novel and emerging technologies in food analysis - · pdf filenovel and emerging technologies...

©2011 Waters Corporation 1

Novel and Emerging Technologies in Food Analysis

Daniel McMillan

Waters UK


RADAR Simultaneous full scan & MRM

Rapid electronics allow instrument to switch between MRM and full scan (FS takes 100ms)

No loss of MRM data quality

Added information can be gained from full scan

— Monitor matrix

— Assess for possibility of matrix effects

— Added information for sample prep method development

— Search for significant non-targeted compounds


MRM

Full Scan

Example - Sewage Sludge Analysis

Spectra from humics and fulvics elution region

Suspected anionic surfactant LAS

Estriol


Diethylstilbestrol

17β estradiol & 17α estradiol

Ethinyl estradiol

Dual Scan-MRM Matrix Monitoring TIC

Elution of matrix components including C11 LAS 311 m/z

Possible matrix effects

Example - Sewage Sludge Analysis


Investigating the LAS Components

m/z 297

m/z 311

m/z 325

m/z 339


Perfluorinated Compounds


Impact of sensitivity RADAR data for complex samples

Original Sample

x10 dilution

x100 dilution

Acquisitions allow levels of matrix to

be monitored during MRM analysis


PFHxA

x100

x10

x 2.

5

decr

ease

x

10.2

de

crea

se

802,268

318,971

31,270

Peak Area

Impact of sensitivity Diluting Away Matrix Effects

Expected RT

Reducing ion suppression

-75% suppression


Grape

Avocado

Marjoram

Ginger

What is in the samples? Ultra trace analysis


Ultra trace analysis Matrix effects

DisQuE Ginger QC extract

pesticides @ 0.01 mg/kg (10 ppb)

Cyromazine

Simetryn

Atrazine

Azinphos-ethyl

Poor peak shape

Low calculated recoveries

High Matrix background


Ultra trace analysis Reduced matrix effects

Cyromazine

Simetryn

Atrazine

Azinphos-ethyl

10 x dilution of Ginger extract

All peaks detected, recoveries good

Reduced Matrix background

Improved peak shape

-54%

-58%

-47%

Reduced ion suppression


Rapid Analysis of Bisphenols (BP) A, B & E

in Baby Food and Infant Formula

using ACQUITY UPLC-Xevo TQD

Food Packaging Seminar Module 1 of 4


BPA Background

What is BPA? – First synthesised in 1905 by Thomas Zincke of the University of Marburg, Germany

– In 1953, Dr. Hermann Schnell of Bayer in Germany and Dr. Dan Fox of General Electric in the United States independently developed manufacturing processes for a new plastic material, polycarbonate, using BPA as the starting material.

– It has been an important industrial chemical not only to make polycarbonate plastic but also epoxy resins; both of which are used in a wide variety of applications.

o Polycarbonate plastics are used in baby bottles, tableware and other food containers

• (Eyeglass lenses, medical equipment, digital media (e.g., CDs and DVDs), cell phones, consumer electronics, computers and other electrical equipment, …)

o Epoxy resins are used in can coatings, industrial floorings, adhesives, industrial protective coatings, powder coatings, and printed circuit boards


BPA Background

What are the potential health issues?

– Can leach into food from the epoxy resin lining of cans and from consumer products such as polycarbonate tableware, food storage containers, water bottles, and baby bottles.

– BPA is a weak "estrogen like" endocrine disruptor which can mimic circulatory endogenous hormones and is suspected to be linked to a higher incidence of certain cancers, reproductive disorders, diabetes and heart disease in humans


International Regulations

Canada - First country to take action, listed it as a toxic substance

US-FDA - Released a report in 2010 expressing some concern regarding exposure of fetuses, infants and young children to BPA. Report encourages further studies into the safety of BPA

European Union - EU directive (2011/8/EU) has banned the use of BPA in infant feeding bottles

o France Bans Bisphenol A in All Food Contact Materials: French National Assembly has been voting to ban BPA from 2014 on. Concerning baby nutrition products the ban will step into force in 2013

Japan - Between 1998-2003, canning industry voluntarily replaced its BPA-containing epoxy resin can liners with BPA-free polyethylene terephthalate (PET) in many of its products


Analysis of BPA Waters Systems Solution


ACQUITY UPLC-Xevo TQD Conditions

UPLC system ACQUITY UPLC System

Column: ACQUITY UPLC BEH C18 2.1 x 50 mm, 1.7 µm

Column temp 40 °C

Mobile phase A 0.5 % NH4OH in H2O

Mobile phase B 0.5 % NH4OH in MeOH

Elution 3 minute linear gradient from 5% (B) to 95% (B)

Flow Rate 0.5 mL/min

Injection volume

50 µL

MS System XEVO TQD

Ionization mode ESI Negative

Capillary voltage 3.5 kV

Cone voltage 30.0 V

Source temp 140 °C

Desolvation temp 350 °C

Desolvation gas 550 L/hr

Cone gas 50 L/hr

Routine targeted quantification

with information rich qualitative data


• Automatically sets up instrument, • Optimises MRMs and performs system suitability

IntelliStart

• Methods generated automatically by QuanPedia, • Clear flagging when data is out of regulatory tolerance

• Reduced risk of errors

TargetLynx

• Long term system monitoring, Lab management tool

TrendPlot

• Generates optimised methods, time scheduled MRMs

Quanpedia

• Real time QC, Intelligent ‘on the fly’ decision making

QCMonitor

Waters QUAN Workflow


Other Related Compounds…?

BPB ( 2,2 Bis(4-hydroxyphenyl)butane)

BPA ( 2,2-Bis(4-hydroxyphenyl)propane)

BPE (1,1-Bis(4-hydroxyphenyl)ethane)


Developing a Suitable Method

Analysis of food samples can be challenging – need to develop a sample preparation that can:

o Reduce matrix effects (e.g. peak distortion, ion suppression…)

o Extract relevant compounds

o Provide a robust, repeatable method

Method development to investigate different extraction techniques can be time intensive

Useful to have tools that support this process and help improve lab efficiency RADAR Matrix Calculator

LC-MS/MS Analysis

Modify analytical method

LC-MS/MS Analysis

Modify sample

preparation


The advantage of RADAR Full scan and MRM in one analysis

1. Protein precipitation 2. SPE – OASIS HLB

1. Protein precipitation 2. DisQUE (QuEChERS) 3. SPE – OASIS HLB


oReduce matrix effects

oDevelop sample prep procedure

oExtract relevant compounds

oProvide a robust, repeatable method

Use results from … to Modify Sample Prep Procedure


Part 3 – OASIS HLB

Part 1 – Protein precipitation Part 2 - DisQUE

Optimised Sample Prep Protocol

Precipitation (10g sample, 10mL CH3CN) Centrifuge and collect supernatant

Add contents from DisQuE tube 1. Shake.

Centrifuge & collect 10 mL supernatant

Add contents from DisQuE tube 2. Shake.

Centrifuge & collect supernatant.

Dilute supernatant with 70 ml H2O. Load on Oasis HLB (3cc)

Wash – 2 mL 40 % MeOH Elute – 1 mL 100 % MeOH

Dilute eluate with 1 mL H2O Inject 50 µL for LC-MS/MS


Spiked Compounds 1 pg/μL in Samples

S/N > 3


oReduce matrix effects

oDevelop sample prep procedure

oExtract relevant compounds

oProvide a robust, repeatable method

Calculating the Matrix effect with the New Sample Prep Method


Correlation coef f icient: r = 0.998164, r^2 = 0.996332

Response type: Internal Std ( Ref 4 ), Area * ( IS Conc. / IS Area )

Curve type: Linear, Origin: Exclude, Weighting: 1/x^2,

Conc-0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Resp

onse

-0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10Bisphenol Infant Formula

(10 pg/μL spike) BPA 102% (3.2%) BPB 95% (5.5%) BPE 81% (4.6%)

Linearity and Repeatability

Bisphenol Baby food (20 pg/μL spike)

BPA 110% (7.8%) BPB 112% (6.7%) BPE 99% (6.1%)


Robustness

TargetLynx Matrix Calculator


Conclusions

BPA – WW very topical compound

Analysis of BPA in food and beverage samples require a robust method

Waters System Solution is rapid and robust

o Sample preparation- DisQUE and OASIS HLB

o Reliable chromatography - ACQUITY UPLC & ACQUITY BEH C18 columns

o Robust and Repeatable results - Xevo TQD

o Matrix monitoring tools - RADAR, Matrix Calculator

o Waters QUAN Workflow - TargetLynx, TrendPlot

©2011 Waters Corporation

Using UPLC-QTof and UPLC-Ion Mobility-Tof

for Food and Environmental Research


Food & Environmental Research

Studies involve the analysis of high and trace level compounds, and transformation products / break-down / metabolised compounds

Target compounds

Quantify compounds

Discover new compounds


Achieving these aims: Key Analytical LC-MS Challenges

Ionise compounds

Separate compounds / Maximise peak capacity / Minimise matrix effect

Confidently detect compounds at high and low levels

Analyse & interpret data acquired

Obtain repeatable results


Food & Environmental Research High Resolution Chromatography

From HPLC separations…. …to ACQUITY UPLC separations


Food & Environmental Research High Resolution MS – Xevo G2 QTof MS

Ionisation flexibility – ESI, APCI, APPI, APGC, MALDI, DESI, DART…

Ability to detect ionised compounds at high and trace level compounds in complex matrix

– Compatible with high resolution UPLC – In spectrum dynamic range in complex matrices – Comprehensive data acquisition with accurate mass data for precursor

and product ions) – MSE – Confidence in mass accuracy and stability

Analyse and interpret data – Variety of apps managers within MassLynx to support targeted and

research-based workflows

Obtain repeatable results – IntelliStart, QuanTof technology

TRIZAIC™ Source with nanoTile Technology. Plug & Play nanoFlow

APGC – Atmospheric Pressure Gas Chromatography

ASAP – Atmospheric Pressure Solids Analysis Probe

MALDI nanoFlow ESI APCi ESCi APPI TRIZAIC ASAP APGC


Ability to detect extracted & ionised compounds - APGC

51

MBT

DOcT

TPhT

TOcT

DPhT

DBT

MOcT

TPrT TBT

MPhT

Organotin family at

100 ppt level


m/z202.050

%

0

100 202.0434

Detection of trace level compounds in complex matrices – Xevo G2 QTof MS

Scan No. Measured Mass ∆M (mDa) ∆M (ppm)

505 202.0434 -0.50 -2.47506 202.0439 0.00 0.00507 202.0436 -0.30 -1.48508 202.0442 0.30 1.48509 202.0437 -0.20 -0.99510 202.0440 0.10 0.49511 202.0439 0.00 0.00512 202.0435 -0.40 -1.98513 202.0441 0.20 0.99514 202.0435 -0.40 -1.98515 202.0433 -0.60 -2.97516 202.0442 0.30 1.48

RMS = 0.30 1.50

m/z202.050

%

0

100 202.0434

m/z202.050

%

0

100 202.0439

m/z202.050

%

0

100 202.0436

m/z202.050

%

0

100 202.0442

m/z202.050

%

0

100 202.0437

m/z202.050

%

0

100 202.0440

m/z202.050

%

0

100 202.0439

m/z202.050

%

0

100 202.0435

m/z202.050

%

0

100 202.0441

m/z202.050

%

0

100 202.0435

m/z202.050

%

0

100 202.0433

m/z202.050

%

0

100 202.0442

202.0757

Thiabendazole 20 ppb

“Confidence in mass accuracy and stability”

Samples provided by SASA, UK

Excellent mass accuracy across peak


Ability to detect extracted & ionised compounds - QTof MSE

• “Comprehensive data acquisition”

Simetryn m/z 96.0559

Desmetryn m/z 172.0650

Time2.40 2.50 2.60 2.70

%

0

100

Simetryn m/z 214.1126

Desmetryn m/z 214.1126

Selective MSE fragments for each compound

m/z50 100 150 200 250

%0

100 172.0650

82.0401124.0617

214.1124

m/z50 100 150 200 250

%

0

100 214.1128

124.0874

96.0559144.0597

215.1148

Desmetryn

Simetryn or

Desmetryn?


However…

Even the highest resolution instruments struggle to find and confirm the closely eluting isobaric compounds

An additional separation dimension would be a plus – On the chromatography side: 2D-LC… – On the MS side: Ion mobility MS…


SYNAPT G2 HDMS Ion mobility MS Food & Environmental Research

SYNAPT G2 HDMS brings an additional dimension for the characterization of complex mixtures:

• Cleaner MS spectra - data interpretation easier

• Isobaric compounds, isomers, conformers to be separated by ion mobility

Studies have shown that utilising the ion mobility data, compounds not previously separated and recorded with other LC-MS approaches can be distinguished

Ionisation flexibility – ESI, APCI, APPI, APGC, MALDI, DESI, DART…

Ability to detect ionised compounds at high and trace level compounds in complex matrix

– Compatible with high resolution UPLC – In spectrum dynamic range in complex matrices – Comprehensive data acquisition with accurate mass data for precursor

and product ions) – MSE – Confidence in mass accuracy and stability

Analyse and interpret data – Variety of apps managers within MassLynx to support targeted and

research-based workflows

Obtain repeatable results – IntelliStart, QuanTof technology


Schematic of the instrument

SYNAPT G2-S High Definition MS (HDMS)

Size

Shape

Charge


Provided As Part of A Study of Environmental Pollutants In Water *

* Study for the ICRA

Analysis of a Unknown Mixture of Pharmaceutical Standards

UPLC MSE

Ion Mobility-MS Drift Time


Experimental Aims ACQUITY UPLC & SYNAPT G2 HDMS

AIMS Identify any pharmaceutical compounds found in environmental samples Quantify the compounds detected in the influent, waste and natural water

– A dilution series of 0.1 to 200 ppb of the standard pharmaceuticals was prepared.

SYSTEM SOLUTION ACQUITY UPLC to produce high resolution chromatography

SYNAPT G2 HDMS offers: – MSE data (high resolution exact mass precursor and fragment ion m/z) – Ion mobility: provides extra dimension of separation to separate out matrix

interferences and isobaric environmental compounds

Process data using – Automatic screen using data base – Use standards analysed to quantify positively detected components


Extracted Mass Chromatogram @ m/z 332.1410

Conventional extracted mass chromatogram m/z 332.1410 shows one peak at 1.92 minutes

But with ion mobility…. (dt = drift time)


There are Two Peaks!! Can only be seen with Ion Mobility Data

4947_214.raw:1

4947_214.raw : 1

XIC of 332.1407 from the LC run

Ion Mobility Data Two components are detected

@ m/z 332.1407

Different Drift Times for the two isobaric compounds at R.T 1.92

TWO COMPONENTS - SAME EXACT MASS!! m/z 332.1407


3D View Mobility Data Co-eluting Isobaric Masses at 1.92 mins

Ciprofloxacin (M+H=m/z 332.1410)

???


CAERULEA EDULIS

INCARNATA ALATA

In collaboration with: C. A. M. Pereira, J. H. Yariwake Universidade de Sao Paulo, Brazil

Utilising the Increased Peak Capacity of

UPLC and SYNAPT HDMS to Overcome Sample

Complexity -

Passiflora Samples UPLC MSE

Ion Mobility-MS (IM-MS)


Marker Profiling of Passiflora Edulis EXTRACT PE 10:1

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

%

0

100EXTRACTTEST RLMM0230410_028 Sm (Mn, 1x2) 2: TOF MS ES+

BPI5.44e4

8.06

7.59

7.141.570.30

0.721.13

2.68

2.541.94

4.403.23

3.464.97

6.896.235.72 6.66

7.84

8.77

8.17

8.9713.63

9.63

9.60

9.12

9.20

10.709.73

10.39

9.95

11.1111.32 13.47

12.3711.97 12.79

13.73

14.9514.28

14.36 15.1715.40

EXTRACTTEST RLMM0230410_028 1: TOF MS ES+BPI

1.77e52.68

1.220.451.13

0.650.72

1.57

2.552.14

7.607.58

2.717.14

2.92

3.75

3.71

3.45

4.39

3.96 6.744.96 5.07 6.636.22

7.16

7.17

8.077.61

8.04

8.38

8.52

8.77

10.399.73

8.78

9.60

9.01

9.74

10.31

10.70

12.70

11.11

11.9711.18

11.89

12.72

13.63

13.14

14.9414.07 14.90 15.17

15.23

15.40

MS

MSe


Co-eluting compounds in a complex data set?

Use ion mobility to help clean up mass spectra


UPLC-SYNAPT HDMS data Without ion mobility

Orientin at 8.3 min region

HDMS not activated

OOH

OHOH

O

OH

OH

OH O

OH

OH


UPLC-SYNAPT HDMS data Ion mobility Activated

Mobility resolved orientin at 8.3 min region

dt 113 ms HDMS activated


Separation of isomers in complex matrices:

Profiling of Super Fruit: Tangerine Juice UPLC

MSE Ion Mobility-MS with TAP -

(Time Aligned Parallel) fragmentation Drift Time

Collisional Cross Section


UPLC/Qtof MS - Tangerine Juice

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

100Exract Mobility MM060710_105 1: TOF MS ES-

BPI2.51e5

BPI for tangerine juice profiling

Conventional BPI plot of sample comprised of flavonoids,

polyphenols, polysaccharides.


UPLC/Mobility profiling of tangerine juice

Conventional MS data indicates one component

present at 3.25 mins

Extracted ion chromatogram

m/z 609


Are there any isomers, conformers, stereoisomers present?

Use additional dimension of ion mobility with MSE Data Viewer


MSE Data Viewer Software: Hesperidin Isomers - 1

MSE Data Viewer filters out components with same mass

& different drift times

MSE data viewer filters out components with same mass retention time and different

drift times

m/z 609.1842

RT 3.25

Drift Time 161.805


MSE Data Viewer Software: Hesperidin Isomers - 2

MSE Data Viewer filters out components with same mass

& different drift times

MSE data viewer filters out components with same mass retention time and different

drift times

m/z 609.1843

RT 3.25

Drift Time 176.204


tangerine conc

m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0

100

m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0

100

Exract Mobility MM060710_105_dt_01 175 (9.396) AM2 (Ar,20000.0,554.26,0.00,LS 3); Cm (162:185) 1: TOF MS ES-1.01e5301.0714

286.0471

302.0740

609.1801325.0710

Exract Mobility MM060710_105_dt_02 161 (8.640) AM2 (Ar,20000.0,554.26,0.00,LS 3); Cm (148:173) 1: TOF MS ES-1.86e5301.0711

302.0744

609.1826325.0716

CH3 H

H

OO O CH3

OHOH

OHOHOH

OHO

Fragment= C16H13O6Mass=301.0712Error ppm= 0.7

Fragment= C16H13O6Mass=301.0712Error ppm= -0.3

M-H=C28H33O15Mass=609.1819Error ppm= -3.0

M-H=C28H33O15Mass=609.1819Error ppm= 1.1

CH3 H

H

OO O CH3

OHOH

OHOHOH

OHO

MSE fragmentation spectra of mobility separated Hesperidin isomers

Fragment: C16H13O6 Theoretical Mass: 301. 0712

Error (ppm): 0.7

M-H: C28H33O15 Theoretical Mass: 609. 1819

Error (ppm): 1.1

Fragment: C16H13O6 Theoretical Mass: 301. 0712

Error (ppm): -0.3

M-H: C28H33O15 Theoretical Mass: 609. 1819

Error (ppm): -3.0


m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0

100Exract Mobility MM060710_109 348 (3.246) AM2 (Ar,20000.0,554.26,0.00,LS 1); Cm (348:350) 2: TOF MS ES-

7.67e4301.0703

164.0101

151.0023

136.0152

134.0361

108.0200

107.0125

242.0566

201.0172

609.1812302.0725

325.0697

603.2435610.1832

693.2762

”TAP” Fragmentation for Hesperidin m/z 609.1812

TAP (time aligned parallel) fragmentation: 609 has fragmented ( ) in trap to produce m/z 301 fragment and then fragmented again in the transfer region to break up the aglycone


3D view TAP fragmentation Hesperidin isomers

Mobility Separated Hi Energy Trap fragments (+ precursor)

Mobility Separated Hi Energy Transfer fragments of fragments

m/z 609.1812

m/z 301.0703

m/z 164.0101


One step further with Ion Mobility: Determining collision cross sections

MS Structural Confirmation can now be based on:

– Retention time - High resolution UPLC separation

– High resolution MSE data

o Exact mass precursor

o Time aligned exact mass fragments

– Ion mobility separation

– Collision cross section

– TAP fragmentation


Benefits of SYNAPT HDMS

High resolution ACQUITY UPLC data with high quality QuanTof MS technology

MSE provides comprehensive record of all precursor & fragment ions

Ion mobility separation – Cleaner MS spectra - data interpretation easier – Isobaric compounds, isomers, conformers to be separated by ion

mobility

Advanced software tools – MSE data viewer: automatically filters the Tof and IMS data for the

analyst – MassFragment: compares theoretical and observed fragmentations to

confirm compound structure – MarkerLynx: statistical data comparison – ChromaLynx find unknowns peaks in a complex chromatogram – Metabolynx: metabolite ID in a complex sample, based on exact

mass information


Summary

Emerging analytical challenges require new technologies

New technologies developed — Separation (UPLC), Limits of detection (MS), Other new ideas (e.g.

RADAR)

Extended capabilities of new instruments

— Complimentary techniques for more information

Working together can provide the best knowledge of — Market requirements (food and environmental issues) — Technology solutions / requirements

novel and emerging technologies in food analysis - · pdf filenovel and emerging technologies...

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