Alcool et toxicomanies

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Testing for a nightmare or for GHB
Pascal Kintz Laboratoire ChemTox France
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GHB ?-hydroxy-butyric acid

• White powder, with no smell, slightly salty
• Can be found under a liquid presentation
• Stable in aqueous medium
• Easy synthesis (Internet - GBL)

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People exposed to GHB

• - Ravers and club attendees (euphoria,
  hallucinations, muscle relaxation after MDMA
  abuse)
• Bodybuilders (stimulation of the release of
  growth hormone)
• Victims of DFSA (sedation, reduced inhibitions,
  increased libido, amnesia)

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GHB analysis in blood

• 20 µl blood 45 µl acetonitrile 500 ng GHB-d6
• Agitation, centrifugation, evaporation of the
  supernatant
• Derivatization (35 µl BSTFA 1 TMCS)
• GC/MS HP 5973
• HP5 MS capillary column
• m/z 233, 204, 147 and 239 (GHB-d6)
• LOQ 1 mg/l
• Derivatives stable gt 24 hours

Villain, J. Chromatogr. B, 2003, 792, 83
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Excretion of GHB in blood
Administration (oral) of a 60 mg/kg dose of GHB
to a volunteer. Analysis by GC/MS (after
precipitation and silylation)
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Excretion of GHB in urine
Administration (oral) of a 60 mg/kg dose of GHB
to a volunteer. Analysis by GC/MS (after
precipitation and silylation)
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Testing for GHB in hair

• Decontamination 2 x 5 ml dichloromethane
• Segmentation of the hair strand
• Incubation in 0.5 ml NaOH 0.01 M 10 ng GHB-d6.
  Neutralisation with HCl 0.1 ml H2SO4 0.01 M
• Extraction with 3 ml ethyl acetate
• Derivatization with BSTFA 1 TMCS
• Analysis by GC-MS/MS (HP5-MS) on a TSQ 700

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(No Transcript)
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GHB in hair
GHB is found in all hair specimens Concentrations
(n24) in the range 0.5 to 12.0 ng/mg Mean
concentration 2.4 ng/mg No influence of sex
No influence of hair color How to interpret a
concentration of GHB in hair ?
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GHB pattern in hair
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A negative rape case
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An authentic rape case
20-year old girl, raped under the influence Claim
to the Police on day 5 - Collection of hair at
1 month
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Screening of post-mortem blood(routine)
LC/DAD GC/MS pharmaceuticals, drugs of abuse,
pesticides CO-oxymeter HbCO ICP/MS
metals GC/FID ethanol HS/GC/MS volatiles,
cyanide LC-MS/MS buprenorphine,
digitalics GC/MS GHB
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A standard case
Woman, 43-year old, no history of drug
abuse Found drowned in the Mediterrannee sea
(strontium ) Post-mortem delay lt 24
hours Found August 31, autopsy 3
days Toxicological analyses 5 days EtOH 0.65
g/l (blood), 0.45 g/l (bile) GHB 95 mg/l
(cardiac blood), 105 mg/l (bile)
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Interpretation of GHB concentrations
Our case GHB in cardiac blood 95 mg/l Usual
positive cut-off 50 mg/l Deaths due to
GHB Marinetti (2000) heart blood 66
mg/l Kalasinsky (2001) femoral blood 330
mg/l Win (2000) heart blood 538 mg/l
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YES, but
1998, Fieler et al GHB is detectable in
post-mortem blood in cases not known to be
GHB-related Blood concentrations 3.2 to 168
mg/l Discrimination with urine, that should be
negative 1999, Stephens et al Confirmation that
urine can differentiate endogenous production
from exposure 2001, Elliott In 13 GHB-unrelated
fatalities, urine tested positive (0 to 217
mg/l) Which specimen can be helpful for
discrimination ?
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Our study basis of discrimination

• Simultaneous collection of cardiac blood, femoral
  blood, bile and vitreous humor in 71 autopsy
  cases (urine is considered as without interest)
• In all cases, GHB exposure was  excluded  by
  the pathologists
• Delay between death and autopsy 12 - 72 hours
• Analysis of GHB by GC/MS within 48 hours after
  autopsy
• GHB analyzed in all the 71 cardiac blood
• When gt 50 mg/l, analysis in other specimens (when
  available)

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GHB results
GHB positive in all the 71 cardiac blood
samples Concentrations 0.4 to 409 mg/l In most
cases GHB in the range 10 to 40 mg/l In 14
cases GHB gt 50 mg/l analysis of other
specimens
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Corresponding GHB concentrations
All the concentrations in mg/l
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Corresponding GHB concentrations
All the concentrations in mg/l
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Corresponding GHB concentrations
All the concentrations in mg/l
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Compendium of the results

• Cardiac blood (lt 409 mg/l) versus bile (lt 238
  mg/l)
• Cardiac blood (lt 409 mg/l) versus femoral blood
  (lt 44 mg/l)
• Cardiac blood (lt 409 mg/l) versus vitreous humor
  (lt 21 mg/l)
• In 2 GHB-related deaths

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Discussion

• GC/MS after precipitation with acetonitrile and
  silylation is applicable to test all biological
  fluids for GHB
• This method is easy and particularly rapid (lt 1
  hour)
• Conversion of GHB to GBL during preparation
  process did not occur
• Post-mortem blood collection with EDTA (as
  achieved in clinical samples) will not prevent
  endogenous formation
• Bile does not fit the requirements for
  discrimination between exposure and post-mortem
  formation

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Interpretation

• Man, 37-year old, killed in a car crash
• Corpse deposited at the Institute at 2H
• Testing for GHB in femoral blood
• at T 3 H 3.5 mg/l
• at T 24 H 9.2 mg/l
• at T 48 H 17.8 mg/l
• at T 72 H 22.9 mg/l
• After immediate freezing, T 2 weeks 3.8 mg/l
• After immediate freezing, T 8 weeks 4.1 mg/l
• Delay between death and sampling

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Interpretation
Doherty (1978) Formation of GHB from
1,4-butanediol, via oxydation by alcohol
deshydrogenase in the liver Miller (1977) GABA
transamination ---gt succinate semialdehyde ---gt
GHB Sakurada (2002) GHB was shown to increase
with time after death in liver of mouse exposed
to GHB GHB is a product of post-mortem
decomposition
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H
ISOTOPE RATIO MASS SPECTROMETRY
O
O
13C
O
O
COMBUSTION
sample (1?L)
SEPARATION
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Observed range d13C-values of endogenous
substances in human
CO2 gas pulses of known d-values
Street GHB X 5
Standard GHB (CerilliantTM)
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Recommendations if possible !
Elian, 2002 Antemortem blood (n240), endogenous
GHB range 0.34-5.75 mg/l (m0.74)
NaF 1 - 20C, 4 C
body discovery
death
autopsy
analysis
re-analysis
- 20C
as short as possible
Proposals to minimize GHB formation
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Conclusion
A positive (gt 50 mg/l) post-mortem blood GHB
concentration cannot support alone drug
exposure The evaluation of multiple specimens
(femoral blood vitreous humor) facilitates
interpretation If femoral blood gt 50 mg/l and
vitreous humor gt 50 mg/l ---gt GHB exposure In
all other situations inconclusive !!!!