1
3
2
d C and d H isotope ratios in amphetamine 1655
cool. Crystal formation was encouraged by scratching the
cooled solution with a glass rod. The crystals were filtered,
washed with water and cold methanol and dried, yielding
a yellow crystalline solid (65.6 g). The solid was identified
by gas chromatography/mass spectrometry (GC/MS),
against a certified reference standard, as 1-phenyl-2-
Amphetamine synthesis by reductive amination of
16
phenyl-2-propanone with NaBH CN
3
To a solution of ammonium acetate (11.42 g) and methanol
(15.5 mL) was added phenyl-2-propanone (2 g) and then
sodium cyanoborohydride (NaBH CN) (1.8 g). The mixture
3
was stirred for 36 h at room temperature after which water
(1000 mL) containing concentrated HCl (5 mL) was added.
The unreacted phenyl-2-propanone was extracted with DCM
(3 ꢁ 100 mL). The aqueous layer was basified to pH 12 with
dilute NaOH solution (1 M) and extracted with DCM
(3 ꢁ 150 mL). The DCM was removed using a rotary
evaporator leaving a yellow oil (ꢂ1.6 g). The oil was
dissolved in cooled isopropanol (10 mL) and acidified with
concentrated hydrochloric acid (18 M) to pH 3. Diethyl ether
(10 mL) was added resulting in precipitation of a crystalline
material. The crystals were filtered, washed with a 50:50
mixture of isopropanol and diethyl ether and dried yielding
white crystals (1.4 g) which were identified by GC/MS,
against a certified reference standard, as amphetamine.
1
3
nitropropene.
Catalytic reduction of 1-phenyl-2-nitropropene to
14
amphetamine with hydrogen gas
1
-Phenyl-2-nitropropene (2 g), ethanol (70 mL), glacial
acetic acid (200 mL) and platinum oxide (1.0 g) were placed
in a hydrogenation flask which was attached to a Parr
hydrogenator. The flask was evacuated and flushed with
hydrogen three times, charged to a pressure of 45 psi with
hydrogen and mechanically shaken for 3 h until uptake of
hydrogen had ceased. The reaction mixture was filtered to
remove catalyst and the residue was washed with ethanol
(
100 mL). Solvent was removed by rotary evaporation
leaving a dark-yellow oil. To the oil was added water
50 mL) which was then acidified to pH 3 with concen-
(
Stable isotope ratio mass spectrometry
trated HCl (10 M). The resulting mixture was washed with
DCM (3 ꢁ 100 mL) and the washings discarded. The
aqueous phase was basified to pH 12 with dilute sodium
hydroxide solution (1 M) and extracted with DCM
Sample preparation
Approximately 0.5 mg of drug material was weighed into tin
foil capsules (3.3 mm ꢁ 5 mm, IVA Analysentechnik, Meer-
1
3
busch Germany) for d C analysis and 0.2 mg into silver foil
(
3 ꢁ 150 mL). The extracts were combined and the DCM
2
capsules for d H analysis. Capsules were crimped and placed
was removed by rotary evaporation leaving a clear
colourless oil (0.8 g). The oil was dissolved in cold ethanol
with forceps into 96-well plates with the position recorded
against the sample identification number.
(
(
10 mL) and acidified with concentrated sulfuric acid
18 M) to pH 3, resulting in a white precipitate. The
precipitate was filtered and washed with a 50:50 mixture
of cold diethyl ether and ethanol leaving white crystals
Isotopic calibration and quality control of EA/TC-IRMS
measurements
(
0.65 g) which were identified by GC/MS, against a
A Flash elemental analyzer (EA) 1112, with dual combustion
and thermal conversion (TC) capabilities (Thermo, Delft, The
Netherlands), connected to a ConFlo IV interface and Delta V
Plus Mass Spectrometer (Thermo, Bremen, Germany), was
certified reference standard, as amphetamine.
Reduction of 1-phenyl-2-nitropropene to amphetamine
with lithium aluminium hydride
15
13
2
used to determine d C and d H values by continuous flow
for samples synthesized in this laboratory. Data was
acquired using ISODAT 2.5 software (Thermo, Bremen,
Germany). Prior to sequence acquisitions, zero enrichment
was performed using each of the two reference gases. The
standard deviation of nine 20-s gas pulses was determined to
1
-Phenyl-2-nitropropene (2 g) in anhydrous THF (15 mL)
was slowly added to stirred mixture of lithium
a
aluminium hydride (2.8 g) in anhydrous THF (35 mL)
under nitrogen at 0 to 58C. The mixture was gently
refluxed for 7 h, allowed to cool and isopropyl alcohol
(
3 mL), dilute NaOH (1 M, 3 mL) and water (6 mL) were
2 2
be less than 0.1% for CO and 0.5% for H .
Sample sequences for d C analysis were bracketed by
1
3
added. The reaction mixture was filtered to remove
particulate matter and the residue washed with THF
triplicates of a certified reference standard of acetanilide
1
3
(
100 mL). The solvent was removed by rotary evaporation
(d C ¼ ꢃ29.5 ꢄ 0.1%). EA-IRMS performance was verified
leaving a yellow oil. To the oil was added water (50 mL)
and it was then acidified to pH 3 with concentrated HCl
by analysis of the high-purity methylamphetamine HCl
1
3
quality control (d C ¼ ꢃ27.9 ꢄ 0.4%) every five samples. The
1
3
(
10 M). The mixture was washed with DCM (3 ꢁ 150 mL)
d C values, reported as per mille (%) differences from the
and the washings were discarded. The aqueous phase was
basified to pH 12 with dilute NaOH solution (1 M) and
extracted with DCM (3 ꢁ 150 mL). DCM was removed from
the combined extracts by rotary evaporation leaving a
clear colourless oil (1.81 g). The oil was dissolved in cold
ethanol (10 mL) and acidified with concentrated sulfuric
acid (10 M) to pH 3, resulting in a white precipitate. The
precipitate was filtered and washed with a 50:50 mixture
of cold diethyl ether and ethanol leaving white crystals
Vienna Pee Dee Belemnite (VPDB) international standard,
2
were measured relative to high purity (>99.5%) CO gas
1
3
(BOC gases, Sydney, NSW, Australia; d C ¼ ꢃ6.8 ꢄ 0.4%)
that had been calibrated against NBS 19 (Environmental
Isotopes Pty. Ltd., Sydney, NSW, Australia).
2
The d H values, reported as per mille (%) differences
from the VSMOW international standard, were
2
measured relative to ultra-high-purity (>99.99%) H gas
2
(BOC gases; d H ¼ ꢃ340 ꢄ 4%) calibrated in-house and
(
1.6 g) which were identified by GC/MS, against a certified
normalized to certified reference materials: hexatriacontane,
2
2
reference standard, as amphetamine.
(d H ¼ ꢃ247 ꢄ 1%); phenanthrene, (d H ¼ ꢃ84 ꢄ 1%); and
Copyright # 2010 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1653–1658
DOI: 10.1002/rcm