Maurer

Transcription

Maurer

© 2015, Univ.-Prof. Dr. Dr. h.c. Hans H. Maurer, Toxikologie, Univ. des Saarlandes, Homburg/Saar
Dieses Handout ist nur zum Gebrauch im DGPT-KlinTox-Weiterbildungskurs in Göttingen 2015 bestimmt.
Eine Weitergabe oder Vervielfältigung insbesondere in elektronischer Form (Internet etc.) ist ausdrücklich untersagt.
Department of Experimental & Clinical Toxicology
Saarland University, Homburg (Saar), Germany
Klinische Toxikologie, DGPT, Göttingen 2015
GC‐MS und LC‐MS
in der Klinischen Toxikologie
Univ.‐Prof. Dr. Dr. h.c. (UGent) Hans H. Maurer
‐ Clinical Toxicologist, GTFCh ‐
‐ Forensic Toxicologist, GTFCh ‐
([email protected])
Teaching
• Pharmacology and Toxicology for Students of Pharmacy
(with the basics of anatomy, physiology, pathophysiology and drug therapy)
• Toxicology for Students of Medicine, Dentistry, Human Biology
• Clinical Toxicology for Master Students of Toxicology, TU Kaiserslautern
Research
• Toxicokinetics (Drug transport, Drug metabolism etc.)
• Analytical Toxicology
Medical Healthcare
• ClinTox Service (24/7)
Everyday service for hospitals
within a radius of 100 km
1
Night and weekend service for
hospitals within a radius of 500 km
Medical Healthcare
Tasks of the ClinTox Service HOM (24/7)
• (Differential) Diagnosis and prognosis of poisonings
• Indication for (invasive) treatment
• Monitoring of the efficiency of detoxication
• Drug determinations for brain death diagnosis
Medical Healthcare
• Monitoring of polytoxicomania patients
(abuse of alcohol, drugs and/or medicaments)
• Monitoring of Munchhausen Syndrome patients
• Detection of adverse drug reactions or interactions
• Monitoring of non-compliant patients (TDM, adherence testing)
2
Forensic Toxicology
• Monitoring of polytoxicomania patients
(abuse of alcohol, drugs and/or medicaments)

Proof of murder by poisoning

Proof of penal responsibility

Elucidation of drug-facilitated crime cases

Monitoring of drivers under the influence of alcohol and drugs

(Forensic doping control)
Biosamples in Clinical Toxicology
Blood/Plasma/Serum
Urine
Gastric content
Detection Windows
3
Which Concentrations can be Analyzed ?
1 kg
1 kg Sugar
1 000 g
1 000 000 mg
1 000 000 000 µg
Bostal Lake, ca. 8 000 000 000 L
Sugar Concentration
= 0.125 µg/L = 0.125 ng/mL
Analytical Toxicology
Analysis
Qualitative
Analysis
Target
Screening
Quantitative
Analysis
Unknown
Screening
Point‐of‐Care Drug Testing
(bedside, roadside, workplaceside ....)
4
Point‐of‐Care Drug Testing
(bedside, roadside, workplaceside ....)
IA vs GC‐MS Study
12 cases with positive Triage®8 not confirmed by GC‐MS (10.8%)
AMP (4)
BAR (1)
TCA (4)
MTD (1)
BZO (2)
THC (1)
OPI (2)
8 cases with negative
Triage®8
despite GC‐MS detection (7.2%)
BZO (3)
AMP (1)
TCA (3)
COC (1)
77 cases with GC‐MS detection of drugs principally not detectable by Triage®8 (69.8%)
Non‐OP Analgesics (43)
Antiepileptics (9)
Non‐TCA AD (16)
Opioids (8)
Neuroleptics (15)
Cardiovascular drugs (6)
Hypnotics (12)
Others (4)
(von Mach/Weber/Meyer/Weilemann/Maurer/Peters, Therap Drug Monitor, 2007)
Confirmation Test by GC‐MS/LC‐MS ‐ Why ?
Confirmation mandatory also for on‐site test results!!
5
Three Pillars of (Bio)analytical Methods
Sample Preparation
Separation
Detection
Pre‐extraction steps
Chromatography
Mass spectrometry
• Homogenization
• Conjugate cleavage
• Gas chromatography (GC)
• Ionization types
‐ EI, PICI, NICI
‐ ESI, APCI
• Mass analyzers
‐ Quadrupole (Q)
‐ Ion Trap (IT)
‐ Orbitrap (OT)
‐ Time of Flight (TOF)
• Liquid chromatography (LC)
Isolation/Extraction
• Protein precipitation
• Liquid‐liquid extraction (LLE)
• Solid phase extraction (SPE)
Post‐extraction steps
Other GC detectors
• Flame ionization detector (FID)
• Concentration
• Derivatization (for GC‐MS)
Other LC detectors
• UV/VIS
• Diode array detector
Techniques for SUSA
GC‐MS (EI, PICI)
Triple Quadrupole (MS/MS)
High Resolution Devices: Q‐Exactive (MS/MS)
Linear Ion Trap (MSn)
Linear Ion Trap‐Orbitrap (MSn)
CT Lab Services in Homburg/Saar
One toxicologist on duty around the clock
• Comprehensive screening in urine by GC-MS (SUSA), LC-MSn, IA
• Limited screening in blood by GC-MS, LC-MSn
• Screenings for rare compounds by Spot Tests
6
Standard Urine Screening Approaches (SUSA)
Drug
Urine
Acid hydrolysis, LLE, AC
Protein precipitation
Turboflow
TF ISQ
GC‐MS (EI, full‐scan)
MPW_tox2015 (AMDIS)
TF LIT LXQ
LC‐MSn (MS2/MS3)
MWW_tox2014
TF Q‐Exactive
LC‐HR‐MS/MS
MHMW_tox2015
(MPW, Mass Spectral and GC Data, 2011; Wissenbach/Meyer/Remane/Weber/Maurer, Anal Bioanal Chem, 2011, Helfer et al., JCA, 2014)
Drug
Urine
Turboflow
TF ISQ
MPW_tox2015 (AMDIS)
TF LIT LXQ
LC‐MSn (MS2/MS3)
MWW_tox2014
TF Q‐Exactive
LC‐HR‐MS/MS
MHMW_tox2015
7
Novel Psychoactive Substances (NPS) ?
350 NPS since 2005
Number of NPS notified for the first time
to the “Early Warning System” since May 2005
EMCDDA–Europol 2012 Annual Report
Novel Psychoactive Substances (NPS) ?
Capsules & Tablets
Bath Salts
Blotters
Incense
Research Chemical
Plant Food
NPS (Stimulants)
8
NPS (Synthetic Cannabinoids)
O
O
N
JWH‐015
O
O
N
N
N
JWH‐018
JWH‐073
JWH‐200
N
O
OH
OH
OH
OH
CP 47,497
CP 55,940
OH
OH
O
OH
Δ9‐Tetrahydrocannabinol (THC)
OH
O
HU‐310
O
O
HU‐210
O
Pharmaco/Toxicokinetics of Drugs of Abuse ?
Drugs of Abuse ??
Study Design for Elucidating Drug Metabolism
Drug
Incubation Mixture
Urine, Blood
GRD/ARS
SPE HCX, AC
GRD/ARS
SPE HCX
PP/SPE C18
TF ISQ
GC‐MS (EI, PICI)
TF Q‐Exactive
LC‐HR‐MS/MS
TF Orbitrap Velos
LC‐HR‐MSn
(Meyer GM/Meyer MR/Wissenbach/Maurer, J Mass Spectrom, 2013)
(Welter/Meyer MR/Wolf/Weinmann/Kavanagh/Maurer, Anal Bioanal Chem, 2013)
9
Identification of Proteins by Tryptic Peptide Mass
Fingerprint Analysis using Orbitrap LC‐HR‐MS2
75
kD
kDa
116
70
60
Trypsin
66
50
40
45
35
30
25
20
Nano LC
10
18.4
14.4
TF Orbitrap Velos
1
Membrane protein
identification
(Fecher‐Trost/ …/Maurer/Flockerzi, J Biolog Chem, 2013)
Study Design for Elucidating Drug Metabolism
• Systematic studies of new drug classes
• Ad hoc studies for particular cases
• Agitation
• Hallucination
• Hyperthermia
• Sweating
10
O
NH
CH3
• Agitation
• Hallucination
• Hyperthermia
• Sweating
CH3
H3C
Mephedrone
O
NH
O
CH3
O
H3C
Butylone
O
NH
O
CH3
CH3
O
Methylone
Cathinone Derivatives: ‐Keto‐(bk‐)AM
O
O
NH2
H
N
Methcathinone
Ephedrone
Cathinone
(alkaloid of Catha edulis, Khat)
O
H
N
Mephedrone
O
O
O
H
N
O
H
N
O
Buthylone
bk‐MBDB
O
Methylone
bk‐MDMA
Metabolism Studies in Vitro
centrifugation
9,000g
centrifugation
100,000g
S9
suspension of
broken cells
Microsomes:
Cytosol:
S9 fraction:
Cell culture:
nuclei,
mitochondria
Cytosol
Microsomes
CYP, FMO, UGT
COMT, SULT, GST, NAT
CYP, FMO, COMT, UGT, SULT, GST, NAT
Enzymes and transporters
11
Metabolism Studies in Vivo
Metabolism in Rats & Humans
O
O
NH
O
R
O
NH2
O
CH3
R
O
Methylone R = CH3
Butylone R = CH2‐CH3
OH
NH2
O
R
O
O
O
O
CH3
NH
HO
HO
NH
H3C
R
CH3
HO
R
O
HO
NH
CH3
H3C
R
O
(Meyer/Wilhelm/Peters/Maurer, Anal Bioanal Chem, 2010)
Detection of Butylone & Methylone in Urine
12
Data Evaluation ‐ AMDIS
Total Ion
Chromatogram
(GC‐MS)
Purified Spectra
“Components”
AMDIS
Identified “Targets” user target
library generated e.g. from:
MPW2011 Library
Detection of Butylone & Methylone in Urine
O
HO
NH
CH3
H3C
R
O
7.5212 Butylone‐M (demethylenyl‐methoxy‐) 2Ac
Unknown spectrum
in patient urine
Reference spectrum HLM studies
MPW_tox2011 target library
Identification of Rare Poisons in Urine
Ions
108
150
192
Methemoglobinemia
and Rhabdomyolysis:
New Designer Drugs?
unknown
Spectrum
13
Identification of Rare Poisons in Urine
peak underlying spectrum
reference library spectrum
O
O
H3C C
C CH3
N
H
N
H
Maurer/Pfleger/Weber MPW_2015 Library
10,300 entries
5,000 metabolites
runs on all
GC‐EI‐MS types!
Drug
Urine
Turboflow
TF ISQ
MPW_tox2015 (AMDIS)
TF LIT LXQ
LC‐MSn (MS2/MS3)
MWW_tox2014
TF Q‐Exactive
LC‐HR‐MS/MS
MHMW_tox2015
14
SUSA Detection by LC‐MSn Using MWW
100
TF LXQ
TIC MS fullscan
fullscan, 100‐800
DDA MS² and MS³
HESI II, positive
Relative Abundance
Hypersil Gold
(1.9 µm, 100 x 2.1 mm)
Mobile Phase
A: NH4+COO‐ 10 mM
B: ACN, 0.1% HCOOH
0
100
Relative Abundance
TIC MS²
0
100
TIC MS³
Relative Abundance
3,000 MS1/ 2/ 3 Spectra per Run
0
0
1
2
3
4
5
6
7
8
9
10
11
Time [min]
12
13
14
15
16
17
18
19
20
21
(Wissenbach/Meyer/Weber/Remane/Maurer, Anal Bioanal Chem, 2011, 2011)
Detection of JWHs by LC‐MSn SUSA II
Detection of JWHs by LC‐MSn SUSA II
2014
15
Authentic Urine Sample (PP), ToxID Result
Actual RT Compound Name
2.4
3.5
3.7
4.2
4.6
5.6
5.7
5.9
6.8
6.9
7.2
7.5
7.5
7.5
7.8
8.6
8.8
8.9
9.3
9.4
9.4
10.0
10.0
10.4
10.6
10.7
11.0
11.0
11.0
11.9
12.0
12.2
12.2
12.6
13.0
13.4
14.0
14.1
14.4
14.4
14.6
14.7
14.9
14.9
Compound Info
m/z
SI
RSI
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Parent
Metabolite
Parent
Metabolite
Metabolite
Parent
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Parent
Parent
Parent
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Parent
Parent
Metabolite
Parent
Parent
Metabolite
Parent
Metabolite
Metabolite
232
218
368
204
232
235
246
332
560
477
350
287
508
508
301
459
487
473
412
370
391
286
464
297
463
283
311
311
332
477
442
477
477
458
471
285
330
504
358
358
281
295
621
311
868
970
976
983
987
951
838
990
984
700
905
984
899
999
935
749
748
707
924
972
926
894
955
941
846
850
636
849
910
996
661
821
887
742
990
906
934
902
865
912
958
943
919
990
897
972
981
986
990
965
860
991
995
887
997
995
911
999
965
944
753
763
934
980
990
904
993
944
944
941
812
861
922
997
784
913
887
822
996
948
942
978
902
935
960
948
945
990
MS2_Paracetamol-M (sulfate)_wideband_35
MS2_Metamizol-M (dealkyl-)_wideband_35
MS2_Ampicillin-M (ampicilloic acid)_wideband_35
MS2_Metamizol-M (bis-dealkyl-)_wideband_35
MS2_Metamizol-M (-SO3H)_wideband_35
MS2_Lidocaine_wideband_35
MS2_Metamizol-M (N-dealkyl-acetyl-)_wideband_35
MS2_Ciprofloxacin_wideband_35
MS2_Pantoprazole-M (glucuronide)_wideband_35
MS2_Promethazine-M (HO- glucuronide) isomer 1_wideband_35
MS2_Ampicillin_wideband_35
MS2_Promethazine-M (nor-HO-) isomer 1_wideband_35
MS2_Bromazepam-M (HO- glucuronide) isomer 1_wideband_35
MS2_Paroxetine-M (demethylenyl-methyl- glucuronide) isomer 2_wideband_35
MS2_Promethazine-M (HO-) isomer 2_wideband_35
MS2_Trimipramine-M (bis-nor-HO- glucuronide)_wideband_35
MS2_Trimipramine-M (HO- glucuronide) isomer 1_wideband_35
MS2_Trimipramine-(nor-HO- glucuronide)_wideband_35
MS2_Chlorprothixene-M (HO- sulfate) isomer 1_wideband_35
MS2_Pantoprazole-M (O-demethyl-)_wideband_35
MS2_Diisooctylphthalate_wideband_35
MS2_Endogenous biomolecule (286)_wideband_35
MS2_Trimipramine-(nor-HO-alkyl)_wideband_35
MS2_Oxazepam-M (glucuronide)_wideband_35
MS2_Trimipramine-M (bis-nor-HO-)_wideband_35
MS2_Trimipramine-M (HO-)_wideband_35
MS2_Trimipramine-M/artifact (311)_wideband_35
MS2_Paroxetine-M (demethylenyl-methyl-) isomer 2_wideband_35
MS2_Diazepam-M (HO-methyl- glucuronide)_wideband_35
MS2_Mirtazapine-M (glucuronide)_wideband_35
MS2_Levomepromazine-M (nor-O-demethyl- glucuronide)_wideband_35
MS2_Temazepam-M (glucuronide)_wideband_35
MS2_Mirtazapine-M (HO-ring- glucuronide)_wideband_35
MS2_Promethazine_wideband_35
MS2_Paroxetine_wideband_35
MS2_Tolazamide-M (HO-aromatic ring- glucuronide)_wideband_35
MS2_Endogenous biomolecule (358) isomer 1_wideband_35
MS2_Trimipramine-M (nor-)_wideband_35
MS2_Trimipramine_wideband_35
MS2_Trimipramine-M/artifact (N-oxide-) dimer_wideband_35
MS2_Trimipramine-M (N-oxide-)_wideband_35
Authentic Urine Sample (PP), ToxID Result
Actual RT Compound Name
2.4
3.5
3.7
4.2
4.6
5.6
5.7
5.9
6.8
6.9
7.2
7.5
7.5
7.5
7.8
8.6
8.8
8.9
9.3
9.4
9.4
10.0
10.0
10.4
10.6
10.7
11.0
11.0
11.0
11.9
12.0
12.2
12.2
12.6
13.0
13.4
14.0
14.1
14.4
14.4
14.6
14.7
14.9
14.9
Compound Info
m/z
SI
RSI
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Parent
Metabolite
Parent
Metabolite
Metabolite
Parent
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Parent
Parent
Parent
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Metabolite
Parent
Parent
Metabolite
Parent
Parent
Metabolite
Parent
Metabolite
Metabolite
232
218
368
204
232
235
246
332
560
477
350
287
508
508
301
459
487
473
412
370
391
286
464
297
463
283
311
311
332
477
442
477
477
458
471
285
330
504
358
358
281
295
621
311
868
970
976
983
987
951
838
990
984
700
905
984
899
999
935
749
748
707
924
972
926
894
955
941
846
850
636
849
910
996
661
821
887
742
990
906
934
902
865
912
958
943
919
990
897
972
981
986
990
965
860
991
995
887
997
995
911
999
965
944
753
763
934
980
990
904
993
944
944
941
812
861
922
997
784
913
887
822
996
948
942
978
902
935
960
948
945
990
MS2_Paracetamol-M (sulfate)_wideband_35
MS2_Metamizol-M (dealkyl-)_wideband_35
MS2_Ampicillin-M (ampicilloic acid)_wideband_35
MS2_Metamizol-M (bis-dealkyl-)_wideband_35
MS2_Metamizol-M (-SO3H)_wideband_35
MS2_Lidocaine_wideband_35
MS2_Metamizol-M (N-dealkyl-acetyl-)_wideband_35
MS2_Ciprofloxacin_wideband_35
MS2_Pantoprazole-M (glucuronide)_wideband_35
MS2_Promethazine-M (HO- glucuronide) isomer 1_wideband_35
MS2_Ampicillin_wideband_35
MS2_Promethazine-M (nor-HO-) isomer 1_wideband_35
MS2_Bromazepam-M (HO- glucuronide) isomer 1_wideband_35
MS2_Paroxetine-M (demethylenyl-methyl- glucuronide) isomer 2_wideband_35
MS2_Promethazine-M (HO-) isomer 2_wideband_35
MS2_Trimipramine-M (bis-nor-HO- glucuronide)_wideband_35
MS2_Trimipramine-M (HO- glucuronide) isomer 1_wideband_35
MS2_Trimipramine-(nor-HO- glucuronide)_wideband_35
MS2_Chlorprothixene-M (HO- sulfate) isomer 1_wideband_35
MS2_Pantoprazole-M (O-demethyl-)_wideband_35
MS2_Diisooctylphthalate_wideband_35
MS2_Trimipramine-(nor-HO-alkyl)_wideband_35
MS2_Oxazepam-M (glucuronide)_wideband_35
MS2_Trimipramine-M (bis-nor-HO-)_wideband_35
MS2_Trimipramine-M (HO-)_wideband_35
MS2_Paroxetine-M (demethylenyl-methyl-) isomer 2_wideband_35
MS2_Diazepam-M (HO-methyl- glucuronide)_wideband_35
MS2_Mirtazapine-M (glucuronide)_wideband_35
MS2_Levomepromazine-M (nor-O-demethyl- glucuronide)_wideband_35
MS2_Temazepam-M (glucuronide)_wideband_35
MS2_Mirtazapine-M (HO-ring- glucuronide)_wideband_35
MS2_Promethazine_wideband_35
MS2_Paroxetine_wideband_35
MS2_Tolazamide-M (HO-aromatic ring- glucuronide)_wideband_35
MS2_Trimipramine-M (nor-)_wideband_35
MS2_Trimipramine_wideband_35
MS2_Trimipramine-M/artifact (N-oxide-) dimer_wideband_35
MS2_Trimipramine-M (N-oxide-)_wideband_35
Applicability: Comparison of Results
100 %
800
LC‐MSn + GC‐MS
89 %
LC‐MSn
84 %
GC‐MS
Compounds detected
600
400
There are two possible conclusion:
• LC‐MS as good as GC‐MS
• GC‐MS as good as LC‐MS
200
0
Sum compounds detected
Compounds by LC-MS
Compounds by GC-MS
16
Applicability: Comparison of Results
100 %
800
LC‐MSn + GC‐MS
89 %
LC‐MSn
84 %
GC‐MS
Compounds detected
600
400
Complementation of
LC‐MSn and GC‐MS screening
200
0
Sum compounds detected
Compounds by LC-MS
Compounds by GC-MS
Drug
Urine
Turboflow
TF ISQ
MPW_tox2015 (AMDIS)
TF LIT LXQ
LC‐MSn (MS2/MS3)
MWW_tox2014
TF Q‐Exactive
LC‐HR‐MS/MS
MHMW_tox2015
Concept for LC‐HR‐MS/MS Screening
Full scan, HESI +/‐
m/z 66.7‐1,000,
Resolution 35,000
DDA MS/MS on the 5 most intense ions
Evaluation of HR‐MS/MS data by TF TraceFinder
Comparison of measured HR data with reference library data:
‐
‐
‐
‐
[M+H]+/[M‐H]‐
isotopic pattern
5 most intense fragment ions
library match
(Helfer/Meyer/Michely/Weber/Maurer, TIAFT, Buenos Aires, 2014)
17
[M+H]+/[M‐H]‐
Isotopic Pattern Score
Five Most Intense Fragment Ions
18
Library Match
Data Evaluation Successful ?
• isotopic pattern of M+/‐H
• 5 most intense fragments
• library match
Adherence Monitoring for Hypertension Therapy
Reasons for inadequate therapy results
• Wrong medication?
• Missing adherence?
• Therapy resistance 
• Renal sympathetic denervation
=> Adherence monitoring by TurboFlow LC‐HR‐MS/MS
19
• 59 y old patient, male
• severe hypertension
24 h blood pressure profile
100
Moxonidine
0
100
Hydrochlorothiazide
0
100
Minoxidil
0
100
Ramipril‐M (deethyl)
0
100
Torasemide
0
100
Doxazosin
0
100
Amlodipine
0
100
Carvedilol
0
0
1
2
3
4
5
6
Time, min
7
8
9
(Linicus/Kindermann/Helfer/Meyer/Maurer/Mahfoud/Böhm, Int J Cardiol, 2014)
Drug Intake Under Supervision
24 h blood pressure profile
100
Moxonidine
0
100
Hydrochlorothiazide
0
100
Minoxidil
0
100
Ramipril‐M (deethyl)
0
100
Torasemide
0
100
Doxazosin
0
100
Amlodipine
0
100
Carvedilol
0
0
1
2
3
4
5
6
Time, min
7
8
9
(Linicus/Kindermann/Helfer/Meyer/Maurer/Mahfoud/Böhm, Int J Cardiol, 2014)
20
Spot Tests, Confirmation & Quantification
Paraquate
Chloral hydrate etc
(Fujiwara)
NaOH, Na Dithionite
NaOH, Pyridine
GC‐MS
(Trichloroethanol)
UV
Quantification ?
“Serum drug levels … can be helpful in gauging the severity of
intoxication and the need to initiate specific interventions and …
can be useful in monitoring and evaluating the effectiveness of the
treatment procedure.”
(Clinical Toxicology Testing: A Guide for Lab Professionals)
• Quantification of solvents in blood and urine by HS-GC or GC-MS
• Quantification of toxic mushroom poisoning amanitin by LC-HR-MS/MS
• Quantification of identified compounds in blood by GC-MS, LC-MS/MS
• Quantification for TDM by LC-MS/MS
21
Determination of Alcohols
and Solvents in Plasma and Urine
0
For alcohols:
Time [min]
Head space, 80°C
GC-MS, HP-5
30m x 0.53mm &
HP-1 12m x 0.2mm
0.33’
Methanol 1g/L
0.83’
1.47’
iso-Propanol 2g/L
0.49’ Ethanol 3g/L
0.67’ Acetone 2g/L
tert.-Butanol (IS) 0.5g/L
Standards
2
For other solvents:
Head space, 80°C, 90°C
GC-MS, HP-5
30m x 0.53mm &
HP-1 12m x 0.2mm,
40-310°C
Target Screening and Quant. of Toxic Glycols,
GHB, etc. in Plasma by GC‐MS (LLE, TMS)
50 µL Urine or plasma + 10 µL IS
Shaking with 50 µL ACN 2 min
Centrifugation 2 min
20 µL Supernatant into vial
10 min sample
preparation
20 µL DMF + 300 µL BSTFA
Microwave 5 min 450 watt
1 µL GC‐MS ThermoFisher DSQ II
(Meyer/Weber/Maurer, Anal Bioanal Chem, 2011)
22
Target Screening and Quant. of Toxic Glycols, GHB,
etc. in Plasma by GC‐MS (LLE, TMS)
ethylene
glycol
lactic acid
1,2-propylene
glycol
100
GHB
IS
glycolic
acid
triethylene
glycol
Anundance, %
diethylene
glycol
tetraethylene
glycol
40% DEG
0
6.0
7.0
8.0
9.0
Time, min
(Meyer/Weber/Maurer, Anal Bioanal Chem, 2011)
GHB als Szene‐Droge
• GHB = gamma‐Hydroxybuttersäure
• in Techno‐Szene als Liquid Ecstasy bekannt
• Wirkung nicht mit der von Ecstasy zu vergleichen
• wirkt zwar u.U. euphorisierend, aber nicht entactogen
• weitere gängige Namen für GHB: Liquid X, Liquid E, G‐Juice
• Missbraucht als KO‐Tropfen
23
Determination of Amanitin in Urine
by ELISA
(Staack/Maurer, Toxichem. Krimtech, 2001)
Determination of Amanitin in Urine
by TLF‐LC‐HR‐MS/MS
High Selectivity by Exclusion of
Isobaric (not isomeric !) Compounds
8.55 8.66 8.85
8.07 8.19
100
Relative Abundance
tMS2: m/z 919.36 ‐> 919.36 (isolation window 0.5)
7.05
7.51
6.13 6.59
alpha‐amanitin 0.01 mg/L
extracted from urine (C18)
TF Q‐Exactive
(low resolution)
5.21
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
‐Amanitin: R = COOH
a‐Amanitin: R = CONH2
OH
OH
HN
CH CO
H
NH C H C O
NH C H C O
OC
H
HO
NH
CH
O
N
OC
C H NH
S
CO CH
R
N
H
HC
OH C O
NH C O C H NH
H
(Meyer/Michely/Helfer/Maurer, J Chromatogr.,2014)
24
8.55 8.66 8.85
8.07 8.19
100
Relative Abundance
7.05
7.51
6.13 6.59
TF Q‐Exactive
(low resolution)
5.21
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
919.62531
100
Relative Abundance
MS/MS spectrum average 5.0 – 5.5 min
various isobaric biomolecules
919.3626
919.0
919.1
919.2
919.3
919.4
m
TF Q‐Exactive
919.6491
(high resolution)
919.5611
919.5188
919.4705
919.5
919.7415
919.6
919.8173
919.7
919.8
919.9
m/z
8.55 8.66 8.85
8.07 8.19
100
Relative Abundance
7.05
7.51
6.13 6.59
TF Q‐Exactive
(low resolution)
5.21
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
919.62531
100
Relative Abundance
MS/MS spectrum average 5.0 – 5.5 min
various isobaric biomolecules
919.6491
919.3626
919.0
919.1
919.2
919.3
919.4
919.5611
919.5188
919.4705
m
919.5
919.7415
919.6
919.8173
919.7
919.8
919.9
m/z
5.21
100
Relative Abundance
tMS2: m/z 919.36 ‐> 919.3614 (2 ppm)
TF Q‐Exactive
(high resolution)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Time (min)
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
25
Emergency Toxicology
(Meyer GM/Weber/Maurer, Drug Test Anal, 2014)
Emergency Toxicology
Standard Qual/Quant Approach in Blood
100
Relative Abundance, %
OPC‐A
100
OPC‐B
5.0
7.0
Time, min
9.0
11.0
Daily one-point calibration with calibrators stored for up to 4 weeks
Certified control samples run with each analysis (RILIBÄK)
(Meyer GM/Weber/Maurer, Drug Test Anal, 2014)
26
Target Screening and Quantification of Toxic Alkaloids
in Plasma by LC‐MS/MS
Delicious Spice or Toxic Plant ?
Ramsons
(Allium ursinum)
Autumn Crocus
(Colchicum autumnale)
LC-ESI-MS/MS (AB 3200 QTrap)
Colcicine
6.0e5
5.0e5
Physostigmine
Cotinine
Cytisine
Atropine
4.0e5
Nicotine
3.0e5
MRM
Trimipramine-d3
Scopolamine
COT-d3
Coniine
2.0e5
Aconitine
1.0e5
0.0
1
2
3
4
5
6
7
8
191
163
177
180
128
304
290
276
293
298
400
646
9
148
132
80
80
69
138
124
162
171
61
152
105
min
(Beyer/Peters/Kraemer/Maurer, J Mass Spectrom, 2007)
Target Screening and Quantification of Toxic Alkaloids
in Plasma by LC‐MS/MS
LC-ESI-MS/MS (AB 3200 QTrap)
MRM
5.0e5
Trimipramine-d3
Scopolamine
6.0e5
Physostigmine
Cotinine
Cytisine
Atropine
4.0e5
Nicotine
3.0e5
COT-d3
Coniine
2.0e5
Colcicine
Aconitine
1.0e5
0.0
1
2
3
4
5
6
7
8
191
163
177
180
128
304
290
276
293
298
400
646
9
148
132
80
80
69
138
124
162
171
61
152
105
min
(Beyer/Peters/Kraemer/Maurer, J Mass Spectrom, 2007)
27
Multi‐Analyte LC‐MS/MS Procedure for
Identification and Quantification in Plasma
>130 analytes
of different drug classes
Screening
O
N
N
Identification
CH 3
Quantification
CH 3
N
Validation
N
Cl
H3 C
Antidepressants
O
OH
Benzodiazepines
Cl
N
F
Neuroleptics
N
O
O
O
S
N
O
O
O
Analytes in the context
of brain death diagnosis
N
HO
O
N
H
N
H
Oral antidiabetics
sulfonylurea‐type
ß‐receptor antagonist
(Remane/Wissenbach/Meyer/Maurer, Anal Bioanal Chem, 2011a, 2011b, Rapid Comm Mass Spectrom, 2010a, 2010b)
Experimental Design for Validation of
LC‐MS(/MS) Procedures
Run
Selectivity
Matrix effect/
Extraction efficiency/
Process efficiency
Processed sample stability
Linearity
0
10 sources of blank
matrix
2 zero samples
X spiked samples
30 samples
(5 neat standards
5 spiked blank extracts
5 extracts. of spiked blanks, 2
conc. each)
16 injections of extracts
(certain time intervals, 8 at
each of 2 conc.)
36 calibration samples
(6 conc. levels
6 replicates, each)
Total
Run
94 (+ x)
Calibration
samples,
6 levels
Validation samples
Low
Medium
P&A
F/T
Total
High
P&A
LLOQ
Dil.
P&A
F/T
P&A
P&A
(6)
2
6
2
2
6
2
2
24 (+4)
2
(6)
2
‐
2
2
‐
2
2
12 (+4)
3
(6)
2
‐
2
2
‐
2
2
12 (+4)
4
(6)
2
6
2
2
6
2
2
24 (+4)
5
(6)
2
‐
2
2
‐
2
2
12 (+4)
6
(6)
2
‐
2
2
‐
2
2
12 (+4)
7
(6)
2
‐
2
2
‐
2
2
12 (+4)
8
(6)
2
‐
2
2
‐
2
2
12 (+4)
Total
optional
1
120 (+32)
(Peters: Method Validation, in Polettini: LC‐MS in AT, Pharmac. Press, 2006)
Applicability (5 internal standard)
Trimipramine‐d3
12000000
12
0
2
4
8
Time (min)
TCA 2/10
1 Nordoxepin
3
2 Doxepin
3 Nortriptyline
4 Amitriptyline
5 Trimipramine
4
5
10
12
14
Zolpidem‐d6
9000000
16
0
2
4
6
TDMA 3/10
8
Time (min)
10
1 9‐OH‐risperidone
Nordazepam‐d5
3
Citalopram‐d6
1 2
18
Proficiency tests
Trimipramine‐d3
Abundance (counts)
0
6
Norclozapine‐d8
0
Proficiency tests
Nordazepam‐d5
Norclozapine‐d8
Citalopram‐d6
Abundance (counts)
Zolpidem‐d6
2 Risperidone
3 Ziprasidone
4 Perazine
4
12
14
16
18
28
• Quality control
Internal Quality Control
• QM System
• Certified control samples in any quantitative analysis run, etc., etc.
External Quality Control
29
External Quality Control
• Quality control
• Interpretation of the analytical result
Interpretation of Toxicological Analysis
Questions for toxicological interpretation
• Does the mode of action of the toxicant fit with the symptoms ?
 Which diseases may also lead to the shown symptoms ?
 Suffer the patient from further diseases in addition to poisoning ?
 PK/metabolism influenced by shock, insufficiency of liver or kidney ?
 Blood levels influenced by interactions/genetic polymorphisms ?
 Reference values available ?
 Risks for long‐term toxicity ?
30
GTFCh Post Graduate Training Programs
 Clinical Toxicologist GTFCh
 Forensic Toxicologist GTFCh
 Forensic Chemist GTFCh
‐ Five years of experience in practical work
‐ Training courses
‐ Examination
Society of Toxicological and Forensic Chemistry
(www.gtfch.org)
 Training Courses for Technicians
I would like to thank my present and former coworkers
for their excellent work…
31

Maurer

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