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The determination of methyprylon and its metabolites in biological fluids by gas chromatography
Forensickience,4(1974) 177-182 0 Elsevier Sequoia S.A., lausanne - Printed in The Netherlands
THE DETERMINATION OF METHYPRYLON AND ITS METABOLITES IN BIOLOGICAL FLUIDS BY GAS CHROMATOGRAPHY
S.J. DICKSON Chemistry Division,Department of Scientific and Industrial Research, PrivateBag, Petone (New Zealand)
SUMMARY
A simple gas chromatographic method for the determination of methyprylon and its metabolites in urine has been developed for the investigation of a post-mortem case.
INTRODUCTION
The carbon monoxide content of the blood of a woman found dead after a house fire was surprisingly low (11 per cent saturation). This level is not appreciably greater than that found for smokers’ ,*, which suggested that the woman may have died prior to the outbreak of the tire. Toxicological analysis revealed the presence of small amounts of 2,4-dioxo-3,3-diethyl5-methylpiperidine (methyprylon), an hypnotic sedative, in the blood and urine samples. Four other compounds in the urine were subsequently identified as methyprylon metabolites. A more extensive study was not possible because of the unavailability of tissue samples. Methyprylon (I) is extensively metabolised in the body (Fig. 1) and it has been reported that only about 3 per cent is generally recovered unmetabolised from the urine of man3. The intake of methyprylon must therefore have been considerably greater than indicated by the urine level. Four metabolites of methyprylon have previously been identified in both man and animals4 7’and it seemed probable that these corresponded to the other four compounds detected in the urine. B6sche6 found that it is necessary to know the combined urine level of methyprylon plus metabolites in order to distinguish between toxic and therapeutic administration, Methyprylon levels in biological specimens have been determined by gas chromatography’, quantitative thin-layer chromatography6 and by a calorimetric method3. The metabolite concentrations have been determined by quantitative thin-layer chromatography6. With the exception of the gas chromatographic method, developed for methyprylon but not its metabolites, the methods lack either specificity or accuracy. A gas
S..l. DICKSOi\
178
Fig. 1. The metabolism of methyprylon.
chromatographic
method
levels of methyprylon
was therefore
and its metabolites
developed
for the accurate determination
of the
in biological fluids.
MATERIALS AND METHODS
Preparation of metabolites 2,4-Dioxo-3,3diethyl-5methyltetrahydropyridine Bromine solution
(0.6 g) in carbon
of methyprylon
(50 ml), lithium
tetrachloride
(0.7g) in carbon
carbonate
(II)
(20 ml) was added drop-wise to a refluxing tetrachloride
(1 .Ig) and lithium
bromide
(20 ml). Dimethylformamide (lg) were then added simulta-
neously to the resulting colourless solution. The mixture was refluxed for 30 minutes and the carbon
tetrachloride
and dimethylformamide
were removed
under
vacuum.
The
residue was dissolved in water and extracted three times with ethyl acetate. The ethyl acetate was removed under vacuum leaving a residue of metabolite II which was purified by recrystallization from petroleum ether (80-100°C). The yield was 90 per cent. The observed melting point was 143-144”C,
lit. value, 142-143°C
(ref. 4).
2,4-Dioxo-3,3diethyl-S-hydroxymethyZtetrahydropy~dine Metabolite
(III)
III was prepared according to the method of Bernhard et al.’ The observed
melting point was 13%140°C,
lit. value, 136-137°C
(ref. 4).
2,4-Dioxo-3,3diethyl-5carboxytetrahydropy;idine (IV) This was prepared according to the method of Bernhard et a1.4 The observed melting point was 165-166’C,
lit. value, 165’C (ref. 4).
2,4,6-Trioxo-3,3diethyl-5methylpiperdine (V) Metabolite V was prepared according to the method melting point was 180-181”C, lit. value, 181’C (ref. 9).
of Rahnert’.
The observed
DETERMINATION
179
OF METHYPRYLON
Analysis of urine and blood (3 ml) were extracted with ether (3 X 25 ml) according to the methods described by Curry’ ’ . An equal volume of concentrated hydrochloric acid was added to the urine which was then refluxed for one hour and re-exAcidified
tracted
urine
(25 ml) and blood
with ether (3 X 25 ml). The ether fractions
were combined,
concentrated
and
separated into weak and strong acid, base and neutral extracts. These extracts were investigated by ultraviolet spectroscopy, gas chromatography and thin-layer chromatography as detailed below. Two blank urine samples were spiked with 0.25 mg I, 0.25 mg II, 0.75 mg III, 0.75 mg IV and 0.25 mg V, extracted as above, and the extracts analysed by gas chromatography as described below. Gas chromatography The gas chromatograms
were recorded
on a Perkin-Elmer
Fll
(FID) gas chromato-
graph fitted with a 3 per cent CHDMS and a 2 per cent 0V17 glass column. These were operated
under the following conditions.
diameter) nitrogen
on 60-80
Three per cent CHDMS (150cm X 3mm internal
mesh AW-DMCS Chromosorb
W; at 205”C, 25 ml per minute
carrier gas. Two per cent OV 17 (150cm X 3mm internal
diameter)
of
on 60-80
mesh AW-DMCS Gas Chrom Q; at 175”C, 25ml per minute of nitrogen carrier gas. Aliquots (1 ~1) of the concentrated extracts (100 ~1) and ethanolic solutions of the authentic metabolites, which all contained butobarbitone as an internal standard, were injected into each column in turn. Thin-layer chromatography Aliquots
of the toxicological
extracts
and authentic
metabolites
were spotted
Schleicher and Schiill F1500 IS 254 plates. The developing solvent was n-butyl saturated with 5N ammonia5. The spots were detected by ultraviolet irradiation spraying with 2N methanolic
potassium
hydroxide
onto
alcohol and by
followed by m-dinitrobenzene5.
Spectroscopy Ultraviolet spectra were recorded spectra on an AEI-MS 30 spectrometer
on a Beckman DK-2A spectrophotometer, mass and nuclear magnetic resonance spectra (NMR) on
an HA-60 spectrometer. RESULTS AND DISCUSSION
The NMR, mass spectroscopy, ultraviolet spectroscopy, gas and thin-layer chromatography results for the synthesized metabolites are listed in Tables I-III. The NMR and
180
S.J. DICKSON
mass spectroscopy
data are consistent
tabolites
in Fig. 1. The ultraviolet
depicted
with the synthesized
compounds,
being the me-
spectra of the post-mortem
extracts
were
consistent with those expected for mixtures of methyprylon metabolites. The gas and thin-layer chromatography data confirmed this finding. The levels of methyprylon and its metabolites in the urine were determined by gas chromatography metabolites
on the 3 per cent CHDMS column
as standards. The different metabolites
did not interfere The ultraviolet of the absorption
with the determination
absorption
with reference
to the synthesized
were well resolved on this column (IV
of III as they appeared in different
observed for the post-mortem
extracts was within 20 per cent
calculated for each extract from the levels determined
and the respective
method
thus provides a simpler and more accurate method for determining
determine
than the thin-layer
of the metabolites.
by gas chromato-
graphy
metabolites
absorbances
extracts).
This gas chromatographic methyprylon
method developed earlier6. It is, of course, not possible to
the levels of methyprylon
and its metabolites
by ultraviolet
spectroscopy
as
their ratios in the urine are not constant6.
TABLE I MASS SPECTRA AND NUCLEAR METHYPRYLON METABOLITES
MAGNETIC RESONANCE
SPECTRA OF SYNTHESIZED
Compound (molecular weight)
Mass spectraa
NMR b
II (181)
41, 55, 69, 83 98,110 124,138,153,166,181
0.74 (triplet,
III (197)
41, 55, 69, 83, 98,109,123,136, 151,168,182,197
IV (211)
45, 55, 69, 83, 93,114,129, 139,152,165,183,196,211
v (197)
41, 55, 69, 83, 98,112,126 139,154,169.182,197
J = 7.5 Hz) (doubIet,J= 1.2 Hz) (quartet, J = 7.5 Hz) (quartet, J = 1.2 Hz) (triplet, J = 7.6 Hz) (quartet, J = 7.6 Hz) (doubIet,J = 1.0 Hz) (triplet, J = 1 .O Hz) (triplet, J = 7.6 Hz) (quartet, J = 7.6 Hz) (singlet) (triplet, J = 7.5 Hz) 1.84(singlet) 1.92 (quartet, J = 7.5 Hz) No signal observed for the proton (Yto the methyl group presumably because of exchange with the solvent via an en01 structure
1.73 1.89 7.25 0.78 1.90 4.25 7.42 0.80 1.97 8.52 0.74
a, Reported according to the method of Finkle et al. 1 ‘The base peak is underlined. b, In ppm relative to tetramethyl silane internal standard. Spectra were recorded in C2Ha02H.
DETERMINATION
181
OF METHYPRYLON
TABLE II ULTRAVIOLET
SPECTRA OF METHYPRYLON
AND ITS METABOLITES _
Compound
a h max (A 1% I cnJ Methanol
O.lN H&?‘,
0. IN NaOH
I
294 (3)
288 (2.3)
II III IV
307 (410) 303 (410) 309 (460)
311 (400) 303 (410) 309 (490)
V
305b 265’
270 (360)
375 285 375 363 358 238 309
(1.5) (2.7) (460) (480) (530) (520) (650)
a, A itFm is the absortion by a 1% solution with a 1 cm path length. b, Relative intensities were very susceptible to acid and base impurities.
Bosche6 established the level of methyprylon and its metabolites in the urine of 38 cases involving methyprylon intoxication by quantitative thin-layer chromatography. Of the two unspecified cases involving fatal dosages, one involved first degree burns. He concluded that although it was possible to distinguish between therapeutic and toxic levels the degree of poisoning could not always be determined. In the present investigation the combined level of methyprylon and its metabolites (85 mg/l) in the urine is certainly higher than a therapeutic concentration reported of 20 mg/l (ref. 6). While this level is several times less than most of the non-fatal levels reported by Bosche, it would result in heavy sedation. In this sedated state the presence of only slightly elevated levels of carbon monoxide in the woman’s blood could have contributed
TABLE III CHROMATOGRAPHIC Compound
DATA FOR METHYPRYLON Thin-layer chromatography
AND ITS METABOLITES
Gas chromatography (Retention time relative to butobarbitone)
(Rfl
I II III IV V
0.80 0.90 0.72 0.20 0.45
2% OVI 7
3% CHDMS
0.64 0.69 2.0 0.72 0.70
0.21 0.36 0.44 0.43 0.53
182
S.J. UIChaUN
TABLE IV LEVELS OF METHYPRYLON AND METABOLITES IN URINE Compound
mg/ Level found
Experimental recovery
1.6
I II III IV V
Corrected level
82 +5% 95 i4%
5.1 25 20 9.5
9 6
73fl%
34
80f6% 83 +7%
25
11
to her death. In fact, if the woman had been a non-smoker levels found would be substantially The drug levels determined state
of intoxication
adaptable meaningful
of the
in the urine do not provide an accurate assessment of the deceased.
The method
to tissue samples and if sufficient picture
then the carbon monoxide
elevated. developed,
however,
should
be
blood or viscera had been available a more
of the level of methyprylon
intoxication
could have been obtained.
With the small blood sample supplied it was possible to detect only traces of methyprylon and 2,4-dioxo-3,3-diethyl-S-methyltetrahydropyridine. REFERENCES J.R. Goldsmith, R.R. Beard and B.D. Dimnan, Epidemiologic appraisal of carbon monoxide effects, in Effects of Chronic Exposure to Low Levels of Carbon Monoxide on Human Health, Behaviour and Performance, National Academy of Sciences and National Academy of Engineering, Washington, D.C., 1969, p. 47. A. Stolman and C.P. Stewart, Gases and vapors (Poisons through inhalation), in C.P. Stewart and A. Stolman (Eds), Toxicology - Mechanisms and Analytical Methods, Vol. 1, Academic Press, New York, 1960, p. 42. G. Xanthaky, A.W. Freireich, W. Matusiak and L. Lukash, Hemodialysis in methyprylon poisoning, J. Am. Med. Assoc., 198 (1966) 1212-1213. K. Bernhard, M. Just, A.H. Lutz and J.P. Vuilleumier, iiber das Verhalten in 5-Stellung methylierter DioxodPthyI-hydropyridine im Stoffwechsel, Helv. Chim. Acta, 40 (1957) 436-444. J. BGsche and G. Schmidt, Der Methyprylon-Metaboht 2,4,6-Trioxo-3,3-diathyl-S-methyl-piperidin, ArzneimitteEForschung,
16 (1966)
548-550.
J. B&che, Konzentrationen von Methyprylon und dessen MetaboIiten im Harn bei Vergiftungsf8Ien, Arzneimittel-Forschrng, 19 (1969) 123-125. L.R. Goldbaum and T.J. Domanski, Detection and identification of micrograms of neutral drugs in biological sampIes,J. Forens. Sci., 11 (1966) 233-242. K. Bernhard, G. Brubacher and A.H. Lutz, Synthese einiger 14C-signierter Dioxodiathyl-hydropyridine und Untersuchungen iiber deren Verteilung, VerweiIzeit und Ausscheidung bei der Ratte, Helv. Chim. Acta, 37 (1954) 1839-1856. H. Rohnert, Substituted 2,4,6&oxopiperidines, Chem. Abstr., 55 (1961) 1663e. 10 A.S. Curry, Poison Detection in Human Organs, Charles C. Thomas, Illinois, U.S.A., 1963, pp. 41 and 50. 11 B.S. Fir&e, D.M. Taylor and E.J. Bonelh, A gc/ms reference data system for the identification of drugs of abuse,J. chromutogr. Sci., 10 (1972) 312-333.
THE DETERMINATION OF METHYPRYLON AND ITS METABOLITES IN BIOLOGICAL FLUIDS BY GAS CHROMATOGRAPHY
S.J. DICKSON Chemistry Division,Department of Scientific and Industrial Research, PrivateBag, Petone (New Zealand)
SUMMARY
A simple gas chromatographic method for the determination of methyprylon and its metabolites in urine has been developed for the investigation of a post-mortem case.
INTRODUCTION
The carbon monoxide content of the blood of a woman found dead after a house fire was surprisingly low (11 per cent saturation). This level is not appreciably greater than that found for smokers’ ,*, which suggested that the woman may have died prior to the outbreak of the tire. Toxicological analysis revealed the presence of small amounts of 2,4-dioxo-3,3-diethyl5-methylpiperidine (methyprylon), an hypnotic sedative, in the blood and urine samples. Four other compounds in the urine were subsequently identified as methyprylon metabolites. A more extensive study was not possible because of the unavailability of tissue samples. Methyprylon (I) is extensively metabolised in the body (Fig. 1) and it has been reported that only about 3 per cent is generally recovered unmetabolised from the urine of man3. The intake of methyprylon must therefore have been considerably greater than indicated by the urine level. Four metabolites of methyprylon have previously been identified in both man and animals4 7’and it seemed probable that these corresponded to the other four compounds detected in the urine. B6sche6 found that it is necessary to know the combined urine level of methyprylon plus metabolites in order to distinguish between toxic and therapeutic administration, Methyprylon levels in biological specimens have been determined by gas chromatography’, quantitative thin-layer chromatography6 and by a calorimetric method3. The metabolite concentrations have been determined by quantitative thin-layer chromatography6. With the exception of the gas chromatographic method, developed for methyprylon but not its metabolites, the methods lack either specificity or accuracy. A gas
S..l. DICKSOi\
178
Fig. 1. The metabolism of methyprylon.
chromatographic
method
levels of methyprylon
was therefore
and its metabolites
developed
for the accurate determination
of the
in biological fluids.
MATERIALS AND METHODS
Preparation of metabolites 2,4-Dioxo-3,3diethyl-5methyltetrahydropyridine Bromine solution
(0.6 g) in carbon
of methyprylon
(50 ml), lithium
tetrachloride
(0.7g) in carbon
carbonate
(II)
(20 ml) was added drop-wise to a refluxing tetrachloride
(1 .Ig) and lithium
bromide
(20 ml). Dimethylformamide (lg) were then added simulta-
neously to the resulting colourless solution. The mixture was refluxed for 30 minutes and the carbon
tetrachloride
and dimethylformamide
were removed
under
vacuum.
The
residue was dissolved in water and extracted three times with ethyl acetate. The ethyl acetate was removed under vacuum leaving a residue of metabolite II which was purified by recrystallization from petroleum ether (80-100°C). The yield was 90 per cent. The observed melting point was 143-144”C,
lit. value, 142-143°C
(ref. 4).
2,4-Dioxo-3,3diethyl-S-hydroxymethyZtetrahydropy~dine Metabolite
(III)
III was prepared according to the method of Bernhard et al.’ The observed
melting point was 13%140°C,
lit. value, 136-137°C
(ref. 4).
2,4-Dioxo-3,3diethyl-5carboxytetrahydropy;idine (IV) This was prepared according to the method of Bernhard et a1.4 The observed melting point was 165-166’C,
lit. value, 165’C (ref. 4).
2,4,6-Trioxo-3,3diethyl-5methylpiperdine (V) Metabolite V was prepared according to the method melting point was 180-181”C, lit. value, 181’C (ref. 9).
of Rahnert’.
The observed
DETERMINATION
179
OF METHYPRYLON
Analysis of urine and blood (3 ml) were extracted with ether (3 X 25 ml) according to the methods described by Curry’ ’ . An equal volume of concentrated hydrochloric acid was added to the urine which was then refluxed for one hour and re-exAcidified
tracted
urine
(25 ml) and blood
with ether (3 X 25 ml). The ether fractions
were combined,
concentrated
and
separated into weak and strong acid, base and neutral extracts. These extracts were investigated by ultraviolet spectroscopy, gas chromatography and thin-layer chromatography as detailed below. Two blank urine samples were spiked with 0.25 mg I, 0.25 mg II, 0.75 mg III, 0.75 mg IV and 0.25 mg V, extracted as above, and the extracts analysed by gas chromatography as described below. Gas chromatography The gas chromatograms
were recorded
on a Perkin-Elmer
Fll
(FID) gas chromato-
graph fitted with a 3 per cent CHDMS and a 2 per cent 0V17 glass column. These were operated
under the following conditions.
diameter) nitrogen
on 60-80
Three per cent CHDMS (150cm X 3mm internal
mesh AW-DMCS Chromosorb
W; at 205”C, 25 ml per minute
carrier gas. Two per cent OV 17 (150cm X 3mm internal
diameter)
of
on 60-80
mesh AW-DMCS Gas Chrom Q; at 175”C, 25ml per minute of nitrogen carrier gas. Aliquots (1 ~1) of the concentrated extracts (100 ~1) and ethanolic solutions of the authentic metabolites, which all contained butobarbitone as an internal standard, were injected into each column in turn. Thin-layer chromatography Aliquots
of the toxicological
extracts
and authentic
metabolites
were spotted
Schleicher and Schiill F1500 IS 254 plates. The developing solvent was n-butyl saturated with 5N ammonia5. The spots were detected by ultraviolet irradiation spraying with 2N methanolic
potassium
hydroxide
onto
alcohol and by
followed by m-dinitrobenzene5.
Spectroscopy Ultraviolet spectra were recorded spectra on an AEI-MS 30 spectrometer
on a Beckman DK-2A spectrophotometer, mass and nuclear magnetic resonance spectra (NMR) on
an HA-60 spectrometer. RESULTS AND DISCUSSION
The NMR, mass spectroscopy, ultraviolet spectroscopy, gas and thin-layer chromatography results for the synthesized metabolites are listed in Tables I-III. The NMR and
180
S.J. DICKSON
mass spectroscopy
data are consistent
tabolites
in Fig. 1. The ultraviolet
depicted
with the synthesized
compounds,
being the me-
spectra of the post-mortem
extracts
were
consistent with those expected for mixtures of methyprylon metabolites. The gas and thin-layer chromatography data confirmed this finding. The levels of methyprylon and its metabolites in the urine were determined by gas chromatography metabolites
on the 3 per cent CHDMS column
as standards. The different metabolites
did not interfere The ultraviolet of the absorption
with the determination
absorption
with reference
to the synthesized
were well resolved on this column (IV
of III as they appeared in different
observed for the post-mortem
extracts was within 20 per cent
calculated for each extract from the levels determined
and the respective
method
thus provides a simpler and more accurate method for determining
determine
than the thin-layer
of the metabolites.
by gas chromato-
graphy
metabolites
absorbances
extracts).
This gas chromatographic methyprylon
method developed earlier6. It is, of course, not possible to
the levels of methyprylon
and its metabolites
by ultraviolet
spectroscopy
as
their ratios in the urine are not constant6.
TABLE I MASS SPECTRA AND NUCLEAR METHYPRYLON METABOLITES
MAGNETIC RESONANCE
SPECTRA OF SYNTHESIZED
Compound (molecular weight)
Mass spectraa
NMR b
II (181)
41, 55, 69, 83 98,110 124,138,153,166,181
0.74 (triplet,
III (197)
41, 55, 69, 83, 98,109,123,136, 151,168,182,197
IV (211)
45, 55, 69, 83, 93,114,129, 139,152,165,183,196,211
v (197)
41, 55, 69, 83, 98,112,126 139,154,169.182,197
J = 7.5 Hz) (doubIet,J= 1.2 Hz) (quartet, J = 7.5 Hz) (quartet, J = 1.2 Hz) (triplet, J = 7.6 Hz) (quartet, J = 7.6 Hz) (doubIet,J = 1.0 Hz) (triplet, J = 1 .O Hz) (triplet, J = 7.6 Hz) (quartet, J = 7.6 Hz) (singlet) (triplet, J = 7.5 Hz) 1.84(singlet) 1.92 (quartet, J = 7.5 Hz) No signal observed for the proton (Yto the methyl group presumably because of exchange with the solvent via an en01 structure
1.73 1.89 7.25 0.78 1.90 4.25 7.42 0.80 1.97 8.52 0.74
a, Reported according to the method of Finkle et al. 1 ‘The base peak is underlined. b, In ppm relative to tetramethyl silane internal standard. Spectra were recorded in C2Ha02H.
DETERMINATION
181
OF METHYPRYLON
TABLE II ULTRAVIOLET
SPECTRA OF METHYPRYLON
AND ITS METABOLITES _
Compound
a h max (A 1% I cnJ Methanol
O.lN H&?‘,
0. IN NaOH
I
294 (3)
288 (2.3)
II III IV
307 (410) 303 (410) 309 (460)
311 (400) 303 (410) 309 (490)
V
305b 265’
270 (360)
375 285 375 363 358 238 309
(1.5) (2.7) (460) (480) (530) (520) (650)
a, A itFm is the absortion by a 1% solution with a 1 cm path length. b, Relative intensities were very susceptible to acid and base impurities.
Bosche6 established the level of methyprylon and its metabolites in the urine of 38 cases involving methyprylon intoxication by quantitative thin-layer chromatography. Of the two unspecified cases involving fatal dosages, one involved first degree burns. He concluded that although it was possible to distinguish between therapeutic and toxic levels the degree of poisoning could not always be determined. In the present investigation the combined level of methyprylon and its metabolites (85 mg/l) in the urine is certainly higher than a therapeutic concentration reported of 20 mg/l (ref. 6). While this level is several times less than most of the non-fatal levels reported by Bosche, it would result in heavy sedation. In this sedated state the presence of only slightly elevated levels of carbon monoxide in the woman’s blood could have contributed
TABLE III CHROMATOGRAPHIC Compound
DATA FOR METHYPRYLON Thin-layer chromatography
AND ITS METABOLITES
Gas chromatography (Retention time relative to butobarbitone)
(Rfl
I II III IV V
0.80 0.90 0.72 0.20 0.45
2% OVI 7
3% CHDMS
0.64 0.69 2.0 0.72 0.70
0.21 0.36 0.44 0.43 0.53
182
S.J. UIChaUN
TABLE IV LEVELS OF METHYPRYLON AND METABOLITES IN URINE Compound
mg/ Level found
Experimental recovery
1.6
I II III IV V
Corrected level
82 +5% 95 i4%
5.1 25 20 9.5
9 6
73fl%
34
80f6% 83 +7%
25
11
to her death. In fact, if the woman had been a non-smoker levels found would be substantially The drug levels determined state
of intoxication
adaptable meaningful
of the
in the urine do not provide an accurate assessment of the deceased.
The method
to tissue samples and if sufficient picture
then the carbon monoxide
elevated. developed,
however,
should
be
blood or viscera had been available a more
of the level of methyprylon
intoxication
could have been obtained.
With the small blood sample supplied it was possible to detect only traces of methyprylon and 2,4-dioxo-3,3-diethyl-S-methyltetrahydropyridine. REFERENCES J.R. Goldsmith, R.R. Beard and B.D. Dimnan, Epidemiologic appraisal of carbon monoxide effects, in Effects of Chronic Exposure to Low Levels of Carbon Monoxide on Human Health, Behaviour and Performance, National Academy of Sciences and National Academy of Engineering, Washington, D.C., 1969, p. 47. A. Stolman and C.P. Stewart, Gases and vapors (Poisons through inhalation), in C.P. Stewart and A. Stolman (Eds), Toxicology - Mechanisms and Analytical Methods, Vol. 1, Academic Press, New York, 1960, p. 42. G. Xanthaky, A.W. Freireich, W. Matusiak and L. Lukash, Hemodialysis in methyprylon poisoning, J. Am. Med. Assoc., 198 (1966) 1212-1213. K. Bernhard, M. Just, A.H. Lutz and J.P. Vuilleumier, iiber das Verhalten in 5-Stellung methylierter DioxodPthyI-hydropyridine im Stoffwechsel, Helv. Chim. Acta, 40 (1957) 436-444. J. BGsche and G. Schmidt, Der Methyprylon-Metaboht 2,4,6-Trioxo-3,3-diathyl-S-methyl-piperidin, ArzneimitteEForschung,
16 (1966)
548-550.
J. B&che, Konzentrationen von Methyprylon und dessen MetaboIiten im Harn bei Vergiftungsf8Ien, Arzneimittel-Forschrng, 19 (1969) 123-125. L.R. Goldbaum and T.J. Domanski, Detection and identification of micrograms of neutral drugs in biological sampIes,J. Forens. Sci., 11 (1966) 233-242. K. Bernhard, G. Brubacher and A.H. Lutz, Synthese einiger 14C-signierter Dioxodiathyl-hydropyridine und Untersuchungen iiber deren Verteilung, VerweiIzeit und Ausscheidung bei der Ratte, Helv. Chim. Acta, 37 (1954) 1839-1856. H. Rohnert, Substituted 2,4,6&oxopiperidines, Chem. Abstr., 55 (1961) 1663e. 10 A.S. Curry, Poison Detection in Human Organs, Charles C. Thomas, Illinois, U.S.A., 1963, pp. 41 and 50. 11 B.S. Fir&e, D.M. Taylor and E.J. Bonelh, A gc/ms reference data system for the identification of drugs of abuse,J. chromutogr. Sci., 10 (1972) 312-333.