Sven Poggensee Agilent Technologies Hamburg · 2016-08-30 · JP Morgan Healthcare Conf. 2011...
Transcript of Sven Poggensee Agilent Technologies Hamburg · 2016-08-30 · JP Morgan Healthcare Conf. 2011...
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6400 Triple Quadrupole Produkte
Sven PoggenseeAgilent Technologies
Hamburg
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6400 Triple Quadrupole Product LineExcellent Value and Performance -
6410 Triple Quadrupole LC/MS
� Robust, Easy-to-Use
� Lowest cost of ownership
� Automate compound optimization
6430 Triple Quadrupole LC/MS� Fast, robust,
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� Fast, robust,
� Targeted Protein Quantitation
� Fast polarity switching
6460 Triple Quadrupole LC/MS� Agilent Jet Stream – sub fg sensitivity!
� Perfect for the most demanding applications
� Fast polarity switching
� Largest mass range for a premium Triple Quad
All compatible with HPLC chip MS interface
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Agilent 6460 QQQ and 6530 QTOF
Rough Pump
Octopole 1
Turbo 1 Turbo 1 Turbo 1
Quad Mass Filter (Q1)
Collision Cell
Lens 1 and 2
Quad Mass Filter (Q3)
10KV Detector
Turbo 2 2nd Turbo
6460 QQQ6530 QTOF
Ion Pulser
Turbo 2
Octopole 1
DC Quad
Rough Pump
Turbo 1 Turbo 1 Turbo 1
Quad Mass Filter (Q1)
Collision CellLens 1 and 2
Octopole 2
*Patent Pending
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New 6490 Triple Quadrupole“The highest sensitivity triple quadrupole on the market”
JP Morgan Healthcare Conf. 2011
Zeptmole Sensitivitymicrodosing
dried blood spotshormone analysispersonal care products in waterpesticides –dilute and shoot
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Dynamic Range of Six Orders Linearityeliminates serial dilution of samplesanalyze trace level impurities in one run
Triggered MRM (tMRM)library searching – NIST data base fit scores
create your own data base for searches
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New 6490 Advances Sensitivity 50 Fold in 4 Years
5000
6000
7000
8000
9000
10000
6490S
NR
1 p
g r
eserp
ine
5
0
1000
2000
3000
4000
2006 2007 2008 2009 2010
64106460S
NR
1 p
g r
eserp
ine
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iFunnel Technology Captures 6x More Ions
Agilent Jet Stream
• Thermal confinement of ESI plume
• Efficient desolvation to create gas phase ions
• Creates an ion rich zone
Hexabore Capillary
• Samples 6 times more ion rich gas from the source with 6 capillaries
• Captures the majority of the gas from the source region
Dual Ion Funnel
• Removes the gas but captures the ions
• Removes neutral noise
• Extends turbo pump life
June 6
region
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Agilent Jet StreamThermal Gradient Focusing Technology
Dramatic Sensitivity Gains for
Premium TOF, Q-TOF, and Triple Quad
Ions Focused in a Collimated
Thermal Confinement Zone
Improved Ionization Efficiency and
Sampling Sampling
Effective Across a Broad Range of
Analyte Classes, including many
APCI compounds
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High
Pressure
Stage 1
Low
Pressure
Stage 2
Line of
Sight
Two Stage Ion Funnel Manages the Gas Load
Stage 1
8-12 Torr
Stage 2
1-3 Torr
Offset ion funnels to prevent neutrals from going straight through to MS
June 8
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Ion Funnel Operation
RF DriveRF Voltage focuses the ions to the center.
UnfocusedFocused
DC Voltage accelerates the ions to the exit. DC DriveDC Drive
UnfocusedIons and GasEnter
FocusedIons Out
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Ion Funnel Construction
Previously, many metal plates made cleaning
a priority because of the large, active surface area…
The high capacitance also required larger power
supplies to provide RF power
Gold Plated
Rim
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The 6490 design uses printed circuit board technology giving a greatly reduces surface area. This low
capacitance enables the use of small power supplies, and enables fast polarity switching
The 6490 design uses printed circuit board technology giving a greatly reduces surface area. This low
capacitance enables the use of small power supplies, and enables fast polarity switching
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Simple and Easy Ion Funnel Cleaning Procedure
• As with all LC/MS systems routine cleaning is necessary periodically.
• The high pressure ion funnel should be cleaned periodically, although this could vary from 3 months to 1 year depending on the quantity and type of sampleson the quantity and type of samples
• The high pressure ion funnel is easily removed
• Clean by sonicating the ion funnel assembly in 100% isopropanol for 15 minutes.
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1000
Abundance
20 femtogram verapamil on-column injection
ConstantResponse
6490 Performance: For Over 2,000 SamplesProtein Precipitated Plasma over Four Days with 5% RSDs
June 12
Number of Injections
0
500 1,000 1,500 2,000
500
Abundance
• Typically 1 – 5 pg injected for such studies• Lower amounts on-column to monitor response
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• Die Software
Agilent 6490 QQQ
• Beispieldaten:
• Anabole Steroide in Urin und Rindfleisch
• Pestizide
• Photoinitiatoren in Verpackungsmaterial
Sven Poggensee
Agilent Technologies
Hamburg
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Steroid hormones
• Steroidhormone werden als wachstumsfördernde Medikamente verwendet, um das Tempo des Gewichtsgewinnes zu steigern und Fütterungseffizienz in Schlachtvieh zu verbessern.
• Estradiol, Progesteron und Testosteron sind natürlich vorkommende Steroidhormone und es wird angenommen, daß Verbraucher wenig Risiko haben, falls Nahrung von behandelten Tieren gegessen wird. (FDA)
• Synthetische Hormone wie trenbolone-Acetat, zeranol, und melengestrol-Acetat werden nicht natürlich produziert. Der Metabolismus erfolgt hier wesentlich langsamer als bei natürlichen Hormonen.
• Es gibt ein Verbot für wachstumsfördernde Hormone in der EU.
• Mitgliedsstaaten der EU sind verpflichtet Steroide und Synthetische Hormone in Lebensmitteln zu analysieren
Sven Poggensee
6490 QQQ Hamburg
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Anabolika in Urin
Target compounds:
• Chlormadinone acetate
• 16-b-Stanozolol
• a/b-Zearalenol
• Ethinyl estradiol
Injection volume: 10 µL
Column: Eclipse PlusC18, 1.8 um, 3.0 x 50 mm
Pump Flow: 0.5 ml/min
Mobile Phase: A = 0.5mM Ammonium Acetate in Water : Methanol (70:30)B = 0.5mM Ammonium Acetate in Water : Methanol (5:95)
Gradient: Time %B
0 251 253 505 756 75
6.01 25 6.01 25
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Steroids – Chromatography 200 ng/ml All compounds
5x10
3.5
3.75
4
4.25
4.5
4.75
5
5.25
5.5
5.75
6
6.25
6.5
6.75
7
7.25
-ESI MRM Frag=380.0V CID@** (295.2000 -> 145.0000) Cal_11-r001.d
1 1
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
3.25
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 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
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5x10
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
4.4
4.6
4.8
-ESI MRM Frag=380.0V CID@** (319.1000 -> 275.1000) Cal_11-r001.d
1 1
Steroids – Alpha and Beta Zearalenol, example of chromatography200ng/ml
Alpha
Beta
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
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 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
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1x10
5.25
5.5
5.75
6
6.25
6.5
6.75
7
7.25
7.5
7.75
-ESI MRM Frag=380.0V CID@** (319.1000 -> 160.0000) Cal_1-1ulr004.d Smooth Noise (PeakToPeak) = 2.66; SNR (3.678min) = 7.6
* 3.67820
1 1
-ESI MRM Frag=380.0V CID@** (319.1000 -> 275.1000) Cal_1-1ulr004.d Smooth
Steroids – αααα and ββββ-Zearalenol 0.05ng/ml (50 fg on column with 1uL inj)
αααα-ZearalenolQual ionS/N = 7.6Peak to peak Based on height
ββββ-ZearalenolQual ionS/N = 8.9Peak to peak Based on height
0.0025 ng/ml urine equivalent
2x10
0.7
0.8
0.9
1
1.1
1.2
-ESI MRM Frag=380.0V CID@** (319.1000 -> 275.1000) Cal_1-1ulr004.d Smooth Noise (PeakToPeak) = 6.34; SNR (3.688min) = 5.7
* 3.68836
1 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 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
αααα-ZearalenolQuant ionS/N = 5.7Peak to peak Based on height
ββββ-ZearalenolQuant ionS/N = 4.4Peak to peak Based on height
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Steroids Low Matrix Spike – Chromatogram
3x10
1
2
3
4
5
6
7
8
-ESI MRM Frag=380.0V CID@** (319.1000 -> 160.0000) UrineSpike_1-r004.d Noise (PeakToPeak) = 28.60; SNR (3.699min) = 253.2
* 3.6997241
* 3.1423768
1 1
-ESI MRM Frag=380.0V CID@** (319.1000 -> 275.1000) UrineSpike_1-r004.d
ββββ-Zearalenol0.04 ng/ml in urine
αααα-Zearalenol0.1 ng/ml in urine
Preconcentration: 20x
4x10
0.2
0.4
0.6
0.8
1
1.2
-ESI MRM Frag=380.0V CID@** (319.1000 -> 275.1000) UrineSpike_1-r004.d Noise (PeakToPeak) = 116.62; SNR (3.699min) = 91.1
* 3.69910619
* 3.1425702
1 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 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
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Steroids – Beta Zearalenol – Quant software Batch at a glance
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1x10
5
5.2
5.4
5.6
5.8
6
6.2
6.4
-ESI MRM Frag=380.0V CID@** (295.2000 -> 143.0000) Cal_2-r001.d Smooth Noise (PeakToPeak) = 2.96; SNR (3.687min) = 2.3
3.6877
1 1
-ESI MRM Frag=380.0V CID@** (295.2000 -> 145.0000) Cal_2-r001.d Smooth
Steroids – Ethynylestradiol 0.1ng/ml (1 pg on column, 10 uL inj)
0.1 ng/ml standardQual ionS/N = 2.3Peak to peak Based on height
0.05 ng/ml urine equivalent
1x10
5
5.2
5.4
5.6
5.8
6
6.2
6.4
6.6
-ESI MRM Frag=380.0V CID@** (295.2000 -> 145.0000) Cal_2-r001.d Smooth Noise (PeakToPeak) = 2.40; SNR (3.678min) = 5.1
3.67812
1 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 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
0.1 ng/nl standardQuant ionS/N = 5.1Peak to peak Based on height
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2x10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
-ESI MRM Frag=380.0V CID@** (295.2000 -> 143.0000) UrineSpike_1-r004.d Noise (PeakToPeak) = 14.00; SNR (3.691min) = 15.9
* 3.691223
1 1
-ESI MRM Frag=380.0V CID@** (295.2000 -> 145.0000) UrineSpike_1-r004.d
Steroids Low Matrix Spike – Chromatogram
Ethinyl estradiol0.2 ng/ml in urine
Preconcentration: 20x
2x10
1
2
3
4
5
6
7
8
-ESI MRM Frag=380.0V CID@** (295.2000 -> 145.0000) UrineSpike_1-r004.d Noise (PeakToPeak) = 21.34; SNR (3.691min) = 22.2
* 3.691473
1 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 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
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Steroids High Matrix Spike – Chromatogram
3x10
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
-ESI MRM Frag=380.0V CID@** (295.2000 -> 143.0000) UrineSpike_2-r001.d Noise (PeakToPeak) = 10.94; SNR (3.691min) = 136.4
* 3.6911493
1 1
-ESI MRM Frag=380.0V CID@** (295.2000 -> 145.0000) UrineSpike_2-r001.d
Ethinyl estradiol1.2 ng/ml in urine
Preconcentration: 20x
3x10
0.5
1
1.5
2
2.5
3
3.5
-ESI MRM Frag=380.0V CID@** (295.2000 -> 145.0000) UrineSpike_2-r001.d Noise (PeakToPeak) = 11.68; SNR (3.691min) = 260.7
* 3.6913045
1 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 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
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Steroids – Ethynylestradiol – Quant software Batch at a glance
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Steroids – Comparison Agilent QQQ 6460 vs. Agilent QQQ 6490200 pg on column
5x10
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3
3.1
3.2
3.3
+/-ESI TIC MRM CID@** (** -> **) 200ng-r001.d
1 1Agilent QQQ 6490Agilent QQQ 6460
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
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 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
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3x10
1
2
-ESI MRM Frag=380.0V CID@** (295.2000 -> 143.0000) VS_Cal_8-r001.d Noise (PeakToPeak) = 9.06; SNR (3.682min) = 206.0
3.6821866
1 1
3x10
2.5
5
-ESI MRM Frag=380.0V CID@** (295.2000 -> 145.0000) VS_Cal_8-r001.d Noise (PeakToPeak) = 6.60; SNR (3.685min) = 603.2
3.6853981
1 1
-ESI MRM Frag=160.0V [email protected] (295.0000 -> 143.1000) 200ng-r001.d
Steroids – Comparison Agilent QQQ 6460 vs. Agilent QQQ 6490200 pg Ethinyl estradiol on column
Agilent QQQ 6490
2x10
0.5
1
-ESI MRM Frag=160.0V [email protected] (295.0000 -> 143.1000) 200ng-r001.d Noise (PeakToPeak) = 2.34; SNR (3.472min) = 23.5
3.47255
1 1
2x10
2
-ESI MRM Frag=160.0V [email protected] (295.0000 -> 144.9000) 200ng-r001.d Noise (PeakToPeak) = 2.54; SNR (3.473min) = 74.4
3.473189
1 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 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
Agilent QQQ 6460
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5x10
1
2
-ESI MRM Frag=380.0V CID@** (319.1000 -> 160.0000) VS_Cal_8-r001.d Noise (PeakToPeak) = 17.74; SNR (3.698min) = 7352.4
3.698130431
3.140126832
5x10
1
2
-ESI MRM Frag=380.0V CID@** (319.1000 -> 275.1000) VS_Cal_8-r001.d Noise (PeakToPeak) = 23.90; SNR (3.700min) = 7183.4
3.700171684
3.141165487
-ESI MRM Frag=162.0V CID@** (319.2000 -> 275.2000) 200ng-r001.d
Steroids – Comparison Agilent QQQ 6460 vs. Agilent QQQ 6490200 pg b/a-Zearalenol on column
AgilentQQQ 6490
4x10
1
2
-ESI MRM Frag=162.0V CID@** (319.2000 -> 275.2000) 200ng-r001.d Noise (PeakToPeak) = 10.42; SNR (3.486min) = 1242.2
3.486129442.911
9412
4x10
0.5
1
-ESI MRM Frag=152.0V [email protected] (319.2000 -> 160.0000) 200ng-r001.d Noise (PeakToPeak) = 3.70; SNR (3.486min) = 1721.7
3.48663702.910
4755
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 3.8 4 4.2 4.4 4.6 4.8 5
AgilentQQQ 6460
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Anabolika in Urin
Target compounds:
• 17-alpha-nortestosterone (aNT)
• 17-beta-nortestosterone (bNT)
• Beta-boldenone (bBOL)
• Norgestrel (NG)
• Trenbolone acetate (TBA)
• Acetoxyprogesterone (AP)
Injection volume: 5 µL
Column: Waters Acquity BEH, 1.8 µm, 100 x 2.1 mm
Pump Flow: 0.4 ml/min
Mobile Phase: A = H2O 0.1% Formic AcidB = Acetonitrile 0.1% Formic Acid
Gradient: Time %B
0.0 101.0 306.5 738.0 100
10.0 10
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3.2. Anabolic Steroids - ResultsThe Signal to Noise for the standard injection was determined and the amount injected which would give a signal to noise of three was calculated.
Noise was calculated peak-to-peak and the noise region is indicated in bold.
aNT: 2.5pg injected - S:N=150.548.8 fg injected for a S:N=3
bNT: 2.5pg injected - S:N= 61.2122.5 fg injected for a S:N=3
bBOL: 2.5pg injected - S:N= 269.727.8 fg injected for a S:N=3
TBA: 5pg injected - S:N= 813.918.4 fg injected for a S:N=3
NG: 12.5pg injected - S:N= 378.299.2 fg injected for a S:N=3
AP: 12.5pg injected - S:N= 430.787.0 fg injected for a S:N=3
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Herausforderung:
> 860 active compounds (Brit. Crop Protection Council)
• > 100 different compound classes
• different physico-chemical properties
• different hetero atoms (halogens, sulfur, nitrogen ...)
• neutral, acidic, and basic pesticides
Pestizide in Nahrung und Umweltproben
• volatile and non-volatile compounds
• complex sample matrices
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• Need for screening methods; confirmation and quantitation of positives
only (e.g. EU water framework directive)
• Need for “ready to use” methods of analysis including sample
preparation (e.g. EPA Method 1694: “Pharmaceuticals and Personal
Care Products in Water, Soil, Sediment, and Biosolids by HPLC/MS/MS”)
• Increased number of samples in labs:
• Need for faster analysis methods with less sample prep
Environmental analysis trends
• Need for multi-component analyses
• Need for a data management solution
• Ideal:
• easy sample prep
• safe identification
• screening and quantitation in
one analytical run
6490 QQQ Introduction, Berlin
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Get started quickly
Column & test mix
LCMS Application Kits
Analysis methods show how to use advanced technologies for fast multi-component screening
Column & test mixApplication Note and Quick start guideCD with method parametersLCMS databases for fast screening:
•Dynamic MRM•Accurate Mass
* Contents may vary based upon the application. Ask your Account Manager about the details for your application of interest.
6490 QQQ Introduction, Berlin
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Pesticide MRM Data Base G1733AA
6490 QQQ Introduction, Berlin
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DMRM Pesticides and Forensic/Tox Database
Analyze medium level standard mixtures
fixed dwell time for each MRM transition using one time segment
MHW Quantitative Data Analysis gets retention times
Load tabular MRM from Database
Compound names, ISTD (optional)
MRM transitions, fragmentorvoltages, collision energies
• Pesticide database contains MRM transitions for more than 600 pesticides
• Forensic/Tox database contains MRM transitions for more than 130 forensic drugs
Create a Dynamic MRM method
Import the results generated in the custom report into acquisition
method
Specify delta retention time window
6490 QQQ Introduction, Berlin
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Comparison of MRM and Dynamic MRM
Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Compounds (10/block)
Cycle Time (sec)
MRM
50 80
0.5 0.8 1
Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4
0.7
100 70
Quantitative analysis of pesticides (dMRM)
Cycle Time (sec)
Max Coincident
Cycle Time (sec) 0.4 0.4 0.4 0.4
20 40 40
Dynamic MRM
0.5 0.8 1
30
0.7
• 2 x shorter cycle times supports narrow chromatographic peaks, more analytes or longer dwell per analyte.
6490 QQQ Introduction, Berlin
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Dynamic MRM Simulation
Abundance
>>
>
Time >>>
ConcurrentCompounds
6490 QQQ Introduction, Berlin
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� Chromatographic conditionsAgilent 1290 Infinity LC system consisting of:
- binary pump
- wellplate sampler
- column compartment
- diode array detector (not used)
HPLC method
Separation column: ZORBAX Eclipse Plus C-18 RRHD column,150 x 2.1 mm, 1.8 µm @ 30°C
4.) Quantitative analysis of pesticides
150 x 2.1 mm, 1.8 µm @ 30°C
Mobile phase: A: 5 mM ammonium formate
B: methanol + 5 mM ammonium formate
Flow: 0.4 ml/min
Gradient: 0.00 min 10 % B0.50 min 10 % B3.50 min 50 % B
12.00 min 100 % B13.00 min 100 % B13.10 min 10 % B15.00 min 10 % B
Inj.Vol.: 1 or 10 µl
6490 QQQ Introduction, Berlin
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� Agilent Jet Stream conditions
Spray chamber conditions:Gas temp.: 200°C
Dry gas: 8 l/min
Nebulizer: 30 psi
Sheath gas temp: 300°C
Sheath gas flow: 11 l/min
Positive NegativeCapVoltage: 4500 V 3000 V
4.) Quantitative analysis of pesticides
CapVoltage: 4500 V 3000 V
Nozzle voltage 300 V 0 V
• Automatic setup of MRM tables based on selected cycle time, retention times and retention time windows for the individual compounds
• Cycle time 500 ms• Interscan delay 3.5 ms• Total No. of MRMs 484• Maximum No. Of concurrent MRMs 68• Minimum Dwell time 3.85 ms• Maximum Dwell time 246.5 ms
6490 QQQ Introduction, Berlin
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� Compounds included in method (242 compounds in total)
4.) Quantitative analysis of pesticides
Acephate
Acetamiprid
Acibenzolar-S-methyl
Acrinathrin
Aldicarb
Aldicarb sulfone
Aldicarb sulfoxide
Amitraz
Atrazine
Avermectin B1a
Azinphos-methyl
Azoxystrobin
Benalaxyl
Bendiocarb
Benfuracarb
Benthiavalicarb
Cinerin 1
Cinerin 2
Clofentezine
Clothianidin
Crufomate
Cyanofenphos
Cyazofamid
Cyflufenamid
Cyhalothrin
Cymoxanil
Cyproconazole
Cyprodinil
Cyromazine
Demeton-S-methyl
Demeton-S-methyl sulfone
Oxydemethon methyl
EPN
Epoxiconazole
Ethiofencarb
Ethiofencarb sulfone
Ethiofencarb sulfoxide
Ethion
Ethofumesate
Ethoprophos
Etofenprox
Etrimfos
Fenamidone
Fenamiphos
Fenamiphos-Sulfone
Fenamiphos-Sulfoxide
Fenarimol
Fenazaquin
Flutriafol
Fluvalinate
Fonofos
Formothion
Fosthiazate
Furalaxyl
Furathiocarb
Haloxyfop acid
Heptenophos
Hexaconazole
Hexazinone
Hexythiazox
Imazalil
Imidacloprid
Indoxacarb
Iprovalicarb
Methidathion
Methiocarb
Methiocarb sulfone
Methiocarb Sulfoxide
Methomyl
Methoxifenozide
Metolachlor
Metolcarb
Metrafenone
Metribuzin
Mevinphos
Monocrotophos
Myclobutanil
Neo-Quassin
Nitenpyram
Nuarimol
Piperonyl butoxide
Pirimicarb
Pirimiphos-ethyl
Pirimiphos-methyl
Prochloraz
Profenofos
Prometryn
Propachlor
Propamocarb
Propanil
Propargite
Propazine
Propetamphos
Propham
Propiconazole
Propoxur
Rotenone
Simazine
Spinosad A
Spinosad D
Spirodiclofen
Spiromefesin
Spiroxamine
Sulfotep
Tebuconazole
Tebufenozide
Tebufenpyrad
Teflubenzuron
Terbufos
Terbufos sulfone
Terbufos sulfoxide
TetrachlorvinphosBenthiavalicarb
Bifenthrin
Bitertanol
Boscalid
Bromuconazole
Bupirimate
Buprofezin
Butralin
Cadusafos
Carbaryl
Carbendazim
Carbofuran
Carbofuran-3-hydroxy
Carbophenothion
Carbosulfan
Chlorfenvinphos
Chlorotoluron
Chlorpyrifos
Chlorthiophos
Oxydemethon methyl
Dialifos
Diazinon
Diclobutrazol
Dicrotophos
Diethofencarb
Difenoconazol
Diflubenzuron
Dimethoate
Dimethomorph
Dimethylvinphos(E)
Dimoxystrobin
Disulfoton-Sulfone
Disulfoton-Sulfoxide
Ditalimfos
Diuron
Dodine
Edifenphos
Emamectin B1a
Fenazaquin
Fenbuconazol
Fenhexamid
Fenoxycarb
Fenpropimorph
Fenpyroximate
Fenthion
Fenthion-sulfon
Fenthion-Sulfoxide
Fipronil
Fluazifop acid
Fluazifop-P-butyl
Fludioxonil
Flufenacet
Flufenoxuron
Fluopicolide
Fluoxastrobin
Fluquinconazole
Flusilazole
Iprovalicarb
Isazofos
Isoprocarb
Isoproturon
Jasmolin 1
Jasmolin 2
Kresoxim-methyl
Lenacil
Linuron
Lufenuron
Malaoxon
Malathion
Mecarbam
Mepanipyrim
Mepronil
Metalaxyl
Metazachlor
Metconazole
Methamidophos
Nuarimol
Ofurace
Omethoate
Oxadixyl
Oxamyl
Paclobutrazol
Paraoxon-ethyl
Paraoxon-methyl
Penconazol
Pencycuron
Pendimethalin
Phenthoate
Phorate Sulfone
Phorate Sulfoxide
Phosalone
Phosmet
Phosphamidon
Phoxim
Picoxystrobin,
Propoxur
Propyzamide
Proquinazid
Prosulfocarb
Pymetrozine
Pyraclostrobin
Pyrazophos
Pyrethrin 1
Pyrethrin 2
Pyridaben
Pyridaphenthion
Pyrifenox
Pyrimethanil
Pyriproxifen
Quassin
Quinalphos
Quinoxyfen
Resmethrin
Tetrachlorvinphos
Tetraconazole
Tetramethrin
Thiabendazole
Thiacloprid
Thiamethoxam
Thiobencarb
Thiodicarb
Tolylfluanid
Triadimefon
Triadimenol
Triazamate acid
Triazophos
Trichlorfon
Trietazine
Trifloxystrobin
Triticonazole
Zoxamide
6490 QQQ Introduction, Berlin
Page 39
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Dynamic MRM viewer – display of concurrent MRMs
Quantitative analysis of pesticides (dMRM)
6490 QQQ Introduction, Berlin
Page 40
-
Quantitative analysis of pesticides
� MRM traces of pesticides in sample EF_Cal 3 (10 ng/ml)
• Final method includes 242 pesticides with 2 MRM transitions each• Variable dwell times, Inter-scan delay 3.5 ms
6490 QQQ Introduction, Berlin
Page 41
-
Quantitative analysis of pesticides
� MRM traces of pesticides in sample EF_Cal 3 (10 ng/ml)
5x10
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
+ESI MRM Frag=130.0V [email protected] (890.6000 -> 567.2000) EF_Cal_3.d
more than 120 MRM
transitions in 1.2 min
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Counts vs. Acquisition Time (min)
9.6 9.7 9.8 9.9 10 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8
6490 QQQ Introduction, Berlin
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Compounds at a glance – peak review in MH Quant
Page 43
6490 QQQ Introduction, Berlin
-
The issue with the matrix …
-100%
-80%
-60%
-40%
-20%
0%
20%
0 5 10 15 20 25 30 35 40 45
ma
trix
eff
ect
s
retentiontime in minutes
� Dilute !!!
,if sensitivity allows ...‘
Page 44
-100%
-80%
-60%
-40%
-20%
0%
20%
0 5 10 15 20 25 30 35 40 45
ma
trix
eff
ect
s
retentiontime in minutes
-100%
-80%
-60%
-40%
-20%
0%
20%
0 5 10 15 20 25 30 35 40 45
ma
trix
eff
ect
s
retentiontime in minutes
1 : 10 1 : 100
Data provided by Stefan Kittlaus, LUA Sachsen
6490 QQQ Introduction, Berlin
-
Pesticides in water
Target compounds:
• Atrazine
• Atrazine-desethyl
• Chlorotoluron
• Cyanazine
• Diuron
• Hexazinone
• Isoproturon
• Linuron
Injection volume: 1 µL
Column: Eclipse PlusC18, 1.8 um, 2.1 x 50 mm
Pump Flow: 0.4 ml/min
Mobile Phase: A = water + 5mM ammonium formate +
0.01% formic acid
B = 95% acetonitrile/5% water + 5mMammonium formate + 0.01% formic acid
Gradient: Time %B
0.00 20
4.00 70
4.10 100
4.50 100• Linuron
• Metazachlor
• Methabenzthiazuron
• Metobromuron
• Metolachlor
• Metoxuron
• Monolinuron
• Sebuthylazin
• Simazine
• Terbuthylazin
6490 QQQ Introduction, Berlin
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-
10 fg On Column(1 uL inj. of 10 pg/mL)
Monolinuron
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 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4
Counts 2x10
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
215.1 -> 126.0 , 215.1 -> 148.1
Ratio = 35.1 (57.0 %)
Acquisition Time (min)
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 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4
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 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9
Counts 2x10
0
1
2
3
4
5
6
7
202.1 -> 132.0 , 202.1 -> 124.1
Ratio = 82.1 (106.1 %)
Simazine
6490 QQQ Introduction, Berlin
Page 46
-
10 fg On Column(1 uL inj. of 10 pg/mL)
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 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4
Counts 2x10
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
278.1 -> 134.1 , 278.1 -> 210.1
Ratio = 58.4 (97.1 %)
Metazachlor
Acquisition Time (min)
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 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4
Acquisition Time (min)
2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4
Counts 2x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
284.1 -> 252.1 , 284.1 -> 176.1
Ratio = 22.4 (111.6 %)
Metolachlor
Qualifier present
Qualifier not present
6490 QQQ Introduction, Berlin
Page 47
-
100 fg On Column(1 uL inj. of 100 pg/mL)
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
Counts 2x10
1
2
3
4
5
6
7
8
188.1 -> 146.0 , 188.1 -> 104.0
Ratio = 29.8 (155.7 %)
Atrazine-desethyl
Acquisition Time (min)
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
Acquisition Time (min)
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 3 3.1 3.2 3.3 3.4 3.5 3.6
Counts 3x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
216.1 -> 174.1 , 216.1 -> 132.0
Ratio = 25.1 (143.4 %)
Atrazine
6490 QQQ Introduction, Berlin
Page 48
-
100 fg On Column(1 uL inj. of 100 pg/mL)
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 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4
Counts 2x10
0
1
2
3
4
5
6
7
259.0 -> 170.0 , 259.0 -> 148.1
Ratio = 57.0 (81.7 %)
Metobromuron
Acquisition Time (min)
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 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4
Counts 2x10
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
259.0 -> 170.0 , 259.0 -> 148.1
Ratio = 55.1 (79.1 %)
10 fg on column
Acquisition Time (min)
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 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4
Acquisition Time (min)
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 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4
Counts 2x10
0.5
1
1.5
2
2.5
3
3.5
4
249.0 -> 160.0 , 249.0 -> 182.0
Ratio = 59.1 (86.2 %)
Linuron
6490 QQQ Introduction, Berlin
Page 49
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Summary of Pesticides Results6490 6460 x-fold improvement
pg on column S/N pg on column S/N
Atrazine-desethyl 0.1 33 1 100 3
Hexazinone 0.01 43 1 350 12
Metoxuron 0.01 42 1 120 35
Simazine 0.1 59 1 75 8
Cyanazine 0.1 23 1 65 4
Methabenzthiazuron 0.01 42 1 125 34
Chlortoluron 0.01 56 1 95 59
Atrazine 0.1 123 1 105 12Atrazine 0.1 123 1 105 12
Isoproturon 0.01 77 1 250 31
Monolinuron 0.01 14 1 80 18
Diuron 0.01 33 1 50 66
Metobromuron 0.1 83 1 30 28
Metazachlor 0.01 43 1 280 15
Sebuthylazin 0.01 29 1 250 12
Terbuthylazin 0.01 38 1 280 14
Linuron 0.1 31 1 20 15
Metolachlor 0.01 65 not recorded
6490 QQQ Introduction, Berlin
Page 50
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Photoinitiators
• More than 1000 compounds are used for printing on packaging materials for food. In 2005 ITX (2-Isopropylthioxanton) a photoinitiator has been found in baby food packed in paperboard containers and had been taken off the shelves in Italy.
S
CH3
CH3
O
• Photoinitiators are compounds which accelerate the drying of colors which undergo a UV induced polymerization.
• ITX is non-mutagenic but it is not acceptable in food products and at concentrations above 0.05 mg/kg food product EFSA requested food producers to react.
• Nestlé has published a list of compounds which are accepted in food packaging, all other compounds need to be tested for food migration and must comply with regulations or must be at concentrations below 0.01 mg/kg.
6490 QQQ Introduction, Berlin
Page 51
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PhotoinitiatorsScreening method
Target compounds:
• 1-(4-((4-Benzoylphenyl)-thio)-phenyl)-2-methyl-2-((4-methylphenyl)-sulfonyl)-1-propan-1-on
• 1-Chlor-4-propoxythioxanthon
• 1-Hydroxycyclohexylphenylketon
• 2-(4-Methylbenzyl)-2-dimethylamino-1-(4-morpholinophenyl)-1-butanon
• 2,2-Bis-(2-chlorophenyl)-4,4,5,5-tetraphenyl-1,2-biimidazol
• 2,2-Diethoxy-acetophenon
• 2,2-Dimethoxy-2-phenylacetophenon
• 2-Hydroxy-4-methoxybenzophenon
• 2-Hydroxy-4-n-octoxy-benzophenon
• 3-Methylbenzophenon
• 4-(4-Methylphenylthio)-benzophenon
• 4,4,4-Methylidynetris-(N,N-dimethylanilin)
• 4,4-Bis-(diethylamino)-benzophenon
• 4,4-Bis-(dimethylamino)-benzophenon
• 4-Dimethylaminobenzophenon
• 4-Hydroxybenzophenon• 2,2-Dimethoxy-2-phenylacetophenon
• 2,4,6-Trimethylbenzophenon
• 2,4,6-Trimethylbenzoylphenylphosphinsäureethylester
• 2,4-Diethylthioxanthon
• 2-Benzyl-2-dimethylamino-4-morpholinobutyrophenon
• 2-Chlorthioxanthon
• 2-Dimethylamino-ethylbenzoat
• 2-Ethylanthraquinon
• 2-Ethylhexyl-4-dimethylaminobenzoat
• 2-Hydroxy-1-(4-(4-(2-hydroxy-2-methylpropanyl)-benzyl)-phenyl-2-methyl-2-propanon
• 4-Hydroxybenzophenon
• 4-Phenoxy-2,2-dichloracetophenon
• 4-Phenylbenzophenon
• Benzophenon
• Bisphenol A
• Butoxyethyl-4-dimethylamino-benzoat
• Diphenyl-2,4,6-trimethylbenzoyl)-phosphinoxid
• Ethyl-4-dimethylamino-benzoat
• Isopropylthioxanthone
• Methyl-2-benzoylbenzoat
• Phenyl-bis-(2,4,6-trimethylbenzoyl)-phosphinoxid
• Thioxanthon
6490 QQQ Introduction, Berlin
Page 52
-
Photoinitiators in cardboard extract
Injection volume: 2 µL
Column: Eclipse PlusC18, 1.8 um, 2.1 x 100 mm
Pump Flow: 0.3 ml/min
Mobile Phase: A = water + 5mM ammonium formateB = acetonitrile
Gradient: Time %B
0 100.5 103.5 609 95
11.5 9511.6 10
6490 QQQ Introduction, Berlin
Page 53
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Photoinitiators – Chromatography 100 ng/ml (all compounds)
6x10
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
+ESI TIC MRM (** -> **) Mix_35_100ppb+BP.d
1 1
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Counts vs. Acquisition Time (min)
0.5 1 1.5 2 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
6490 QQQ Introduction, Berlin
Page 54
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Photoinitiatoren – Quant software Batch at a glance
2-Hydroxy-1-(4-(4-(2-hydroxy-2-methylpropanyl)-benzyl)-phenyl-2-methyl-2-propanon
6490 QQQ Introduction, Berlin
Page 55
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Photoinitiatoren – Quant software Batch at a glance
Methyl-2-benzoylbenzoat
6490 QQQ Introduction, Berlin
Page 56
-
Photoinitiatoren – Quant software Batch at a glance
1-(4-((4-Benzoylphenyl)-thio)-phenyl)-2-methyl-2-((4-methylphenyl)-sulfonyl)-1-propan-1-on
6490 QQQ Introduction, Berlin
Page 57
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Bisphenol A and benzophenone – Comparison Agilent 6460 vs. 6490100 ng/ml in muesli extract, 5 ul
Agilent QQQ 6490Agilent QQQ 6460
6490 QQQ Introduction, Berlin
Page 58
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Bisphenol A – Comparison Agilent QQQ 6460 vs. 6490100 ng/ml in muesli extract, 5 ul
30 X Signal12 X SNR
Agilent QQQ 6490
Agilent QQQ 6460
6490 QQQ Introduction, Berlin
Page 59
LOQ 2.5 pg o.c.(S/N 10, Peak-to-Peak)
-
Benzophenon – Comparison Agilent QQQ 6460 vs. 6490100 ng/ml in muesli extract, 5 ul
9 X Signal6 X SNR
Agilent QQQ 6490
Agilent QQQ 6460
6490 QQQ Introduction, Berlin
Page 60
LOQ 2.5 pg o.c.(S/N 10, Peak-to-Peak)
-
New triggered MRM functionality for confirmation
6490 QQQ Introduction, Berlin
Page 61
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Identification of compounds by library search - Qual
Library search functionality in MassHunter Qual
Excellent peak shapes for primary transitions!
6490 QQQ Introduction, Berlin
Page 62
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Identification of compounds by library search - Quant
6490 QQQ Introduction, Berlin
Page 63
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Qualifier ratios in triggered spectra
0.0
2000.0
4000.0
6000.0
8000.0
10000.0
12000.0
14000.0
16000.0
105.1 133.1 139.00 146.00 331.1 360.00
1-(4-((4-Benzoylphenyl)-thio)-phenyl)-2-methyl-2-((4-methylphenyl)-sulfonyl)-1-
propan-1-on
m/zin spectra
ratioRSD
Peak Area ratio
105.1 100.0 0.0
133.1 30.4 18.5
139.0 25.1 19.6
146.0 54.4 4.4
331.1 89.7 5.7 80.6
360.0 47.6 6.8
0.0
10000.0
20000.0
30000.0
40000.0
50000.0
60000.0
127.00 171.00 200.00 228.00 234.00 263.00
1-Chlor-4-propoxythioxanthon
m/zin spectra
ratioRSD
Peak Area ratio
127.0 47.0 17.6
171.0 74.8 0.7 74.9
200.0 100.6 16.4
228.0 175.4 19.7
234.0 9.6 22.8
263.0 100.0 0.0
6490 QQQ Introduction, Berlin
Page 64
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0.0
10000.0
20000.0
30000.0
40000.0
50000.0
60000.0
70000.0
80000.0
90000.0
100000.0
89.10 165.10 191.00 226.00 329.10
2-(2,2-Bis-(2-chlorophenyl)-4,4,5,5-tetraphenyl-1,2-biimidazol
Qualifier ratios in triggered spectra
m/zin spectra
ratioRSD
Peak Area ratio
89.10 21.9 0.86 21.8
165.10 46.1 19.76
191.00 18.3 20.14
226.00 16.5 21.99
329.10 100.0 0.00
0.0
20000.0
40000.0
60000.0
80000.0
100000.0
120000.0
140000.0
77.10 105.10 165.10 197.10
2-(2,2-Dimethoxy-2-phenylacetophenon
m/zin spectra
ratioRSD
Peak Area ratio
77.10 100.0 0.00
105.10 377.8 18.22
165.10 185.5 17.22
197.10 93.0 0.79 93.7
6490 QQQ Introduction, Berlin
Page 65
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5. Summary and Conclusions
• It was demonstrated that the 1290 Infinity UHPLC coupled with a 6490 QQQ gives excellent sensitivity for the three analyte groups.
• The detection limit of almost all analytes was improved over the 6460 QQQ, in many cases by more than a factor of 10.
• Although highly sensitive, the 6490 QQQ performs with great robustness, which shows in the good reproducibility of the peak area.
6490 QQQ Introduction, Berlin
Page 66
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Vielen Dank für Ihre Aufmerksamkeit
Sven PoggenseeAgilent TechnologiesSales & Service GmbH & Co KG
?
Sales & Service GmbH & Co KG
Page 67
6490 QQQ Introduction, Berlin