the evolution of liquid chromatography coupled to mass...
TRANSCRIPT
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
• 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
© 060207, Kantonales Labor Zürich, 8032 Zürich
• 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
Food safety KLZH
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
• 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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
Limitation of LC-MS/MS for multiresidue methods
Multiple compounds can be monitored,
But how far can or should we go ?
.
© 060207, Kantonales Labor Zürich, 8032 Zürich
Single MRM
(one analyte)
Retention time
Analyte:
A
5
© 060207, Kantonales Labor Zürich, 8032 Zürich
Multiple MRM
(several analytes)
Retention time
Analyte:
A
B
C
D
E
F
Dwell time versus
number of data points
across a peak
© 060207, Kantonales Labor Zürich, 8032 Zürich
MRM windows
(about hundred analytes)
Retention time
Analyte:
A
B
C
D
E
F
6
© 060207, Kantonales Labor Zürich, 8032 Zürich
Scheduled MRM
(one thousend analytes)
Retention time
Analyte:
A
B
C
D
E
F
G
H
I
J
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
Limitation of LC-MS/MS for multiresidue methods
Monitoring dozens of MRMs reduces sensitivity.
Managing these MRMs can become a difficult task.
.
© 060207, Kantonales Labor Zürich, 8032 Zürich
• The challenge
• New analytical strategies (Multiresidue methods)
• High resolution liquid chromatography
& mass spectrometry
New analytical strategies in veterinary drug control
• The Future: Generic detection
8
© 060207, Kantonales Labor Zürich, 8032 Zürich
• 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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
Extraction Recoveries
Chinolones
BenzimidazolesPenicillins
apolar
Recovery
Tetracyclines
Sulfonamides
IonophoresAvermectines
polar
100 %
0 %
muscle
liver
Extraction solvent: 70 % acetonitrile
29 % water
1 % acetic acid
© 060207, Kantonales Labor Zürich, 8032 Zürich
Variety of analytes
More compounds and lower concentrations
AminoglycosidesTetracyclines
Nitrofurans
Avermectines Nitroimidazoles Penicillines
Chinolones
10
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
Concentration range (MRL&MRPL):
More compounds and lower concentrations
• 0.3 µg/kg Chloramphenicole
• 20 µg/kg Tetracycline in honey
• 600 µg/kg Tetracycline in kidney
• 5000 µg/kg Neomycin in kidney
11
so einfach; so anspurchsvoll
© 060207, Kantonales Labor Zürich, 8032 Zürich
Analyte loss I: Extraction
Extracting solvent polarity does not correspond to analyte
so einfach; so anspurchsvoll
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
so einfach; so anspurchsvoll
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
so einfach; so anspurchsvoll
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
so einfach; so anspurchsvoll
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
so einfach; so anspurchsvoll
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
The analytical method
© 060207, Kantonales Labor Zürich, 8032 Zürich
N2
Bi-Polarity
Extraction
Centrifugation
Evaporation
of organics
Centrifugation
SPE
OASIS HLB
Evaporation
of organics
Centrifugation
Workflow of the method
The analytical method
15
© 060207, Kantonales Labor Zürich, 8032 Zürich
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)
© 060207, Kantonales Labor Zürich, 8032 Zürich
A combined approach
• New extraction and clean-up method
• High LC separation power (UPLC)
• Fast, flexible and sensitive detection (TOF)
More compounds and lower concentrations
16
History of drug screening at Kantonales Labor Zürich
© 060207, Kantonales Labor Zürich, 8032 Zürich
Good chromatographic resolution
Pork liver extract spiked with 100 vet. drugs
TIC
Analyte trace
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
• The challenge
• New analytical strategies (Multiresidue methods)
• High resolution liquid chromatography
& mass spectrometry
New analytical strategies in veterinary drug control
• The Future: Generic detection
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
Confirmation of a Chloramphenicol finding
(urine sample)
Chloramphenicol trace (ESI neg.)
Standard 100 µg/l
Urine with Chloramphenicol or simply noise ?
Confirmation CAP B
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
Confirmation of positive findings
TIC
m/z =497.037Exact mass of C17H19N2O11Cl2
A-posteriori („inject now, ask later“)
Confirmation CAP D
© 060207, Kantonales Labor Zürich, 8032 Zürich
Isotopic pattern of chloramphenicol glucuronide
calculated
suspected peak
A-posteriori („inject now, ask later“)
20
© 060207, Kantonales Labor Zürich, 8032 Zürich
How much “high resolution” do we need ?
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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 )
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
Liver extract spiked with marbofloxacin (m/z = 363.14628 window: 50 mD)
Second Generation TOF Resolution
10’000 FWHM
22
© 060207, Kantonales Labor Zürich, 8032 Zürich
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: 50 mD)
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
Second Generation TOF Resolution
10’000 FWHM
© 060207, Kantonales Labor Zürich, 8032 Zürich
Second Generation TOF Resolution
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
09_10_08_79 #4125 RT: 6.39 AV: 1 NL: 4.84E4T: 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
320.13986
09_10_08_79 #4126 RT: 6.39 AV: 1 NL: 3.66E4T: 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
320.13956
09_10_08_79 #4127 RT: 6.39 AV: 1 NL: 4.27E4T: 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
320.14017
09_10_08_79 #4128 RT: 6.39 AV: 1 NL: 4.90E4T: 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
320.13965
09_10_08_79 #4129 RT: 6.39 AV: 1 NL: 3.58E4T: 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
320.13837
320.17184
09_10_08_79 #4130 RT: 6.39 AV: 1 NL: 3.41E4T: 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
320.14005
09_10_08_79 #4131 RT: 6.39 AV: 1 NL: 4.43E4T: 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
320.13855
Second Generation TOF Resolution
10’000 FWHM
> 2 mD
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
09_10_08_79 #4131 RT: 6.39 AV: 1 NL: 4.43E4T: 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
bun
dan
ce
320.13855
FWHM = 10‘000
FWHM = 100‘000
Norfloxacin
Matrix
Quad peak width
© 060207, Kantonales Labor Zürich, 8032 Zürich
Switching to single stage Orbitrap
(Exactive)
25
© 060207, Kantonales Labor Zürich, 8032 Zürich
• Adapting an existing analytical method
• Validation HRMS versus MS/MS
• Resolution to obtain MS/MS selectivity
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
Comparing apples to oranges
Comparing
2 dimensional data (MS/MS)
versus
1 dimensional data (HRMS)
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
HRMS surpasses MS/MS selectivity
at 50’000 FWHM
2 data points per second (UPLC capable)
30
© 060207, Kantonales Labor Zürich, 8032 Zürich
Extracts which were ok for the TOF are
not for the Orbitrap
Another gate to be opened
© 060207, Kantonales Labor Zürich, 8032 Zürich
• Adapting an existing analytical method
• Validation HMRS versus MS/MS
• Resolution to obtain MS/MS selectivity
31
© 060207, Kantonales Labor Zürich, 8032 Zürich
0 2 4 6 8 10 12 14
0
50
Signal suppression
8.81
9.73
10.21
11.05
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
Low mass ion discrimination
09_11_27_04 #2373 RT: 8.02 AV: 1 NL: 2.39E5T: FTMS {0,0} + p ESI Full ms [190.00-2000.00]
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
09_11_27_05 #2373 RT: 8.02 AV: 1 NL: 6.72E4T: FTMS {0,0} + p ESI Full ms [190.00-2000.00]
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
vacuum
vacuum
C-Trap
Orbitrap
Octapole
Interface
Principle of Orbitrap Operation
© 060207, Kantonales Labor Zürich, 8032 Zürich
+
+
++
++++
++
++
C-Trap
Orbitrap
Postulated phenomena:
post interface signal suppression
35
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
• Adapting an existing analytical method
• Validation HMRS versus MS/MS
• Resolution to obtain MS/MS selectivity
37
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
• The challenge
• New analytical strategies (Multiresidue methods)
• High resolution liquid chromatography
& mass spectrometry
New analytical strategies in veterinary drug control
• The Future: Generic detection
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
The generic detector
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
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:
© 060207, Kantonales Labor Zürich, 8032 Zürich
MS/MS
Precursor scan sulfonamides
Urin blank
Spectrastandard mix
honey containing50 µg/kg sulfadimidine
41
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
© 060207, Kantonales Labor Zürich, 8032 Zürich
Drug group specific screening
Sulfonamides
Suspected [M+H]+
Proposed composition
Merck Index:
„Sulfamethoxazole“
42
© 060207, Kantonales Labor Zürich, 8032 Zürich
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
10_08_06_21 #1203 RT: 9.55 AV: 1 NL: 6.53E4T: FTMS {1,1} + p ESI Full ms [140.00-1000.00]
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)
© 060207, Kantonales Labor Zürich, 8032 Zürich
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