Ultrasensitive LC MS/MS: Agilent 6470 and 6495 LC …...Brief History of Agilent 6400 QQQ LC/MS 2...

Ultrasensitive LC MS/MS: Agilent 6470 and 6495 LC-QQQ

ROCK SOLID Performance

for Trace-Level Quantitation

Agilent Technologies

6/27/2016

Customer Facing Presentation - The New 6470 QQQ LC/MS

System

1

Brief History of Agilent 6400 QQQ LC/MS

2

6410

2006 6460 Agilent Jet Stream

2008

6430

2009

2010

6490 iFunnel

2011

6420

2012

6460 ESI

2014

6495 iFunnel

2015

6470 AJS

Agilent 6470 and 6495 Triple Quadrupole LC/MS

• It all starts with the source!

• Improved Robustness

• Improved performance at Intrument Detection Limit (IDL Specification)

• Up to 4000 MRM transitions per method with DynamicMRM

• Simultaneous Quantitation and ID Confirmation with TriggeredMRM

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System

3

Higher Throughput

Higher Confidence

Higher Productivity

+

=







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System

4

Higher Throughput

Higher Confidence

Higher Productivity

+

=

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System

5

6470 - 6495 QQQ Technologies Enhanced Performance in a Smaller Space

Agilent Jet Stream Technology

• Thermal gradient focusing

• Efficient desolvation

• Creates an ion rich zone

• Up to 10x gains in sensitivity

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System

6


Unique to 6495: Proven iFunnel Technology

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Agilent Restricted

7

Agilent Jet Stream Hexabore Capillary Dual Ion Funnel




• Six capillary inlets

• Samples x10 times more ion rich gas • Removes the gas but captures the

ions

• Removes neutral noise







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System

8

Higher Throughput

Higher Confidence

Higher Productivity

+

=

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System

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6470 - 6495 QQQ Technologies Enhanced Performance in a Smaller Space





• Up to 10x gains in sensitivity

• An Ion Detector with High Energy Conversion Dynode and Low Noise

• Improved ion detection 3

• A Curved and Tapered Hexapole Collision Cell

• Effective ion collection 2

• Enhanced Q1 Ion Optics

• Improved ion transmission 1







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System

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Higher Throughput

Higher Confidence

Higher Productivity

+

=

Many Ways to Manipulate S/N

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Agilent 6495 QQQ LC/MS System

11

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

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IDLLCMS = 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 Add IDL Specs for QQQ LC/MS?

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S/N

Many factors impact S/N: - Lower noise - Increase signal

Significant variation in S/N measurements

A relatively high level is used for S/N measurement

Fails to estimate the true limits of detection and quantitation (LLOQ)

Not a good metric of sensitivity performance

IDL (%RSD)

Access sensitivity performance from area %RSD (precision) of replicate injections

Based on a well established statistical formula – follows IUPAC / EPA guidelines

An analytical low level is used for IDL measurement – determined using calibration curve

Accurate assessment of the true limits of detection and quantitation (LLOQ)

A better and more rigorous sensitivity performance metric

Improved Sensitivity and Precision – 6495 QQQ IDL

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1 fg of reserpine used to measure IDL (+) 1 fg of chloramphenicol used to measure IDL (-)

IDL = t x (%RSD / 100) x Amount measured

= 2.821 x (7.2 / 100) x 1 fg

= 0.20 fg

IDL = t x (%RSD / 100) x Amount measured

= 2.821 x (9.7 / 100) x 1 fg

= 0.27 fg

6495 QQQ IDL Amount measured Replicates Area %RSD t (99%) IDL

Reserpine (+) 1 fg n = 10 7.2 2.821 0.20 fg

Chloramphenicol (-) 1 fg n = 10 9.7 2.821 0.27 fg

IDL for the Agilent 6495 QQQ LC/MS System

1 x10

5.4

5.6

5.8

6

6.2

6.4

6.6

6.8

7

7.2

7.4

7.6

7.8

3.6 3.7 3.8 3.9 4 4.1 4.2 4.3

… 1 x10

5.4

5.6

5.8

6

6.2

6.4

6.6

6.8

7

7.2

7.4

7.6

7.8

Acquisition Time (min) 1.45 1.55 1.65 1.75 1.85 1.95 2.05

Acquisition Time (min)

IDLLCMS = t x SD = t x (%RSD / 100) x amount measured

IDL Specifications for 6400 QQQ LC/MS Agilent Differentiator!

QQQ Model 6420A 6460C 6470A 6495A

Positive

Using Reserpine

60 fg

Measured @ 100 fg

12.5 fg

Measured @ 20 fg

4.0 fg

Measured @ 10 fg

0.75 fg

Measured @ 1 fg

Negative

Using Chloramphenicol

60 fg

Measured @ 100 fg

12.5 fg

Measured @ 20 fg

4.0 fg

Measured @ 10 fg

0.75 fg

Measured @ 1 fg







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Higher Throughput

Higher Confidence

Higher Productivity

+

=

Dynamic MRM (dMRM)

6/27/2016

Agilent Restricted

17

Dynamic MRM, Fast MRM Speed and Polarity Switch

• Group MRMs in RT windows instead of

time segments

• 2x shorter cycle time supports narrow

UHPLC peaks

• Supported by very fast MRM speed (1 ms

dwell) and polarity switch (20 ms)

Dynamic MRM (dMRM): Up to 4000 MRM Transitions per Run in Complex Matrix!

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Agilent Restricted

18







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Higher Throughput

Higher Confidence

Higher Productivity

+

=

Triggered MRM (tMRM)

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• tMRM database accelerates

acquisition method setup

• tMRM library – Match Score

for confirmation

• tMRM is Fast and Sensitive

• UHPLC compatible

• Simultaneous Quantitation,

Screening and Confirmation

Triggered MRM, tMRM Database and Library

Match

Score

95.1 Trigger

Threshold

tMRM Product

Ion Spectrum

Apex

Enhanced Optimizer Workflow in MassHunter B.07 (to Better Support tMRM)

• Find 10 MRM transitions in Optimizer

• Injection volume flexibility

• Improved method editing

MassHunter

Optimizer B.07

Triggered MRM (tMRM)

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Agilent Restricted

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Threshold

Secondary MRM

Transitions are “Triggered”

Primary cycle (below threshold)

Triggered cycle (above threshold)

Compound Precursor Product

Analyte 1 334.0 145.0

Analyte 1 334.0 117.0

Compound Precursor Product

Analyte 1 334.0 145.0

Analyte 1 334.0 117.0

Analyte 1 334.0 132.1

Analyte 1 334.0 105.1

Analyte 1 334.0 91.1

Analyte 1 334.0 76.0

Analyte 1 334.0 119.0

QTrap Approach

• Scan the entire finger print

• Not very fast and sensitive

QQQ tMRM Approach

• Focus on ten fingerprint features (10 MRMs)

• Maintains good MDL

• Fast – UHPLC compatible

tMRM Composite Product Ion Spectrum

6/27/2016

6400 QQQ LC/MS Overview

22

x103

8

7

6

5

4

3

2

1

0 80 100 120 140 160 180 200 220 240 260 280 300 320 340

334.2 171.1

145.0

132.1

117.0

105.1 91.1 76.0

tMRM Product Ion

Spectrum

x103 8

7

6

5

4

3

2

1

0 80

76.0

x103 8

7

6

5

4

3

2

1

0 80 100

91.1

x103 8

7

6

5

4

3

2

1

0 80 100 120

105.1

x103 8

7

6

5

4

3

2

1

0 80 100 120

117.0

x103 8

7

6

5

4

3

2

1

0 100 140

132.1

x103 8

7

6

5

4

3

2

1

0 80 100 120

119.0

x103 8

7

6

5

4

3

2

1

0 100 140 180

145.0 x103

8

7

6

5

4

3

2

1

0 100 140 180

147.0

x103 8

7

6

5

4

3

2

1

0 80 100 120 140 160 180

171.1

334 > 76 334 > 91 334 > 105 334 > 117 334 > 119

334 > 132 334 > 145 334 > 147 334 > 171

tMRM Library Searching – Match Score for Confirmation

6/27/2016


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Library match score: 96.75

LC/MS Application

Kits

Method

Standards

On Site Trainin

g

LC Column

tMRM Database & Library

tMRM Application Kits For LC/MS Targeted Screening & Confirmation with QQQ

Pesticides

Test Mix: 254 compounds

DB: 700+ compounds

Library: 200+ compounds

Veterinary Drugs


DB: 500+ compounds


Forensic Toxicology


DB: 2500+ compounds


• Agilent unique data dependent acquisition for fast and sensitive compound

screening, quantitation and confirmation.

6/27/2016


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Applications for 6470 and 6495 QQQ LC/MS Which one is needed?

6470 6495

6/27/2016


System

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Food Safety - Pestcides

Large Panels, High Throughput

Environmental – Water Analysis

PPCPs in surface water using direct injection

Pharmaceutical –

High-throughput analysis of drugs & metabolites in plasma

Environmental - Water Analysis

Ultra-trace level hormones (EDCs) in drinking water using direct injection

Clinical - Peptide Quantitation

Quantitation of peptides at sub-attomole level using nanoflow and standard flow chromatography

Food Safety - Pesticides

Tough matrices, ultra-trace levels

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Summary: 6470 and 6495 QQQ LC/MS Rock Solid Performance for Confident Quantitation and Highest Lab Productivity

Additional Robustness

Improved Sensitivity

Improved Precision

Faster Speed

Smaller Footprint

6470 Upgradeable

to the 6495

Ion Optics

20KV Detector

Curved Collision Cell

Less Maintenance Cleaning

Reliable Consistent Results

over Longer Time (24/7)

Streamlined Analytical

Workflow

Reproducible

High Quality Data

Higher Throughput

at UHPLC Speed

Bench space can be optimized

Protection of

Your Investment


System

6/27/2016


System

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Merci pour votre attention!

QUESTIONS??

Enhanced Q1 Ion Optics with Optimized Prefilter Improved transmission of precursor ions and additional system robustness

6/27/2016


System

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1. Reduced Octopole length 1

1. New prefilter 2

1

2

• New optimized MS 1 prefilter geometry for improved precursor ion transmission

– Improved peak area response and peak area %RSD, more sensitive and precise

• New optical lens elements for reduced the probability of contamination

– More reliable and robust performance

Curved and Tapered Hexapole Collision Cell Effective collection and transmission of product ions and smaller footprint

6/27/2016


System

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• Curved and Tapered Hexapole Assembly for efficient collection and transmission of

product ions

• Designed for consistent collision energies across all QQQ platforms

• A compact, smaller benchtop footprint

6460 QQQ LC/MS

- Linear Collision Cell

6470 QQQ LC/MS

- Curved Collision Cell

30% smaller

New Detector with High Energy Conversion Dynode More efficient detection and quantitation of ions with low noise characteristics

6/27/2016


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• Improved ion detection efficiency with High Energy Dynode (HED) voltage up to 20 kV

- Improved peak area response and peak area %RSD in positive & negative ion mode

- Improved sensitivity and precision for a wide mass range

• Low noise level at 20 kV

- Improved signal to noise

40 – 113% increase in

response to 20kV vs. 10kV

iFunnel Express iFunnel Express – Source and iFunnel Optimization

Method Generation Acquisition Optimization

• Automate the source/iFunnel

optimization process

Create

Methods

& Worklist

MH Source

and Funnel

Optimizer

Acquire Data

for Each

Parameter

MassHunter

Quant

Data

Files Integrate for

Peak Data

Excel File for

Optimized

Parameter

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Ultrasensitive LC MS/MS: Agilent 6470 and 6495 LC …...Brief History of Agilent 6400 QQQ LC/MS 2...

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