LC-MS/MS Technology and Applications Transform your routine science

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LC-MS/MS Technology and Applications

“ Transform your routine science ”

Jonathan Beck, Ph.D Marketing Scientist

Environmental & Food Safety October 20th, 2015

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Outline

•

High Resolution Accurate Mass for Environmental Analysis

•

EQuan MAX Plus online sample preconcentration and

cleanup

•

New Triple Quadrupole developments from Thermo Scientific

•

Project with Duke University for Emerging contaminants

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Pharmaceuticals, Pesticides, Endocrine Disruptors, Personal Care Products

N O F O H C l NH N N N Cl OH O H N H N H I I O I O H O O O H N N O O O O OH OH O O O O O O O OH OH O O O O O H N O

What is Environmental Analysis Challenge?

•

Analysis of thousands of pollutants

•

High diversity of chemical

characteristics

•

Different matrices

•

Screening Workflows with Quan/Qual

capabilities needed

•

New issues: Hydraulic fracturing

•

Regulatory

• EU Water Framework Directive

•

Contamination of ground and drinking water is a risk for

human health

Emerging Contaminants in The Environment – USGS study

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What’s in our water supplies?

Pharmaceuticals, Personal Care Products, Pesticides

*Image from National Geographic, April 2010

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Sample preparation/analyte detection strategy

Clean-up efficiency Detection selectivity

MS/MS HRAM-MS/MS HPLC-UV HPLC-FLD Simple extraction SPE Dispersive SPE LC-MS GC-MS, ECD GPC LLE

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Major Challenges in EFS Analysis

Low

Concentration

High throughput for routine labs.

Identifying low abundance analytes

obscured by high abundance compounds.

Matrix

Complexity

Getting the most accurate information

from precious samples

Small Sample

Volumes

Being able to detect and identify trace level

analytes in simple or complex samples.

Large Sample

Numbers

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Analytical Challenges for Traditional Mass Spectrometry

Detect and identify isobaric compounds

Isolate compounds from matrix components

High Resolution

High mass accuracy (HRAM) MS can do the job

Searching for unknowns

Here targeted data acquisition (SIM, SRM…)

fails

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Resolution (resolving power)

•

Resolutio

n

m

R





m

m (FWHM) m 10%

m

500

•

Quadrupole MS

R



833



m

0.6

m

500

•

Orbitrap MS

R



100000



m

0.005

8

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Detection of analytes in heavy matrix

Pesticide in Matrix

239.00 239.05 239.10 239.15 239.20 239.25 0 20 40 60 80 100 Relative Abunda nce 239.15181 C 11 H19 O2 N 4 6.50 ppm

R = 17,500

Error = 6.50 ppm

239.00 239.05 239.10 239.15 239.20 239.25 0 20 40 60 80 100 Relative Abunda nce H O Pirimicarb m/z m/z 239.15033 C11H19O2 N4 0.32 ppm

R = 70,000

Error = 0.32 ppm

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35 40 45 50 55 60 65 R el at iv e A bundan c e

Resolution influence (17.500 vs. 70.000)

Resol uti on_17_5K #1823 RT : 2.30 AV: 1 NL: 1.60E6 Resol uti on_70K #123 RT : 0.54 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 222.00 222.05 222.10 222.15 222.20 222.25 T : FT M S + p ESI Ful l m s [120.00-1000.00] 222.1263 222.1463 222.1838 AV: 1 NL: 1.80E6 T : FT MS + p ESI Ful l m s [120.00-1000.00] 0 5 10 15 20 25 30 70 75 80 85 90 95 100 222.1238 222.1129 222.1465 222.1854 Name Molecular Formula M+H Carbofuran C12H15NO3 222.1125 Formetanate C11H15N3O2 222.1237 222.0 222.1 222.2 222.3 m /z m /z R el a ti v e A b undan ce 10

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Mass accuracy



Mass accuracy

m

meas



m

true



m

/

z





10

6

m

true

500.1



500.0

•

Quadrupole MS



m

/

z





10

6



200

ppm

500

500.10314



500.10214

•

Orbitrap MS



m

/

z





10

6



2

ppm

TOF MS

500.10314

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Selectivity increases with higher mass accuracy

Beer extract, T-2 toxin 5 µg/L

Re la tiv e Re la tiv e RT: 8.45 NL: 1.20E5 100

2 ppm

7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 Time (min) 30 40 50 60 Abundance 8.55 7.99 7.49 7.67 7.76 7.91 10.61 7.41 7.26 9.46 10.16 10.31 10.50 m/z= 484.25313-90 _{484.25507 F: FTMS} {1,0} + p APCI Full 80 _{ms [300.00-850.00]} 70 MS pivo_5_UH 20 10 0 RT: 8.45 NL: 1.20E5 100

20 ppm

7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 Time (min) 30 40 50 60 Abundance 8.21 7.62 7.85 7.59 7.40 7.37 7.81 7.92 7.99 8.25 8.61 7.30 7.26 8.67 8.88 9.11 9.46 10.09 10.16 10.31 10.5010.61 m/z= 484.24441-90 _{484.26379 F: FTMS} {1,0} + p APCI Full 80 _{ms [300.00-850.00]} 70 MS pivo_5_UH 20 10 0 Re la tiv e RT: 8.45 NL: 1.25E5 100 30 40 50 Abundance 9.29 8.72 8.67 8.99 8.78 9.11 8.21 7.85 10.09 10.16 10.34

100 ppm

8.83 8.13 7.62 7.59 7.81 8.10 8.35 7.38 7.33 7.27 9.46 _{9.50 9.64} 10.50 10.61 m/z= 484.20567-90 _{484.30253 F: FTMS} {1,0} + p APCI Full 80 _{ms [300.00-850.00]} 70 MS pivo_5_UH 60 20 10 0 7.2 7. 7. 7.8 8. 8. 8. 8. 8. 9. 9. 9. 9. 9. 10.0 10.2 10.4 10.6 4 6 0 2 4 6 8Time (min) 0 2 4 6 8

*Zachariasova M et al, Anal. Chim. Acta

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288.0441 C9H21O2P1S3 Terbufos 288.0949 C13H21O3P1S1 Iprobenfos 288.1142 C15H17N4Cl1 Myclobutanil 288.1256 C11H20N4O3S1 Epronaz 288.1351 C11H21N4O3P1 Pirimethaphos 288.1474 C16H20N2O3 Imazamethabenz 289.00 289.05 289.10 289.15 289.20 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abunda nc e 289.1314 289.0513 289.1022 289.1215 289.1329 289.1424 289.00 289.05 289.10 289.15 289.20 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abunda nc e 289.1390 289.1021 289.0514 289.1215 289.1329 289.1424

Detection of Isobaric Compounds in mixtures

Element Exact Mass

H 1.007825 C 12.000000 N 14.003074 O 15.994915 Is a simultaneous measurement possible?

0.1033 amu

289.00 289.05 289.10 289.15 289.20 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abunda nc e 289.1424 289.1547 289.1329 289.1022 289.0514 289.1215 R = 140,000

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The Exactive Plus Orbitrap LCMS system

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Q Exactive: Orbitrap MS/MS system

 Higher ion transmission with rugged optics

 Increased sensitivity, selectivity, true MS/MS

 Directly interfaced to HCD increases spectrum quality

 Multiple fills for spectrum multiplexing increases duty cycle

 Predictive automatic gain control for parallel filling & detection brings more speed

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Caffeine

What do we gain by using the quadrupole ?

195.0876 N=248402.81 NL: 1.94E8 100 [150.00-2000.00] NL: 1.12E8 Full MS S/N = 745 Lowest detected 250330 80 signal/scan 60 40 20 0 195.0877 N=20741.58 100 Relativ e Abundance S/N (spectrum) 4000 3000 2000 1000 0 195.082195.084195.086195.088 195.09 195.092195.094 S/N(FMS) S/N(SIM10)

Sensitivity gain 5 – 10 x with SIM mode

80 60 40 20 0 [190.10-200.10] Lowest detected

SIM (10amu) _signal/scan Gain in sensitivity (7x)

S/N = 5400 28240 6000 5000

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Data dependent MS/MS confirmation

158.9762 Diuron Azoxystrobin Fluridone Benoxacor Fenamidone Desmedipham Imazalil 2 4 6 8 10 12 14 Time (min) 0 50 100 0 50 100 0 50 100 0 50 100 Relative Abundance 0 50 100 0 50 100 0 50 100 6.96 7.35 7.35 7.44 7.55 7.58 7.80 NL: 1.98E7 m/z= 297.0541-297.0571 F: FTMS + p ESI Full ms [50.00-750.00] MS 4xloq_02 NL: 4.67E6 m/z= 301.1163-301.1193 F: FTMS + p ESI Full ms [50.00-750.00] MS 4xloq_02 NL: 8.97E7 m/z= 233.0231-233.0255 F: FTMS + p ESI Full ms [50.00-750.00] MS 4xloq_02 NL: 1.15E7 m/z= 260.0226-260.0252 F: FTMS + p ESI Full ms [50.00-750.00] MS 4xloq_02 NL: 2.21E7 m/z= 330.1084-330.1118 F: FTMS + p ESI Full ms [50.00-750.00] MS 4xloq_02 NL: 1.42E7 m/z= 404.1221-404.1261 F: FTMS + p ESI Full ms [50.00-750.00] MS 4xloq_02 NL: 4.23E7 m/z= 312.1149-312.1181 F: FTMS + p ESI Full ms [50.00-750.00] MS 4xloq_02 0 50 100 0 50 100 0 50 Relative Abundance 100 0 50 100 297.0551 69.0455 200.9867 255.0082 109.0763 316.1774 72.0451 0 233.0241 119.0856159.1165 307.0625 100 50 139.9895 _271.0859 343.0392 112.0395165.0904 149.0834 50 0 100 260.0236 120.0445 188.0704 59.0500 281.7393 344.1026 372.0974 329.0791 316.1078 216.0655 172.0391 _405.2731 81.0705 92.0500 236.1180 103.0546_165.0481 267.0951312.1149 100 200 300 400 m/z

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Q Exactive – “Quanfirmation” what is it?

•

High performance HRAM

Quan

titation and Con

firmation

bench top LCMS system, capable of :

•

Multi-residue quan performance similar to mid-high end Triples

•

Ideal for targeted and general unknown screening

•

Highest confidence confirmation with R = 140K, and MS/MS

•

UHPLC compatible

•

Let‘s use the QExactive to combine these 2 experiments into one.

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Outline

•

What is EQuan MAX Plus?

•

EQuan MAX Plus

•

How can EQuan MAX Plus help your workflow?

•

Customer testimonials

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Low Sample Concentration

Challenge

TSQ Quantiva

Demanding assays which require

the absolute lowest limit of

detection

Powered by AIM technology, the

TSQ Quantiva MS is the world’s

most sensitive triple quadrupole

MS, detecting compounds at the

ppt level.

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Environmental Analysis (Water)

EQuan MAX

Plus

For targeted quantitation (TSQ)

or

Targeted/non-targeted screening and quantitation using High Resolution Accurate Mass (Orbitrap platform)

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EQuan MAX Plus: What is it ?

•

Turnkey method for

assaying environmental

water samples (pesticides,

antibiotics, etc.) at low ppt

levels

•

On-line sample clean-up and

preconcentration

• 2 Columns : Loading and

Analytical

• 2 pumps

•

High injection volumes

• 1mL-20mL

•

Standard injection volumes

• 1-100 uL

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EQuan MAX Plus: Targeted Quantitation

•

Couple EQuan MAX Plus

with any TSQ Quantum

from Thermo Scientific for

the most sensitive and

selective experiments.

•

Fast Positive Negative

Switching

•

TraceFinder Software

•

Built in SRM parameters

•

Built in EQuan Methods

•

Quantum Access MAX

•

Quantum Endura

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EQuan MAX Plus: Non-targeted screening and

Quantitation

•

Couple EQuan MAX Plus with

the Exactive Orbitrap

instruments (Exactive Plus or

Q-Exactive).

•

High Resolution Accurate

Mass (HRAM)

•

All ions are collected in every

experiment.

•

Re-interrogate your data at a

later time

•

Quantitation and screening

methods are easy to set up

since there are no compound

dependant parameters to

optimize.

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SPE - standard enrichment procedure

7 Detection:

LC-MS LC-MSMS

1 Sample preparation: 1L sample filtration internal Std. Filtration 6 Elution: 5 Drying: 4 Washing: 3 Extraction: 2 Conditioning: Enrichment MeOH / H₂O 60mg Oasis H₂O/MeOH (95/5) air MeOH

40 samples

~ 5h ~ 2d ~ 2d

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EQuan MAX Plus Solution – Gain Vs. Conventional Injections

T r i a z i n e P e s t M i x _ 1 0 p p t _ 1 0 0 0 u l _ 0 4 3 / 2 4 / 2 0 0 5 1 : 1 0 : 0 1 A M R T : 5 . 9 8 - 8 . 0 0 N L : 8 . 1 0 E 4 1 0 0 10 ul injection T I C F : + c E S I si d = - 1 0 . 0 0 S R M m s2 2 1 6 . 1 3 @ - 1 8 . 0 0 [ 9 5 _{1 7 4 . 0 0 ] M S} t r i a z i n e p e st m i x _ 1 0 p p t _ 9 0 1 0 u l _ 0 5 8 5 8 0 7 5 7 0 6 5 6 0 R el a ti v e A bundan c e 5 5 5 0 R T : 5 . 9 8 - 8 . 0 2 R T : 6 . 9 1 N L : 8 . 1 0 E 4 A A : 3 1 9 9 9 9 T I C F : + c E S I si d = - 1 0 . 0 0 S N : 1 1 6R M S S R M m s2 2 1 6 . 1 3 @ - 1 8 . 0 0 1 0 0 _{[ 1 7 4 . 0 0 ] M S I C I S} T r i a z i n e P e st M i x _ 1 0 p p t _ 1 9 5 0 0 0 u l _ 0 4 9 0 8 5 8 0 7 5 7 0 1000 ul injection 6 5 6 0 R el a ti v e A bundan c e 5 5 5 0 4 5 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 0 6 . 0 6 . 5 7 . 0 T i m e ( m 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 0 7 . 5 8 . 0 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0 i n ) T i m e ( m i n )

10 ppt Atrazine in ground water

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Sensitivity, Speed & Robustness for EFS high throughput

laboratories

TSQ Endura

Extreme quantitative value • Designed for non-stop operation. • For scientist who need to run routine

samples day in and day out.

TSQ Quantiva

Extreme quantitative performance • Designed for the most challenging

assays.

• For scientists needing to stay at the forefront of analytical technology

TSQ Quantiva TSQ Endura

Mass Range 10-1850 10-3400

Max SRM Number 30,000 SRMs 30,000 SRMs

SRM/Sec 500 SRMs/sec 500 SRMs/sec

Ion Optics Active Ion Management (AIM)

• Ion Max NG source • Electrodynamic ion

funnel

• ion beam guide with neutral blocker • 6 mm HyperQuad

quadrupoles with asymmetric RF drive

S- LENS with Beam Blocker Technology

Quadrupole Design 4mm Quadrupoles with Asymetric RF Reserpine Specification 100,000 :1 S/N for 1 pg Reserpine 10,000 :1 S/N for 1 pg Reserpine

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elos

™

From Identification...

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Challenge

Orbitrap V

MS

Confidently identify unknown

emerging contaminants…………

High-Resolution Accurate Mass for

identification of unknown emerging

contaminants.

?

? ?

?

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To Routine Quantitation

Challenge

EQuan MAX Plus and

TSQ Quantiva

™

MS

Followed by routine quantitation

.

Online sample prep LC/MS for

quantitation of emerging

contaminants identified by the

Orbitrap Velos

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Analysis of Targeted and

Non-targeted Contaminants in

Storm Water Retention Ponds

Emerging Contaminants, from ID to Quantitation. The Total Thermo Scientific Solution

Gordon Getzinger – Duke University Jonathan Beck, PhD –

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UHPLC-HRMS: An Emerging Technique for “Helping

Contaminants Emerge”

• Target screening:

―

Monitor known contaminants using reference standards.

• Suspect screening

―

Screen high-resolution accurate-mass data against

molecular databases of suspected contaminants.

• Non-target screening

―

Assign molecular and structural formula to

chromatographic features without previous knowledge of

contaminant presence or identity.

Krauss et al. LC-high resolution MS in environmental analysis: from target screening to the identification of unknowns. (2010)

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Emerging Contaminant ID to Quantitation Workflow

• Reclaimed waste water used for irrigation on a golf course for emerging contaminants (PPCPs) • Lee Ferguson and Gordon Getzinger, Environmental Chemistry, Duke University, USA

Acquire Fast

Identify

Develop

Routine

HRAM Data

Contaminants

Quantitative

Quantitation

Assay

ExactFinder Software Etofenprox Benzotriazole Propanamide Acephate Allethrin 32

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Kiawah Island, SC, USA

WWTP Land application of WW

for golf course irrigation

Question: Which microcontaminants are

entering the aquatic environment as a result of wastewater reuse practices?

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Typical Sampling Site

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Variety of Sampling Sites

Sample Site Inputs

Pond 5

Golf course runoff Pond 25

Golf course runoff Pond 43

Residential stormwater Wastewater lagoon

Treated municpal wastewater Wastewater Composite

24hr composite effluent Well 1

Infiltration from pond 25 Well 7

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Quantitation of Samples

•

Use a triple quad for quantitation.

•

Analyze samples directly, no offline SPE to save time and

money.

•

Remove variables and sources of experimental error by

automated extraction and preconcentration.

•

An ideal solution for this challenge is…

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What Did We Want to See in the Quantitation

Experiment?

•

Quantitative results at low concentrations… low and sub

PPT levels

•

Calibration range:

•

0.06 pg/mL – 1000 pg/mL

RT :4.00-13.57

•

With a 1mL injection, that’s 60 fg – 1000 pg on column.

9.68 NL: 1.07E6

100 90 80 70 60

TIC of 1.75 pg/mL injected standard ₅₀ 1.75 pg on column 40 30 20 10 0 9.79 10.26 6.87 10.37 11.26 9.46 8.17 7.85 6.63 _11.51 6.01 _12.42 5.84 4.34 4 5 6 7 8 9 10 11 12 13 TIC MS 1,75pgmL Relat iv e Abundu anc e

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Calibration range, 0.06-1000 pg/mL - Flutolanil

60fg On Column Ion Ratio overlay 4.5 orders of magnitude

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Identification of Emerging Contaminants

Data Analysis Workflow

Target screening

Are compounds x, y,

& z present in this

sample?

Non-target screening

Which compounds are present in this sample?

Suspect screening

Which compounds of a defined list are present in this sample?

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LC/MS Workflows for Environmental and Food Safety

Tools to Develop methods for Known and Emerging

contaminants

Raw Sample

• Accelerated Solvent Extraction (ASE)

Dionex ASE

Dionex UHPLC 3000

EQuan MAX Plus

• Equan MAX Plus online sample prep LCMS

Customized Reporting

• Custom software to meet the needs of your workflow

• TraceFinder, SIEVE, mzCloud

TSQ Quantiva TSQ Endura

• TSQ Series Mass Spectrometers: triple stage quadrupole LC-MS/MS and GC MS/MS for high sensitivity quantitation • Exactive Plus and Q Exactive Mass

Spectrometers: High Resolution Accurate Mass to identify, quantify and confirm

Orbitrap HRAM

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