New Triple Quadrupole LC/MS Developments

Bring sensitivity and precision to a new dimension

Thomas Glauner Senior LC/MS scientist Market Development Group

7/4/2014

EPRW Lunch Seminar - IDL and 6495

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7/4/2014

EPRW Lunch Seminar - IDL and 6495

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6495 QQQ Technologies Continued Development

1 2

3

• New Detector with High Energy Conversion Dynode (up to 20 kV)

• Improved NEG ion detection with low noise 3

• New Tapered Hexapole Collision Cell

• Effective ion collection and transmission 2

• New Enhanced Q1 Ion Optics

• Improved ion transmission 1

Proven iFunnel Technology • Agilent Jet Stream

• Hexabore Capillary

• Dual Ion Funnel

• Increased ion generation

• Enhanced ion sampling

New Enhanced Q1 Ion Optics Improved transmission of ions and system robustness

7/4/2014

EPRW Lunch Seminar - IDL and 6495

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1. Octopole 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 contamination and easier autotune

– More reliable and robust performance

New Detector with 20 kV High Energy Conversion Dynode More efficient detection of ions with low noise characteristics

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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 m/z range of product ions

• Low noise level at 20 kV

- Improved signal to noise

Gain in Signal with Higher HED Voltage: Anti-EHR2/neu mAB

Peptides

60 – 120% increase

in response at 20kV

EPRW Lunch Seminar - IDL and 6495

7/4/2014

EPRW Lunch Seminar - IDL and 6495

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6495 QQQ Technologies Continued Development

1 2

3

• New Detector with High Energy Conversion Dynode

• Improved NEG ion detection with low noise 3

• New Tapered Hexapole Collision Cell

• Effective ion collection and transmission 2

• New Enhanced Q1 Ion Optics

• Improved ion transmission 1

Proven iFunnel Technology • Agilent Jet Stream

• Hexabore Capillary

• Dual Ion Funnel

• Increased ion generation

• Enhanced ion sampling

Quantitative Applications

• Enhanced peak area response

• Improved peak area %RSD

• More sensitive and precise

• Lower Limits of Detection

(IDL)and Quantitation (LLOQ)

• More reliable and robust

6495 QQQ LC/MS - Premium Performance

7/4/2014

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Improved sensitivity and lower LLOQs – 3x in S/N and IDL specifications

Improved precision and excellent accuracy at the lowest levels – 3x in IDL specifications

Proven 6 orders of linear dynamic range

Proven robustness in complex matrix – Food matrix and biological matrix (plasma)

Improved mass range, fast scan speed and MRM acquisition rate

Improved Sensitivity – Peak Area Response and S/N Reserpine (+) and Chloramphenicol (-), 1 pg on-column

7/4/2014

EPRW Lunch Seminar - IDL and 6495

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6495 QQQ

Avg. Area = 14,519

Avg. S/N = 393,000:1

6490 QQQ

Avg. Area = 5,581

Avg. S/N = 157,000:1

6495 QQQ

Avg. Area = 8,808

Avg. S/N = 315,000:1

6490 QQQ

Avg. Area = 3,318

Avg. S/N = 125,000:1

4 x10

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Counts vs. Acquisition Time (min)

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8

3 x10

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Counts vs. Acquisition Time (min)

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6

1 pg reserpine (+) (n=10) 1 pg chloramphenicol (-) (n=10)

2.6 x higher area response

2.5x higher S/N

2.6 x higher area response

2.5x higher S/N

• The 6495 QQQ LC/MS system shows improved peak area response and S/N, in both positive

and negative ion modes compared to previous designs

Improved Sensitivity and Precision – Peak Area %RSD Reserpine (+) and Chloramphenicol (-) at low levels, 5 fg on-column

7/4/2014

EPRW Lunch Seminar - IDL and 6495

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6495 QQQ

Avg. Area = 157

Area %RSD = 6.1

6490 QQQ

Avg. Area = 47

Area %RSD = 10.4

6495 QQQ

Avg. Area = 121

Area %RSD = 2.7

6490 QQQ

Avg. Area = 40

Area %RSD = 5.6

5 fg reserpine (+) (n=10) 5 fg chloramphenicol (-) (n=10)

3 x higher area response

Lower area %RSD

3 x higher area response

Lower area %RSD

• The improved sensitivity of the 6495 QQQ LC/MS system results in enhanced peak area

response and improved area precision (%RSD), especially at the low levels

• This ultimately leads to lower instrument detection limits (IDLs) compared to previous designs

2 x10

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Counts vs. Acquisition Time (min) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

2 x10

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Counts vs. Acquisition Time (min) 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6

The curve is the theoretical fitting of RSD [%]

based on ion statistics

IDL = based on area precision, minimum amount

of analyte that is detectable and distinguishable

from background noise with a confidence level

IDL based on area precision

RSD [%] observed = 100 / √ N,

where N = number of ions

Different ways of measuring instrument performance Instrument Detection Limit (IDL) versus S/N

7/4/2014

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Signal-to-Noise

S/N is susceptible to manipulation:

Lower Noise:

Different noise definitions and situations

Increase signal:

- Increase detector gain

- Narrow chromatographic peak width

Peak-to-Peak

RMS Smoothing

Baseline setting

Sensitivity of Triple Quadrupole LC/MS Goal of Quantitative Analysis – Reporting Detectable AMOUNT

Limit of Detection - LOD

Lower Limit of Quantitation - LLOQ

S/N > 3 for LOD

S/N > 10 for LLOQ

Statistical criteria are met at LLOQ

Precision %RSD <20 %Accuracy <±20

Perform calibration curve with replicate injections per level

Validated by analysis of standards at or close to Quantitation Limit

Sensitivity Performance Standard

Signal to Noise (S/N)

Instrument Detection Limit (IDL)

Statistically defined

Based on precision %RSD of replicates

Measured at close to detection limit

EPRW Lunch Seminar - IDL and 6495

7/4/2014

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Instrument Detection Limit (IDL) is based on %RSD

7/4/2014

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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 by a particular instrument

IDL

• Student “t” value, for

• 99% confidence level

• Number of replicates (n – 1 degree of freedom) t

• Relative standard deviation (%RSD) / precision of peak area at the amount measured

• From a series of replicate injections (n) %RSD

• Limited to 2 – 5 x times higher than the Detection Limit (DL) Amount

measured

Improved Sensitivity and Precision – 6495 QQQ IDL

7/4/2014

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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

5 fg

5 fg

1.02 fg

1.25 fg

7.2

8.9

Improved precision (%RSD) and Instrument Detection Limit (IDL) achieved on the 6495 vs. the 6490

IDL for the Agilent 6495 QQQ LC/MS System

1 x10

5.4

5.6

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3.6 3.7 3.8 3.9 4 4.1 4.2 4.3

… 1 x10

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

Acquisition Time (min)

IDL level verified by

Injection of 200 attog.

on-column

n=10

Area RSD [%] = 19

Accuracy [%] = 108

IDL level verified by

Injection of 300 attog.

on-column

n=10

Area RSD [%] = 16

Accuracy [%] = 118

Applications for a new highly sensitive triple quad

Food Safety - Pesticides

Analysis of >250 trace level pesticides in food matrices (e.g. black tea) with extensive sample dilutions

Food Safety - Estrogens

Determination of ultra-trace level estrogens in milk product

Environmental - Water Analysis

Quantitation of ultra-trace level hormones (EDCs) in drinking water using direct injection

Peptide Quantitation

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

6495 Data Sheet: 5991-4704EN

6495 App Note: 5991-4687EN

6495 App Note: 5991-4686EN

6495 App Note: 5991-4685EN

EPRW Lunch Seminar - IDL and 6495

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Why do you want a 6495? Utmost sensitivity is required for …

• the analysis of a large number of relevant compounds with a single

injection

• dilution of samples for reduced matrix effects, better accuracy and

improved robustness

• improved LOQs for pesticides in complex

matrices

• highest precision

Experimental setup

• Small validation with tomato, orange, black tea

• QuEChERS extraction

• Dilution of black tea samples with acetonitrile prior injection

• Quantitation with solvent calibration – one calibration for all matrices?

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EPRW Lunch Seminar - IDL and 6495

7/4/2014

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4 x10

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Counts vs. Acquisition Time (min) 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17

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UHPLC/MS/MS Setup: application kit

1290 Infinity UHPLC • column: Zorbax RRHD Eclipse Plus C18

• A: H2O / 0.1% HCOOH / 5mM NH4COOH

• B: MeOH / 0.1% HCOOH / 5mM NH4COOH

6495 LC/MS system • single run, fast polarity switching, dynamic MRM

Spiked and diluted tea extract (0.1 ng/ml)

EPRW Lunch Seminar - IDL and 6495

7/4/2014

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Bentazon: Superb Precision & Sub-fg IDL in Black Tea

7/4/2014

EPRW Lunch Seminar - IDL and 6495

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Amount measured Replicates %RSD t (99%) IDL

1 pg/mL, 2.0 fg (LLOQ) n = 7 injections 11.4 3.143 0.36 pg/mL, 0.72 fg

IDL = t x (%RSD/100) x Amount = 3.143 x (11.4/100) x 2.0 fg = 0.72 fg

Inj # Peak

Area

S/N (Peak to peak)

1 82 20.9

2 96 13.2

3 70 12.7

4 86 13.4

5 92 10.3

6 72 12.7

7 83 29.4

%RSD 11.4 Ave 16.1

Injection volume = 2 µL

• Excellent peak area precision (%RSD) are observed at the lowest level in black tea matrix

1 pg/mL

1 pg/mL

1 pg/mL

1 pg/mL

1 pg/mL

1 pg/mL

1 pg/mL

Signal Improvements for High Relevance Pesticides

7/4/2014

EPRW Lunch Seminar - IDL and 6495

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6495 6490 6495 6490

Methidathion

Area Gain 3.7x

Methomyl

Area Gain 3.5x

Buprofezine

Area Gain 4.2x

Monocrotophos

Area Gain 2.9x

Thiabendazole

Area Gain 3.2x

Spiromesifen

Area Gain 2.1x

6495 6490 6495 6490

6495 6490 6495 6490

• Improved sensitivity (peak area gain of 3x) are observed on the new 6495 vs. the 6490

Improved Precision & Lower LLOQs in Black Tea

7/4/2014

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• Improved precision (%RSD) are observed, particularly at the lowest levels (LLOQs)

• The enhanced peak area response and improved precision (%RSD) means pesticides

achieve lower LLOQs ( MRL level) using the new 6495.

Comparison of Area %RSD for 50 Pesticides at MRL # of pesticides detected at MRL

• 67% of pesticides were easily detected

with a %RSD < 20 with 1:100 dilution

• This means LLOQs < 20 pg/mL (ppt)

• %RSD < 20 meets SANCO guidelines

6495

Dilution allows More Efficient Ionization of Pesticides

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1 : 5 1 : 10 1 : 20 1 : 100 1 : 50

1 : 5 1 : 10 1 : 20 1 : 100 1 : 50

Alanycarb, 10 µg/kg in Black Tea

No

Dilution

Oxamyl, 10 µg/kg in Black Tea

No

Dilution

Recovery (%) in Black Tea with Different Dilutions

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Pesticides No dilution

(n = 5)

Dilution 1:5

(n = 5)

Dilution 1:10

(n = 5)

Dilution 1:20

(n = 5)

Dilution 1:50

(n = 5)

Dilution 1:100

(n = 5)

Acetamiprid 29.4 ± 0.8 57.3 ± 1.4 67.5 ± 3.7 79.9 ± 2.9 91.8 ± 5.2 109.5 ± 3.4

Alanycarb 10.4 ± 1.3 73.9 ± 2.2 81.5 ± 14.3 85.7 ± 11.1 87.6 ± 4.7 121.7 ± 10.8

Aldicarb 36.9 ± 1.0 69.9 ± 1.4 78.0 ± 3.5 91.0 ± 4.2 95.2 ± 8.8 104.9 ± 14.1

Carbaryl 56.9 ± 1.8 80.1 ± 3.8 80.8 ± 4.1 96.1 ± 7.2 102.6 ± 6.6 116.4 ± 9.6

Dimethoate 33.9 ± 1.7 68.6 ± 2.4 84.1 ± 5.4 89.0 ± 7.9 88.2 ± 8.8 84.7 ± 7.5

Diuron 79.7 ± 4.0 90.4 ± 7.0 91.7 ± 4.9 94.9 ± 7.2 89.2 ± 7.3 100.9 ± 13.5

Flufenoxuron 95.4 ± 1.1 88.8 ± 1.6 89.4 ± 3.8 93.3 ± 5.8 100.0 ± 6.1 119.2 ± 13.9

Monocrotophos 4.6 ± 0.3 13.9 ± 0.3 21.8 ± 0.8 33.8 ± 1.1 58.5 ± 2.0 95.1 ± 5.7

Oxamyl 20.8 ± 0.7 52.6 ± 1.9 65.0 ± 2.0 79.7 ± 3.0 91.2 ± 4.6 110.6 ± 5.2

Thiamethoxam 40.0 ± 1.4 45.9 ± 0.9 46.6 ± 3.8 52.2 ± 1.7 70.9 ± 2.9 97.3 ± 2.0

• Cells shaded in green shows %recovery of 80 – 120, which are in compliance with

SANCO requirements.

• Pesticides show full recovery and no signal suppression with 1:100 dilution

• Neat solvent calibration curve can be used for pesticides quantitation with 1:100 dilution

7/4/2014

EPRW Lunch Seminar - IDL and 6495

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Summary: The New 6495 QQQ LC/MS

Increased sensitivity

Lowest Limits of Quantitation

Reproducible, High Quality Data

- More Confident Results

Ease of Operation & Fast Set-up

- Get Answers Quickly!

Screen, Confirm and Quantify

with UHPLC Speed

Streamlined Analytical Workflow

- More Productive Lab

Simpler Methods & Faster

Results

Less Maintenance & Downtime

Excellent precision & accuracy

at the Lowest Levels Wide linear dynamic range

Fast MRM speed, dynamic and

triggered MRM

Ease to use, workflow driven

software, kits and solutions

Proven robustness and

stability in complex matrices

Sample dilution

Direct injection, LC…

Confident quantitation and confirmation for the most demanding applications