Advances in Analytics of Chemical Hazards in...

Advances in Analytics of Chemical Hazards in Foods

Manoj Pillai. Ph.D; Director Application Support (India), Sciex

Agenda

• Introduction

• Analytes and Analytics

• Hyphenated technology – LC MS

• Mass analysers

• Scan function for Quantitation

• Applications of LC MS

• Where MRM can fail

• Hybrid Linear Ion traps

• Use of Library search in LC MS/MS applications

• HRMS for targeted and non targeted screening of hazards

3 © 2015 AB Sciex

Food Analysis

• Continuous demand for more robust, efficient, sensitive and cost effective methodologies

• Guarantee the safety, Quality and traceability of foods in compliance with legislation and consumers demand

Old methods – concepts based

on Wet chemistry

Powerful instrumental

techniques used in food labs

Sample throughput

Analytical Accuracy

Precision

Specificity

Detection limits

4 © 2015 AB Sciex

Broad areas Analysis

Chemical Constituents Instrumental analysis Microbiological parameters

Sample

Moisture , Crude protein Crude fat, Crude fiber

Dry matter, Organic Matter Inorganic Matter, Protein

Non Protein Nitrogen Lipids, Pigments, Starch,

Sugards Organic acids, Pectins,

Hemicellulose, Cellulose, lignin, Fiber bound

nitrogen, insoluble Ash, Soluble Ash, neutral detergent fiber, acid

detergent fiber, Silica

Pesticides, Drug residues,

Mycotoxins, Allergens,

Dyes Antioxidants

Bioactive principles Dioxins

Organic pollutants Heavy metals

Vitamins Amino acids

Fatty acid analysis Lipid analysis

Hormones Antiparasites Plasticizers

Pathogens and Toxins Listeria monocytogenes

Salmonella Legionella pneumophila Escherichia coli O157

(E.coli) Vibrio spp.

Clostridium perfringens Bacillus cereus

Enterobacter sakazakii Campylobacter

Enterotoxins Staphylococcus aureus

5 © 2015 AB Sciex

Analytics

Analytical techniques – classification according to their working principle

• Biological • Spectroscopic • Electrochemical • Separation Based • Hyphenated

6 © 2015 AB Sciex

High-risk compounds commonly targeted in routine food testing :

Incr

easi

ng M

W

Increasing polarity

Other chemical properties of interest: - Solubility - Structure - Stability - Charge

Quaternary ammonium salts

Acidic/polar herbicides

Macrolide antibiotics

Acrylamide

Melamine

Packaging migrants

Mycotoxins

Marine biotoxins

Assorted pesticides

Heterocyclic antibiotics

Aminoglycoside antibiotics

Assorted antibiotics

Why the trending workflows?

7 © 2015 AB Sciex

Other contaminants emerge almost daily

Incr

easi

ng M

W

Increasing polarity

Quaternary ammonium salts

Acidic/polar herbicides

Macrolide antibiotics

Acrylamide

Melamine

Packaging migrants

Mycotoxins

Marine biotoxins

Assorted pesticides

Heterocyclic antibiotics

Aminoglycoside antibiotics

Assorted antibiotics

Assorted

pesticide metabolites

Other unknown adulterants / chemical contaminants

Allergens

Steroids & Hormones

‘Gutter oil’

Recycled chemical

migrants

But, to further complicate matters…

8 © 2015 AB Sciex

The analytical challenges for food analysis

• Need to be able to deal with complex matrixes like meat, tissue and

organs

• As with any method well characterized sample preparation methods are

required ‒ In many cases the throughput demands simple, almost universal sample preparation

techniques

• The detection system must exhibit both selectivity and high sensitivity ‒ For many contaminants, low or zero tolerance of residues presents a challenge to

detector response

• In many cases simultaneous quantitation and confirmation of

contaminants is desired

• Need to be able to screen for a wide range of contaminants in a single

analysis to maximize the throughput

• In general, methods must be robust, precise and accurate ( and

validated)

Non Targeted Analysis

(Unknowns)

9 © 2015 AB Sciex

Is it possible to do Multiresidue analysis efficiently using UV or FL detector?

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0Time, min

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

Inte

nsity

, cps

2.9

Detection of >750 compounds in a forensic screening method

10 © 2015 AB Sciex

Why use LC/MS/MS for Screening and Confirmation?

• Spectrometry – LC/UV/FL, GC/MS, HPTLC ‒ Insensitive, non-specific

‒ Rigorous sample prep. & derivatisation is often required

‒ Difficulty to analyse multiple analytes in a single run

• Immunoassay – RIA, EIA ‒ Not very specific, issues with cross contamination, false positive

‒ Expensive and only available for few compound classes

‒ No way to analyse multiple analytes in a single run

• LC/MS/MS ‒ Highly selective, sensitive and accurate

‒ Reduced sample preparation

‒ Option to differentiate the false positives/false negatives

‒ Provides both qualitative and quantitative analysis in a single run

‒ Meet all requirements for modern residue analysis

‒ Technique recommended by EU for specific analysis

11 © 2015 AB Sciex

Weighing Atoms

Mass spectrometry (MS) is the science of "weighing" individual atoms and molecules

A mass spectrometer is an instrument that measures the

masses of individual molecules that have been

converted to ions; i.e., molecules that have been electrically charged.

A machine used to weigh molecules.

At molecular scale.

12 © 2015 AB Sciex

Liquid Chromatography Mass spectrometry

Chromatography helps in separation of analytes

Mass spectrometry helps in identification , confirmation and Quantitation of the analytes

Mass spectrometer identifies analyte masses only if they are in the form of ions in gaseous state

Need to convert analyte in solution to ions in gaseous state

13 © 2015 AB Sciex

MASS ANALYSERS

Time of Flight

GOLD STANDARD

FOR QUANTITATION

•Can Select a mass

•Scan a mass range

•Transfer Ions

UNIT RESOLUTION

Good for Scanning

Moderate Resolution

High efficiency for Scanning

High Resolution

IONISATION TECHNIQUES GENERALLY USED WITH LC MS INSTRUMENTATION

ELECTROSPRAY, APCI, APPI, MALDI

Qualitative Analysis

ION TRAPS

Quadrupole

14 © 2015 AB Sciex

What is LC/MS/MS Technology?

• Liquid Chromatography Coupled to a Mass Spectrometer

• (In this case the Mass Spectrometer is a QQQ instrument)

HPLC Column

Q1 Q2 Q3

923 765

923/765

15 © 2015 AB Sciex

O CH3

H2N

O

Benzocaine

NH2

O CH3

OEthenzamide

- Same Formula : C9H11NO2 - Same Mass : 165.19 daltons - Same number of double bonds : 4

Why LCMSMS and not LCMS?

Different fragmentation pattern

16 © 2015 AB Sciex

Typical fragmentation spectra m/z 200

Qualifier Quantifier

17 © 2015 AB Sciex

Selected Ion Monitoring (SIM) vs. Multiple Reaction Monitoring (MRM) MS detection schema for MS vs. MS/MS

• MRM provides superior selectivity and sensitivity for quantitation of targeted compounds (double-mass filtering)

• MRM ratio calculation for multiple ions provides good compound identification (qualifier/quantifier)

• MRM is highly efficient to enable multi-target screening for hundreds of compounds in a single injection

SIM (MS) MRM (MS/MS)

filter filter fragment filter

18 © 2015 AB Sciex

Single MS Analysis of Clonazepam – m/z 316.1 + other interferences with m/z 316 = multiple peaks

316.1 Extracted

MS/MS Analysis of Clonazepam – MRM of 316.1/270.1 Interferences present, but MRM is highly specific (unique Parent to Fragment transition)

316.1/270.1 Extracted

MS/MS Spectrum

Confirmation: Selectivity of MRM

19 © 2015 AB Sciex

1 2 3 4 5 6 7 8 9 10 11 12 13 14Time, min

0.0

1.0e4

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Inte

nsi

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cps

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7.1

9.6

0.6

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0

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Inte

nsi

ty,

cps

6.9

Single Quadrupole vs. Triple Quadrupole

• Higher selectivity in MRM results in better S/N for quantitation

• Better accuracy and reproducibility in MRM detection

• MRM detection provides wider linear range for quantitation

• More reliable identification using MRM ratios over SIM

MRM SIM

20 © 2015 AB Sciex

Confirmation: 3-5 Criteria

XIC of +MRM (89 pairs): 194.137/163.200 amu Expected RT: 2.8 ID: MDMA 1 from Sample 5 (Cal4) of 10460 pos AB4.wiff (Turbo Spra... Max. 9.2e4 cps.

1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8Time, min

0.0

5000.0

1.0e4

1.5e4

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3.5e4

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5.0e4

5.5e4

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6.5e4

7.0e4

7.5e4

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8.5e4

9.0e4

In

te

ns

ity

, c

ps

2.79

1. Retention time 2. Q1 (parent) mass 3. Q3 (fragment mass) #1 4. Q3 (fragment mass) #2 5. Ratio of peak areas

21 © 2015 AB Sciex

Triple Quadrupole MS Multiple Reaction Monitoring (MRM)

Continuous Ion Flow

Quadrupole 1 filters the desired precursor ion

Precursor ions fragment in Q2 LINAC Collision Cell (filled with Nitrogen CAD gas)

Quadrupole 3 filters the desired product ion

Product ion detection and registration

Selective quantitative information

Q3= Fixed

Q1= Fixed

22 © 2015 AB Sciex

Q1 Q3 Dwell time

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2

3

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8

453 254

685 885

453 254 396 274

1098 870

20ms

20ms

20ms 20ms

20ms

464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3

Multiple Reaction Monitoring (MRM)

23 © 2015 AB Sciex

Q1 Q3 Dwell time

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8

453 254

685 885

453 254 396 274

1098 870

20ms

20ms

20ms 20ms

20ms

464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3


24 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254

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464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3


25 © 2015 AB Sciex

Q1 Q3 Dwell time

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20ms

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464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3


26 © 2015 AB Sciex

Q1 Q3 Dwell time

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464 222 987 274

887 870

20ms 20ms

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CE(V)

Q0 Q1 Q2 Q3


27 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254 396 274

1098 870

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464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3


28 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254

685 885

453 254 396 274

1098 870

20ms

20ms

20ms 20ms

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464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3


29 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254

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1098 870

20ms

20ms

20ms 20ms

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464 222 987 274

887 870

20ms 20ms

20ms

Q0 Q1 Q2 Q3


30 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254

685 885

453 254 396 274

1098 870

20ms

20ms

20ms 20ms

20ms

464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3


31 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254

685 885

453 254 396 274

1098 870

20ms

20ms

20ms 20ms

20ms

464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3


32 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254

685 885

453 254 396 274

1098 870

20ms

20ms

20ms 20ms

20ms

464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3


33 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254

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20ms

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887 870

20ms 20ms

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CE(V)

Q0 Q1 Q2 Q3


34 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254

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20ms

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887 870

20ms 20ms

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CE(V)

Q0 Q1 Q2 Q3


35 © 2015 AB Sciex

Q1 Q3 Dwell time

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CE(V)

Q0 Q1 Q2 Q3


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Q1 Q3 Dwell time

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CE(V)

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38 © 2015 AB Sciex

Q1 Q3 Dwell time

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CE(V)

Q0 Q1 Q2 Q3


39 © 2015 AB Sciex

Q1 Q3 Dwell time

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40 © 2015 AB Sciex

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Q1 Q3 Dwell time

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Q1 Q3 Dwell time

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20ms

20ms

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464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2

Q3


44 © 2015 AB Sciex

Q1 Q3 Dwell time

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453 254

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20ms

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464 222 987 274

887 870

20ms 20ms

20ms

CE(V)

Q0 Q1 Q2 Q3


45 © 2015 AB Sciex

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464 222 987 274

887 870

20ms 20ms

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Q0 Q1 Q2 Q3


46 © 2015 AB Sciex

The Key for Multi-Target Analysis – Fast MS/MS

• Linear Accelerator (LINAC® Collision Cell): tilted quadrupole rods of the collision cell cause an electrical field gradient and accelerate product ions after fragmentation

• Faster MS/MS experiments without loss in sensitivity and without false positive results due to cross talk

1.31 1.13 1.14 1.08 1.13 1.10 1.13 1.11 1.12 1.15 peak area x10e6 counts

1ms 2ms 3ms 4ms 5ms 10ms 15ms 20ms 25ms 50ms Sample Name: "1msec" Sample ID: "01" File: "Data d...Peak Name: "Atrazine" Mass(es): "216.1/174.0 amu"Comment: "" Annotation: ""

7.0 7.5 8.0 8.5 9.0 9.5Time, min

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1.8e57.9

Sample Name: "2msec" Sample ID: "02" File: "Data d...Peak Name: "Atrazine" Mass(es): "216.1/174.0 amu"Comment: "" Annotation: ""

7.0 7.5 8.0 8.5 9.0 9.5Time, min

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ty, cps

8.1


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ty, cps

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ty, cps

8.1


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ty, cps

8.1

Sample Name: "10msec" Sample ID: "10" File: "Data ...Peak Name: "Atrazine" Mass(es): "216.1/174.0 amu"Comment: "" Annotation: ""

7.0 7.5 8.0 8.5 9.0 9.5Time, min

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ty, cps

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ty, cps

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7.0 7.5 8.0 8.5 9.0 9.5Time, min

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Intensi

ty, cps

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7.0 7.5 8.0 8.5 9.0 9.5Time, min

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ty, cps

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ty, cps

8.1

47 © 2015 AB Sciex

XIC of +MRM (378 pairs): 194.0/122.0 amu Expected RT: 3.2 ID: 3,4,5-Trimethacarb 1 from Sample 27 (apple 60564) of Eurofins sMRM on 5... Max. 1774.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5Time, min

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Inte

nsi

ty,

cps 2. LC-MS/MS

Routine in a Food Testing Laboratory

3. Quantitation and Identification

1. Extraction 1. Extraction

48 © 2015 AB Sciex

LC-MS/MS data evaluation

• For quantitative evaluation of the data the following criteria are applied: – Correlation coefficient ( r value) – intercept – relative standard deviations of average and single response factors

49 © 2015 AB Sciex

For qualitative evaluation the following criteria are applied:

– Retention time

– Peak area ratio of quantifier and qualifier mass transition (MRM ratio)

LC-MS/MS data evaluation

The MRM ratio of the selected analyte specific mass transitions obtained in the

sample should be the same in standards Due to the characteristic physico-chemical behaviour of the analyte during the

collision induced fragmentation

50 © 2015 AB Sciex

EU Criteria for Confirmation

• Guideline 2002/657/EG

‒ SIM and MRM (4 identification points) – MS precursor 1.0 – MSn product 1.5

‒ Full scan spectra (ions > 10% of base peak, mass spectral libraries)

‒ High resolution MS (>10 000)

2 MRMs = 4

51 © 2015 AB Sciex

…but every year is a big year in food & environmental topics!

Consumer topics and trends

Health impacts of vitamins & supplements

Fungicides in water Overuse of antibiotics in feed animals Pesticides in bee colonies

Undeclared allergens

Bans on brominated flame retardants

Horsemeat contamination in beef products What’s

next?

52 © 2015 AB Sciex

Developing new workflows that address pertinent topics & trends

• Meat speciation by LC-MS/MS

• Allergen testing by LC-MS/MS

• Vitamin testing by LC-MS/MS

53 © 2015 AB Sciex

Meat speciation & allergens testing by LC-MS/MS The world of ‘foodomics’

The most accurate and reliable approach to finding allergens or identifying meat species in food samples:

• Detect and quantify the proteins associated with the allergen or species

Intact protein

Enzyme - Trypsin

Peptide fragments

200 400 600 800

m/z

0

MS/MS – Q3 m/z

* *

* *

Peptide Q1 m/z

MRM Method

54 © 2015 AB Sciex

Allergen testing by LC-MS/MS workflow

Analytical analysis details:

Weigh 5 g homogenized food sample

Extract proteins with extraction buffer (tris, urea, & ACN recipe)

Shake, ultrasonicate (15 min), & mix (45 min)

Centrifuge & collect 0.5 mL aliquot

Add buffers to reduce, alkylate, & digest proteins (150 min, 40C)

Filter and analyze using online SPE

Eksigent ultraLC 110

4000 Q TRAP® system

Sample preparation details:

LC-MS/MS conditions

Column Phenomenex Synergi Hydro-RP 4um, 150 x 2.1 mm

Injection volume 10 uL

Flow rate 300 uL/min

Ionization Positive ESI, MRM detection

55 © 2015 AB Sciex

Results Monitoring multiple allergenic peptides simultaneously – Detection of milk, egg, wheat, & peanut allergens in bread

56 © 2015 AB Sciex

Meat speciation by LC-MS/MS workflow


Weigh 2 g homogenized meat tissue sample

Extract proteins with 10 mL extraction buffer (tris, urea, & ACN recipe)

Shake, ultrasonicate (15 min), & mix (45 min)


Add buffers to reduce, alkylate, & digest proteins (60 min, 40C)

Purify filtrate using polymeric SPE cartridge (PHX)

ekspert ™ microLC 200

QTRAP® 5500 system


LC-MS/MS conditions

Column Halo C18 2.7 um, 90Å, 50 x 0.5 mm


Flow rate 20 uL/min

Mobile phases 0.1% formic acid in water (A) or acetonitrile (B) – gradient


57 © 2015 AB Sciex

Results Monitoring 3 unique horse meat peptide markers – Can detect as low as 1% horse meat contamination in beef

58 © 2015 AB Sciex

Vitamin testing by LC-MS/MS Example: Vitamin B series testing

• Verifying what’s on these labels:

• Analyzing for water-soluble B vitamins by LC-MS/MS provides accurate & reliable quantitation

Vitamin DV (mg)

Thiamine Vitamin B1 1.5

Riboflavin Vitamin B2 1.7

Niacin Vitamin B3 20

Pyridoxal HCl Vitamin B6 2

Folic acid Vitamin B9 0.4

Cyanocobalamin Vitamin B12 0.006

Biotin Vitamin B7 0.3

Pantothenic acid Vitamin B5 10

59 © 2015 AB Sciex

Vitamin B series by LC-MS/MS workflow


Weigh 1 g homogenized sample

Mix with 9 mL 50% methanol in water

Shake, ultrasonicate (5 min), or mix (20 min)


Dilute aliquot 100x with 0.1% TFA in water

Analyze by LC-MS/MS

QTRAP® 5500 system


LC-MS/MS conditions

Column Phenomenex Synergi Hydro-RP 4um, 150 x 2.1 mm


Flow rate 20 uL/min

Mobile phases 0.1% formic acid in water (A) or acetonitrile (B) – gradient


Eksigent ultraLC 110

60 © 2015 AB Sciex

Results Monitoring B vitamins in dog food – All claimed vitamin additives listed on the label were detected

XIC of +MRM (39 pairs): 220.106/202.100 amu Expected RT: 4.6 ID: VITAMIN B5 1 from Sample 3 (DOOG FOOD) of WATER SOL.wi... Max. 8.1e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

1.10e6

1.15e6

1.20e6

1.25e61.29e6

Inte

ns

ity

, c

ps

4.38

B1

B3

B5

B2

61 © 2015 AB Sciex

Results Monitoring B vitamins in infant formula – Comparing LC-MS/MS results to NIST standard reference material

Vitamin

NIST SRM 1849a assigned value (mg/kg)

AB SCIEX results (mg/kg) Accuracy

B1 (Thiamine) 12.57 17.8 142%

B2 (Riboflavin) 20.37 23.3 114%

B3 (Niacin) 109 102 94%

B5 (Pantothenic acid) 68.2 73.6 108%

B6 (Pyridoxal HCl) 13.46 13 96%

B7 (Biotin) 1.99 2.02 102%

B9 (Folic acid) 2.293 3.2 143%

B12 (Cyanocobalamin) n/a n/a n/a

62 © 2013 AB SCIEX

Antibiotics and veterinary drug residues

Natural toxins Vitamin and nutritional content

Pesticide residues

Genetically modified organisms (GMO)

Bacteria and microbial contaminations

Heavy metals

Production & packaging contaminants

Chemical adulterants

Allergens

Authenticity and integrity

AB SCIEX and Food & Beverage Testing Food & Beverage testing covers a wide scope of areas

63 © 2015 AB Sciex

XIC of +MRM (158 pairs): 130.1... Max. 1.8e5 cps.

2 4 6 8 10Time, min

0.0

5.0e4

1.0e5

1.5e5

1.8e5

Inte

ns

ity

, c

ps

0.6

+EPI (130.10) Charge (+0) CE ... Max. 1.2e6 cps.

80 100 120 140 160 180 200m/z, Da

0.00

2.00e5

4.00e5

6.00e5

8.00e5

1.00e6

1.20e6

Inte

ns

ity

, c

ps

88.9

70.9

68.9112.0

83.975.8

130.2

XIC of +MRM (158 pairs): 130.1... Max. 2.7e4 cps.

2 4 6 8 10Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

Inte

ns

ity

, c

ps

0.7

+EPI (130.00) CE (20): Exp 1, ... Max. 1.1e6 cps.

80 100 120 140 160 180 200m/z, Da

0.00

2.00e5

4.00e5

6.00e5

8.00e5

1.00e6

1.14e6

Inte

ns

ity

, c

ps

71.0

130.2

85.168.0

113.0

66.4 132.0

Standard 1μg/L

MRM ratio 0.083

Water sample 1μg/L

MRM ratio 0.082

Library spectrum

(Metformin)

Unknown spectrum with same MRM

ratio

Early eluting compound

where a background ion gave the same MRM ratio and the MRM ratio was low due to

poor MSMS fragmentation

Where MRM Ratios Can Fail

64 © 2015 AB Sciex

Quantitation Using MRM

• Positives: ‒ Fast – can analyse hundreds of analytes in a single injection ‒ Sensitive – specific Q1 and Q3 mass maximises signal/noise ‒ Best solution for quantitation ‒ Ion ratio helps confirm compound identity ‒ Wide dynamic range

• Negatives ‒ No detailed structural information ‒ MRM ratio can fail

‒ Concentration outside linear range ‒ Possibility of interfering peaks

• Need a technique complementary to MRM . . . . ‒ Detailed product ion spectra gives structural information ‒ Can then compare against library for confirmation ‒ Triple quads have poor full scan sensitivity ‒ Ion trap needed

65 © 2015 AB Sciex

Full scan Using Quadrupole – Why Not?

Ions are continuously fed into the quadrupole

Only one ion pair is stable at any one time

Consequently ions are lost

Low Duty Cycle

Continuous Ion Flow

Quad = Scanning

67 © 2015 AB Sciex

Q0 Q1 Q2 Q3

Q TRAP® System - Enhanced Product Ion Scanning

1. Precursor ions selection in Q1-No Isolation time.

N2 CAD Gas linear ion trap

3x10-5 Torr

Precursor ion selection

Ion accumulation

Fragmentation

Exit lens Steps MS2: 1 2 3 &4

2. Fragmentation in Q2-for a richer fragmentation pattern, no low mass cutoff

3. Trap products in Q3-short, efficient fill

4. Mass scan.

High sensitivity , richer fragmentation pattern - EPI

68 © 2015 AB Sciex

QTrap® – What Does It Give? 1. Sensitivity!

QqQ – Product ion scan

Enhanced product ion spectra More sensitivity

No LOW MASS CUT OFF

69 © 2015 AB Sciex

IDA – Information Dependent Acquisition

• Hybrid instrument combining MRM quantitation and ion trap full scan capabilities

• Individually, these techniques can be useful, but need a way of linking them together in a useful way

• Software solution is IDA – information dependent acquisition

• Added capability of having combination of scan functions in one single run

70 © 2015 AB Sciex

Information Dependent Acquisition

On the fly acquisition of masses eluting along with the simultaneous generation of Enhanced resolution data and enhanced product ion spectra

Will help in carrying out the combination of operating modes in a Linear ion trap

72 © 2015 AB Sciex

Screening and Confirmation:

Workflow: MRM survey –MS/MS – Library Search

Collection of MS/MS…(full scan confirmation)

Library Search Results…

Quantification… In case of questionable or contradictory results: –EPI always gives better certainty than ion ratio –No other technique offers MS/MS scans at the same sensitivity level

73 © 2015 AB Sciex

QTRAP® Systems: MRM – IDA – EPI Experiments XIC of +MRM (10 pairs): Exp 1, 216.1/174.0 amu from Sample 7 (10) of Data EPI vs MS2 - EPI dynFT 216.wiff (Turbo Spray) Max. 1.4e4 cps.

1 2 3 4 5 6 7 8 9 10 11 12 13 14Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

In

te

ns

it

y,

c

ps

7.20

+EPI (216.10) CE (35): Exp 2, 7.073 to 7.299 min from Sample 7 (10) of Data EPI vs MS2 - EPI dynFT 216.wiff (Turbo Spray) Max. 6.0e5 cps.

70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230m/z, amu

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

In

te

ns

it

y,

c

ps

174.0

103.9132.0 146.096.0

216.079.0

138.0110.0

Quantitation: Hundreds of MRM

Confirmation using EPI spectra Library search – PUR 95.6%

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200Concentration, ng/mL

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.4e5

Ar

ea

,

co

un

t

74 © 2015 AB Sciex

Benefits of QTRAP® – Overcome matrix ambiguity

A. Enhanced Product Ion (EPI) scanning with QTRAP® is basically like having more than 4 MRMs for any detected compounds in your sample.

s. XIC of +MRM (202 pairs): 297.0/... Max. 7300.0 cps.

7 8 9 10 11 12Time, min

0

1000

2000

3000

4000

5000

6000

7000

Inte

ns

ity

, c

ps

9.1


7 8 9 10 11 12Time, min

0

200

400

600

800

1000

Inte

ns

ity

, c

ps

9.3

10ppb Imazalil MRM ratio = 0.63 0.093

Imazalil in sample?? (10x diluted extract) MRM ratio = 0.50

?

Example

75 © 2015 AB Sciex

Q: Are matrix interferences complicating my results?

Benefits of QTRAP® – Overcome matrix ambiguity

A. Enhanced Product Ion (EPI) scanning with QTRAP® is basically like having more than 4 MRMs for any detected compounds in your sample.


7 8 9 10 11 12Time, min

0

1000

2000

3000

4000

5000

6000

7000

Inte

ns

ity

, c

ps

9.1


7 8 9 10 11 12Time, min

0

200

400

600

800

1000

Inte

ns

ity

, c

ps

9.3

10ppb Imazalil MRM ratio = 0.63 0.093

Imazalil in sample?? (10x diluted extract) MRM ratio = 0.50

?

s. +EPI (297.00) Charge (+0) CE (3... Max. 8.0e5 cps.

50 100 150 200 250 300 350m/z, amu

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

Inte

ns

ity

, c

ps

201.0

297.2159.1

69.0173.2

109.2 176.2 255.281.1

MS/MS Library fit = 90.4%

• More fragments to confirm • Ability to compare to a library spectrum

The EPI data can help to de-complicate any effects that the matrix might have on the MRM ratio alone for better confirmation of positives.

Example

76 © 2015 AB Sciex

Q: Am I sure of the positive hits? A: Your QTRAP® can give you that extra sense of security when confirming positives.

Benefits of QTRAP® – Added confidence

XIC of +MRM (150 pairs): 280.2/2... Max. 1.7e4 cps.

2 4 6 8 10 12 14Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

Inte

ns

ity

, c

ps

7.7

6.2

XIC of +MRM (150 pairs): 280.2/... Max. 2100.0 cps.

2 4 6 8 10 12 14Time, min

0

500

1000

1500

2000

Inte

ns

ity

, c

ps

7.9

10 ppb Metalaxyl Standard MRM ratio = 0.74

Metalaxyl in sample? (10x diluted extract)

?

. +EPI (280.00) CE (35) CES (15):... Max. 6.6e7 cps.

50 100 150 200 250 300 350 400m/z, amu

1.0e7

2.0e7

3.0e7

4.0e7

5.0e7

6.0e7

6.6e7

Inte

ns

ity

, c

ps

160.1

145.1 192.2

132.1 148.1 162.2220.3

134.0105.1248.3 280.3

. +EPI (280.20) Charge (+0) CE (3... Max. 3.3e5 cps.

50 100 150 200 250 300 350 400m/z, amu

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.3e5

Inte

ns

ity

, c

ps

280.3

220.3

192.4

148.3

202.3134.0

Library spectrum Metalaxyl

Unknown sample spectrum FIT for Metalaxyl = 23.6%

Calculated concentration = 13.3μg/kg MRM ratio = 0.72

MRM ratio would suggest this sample was positive for Metalaxyl RT was slightly off (but not unusual in food matrices)

Am I sure??

Example 1

77 © 2015 AB Sciex

Q: Am I sure of the positive hits? A: Your QTRAP® can give you that extra sense of security when confirming positives. Benefits of QTRAP® – Added confidence

XIC of +MRM (150 pairs): 280.2/2... Max. 1.7e4 cps.

2 4 6 8 10 12 14Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

Inte

ns

ity

, c

ps

7.7

6.2

XIC of +MRM (150 pairs): 280.2/... Max. 2100.0 cps.

2 4 6 8 10 12 14Time, min

0

500

1000

1500

2000

Inte

ns

ity

, c

ps

7.9

10 ppb Metalaxyl Standard MRM ratio = 0.74

Metalaxyl in sample? (10x diluted extract)

?

. +EPI (280.00) CE (35) CES (15):... Max. 6.6e7 cps.

50 100 150 200 250 300 350 400m/z, amu

1.0e7

2.0e7

3.0e7

4.0e7

5.0e7

6.0e7

6.6e7

Inte

ns

ity

, c

ps

160.1

145.1 192.2

132.1 148.1 162.2220.3

134.0105.1248.3 280.3

. +EPI (280.20) Charge (+0) CE (3... Max. 3.3e5 cps.

50 100 150 200 250 300 350 400m/z, amu

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.3e5

Inte

ns

ity

, c

ps

280.3

220.3

192.4

148.3

202.3134.0

Library spectrum Metalaxyl

Unknown sample spectrum FIT for Metalaxyl = 23.6%

Calculated concentration = 13.3μg/kg MRM ratio = 0.72

The EPI data can help to de-complicate the results for better confirmation of positives – or, in this case, confirmation that this peak is not Metalaxyl.

Example 1

79 © 2015 AB Sciex

High Resolution Mass Spectrometry

Resolution measures of the ability to distinguish two peaks of slightly different mass-to-charge ratios ΔM, in a mass spectrum.

From Current protocols in Protein Science, J.Wiley and Sons 1996

Triple Quadrupole - Unit resolution

80 © 2015 AB Sciex

• HRMS instruments can be used for ‒ Accurate mass Measurements

‒ Helps in determining the elemental formula ‒ Collection of Full Scan Spectra

‒ Greater insight in to the m/z present in the sample ‒ High resolution Product Ion Spectra

‒ Insight in to the structural information ‒ Freedom to measure compounds without previous compound specific tuning

‒ Targeted Screening ‒ Identification of known molecules and confirmation using MS/MS

‒ Non Targeted Screening ‒ Identification of Unknown molecules

‒ Similarity check or cross sample comparison using HRMS data and statistical tools like PCA

‒ Possibility to retrospective analysis ‒ Possibility of high resolution quantitation

• Excellent technique to analyse multi-components in complex matrix

Role of High Resolution Mass Spectrometry

98 © 2015 AB Sciex

What needs to be accomplished?

Needs to find the contaminants in Food (with out any false negatives)

What are the impacts of false negatives?

major Health crisis, expensive recall, negative press, tarnished reputation etc

Need to accurately ID contaminants (No false Positives)

What are the impacts of false positives

More time required for sample analysis, reduced throughput, higher cost per analysis,

tarnished reputation

99 © 2015 AB Sciex

Improving confidence in results

Workflows Advantages

MRM based analysis – Triple Quads

•Gold standard for Quantitation •MRM is highly selective and sensitive •Targeted Quantitation applications

•Do not have Full Scan capabilities ( (no good sensitivity to generate Full Scan product Ion spectra on an LC scale) •Limited to only MRM Quant or targeted screening using MRM only

TRAP analysers Full scan capabilities are good for not used for high throughput Quantitation

Hybrid QTRAP 1. Work as a Triple Quad and as a TRAP, 2. Switch scan functions of QQQ to TRAP in 2 ms 3. MRM Quantitation Possible 4. High sensitive Product Ion Spectra on LC scale triggered on MRM

identification possible 5. Library Search using the Product ion Spectra Possible for Known

molecules 6. MRM based screening along with Library Search Possible for Known

molecules

QqTOF •Acquisition of Highly sensitive Full Scan MS spectra with high resolution and Mass accuracy •IDA can be used to collect high resolution MS/MS spectra and reliable compound identification using Library searching •Screening of Unknown compounds - possible

100 © 2015 AB Sciex

COE Focus Areas

• LC MS Training Programs

• Hands-on Workshops

• Method development assistance & Troubleshooting support

• Joint Research activities for qualified applications.

• Presale Demos and Sample analysis

Five key focus areas for the COE

101 © 2015 AB Sciex

LC-MS/MS method is a combination of things

Data Processing

Success of an LC/MS method

Is a combination of things

MS HPLC Sample Processing

Analytical Scientist

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