New Triple Quadrupole LC/MS Developments · 2016. 9. 4. · New Enhanced Q1 Ion Optics Improved...
Transcript of New Triple Quadrupole LC/MS Developments · 2016. 9. 4. · New Enhanced Q1 Ion Optics Improved...
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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6495 QQQ Technologies Continued Development
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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
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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
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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
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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
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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
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Counts vs. Acquisition Time (min)
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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
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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
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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
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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
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Instrument Detection Limit (IDL) is based on %RSD
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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
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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
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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
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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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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
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Bentazon: Superb Precision & Sub-fg IDL in Black Tea
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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
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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
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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
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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