted to 1 % in IPA with 10 mM Ammonium acetate then SAMPLED DIRECTLY ON MESH
Rapid Authenticity Checks - Olive Oil
Profile of fatty acids and their ratios can be used to identify
olive oil, other edible oils and any potential
adulteration.
A recent study showed 70% of olive oils did not meet their
label claims Oleic acid exhibits the highest response L/O = 0.18 Ln/O = 0.017
18
No chromatography, Little Sample Preparation, results in 30 seconds
Soya oil diluted to 1 % in IPA with 10 mM Ammonium acetate then SAMPLED DIRECTLY ON MESH
Rapid Authenticity Checks - Olive Oil
The ratio of Linoleic acid to Oleic Acid
response can be used as a way to detect
adulteration of olive oil.
Cheaper vegetable based oils are a
common adulterant. Linoleic acid has highest response in soy based vegetable oil. L/O = 1.86 Ln/O = 0.29
Olive oil is composed of ~ 75% mono-
unsaturated oleic acid. Soy based veg oil
consists of polyunsaturated
linoleic acid, ~ 55 % & linolenic acid ~ 5%.
19
No chromatography, minimal sample preparation
RAPID CONFIRMATION OF OLIVE OIL ADULTERATION – RESULTS IN SECONDS
Rapid Authenticity Checks - Olive Oil
The ratio of Linoleic acid to Oleic Acid response can be used as a way to
detect adulteration of olive oil.
Effect of Soy based Vegetable oil adulteration in olive oil on Linoleic to Oleic acid response
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 20 40 60
Ra
tio
Re
spo
nse
of Li
no
leic
Ac
id
to O
leic
Ac
id
Vegetable Oil Adulteration in Olive Oil/%
Mass Spectra of Olive oil adulterated with 10 % Soy based Vegetable oil
Olive oil is composed of ~ 75% mono-
unsaturated oleic acid. Soy based veg oil
consists of polyunsaturated
linoleic acid, ~ 55 % & linolenic acid ~ 5%.
L/O = 0.30 Ln/O = 0.031
20 10/1
Adulteration of Fraud of Spices?
• Potential food safety issue with allergens
present
• The opportunity for adulteration?
◦ Poor cumin harvest
◦ Abundance of nut shells
◦ Bulking out of raw material
• Products recalled
◦ Incumbent method ELISA
◦ Tests showed positive for peanut
and almond
21
Accurate mass and isotope ratio data
for147mw using EC ID gave a hit for
coumarin; 177mw is methoxycoumarin.
MeOH EXTRACTION, LIQ ANALYSED ANSWER IN <30 SECONDS
INVESTIGATING QUANTITATION BY GCMS
Deterring Fraud – Screening for Unknown Adulterants
Potential adulteration of ground cumin following a bad
harvest .
Power of the TOF to detect unknown
unknowns used to identify markers of
adulteration.
Cumin
Mehlab
y = 3415.8x + 2404.3
R² = 0.9994
0.000
5000.000
10000.000
15000.000
20000.000
25000.000
30000.000
35000.000
40000.000
45000.000
50000.000
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Pe
ak
Are
a
Conc (ppm)
Non-target analysis of pesticides
Utilizing LC TOF analysis with AxION EC ID Software
Inputting accurate mass and isotope
ratio data using EC ID gave a list of
potential compounds.
PESTICIDE CARBARYL IDENTIFIED AS THE UNKNOWN
Ensuring Food Safety – Detecting Contamination
Pesticides linked to cancer, birth defects, liver, kidney damage
EU directive: EU 2002/657/EC
regulation on MRL of pesticides in or on food
and feed of plant intended for human
consumption
2 0 2 .0 8 6 6
2 0 2 .5 7 1 1
2 0 3 .0 8 8 7
2 0 4 .0 9 1 2 2 0 4 .1 5 8 3
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
6 0 0 0
7 0 0 0
8 0 0 0
9 0 0 0
1 0 0 0 0
1 1 0 0 0
2 0 1 .5 2 0 2 .0 2 0 2 .5 2 0 3 .0 2 0 3 .5 2 0 4 .0 2 0 4 .5
M a s sR E T ( 0 2 .8 8 6 0 0 )
Am
pli
tu
de
m / z
Identifying an unknown analyte in QuEChERS extract of artichokes
Unknown peak
eluting at 3mins
Accurate mass of unknown peak in artichoke extract
Based on accurate mass information and AxION EC ID prediction the likely
candidate is Spinosad A.
Retention time matching of the celery extract with a standard
confirms the pesticide indentity.
Ensuring Food Safety – Contaminated Organic Produce?
Spinosad A is an insecticide sprayed
on vegetables
EPA registered pesticides are
prohibited in organic production; however residues up to 5% EPA
tolerance allowed (environmental effects)
Identifying an unknown analyte in QuEChERS extract of organic celery
Accurate mass of extract of organic celery
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
0 1 2 3 4 5 6 7
E I Cm / z ( 7 3 2 . 3 0 0 : 7 3 2 . 5 0 0 )
Am
pli
tu
de
T i m e ( m i n )
7 3 2 . 4 7
7 3 3 . 4 7
7 3 4 . 5 3
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
7 2 5 . 0 7 2 7 . 5 7 3 0 . 0 7 3 2 . 5 7 3 5 . 0 7 3 7 . 5 7 4 0 . 0 7 4 2 . 5
M a s sG E N ( 0 3 . 8 3 5 2 5 : 0 4 . 2 0 1 9 1 )
Am
pl
it
ud
e
m / z
C u
31
Summary
• DSA is an effective tool for rapid screening
◦ Real time detection for authenticity
◦ Fast screen for contamination detection and identification
◦ Targeted and non-targeted analysis
• Continued focus on food adulteration and food safety
• Innovation is key to support customers
• Extending collaborations with food companies
• Solutions and applications