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Tackling the

challenge in food
safety analysis
Dian Maulana
Sales Engineer
PT. Berca Niaga Medika
GC Overview
Agilent Technologies

Company Profile
BNM is a member of CCM Group
GC Overview
Agilent Technologies

Authorized Distributor
GC Overview
Agilent Technologies

Our Key Products Offering
Liquid Chromatography (HPLC)
Gas Chromatography (GC)
Mass Spectrometry (MS)
LC-MS, LC-MS/MS, GC-MS, GC-MS/MS
ICP-MS
Microbial Identification System (MIDI)
Agilent Sample Workbench
UV-Visible Spectroscopy
Columns and Supplies
Training, Application, Consulting, and support Services
Food Industry Testing Segmentation


Residues
Additives,
Natural products
Contaminants

Other
Pesticides (Biocides)
in fruits and
vegetables
Veterinary drugs in
meat and fish
Hormones in plants
and meat
Residues in animal
feed
Food and Feed authenticity
(origin and adulteration) i.e.
Fish and meat speciation;
Feed speciation
Functional (Health) Foods
(Metabolomics)
Food and feed hygiene
(pathogens)
Food and Feed Quality
testing (incl. OOL)
Animal and Plant health
GMO
Food terrorism (incl. OOL)


Food contact
materials
Trace Metals
Mycotoxins
Food allergens
Acrylamide
Dioxins, PAHs
PFOA and PFOS
Contaminants in
animal feed
Human health impact
of contaminants
(metabolomics)

Flavors
Colorants
Vitamins and
minerals
Sugars
Fats (i.e. transfats)
Protein content
Nutritional labeling
Food enzymes





Agilent Provide Total Solution for Food Customers


Residues


Additives,
Natural products


Contaminants



Other

Private Contract Labs
Government Labs
Academia/University
Food industry

Sample
Preparation
GC & GC/MS
GC/QQQ
LC & LC/MS
LC/SQ
LC/TOF
LC/QQQ
Columns &
Suppliers
Services
Applications and Collaborations
ICP/MS Bioanalyzer
The world of mass spectrometry
LC/MS
Polar
Labile
Involatile
GC/MS
Non-polar
Stable
Volatile
ICP/MS
Elemental
June 26, 2014
7
Data
Acquisition
and Analysis
HPLC -
Separation
Ion Source
Sample
Introduction
Mass
Spectrometer
Separation and
Measurement
Separation Ionization Detection
LC + MS = LCMS
June 26, 2014
8
Advantages
High Specificity Confidence in results
Excellent Quantitation Wide dynamic range
Sensitivity Lower detection limits

No derivatization Minimum sample preparation
One instrument suitable for many methods
High throughput: multi-analyte analysis

Food Safety Analysis: Meet regulatory requirements

LC-Triple Quad
Method of Choice for Food Safety Analysis

Reduced
Costs
a
Collision Cell
MS MS
June 26, 2014
10
6495 Triple Quadrupole LC/MS
The most sensitive triple quadrupole in the market
Greater ion sampling and ion transmission efficiency yields better
assay performance for your most demanding applications

6490 Triple Quadrupole LC/MS
Agilent iFunnel Technology Attogram zeptomole Sensitivity!
Performance Ultra-Trace Analysis: Ideal for Biomarker and most
Challenging Bioanalytical/Clinical Assays, Steroids, Hormones,
Complex matrices

6460 Triple Quadrupole LC/MS
Agilent Jet Stream sub femtogram sensitivity!
Routine Trace Analysis: Ideal for Multi-Residue Pesticide Analysis,
Mycotoxins, Veterinary drugs

6420 Triple Quadrupole LC/MS
Lowest cost of ownership
Routine Analysis: Ideal for clinical and drug analysis, environmental
analysis















Agilent 6400 Triple Quadrupole Product Line
Excellent Value and Performance

6420
QQQ
6495
QQQ
P
e
r
f
o
r
m
a
n
c
e

646
0
Trip
le
Qua
6460
QQQ
6490
QQQ
June 26, 2014
11
6495 QQQ - Premium Performance
Improved sensitivity and lower LLOQs
Average 3x in S/N specifications and applications
Improved precision and excellent accuracy at
the lowest levels 3x in IDL specifications
Proven 6 orders of linear dynamic range
Proven robustness in complex matrix
Food matrix and biological matrix (plasma)
Improved mass range, fast scan speed and MRM
acquisition rate
Proven iFunnel Technology
10/6/2014
Internal Sales Training - The new 6495 QQQ LC/MS
13
Agilent Jet Stream Hexabore Capillary Dual Ion Funnel
Thermal gradient focusing
Efficient desolvation
Creates an ion rich zone
Six capillary inlets
Samples x10 times more
ion rich gas
Removes the gas but
captures the ions
Removes neutral noise
Many Ways to Manipulate S/N
Variation in S/N measurements makes direct assessment difficult
Increase signal
Increase the gain
Narrow chromatographic peak
width
Increase scan averaging
Lower noise
Select noise region
Narrow the width of noise region
Adjust baseline
Apply peak smoothing & noise
filtering
Vary noise calculation algorithms:
Peak-to-Peak, RMS, and ...
a. Peak-to-peak noise
b. RMS noise
c. Smoothing
d. Baseline noise filtering
Instrument Detection Limit (IDL) is Defined by Statistics
IDL
LCMS
= t x SD = t x (%RSD / 100) x amount measured
Based on a well-established statistical formula, follows regulatory guidelines
The minimum amount of analyte that is
detectable and distinguishable from
background noise with a confidence level
IDL
Student t value, for
99% confidence level
n 1 degree of freedom
t
Relative standard deviation / precision
of peak area at the amount measured
From n replicate injections
%RSD
Limited to 2 5 x times higher than the
Detection Limit (DL)
Amount
measured
Theoretical fitting of %RSD is
based on ion statistics
%RSD increases at lower injected
amount
%RSD vs. Injected Amount
Why Change from S/N to IDL for QQQ LC/MS?
Goal of Quantitative Analysis Reporting Detectable AMOUNT
Sensitivity Performance of QQQ LC/MS
Lowest detectable amount Detection Limit (DL)
Lowest quantifiable amount Limit of Quantitation (LLOQ)
Criteria of precision and accuracy are met at LLOQ
Signal to Noise (S/N) NOT AMOUNT
Variation associated with S/N (noise) measurement
Fails to estimate the true detection limit and LLOQ, when measured
at a high level of S/N > 50:1
Not a good metric of sensitivity performance
Instrument Detection Limit (IDL) AMOUNT
A rigorous metric based on statistical analysis of precision (%RSD)
Measured at an analytical low level close to the detection limit
Accurate assessment of the true detection limit and LLOQ
A practical means of evaluating sensitivity performance
Sensitivity and Precision Instrument Detection Limit
1 fg of Reserpine used to measure IDL (+) 1 fg of Chloramphenicol used to measure IDL (-)
IDL = t x (%RSD / 100) x Amount
= 2.821 x (10.9 / 100) x 1 fg = 0.31 fg
IDL = t x (%RSD / 100) x Amount
= 2.821 x (10.0 / 100) x 1 fg = 0.28 fg
6495 IDL Amount measured Replicates Area %RSD t (99%) IDL
Reserpine (+) 1 fg n = 10 10.9% 2.821 0.31 fg
Chloramphenicol (-) 1 fg n = 10 10.0% 2.821 0.28 fg
1.4
Average of 13x 6495 units
Area %RSD = 10.9
n = 10 injections
Area %RSD = 10.0
n = 10 injections
Multiple Reaction Monitoring (MRM)
June 26, 2014
18
LC/MS in food safety analysis
Typical food matrices:
Fruit and vegetables
Rice, maize, nuts
Meat, poultry, fish

Main analytes of interest:
Pesticides
Antibiotics
Mycotoxins


Method of choice:
LC-Triple quad, MRM mode

June 26, 2014
19
Pesticides screening the state of the art
Pesticide ranking published by EURL for pesticides

Check your scope ranking for pesticides
Listing includes more than 1100 compounds
- Pesticides currently used or used in the past
- Pesticide metabolites of importance
Ranking of pesticides based on:
Toxicological data (toxicological endpoints and endocrine disruptive activity)
Residue situation in crops (reporting from labs in Europe and RASFF notifications)
Agricultural usage (including potential for misuse and persistent pesticides)
Analytical information
Compounds Exclusive method
LCMS amenable 683 212
GCMS amenable 552 156
GCMS and LCMS amenable 381
No good method mentioned* 252
June 26, 2014
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Agilent 1290 Infinity LC + 6460A Triple Quad with Agilent Jet Stream Technology
~600 Dynamic MRM Transitions
9 replicates analyzed at 200 pg level
Mean area %RSD = 3.2, Mean height %RSD= 3.6
Application: Analysis of 300 Pesticides in 15 min.
The combination of UPLC-QQQ with Dynamic MRM acquisition results in high sensitivity,
quantitative data and very fast analysis times
June 26, 2014
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Unknown Screening
whats in the
sample?
Qual/Quan:
Confirmation and
Quantitation?
Quantitation how
much compound(s) is
in the sample?
Non-Selective Selective
Is it Possible to Simultaneously Obtain Qualitative and
Quantitative Results?
June 26, 2014
23
A complete, guaranteed
solution
Validated method from
sample preparation to
sample analysis
Guaranteed LOQ and LOD
Complete SOP
Training (optional)
Also available for GC/MS
Also available for other
matrices, example: tobacco

June 26, 2014
25
Target analysis of porcine peptide biomarkers in
processed food commodities with Agilent 6490 QQQ
Jelly
Marshmallow
Soft Gummy
Capsule
Sample preparation
Enzymatic digestion
Agilent 6490 LC/QQQ
Target analysis:
Can we find the porcine
peptide biomarkers in these
food samples?
Confirmation of the targeted porcine peptides by MS/MS
Porcine Marshmallow
Product ion 1
Product ion 2
Product ion 3
Product ion 4
Product ion 1
Product ion 2
Product ion 3
Product ion 4
Bovine Marshmallow
CL EARLY BE T T E R L C - MS S OL UT I ONS
Agilent provides the complete solutions
June 26, 2014
28
Agilents MassHunter Workstation
Instrument Control
Real-time monitoring
Method set-up
Autotune

Qualitative Analysis
Chromatographic results
Spectral results
Find compounds

Quantitative Analysis
User filters
Compound results
Calibration curve
Batch-at-a-Glance
Powerful Navigation
Single Vendor Solutions!
Agilent is a Mass Spec Company
A complete portfolio of Analyzers and inlets
Continuous Innovations
Agilent is a Chromatography Company
A complete portfolio of LC components
Continuous Innovations
Agilent Solutions with Pesticide /
Forensics Application Kits
Customizable to meet YOUR needs
Easy to use but POWERFUL
Single Vendor Solutions!
240 IT
7000 TQ
220 IT
5975E SQ 5975T LTM SQ
5975C SQ
7200 Q-TOF
More Choices Better Solutions
Agilent GC/MS & GC/MS/MS
ASMS 2011




Agilent GC/MS Analyzers


Factory setup and
chemically tested
GC/MS/MS Analyzers
for high-sensitive multi-
residue pesticide analysis




GC/MS SP-1 Pesticide Analyzer
Two Configuration Methods
Based on 7890A/7000B GC/MS/MS system, configured with backflush using
Capillary Flow Technologies. MRM database is included with the method, which is
set up and chemically tested in the factory.

Two configuration methods are offered to meet customers need:
SP1 7890-0501: Method based upon Agilents Retention Time Locked (RTL) GC/MS/MS
MRM database (500+ pesticides) set up at Constant Pressure mode with Post-column
backflush. It provides flexibility to add GC detectors and easily scaled for shorter runtime.
SP1 7890-0502: Method based upon USA targeted pesticide list RTL GC/MS/MS MRM
database (200+ pesticides) setup at Constant Flow mode with Mid-column backflush. The
method provides ultimate performance and shortest cycle time with reduced carrier gas
consumption.
New !!!




Individual
Components
produced
Individual
Components
Tested (OFN)
System
assembled
App. method
developed on
cust. system
System checkout
on App. mix
Run 1
st
customer
sample
customization or
optimization
method
On-going
maintains/service
Factory CE Setup; I&F Optional Consulting Aftermarket
Without Ordering Analyzer
Factory Setup
Optional
Consulting
Aftermarket
Order Analyzer CE specialist
I&F
Application
report
System w.
Application setup
App. optimized
Consumables
SP1: Factory Pre-setup Analyzer
Familiarization
& consulting
Std. HW, SW
What is included with the Analyzer?
7200 Series Q-TOF for GC/MS
7200 Analyzer
Doug King
7000B TQ 6500 Q-TOF
NEW
Optics
New . . . Yet Totally Proven
Dual-stage ion mirror improves
second-order time focusing for
high mass resolution.
Hexapole collision cell accelerates
ion through the cell to enable faster
generation of high-quality MS/MS
spectra without cross-talk
Split-flow turbo differentially
pumps the ion source and
quadrupole analyzer compartments
4GHz ADC electronics enable a high
sampling rate (32 Gbit/s) which improves
the resolution, mass accuracy, and
sensitivity for low-abundance samples.
Dual gain amplifiers simultaneously
process detector signals through both low-
gain and high gain channels, extending the
dynamic range to 105.
Analog-to-digital (ADC) Detector:
Unlike time-to-digital (TDC) detectors
which record single ion events, ADC
detection records multiple ion events,
allowing very accurate mass
assignments over a wide mass range
and dynamic range of concentrations.
Proprietary INVAR flight tube
sealed in a vacuum-insulated
shell eliminates thermal mass
drift due to temperature changes
to maintain excellent mass
accuracy, 24/7. Added length
improves mass resolution.
Hot, quartz monolithic quadrupole
analyzer and collision cell identical
to the 7000 Quadrupole MS/MS
Two 300L/s t urbos pump the
focusing optics and flight tube
From 3 pg of brochure
OFN Results
theoretical masss = 271.986677 error = -0.5 ppm
1pg OFN
Theoretical Mass = 271.986677 error = -0.5 ppm
Resolving Power = 13464
RIS, Tom Doherty
0.25 u
Resolving Power
Resolving Power, R=Mass/PW
R = 614/0.68 = 903
Pw=0.68
Mz=614
SQ
IT
TQ
TOF
Accurate Mass
Pw=0.68
Mz=614
mz < 1ppm
mz = 0.1/614
= 160 ppm scan
SQ
IT
TQ
TOF
240.1218
240.0785
Flurenol methyl ester
m/z = 240.0781
Mass error = 1.7 ppm
Dimetilan
m/z = 240.1217
Mass error = 0.4 ppm
~13,500 resolution FWHM
High Res TOF to Eliminate Isobaric Interferences
Appears as Single 240 u Mass Peak for Any Unit Mass Resolution MS
43
No Internal Reference Mass (IRM)
corrections applied
m/z = 0.0433
Q-TOF MS/MS for Structural Elucidation
C16H14O4 (Rings + Double Bonds = 10)
44
(M H)
+

269.0802
Candidate
structures
m/z
(experimental
)
Formula Error
(ppm)
Score
269.0802 C
16
H
13
O
4
2.2 80.7
193.0494 C
10
H
9
O
4
0.6 96.7
167.0334 C
8
H
7
O
4
3.0 N/A
166.0259 C
8
H
6
O
4
0.6 N/A
138.0310 C
7
H
6
O
3
1.1 98.1
110.0359 C
6
H
6
O
2
3.0 N/A
95.0127 C
5
H
3
O
2
0.9 99.5
CH
2
=CHC
6
H
5
CO

CH
3
CO

H

C
6
H
5
CH=CHC
6
H
5
Fragment Sequence from
Multiple MS/MS Experiments
Candidate structure #4 fits the
fragmentation information the best
Agilent Technologies
Innovations in ICP-MS
Slide #45
Agilents ICP-MS Innovations
7900 is a successor to 7700 in Single Quad ICP-MS history
1987 2000 2009 1994
First computer-controlled ICP-MS
PMS series
4500
7500
7700
Enabling high
sensitivity
metal analysis
Enabling routine
robust ICP-MS
analysis
Enabling a new level
of interference
management
World's first benchtop system.
Hyperbolic profile quad, motorized torch XYZ,
cool plasma
Enabling a new
level of ease of use
in ICP-MS
9 orders detector range
Octopole Reaction System
HMI
ISIS-DS
3
rd
Generation ORS
MassHunter SW

Enabling a new era
in ICP-MS analysis
8800 ICP-QQQ
2012 2014
7900
Worlds First
ICP-QQQ
ICP-QMS Redefined
New UHMI

New Plasma interface
and vacuum design
New Detector system
New ISIS 3
New MassHunter 4.1
Slide #46
Agilent 7900 ICP-QMS
Continuing on the success of the Agilent 7700 series ICP-MS:
SMALL in Size, but BIG on Performance
Slide #47
3 Key Benefits of Agilent 7900 ICP-MS
Advancing the Analytical Dynamics of Food Analysis
1) Matrix Tolerance 2) Interference Removal

3) Dynamic Range
Ultra High Matrix
Introduction System (UHMI)
Handles tough sample
matrices better than any
other ICP-MS. Highest total
dissolved solids(TDS)
capability up to 25%
Reduces sample prep time
and error
Better long-term stability
Octopole Reaction System
(ORS
4
) He Mode
He Mode effectively removes
common polyatomic
interferences in the samples

Easy to use - He collision
mode only
Effective for a wide range of
sample types
Accurate measurements

Wider Dynamic Range
11 orders dynamic range:
0.1ppt(DL) to 10,000ppm

Simplifies method
development
Easy sample prep.
Improves productivity
Facilitates fast TRA
signals, e.g. SP-ICP-MS

Slide #48
Faster TRA mode facilitates SP-ICP-MS

Minimum dwell time is shortened from 3 msec. on the 7700 Series to 0.1
msec to allow faster sampling of transient signals.
0.1 msec
This enables the
measurement of the
single NP peak signal
arriving on the detector.
30 nm Au Nanoparticle
Slide #49
Agilent 8800 ICP-QQQ
Best New Spectroscopy Product of 2012
Outstanding New Scientific Instrument Product of 2012
R&D100 2013
ACCSI Outstanding
New Scientific
Instrument Product of
2012 IBO award
ASMS 2012
Select Science 2012
Award Best
Spectroscopy Product
Slide #50
ICP-MS/MS: How Does it Work?

ICP (plasma) and Interface:
Forms and extracts ions from
the sample (just like the 7700)
EM (detector): Measures the
ions that are scanned by Q2
(just like the 7700)
Q1 controls ions
that enter the cell
Consistent reactions
even if sample
composition changes
ORS
3
collision/
reaction gas added

Ions react and are
neutralized or moved
Product ions are formed
Q2 selects the
target analyte mass
Interference-free analyte
ions passed to EM
Slide #51

MS/MS reaction cell having high selectivity
MS/MS reaction cell removes the most challenging
interferences.
Phosphorus can be quantified below ppb DL<50ppt for P is
spec..
High sensitivity and Low noise
Sensitivity of Agilent 8800 is twice the Agilent 7700
Random noise is extremely low, < 0.2 cps.
Excellent Abundance Sensitivity
MS/MS scan achieves < 1x10
-10
abundance sensitivity.
Key Features & Benefits of Agilent 8800 ICP-QQQ
Advancing the Analytical Dynamics of Food Analysis
Slide #52
Applications
(Routine Emerging Trends)
Slide #53
Trace Elemental Constituents in Foods
Method Automation Function (Agilent 7900)
K. Sakai et al.,, Agilent Appl. Note: 5991-4556EN, 30 May 2014..
Slide #54
Challenge
The adoption of new analytical instrumentation can be challenging for food
testing laboratories that have previously used techniques such as ICP-OES and
AAS. Trace level analysis requires tighter control of general laboratory practice
to ensure that the low detection limits can be achieved routinely. ICP-MS, while
offering high sample throughput and low limits of detection, is generally
considered more difficult to use than the well-established ICP-OES and AAS
techniques, with one perceived drawback being the greater complexity of
developing a reliable analytical method for new sample types.
Solution
The Agilent 7900 ICP-MS uses ICP-MS MassHunter software, which includes a
new Method Automation function to simplify the process of method
development, making it easier for users of all levels of experience to develop
reliable methods for their particular sample type.
Applications
(Advanced Emerging Trends)
Slide #55
Hg Speciation in Marine Biological Tissues
S. Sannac et al., Benefits of HPLC-ICP-MS Coupling for Hg Speciation in Food, Agilent Handbook
of Speciation, 2
nd
Ed., 5990-9473EN (2012).

Gradient LC (1260) conditions:
Start at 2% MeOH
Reaching 90% MeOH in 1 min
90% MeOH till 3 min
Going back at 2% MeOH
7700x ICP-MS conditions:
RF: 1,600 W; 8 mm
sampling depth
CRG: 0.54 L/min; MUG:
0.10 L/min
Option Gas: 9% (20% O
2
in
Ar)
Spray Chamber: -5
o
C
As Speciation in Rice
Background
Rice (Oryza sativa):
Major food source of the world staple
diet of Asians
> 8,000 different species
Recent interest is on accumulation of As
and its potential impact on humans
Conc of > 2 ppm dry weight have been
reported
Different forms of As:
Toxic inorganic forms, As(III) and
As(V)
Less toxic organic forms, DMAA
Current interest is to investigate the
species of As within various rice species
to determine the safest species to be
consumed
Slide #57
Current CODEX Regulatory Levels*
(Total & Inorganic As)
*ftp://ftp.fao.org/codex/meetings/cccf/cccf5/cf05_10e.pdf, viewed 17
th
Jan 2014
Slide #58
Speciation of As in Rice
Synergy of Separation Science and Elemental
Detection
H.R. Hansen, et al. (2012), Arsenic Speciation in Rice A Routine HPLC-ICP-MS
Method, Agilent Handbook of Speciation, 2
nd
Ed., 5990-9473EN.
Sample Preparation
0.2 g of dried and milled acidic extraction (1% HNO
3
)
overnight
Microwave extraction
Centrifuged
Analysis by LC-ICP-MS using
75
As with
115
In as ISTD
Calibration from 0.5 to 25 ppb
LODs: 0.1-0.3 ppb.
As Speciation in Rice
H.R. Hansen, et al. (2012), Arsenic Speciation in Rice A Routine HPLC-ICP-MS
Method, Agilent Handbook of Speciation, 2
nd
Ed., 5990-9473EN.
Species found
in rice were
DMA(V) and
As(V)
Total
inorganic As
is the sum of
peak areas for
As(III) and
As(V)

Slide #61
March 11, 2011
Agilent Hachioji
Fukushima
131
I
137
Cs
90
Sr
129
I
Radioactive
Contamination
Summary
Foods analysis is constantly evolving to keep up with
emerging contaminants in life sciences and chemical
analysis
ICP-QMS and ICP-QQQ are now indispensable tools in
modern food testing laboratories due the analytical
dynamics of this technique (sensitivity and specificity).
Emerging frontiers in food analysis will see the analytical
dynamics ICP-MS being used to address both routine and
advanced food analysis applications





Slide #62
Agilent Technologies
Continued evolution as a leading chemical and bio-analytical measurement
company

T N K
U O Y
H A