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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
6/30/2014
Internal Sales Training - The new 6495 QQQ LC/MS
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Proven iFunnel Technology
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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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
6/30/2014
Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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Sensitive Quantitation of Estrogens in Cow Milk
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Internal Sales Training - The new 6495 QQQ LC/MS
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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
Estriol: Superb Precision and IDL at Low-fg Level
6/30/2014
Internal Sales Training - The new 6495 QQQ LC/MS
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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
Injection volume = 5 µL
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
6/30/2014
Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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Analysis of Hormones
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Internal Sales Training - The new 6495 QQQ LC/MS
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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)
Highest Sensitivity and Excellent Precision LLOQs and IDLs at sub-ng/L Level
6/30/2014
Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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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
6/30/2014
Internal Sales Training - The new 6495 QQQ LC/MS
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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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Internal Sales Training - The new 6495 QQQ LC/MS
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Outstanding Sensitivity with Standard Flow Chromatography
6/30/2014
Internal Sales Training - The new 6495 QQQ LC/MS
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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
Injection volume = 1 µL
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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Internal Sales Training - The new 6495 QQQ LC/MS
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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