Sviluppo di Nuove Tecnologie in LC-ESI-MS - HOME … · Agilent Technologies Italia S.p.A. Sviluppo...
Transcript of Sviluppo di Nuove Tecnologie in LC-ESI-MS - HOME … · Agilent Technologies Italia S.p.A. Sviluppo...
Fabio Fabris
Agilent Technologies Italia S.p.A.
Sviluppo di Nuove Tecnologie in LC-ESI-MS
Modena, 27 Maggio 2009
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.
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
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
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
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
La Spettrometria di Massa in HP/Agilent
1971
1976
Primo spettrometro di massa “benchtop”
1984
LC/MS
Interfaccia LCMS API-ELECTROSPRAY
Fenn & Whitehouse 1985
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
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
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
Page 12
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)
Residui di Farmaci e Prodotti per la cura della
persona (PPCPs) in Acqua Potabile
LC-MS triplo Q
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
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)
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
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
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
Page 19
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
Page 20
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
La Tradizione HP/Agilent nella microfluidica
Hewlett Packard Inkjet CartridgeAgilent LabChip for RNA, DNA
and protein analysis
Page 22
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 !
Page 23
Soluzioni Agilent HPLC-Chip-MS HPLC-Chip
MSD Ion Trap QTOF QQQ
Page 24
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
Sistema Nano-HPLC-Chip per NanosprayLC/MS
1200 NanoLC System6000 Series Mass Spectrometer (Ion trap, SQ, QQQ, TOF, Q-TOF)
HPLC-Chip/MS interface
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
Page 27
Alcune applicazioni HPLC-chip-MS
Biomarker Discovery & Validation
Metaboliti e Farmacocinetica
Analisi quantitativa residui inquinanti ambientali
Page 28
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
Page 29
Agilent Proteomics Biomarker Workflow
Candidates
Proteolytic Digest
Extraction
Depletion
Fractionation
Extraction
Identification
6520 QTOF
6410 QQQ
Candidate Biomarker
Identification
Validation
SAMPLE1 DATA
SAMPLE2
Page 30
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
Digestione Triptica in linea : Citocromo C
Page 31
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
Q-TOF Serie 6520
6520 Accurate Mass Q-TOF
Fall 2007
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
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
L’importanza della massa accurata
Identificazione dei picchi molecolari
Page 37
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)
Page 38
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.
Page 39
Analisi ed identificazione prodotti incogniti
incognito
Extracted mass spectrum peak 5.29
33
Page 40
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
Page 41
Elemental composition calculator
Analisi ed identificazione prodotti incogniti
35
Page 42
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
Page 43
Grazie per l’attenzione.