FORMATION OF AN INTERFERING SUBSTANCE, 3,4-DIMETHYL-5-PHENYL-1,3-
OXAZOLIDINE, DuRING A PSEUDOEPHEDRINE URINALYSIS

INTRODUCTION

In fatal aircraft accident investigations, postmor-
tem samples collected from the accident victims at
autopsy are submitted to the Federal Aviation
Administration’s (FAA’s) Civil Aeromedical Insti-
tute (CAMI) for toxicological evaluation (Public
Law, 1988). The submitted samples are analyzed for
the presence of drugs, including sympathomimetic
amines (SMAs), suchas ephedrine, pseudoephedrine,
and phenylpropanolamine. An unknown substance,
which interfered with the analysis of pseudoephe-
drine, was found during the analysis ofa urine sample
from a pilot who died in an aviation accident. Under
our gas chromatographic conditions, this substance
had the same retention time as that of pseudoephe-
drine, but its infrared and mass spectra were differ-
ent. The pseudoephedrine’s characteristic O-H and
N-H bond stretchings were missing in the infrared
spectrum of the substance, and its molar mass was 12
atomic mass units (amu) more than pseudoephedrine.

It is known that ephedrine-like amines undergo
cyclization with aliphatic, as well as aromatic, alde-
hydes, forming oxazolidines (Neelakantan, 1971;
Beckett, Jones, and Hollingsbee, 1978; Nishiyama
and Yamada, 1989; and Walker, Wood, Akmal, and
Sharks, 1992). This cyclization reaction involves the
condensation of the amines with an aldehyde, with a
resulting loss of one water molecule (see Fig. 1).
Therefore, the molar mass of the oxazolidine is the
total of the masses of the amine plus the aldehyde,
minus the mass of water. Since the molar mass of the
unknown compound was only 12 amu more than
that of pseudoephedrine, the increase was potentially
accounted for by the addition of a carbon atom
through the reaction between pseudoephedrine and
formaldehyde.

The problem of aldehydic impurities in solvents is
not new—for example, the presence of trace amounts
of formaldehyde, acetaldehyde, and/or pro-
pionaldehyde has been reported in diethyl ether and
ethyl acetate (Beckett et al., 1978; TOXI-NEWS,
1996). Formaldehyde is also present in some com-
mon grades of methanol. During the analysis of

ephedrine, while using diethyl ether as an extraction
solvent, as many as three peaks corresponding to
oxazolidines of these three aldehydes were observed
by Beckett et al. (1978). Similarly, acetaldehyde as a
contaminant in ethyl acetate has been reported to
potentially react with ephedrine, pseudoephedrine,
and phenylpropanolamine, thereby potentially
affecting their analyses (TOXI-NEWS, 1996). It
appears that a chemical, such as methanol, containing

OH
«_-NHCH,

-HCl

CH,

(+)-Pseudoephedrine-HCI

HCHO

-H,O

(+)-3,4-Dimethyl-5-phenyl-1,3-oxazolidine-HCl

FIG. 1—The chemical scheme for the syn-
thesis of the oxazolidine using (+)-
pseudoephedrine-HCI and formaldehyde
(HCHO). The asterisks indicate chiral cen-
ters (asymmetric carbons) in the molecules.


Carbon 13 NMR in DMSO

Atom Observed (PPM) Calculated (PPM)
C3) 10.6 12.6

cq) 35.6 32.8

C(2) 66.7 68.4

C(5) 84.0 85.6

c(4) 85.3 O17

C9) 127.1 125.8

C(7) 128.5 128.3

C(8) 129.2 128.6

C(6) 135.9 138.8
NMR in DMSO

Atom Observed (PPM) Calculated (PPM)
CH,;(3) 1.00 1.10
CH;(1) 2.12 2.27
CH(2) 3.10 3.50
CH(5) 4.45 4.35
CH(4) 4.48 4.44
CH(9) 7.04 7.19
CH(7) 7.06 TAX
CH(8) TAL 719

C(8)=C(7) O
4 J C5)
ce GO-CW) |
dech Ya
Cd)
C3)

FIG. 5—Comparison of observed NMR spectra with
calculated NMR spectra.


column (12 m x 0.2-mm i.d.; 0.33-mm film thick-
ness) was used. For both analyses, the injection
volume was 1 wL in the splitless mode, with a purge
time of 0.5 min. The GC oven temperature was
increased from 70°C to 160°C at 15°C/min and then
to 290°C at 40°C/min. The final temperature of
290°C was maintained for 1.75 min, totaling an 11-
min run time. Helium was the carrier gas with a flow
of 1 mL/min. The injector temperature was main-
tained at 250°C. The transfer line was set at 280°C.
The retention time of the oxazolidine was 6.4 min
under the GC/FTIR conditions, whereas it was 4.07
min under the GC/MS conditions.

RESULTS AND DISCUSSION

Initial screening of the urine specimen by TOXI-
LAB revealed a spot consistent with the characteristic
Rf value (* 0.14) and the color of pseudoephedrine/
ephedrine. Based on the retention time, the GC/
FID/NPD analysis of the liquid-liquid extract fur-
ther concluded that pseudoephedrine was present in
the specimen. The retention time for the analyte of
interest from the solid-phase extract matched that of
pseudoephedrine during the GC/FTIR and GC/MS
analyses. Gas chromatography-Fourier transform
infrared spectrometry (GC/FTIR) revealed the pres-
ence of characteristic absorptions of aromatic C-H,
monosubstituted benzene, -CH,-, ~CH,, and C-
O-C groups. The characteristic pseudoephedrine
O-H stretch at 3600 cm" and the N-H stretch at
3400 cm" were absent in the analyte spectrum (Figs.
2A & 2B). However, a strong absorbance associated
with an ether (C-O-C) stretch at 1100 cm? was
observed (Fig. 2A). The gas chromatography-mass
spectrometry (GC/MS) electron ionization (EI) spec-
tra disclosed a base peak of 71 amu and positive
chemical ionization (PCI) gave a molar mass of 178
(M + 1). As is shown in Fig. 3A, the unknown
substance has a mass spectrum with a base peak of 71
amu, whereas pseudoephedrine has a base peak at 58
amu (Fig. 3B). PCI mass spectrometry of the un-
known substance exhibited a mass of 178 amu (M +
1), indicating its actual molar mass to be 177 amu
(Fig. 4), which is 12 amu more than the molar mass
of pseudoephedrine (165 amu). The synthesized com-
pound had similar spectral characteristics as that of
the unknown, interfering substance. The 'H and '3C
nuclear magnetic resonance spectra obtained at the
University of Southern Mississippi, Hattiesburg, MS,

were consistent with the nine different peaks of
protons and carbons corresponding to the oxazolidine
molecule (Fig. 5). The calculated elemental analysis
for the artifact, C,,H,,CINO, is: C, 61.82%; H,
7.55%; N, 6.55% and the observed elemental analy-
sis is: C, 61.44%; H, 7.61%; N, 6.55%. The Mass
Spec for MS (70 eV) is M/Z (relative intensity): 71
(100%); 56 (26%); 91 (10%); 117 (7%).

Findings from this study revealed that the presence
of formaldehyde as an impurity in the chemicals used
for analysis has a potential to form a substance that
could interfere with the pseudoephedrine analysis.
Identified as 3,4-dimethyl-5-phenyl-1,3-oxazolidine
(Fig. 1), the substance is formed by a reaction be-
tween formaldehyde and pseudoephedrine, involv-
ing the-OH and —-NH- groups of pseudoephedrine.
This type of reversible reaction has been reported
with ephedrine-like amines and aldehydes, forming
oxazolidines (Neelakantan, 1971; Beckett, Jones, and
Hollingsbee., 1978; Nishiyama and Yamada, 1989;
and Walker and Wood, 1992). The reaction is a
condensation between an aldehyde and a secondary
amine leading to the formation of an intermediate
iminium ion, which subsequently reacts with an
active hydrogen atom forming an oxazolidine. Since
pseudoephedrine has an -NH— group (a secondary
amine) as well as an —OH group (an active hydrogen
atom), the reaction could be referred to as an in-
tramolecular reaction, leading to the formation of the
five-membered cyclic oxazolidine with a 12-amu
increase (Fig. 1). In this reaction, the chirality of the
pseudoephedrine’s two asymmetric carbons does not
change, suggesting that the stereochemistry of the
formed compound remains the same as that of the
starting material. In those SMAs—for example,
amphetamine and methamphetamine—wherein the
—-OH group is absent, the formation of the
oxazolidines is not possible.

Findings from this study clearly emphasize the
importance of using high-quality reagents and of the
identification of unknowns by multi-analytical ap-
proaches, including spectral analyses. The presence
of contaminants in extraction solvents, or in any
other chemical, can result in a false negative or false
positive analytical finding. If the substance formed
from the contaminant is a known drug, then the
findings could be misleading. Therefore, it is prudent
that chemicals of known purity be used during the
drug analysis to maintain a high degree of quality
assurance for a laboratory.


formaldehyde as an impurity might have been used
during the pseudoephedrine urinalysis, causing the
formation of the oxazolidine.

In view of the initial spectral findings and the
potential aldehydic impurity, the oxazolidine was
synthesized using pseudoephedrine and formalde-
hyde. Furthermore, analytical and chemical proper-
ties of the synthesized oxazolidine were compared
with those of pseudoephedrine to elucidate the chemi-
cal structure of the unknown interfering substance.

MATERIALS AND METHODS

Materials

All reagents and solvents were of analytical grade
and were of the highest available purity. These
chemicals, standards, and other agents were obtained
from commercial sources. (+)-Pseudoephedrine as
hydrochloric acid salt was supplied by Sigma Chemi-
cal Co., St. Louis, MO. Supplies for the thin layer
chromatography were provided by TOXI-LAB, Inc.,
Irvine, CA. CS ChemOffice software for the calcu-
lation of theoretical NMR spectra was supplied by
CambridgeSoft Cambridge, MA.

Oxazolidine—(+)-3,4-Dimethyl-5-phenyl-1,3-
oxazolidine-HCl was synthesized (Fig. 1) in our labo-
ratory and characterized by its melting point,
elemental analyses (Galbraith Laboratories, Inc.,
Knoxville, TN), and spectral analyses. To a 50 mL
portion of methanol, 2.0 g (9.9 mmol) of
(+)-pseudoephedrine-HCI and 1.2 g (40 mmol) of
formaldehyde (3 mL of 37%) solution were added.
This homogeneous mixture was then stirred for 15
min at the ambient temperature, After stirring, the
volume of the mixture was reduced to 2-3 mL, using
a 40°C water bath and a stream of nitrogen gas. To
this concentrate, 2 mL of water was added, and the
solution was extracted three times with 8 mL aliquots
of chloroform. After drying over sodium sulfate, the
chloroform extract was transferred and then evapo-
rated to dryness. The white solid was recrystallized
from a diethyl ether-methanol solvent system. The
product was filtered and dried under vacuum at 40°C
for 10 min, yielding a white crystalline powder (1.7
g; 82%) melting at 173-174°C (dec.).

Screening

Thin layer chromatography: The urine sample
was screened for the presence of drugs following the
TOXI-LAB’s standard recommended procedure for
the analysis of basic drugs.

Gas chromatography: A 5-mL portion of urine was
mixed with 500 ng of the internal standard
propylamphetamine and was then subjected to liq-
uid-liquid basic extraction with ammonium hydrox-
ide (>10.00 pH) followed by extraction with 5 mL of
chloroform. The chloroform layer was transferred to
another test tube and was washed with 5 mL of 0.1N
hydrochloric acid. The acidic aqueous layer was
transferred and made basic (>10.00 pH) with ammo-
nium hydroxide. To this mixture, an equal volume
of chloroform was added and gently mixed. The
chloroform layer was removed and evaporated down
to approximately 25 pL using a stream of nitrogen.
One pL of the evaporate was injected into a 5890
Model Series II Hewlett Packard gas chromatograph
(GC), functioning under our standard laboratory
GC screening conditions. The GC was equipped
with flame ionization and nitrogen phosphorus de-
tectors (FID/NPD). A crosslinked 5% phenyl me-
thy silicone column (15 mx 0.25-mm i.d.; 0.25-mm.
film thickness) was used.

Confirmatory Analysis

A reaccessioned urine sample was subjected to
solid phase separation, following the manufacturer’s
(Bond Elut CertifyTM, Varian Sample Preparation
Products, Harbor City, CA) recommended proce-
dure for isolation of basic drugs. During this proce-
dure, methanol was used in various steps. The eluates
were evaporated and subjected to analyses using a
Hewlett Packard 5890 Series II Gas Chromatograph
equipped with a HP 5965B infrared detector (GC/
FTIR) and using a Hewlett Packard 5890 Series II
Gas Chromatograph in combination with a 5989A
Mass Spectrometer (GC/MS). Further analysis was
performed on the extract using the GC/MS PCI
technique.

The column used for the GC/FTIR analysis was a
Hewlett Packard HP1 crosslinked 100% methyl si-
loxane column (15 m x 0,.32-mm i.d.; 1-mm film
thickness). For the GC/MS analysis, a Hewlett
Packard ULTRA 1 crosslinked 100% methyl siloxane


Abundance
71

40000

30000
20000
10000 43

0 |

91

iL

ll
105 +
f

7
132
£

a

162
148
i

40 60

80

100

120 140

160

180 200

FIG. 3A—The mass spectrum obtained after the GC/MS
analysis of the urine extract.

58
Abundance: a
40000
30000.
20000:
aT
40000: 405
5 a, 132
417 148
ee | are a PL a
40 60 80 100 420 140 160 180, 200
FIG. 3B—The mass spectrum of (+)-pseudoephedrine
subjected to the GC/MS analysis.
‘Abundance 178
40000
30000
20000
148
10000. n (
100
3 i 7 107 | 162 |
L? ’
0 T T t T T T T —
60 30 100 120 140 160 180 200220

FIG. 4—The PCl-based GC mass spectrum obtained for the urine extract.


DOT/FAA/AM-99/15 Formation of an Interfering

Office of Aviation Medicine Substance, 3 4-D imethyl-5-
Washington, D.C. 20591 Phenyl-1,3-Oxazolidine, During
A Pseudoephedrine Urinalysis

Russell J. Lewis
Edwin F. Huffine
Arvind K. Chaturvedi
Dennis V. Canfield
Jerry Mattson

Federal Aviation Administration
Civil Aeromedical Institute
Oklahoma City, Oklahoma 73125

May 1999

Final Report

This document is available to the public
through the National Technical Information
Service, Springfield, Virginia 22161.

U.S. Department
of Transportation

Federal Aviation
Administration




REFERENCES

Beckett AH, Jones GR, Hollingsbee DA (1978). Deg-
radation of (-)-ephedrine in solution and during
extraction with diethyl ether. J Pharm Pharmac
30:15-9.

Finar IL (1973). Organic chemistry: the fundamental
principles, vol 1, Gth ed. Longman Group Lim-
ited: London, p 375.

Gould ES (1959). Mechanism and structure in organic
chemistry. Holt, Rinehart and Winston: New
York, p 410.

Neelakantan L (1971). Asymmetric synthesis. II. Syn-
thesis and absolute configuration of oxazolidines
derived from (-)-ephedrine and aromatic alde-

hydes. J Org Chem 36:2256-60.

Nishiyama T, Nishikawa T, Yamada F (1989). A
stereochemical investigation of 2-methyl-and 2,5-
dimethy!-3-phenyl-1,3-oxazolidines using NMR.
J Heterocyclic Chem 26:1687-90.

Public Law (1988). Aviation Safety Research Act of
1988, Public Law 100-591 [H.R. 4686]. 100th.
U.S. Cong., 2nd Sess., November 3, 1988.

TOXI-NEWS (1996). Ethyl acetate contamination.
TOXI-NEWS. Irvine, CA: TOXI-LAB, Volume
15, Number 1, Winter 1996, pp. 5 & 7.

Walker RB, Wood DM, Akmal MM, Sharks E (1992).
Effects of oxazolidines derived from (-) ephedrine
in the rat. Gen Pharmac 23:729-32.


100.0 +
99.5
99.0
98.5
98.0
975

97.0: T 7 T +
4000 3500 3000 2800, = 2000 1500 1000
Frequency [ori ]

Transmittance {%T}

FIG. 2A—The infrared spectrum obtained in the process of the
urine extract GC/FTIR analysis.

100.0
99.5
99.0
98.5
98.0

Transnittance (*%T)

97.5

97.0 } T r
4000 3500 3000 2500 2000 1500 1000
Frequency (cm)

FIG. 2B—The infrared spectrum of (+)-pseudoephedrine
subjected to the GC/FTIR analysis.




Technical Report Documentation Page

1. Report No.
DOT/FAA/AM-99/15

2. Government Accession No.

3. Recipient's Catalog No.

4. Title and Subtitle
Formation of an Interfering Substance, 3,4-Dimethyl-5-Phenyl-1,3-

Oxazolidine, During a Pseudoephedrine Urinalysis

8, Report Date

May 1999

6. Performing Organization Code

7, Author(s)

Lewis, RJ., Huffine, E.F., Chaturvedi, A.K., Canfield, D.V.,
and Mattson, J.

8. Performing Organization Report No.

9. Performing Organization Name and Address
FAA Civil Aeromedical Institute
P.O. Box 25082

Oklahoma City, Oklahoma 73125

10. Work Unit No. (RAIS)

11. Contract or Grant No.

12. Sponsoring Agency name and Address

Office of Aviation Medicine
Federal Aviation Administration
800 Independence Ave., S.W.
Washington, D.C. 20591

13. Type of Report and Period Covered

14, Sponsoring Agency Code

15. Supplemental Notes

This work was accomplished under the approved task AM-B-98-TOX-202.

16, Abstract

During fatal aviation accident investigations, bio-samples from the victims are submitted to the FAA Civil
Aeromedical Institute for drug analysis. In the process of one such analysis, an unknown substance was found in a
urine sample. Its initial analyses by thin layer chromatography and by liquid-liquid extraction/gas chromatography
(GC) disclosed the presence of pseudoephedrine. Subsequent analyses of the reaccessioned sample by solid phase
separation/GC Fourier transform infrared/GC mass spectrometry indicated that the retention times of the unknown
substance matched with those of pseudoephedrine. However, its infrared and mass spectra were different—the -OH
and —NH- groups were missing, a C-O-C group was present, and the molar mass was 12 atomic mass units (amu)
more than that of pseudoephedrine. A subsequent literature search suggested that ephedrine-like amines react with
aldehydes to form oxazolidines. Therefore, the 12-amu increase could be accounted for by condensation of
pseudoephedrine with formaldehyde. Since this aldehyde is present in various grades of methanol, and methanol was
used during the solid phase separation, 3,4-dimethyl-5-phenyl-1,3-oxazolidine was synthesized by using (+)-
pseudoephedrine-HCI and formaldehyde. The analytical and spectral findings of the synthesized compound were
consistent with those of the unknown interfering substance, confirming that it was the oxazolidine. Aldehyde
contaminants can transform the drug of interest and may result in misidentification of a compound not originally
present in specimens. Therefore, chemicals used in analyses should be of the highest available purity, and a multi-
analytical approach should be adopted to maintain a high degree of quality assurance.

17, Key Words

Forensic Science, Toxicology, Interfering Substance,
Oxazolidine, Pseudoephedrine, Urinalysis,

Aircraft Accident Investigation

76. Distribution Statement

Document is available to the public through the
National Technical Information Service
Springfield, Virginia 22161

19. Security Classif. (of this report) 20. Security Classif, (of this page)

Unclassified Unclassified

21. No. of Pages 22. Price

10

Form DOT F 1700.7 (8-72)

Reproduction of completed page authorized



NOTICE

This document is disseminated under the sponsorship of
the U.S. Department of Transportation in the interest of
information exchange. The United States Government
assumes no liability for the contents or use thereof.