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Maciej Bromirski Q Exactive Product Marketing Manager NACRW, July 21, 2014
Quick and Sensitive Analysis of Multiclass Veterinary Drug Residues in Meat Products, Plasma and Urine Using Fast Chromatography and a Bench Top Orbitrap Mass Spectrometry System
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Agenda
• Instrument & method setup – illustrate the ease of use
• Application – a generic method works for all
• Why DIA? – DIA because of full record of data (isotope match, full screening capabilities)
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Q Exactive Application Details
• Animal product samples came from CVUA MEL, Muenster, Germany • A simplified sample preparation method was applied to the samples • A generic LC method was used for all samples:
• Thermo Scientific™ Accucore™ C18 aq 100x2.1mm, 3.5µm column • 6 min gradient from 5% B to 95% B
• A: H2O + 0.1% formic acid; B: ACN + 0.1% formic acid
• 15 min total chromatographic cycle • A generic MS method on a Thermo Scientific™ Q Exactive™ MS system
was used for all samples: • Full scan with wide isolation variable data independent acquisition (FS-vDIA)
• Full scan resolution setting of 70,000 • Variable DIA resolution setting of 17,500
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Q Exactive Instrument Layout
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Q Exactive Instrument Layout
RF-Lens for higher transmission with rugged optics
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Q Exactive Instrument Layout
Transfer Multipole optimal ion transmission
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Q Exactive Instrument Layout
RF-Lens for higher transmission with rugged optics Transfer Multipole optimal ion transmission
C-Trap directly interfaced to HCD increases spectrum quality
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Q Exactive Instrument Layout
RF-Lens for higher transmission with rugged optics Transfer Multipole optimal ion transmission
C-Trap directly interfaced to HCD increases spectrum quality
HCD cell for MS/MS and DIA excellent confirmation
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Enhanced automatic gain control, parallel filling & detection more speed Advanced signal processing for Orbitrap™ data speed & resolution
increase
Q Exactive Instrument Layout
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Scan-to-Scan Mass Accuracy: Carbaryl, C12H11NO2
RMS = 0.62 ppm across the peak
Resolution @ 70k 5 sec base peak
18 Pos scans
2.00 2.02 2.04 2.06 2.08 2.10 2.12 2.14 Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
0.84 ppm
0.62 ppm 0.62 ppm
0.69 ppm
0.84 ppm
0.62 ppm
0.47 ppm
0.77 ppm
0.54 ppm
0.32 ppm
0.24 ppm 0.16 ppm
0.54 ppm
0.69 ppm
0.69 ppm
0.69 ppm
0.24 ppm
0.84 ppm
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Scan-to-Scan Mass Accuracy : Azoxystrobin 404.1241
Positive/Negative Switching at Resolution Setting of 70K
5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
5.74
5.72
5.75
5.77
5.79 5.70
5.81 5.8 5.85 5.68 5.89 6.12 6.05
RT m/z ppm
+ 5.68 404.1242 -0.29694 - 5.70 404.1243 -0.54439 + 5.72 404.1242 -0.29694 - 5.74 404.1242 -0.29694 + 5.75 404.1241 -0.04949 - 5.77 404.1242 -0.29694 + 5.79 404.1243 -0.54439 - 5.81 404.1243 -0.54439 + 5.83 404.1241 -0.04949 - 5.85 404.1241 -0.04949
True Std = 0.2020
RMS = 0.3534 across the peak
10 sec base peak • 5 Pos scans • 5 Neg scans
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Antibiotics: Sensitivity
Component Elemental Composition Conc.
LOD [ppb]
LOQ [ppb]
Ampicillin C16H19N3O4S 0.1 0.5 Amoxicillin C16H19N3O5S 0.1 0.5 Benzylpenicillin (G) C16H18N2O4S 0.1 0.5 Cefalexin C16H17N3O4S 0.1 0.1 Cefalonium C20H18N4O5S2 0.1 0.5 Cefaperazone C25H27N9O8S2 0.5 1 Cefapririm C17H17N3O6S2 0.1 0.5 Cefquinome C23H24N6O5S2 5 5 Chlortetracycline C22H23ClN2O8 0.5 5 Ciprofloxacin C17H18FN3O3 0.5 0.5 Cloxacillin C19H18ClN3O5S 0.1 0.5 Danofloxacin C19H20FN3O3 0.5 5 Dapsone C12H12N2O2S 0.1 0.1 Difloxacin C21H19F2N3O3 0.1 0.5 Doxycyclin C22H24N2O8 0.1 0.5 Enrofloxacin C19H22FN3O3 0.5 0.5 Erythromycin C37H67NO13 0.5 0.5 Flumequin C14H12FNO3 0.5 0.5
Component Elemental Composition Conc.
LOD [ppb]
LOQ [ppb]
Marbofloxacin C17H19FN4O4 0.1 1 Nafcillin C21H22N2O5S 0.1 0.1 Oxacillin C19H19N3O5S 0.1 0.5 Penicillin (V) C16H18N2O5S 0.5 0.5 Sarafloxacin C20H17F2N3O3 0.5 0.5 Sulfadiazin C10H10N4O2S 0.1 0.1 Sulfadimethoxin C12H14N4O4S 0.1 0.1 Sulfadimidin C12H14N4O2S 0.1 0.1 Sulfadoxin C12H14N4O4S 0.1 0.1 Sulfamerazin C11H12N4O2S 0.1 0.1 Sulfamethoxazole C10H11N3O3S 0.1 0.1 Sulfamethoxypyridazine C11H12N4O3S 0.1 0.1 Sulfathiazole C9H9N3O2S2 0.1 0.5 Tetracycline C22H24N2O8 0.1 0.5 Thiamphenicol C12H13Cl2NO5S 0.5 0.5 Tilmicosine C46H80N2O13 n.d. n.d. Trimethoprim C14H18N4O3 0.1 0.1 Tylosin C46H77NO17 0.5 1
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Avermectins, Imidazoles: Sensitivity
Component Elemental Composition Conc.
LOD [ppb]
LOQ [ppb]
Dimetridazol C5H7N3O2 0.5 5 Ipronidazol-OH C7H11N3O3 0.5 1 Metronidazol-OH C6H9N3O4 0.5 1 Ronidazol C6H8N4O4 0.1 0.5
Component Elemental Composition Conc.
LOD [ppb]
LOQ [ppb]
Abamectin C48H72O14 5 10 Doramectin C50H74O14 5 10 Eprinomectin C50H75NO14 1 5 Moxidectin C37H53NO8 0.5 1
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Ampicillin @ 10 ppb in Pig Muscle
Target Peak Isotope Pattern Match Library Match Cal Curve
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Ipronidazol-OH @ 0.1 ppb in Pig Plasma
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Linearity Ampicillin
Metronidazol Cefalonium
Dimetridazol
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Subsequent Suspect Screening
• Same data set were used • Built in compound database with fragment information were
used • Built in HRAM spectra library with 1500 components were
used • Identification criteria applied:
• fragment search • isotope pattern match • library search
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Additional Suspect Screening
An additional suspect screening with built-in databases and spectral libraries can yield additional identifications like nicotinamide here
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Steps of Confirmation
extracted ion chromatogram
library search result fragment search result
isotope pattern match
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What is DIA?
• Data independent acquisition (DIA) acquires MS2 spectra systematically and independent of previously acquired scans
• You obtain: • complete record of data • very good reproducibility • high throughput comprehensive quantification acquisition methods with
quantitative information • no requirement for detailed sample knowledge prior to data acquisition
• Your routinely acquired data is ready to go for any non-target
screening approach
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How to run variable DIA (vDIA) experiments
• For data independent data acquisition, a full scan is interchanging with a series of MS2 scans
• These MS2 scans have: • a wide isolation window of typically 100 Da or more • variable isolation range
• A typical vDIA experiment can look like this:
vDIA m/z 500 – 1000
full scan m/z 100 - 1000
vDIA m/z 100 - 200
vDIA m/z 200 - 300
vDIA m/z 300 - 400
vDIA m/z 400 - 500
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How to run variable DIA (vDIA) experiments
• For data independent data acquisition, a full scan is interchanging with a series of MS2 scans
• These MS2 scans have: • a wide isolation window of typically 100 Da or more • variable isolation range
• A typical vDIA experiment can look like this:
vDIA m/z 500 – 1000
full scan m/z 100 - 1000
vDIA m/z 100 - 200
vDIA m/z 200 - 300
vDIA m/z 300 - 500
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Why Use Wide Isolation Variable DIA?
• vDIA fills a gap between data dependent MS2 acquisition (ddMS2) and all ion fragmentation (AIF) • ddMS2 has the best selectivity, but it offers confirmation only for target
compounds, thus screening capabilities are limited • AIF is limited in selectivity (multiple precursors / interferences may have
the same fragment) and dynamic range
• By “segmenting the AIF scans” vDIA enhances selectivity and dynamic range without limiting the ability to use the same data for suspect screening and general unknown screening purposes
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Comparison to AIF
• For comparison, the same application setup was used to acquire data in full scan – all ion fragmentation mode (FS-AIF) and data independent acquisition (FS-DIA) • Full scan resolution setting 70,000 • AIF scan resolution setting 35,000
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AIF vs vDIA
AIF
vDIA
Ampicillin, 0.5 ppb Neat Standard background
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AIF vs vDIA
AIF
vDIA
Cefalexin in Pig Muscle @ 2 ppb background
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AIF vs vDIA
AIF
vDIA
Oxacillin in Pig Kidney @ 2 ppb background
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Conclusion
• One generic methods for all samples and compounds • Easy to setup and operate • Integrated software for data acquisition, processing and
reporting • Variable DIA: sensitive, selective and ready for screening
applications (used for retrospective analysis)
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Emile DeLeeuw Jean-Jacques Dunyach Margarita Fernandez Paul Gregory Suvarna Bhakara Eric Hemenway Jenny Berryhill Marcus Kellmann Paul Huang Syed Rizvi
Eric Stover Jens Grote Mark Hardman Paul Johnson Thomas Cheng Ethan Chan Jesse Canterbury Mari Preito Connaway Paul Van Hoorickx Thomas Moehring
Eugene Zhuk Jessica Lin Marta Kozak Philip Remes Thomas Rietpitsch Farhana Afroz Jessica Wang Martin Kast Pixie Kemple Thomas Ng
Fishaye Gebrehiwet Jew-Dong Kuo Martin Zeller Qiming Wan Tim Stratton
F. Grosse-Coosmann Jill Hagaman Mary Blackburn Qingyu Song Tom McClure
Frank Czemper Jim Lonero Matthew Kump Rajesh Raina Tonya Second Frank Marcos Jim Shofstahl Michael Athanas Ralf-Achim Purrmann Torsten Ueckert
Agus Ong Charles Yang Fred Ayres Jim Sklenar Michael Belford Raman Mathur Tracy Pham Alan Schoen Chris Mullen Ganka Canagasaby Jimmy Tran Michael Blank Ray Chen Trevor Hall
Alen Cileli Chrisanne Gordon Gary Mahany Joe Senteno Michael Chen Reiko Kiyonami Tricia Gordon Alexander Kholomeev Cindy Taylor Gerry Reynolds John Smith Michael Senko Rex Heller Tuan Bi
Alfredo Alvarez Clay Campbell Grace Li John Syka Mike Antonczak Richard Hartford Udeni Dharmawardana Alison Cook Dan Schaeffer Hai Nguyen Jon Moeller Mike Giusti Rob Grothe Vane Sanghvi Alvin Abdon Dan Valk Haishan Wei Jonathan Beck Mike Konicek Rob Heather Viatcheslav Kovtoun
Andreas Huhmer Daryl Baer Hans Schweingruber Jose Cruz Medina Misael Martinez Rob Worley Vlad Zabrouskov
Andreas Boegehold Dave Horn Hao Tran Joseph Guerrero Mohsen Tadjvar Roberta Pino Wayne Dewey Andreas Wieghaus Dave Minkler Hao Truong Josh Maze Nan Huang Robert Malek Wilko Balschun
Andreas Kuehn David Fisher Harald Oser Julie Lin Neal Borelli Rosa Viner Ya-Mei Liu Andrew Schirmer David Wright Helen Tran Julie Horner Nick Cairns Ryan Hermezian Yihan Bao
Andy Jacobs DE Lee Hetal Rajput June Altieri Nick Izgarian Sachin Deshpande Yingying Huang Andy Le Diane Cho Hien Nguyen Junhua Wang Nigama Ekkad Sally Webb Yitman Liang
Andy tai Dirk Nolting Hilda Li Ken Miller Oleg Silivra Sanjeev Tanna Yufan Zhu Arthur Shwarts Dipankar Ghosh Hossein Arjomand Koshy Panicker Oleh Bondarenko Satendra Prasad Yvonne Arenas August Specht Doug Miller Howard Tran Kerstin Strupat Oliver Lange Sergio Maluendes Yvonne Lu
Balaram Barange Erkan Diri Huy Nguyen Kristine Van Natta Omar Kurdi Shannon Eluk Zesmaa Morimoto Barbara Gibson Ed Gonzalez Iain Mylchreest Ksheeraj Kumud Pabla Singh Kulwinder Shelley Hoyt
BC Cha Ed Mantilla Ian Jardine Lalasa Ala Pascual Cardenas Shijun Li Beena Raman Ed Yuen Iman Mohtashemi Lee Earley Pat Bennett Sree Paruchuri Berg Tehlirian Eduard Denisov Jackson Louie Lenis Doan Paul Atherton Stevan Horning Beth Anderson Eloise Blanchard Jae Schwartz Linda Duvall Paul Chen Steve Fenske
Brenda Li Eloy Wouters Jan- Peter Hauschild Linda Lin Paul Gazis Sunandini Yedla Cesar Nepacena Ernst Schroeder Jane Razumovskaya Manish Doshi Bjorn Rose Susan Yung
The Orbitrap Team
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Thank you !
