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.