the evolution of liquid chromatography coupled to mass...

1

© 060207, Kantonales Labor Zürich, 8032 Zürich

The evolution of liquid chromatography

coupled to mass spectrometry from single

analyte detection towards non-target

screening

Anton Kaufmann

Official Food Control Authority of the Canton of Zurich

(Kantonales Labor Zürich)

Switzerland


• The challenge

• New analytical strategies (Multiresidue methods)

• High resolution liquid chromatography

& mass spectrometry

New MS based analytical strategies in veterinary

drug residue control

• The Future: Generic detection

2


• The challenge



& mass spectrometry

New MS based analytical strategies in veterinary

drug residue control


Food safety KLZH


Veterinary drug analysis is a moving target

2002 2003 2004 2005 2006 2007year

Percentage

Positive samples

100%

0%

Positive Pangasius samples (Aquaculture Vietnam)

NitrofuransMalachite green

Enrofloxacine

?

3


• New emerging strains of bacteria can ruin aquaculture

within a whole region or country

• Anything which reduces mortality of livestock

might be used

• There are a lot of banned, yet very effective drugs

(e.g. chloramphenicol, nitrofurans etc.)

Not registered drugs in veterinary medicine?

• There are even more analogues which were abandoned

in human medicine clinical trials


Limitations of MS-MS based multiresidue methods

� MS-MS finds only analytes which have been

previously tuned

� MS-MS is a victim of its own selectivity

4


Limitation of LC-MS/MS for multiresidue methods

Multiple compounds can be monitored,

But how far can or should we go ?

.


Single MRM

(one analyte)

Retention time

Analyte:

A

5


Multiple MRM

(several analytes)

Retention time

Analyte:

A

B

C

D

E

F

Dwell time versus

number of data points

across a peak


MRM windows

(about hundred analytes)

Retention time

Analyte:

A

B

C

D

E

F

6


Scheduled MRM

(one thousend analytes)

Retention time

Analyte:

A

B

C

D

E

F

G

H

I

J


Can we or our software manage all these transitions?

• An analyte might produce more than one peak

• Some analytes might produce the same MRM

• A labile analyte might have degraded in the standard solution

• A pH sensitive analyte might move out of a window

.

7


Limitation of LC-MS/MS for multiresidue methods

Monitoring dozens of MRMs reduces sensitivity.

Managing these MRMs can become a difficult task.

.


• The challenge



& mass spectrometry

New analytical strategies in veterinary drug control


8


• Provides similar selectivity as MS/MS

• Sensitivity becomes comparable to MS/MS

• Full scan data

Ultra performance liquid chromatography coupled

to high resolution mass spectrometry can be the

answer

• Qualitative work without standards


More selectivity and sensitivity by higher

chromatographic separation power (UPLC)

LC

3-5 µm

particles

UPLC

1.7 µm

particles

9

so einfach; so anspurchsvoll


Extraction Recoveries

Chinolones

BenzimidazolesPenicillins

apolar

Recovery

Tetracyclines

Sulfonamides

IonophoresAvermectines

polar

100 %

0 %

muscle

liver

Extraction solvent: 70 % acetonitrile

29 % water

1 % acetic acid


Variety of analytes

More compounds and lower concentrations

AminoglycosidesTetracyclines

Nitrofurans

Avermectines Nitroimidazoles Penicillines

Chinolones

10


Conflicting extraction & clean-up

requirements

• Aminoglyocsides require low pH extraction for

sufficient recoveries

• Penicillines degrade at low and high pH values

• Tetracyclines require complexing agents for

good recoveries

The analytical method


Concentration range (MRL&MRPL):


• 0.3 µg/kg Chloramphenicole

• 20 µg/kg Tetracycline in honey

• 600 µg/kg Tetracycline in kidney

• 5000 µg/kg Neomycin in kidney

11



Analyte loss I: Extraction

Extracting solvent polarity does not correspond to analyte



Extraction Recovery

� One single solvent can not extract the whole

analyte polarity range

� Bi-polarity Extraction:

Emulsion of two immiscible solvents permits

parallel extraction

(acetonitrile/aqueous buffer with high concentration

of ammonium sulfate to induce phase separation)

12



Extraction Recovery

Extraction

Rel

ativ

e R

ecov

ery

[%]

Ace

prom

azin

Chl

orpr

omaz

in

Pro

bion

ylpr

omaz

in

Meb

enda

zol

Oxf

enda

zol

Am

pici

llin

Cep

hale

xin

Cep

hapi

rin

Dox

ycyc

lin

Oxy

tetr

acyc

line

Tet

racy

clin

Cip

roflo

xaci

n

Sul

fam

etho

xazo

le

Analyte

-20

0

20

40

60

80

100

120

140

160

A: Bi-Polarity B: Acetonitrile



Analyte loss II: SPE

Solid phase extraction breakthrough and irreversible retention

� Polymer reversed Phase SPE (OASIS HLB)

� Solvent free loading solution

� pH control of loading solution

� Two elution steps (solvent and buffer in solvent)

13



Analyte loss III: Adsorption

Adsorption by precipitating proteins and on vessel walls

� High ionic strength

� Rinsing of vessels and residues with aqueous dimethyl-

sulfoxide

� Avoidance of glassware

� No evaporation till dryness, use keeper



Analyte loss III: Adsorption

Use of DMSO

R

elat

ive

Rec

over

y [%

]

Ace

prom

azin

Chl

orpr

omaz

in

Pro

bion

ylpr

omaz

in

Meb

enda

zol

Oxf

enda

zol

Am

pici

llin

Cep

hale

xin

Cep

hapi

rin

Dox

ycyc

lin

Oxy

tetr

acyc

line

Tet

racy

clin

Cip

roflo

xaci

n

Sul

fam

etho

xazo

le

Analyte

-20

0

20

40

60

80

100

120

140

A: DMSO for rinsing and as keeper B: DMSO as keeper only C: DMSO for rinsing only

14


New extraction and clean-up method

• Bi-polarity extraction

• Generic solid phase extraction (OASIS HLB)

• Avoiding extreme pH

• Washing of precipitates and glassware to

minimize analyte loss



N2

Bi-Polarity

Extraction

Centrifugation

Evaporation

of organics

Centrifugation

SPE

OASIS HLB

Evaporation

of organics

Centrifugation

Workflow of the method


15


Instrumentation

� LC

UPLC (Waters)

Column: T3; 100 * 2.1 mm 1.8 µm (Waters)

Mobile Phase: Gradient Water/Acetronitrile + Formic acid

� MS

LCT Premium (Waters)

Positive mode (ESI)

Dynamic range enhancement “DRE”

Data processing: QuanLynx (Waters)


A combined approach

• New extraction and clean-up method

• High LC separation power (UPLC)

• Fast, flexible and sensitive detection (TOF)


16

History of drug screening at Kantonales Labor Zürich


Good chromatographic resolution

Pork liver extract spiked with 100 vet. drugs

TIC

Analyte trace


Multiple target analysis

Chinolones: Ciprofloxacin; Danofloxacin; Enrofloxacin; Flumequin; Oxolinic acid; Enoxacin; Lomefloxacin;

Nalidixic acid; Norfloxacin; Ofloxacin; Difloxacin; Sarafloxacin; Piromidic acid; Sparfloxacin; Azithromycin

Sulfonamides: Sulfagunaidin; Sulfanilamid; Sulfadiazin; Sulfathiazol; Sulfapyridin; Sulfamerazin;

Sulfamethizol; Sulfadimidin; Sulfamethoxypyridazin; Sulfachlorpyridazin; Sulfachlorpyrazin; Sulfadoxin;

Sulfadimethoxin; Sulfacetamid; Sulfamethoxazol; Sulfisoxazol; Sulfabenzamid; Sulfameter; Sulfamomomethoxin;

Sulfamoxol; Sulfanitran; Sulfaquinoxalin; Sulfasalazin; Sulfaisomidin;

Tetracyclines: Oxy-Tetracyclin; Tetracyclin; Chlor-Tetracyclin; Minocyclin; Doxycyclin; Demeclocyclin

Nitroimidazoles: Ipronidazol-OH; Metronidazol-OH; Dimetrinidazol; Ronidazol; Metronidazol; Ipronidazol;

HMMNI; Tinidazol

Cephalosporines: Cefaperazon; Cefazolin; Cephalexin; Cephapirin

Avermectins: Avermecting: Ivermectin; Doramectin; Emamectin; Eprinomectin

Macrolides: Roxithromycin; Tylosin A; Erythromycin A; Tilmicosin; Spiramycin I; II; III; Oleandomycin;

Josamycin; Tulathromycin;

Lincosamides: Lincosamide: Lincomycin; Pirlimycin; iso-Pirlimycin; Clindamycin; Tiamulin

Benzimidazoles: Febantel; Albendazol; Fenbendazol; Fleroxacin; Flubendazol; Mebendazol; Ofendazol;

Oxibendazol; Parabendazol; Thiabendazol; Triclobendazol;

Penicillines: Ampicillin; Nafcillin; Penicillin G; Penicillin V; Dicloxacillin; Cloxacillin; Amoxicillin; Oxacillin

Tranquilizers: Acepromazin; Azaperol; Azaperon; Carozolol; Chlorpromazin; Propionylpromazin; Xylazin

Various: Acriflavin; Praziquantel; Trimethoprim; Diaveridin; Rifampicin; Pyrimethamin; Rifamixin; Virginiamycin

17


• The challenge



& mass spectrometry




Benefits of High Resolution Mass Spectrometry

(TOF or Orbitrap)

• High selectivity (approaching MS-MS)

• Full scan data “a posteriori hypothesis”

• Retrospective analysis

• “No need for reference substances”

18

Confirmation CAP A


Confirmation of a Chloramphenicol finding

(urine sample)

Chloramphenicol trace (ESI neg.)

Standard 100 µg/l

Urine with Chloramphenicol or simply noise ?

Confirmation CAP B


Confirmation of positive findings with TOF

Chloramphenicol is known to be metabolized to

Chloramphenicol glucuronide

Commercially not available as reference substance

19

Confirmation CAP C


Confirmation of positive findings

TIC

m/z =497.037Exact mass of C17H19N2O11Cl2

A-posteriori („inject now, ask later“)

Confirmation CAP D


Isotopic pattern of chloramphenicol glucuronide

calculated

suspected peak

A-posteriori („inject now, ask later“)

20


How much “high resolution” do we need ?


m/z0

20

40

60

80

100

120

140

160

Required resolution

199 200 201

Mass axis [m/z]

nu

mb

ero

f p

oss

ible

elem

enta

lco

mp

osi

tio

ns

HNO3SCl3 C13H30N

TOF Resolution

void void

Isobaric compositions

21


Second Generation TOF Resolution

10’000 FWHM

RT: 0.00 - 15.60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

10.41

11.031.26

1.451.83 4.73 10.20

5.07 6.287.814.453.012.10 10.156.15

6.833.37 3.79 5.749.097.32 7.98

13.699.369.01 13.47 13.799.461.13 14.3513.2111.85

NL:3.88E8

TIC MS 09_10_08_79

RT: 0.00 - 15.60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

2.46

1.76

5.44

6.18

5.334.47 13.08

1.08

5.88 11.896.54

13.4013.026.93 13.793.21 3.88 10.58 11.809.477.24 14.508.740.99

NL:1.51E6

m/z= 362.64628-363.64628 MS 09_10_08_79

Liver extract spiked with marbofloxacin (TIC)

Liver extract spiked with marbofloxacin (m/z = 363.14628 window: 500 mD )


RT: 0.00 - 15.60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

2.46

1.76

5.44

6.18

5.334.47 13.08

1.08

5.88 11.896.54

13.4013.026.93 13.793.21 3.88 10.58 11.809.477.24 14.508.740.99

NL:1.51E6

m/z= 362.64628-363.64628 MS 09_10_08_79

Liver extract spiked with marbofloxacin (m/z = 363.14628 window: 500 mD)

RT: 0.00 - 15.60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

da

nce

2.46

1.76

6.18

5.741.53 4.49 7.243.27 13.0810.90 13.566.41 14.507.57 11.469.42 10.268.68

NL:1.51E6

m/z= 363.09628-363.19628 MS 09_10_08_79



10’000 FWHM

22


RT: 0.00 - 15.60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

da

nce

2.46

1.76

6.18

5.741.53 4.49 7.243.27 13.0810.90 13.566.41 14.507.57 11.469.42 10.268.68

NL:1.51E6

m/z= 363.09628-363.19628 MS 09_10_08_79


Liver extract spiked with marbofloxacin (m/z = 363.14628 window: 2 mD)RT: 0.00 - 15.60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

6.18 NL:8.21E5

m/z= 363.14428-363.14828 MS 09_10_08_79


10’000 FWHM



10’000 FWHM

Liver extrakt spiked with norfloxacin (m/z = 320.14047 window: 2 mD )

RT: 0.00 - 15.60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

3.72

6.40

5.00

5.9510.45

9.928.07

NL:9.95E4

m/z= 320.13847-320.14247 MS 09_10_08_79

RT: 6.08 - 6.55

6.10 6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 6.55

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

6.40

6.396.39

6.40

6.41

NL:5.30E4

m/z= 320.13847-320.14247 MS 09_10_08_79

RT: 3.54 - 6.59

3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

danc

e

3.72

3.70

3.75

3.76

6.406.393.68

3.79 5.00

6.415.954.98

NL:9.95E4

m/z= 320.13847-320.14247 MS 09_10_08_79

23


Norfloxacin spectrum (m/z = 320.14047)

09_10_08_79 #4124 RT: 6.38 AV: 1 NL: 4.64E4T: FTMS {0,0} + p ESI Full ms [85.00-1000.00]

319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

320.13931


319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

320.13986


319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

320.13956


319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

320.14017


319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

320.13965


319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

320.13837

320.17184


319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

320.14005


319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

320.13855


10’000 FWHM

> 2 mD


Maximum resolution

FWHM = 100’000

RT: 0.00 - 15.62

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

6.38 NL:3.02E4

m/z= 320.13947-320.14147 MS 09_10_08_28

Liver with norfloxacine m/z = 320.14047 1 mDa

RT: 5.79 - 7.24

5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

6.38 NL:3.02E4

m/z= 320.13947-320.14147 MS 09_10_08_28

24


Comparison of Spectra

09_10_08_28 #473-476 RT: 6.37-6.41 AV: 4 NL: 1.75E4T: FTMS {0,0} + p ESI Full ms [85.00-1000.00]

319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce320.14050

320.16062

319.70224319.51269 319.92961


319.5 319.6 319.7 319.8 319.9 320.0 320.1 320.2 320.3 320.4

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

320.13855

FWHM = 10‘000

FWHM = 100‘000

Norfloxacin

Matrix

Quad peak width


Switching to single stage Orbitrap

(Exactive)

25


• Adapting an existing analytical method

• Validation HRMS versus MS/MS

• Resolution to obtain MS/MS selectivity


Required resolution to compete with MS/MS ?

The Orbitrap permits resolutions from 10’000 – 100’000 FWHM

Generic answer not particular analyte matrix combination

26


Comparing apples to oranges

Comparing

2 dimensional data (MS/MS)

versus

1 dimensional data (HRMS)


The approach

• Producing extracts from blank matrices

• Separating the extracts by UPLC

• Monitoring dummy transitions (MS/MS)

dummy exact masses (HRMS)

• Make observed peak areas comparable

27


Dummy transitions (MS/MS)

Some of these transitions are probably impossible

(not existing elemental composition for neutral loss)

50

150

250

350

450

550

50 150 250 350 450 550

Reihe1

>=18 Dalton

m/z (precursor ion)

m/z

(product

ion)

two dimensions

m/z: 350>172


Dummy exact masses (HRMS)

Only a relatively narrow mass defect range was tested

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

100 200 300 400 500 600

Reihe1

one dimensionm/z nominal mass

mass

defect

m/z: 348.2014

28


Comparing apples and oranges

• Select 6 drugs (tetracyclins, chinolons and sulfonamides)

• Determine their response “peak area / concentration”

• Calculate average response for these 6 drugs

• Divide each dummy peak area by the average response

• Do this for MS/MS and HRMS dummy peaks


Effect of resolution

rt

con

c FWHM = 10'000

0 2 4 6 8 10 12 14 160

1000

2000

3000

4000

5000

6000

FWHM = 25'000

0 2 4 6 8 10 12 14 16

FWHM = 50'000

0 2 4 6 8 10 12 14 160

1000

2000

3000

4000

5000

6000

FWHM = 100'000

0 2 4 6 8 10 12 14 16

Honey matrix

elution region

of analytes

29


Liver

(difficult matrix)

HRMS versus MS/MS

rt

con

c FWHM = 10'000

0 2 4 6 8 10 12 14 160

10

20

30

40

50

60

70

FWHM = 25'000

0 2 4 6 8 10 12 14 16

FWHM = 50'000

0 2 4 6 8 10 12 14 16

0

10

20

30

40

50

60

70

FWHM = 100'000

0 2 4 6 8 10 12 14 16

0 2 4 6 8 10 12 14 16rt

0

10

20

30

40

50

60

70

conc

MS/MS


HRMS surpasses MS/MS selectivity

at 50’000 FWHM

2 data points per second (UPLC capable)

30


Extracts which were ok for the TOF are

not for the Orbitrap

Another gate to be opened



• Validation HMRS versus MS/MS


31


0 2 4 6 8 10 12 14

0

50

Signal suppression

8.81

9.73

10.21

11.05


0 2 4 6 8 10 12 14

0

50

Signal suppression

Signal termination

8.81

9.73

10.21

11.05

6.85

6.85

6.85

32


Standard: 10 µl injection volume

m/z: 285.0107

m/z: 311.0808

m/z: 199.0290

C:\Publikation\...\09_11_27_06 11/27/2009 1:12:10 PM Std 500µg/lPositiv

RT: 0.00 - 15.62

0 2 4 6 8 10 12 14Time (min)

0

50

1000

50

100

Rel

ativ

e A

bund

ance

0

50

1008.12

6.57

5.49 7.04 8.301.24 9.336.98 8.34

7.13 8.535.624.952.621.00

4.41 6.174.04 7.69 8.022.96 9.00 9.51 10.90 11.75 14.00

NL:2.55E6

m/z= 285.01873-285.02273 MS 09_11_27_06

NL:7.18E6

m/z= 311.07882-311.08282 MS 09_11_27_06

NL:4.01E6

m/z= 199.02700-199.03100 MS 09_11_27_06

sulfachlorpyridazin

sulfachlorpyrazin

sulfadoxin sulfadimethoxin

background signal


Spiked kidney extract: 10 µl injection volume

C:\Publikation\...\09_11_27_04 11/27/2009 12:35:17 PM Probe +B

RT: 0.00 - 15.61

0 2 4 6 8 10 12 14Time (min)

0

50

1000

50

100

Rel

ativ

e A

bund

ance

0

50

1006.58

5.501.77

6.98

2.69 14.653.550.13

0.61

3.933.70 4.092.754.79 5.25 5.57 6.54 12.20 12.74

NL:8.39E4

m/z= 285.01873-285.02273 MS 09_11_27_04

NL:5.37E5

m/z= 311.07882-311.08282 MS 09_11_27_04

NL:4.86E5

m/z= 199.02700-199.03100 MS 09_11_27_04

sulfachlorpyridazin

sulfachlorpyrazin ??

sulfadoxin sulfadimethoxin ??

33


Spiked kidney extract: 1 µl injection volume

C:\Publikation\...\09_11_27_05 11/27/2009 12:53:11 PM Probe +B50 ml inj time 1 ul

RT: 0.00 - 15.61

0 2 4 6 8 10 12 14Time (min)

0

50

1000

50

100

Rel

ativ

e A

bund

ance

0

50

1008.04

6.48

1.22 5.645.40 9.49

6.89

8.25

14.395.640.34 3.54 7.283.03 10.350.12

0.573.23 3.19 4.873.11 5.22

6.187.137.79

8.18 8.70 12.11 12.3211.81 14.12

NL:2.93E4m/z= 285.01873-285.02273 MS 09_11_27_05

NL:1.42E5

m/z= 311.07882-311.08282 MS 09_11_27_05

NL:3.88E5

m/z= 199.02700-199.03100 MS 09_11_27_05

sulfachlorpyridazin

sulfachlorpyrazin

sulfadoxin sulfadimethoxin


Low mass ion discrimination


200 400 600 800 1000 1200 1400 1600 1800 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

1119.09912

1007.09027

1258.98328

743.66467

915.81134

694.29834

588.33295

1438.56274529.50006

1678.58350475.17117


200 400 600 800 1000 1200 1400 1600 1800 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

1118.98682

1258.72925

1007.29095

755.27191302.10037 847.66498

660.98608 1438.55981479.29965

10 µl sample spectum

1 µl sample spectum

multiple charged proteins

34


vacuum

vacuum

C-Trap

Orbitrap

Octapole

Interface

Principle of Orbitrap Operation


+

+

++

++++

++

++

C-Trap

Orbitrap

Postulated phenomena:

post interface signal suppression

35


How to reduce post interface signal suppression

• Resolution >= 50‘000 FWHM

low scan start. e.g.: m/z = 50

reduced number of ion in C-trap. e.g.: < = 1’000’000

limited C-trap filling time. e.g. <= 50 ms

• less injection volume

more extensive protein removal


Low protein method

• Multiresidue methods do not like extensive clean-up

(more than 100 analytes in different matrices)

• Testing almost every known protein removal method

36


Key elements of modified method

• High ammonium sulphate concentration in extraction buffer

• Bipolarity extraction (buffer and acetonitrile)

• Evolute ABN SPE (Biotage) narrow pore size

• Kinetex Core-Shell column 150*2.1 mm; 2.6 µm



• Validation HMRS versus MS/MS


37


Comparison among different techniques

Coefficient of determination (r2)

0.985590.979160.99684MS/MS

0.934740.929020.82102TOF

0.991880.995840.99670Orbitrap

21.51

0

20

40

60

80

100

120

0 20 40 60 80 100 120

order of magnitude

Levels:

1/10 µg/l

3.3/33 µg/l

10/100 µg/l

33/333 µg/l

100/1000 µg/l


Unified multiresidue veterinary drug analysis

� Screening

(sensitive; non-target)

� Quantification

(comparable to MS/MS)

� Confirmation

(FWHM > 20‘000 produces 2 identification points

Cone induced fragmentation and adducts produce

additional ions: 4 to 6 identification points )

38


• The challenge



& mass spectrometry




Our aim

• Food scandal ready method

• Capability to monitor metabolites or drugs where no

reference substances are commercially available

• Impossible dream:

“No drugs at relevant levels are present in this sample”

39


The generic detector


Non-target drug group specific screening

Penicillines

N

S

O

CH3

CH3

H

O

OH

HN

H2N

H

O

N+

SCH3

CH3

H

O

OH

H

H

C6H10NO2S

[M+H]+ = 160.043

(variable)

Amoxicillin

CID Fragment:

40


Non-target Drug group specific screening

Sulfonamides

NH2 SHN

N O

CH3

O

O

R (variable)

C6H6NO2S

[M+H]+ = 156.012+NH2 S

O

O

Sulfamethoxazole

CID Fragment:


MS/MS

Precursor scan sulfonamides

Urin blank

Spectrastandard mix

honey containing50 µg/kg sulfadimidine

41


HRMS

at 50‘000 FWHM

c:\xcal ibur\...\10_10_01\10_10_01_36 10/2/2010 12:38:33 AMHCD 18eV

RT: 0.00 - 10.55

0 1 2 3 4 5 6 7 8 9 10

Time (min)

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

0

20

40

60

80

100R

elat

ive

Abu

ndan

ce6.95

5.87

7.988.164.85

6.697.464.73

4.321.21

3.838.42 9.092.481.49 9.753.40

5.48

NL:7.16E5

m/z= 156.00964-156.01276 MS 10_10_01_32

NL:2.21E4

m/z= 156.00964-156.01276 MS 10_10_01_36

standard mix

honey containing50 µg/kg sulfadimidine


Drug group specific screening

Sulfonamides

Suspected [M+H]+

Proposed composition

Merck Index:

„Sulfamethoxazole“

42


HRMS

Searching for chlorine isotopic patterns

C:\Xcalibur\...\10_08_06\10_08_06_21 8/6/2010 6:38:49 PM

RT: 0.00 - 13.51

0 2 4 6 8 10 12

Time (min)

0

20

40

60

80

100

Rel

ativ

e A

bun

dan

ce

9.55 NL:2.15E4

m/z= 470.03183-470.03583 MS 10_08_06_21


470 475 480 485 490 495 500 505

m/z

0

20

40

60

80

100

Re

lativ

e A

bun

da

nce

502.06064

504.05737

470.03433

472.03168506.05374

478.33014494.81219485.70322

[M+Na]+[M+H]+

m/z = 470.0338(Dicloxacillin)


Conclusion

• High resolution full scan MS Data is an alternative

to LC-MS/MS

• Appropriate sample clean up method is required

• Generic, intelligent detection is needed

• Hardware is an issue, but software is the real limitation

43


We are not yet there !

But we keep moving !

the evolution of liquid chromatography coupled to mass...

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