simplified workflow and improved confidence in screening ... · ms. e ⇒ hdms. e ms. e – high...

©2016 Waters Corporation 1

Simplified Workflow and Improved Confidence in Screening Methods on Time of

Flight Mass Spectrometry

Steve Preece MS Business Manager

Waters Wilmslow


Overview

Use of high resolution MS (Q-Tof) for suspect screening

Key criteria for identification

Case study with Xevo G2-XS QTof

What else can I do with the data?

Using ion mobility MS for screening


Advantages of accurate mass screening? Over recent years high resolution mass spectrometry has gained in popularity as

a screening tool in the food and environmental sector Ability to perform non-targeted analysis

• The freedom to measure compounds without prior compound specific tuning

Ability to perform full spectral analysis • Providing greater insight into the composition of a complex sample

Ability to perform historical (retrospective) data review • The capability of performing structural elucidations of unknowns or suspected compounds

Ability to screen for larger number of compounds and adducts

• Compared to QqQ based screening Increased specificity & selectivity in complex matrices

• Sensitivity in line with Regulations. Accurate mass, diagnostic fragment ions…

“HRMS strongly competes with classical tandem mass spectrometry in the field of quantitative multiresidue methods (e.g., pesticides and veterinary drugs). It is one of the most promising

tools when moving towards nontargeted approaches...” Kaufman A., Anal BioAnal Chem 2011 Dec 17


HRMS UNIFI Screening Key Benefits

Are these compounds in my sample?

Screening

Scientific Library RT, MW, Structure, Fragments & CCS

Workflows Simplify data review

How much is in my sample?

Quantitation

Quan/Qual Workflow MSE Single Analysis

Alternately Tof MRM Ultimate Sensitivity

What else is in my sample?

Elucidation

Discovery Tools Anything Interesting? Elucidation tools

Identify what is interesting

What is the difference between my sample and another one?

Comparison

Binary Compare/Multivariant Analysis Statistical Comparison of Samples


Atrazine: TQ

216.1 > 174.1

216.1 > 96.1

Identification from RT and ion ratios


Advantages of Non-Target ToF Screening over MRM Target Screening

MRM #1

MRM #2

MRM #3

MRM #4

MRM #5

MRM #6

MRM #7


Accurate Mass Screening

Acquiring full MS spectral data – See ‘everything’ unlike specific MRM acquisition

Using 20mDa extraction window for mass chromatograms

– Specificity from accurate mass data

Acquiring MSE data – Molecular weight and fragment data from the same run

Using multiple data features to confirm or identify – Accurate mass, retention time, fragments, adducts, isotopes


Xevo G2-XS QTof : MSE

MSE

– High and low energy spectra – Precursor and fragment ions – Single acquisition – Data independent acquisition

Data automatically aligned – Requires partial LC peak resolution – Links associated precursors and fragments


Atrazine: HRMS


Mass accuracy alone …

0

50

100

150

200

250

300

5ppm 3ppm 1ppm

272

151

48

Number of Identified Pesticides (mass accuracy alone)

…. provides insufficient specificity


Retention time…..

…. helps reduce false detects

1 min window - better than 1ppm + reduced risk of false negatives

0

50

100

150

200

250

300

± 0.25 min ± 0.4 min ± 0.5 min no Rt

9 15 20

272

Number of Identified Pesticides (using Rt and m/z (5ppm))

±0.1 min


Fragment ions

0

1

2

3

4

5

± 0.25 min ± 0.4 min ± 0.5 min no Rt

2 2 2

5

Number of Identified Pesticides (Rt, m/z and at least 1 fragment)

…. provide additional specificity


Pesticide-Based Installation

Specifications


UNIFI: Raw data is processed once Tabulated in oracle database Data is componentised

– Spectral features are already associated – Background ions are excluded

Extracted Spectrum

Component Spectrum


Real Life Example: XEVO G2 XS

(strawberry pomace)

Pesticide Screening Application Solution using Xevo G2-XS QTof

Sample supplied as extracted by the customer

Sample information – approx 20 compounds >10 ppb – approx 10 compounds < 10ppb

What else can you see?

Identified, at least 1 fragment ion Identified, no fragment ion(s)


Non-Targeted Screening with Xevo G2-XS QTof

Higher instrument sensitivity means : – Lower screening detection limits – Better confidence in data – Better isotope match scores – Better ability to detect adducts – Greater ability to detect fragments

More pesticides being detected…

…and they are now being detected with fragments – Fragments are considered to be confirmatory – Other MS information is used as additional evidence


Example 3: Non targeted Screening

January 2014 – Analysis of more samples requested (

Sample information

– approx 20 compounds >10 ppb – approx 10 compounds < 10ppb

What else can you see?

(strawberry pomace)

Are these compounds in my sample?

Screening

How much is in my sample?

Quantitation

What else is in my sample?

Elucidation

What is the difference between my sample and another one?

Comparison


Region of interest Red = Blank/Reference Blue = Unknown Green = Difference

Qualitative Screening: Binary Compare Spinach


Highlight Masses of Interest Send to Batch Elucidation Tool

Reference sample

Control sample

Qualitative Screening: Binary Compare Spinach


Beyond Resolution


Ion Mobility-Mass Spectrometry

Why ion mobility for complex samples?

How does it work? – Orthogonal separations – Calculation of collision cross section

What effect does it have on analyses? – Drug metabolism data – Pesticide screening data


Ion Mobility Spectrometry LC-IMS-MS

IMS provides greater peak capacity than LC-MS alone – Reveals compounds that would have been missed – Provides greater coverage for sample profiling

IMS generates cleaner data – Removes background ions from spectra – Allows easier and faster data interpretation

IMS reduces errors in compound confirmation – Additional collision cross section (CCS) information – Reduces false positives and negatives for screening


The Basic Principal

Ions are driven through a tube by an electric field – Velocity of ions is determined by mass and charge

Motion of the ions is resisted by a drift gas – Resistance is determined by size (mass) and shape of the ion

Ions are separated based on their charge, mass and shape

With chromatography and MS, this provides additional separation power

N2


LC-IM-MS

LC

time

MS

IMS


IMS drift time ⇒ CCS Additional identification point Matrix independent System independent

The ability to see more Resolution of isomers Cleaner spectra

IMS

Orthogonal Separation Collision Cross Section

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What is CCS?

Important differentiating characteristic of an ion

— Chemical structure (mass, size)

— Dimensional information (shape)

Precise physicochemical property of an ion


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Axis Title

Synapt WIV ISP/Vion IMS Q ToF Comparison April 2015

Pesticide

WIV ISP Study: SYNAPT/Vion Comparison for Pesticides

• Consistent CCS values • Different instruments in different countries


MSE ⇒ HDMSE

MSE

– High and low energy spectra – Precursor and fragment ions – Single acquisition – Data independent acquisition

Data automatically aligned – Requires partial LC peak resolution – Links associated precursors and fragments

HDMSE

– IMS separation in front of MSE

– Can separate ions that co-elute in LC – Cleaner spectra due to mobility resolution


Spectral Cleanup Pentanochlor at 1 µg.kg-1

courtesy of Dr. Jens Luetjohann, GALAB, Germany, 2015


Screening

Detection of low level compounds in complex samples – Requirement for efficient separation – Need not to miss peaks

Confident confirmation of presence or absence of targets – No false negatives – No false positives

Identification Points -Exact mass -Retention time -Fragment ions -Isotope peaks

Matrix and system dependent

Can be lost near limit of detection

Increased likelihood of false positives with large library

Use additional identifier - CCS


Mandarin EU PT Sample

29 Observed using a “wide screen”

8 Expected to be detected


Results summary for EU RL proficiency sample FV-13

9 observed after 10ppm accurate mass and fragment filtering

8 Expected to be detected

False +ve

False -ve

8 observed with 2% CCS filter


Vion and UNIFI: Providing routine ion mobility

• Just press START

• Simply Acquire

• Get to the Answers

Seamlessly from sample to answers


UNIFI screening workflow for Vion


1.4 -0.03


UNIFI screening workflow for Vion



Sensitivity: 0.001 mg/kg pentanochlor in tomato extract

HDMSE



Conclusions

Targeted, suspect and unknown screening require sensitivity and selectivity for successful outcomes – Xevo G2-XS QTof

Data processing and review have evolved

– Beyond 2D extracted ion chromatograms – Using all of the data for confirmation

CCS adds another dimension for separation and identification

– Matrix and system independent – Additional confidence in decisions


Acknowledgements US FDA Boethell Lab

GALAB, Germany WIV-ISP, Belgium

www.waters.com

Thank you for your attention

Any questions

simplified workflow and improved confidence in screening ... · ms. e ⇒ hdms. e ms. e – high...

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