Preparation • PCP and pethidine solutions: the compounds are separately dissolved in appropriate amounts of methanol; a 10–20 µL aliquot is spiked into 1 mL of a whole blood or urine spe
Trang 1© Springer-Verlag Berlin Heidelberg 2005
by Akira Ishii and Yoshinao Katsumata
Introduction
Phencyclidine ( PCP) (> Fig 8.1), a synthetic arylcyclohexylamine hallucinogen, had been
fi rst applied as an anaesthetic to animals and then to humans for a short period PCP is known
by street names of “ angel dust” and “ crystal” Illicit use of PCP fi rst appeared during mid-1960s along the West Coast, and then peaked in the United States in 1979; illicit PCP use declined by
1992 However, daily use of PCP has remained stable among young school seniors over the past decade; PCP is thus being an important drug of abuse [1–3] It is being regulated as a subclass compound of narcotics and stimulants (DEA Class II)
PCP in both antemortem and postmortem specimens is being analyzed by immunoassays [4–9], GC [10–13], GC/MS [14–18], GC/MS/MS [19, 20], HPLC [21] and CE [22] In this chapter, a detailed procedure for simple GC/MS analysis of PCP in blood and urine is pre-sented
Structures of PCP and pethidine (IS).
⊡ Figure 8.1
Reagents and their preparation
i Reagents
PCP hydrochloride and pethidine ( meperidine) hydrochloride (internal standard, IS) can be purchased from Sigma (St Louis, MO, USA) with suitable legal documentation Bond Elut Glass columns are obtained from Varian (Harbor City, CA, USA) Other chemicals to be used are of analytical grade
Trang 2242 Phencyclidine
ii Preparation
• PCP and pethidine solutions: the compounds are separately dissolved in appropriate amounts
of methanol; a 10–20 µL aliquot is spiked into 1 mL of a whole blood or urine specimen
• 1 M NaHCO3 solution: 8.4 g of NaHCO3 is dissolved in distilled water to prepare 100 mL solution
• Chloroform/methanol (9:1, v/v): a 100-mL volume of the mixture is prepared
GC/MS conditions
GC columna: a FactorFour VF-5ms fused-silica capillary column (30 m × 0.25 mm i d., fi lm thickness 0.25 µm, Varian, Harbor City, CA, USA)
GC conditions; instrument: a Varian CP-3800 gas chromatograph with a split-splitless in-jector (Walnut Creek, CA, USA); column (oven) temperature: 100 °C (1 min)→ 20 °C/min→
300 °C; injection temperature: 250 °C; carrier gas: He; its fl ow rate: 1.0 mL/min; injection: splitless mode for 1 min, followed by the split mode (split ratio: 50)
MS conditions; instrument: a Varian Saturn 2000 ion-trap tandem mass spectrometer b connected with the above GC; ionization: positive ion EI; electron energy: 70 eV; emission cur-rent: 10 A; multiplier off set: 230 V; detector voltage: 1.6 kV; scan time: 0.6 s; transfer tempera-ture: 240 °C; manifold temperatempera-ture: 45 °C; trap temperatempera-ture: 210 °C
Procedure [18]
i To 1 mL of whole blood or urine specimen containing PCP, are added 100 ng of pethidine (IS, methanolic solution) and 8 mL distilled water, followed by mixing well For a whole blood specimen, it is necessary to confi rm the complete hemolysis A 1-mL volume of 1 M NaHCO3 solution is added to the above mixture to bring its pH to about 8
ii A 10-mL volume of methanol and 10 mL distilled water are passed through a Bond Elut Glass column to activate it Th is procedure is repeated at least twice
iii Th e above mixture is loaded onto the column, and the column is washed with 20 mL dis-tilled water
iv PCP and IS are slowly eluted with 3 mL of chloroform/methanol (9:1) into a glass vial
v A small amount of upper aqueous layer is carefully removed by aspiration with a Pasteur pipette Th e organic layer (chloroform) is evaporated to dryness under a stream of nitro-gen Th e residue is dissolved in 50 µL methanol; a 2-µL aliquot of it is injected into GC/MS being operated in the mass chromatographic mode
vi Combined ions at m/z 242 plus 200 are analyzed for PCP and those at m/z 246 plus 232
plus 218 are analyzed for IS from 4 to 12 min of retention time
vii A calibration curve is constructed by adding various concentrations of PCP and 100 ng IS
to the vials containing 1 mL of blank whole blood or urine and 8 mL distilled water each, followed by the above procedure Th e number of diff erent concentrations of PCP should not be smaller than 4 Th e calibration curve is composed of PCP concentration
on the horizontal axis and peak area ratio of PCP to IS on the vertical axis Th e peak
ar-ea ratio of a test specimen is applied to the calibration curve to calculate its concentra-tion
Trang 3243 Phencyclidine
Assessment of the method
> Table 8.1 shows EI mass spectra of PCP and IS In this method, combined ions at m/z 242
([M–1]+) plus 200 and those at m/z 246 ([M–1]+) plus 232 plus 218 are used for PCP and IS, respectively Th e mass chromatograms of PCP and IS are shown > Fig 8.2.
Th e detection limit (S/N = 3) was about 5 ng/mL for PCP According to NIDA guidelines, the cutoff level of PCP in urine samples is 25 ng/mL Th e toxic concentrations of PCP in blood were reported to be 7–240 ng/mL; the fatal blood levels were 1–5 µg/mL [23] Th us, the present
meth-od can be suffi ciently applicable for detection and quantitation of toxic levels of PCP in blood
Th e recoveriy of PCP using the Bond Elut Glass column was about 100 % for whole blood [18]
⊡ Table 8.1
EI mass spectra of PCP and pethidine (IS)
Compound m/z (% peak intensitiy)
PCP 200 (100) 242 (84) 84 (24) 91 (18) 186 (13)
IS 246 (100) 71 (98) 172 (84) 232 (63) 218 (46)
Mass chromatograms for PCP and pethidine (IS) extracted from whole blood In this system,
ions at m/z 200 plus 242 (PCP) and at m/z 246 plus 232 plus 218 (IS) were used at the retention
time of 4–12 min The amounts of PCP and IS spiked into 1 mL blank whole blood were 25 and
100 ng, respectively.
⊡ Figure 8.2
Poisoning case, and toxic and fatal concentrations
A 28-year-old white man [24], who had had a history of drug abuse, exhibited bizarre behav-ior on an airline fl ight; he was transferred to the University of California, San Diego Medical
Trang 4244 Phencyclidine
Center At admission, he stared straight ahead, following commands but not responding verbally; the levels of serum creatinin kinase and aspartate aminotransferase were more than 100-times the normal limits On hospital day 2, he became rigid, diaphoretic and had
a temperature reaching 39.2 °C; he was treated for neuroleptic malignant syndrome On day 4, the serum PCP concentration reached 1,879 ng/mL, the highest level during the course
On day 8, he required intubation due to respiratory failure; his temperature increased to 41.4 °C On day 11 (13 days aft er ingestion), he was found to pass two plastic bags through his rectum; one bag had been ruptured He had probably swallowed the two plastic bags contain-ing PCP powder, one of which had been ruptured to cause the PCP poisoncontain-ing On hospital day 12, he made a rapid neurologic recovery; he was discharged with clear consciousness on day 24
A similar case of protracted coma, caused by an intestinal deposit containing PCP, was also reported; the highest PCP concentration in serum reached 1,690 ng/mL [25]
A fatal PCP poisoning case associated with hypertensive crisis [26], and two sudden death cases during arrest associated with PCP poisoning [27] were reported
Th ree death cases, resulting from the PCP use, were reported in Los Angeles County, 1976; PCP concentrations in blood and the liver ranged from 2.0 to 19.0 µg/mL and from 1.7 to 32.7 µg/g, respectively [28]
Cravey et al reported nine PCP-related deaths; the concentrations in blood and the livers ranged from 0.3 to 12 µg/mL (average: 2.4 µg/mL) and from 0.9 to 80 µg/g (average: 20.1 µg/g), respectively [29]
Notes
a) Any capillary column of 5 % phenylsiloxane/95 % dimethylsiloxane stationary phase can
be used, regardless of manufacturers; but GC/MS grade columns are recommendable b) Any modern type of GC/MS instruments can be used Th e present instrument can be used
as a GC/MS/MS system; the better selectivity can be obtained in the tandem mode
References
1) Zukin SR, Sloboda Z, Javitt DC (1997) Phencyclidine (PCP) In: Lowinson JH, Ruiz P, Millman RB (eds) Substance Abuse: A Comprehensive Textbook, 3rd edn Williams & Wilkins, Baltimore, pp 238–246
2) Gorelick DA, Balster RL (1996) Phencyclidine (PCP) In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: The Forth Generation of Progress Raven Press, New York, pp 1767–1776
3) Schneider S, Kuffer P, Wennig R (1998) Determination of lysergide (LSD) and phencyclidine in biosamples J Chromatogr B 713:189–200
4) ElSohly MA, Stanford DF (1990) Cutoff of 25 ng/mL for the EMIT d.a.u phencyclidine assay J Anal Toxicol 14:192–193
5) Armbruster DA, Krolak JM (1992) Screening for drugs of abuse with Roche ONTRAK assays J Anal Toxicol 16:172–175
6) Asselin WM, Leslie JM (1992) Modification of EMIT assay reagents for improved sensitivity and cost effective-ness in the analysis of hemolyzed whole blood J Anal Toxicol 16: 381–388
7) Sneath TC, Jain NC (1992) Evaluation of phencyclidine by EMIT ® d.a.u.™ utilizing the ETS® analyzer and a 25-ng/
mL cutoff J Anal Toxicol 16:107–108
8) Diosi DT, Harvey DC (1993) Analysis of whole blood for drugs of abuse using EMIT d.a.u reagents and a
Trang 5Mon-245 Phencyclidine
9) Parsons RG, Kowal R, LeBlond D et al (1993) Multianalyte assay system developed for drugs of abuse Clin Chem 39:1899–1903
10) Kandiko CT, Browning S, Cooper T et al (1990) Detection of low nanogram quantities of phencyclidine
extract-ed from human urine preparation of an acetylatextract-ed column packing material for use in gas chromatography with nitrogen-phosphorus detection J Chromatogr 528:208–213
11) Werner M, Hertzman M, Pauley CJ (1986) Gas-liquid chromatography of phencyclidine in serum, with nitrogen-phosphorus detection Clin Chem 32:1921–1924
12) Ishii A, Seno H, Kumazawa T et al (1996) Simple and sensitive detection of phencyclidine in body fluids by gas chromatography with surface ionization detection Int J Legal Med 108:244–247
13) Ishii A, Seno H, Kumazawa T et al (1996) Simple extraction of phencyclidine from human body fluids by head-space solid-phase microextraction (SPME) Chromatographia 43:331–333
14) Nakahara Y, Takahashi K, Sakamoto T et al (1997) Hair analysis for drugs of abuse X VII Simultaneous detection
of PCP, PCHP, and PCPdiol in human hair for confirmation of PCP use J Anal Toxicol 21:356–362
15) ElSohly MA, Little TL Jr, Mitchell JM et al (1988) GC/MS analysis of phencyclidine acid metabolite in human urine J Anal Toxicol 12:180–182
16) Slawson MH, Wilkins DG, Foltz RL et al (1996) Quantitative determination of phencyclidine in pigmented and nonpigmented hair by ion-trap mass spectrometry J Anal Toxicol 20:350–354
17) Stevenson CC, Cibull DL, Platoff GE et al (1992) Solid phase extraction of phencyclidine from urine followed by capillary gas chromatography/mass spectrometry J Anal Toxicol 16:337–339
18) Ishii A, Seno H, Watanabe-Suzuki K et al (2000) Ultrasensitive determination of phencyclidine in body fluids by surface ionization organic mass spectrometry Anal Chem 72:404–407
19) Kidwell DA (1993) Analysis of phencyclidine and cocaine in human hair by tandem mass spectrometry J Forensic Sci 38:272–284
20) Moore CM, Lewis DE, Leikin JB (1996) The determination of phencyclidine in meconium using ion trap mass spectrometry J Forensic Sci 41:1057–1059
21) Cook CE, Brine DR, Jeffcoat AR et al (1982) Phencyclidine disposition after intravenous and oral doses Clin Pharmacol Ther 31:625–634
22) Chen F-TA, Evangelista RA (1994) Feasibility studies for simulatneous immunochemical multianalyte drug as-say by capillary electrophoresis with laser-induced fluorescence Clin Chem 40:1819–1822
23) Schulz M, Schmoldt A (2003) Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics Pharmazie 58:447–474
24) Jackson JE (1989) Phencyclidine pharmacokinetics after a massive overdose Ann Intern Med 111:613–615
25) Young JD, Crapo LM (1992) Protracted phencyclidine coma from an intestinal deposit Arch Intern Med 152:859–860
26) Eastman JW, Cohen SN (1975) Hypertensive crisis and death associated with phencyclidine poisoning JAMA 231:1270–1271
27) Pestaner JP, Southall PE (2003) Sudden death during arrest and phencyclidine intoxication Am J Forensic Med Pathol 24:119–122
28) Noguchi TT, Nakamura GR (1978) Phencyclidine-related deaths in Los Angeles County, 1976 J Forensic Sci 23:503–507
29) Cravey RH, Reed D, Ragle JL (1979) Phencyclidine-related deaths: a report of nine fatal cases J Anal Toxicol 3:199–201
Trang 6© Springer-Verlag Berlin Heidelberg 2005
Introduction
γ-Hydroxybutyric acid ( GHB) is an endogenous compound present in the central nervous system (CNS) and peripheral tissues [1] It is a minor metabolite and precursor of γ-aminobu-tyric acid (GABA), and also a potent inhibitory neurotransmitter [2] GHB has a distinct pre-synaptic receptor in the brain [3], and is an agonist of GABAB receptors [4–8] GHB was fi rst synthesized in 1960 and used as an anaesthetic adjuvant or induction agent [9] Th is applica-tion is still in use in Europe In the United States, GHB has been being evaluated for the treat-ments of narcolepsy and alcohol or opiate withdrawal since the 1970s Recently, Xyrem®, the
fi rst medically formulated GHB, has been approved by the FDA
Th e sodium salt of GHB is an odorless white powder miscible with water and alcohol beverages It is rapidly absorbed from the gastrointestinal tracts, and strongly depresses the CNS, depending on its doses [2] Because of its unique pharmacological actions on the CNS, GHB has become one of many popular “ club drugs” [2] GHB has a strong amne-siac action and is being implicated in a number of drug-facilitated sexual assaults [10] Since
it also has growth hormone releasing eff ects [11], many of body builders are using it as
a steroid alternative; but its eff ects on muscle growth are questionable [12] Because of the popularity of GHB, it became a federally controlled Schedule I substance in the United States in March, 2000 In Japan, the use of GHB has been strictly regulated by the Narcotics and Psychotropic Substances Control Law since October, 2001 In addition to GHB, abuse of γ-butyrolactone ( GBL) and 1,4-butanediol ( 1,4-BD) is on the rise, because both are rapidly biotransformed to GHB [2] Structures of these substances are shown
in > Fig 9.1.
Structures and molecular weights of GHB, GHB sodium salt, GBL and 1,4-BD.
⊡ Figure 9.1
Trang 7248 γ-Hydroxybutyric acid
GHB rapidly disappears from blood with non-linear kinetics Its half-life depends on doses, but usually ranges from 0.3 to 1.0 h [13] As a result, GHB is undetectable within 12 h even aft er a large dose [13] It is diffi cult to determine if GHB comes from an exogenous or endog-enous source, when small amounts are found in blood and urine
In this chapter, a simple and reliable headspace gas chromatographic (GC) method for de-tecting GHB from body fl uids is described a
Reagents and their preparation
Sodium salt of GHB can be purchased from Sigma (St Louis, MO, USA) with suitable legal documentation A 100-mg aliquot of the compound is accurately weighed and dissolved in
100 mL methanol to prepare 1 mg/mL standard solution b in a volumetric fl ask
A 100-mg aliquot of α-methylene-γ-butyrolactone (AMGBL, Aldrich, Milwaukee, WI, USA) is accurately weighed and dissolved in 100 mL methanol to prepare its 1 mg/mL solution (internal standard, IS) b
GC analysis
GC column c: DB-624 (30 m × 0.545 mm i d., fi lm thickness 3 µm, J&W Scientifi c, Folsom,
CA, USA)
GC conditions; instrument: GC-14B (Shimadzu Corp., Kyoto, Japan); detector: fl ame ioni-zation detector (FID); column (oven) temperature: 50 °C (3 min) → 20°C/min → 150 °C (2 min); temperature of the injection port and detector: 150 °C; carrier gas: N2 (100 kPa)
Procedures d
i Body fluids
i A 1-mL volume of each fl uid is mixed with 1 mL distilled water and 100 µL IS solution in a test tube with a screw cap
ii A 300-µL volume of concentrated sulfuric acid is added to the mixture little by little on a Vortex mixer to convert GHB into its cyclized form (cyclized GHB)e
iii Th e acidifi ed mixture is left at room temperature until it becomes cool (approximately
15 min) and then extracted by vigorous shaking with 6 mL of dichloromethane for 15 min using a mechanical shaker
iv Th e upper aqueous phase is discarded by aspiration and the lower organic phase is trans-ferred to a new disposable test tube f
v Th e organic phase is evaporated to 50–100 µL at 35°C under a gentle stream of nitrogen and transferred to a 15-mL glass vial g
vi Th e vial is capped with a Tefl on-coated silicone rubber stopper and sealed with an alu-minum cap
vii Th e vial is heated at 100 °C for 15 min on a heating block and 1 mL of the headspace gas is injected into GC
Trang 8ii Calibrators
i Various volumes (10–100 µL) of the standard solution of sodium salt of GHB are placed in test tubes with screw caps, and evaporated to dryness under gentle streams of nitrogen at room temperature
ii A 1-mL volume of blank blood or distilled water is added to each test tube and mixed on the vortex mixer h
iii Each mixture is then processed according to the procedure for body fl uids mentioned above
Assessment of the method
i Advantages of the method
Th e analytical procedure is simple
No interfering peaks appear even for body fl uids obtained from corpses with moderate decomposition i
Th e GC injection port and column are kept clean by introducing headspace gas to the GC
ii Disadvantages of the method
Harmful concentrated sulfuric acid is used
A caution must be taken not to evaporate dichloromethane containing cyclized GHB to dryness, when it is concentrated
iii Sensitivity and accuracy of the method
A minimum limit of detection (S/N = 3) for GHB was about 0.5 µg/mL Calibration curves
prepared by plotting GHB concentrations versus peak height ratios of GHB to IS were linear in
the range of 0–83 µg/mL Th e regression equations were y = 69.4 x – 1.28 (r = 0.9996) for blood, and y = 115 x + 1.80 (r = 0.9990) for distilled water Th e coeffi cients of variation for blood and distilled water were in the ranges of 6.46–7.38 % and 4.54–6.59 %, respectively Gas chromato-grams for blank blood and blood spiked with 83 µg/mL GHB are shown in > Fig 9.2.
Poisoning cases, and toxic and fatal concentrationsj
In a series of study with sixteen adult patients, Helrich et al [14] found that blood GHB levels
at 244–395 µg/mL were associated with deep sleep; those at 151–293 µg/mL with medium sleep; those at 63–265 µg/mL with light sleep; and those at less than 100 µg/mL with wakeful-ness Sporer et al [15] analyzed serum and urine specimens of fi ft een GHB overdose patients, who had been transferred to a hospital with a Glasgow Coma Scale score of 8 or even lower Serum GHB levels were in the range of 45–295 µg/mL (average: 180 µg/mL) at the time of ad-mission Eleven patients, who showed a Glasgow Coma Scale score of 3, had peak serum GHB levels at 72–300 µg/mL (average: 193 µg/mL) Th e time required for awakening ranged from 30
to 190 min (average: 120 min) Serum GHB levels did not correlate with the degree of coma or the time until awakening GHB levels in urine collected within 30 min aft er arrival were in the range of 432–2,410 µg/mL (average: 1,260 µg/mL) In a male patient who lost consciousness for several hours aft er ingesting GBL, serum GHB concentration was 133 µg/mL [13]
γ-Hydroxybutyric acid
Trang 9250 γ-Hydroxybutyric acid
In six fatal GHB overdose cases, postmortem blood GHB concentrations were in the range
of 27–1,030 µg/mL (average: 228 µg/mL) [13] In a male subject who had committed suicide by ingesting GBL, postmortem blood GHB concentration was 538 µg/mL [13] In two fatalities aft er consuming 1,4-BD, GHB was detected from potmortem blood at concentrations of 280 and 432 µg/mL [13]
Notes
a) GHB determination was also accomplished by GC/MS methods using GHB-d6 as IS [16–21] GHB was either converted to its cyclized form or derivatized with
N,O-bis-(tri-methylsilyl)trifl uoroacetamide (BSTFA) containing 0.1 % trimethylchlorosilane (TMCS)
Th ese methods employ liquid-liquid extraction, solid-phase extraction or solid-phase microextraction HPLC is not in routine use for GHB analysis
b) Th ese standard solutions are stable in capped brown glass bottles at room temperature for
a long period
c) A TC-1 capillary column (dimethylsilicone, 15 m × 0.53 mm i d., fi lm thickness 1.5 µm, GL Sciences Inc., Tokyo, Japan) can be also used for identifying cyclized GHB by GC/MS [22]
Gas chromatograms for blank blood and blood spiked with GHB at a concentration of 83 µg/mL 1: cyclized GHB; 2: IS (AMGBL).
⊡ Figure 9.2
Trang 10d) Th e analytical method described by LeBeau et al [16] has been modifi ed
e) Th e use of a liquid dispenser for adding concentrated sulfuric acid is convenient To pre-vent a blood sample from coagulation, concentrated sulfuric acid should be dispensed while the test tube is vortexed Th e cyclized GHB is identical with GBL If the intake of GBL
is suspected, two specimens should be prepared for detecting the total GBL (unchanged GBL plus GHB biotransformed from GBL) and unchanged GBL For unchanged GBL, the cyclization step with sulfuric acid for GHB is skipped Th e concentration of GHB biotrans-formed from GBL can be calculated by subtracting unchanged GBL concentration from the total GBL concentration
f) For blood samples, denatured proteins cannot be removed by aspiration completely Th e clear organic phase is drawn through the layer of denatured proteins A 3.5-mL polyethyl-ene disposable pipette with a 15-cm length (ELKAY LIQUIPETTE™, Tyco Healthcare Group LP, Mansfi eld, MA, USA) is convenient for the manipulation
g) If the remaining volume of dichloromethane containing cyclized GHB is more than 100 µL
in the vial, the proportion of dichloromethane gas, being contained in the headspace vapor
to be injected aft er heating, becomes too high; this results in much less sensitivity for de-tecting cyclized GHB However, the organic phase should not be evaporated to dryness, because there is a signifi cant loss of cyclized GHB and IS under such conditions
h) Since liquid-liquid extraction of cyclized GHB and IS is moderately aff ected by matrix eff ects, calibration curves should be prepared using blank blood and distilled water for determining GHB levels in blood and other fl uid specimens, respectively
i) When putrefaction advances, interfering peaks appear
j) Endogenous GHB levels in blood of healthy humans are usually very low [22–24] Elian [24] detected 0.17–1.51 µg/mL (average: 0.74 µg/mL) of endogenous GHB from blood specimens of 240 subjects; 0.34–5.75 µg/mL (average: 3.08 µg/mL) of GHB from urine specimens of 670 subjects Similar results were reported for urine of antemortem subjects
by LeBeau et al [25], and Yeatman and Reid [26] GHB levels at 5 µg/mL in blood and
10 µg/mL in urine obtained from antemortem subjects have been proposed as the cutoff limits for judging exogenous use of GHB, GBL or 1,4-BD
When antemortem citrate-buff ered blood specimens were stored at –20 °C for 6–32 months,
GHB was produced de novo at levels of 4–12 µg/mL (average: 9 µg/mL) However, such
de novo production of GHB in blood could not be observed in the presence of EDTA as
anticoagulant during storage at the same temperature [27]; citrate should not be used as anticoagulant for blood samples for GHB analysis Postmortem blood oft en contains higher levels of endogenous GHB [28–30] Substantial amounts of GHB are produced in blood inside a corpse as early as several hours aft er death [30] Sometimes, postmortem blood GHB concentrations reach therapeutic levels at 24–88 µg/mL A gas chromatogram of blood obtained from a deceased, who was not a user of GHB, is shown in > Fig 9.3
Post-mortem enzymatic conversion of succinic acid or putrescine into GHB is suspected [2]
A large portion of endogenous GHB in postmortem blood may be produced during the interval between death and autopsy (sample collection), rather than during storage of blood at 4 °C until analysis [30] Th e author has found that postmortem production of GHB is slower in cerebrospinal fl uid, vitreous humor and urine than in blood Villain et al [21] and Elliott [31] have tentatively set positive cutoff limits of GHB at 50 and 30 µg/mL for postmortem blood, and 10 and 20 µg/mL for postmortem urine, respectively
γ-Hydroxybutyric acid