the new 6495 triple quadrupole lc/ms - agilent · 2016-09-11 · automation 6/30/2014 internal...

The New 6495 Triple Quadrupole LC/MS

Experience A New Level of

Confidence

Martin Haex

Product Specialist MS and

Automation

6/30/2014

Internal Sales Training - The new 6495 QQQ LC/MS

1

Overview of Topics

The New 6495 QQQ LC/MS

• Technology Innovations

• Quantitation Performance - New performance standard IDL

• Key Applications

• New Software Capabilities

Summary

The New 6560 IM-QTOF LCMS

• Technology innovations

• Applications.

Summary

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Proven iFunnel Technology

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Agilent Jet Stream Hexabore Capillary Dual Ion Funnel

• Thermal gradient focusing

• Efficient desolvation

• Creates an ion rich zone

• Six capillary inlets

• Samples x10 times more

ion rich gas

• Removes the gas but

captures the ions

• Removes neutral noise

6495 QQQ Technologies Continued Development

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1 2

3

• New Detector with High Energy Conversion Dynode

• Improved NEG ion detection with low noise 3

• New Curved and Tapered Hexapole Collision Cell

• Effective ion collection and transmission 2

Proven iFunnel Technology • Agilent Jet Stream

• Hexabore Capillary

• Dual Ion Funnel

• Increased ion generation

• Enhanced ion sampling

• New Enhanced Q1 Ion Optics

• Improved ion transmission 1

6495 QQQ - Premium Performance

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

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

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6495

Avg. Area = 14,519

Area RSD [%] = 1.5

Avg. S/N = 393,000:1

6490

Avg. Area = 5,581

Area %RSD = 1.7

Avg. S/N = 157,000:1

6495

Avg. Area = 8,808

Area RSD [%] = 1.1

Avg. S/N = 315,000:1

6490

Avg. Area = 3,318

Area %RSD = 2.5

Avg. S/N = 125,000:1

4 x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

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

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

3.25

3.5

3.75

4

4.25

4.5

4.75

5

5.25

5.5

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 the previous designs

Many Ways to Manipulate S/N

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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 Change from S/N to IDL for QQQ LC/MS? Goal of Quantitative Analysis – Reporting Detectable AMOUNT

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Sensitivity Performance of QQQ LC/MS

• Lowest detectable amount – Detection Limit (DL)

• Lowest quantifiable amount – Limit of Quantitation (LLOQ)

• Criteria of precision and accuracy are met at LLOQ

Signal to Noise (S/N) – NOT AMOUNT

• Variation associated with S/N (noise) measurement

• Fails to estimate the true detection limit and LLOQ, when measured at a high level of S/N > 50:1

• Not a good metric of sensitivity performance

Instrument Detection Limit (IDL) – AMOUNT

• A rigorous metric based on statistical analysis of precision (%RSD)

• Measured at an analytical low level close to the detection limit

• Accurate assessment of the true detection limit and LLOQ

• A practical means of evaluating sensitivity performance

6495 QQQ Performance Specifications

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Bid Specifications Agilent

6490

Agilent

6495

Sensitivity, S/N

Positive, 1 pg Reserpine 50,000:1 150,000:1

Sensitivity, S/N

Negative, 1 pg chloramphenicol 50,000:1 150,000:1

Sensitivity, IDL

Positive, Reserpine 2.5 fg 0.75 fg

Sensitivity, IDL

Negative, chloramphenicol 2.5 fg 0.75 fg

Linear dynamic range 106 106

Mass range 5 – 1,400 Da 5 – 2,250 Da

Maximum scan speed 12,500 Da/sec 15,000 Da/sec

Maximum MRM rate 250

MRMs/sec

500

MRMs/sec

Minimum MRM dwell time 1 ms 1 ms

Polarity switching 30 ms 30 ms

Installation Specifications 6490 6495

Sensitivity, S/N

Positive, 1 pg Reserpine 25,000:1 75,000:1

Sensitivity, S/N

Negative, 1 pg chloramphenicol 25,000:1 75,000:1

• Improved sensitivity specs (3x)

- S/N - IDL

• Improved mass range

• Improved full scan and MRM speed

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

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6495

Avg. Area = 157

Area %RSD = 6.1

6490

Avg. Area = 47

Area %RSD = 10.4

6495

Avg. Area = 121

Area %RSD = 2.7

6490

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 LC/MS system results in enhanced peak area response

and improved area precision especially at the low levels

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

2 x10

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

1.35

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

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15 1

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

Sensitivity and Precision – Instrument Detection Limit

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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

= 2.821 x (10.9 / 100) x 1 fg = 0.31 fg

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

= 2.821 x (10.0 / 100) x 1 fg = 0.28 fg

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

Reserpine (+) 1 fg n = 10 10.9% 2.821 0.31 fg

Chloramphenicol (-) 1 fg n = 10 10.0% 2.821 0.28 fg

5 fg

5 fg

1.3 fg

1.3 fg

9.2%

9.2%

• Better precision and IDL (> 3x) on the

6495 vs. the 6490

• Better precision and IDL (> 3x) on the

6495 vs. the 6490

1.4

Average of 13x 6495 units

Area %RSD = 10.9

n = 10 injections

Area %RSD = 10.0

n = 10 injections

6495 QQQ Key Applications

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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

Peptide Quantitation

Quantitative analysis of microcystins (cyclic nonribosomal peptides) in liver extract samples using stand flow chromatography

• Application tests are designed based on the requirements of the key high-end QQQ markets

FOOD SAFETY DETERMINATION OF ESTROGENS IN MILK

Sensitivity, Precision, Accuracy, Linear Dynamic Range and Matrix Recovery

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https://www.google.com/url?q=http://www.anh-usa.org/raw-milk-is-good-milk/&sa=U&ei=elMfU9uyDsPekgXuh4HADQ&ved=0CEEQ9QEwCg&sig2=SPEt5_PRKwiNck_2cwiqZQ&usg=AFQjCNErSA0Z2qVskftnULA3Ki3du8Gq7w

Sensitive Quantitation of Estrogens in Cow Milk

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Eight estrogens spiked in cow milk at 0.1 µg/kg, corresponding to 0.1 ng/mL

• Estrogen levels in milk products are regulated by government agencies down to 1 µg/kg

• Highly sensitive and precise quantitation of estrogens was achieved using the new 6495

with a simple QuEChERS procedure without derivatization or enrichment.

• LLOQs for all estrogens (<< 0.1 µg/kg) are far below the regulatory requirement (1 µg/kg)

ID Compounds

1 Estriol (-)

2 17-α-Estradiol (-)

3 17-β-Estradiol (-)

4 Ethynyl estradiol (-)

5 Estrone (-)

6 Diethylstilbestrol (-)

7 Hexestrol (-)

8 Dienestrol (-)

2 3

4

5

6

7,8

1

--- 0.1 ng/mL estrogens in cow milk

--- Milk blank

Injection volume = 5 µL

http://www.google.com.hk/url?q=http://hudabeauty.com/2012/05/16/vitamin-hb-why-you-muuust-start-drinking-almond-milk/&sa=U&ei=FNAnU7HRH4j8iAf4z4DoDA&ved=0CC4Q9QEwAQ&usg=AFQjCNEydYEuppf1a5V5OCVjSS--_XOGCw

Estriol: Superb Precision and IDL at Low-fg Level

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Estriol Amount measured Replicates %RSD t (99%) Estriol IDL

5 pg/mL, 25 fg (LLOQ) n = 7 injections 11.3 3.143 1.8 pg/mL, 8.9 fg

IDL = t x (%RSD/100) x Amount = 3.143 x (10.4/100) x 25 fg = 8.9 fg

Inj # Peak

Area S/N

(Peak to peak)

1 44 11.1

2 54 11.1

3 41 9.8

4 46 9.8

5 50 10.5

6 54 8.6

7 52 9.9

%RSD 11.3 Ave. 10.1


5 pg/mL

5 pg/mL

5 pg/mL

5 pg/mL

5 pg/mL 5 pg/mL

5 pg/mL

• Excellent peak area precision (%RSD) are observed at the lowest levels (LLOQs)

Confident Estrogen Quantitation in Milk, 0.1 – 10 µg/kg

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Levels (µg/kg) %RSD (n = 7) %Accuracy

0.1 5.33 106.0

0.2 2.96 101.2

0.5 2.99 94.6

1 2.75 95.3

2 0.94 100.3

5 1.68 104.1

10 1.64 98.5

Levels (µg/kg) %RSD (n = 7) %Accuracy

0.1 3.86 113.8

0.2 2.61 102.5

0.5 3.68 91.1

1 1.74 92.5

2 1.45 97.9

5 3.64 101.6

10 2.14 100.6

• Excellent linearity (R2 ≥0.998), accuracy and precision are achieved at all levels

17 -Estradiol in milk, 0.1 – 10 µg/kg

0.1 – 10 ng/mL

R2 = 0.999

Estriol in milk, 0.1 – 10 µg/kg

0.1 – 10 ng/mL

R2 = 0.999

Summary of Estrogen Quantitation in Milk Excellent linearity, precision, accuracy and matrix recovery

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Compounds R2 Precision

%RSD, n = 7 %Accuracy

%Recovery

0.2 µg/kg 1 µg/kg 5 µg/kg

Estriol 0.9988 0.94 – 5.33 94.6 – 106.0 92.9 102.8 85.8

17-α-Estradiol 0.9973 1.32 – 5.31 90.6 – 112.0 92.2 99.4 93.4

17-β-Estradiol 0.9985 1.45 – 3.86 91.1 – 113.8 92.9 106.0 96.9

Ethynyl estradiol 0.9984 1.59 – 6.61 90.1 – 114.5 90.2 104.9 94.0

Estrone 0.9987 1.45 – 3.76 92.4 – 110.7 94.0 101.7 96.3

Diethylstilbestrol 0.9985 2.27 – 5.04 94.9 – 104.6 83.5 98.6 91.0

Hexestrol 0.9981 0.88 – 4.58 90.1 – 112.8 86.1 98.4 91.8

Dienestrol 0.9995 1.24 – 4.34 95.6 – 102.7 82.9 92.8 85.3

ANALYSIS OF HORMONES

Sensitivity, Precision, Accuracy, Linear Dynamic Range, and Direct Injection

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Analysis of Hormones

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• Endocrine Disrupting Chemical (EDCs) levels in

municipal water supplies are regulated by government

agencies down to ng/L (ppt) level

• The highly sensitive 6495 allows the quantitation of

EDCs at sub ng/L using direct injection

• No need for time consuming offline SPE or enrichment

Estriol 17-β-Estradiol

Testosterone

17-α-Ethynylestradiol Equilin Estrone

Androstenedione

Estrone (-)

Estriol (-)

Testosterone (+)

Androstenedione (+)

Equilin (-)

17--Ethynyl estradiol (-)

17--estradiol (-)

Hormones in drinking water, 5 ng/L (testosterone) to 17.5 ng/L (EE)

http://en.wikipedia.org/wiki/File:Estradiol.svg

http://en.wikipedia.org/wiki/File:Estriol.svg

http://en.wikipedia.org/wiki/File:Estron.svg

http://en.wikipedia.org/wiki/File:Ethinylestradiol-2D-skeletal.svg

Highest Sensitivity and Excellent Precision LLOQs and IDLs at sub-ng/L Level

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Estriol

2 ng/L

Testosterone

0.1 ng/L

Equilin

0.2 ng/L

17-

Ethynylestradiol

1.75 ng/L

17β-Estradiol

0.5 ng/L

Estrone

0.2 ng/L

Androstenedione

0.2 ng/L

Compounds LLOQ

(ng/L)

%RSD

n = 8

IDL

(ng/L)

%

Accuracy

Estriol 1 5.6 0.168 94.8

17β-Estradiol < 0.5 13.5 0.202 102.2

Testosterone 0.1 10.3 0.031 114.5

17α-Ethynylestradiol 1.75 14.8 0.78 98.4

Equilin < 0.2 3.7 0.022 115.1

Estrone < 0.2 7.2 0.043 115.1

Androstenedione < 0.2 4.3 0.026 107.2

17-Estradiol: Superb Precision and Ultra-Low IDL

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Estriol Amount measured Replicates %RSD t (99%) IDL

0.5 ng/L (LLOQ) n = 8 injections 13.5 2.998 0.202 ng/L

MDL = t x (%RSD/100) x Amount = 2.998 x (13.5/100) x 0.5 ng/L = 0.202 ng/L

Inj # Peak

Area

S/N (Peak to peak)

1 869 9.1

2 925 10.0

3 671 6.7

4 699 6.8

5 950 11.5

6 946 16.0

7 857 8.2

8 762 12.2

%RSD 13.5 10.1

0.5 ng/L

0.5 ng/L

0.5 ng/L

0.5 ng/L

0.5 ng/L 0.5 ng/L

0.5 ng/L

0.5 ng/L

• Excellent peak area precision (%RSD) are observed at the lowest levels (LLOQs)

Confident Quantitation of EDCs in Drinking Water

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• Excellent linearity (R2 ≥0.995), assay accuracy and precision are achieved at all levels

Testosterone (+)

0.1 – 10 ng/L

R2 = 0.996

17-estradiol (-)

0.1 – 10 ng/L

R2 = 0.996

Levels (ng/L) %RSD (n = 8) %Accuracy

0.1 10.3 114.5

0.2 4.89 104.2

0.5 2.71 88.8

1 4.34 92.8

5 4.49 97.4

10 2.09 104.7

Levels (ng/L) %RSD (n = 8) %Accuracy

0.1 13.7 120.3

0.2 13.5 102.2

0.5 3.78 87.5

1 4.14 89.1

5 2.66 98.8

10 0.73 104.2

PEPTIDE QUANTITATION

Sensitivity, Precision, Accuracy, Linear Dynamic Range and

Robustness in Matrix

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Outstanding Sensitivity with Standard Flow Chromatography

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Levels %RSD

(n = 10)

%

Accuracy

RT %RSD

(n = 100)

5 amol 14.0 109.8

0.12

7.5 amol 16.0 108.7

15 amol 9.4 105.0

30 amol 9.0 87.1

300 fmol 1.6 85.2

3 fmol 1.2 81.4

30 fmol 0.6 86.4

300 fmol 0.7 87.4

3 pmol 2.1 105.6

5 pmol 10. 97.5

Zoom-in 5 – 30 amol

LVNEVTEFAK

5 amol – 5 pmol

6 Orders

R2 = 0.998

• Low attomole sensitivity for synthetic peptide spiked in tryptic

digest using the 6495 with standard flow chromatography

• Six orders of linear dynamic range

• Excellent precision and accuracy are observed at all levels

including the LLOQ level

Blank

3 amol

5 amol

Area %RSD = 14.0, n = 10

%Accuracy = 109.8

7.5 amol

LOD

LLOQ


Ultimate Sensitivity with Nanoflow LC

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• Sub-attomole sensitivity for synthetic peptide spiked in

tryptic digest using the 6495 with nanoflow chromatography

• Wide (over 5 orders) linear dynamic range

• Excellent precision and accuracy are observed at all levels

including the LLOQ level

LVNEVTEFAK

0.5 amol – 0.1 pmol

> 5 Orders

R2 = 0.99996

Levels

(amol)

%RSD

n = 5

Accuracy

[%]

RT %RSD

n = 35

0.5 4.52 103.2

1.4

1 8.50 80.2

10 5.75 84.6

100 4.71 88.9

1000 1.23 85.6

10,000 1.09 97.6

100,000 1.69 100.4

Zoom-in 0.5 – 10 amol

Blank

0.25 amol

0.5 amol

Area %RSD = 4.52, n = 5

%Accuracy = 103.2

1.0 amol

MassHunter Acquisition B.07

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Acquisition

Quant

Support for 6495 QQQ LC/MS

New diagnostics tool – Nebulizer, PCA main board, quad driver, detector

Faster and accurate autotune

Improved workflows

Optimizer to better support tMRM

dMRM, tMRM, iFunnel express, Study Manager, Skyline automation, application kits and solutions

New Diagnostics Tool – Lab Advisor Improve Serviceability and Reduce Warranty Costs

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Ion Source Quads / Quad Drivers Main PCA board Detectors

Nebulizer Test Quad transmission and Quad Stability

RFPA

Quad frequency ramp

Ramp gain and Ramp offset

Faults

Power supplies

Ramp EMV power

Ramp HED power

Detector noise

• Currently for MEs, FSEs and AEs

+

Mind map tool to guide

users to diagnostic tests

Lab Advisor tool to run tests

and display results and reports

the new 6495 triple quadrupole lc/ms - agilent · 2016-09-11 · automation 6/30/2014 internal...

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