1 "The impact of enantioselective analyses in clinical and forensic toxicology" Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012 C * A D C B C * A B C D Isomers Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012 Isomers Chemical identities with the same molecular formula C x H y O z Constitutional isomers The same atoms attached in different sequences Stereoisomers Isomers with the same connectivety Differs in their spatial arrangement Enanatiomers Stereoisomers which are mirror images Diasteriomers Stereoisomers which not are mirror images
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The impact ofenantioselectiveanalyses in clinical …..."The impact ofenantioselectiveanalyses in clinical and forensic toxicology" Martin Josefsson, Research Chemist (Ph.D.)Waters
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"The impact of enantioselective analyses in clinical and forensic toxicology"
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
C*
A
D
C
B
C*
A
B
C
D
Isomers
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
IsomersChemical identities with the
same molecular formulaCxHyOz
Constitutional isomersThe same atoms attached
in different sequences
StereoisomersIsomers with the same connectivetyDiffers in their spatial arrangement
EnanatiomersStereoisomers which
are mirror images
DiasteriomersStereoisomers which not are mirror images
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
Pharmacodynamics-what a drug do with the human body
Chirality in pharmacology and toxicology 2:2
Absorption- active transportDistribution- protein bonding- tissue distributionMetabolism- phase I (oxidation/reduction/hydrolysis)- phase II (conjugation)Excretion
metabolism is often highlystereoselective
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
Pharmacokinetics-what the human body do with a drug
passive diffusion is notstereroselective
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Enantioselective bioanalysis
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
more complicated than conventional analyses “a four to five time factor in cost, time and effort”
• Detection no sensitive on-line detectors are stereoselective (UV, Fluorescence, MS)
• Chromatography (or Capillary Electrophoresis)separation by differences in chemical and physically propertiesenantiomers are equal while diasteromers differs
enantiomers can not be separated by conventional chromatography (RP/NP) but diastereomers can
the rule of three point interaction (attractive or repulsive), x-y-z
Stereoselective (chiral) analysis 1:2
Prosreagents applicable for many analytesseparation on conventional chromatographyor by capillary electrophoresis
Consrequires highly (enantiomeric) pure reagents risk for racemisation during derivatisation
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
indirect methods• Conversion to diastereomers
ion-pairing (dynamic)derivatisation (static)
R-isomer R,R-derivative+ R-reagent
S-isomer S,R-derivative
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Stereoselective (chiral) analysis 2:2Pros
many different stationary phases are available no chemical conversion is neededreduced risk for racemisation
Consexpensive columns (1000-3000 euro) often poor column efficiency, N (long analyses)often hard to predict the stereoselectivity
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
direct methods• Chiral GC
chemically modified capillary columns(less common compared to LC)
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
Chiral Chromatography by HPLC
B-cyclodextrin (Cyclobond I, Astec)250 x 4.6 mm, isocratic within 30 minMeOH/100mM citric acid TEA (55:45, pH 6.3)Fluorescence detection (240/300 nm)
Carlsson B et. al, Linköping Sweden
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Impact of stereoselectivity in toxicology 2:3
do we know what drug thatwas taken? (prohibited or legal)
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
• Illicit drugslegal drug prescribed as a pure enantiomer“home made” copy drugs as racemic mixtures
the true identity of a new designer drug (RC)
Illicit drugs – amphetamines
ü S-amphetamine,Metamina – ADHDü R-amphetamine, metabolite for Seliginine – Parkinson’s diseaseü rac-amphetamine (or other ratios) from illicit drugs - Abuse
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
H
N
CH 3
CH 3
3
H
N
CH3
CHH
H
N
CH3
HH
H CH3NH
Selegilin
Dm-selegilin
R-amfetamin
R-metamfetamin
H
N
CH 3
CH 3
3
H
N
CH3
CHH
H
N
CH3
HH
H CH3NH
Selegilin
Dm-selegilin
R-amfetamin
R-metamfetamin
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amphetamines cont…
Samples R/S-ratio
Case 1,25
Ref. (abuse case) 1,34
Metamina patients 0.10 (S-)
Selegilin patients 11.2 (R-)
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
Chiral derivatisation and GC-MS
N-trifluoroacetyl-l-prolylkloridHP5-MS 30 m 0.25mm/0.25µm
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
• Reference materialmany drugs are not commercially available as pure enantiomershigh costs
• Chromatographic selectivitycomplete baseline separation could be hard to achievesmall changes in mobile phase composition could give drastic change in chromatography
• Requires sensitive detection the “pure” enantiomer is 50% of the racemate (often other ratio 75:25, 90:10)detection under non-optimized conditions (e.g. pH, type and content of organic solvent)MS-detection not always applicable (e.g. normal phase conditions)
• Complex bioanalytical methods chiral parent drug and chiral metabolites must be separated under the same conditionschiral phases often more sensitive for long term matrix effects
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Thank you for your attention!
Martin Josefsson, Research Chemist (Ph.D.) Waters Nordic MS Symposium, Jurmala 11-12/9 2012
Further reading
Chiral Toxicology: It’s the Same Thing…Only Different (Review)Silas W. Smith (2009) Tocixol Sci 110(1)4-30
Chiral recognition by enantioselective liquid chromatography: Mechanisms and modern chiral stationary phases (Review)Michael Lämmerhofer (2010) J Chrom A 1217:814-856