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![Page 1: Sviluppo di Nuove Tecnologie in LC-ESI-MS - HOME … · Agilent Technologies Italia S.p.A. Sviluppo di Nuove Tecnologie in LC-ESI-MS Modena, 27 Maggio 2009. ... •#1 in LC/MS SingleQ](https://reader031.vdocuments.net/reader031/viewer/2022022016/5b6360737f8b9a3c5e8be914/html5/thumbnails/1.jpg)
Fabio Fabris
Agilent Technologies Italia S.p.A.
Sviluppo di Nuove Tecnologie in LC-ESI-MS
Modena, 27 Maggio 2009
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Agilent Technologies…la storia
• Agilent dates back to the earliest
days of Hewlett-Packard, which
started as a test and
measurement company in 1939.
• On Nov. 1, 1999, Agilent started
operating as an independent
company.
• Agilent embodies historical
commitment to innovation and
contribution, uncompromising
integrity, teamwork, trust and
respect for the individual.
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Agilent at a Glance
$5.8 billion annual
revenue (FY2008)
19,100 employees
Customers in 110+ countries
60+ year heritage of
invention and innovation
Corporate Headquarters Manufacturing Centers
More than 60% of revenue generated outside U.S.
Global manufacturing and R&D
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Agilent Market Focus
Bio-Analytical
Measurement•Life science
•Chemical analysis
•Materials science
•Communications
•Aerospace/Defense
•General purpose
Electronic
MeasurementBio-Analytical
Measurement
•Applied research
•Research in existing businesses
•Research in new businesses
Agilent Laboratories
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Technology and Market Leader
• Broadest product and service offering
• Addresses entire electronics product life cycle
• End-to-end communications and computation test
• #1 in overall test and measurement
• #1 in wireline and wireless test
• #1 in signaling monitoring
• #1 in VOIP assurance monitoring
• #1 in passive & active probe systems
• #1 in parametric test
• #1 in service assurance
• Leadership in chemical and pharmaceutical analysis
• Leverages expertise into emerging life science solutions
• #1 in LC
• #1 in GC
• #1 in GC/MS
• #1 in ICP-MS
• #1 in LC/MS SingleQ
• #2 in LC/MS QQQ
• #1 in LC/MS TOF and QTOF
• #2 in microarrays
Electronic Measurement
Life Sciences and Chemical Analysis
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Spettrometria di Massa
Page 6
La spettrometria di massa e’ una tecnica analitica “indispensabile” per :
1) Identificare Prodotti Incogniti
2) Chiarire le proprieta’ strutturali e chimiche delle molecole
3) Determinazioni quantitative di composti noti (analisi in tracce)
LC-MS ion trap
LC-MS triplo Q Q-TOF
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La Spettrometria di Massa in HP/Agilent
1971
1976
Primo spettrometro di massa “benchtop”
1984
LC/MS
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Interfaccia LCMS API-ELECTROSPRAY
Fenn & Whitehouse 1985
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Interfaccie LCMS /Ionizzazione a Pressione Atmosferica API
AP El ect rospray
APCI
Molecular W
eight
Anal yt e Pol ari t y
1000
100,000
10,000
nonpolar very polar
GC/MS
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Q-TOF
Agilent Restricted
Bioscienze
Pharma, Biotech, CRO, CMO
DiagnosticsAcademia & Government
Chemical QA/QC
Environmental Testing
Food Testing
Forensics Testing
Applicazioni LC-MS
- identificazione di proteine e biomarkers
-analisi quali -quantitativa di farmaci
-caratterizzazione prodotti di sintesi
-Identificazione di impurezze
-Analisi e caratterizzazione metaboliti
- studi chimica clinica
Analisi Chimica
- Analisi quantitativa in matrici complesse
- Screening pesticidi in prodotti alimentari
-analisi quantitativa target su molecole “proibite”
-Doping & Tossicologia
-Analisi e caratterizzazione metaboliti
LC-MS triplo QLC-MS ion trap
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Page 11
Mean area %RSD = 3.2, Mean height %RSD= 3.6
Analisi di 300 Pesticidi in 15 minuti
LC-MS triplo Q
10 pg/ul in Matrice
Modalita’ MRM
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Desisopropyl atrazine
10 pg
10 pg
Cymoxanil
Butylate
10 pg
Bromuconazol (diastereomers)
10 pg
R2= 0.99883
R2= 0.99830
R2= 0.99873
R2= 0.99859
Curve di Calibrazione (10 pg – 1 ng)
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Residui di Farmaci e Prodotti per la cura della
persona (PPCPs) in Acqua Potabile
LC-MS triplo Q
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EPA 1694 - analisi PPCPs in Acqua Potabile
IbuprofenIsochlortetracycline (ICTC)LincomycinLomefloxacinMetforminMiconazoleMinocyclineNaproxenNorfloxacinNorgestimateOfloxacinOrmetoprimOxacillinOxolinic acidOxytetracycline (OTC)Penicillin VPenicillin GRanitidineRoxithromycinSarafloxacinSulfachloropyridazineSulfadiazineSulfadimethoxineSulfamerazineSulfamethazineSulfamethizoleSulfamethoxazoleSulfanilamideSulfathiazoleTetracycline (TC)ThiabendazoleTriclocarbanTriclosanTrimethoprimTylosinVirginiamycinWarfarinOther standards
AcetaminophenAlbuterolAmpicillinAnhydrochlortetracycline (ACTC)Anhydrotetracycline (ATC)AzithromycinCaffeineCarbadoxCarbamazepineCefotaximeChlortetracycline (CTC)CimetidineCiprofloxacinClarithromycinClinafloxacinCloxacillinCodeineCotinineDehydronifedipineDemeclocyclineDigoxigeninDigoxinDiltiazem1,7-DimethylxanthineDiphenhydramineDoxycyclineEnrofloxacin4-Epianhydrochlortetracycline(EACTC)4-Epianhydrotetracycline (EATC)4-Epichlortetracycline (ECTC)4-Epioxytetracycline (EOTC)4-Epitetracycline (ETC)ErythromycinErythromycin anhydrateFlumequineFluoxetineGemfibrozil
Antibiotici
Birth Control Steroids
Anti-Depressivi
Pain Killers
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Aggiunta 1 pg in acqua potabile
Column:Zorbax Eclipse Plus C18(2.1 x 100mm, 1.8um)
Flow Rate = 1ml/min
On column amount = 1pg
65 EPA 1694 + ve analytes(112 total transitions)
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0
20
40
60
80
100
150 200 250 300 350 400 450
163.1 203.3 231.4
255.3
313.4
339.5397.5
423.5
467.5
493.4
m/z
% R
ela
tive
Ab
undance
Vitamin D3 in
Poultry Feed Extract
10 µL loop injection
APCI Full Scan MS
383.4385.4
Vitamin D3 ?
[M+H]+ 385
500
LC(MS)n Trappola Ionica
LC-MS ion trap
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0
20
40
60
80
100
150 200 250 300 350 400 450
199.3 219.0 255.2 287.3325.4
367.4
500
m/z
% R
ela
tive
Ab
un
dance
Poultry Feed Extract
10 µL loop injection
APCI MS/MS of 385.4
[M+H]+ - H2O
MSn
LC-MS ion trap
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0
20
40
60
80
100
150 200 250 300 350 400 500
185.0
213.1
255.1
285.4311.2
353.2
241.2
271.1325.5
227.2
158.5
Poultry Feed Extract
MS3
385 367
m/z
0
20
40
60
80
100
150 200 250 300 350 400 500
158.6
185.0
213.0
255.2
271.1
325.5
349.2 367.6
241.3
285.3
227.2
Pure Standard
Vitamin D3
MS3385 367
% R
elati
ve A
bundance
% R
ela
tive
Ab
un
da
nce
MSn
LC-MS ion trap
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Agilent Labs & le sfide e nuove frontiere nella Misura
• Elevata’ densita’ di segnali
• Matrici e campioni complessi
• Nanoscale
• Segnali ad elevata velocita’
• Costo test/analisi
• Frequenza campionamento elevatissime
The list goes on …
• Alta Velocita’
• Segnali ridotti
• Complessita’
• Risoluzione
• Miniaturizzazione
Difficolta’ Tecnologiche/AmbientaliCondizioni Estreme
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Le frontiere della Misura nel 21.mo Secolo Elettronica e Biologia convergono nelle dimensioni “Nano-”
Biologia
Nanotecnologie
Elettronica Integrazione e
Miniaturizzazione
Complessita’
Molecolare
Nanomotor
ATP Synthase
Nanotransistor
LETI
10nm
20nm
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La Tradizione HP/Agilent nella microfluidica
Hewlett Packard Inkjet CartridgeAgilent LabChip for RNA, DNA
and protein analysis
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Perche’ Nanotecnologie in HPLC/MS?
• Riduzione quantita’ di campione necessaria
• Riduce l’utilizzo di reagenti e fasi mobili
• Aumento di sensibilita’ .
• Aumento efficienza e potere risolutivo
• Velocita’ di analisi
Ed anche
facilita’ d’uso ed automazione !
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Page 23
Soluzioni Agilent HPLC-Chip-MS HPLC-Chip
MSD Ion Trap QTOF QQQ
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Perche’ utilizzare separazioni lcms a nanoflussi?
Flusso (µL/min)
Diam. colonna (mm)
0.1 1 10 100 1,000 10,000 100,000
Prep LCHPLCMicro LCCapillary LCNano LC
0.05 0.2 1 2 4 50
Guadagno sensibilita’ 6400 400 16 4 1
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Sistema Nano-HPLC-Chip per NanosprayLC/MS
1200 NanoLC System6000 Series Mass Spectrometer (Ion trap, SQ, QQQ, TOF, Q-TOF)
HPLC-Chip/MS interface
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Caratteristiche del Chip
µ-fluidic holes
µ-filter as column packing material retaining device
Top View from Rotor Side C634
Analytical column 75 x 50µm2 43 mm long Zorbax 5µm C18 SB 300
Laser Ablated Cylindrical ES-Tip
o.d. 40µm 0.5mm long tip channel
10 x 14 µm2
integrated 2x5 channel frit
Enrichment Column 40nl Zorbax 5µm
C18 SB 300A
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Alcune applicazioni HPLC-chip-MS
Biomarker Discovery & Validation
Metaboliti e Farmacocinetica
Analisi quantitativa residui inquinanti ambientali
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Analysis
Protein Biomarker Workflow: Protein Identification
Approach
Protein
Fractionation
Sample Preparation
Enzymatic Digestion
Peptide
Fractionation
Identification
Proteins identified in sample 1
Identify proteins in each sample
Compare identified proteins across multiple samples
Proteins identified in sample 2
Comparison of samples
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Agilent Proteomics Biomarker Workflow
Candidates
Proteolytic Digest
Extraction
Depletion
Fractionation
Extraction
Identification
6520 QTOF
6410 QQQ
Candidate Biomarker
Identification
Validation
SAMPLE1 DATA
SAMPLE2
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Ultrasensibilita’ con HPLC-Chip/QQQ
0.04 fmol/µL * 1µL injection amolALEFLR*
Triplicate runs
Pept i de pr ecur sor f r agment quant / qual
LFTGHPETLEK 636. 3 716. 4 quant
LFTGHPETLEK 636. 3 1011. 6 qual
LFTGHPETLEK* 640. 3 724. 4 quant
LFTGHPETLEK* 640. 3 1019. 9 qual
ALELFR 374. 7 564. 3 quant
ALELFR 374. 7 435. 3 qual
ALELFR* 379. 7 574. 3 quant
ALELFR* 379. 7 445. 3 qual
HPLC Chip: 43mm Zorbax SB C18
nanoPump: 400nL/min, A. water+0.1%FA, B. 95%ACN 0.1%FA
0 min 5%B, 10min 30%B, 12min 80%B
CapPump: 3µL/min, 2%ACN water
Sample: 0.04fmol/µL * 1µL injection, sample is in 240ng/µL digested serum
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Digestione Triptica in linea : Citocromo C
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m/z400 600 800 1000 1200 1400
0
20
40
60
80
100
*MSD1 SPC, time=3.000:5.000 of F:\MAY16-A\DIG_01.D
Max: 20395
8
17
.7
7
79
.7
6
34
.5
5
85
.0
7
80
.5
6
78
.5
1
00
6.0
7
93
.2
7
29
.0
1
07
0.1
5
89
.3
6
35
.5
9
64
.7
8
01
.5
6
79
.5
7
13
.7
4
89
.4
5
90
.3
1
16
8.8
8
28
.7
1
01
4.3
5
62
.5
m/z400 600 800 1000 1200 1400
0
20
40
60
80
100
*MSD1 SPC, time=8.000:19.000 of F:\JULY-2\DIG-OC5.D
Max: 4268
8
17
.5
7
79
.4
5
84
.9
6
34
.3
6
78
.3
7
28
.9
7
82
.1
8
39
.5
1
07
0.0
7
80
.4
1
00
5.6
7
02
.9
5
89
.2
6
49
.0
8
23
.3
9
33
.6
7
50
.7
7
13
.4
5
91
.1
8
50
.4
5
62
.2
1
01
2.1
1
09
9.5
6
53
.9
5
34
.4
7
88
.0
9
64
.5
1
05
4.0
Digest conditions: 2.5 mM Ammonium Bicarbonate buffer, pH 8.2 + 20% methanol
In-solution- 24 hr
5 mM cytochrome C
On-chip- 14 sec
5 mM cytochrome C
Protein ID TrypProtein ID
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Q-TOF Serie 6520
6520 Accurate Mass Q-TOF
Fall 2007
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Come funziona il QTOF?
a
Quad Mass Filter (Q1)
Collision Cell
Lens 1 and 2
Quad Mass Filter (Q1)
Collision Cell
Lens 1 and 2
m3
m1
m4
m2
Primo quadrupolo in SIM!
Spettri MSMS accurati!
• No 1/3 cutoff
•ITraq
•No limite capacità
•De Novo
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Nuovo sistema acquisizione 4GHz up to 5 Decades of In-Spectrum Dynamic Range
In-Scan Dynamic Range
This Example
9x106 / 250 = 3.6 104
Maximum
7 1.2x 105
Max Abd
3x107Erythromycin, 500 fg/µL
[M+H]+ = 734.468518 m/z
Error = +0.6 ppm
Niacinamide, 10 ng/µL
[M+H]+ = 123.055289 m/z
Error = +0.8 ppm
250 cts
9x106 cts
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L’importanza della massa accurata
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Identificazione dei picchi molecolari
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Triazines Spiked Into Tomato Matrix, 100 ppt
6530 QTOF, Targeted MS/MS Mode
Compound Transitions for EICs % RSD
(3 inj.)
Simazine 202.0849124.0868+132.0317 13.2
Prometon 226.1662142.0717+184.1189 1.5
Atrazine 216.1010174.0539 16.0
Ametryn 228.1277186.0803 10.1
Propazine 242.1435158.0487+200.0962 17.2
Prometryn 230.1177146.0226+188.0692 14.2
Terbutryn 242.1435186.0803 12.6
3x10
0
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)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 8 8.2 8.4
Ametryn
Propazine
Prometon
Simazine
Prometryn
Terbutryn
Atrazine
Product Ion EICs (10 µL injection of 100 ppt)
3x10
0
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)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 8 8.2 8.4
Ametryn
Blanks (triplicates)
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QqTOF Protein Digest Sensitivity Check-
MS (precursor) Ion Traces - 200 attomol BSA Digest
K)HLVDEPQNLIK(Q)
200 attomole bSA digest near the limits of detection in MS (precursor) mode.
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Analisi ed identificazione prodotti incogniti
incognito
Extracted mass spectrum peak 5.29
33
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Unknown [M+H+]
Adduct (Unknown + Na)
[M+Na+] 332 + 23
Adduct (2*unknown + Na)
[2*M+Na+] 2 * 332 + 23
Analisi ed identificazione prodotti incogniti
34
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Elemental composition calculator
Analisi ed identificazione prodotti incogniti
35
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Search on the internet molecular Formula
Conclusion compound is of the group steroids
The name is 17 oxo-dexamethason
Molecular Weight: 504.588 g/molMolecular Formula: C28H37FO7
Molecular Weight: 332.409 g/molMolecular Formula: C20H25FO3
IUPAC Name: 9-fluoro-11-hydroxy-10,13,16-trimethyl-7,8,9,10,11,12,13,14,15,16-decahydro-6H-cyclopenta[a]phenanthrene-3,17-dione
Dexamethason Dipropionate
36
Analisi ed identificazione prodotti incogniti
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Grazie per l’attenzione.