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Journal of Analytical Toxicology, Vol. 35, January/February 2011
Differences in Binding Affinities of MDA, MDMA,
MDEA, Amphetamine, Methamphetamine,
and their Deuterated Analogues to Solid-Phase
Extraction Cartridges*
R.W. Romberg†, A.G. Ntamack, L.J. Blacik, C.M. Kazarian, J. Jacob Snyder, and E.R. Welsh
Navy Drug Screening Laboratory, 320B B Street, Great Lakes, Illinois
Abstract
This study evaluated the potential for partial separation of drugs
from their deuterated internal standards using Cerex® Polycrom™
CLIN II solid-phase extraction (SPE) cartridges. After elution from
the column and derivatization, gas chromatography–mass
spectrometry results showed that the target compound eluted from
the SPE cartridge prior to its deuterated form. This elution
separation effect was greater for 3,4-methylenedioxy-
methamphetamine (MDMA) and methamphetamine (MAMP) than
for the other drugs studied. When the drugs were eluted in 0.5 mL
increments from a 50 mg sorbent bed, no drug appeared in the
first fraction. The drug to internal standard ratios (expected value
1.00) for subsequent fractions collected were 1.30, 1.07, and 0.83
for MDA/MDA-d5; 1.65, 1.18, 0.67, and 0.56 for MDMA/MDMA-
d5; and 1.37, 1.18, and 0.95 for MDEA/MDEA-d6. For d-AMP and
d-MAMP, the expected ratio was 0.40. The subsequent ratios were
0.63, 0.46, 0.35, and 0.34 for d-AMP/d-AMP-d11; and 1.00, 0.59,
0.25, and 0.18 for d-MAMP/d-MAMP-d14. The affinity of d-MAMP-
d14 was shown to be greater than that of d-MAMP-d5, and
deuteration at the propyl end of the molecule was shown to
increase binding more than deuteration on the phenyl group.
Introduction
Amphetamine (AMP) and methamphetamine (MAMP) are
stimulants of the central nervous system (1). Their designer
methylenedioxy analogues, 3,4-methylenedioxyamphetamine
(MDA), 3,4-methylenedioxymethamphetamine (MDMA), and
3,4-methylenedioxyethamphetamine (MDEA) are Schedule I
drugs that cause the user to experience euphoria, sociability,
and a sense of general well-being (2,3). Because of their ad-
* The views expressed in this article are those of the authors and do not necessary reflect the
official policy or position of the Department of the Navy, Department of Defense, or the U.S.
Government. “I am military service member (or employee of the U.S. Government). This work
was prepared as part of my official duties. Title 17 U.S.C. 105 provides that ‘Copyright
protection under this title is not available for any work of the United States Government.’ Title
17 U.S.C. 101 defines a United States Government work as a work prepared by military service
member or employee of the United States Government as part of that person’s official duties.”
† Author to whom correspondence should be addressed. E-mail: robert.romberg@med.navy.mil.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
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dictive nature and adverse health effects, illegal use of these
drugs by members of the United States Armed Forces is detri-
mental to military readiness, due to compromised service
member health and mission performance.
An effective approach to deter the use of illicit drugs, in-
cluding AMP, MAMP, and their designer methylenedioxy ana-
logues in the workforce is to monitor employees and military
personnel through random drug testing (4,5). Forensic analysis
of urine specimens for these amphetamines by the Depart-
ment of Defense (DoD) (6,7) or Department of Health and
Human Services guidelines (8) requires initial immunoassay
test(s) followed by confirmation and quantification by gas chro-
matography–mass spectrometry (GC–MS).
Accurate quantification of AMP, MAMP, MDA, MDMA, MDEA,
and other drugs of abuse has been achieved by the use of
deuterated internal standards that have chemical and physical
properties that are almost identical to that of the parent drug.
The only difference between the drug and its deuterated in-
ternal standard is that one or more hydrogen atoms has/have
been substituted with deuterium. MS is ideally suited for iden-
tification and quantification because the drug and internal
standard can easily be distinguished from one another by the
increased mass resulting from the substitution of deuterium
for one or more hydrogens.
Although the use of deuterated internal standards is the
method of choice in most forensic urine testing laboratories,
some limitations have been reported. MS fragmentation can in-
clude [M – Hn] processes that generate a series of “cluster
ions”, where n is the number of hydrogens involved in the
process. Deuterated internal standards can also include [M –
Dn] processes that are likely to generate ions interfering with
the intensities of some of the ions attributed to unlabeled drug
(9,10). Additionally, ionization efficiencies of a drug and its
deuterated analogue may differ because of differences in con-
centrations and retention times. These effects would not be
expected with other isotopically labeled analogues such as 13C
internal standards (10).
In order to perform quantitative analysis by GC–MS or other
analytical methods, the drug and internal standard were ex-
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Journal of Analytical Toxicology, Vol. 35, January/February 2011

tracted from a sample matrix using procedures such as liquid–
liquid and solid-phase extraction (SPE) (11,12). Various studies
have reported the success of SPE applied to the analysis of
many drugs, including AMP, MAMP, and their methylenedioxy
derivatives (13–20).
There are several commercially available brands of SPE
columns with different resin beds that have been used for ex-
traction of amphetamines (13,14,21). Selecting the appropriate
SPE column depends upon the volume of the specimen, the
specimen matrix, the sensitivity of the analytical method and
the characteristics of the analyte. The Cerex® Polycrom™
CLIN II SPE columns evaluated in this study are mixed mode
with a hydrophobic highly cross-linked divinylbenzene (DVB)
polymer and strong cation exchanger in the form of bound sul-
fonic (SO3–) groups. This column does not need pre-condi-
tioning, and the small 10-µm particle size provides sufficient
urine from Roche Diagnostics (Indianapolis, IN). Cerex Poly-
crom CLIN II with 50 mg (6-mL capacity) of sorbent were
purchased from SPEware (Baldwin Park, CA).
Extraction
All extraction steps were done using Speedisk® 48 positive-
pressure extraction manifolds (SPEware, San Pedro, CA) and
polymer-based cation exchange/mixed-bed extraction columns
(Cerex Polycrom CLIN II, 50-mg resin bed), as described by
Klette et al. (22).
Samples were prepared by pipetting 2.0-mL aliquots of stan-
dards or controls containing MDA, MDMA, and MDEA into 15-
mL glass centrifuge tubes (Kimble Chase, Vineland, NJ). A
100-µL aliquot of internal standard solution containing 0.01
mg/mL each of MDA-d5, MDMA-d5, and MDEA-d6 and 0.75
mL of 2.3 M phosphate buffer (pH 9.0) was added to each

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surface area for drug binding in a 35- or 50-mg resin bed.
The purpose of the current study was to conduct compara-
tive elution analysis of AMP, MAMP, MDA, MDMA, and MDEA
and their respective internal standards using strong cation-ex-
change SPE Cerex PolyCrom CLIN II columns with a 50-mg
resin bed. Experiments were devised to challenge the assump-
tion that amphetamine-like drugs and their internal standards
are eluted equally from SPE columns. Results from this study
can help in determining the appropriate elution volume and
extraction procedures for eluting drugs of abuse from SPE
columns. It was not the intention of this study to provide a
comprehensive comparison of SPE columns from alternate
vendors, but rather to assess the general
elution characteristics of amphetamine-
like drugs on Cerex Polycrom CLIN II
columns.
sample. In a similar manner, 100 µL of internal standard
solution containing 0.01 mg/mL each of AMP-d11 and MAMP-
d14, 1.0 mL of 2.3 M phosphate buffer (pH 9.0), and 3.0 mL of
0.1 M sodium periodate were added to 2.0 mL of urine sample
that contained AMP and MAMP. All samples were vortex mixed
for 5 s. Specimens that were treated with periodate were
capped and incubated for 10 min at room temperature. Peri-
odate oxidation prevents potential interference from over-
the-counter medications containing pseudoephedrine and
ephedrine which may be present in urine specimens (23). All
specimens were centrifuged at 2000 rpm for 5 min to remove
any precipitate.

Experimental Procedures

Materials
Methanol, ethyl acetate, hydrochloric
acid, sodium periodate, sodium hy-
droxide, ammonium hydroxide, and
dibasic potassium phosphate were all ACS
grade purchased from Fisher Scientific
(Fairlawn, NJ). The derivatizing reagents,
heptafluorobutyric anhydride (HFBA) and
R-(–)-α-methoxy-α-(trifluoromethyl)
phenylacetyl chloride (R-MTPA), were ob-
tained from Sigma Aldrich-Fluka (St.
Louis, MO). Stock solutions of d-AMP and
d-MAMP and racemic solutions of MDA,
MDMA, AMP-d6, AMP-d11, AMP-d5 (ring),
AMP-d5 (sidechain), MAMP-d5, MAMP-d14,
MDA-d5, and MDMA-d5 were purchased
from Cerilliant (Round Rock, TX). The
structures of these drugs are shown in
Figure 1. d-AMP, d-MAMP, MDA, MDMA,
and MDEA controls, samples and stan- Figure 1. Chemical structures of the deuterated drugs used in this study.
dards were prepared in certified drug-free

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Journal of Analytical Toxicology, Vol. 35, January/February 2011

Samples were then poured into 50 mg Cerex Polycrom
CLIN II SPE columns and placed onto the Speedisk® appa-
ratus. A low nitrogen flow of 2–3 standard cubic feet/h (SCFH)
was applied to load the columns. Next, 2.0 mL of distilled
water was applied to the columns followed by 2.0 mL 0.1M HCl
(For MDA, MDM, and MDEA) or 0.1M acetic acid (for AMP and
MAMP). Nitrogen gas (2–3 SCFH) was applied after addition of
each reagent, and then the columns were dried for 2–3 min
at 25 psi. The columns were then washed with 2.0 mL of
methanol and 2.0 mL of ethyl acetate. Nitrogen gas (2–3
SCFH) was applied after the addition of each reagent and the
columns were dried for 2–3 min at 25 psi. A solution of
ethyl acetate/ammonium hydroxide (98:2) or methylene
chloride/acetone/triethylamine (80:20:2) was used to elute
AMP and MAMP, whereas a solution of ethyl acetate/
methanol/ammonium hydroxide (80:20:2) was used to elute
mode using an Agilent Technologies (Palo Alto, CA) 6890N
GC and a 5973N mass selective detector. GC injections were
performed with a 7683 automated liquid sampler. Drug to in-
ternal standard ratios were calculated directly from the area
abundance printed on the chromatograms.
MDA, MDMA, and MDEA method
GC separations were performed using an Agilent J&W
(Wilmington, DE) DB-5ms bonded-phase capillary column (15
m × 0.25-mm diameter, 0.25-µm film thickness). The injection
port temperature was 245°C. Initial oven temperature was
maintained at 150°C for 0.10 min then ramped at 30°C/min to
250°C and held for 1 min. All injections were performed with
a split ratio of 12:1 using helium as a carrier gas and a column
flow rate of 0.6 mL/min. The transfer line temperature was
295°C.

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MDA, MDMA, and MDEA. The target compounds were eluted
by gravity flow either directly into 1.5-mL capacity auto-
sampler vials (ALS) vials for MDA, MDMA, and MDEA, or
into 15.0-mL conical tubes for AMP and MAMP. Fractions
with 0.5 mL elution volume were incrementally collected
until the drugs had fully eluted from the column. In some
cases, a low nitrogen flow of 2–3 SCFH was applied for the
collection of the first 0.5 mL fraction. After addition of
0.050 mL of 1% concentrated HCl in methanol to each
eluate, the eluates were evaporated to dryness.
AMP and MAMP method
GC separations were performed using an Ultra 1 (methyl
siloxane bonded-phase) capillary column (25 m × 0.200-mm,
0.33-µm film thickness). The injection port temperature was
220°C. GC separations were performed under isothermal con-
ditions with an oven temperature of 210°C. Samples were in-
jected under split mode using a split ratio of 10:1 using helium
as a carrier gas and a column flow rate of 0.9 mL/min. The
transfer line temperature was maintained at 280°C.

Derivatization
MDA, MDMA, and MDEA samples were Table I. Summary of Assay Parameters
derivatized by adding 0.035 mL HFBA and
0.100 mL ethyl acetate to the ALS vials Confirmation
that were then capped, mixed and incu- Ions Quantitation Qualifier Cutoff Drug/IS Ratio
bated for 10 min at 60–70°C. Samples Compound (m/z) Ion Ratios (ng/mL) (Theoretical)
were removed from the heating block, al-
lowed to cool, and then evaporated to dry- AMP 260, 162, 118 260 118/260, 162/260 100 0.4
ness at 50–60°C under a stream of ni- AMP-d11 264, 130 264 or 130* 130/264 – –
trogen. Samples were reconstituted in 100
AMP-d6† 264, 125 125 –
µL of ethyl acetate for GC–MS analysis. d-
AMP and d-MAMP samples were deriva- AMP-d5 † 265,264 264 265/264 – –
tized by adding 20 µL of R-MTPA solution (side chain)
(500 mg R-MTPA in 8.0 mL acetonitrile) AMP-d5† 260,234 260 234/260 – –
and 200 µL chlorobutane/triethylamine (ring)
(100:7.5) to the 15.0-mL collection glass
tubes. Samples were vortex mixed, MAMP 275, 274, 176 274 275/274, 176/274 100 0.4
capped, and incubated in a heating block MAMP-d14 281, 98 281 98/281 – –
at 60°C for 30 min, at which time 0.100
MAMP-d5† 278, 92 278 92/278 – –
mL anhydrous ethanol was added. Sam-
ples were vortexed for approximately 15 s, MDA 375, 240, 162 240 162/240, 375/240 500 1.0
and then reincubated in a heating block MDA-d5 380, 244 244 380/244 – –
for 30 min at 60°C. Tubes were removed
and evaporated to dryness at 30°C under a MDEA 403, 268, 240 268 240/268, 403/268 500 1.0
stream of nitrogen. Samples were recon- MDEA-d6 409, 274 274 409/274 – –
stituted in 200 µL of ethyl acetate prior to
MDMA 254, 210, 389 254 210/254, 389/254 500 1.0
transfer to ALS vials for GC–MS analysis.
MDMA-d5 258, 394 258 394/258 – –
Analysis
* Used when analyzed with AMP-d6.
All samples were analyzed by GC–MS † Used for relative binding studies, not used in quantitative assays.

under selected ion monitoring (SIM)

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Journal of Analytical Toxicology, Vol. 35, January/February 2011

All quantifications were made using Agilent MSD ChemSta- and its internal standard exhibited elution profiles analogous
tion version E.01 software. Analyte concentrations for speci- to those of MDA and MDA-d5 (Figure 3). Again, early fractions
mens and controls were determined by single-point calibration favor drug elution over internal standard and the cumulative
at the current DoD cutoff. For d-AMP and d-MAMP, the cali- DI ratio after 2.5 mL of elution buffer was 1.16.
bration standard contained 100 ng/mL of each drug. The MDA,
MDMA, and MDEA calibration standard contained 500 ng/mL
of each drug. Table I shows the ions and ion ratios used for
analysis.

Results and Discussion

Because the drug to internal standard ratio is the basis for

quantification, recovery of both the analyte of interest and its
corresponding deuterium labeled internal standard during the

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extraction process is critical. Elution profiles show the amount
of drug eluting off of the column versus volume. The internal Figure 2. Elution of MDA and MDA-d5 on a 50-mg resin bed Cerex Poly-
standard and drug should elute from the SPE cartridge at the crom CLIN II SPE column. The MDA/MDA-d5 ratios for the 2nd, 3rd, and
same time or in the same proportion in order to have consis- 4th fractions collected were 1.30 ± 0.02, 1.07 ± 0.06, and 0.083 ± 0.08;
tent recoveries and quantification. Differences in the binding n = 5.
of the drug and internal standard to the column resin have the
potential to produce quantification errors if all of the drug
and internal standard are not eluted uniformly from the
column.

MDA, MDMA, and MDEA

In order to assess the elution profiles during SPE, urine
specimens containing 500 ng/mL MDA, MDA-d5, MDMA,
MDMA-d5, MDEA, and MDEA-d6 (Figure 1) were applied to
Cerex Polycrom CLIN II SPE columns with a sorbent bed size
of 50 mg and eluted in 0.5-mL increments. Ten 0.5-mL frac-
tions were collected, but over 99% of the drugs were eluted in
the first six or seven fractions. Aliquots containing less than 1%
of drug are not shown in the figures. After derivatization and
analysis by GC–MS, the fraction of drug and internal standard
eluted in each aliquot was calculated from the ion abundances Figure 3. Elution of MDEA and MDEA-d6 on a 50-mg resin bed Cerex Poly-
crom CLIN II SPE column. The MDA/MDA-d5 ratios for the 2nd, 3rd, and
of the quantitative ions for the drug and corresponding internal
4th fractions collected were 1.37 ± 0.03, 1.18 ± 0.04, and 0.95 ± 0.09;
standard (Table I). The theoretical ratio of drug to internal n = 2.
standard (DI ratio) is 1.00; however, the actual DI ratio may be
greater or less than unity because of the actual concentration
of the starting material and any experimental error in the pro-
cedure. Differences in DI ratios between the collected 0.5-mL
fractions indicate non-uniform elution and, therefore, differ-
ences in partitioning of the drugs between the column sorbent
and the elution solvent.
The DI ratios for MDA/MDA-d 5, MDMA/MDMA-d 5, and
MDEA/MDEA-d6 (Figures 2–4, respectively) were greater than
unity for the early fractions and less than unity in the later frac-
tions, indicating that the drug is eluted prior to its deuterated
analogue. For example in Figure 2, a ratio of 1.30 for
MDA/MDA-d5 revealed that the second 0.5-mL fraction con-
tained 57% MDA and 43% of MDA-d5. The third 0.5-mL frac-
tion from this column had a ratio of 1.07 corresponding to 52%
Figure 4. Elution of MDMA and MDMA-d5 on a 50-mg resin bed Cerex
of MDA and 47% of MDA-d5. If elutions were uniform, the DI Polycrom CLIN II SPE column. The MDMA/MDMA-d5 ratios for the 2nd
ratios of these fractions would be unity with each analyte pre- through the 5th fractions collected were 1.65 ± 0.024, 1.18 ± 0.11, 0.67
sent at 50%. Both analytes were fully eluted with 2.5 mL of elu- ± 0.17, and 0.56 ± 0.01; n = 5.
tion buffer with a cumulative DI ratio of 1.12. Similarly, MDEA

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Journal of Analytical Toxicology, Vol. 35, January/February 2011
In contrast, MDMA shows an even more pronounced differ-
ence in elution profile from its internal standard than for MDA
or MDEA (Figure 4). The early fractions again contain more
drug, and the latter contain more internal standard. Specifi-
cally, the successive fractions had DI ratios above and then
below unity, corresponding to 62%, 56%, 45%, and 36%
MDMA for fractions 2–5, respectively. More elution buffer was
required for full elution of analytes with a cumulative DI ratio
of 1.07.
The difference in binding affinity between MDMA and the
other two designer amphetamines implicates the methyl- and
ethyl- groups bound to the amine as significant to such inter-
actions. Because the apparent order of elution does not follow
expected trends in increased hydrophobicity imparted by these
substituents and proportional interaction with the same phase
in the resin (24), the stronger ionic interactions of the basic
moiety with the sulfonate groups of the resin must predomi-
nate. Alkylation of the amine modulates interactions for methyl
and less so for ethyl or no alkyl group for MDMA, MDEA, and
MDA. This follows the expected trend where additional alkyl
groups, in general, increase basicity of amines through induc-
tive effects, yet steric effects prevent such trends from in-
creasing in parallel with increases in the number of sub-
stituents on the amine or their relative sizes. Consequently, the
observed affinities are most likely due to perturbations in ba-
sicity of the amines, where the inductive effect from the amino
methyl group causes MDMA to be more basic than MDA, but
the binding of MDEA is reduced compared to MDMA due to a
steric interaction of the larger amino ethyl group. For all of the
drugs, deuteration caused higher affinity for the packing ma-
terial as discussed later, but the binding of MDMA-d5 was ad-
ditionally increased because of the proximity of the deuteriums
to the nitrogen.
Amphetamine and methamphetamine
Urine specimens containing d-AMP, AMP-d11, d-MAMP, and
MAMP-d14 were applied to 50-mg Cerex Polycrom CLIN II SPE
columns and eluted with 0.5-mL aliquots of ethylacetate/am-
monium hydroxide (98:2). After derivatization and analysis by
GC–MS, the fraction of drug and internal standard eluted in
each aliquot was calculated from the ion abundances of the
quantitative ions for the drug and corresponding internal stan-
dard (Table I). The theoretical DI ratio was 0.40 (100 ng/mL
drug to 250 ng/mL internal standard) with any deviation indi-
cating non-uniform elution and, therefore, differences in
binding affinities to the column sorbent.
As with the designer amphetamines, the drugs and internal
standards were present in different proportions in successive
fractions and the methyl analogue eluted later than non-
methylated compounds, presumably due to the previously dis-
cussed differences in the affinity of each drug to the column
packing material (Figure 5). d-AMP eluted before d-AMP-d11,
and d-MAMP eluted before d-MAMP-d14, indicating that the
deuterated forms of the drug have a greater affinity to the
column packing material. After five fractions, the cumulative
DI ratio for d-AMP/d-AMP-d11 was 0.43 and that for d-MAMP/d-
MAMP-d14 was 0.40.
When the polarity of the elution solvent was changed, there
Page 5
were still differences in the elution profiles between the drug
and its corresponding internal standard. Using methylene chlo-
ride/acetone/triethylamine (80:20:2) as the elution solvent
caused the drugs to elute later and increased the separation be-
tween the drugs and their deuterated analogues compared to
ethylacetate/ammonium hydroxide (98:2). With methylene
chloride/acetone/triethylamine (80:20:2), 8 fractions were re-
quired to collect 99% of d-AMP and d-AMP-d11, and 10 fractions
were required to collect 99% of d-MAMP and 97% of d-MAMP-
d14 (data not shown).
Degree and position of deuterium substitution
Molecules where hydrogen has been replaced by deuterium
have been shown to have slightly different properties com-
pared to the non-deuterated molecules (25). The C-H bond
has a higher vibrational frequency, a longer bond length and is
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weaker compared to the C-D bond. The shorter C-D bond
length results in a more compact electron distribution, a de-
crease in electronic polarizability and a slightly increased
ability of the C-D bond to donate electron density by an in-
ductive effect. Steric effects are expected to be less with deuter-
ated compounds because of the more compact size imparted by
the shorter C-D bonds compared to C-H bonds (26). These
properties can cause small but measurable differences in
binding affinities of the drug and its deuterated internal stan-
dard. Cody et al. (11) reported gas-phase separation between an
analyte and its deuterated analogue using HP-1, HP-5, and
DB-17 GC columns, and concluded that the observed separa-
tions were correlated to the amount of deuteration
To investigate the effect of the number of deuterium atoms
on the binding of AMP and MAMP to Cerex Polycrom CLIN II
SPE columns, the binding of two other deuterated drugs, AMP-
d6 and MAMP-d5 was investigated. Spiked urine specimens
containing d-AMP, d-MAMP, AMP-d6, AMP-d11, MAMP-d5, and
MAMP-d14 were applied to 50-mg Cerex Polycrom CLIN II SPE
Figure 5. Elution profile of d-AMP, d-MAMP, d-AMP-d11, and d-MAMP-d14
on a 50-mg resin bed Cerex Polycrom CLIN II SPE column eluted with
ethyl acetate/ammonium hydroxide (98:2). Drug to internal standard ratios
for the first five fractions were na, 0.63 ± 0.08, 0.46 ± 0.07, 0.35 ± 0.01,
0.34 ± 0.04, 0.35 ± 0.02 for d-AMP/d-AMP-d11 with a cumulative ratio of
0.43 and na, 1.00 ± 0.29, 0.59 ± 0.23, 0.25 ± 0.05, 0.18 ± 0.04 for d-
MAMP/d-MAMP-d14 with a cumulative ratio of 0.40 (n = 3, theoretical DI
ratio = 0.40).
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columns and eluted with 0.5-mL fractions of ethylacetate/am-
monium hydroxide (98:2), as shown in Figures 6 and 7. d-
AMP eluted before d-AMP-d6 and d-AMP-d11 and the drugs
eluted in the following order: d-AMP > d-AMP-d6, d-AMP-d11, d-
MAMP > d-MAMP-d5 > d-MAMP-d14. Relative binding was cal-
culated using the d-AMP-d11/d-AMP-d6 ratio as measured by the
130/125 abundance ratios and d-MAMP-d14/d-MAMP-d5 as mea-
sured by the 281/278 abundance ratios. No drugs were eluted
in the first 0.5-mL aliquot. For the second through sixth frac-
tions, the internal standard ratios were 0.89, 0.94, 0.92, 0.88,
and 0.76 for d-AMP-d11/d-AMP-d6 and 0.79, 0.90, 1.13, 1.28, and
1.36 for d-MAMP-d14/d-MAMP-d5. The increasing d-MAMP-
d14/d-MAMP-d5 ratio with each successive fraction indicates d-
MAMP-d14 has a greater affinity to the packing material than d-
MAMP-d5. The pattern was less apparent for d-AMP-d6 and
d-AMP-d11; however, the d-AMP-d11/d-AMP-d6 ratio was re-
duced for the last fraction which contained 1.5% of the drug.
Figure 6. The fraction of d-AMP, d-AMP-d11, and d-AMP-d6 present in each
0.5-mL fraction eluted from a 50-mg resin bed CEREX Polycrom CLIN II with
ethylacetate/ammonium hydroxide (98:2). For the first five 0.5-mL fractions,
the drug to internal standard ratios were na, 0.55 ± 0.04, 0.39 ± 0.02, 0.30
± 0.01 and 0.29 for d-AMP/d-AMP-d11 and na, 0.55 ± 0.02, 0.42 ± 0.01,
0.32 ± 0.004 and 0.29 ± 0.003 for d-AMP/d-AMP-d6 (DI ratios were nor-
malized to a theoretical DI ratio of 0.40, n = 2).
Figure 7. The fraction of d-MAMP, d-MAMP-d14, and d-MAMP-d5 present
in each 0.5-mL fraction eluted from a 50-mg resin bed CEREX Polycrom
CLIN II with ethylacetate/ammonium hydroxide (98:2). For the first five 0.5-
mL fractions, drug to internal standard ratios were na, 1.04 ± 0.04, 0.54
± 0.01, 0.21 ± 0.01 and 0.15 ± 0.002 for d-MAMP/d-MAMP-d14 and na,
0.82 ± 0.03, 0.48 ± 0.01, 0.24 ± 0.01, and 0.19 ± .001 for d-MAMP/
d-MAMP-d5 (theoretical DI ratio is 0.40, n = 2).
20
Journal of Analytical Toxicology, Vol. 35, January/February 2011
These data indicate that analyte affinity for the column resin
increases with increasing deuteration and more so for metham-
phetamine.
Although the difference of nine deuteriums between d-
MAMP-d5 and d-MAMP-d14 resulted in a lower affinity of the
former for the column material, the difference of five deu-
teriums between d-AMP-d6 and d-AMP-d11 did not cause a sig-
nificant change in binding properties. The smaller effect of
deuterium substitution on d-AMP may be related to the posi-
tion of the deuteriums as well as to the number of substitu-
tions. The only difference between d-AMP-d6 and d-AMP-d11 is
the five deuterium substitutions on the phenyl ring. In com-
parison, the differences between d-MAMP-d5 and d-MAMP-d14
include the five deuterium substitutions on the phenyl ring,
one deuterium on the beta carbon, and three deuteriums on
the methyl group attached to the alpha carbon. Taken together,
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these data indicate that deuteriums either enhance the ba-
sicity of the proximal amine through increased induction from
the higher electron density imparted by the shorter C-D bond
length, or enhance hydrophobic affinity of the propyl moiety,
with aryl interactions being less significant. That the former
enhancement to basicity is more significant is supported by the
greater discrimination between d-AMP and d-MAMP, and MDA
and MDMA, due to induction from the methyl group, as dis-
cussed previously.
Several studies (11,25,27–29) have shown that, in general,
deuteration of hydrocarbons will produce a more polar
molecule, but if heteroatoms such as N, O, or S are present,
deuteration may either increase or decrease the hydropho-
bicity depending upon the position of the deuterium substitu-
tion (25). Mráz et al. (27) found that, if deuterium was bound
to the formyl group of N,N-dimethylformamide, N-methylfor-
mamide, or formamide, the deuterated forms eluted later than
the unlabeled molecules, indicating the deuteration of the
formyl group caused the molecule to be more lipophilic than
the unlabeled molecule (27). In a similar manner, deuteration
of MDA, MDMA, MDEA, AMP, and MAMP near the amine would
be expected to cause the deuterated drug to be more lipophilic
than the non-deuterated drug.
Figure 8. The fraction of d-AMP-d5 (side chain), and d-AMP-d5 (ring) pre-
sent in each 0.5-mL fraction eluted from a 50-mg resin bed CEREX Poly-
crom CLIN II with ethylacetate/ammonium hydroxide (98:2). For the first
five 0.50-mL fractions, d-AMP-d5 (side chain)/d-AMP-d5 (ring) ratios were
0.73 ± 0.04, 0.92 ± 0.04, 1.13 ± 0.090, 1.17 ± 0.11, and 1.15 ± 0.04. The
cumulative ratio for the first six fractions was 0.94.
Romberg_jf11.qxd:JATLynneTemplate 12/20/10 11:18 AM
Journal of Analytical Toxicology, Vol. 35, January/February 2011
To investigate the effect of the position of the deuteriums on
binding affinity, two different AMP-d5 internal standards were
analyzed using the fractionation method described in this
study. d-AMP-d5 (ring) has five deuteriums on the phenyl ring,
and d-AMP-d5 (side chain) has five deuteriums on the propyl
moiety as shown in Figure 1. If deuteration on the side chain
is more influential in binding of the drug to the column ma-
terial compared to deuteration on the ring, then d-AMP-d5
(ring) would be expected to elute before d-AMP-d5 (side chain).
Figure 8 shows the elution profile of urine specimens con-
taining 500 ng/mL of AMP-d5 (ring) and 500 ng/mL of AMP-d5
(side chain) from a 50-mg CEREX Polycrom CLIN II SPE that
were eluted sequentially with 0.5-mL aliquots of ethyl acetate/
ammonium hydroxide (98:2). The ratios of d-AMP-d5 (side
chain)/d-AMP-d5 (ring) as measured by the 264/260 abundance
ratios increased from 0.73 to 1.13 for the second through the
fourth fractions before leveling off at 1.17 and 1.15 for fractions
five and six. The data show that d-AMP-d5 (ring) is eluting ear-
lier and indicate that the binding of d-AMP-d5 (side chain) is in-
creased due to increased electron density on the nitrogen from
the deuteriums on the propyl moiety.
Quantification errors
Separation of drug and internal standard during SPE is a po-
tential source for inaccurate quantification of drug for a sample
of unknown concentration. As long as the same amount of
elution solvent is used for all analytical runs, the elution dis-
parity is not expected to cause issues, because the DI ratio will
be uniformly disparate for all specimens. However, for a sce-
nario where drug elutes before its internal standard, insuffi-
cient elution solvent used to establish a DI ratio upon calibra-
tion will lead to all subsequent quantifications being
underestimated. In contrast, if less elution solvent is used for
unknowns, relative to that used for the calibrator, quantities
will be overestimated. The data in Table II illustrate this latter
scenario, wherein insufficient volumes of the elution solvent
ethyl acetate/ammonium hydroxide (98:2), were added to an
SPE column containing a sample of 100 ng/mL of d-AMP and
100 ng/mL of d-MAMP relative to a calibration standard eluted
with 2.0 mL of the same solvent.
Figure 9 shows the relative recovery of d-MAMP and d-
Table II. Quantification of a 100 ng/mL Sample d-AMP
and d-MAMP from a 50-mg Resin Bed CEREX Polycrom
CLIN II SPE Column with Different Volumes of
Ethylacetate/Ammonium Hydroxide (98:2)*
Elution [d-AMP] ± [d-MAMP] ±
Volume Standard Deviation Standard Deviation
1.00 112 ± 3 136 ± 9
1.25 104 ± 0.3 113 ± 1
1.50 102 ± 0.4 106 ± 3
1.75 100 ± 1 104 ± 1
2.00 100 ± 1 100 ± 1
2.50 101 ± 1 101 ± 1
* The internal standard was 250 ng/mL of d-AMP-d11 and d-MAMP-d14.
Page 7
MAMP-d14 as a function of elution volume. Using 1.50 mL (i.e.,
0.50 mL less than was used for the calibration standard) did not
affect the DI ratio, but when the elution volume was reduced
to 1.00 mL, there was a 12% and 36% increase in the quan-
tification of d-AMP and d-MAMP, respectively. This increase
was due to a change in the DI ratio because of the differences
in elution profiles between the drug and internal standard.
These types of errors can be avoided by accurate addition of elu-
tion solvent to all of the calibrators, controls, and specimens in
the analysis. Additional assurance can be achieved by using a
procedure that uses sufficient excess elution solvent where
small changes of elution solvent do not affect quantification or
to use a procedure that has a minimal deuterium isotope effect.
Conclusions
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The work described herein shows that the different binding
affinities and solubilities of deuterated drug and drug produce
different elution profiles for MDA, MDMA, MDEA, d-AMP, and
d-MAMP and MDA-d5, MDMA-d5, MDEA-d6, d-AMP-d11, d-AMP-
d6, d-MAMP-d14, and d-MAMP-d5. Deuteration of these basic
drugs increased the affinity of the drug for the SPE packing
material. This difference is most likely due to an increase in ba-
sicity of the amine imparted by deuteration. The difference in
binding between drug and deuterated drug was greater when
there was a deuterated methyl group on the amine. The rela-
tive difference in binding of drug and its deuterated analogue
to DVB mixed phase SPE columns may result in quantification
errors if disproportionate amounts of drug and internal stan-
dard are left on the SPE column. Adequate recovery of drug
and internal standard for analysis can be achieved by applying
sufficient elution solvent to assure that essentially all of the
drug and internal standard are eluted from the SPE column.
Because commercial SPE columns differ in their sorbent
characteristics, particle size, packing, and physical character-
istics, validation procedures should evaluate elution volume
and elution profiles of all analytes to assure that dispropor-
tionate losses of drug and internal standard are not occurring
during the extraction process. Further study in this area with
Figure 9. Elution of a 100 ng/mL sample of d-MAMP and 250 ng/mL of
d-MAMP-d14 from a 50-mg resin bed CEREX Polycrom CLIN II SPE column
with different volumes ethylacetate/ammonium hydroxide (98:2) The
d-MAMP/d-MAMP-d14 ratio is shown above the d-MAMP column.
21
Romberg_jf11.qxd:JATLynneTemplate 12/20/10 11:18 AM Page 8

Journal of Analytical Toxicology, Vol. 35, January/February 2011

different deuterated analogues or 13C analogues, which would
not be expected to have a significant inductive effect, would be
beneficial in characterizing the binding of drugs to mixed
phase and other column sorbents.
Acknowledgments
The authors would like to acknowledge the efforts of
Mr. Florencio Martinez, Ms. Patricia Johnson, Mr. Kiyosi Tang,
Ms. Maribel Sagucio, and Ms. Marnie Whitlock for the pro-
cessing of samples for these studies.
References
mination of amphetamine, methamphetamine and their
methylenedioxy derivatives in urine by automated in tube solid-
phase microextraction coupled with liquid chromatography ion-
ization mass spectrometry. J. Anal. Toxicol. 24: 257–264 (2000).
16. D.-L. Lin, H.-C. Liu, R.-M. Yin, D.-T.Chen, S.-J. Soong, and
R.H. Liu. Effectiveness of multiple internal standards: deuterated
analogues of methylenedioxymethamphetamine, methylene-
dioxyamphetamine, methamphetamine and amphetamine. J. Anal.
Toxicol. 28: 650–654 (2004).
17. C. Jurado, M.P. Gimerez, T. Soriano, M. Menendez, and
M. Repetto. Rapid analysis of amphetamine, methamphetamine,
MDA and MDMA in urine using solid-phase microextraction,
direct on-fiber derivatization and analysis by GC–MS. J. Anal.
Toxicol. 24: 11–16 (2000).
18. S.O. Pirnay, T.T. Abraham, and M.A. Huestis. Sensitive gas chro-
matography–mass spectrometry method for simultaneous mea-
surement of MDEA, MDMA and metabolites HMA, MDA, and
HMMA in human urine. Clin. Chem. 529: 1728–1734 (2006).
19. H.K. Nordgren and O. Beck. Direct screening of urine for MDMA
and MDA by liquid chromatography–tandem mass spectrometry.

Downloaded from https://academic.oup.com/jat/article/35/1/15/757948 by guest on 01 May 2022

J. Anal. Toxicol. 27: 15–18 (2003).
20. C.K. Horn, K.L. Klette, and P.R. Stout. LC–MS analysis of 2 oxo-
1. M. Perez-Reyes, W.R. White, S.A. McDonald, J.M. Hill, A.R. Jeff-
3-Hydroxy LSD from urine using a Speeddisk® positive-pressure
Coat, and E. Cook. Clinical effects of methamphetamine vapor in-
processor with Cerex® PolyCrom™ CLIN II columns. J. Anal. Tox-
halation. Life Sci. 49: 953–959 (1991).
icol. 27: 459–463 (2003).
2. J.F. Buchanan and C.R. Brown. ‘Designer drug’. A problem in clin-
21. K.M. Jenkins, M.S. Young, and C.R. Mallet. Mixed-mode solid-
ical toxicology. Med. Toxicol. Adverse Drug Exp. 3: 1–17 (1988).
phase extraction procedure for the determination of MDMA and
3. J.D. Ropero-Miller and B.A. Goldberger. Recreational drugs. Cur-
metabolites in urine using LC–MS, LC–UV, or GC–NPD. J. Anal.
rent trends in the 90s. Clin. Lab. Med. 18: 727–746 (1998).
Toxicol. 28: 50–58 (2004)
4. S.B. Needleman and R.W. Romberg. Comparison of drug abuse
22. K.L. Klette, M.H. Jamerson, C.L. Morris-Kukoski, A.R. Kettle, and
in different military populations. Forensic Sci. 34: 848–857 (1989).
J.J. Snyder. Rapid simultaneous determination of amphetamine,
5. 2002 Department of Defense Survey of Health Related Behaviors
methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-
Among Military Personnel. http://www.tricare.mil/main/news/
methylenedioxymethamphetamine, and 3,4-methylene-
2002WWFinalReport.pdf (accessed December 2010).
dioxyethylamphetamine in urine by fast gas chromatography–
6. Department of Defense Directive 1010-1 Military Personnel Drug
mass spectrometry. J. Anal. Toxicol. 29: 669–674 (2005).
Abuse Testing Program. 09 December 1994.
23. M.A. ElSohly, D.F. Stanford, D. Sherman, H. Shah, D. Bernot,
7. Department of Defense Instruction 1010.16. Technical Proce-
and C.E. Turner. A procedure for eliminating interferences from
dures for Military Personnel Drug Abuse Testing Program. 09
ephedrine and related compounds in the GC/MS analysis of am-
December 1994.
phetamine and methamphetamine. J. Anal. Toxicol. 16: 109–111
8. Mandatory Guidelines for Federal Workplace Drug Testing Pro-
(1992).
grams. Fed. Regist. 69(71): 19659–19673 (2004).
24. A.V. Pirogov, M.V. Chernova, D.S. Nemtseva, and O.A. Shpigun.
9. F.M. Urry, M. Kushnir, G. Nelson, M. McDowell, and T. Jennison.
Sulfonated and sulfoacylated poly (styrene-divinylbenzene)
Improving ion mass ratio performance at low concentrations in
copolymers as packing materials for cation chromatography. Anal.
methamphetamine GC–MS assay through internal standard
Bioanal. Chem. 376: 745–752 (2003).
selection. J. Anal. Toxicol. 20: 592–595 (1996).
25. D. Wade. Deuterium isotope effects on noncovalent interactions
10. R.H. Liu, D.L. Lin, W.T. Chang, C. Liu, W.I. Tsay, J.H. Li, and
between molecules. Chem. Biol. Interact. 117: 191–217 (1999).
T.L. Kuo. Issues to address when isotopically labeled analogues of
26. Y. Cherrah, R. Zini, J.B. Falconnet, M. Desage, J.P. Tillement, and
analytes are used as internal standards. Anal. Chem. 74: 618A–
J.L. Brazier. Study of deutero-isotopomer-induced inhibition of caf-
626A (2001).
feine and phenobarbitone binding to human serum albumin.
11. S. Valtier and J.T. Cody. Evaluation of internal standards for the
Biochem. Parmacol. 37: 1311–1315 (1988).
analysis of amphetamine and methamphetamine. J. Anal. Tox-
27. J. Mráz, P. Jheeta, A. Gescher, and M.D. Threadgill. Unusual deu-
icol. 19: 375–380 (1995).
terium isotope effects on the retention of formamides in gas-
12. J.T. Cody, S. Valtier, and S.L. Nelson. Amphetamine enantiomer
liquid chromatography. J. Chromatogr. 641: 194–198 (2993).
excretion profile following administration of Adderall. J. Anal.
28. J. Bermejo, C.G. Blanco, and M.D. Guillen. Gas chromatography
Toxicol. 27: 485–492 (2003).
of deuterated and protiated chloro derivatives of 1,4-dimethyl-
13. Z. Huang and S. Zhang. Confirmation of amphetamine, metham-
benzene. J. Chromatogr. 351: 525–432 (1986).
phetamine, MDA and MDMA in urine samples using disk solid-
29. M.G. Horning, J.P. Thenot, O. Bouwsma, J. Nowlin, and K. Ler-
phase extraction and GC–MS after immunoassay screening.
tratanangkoon. Changes in chemical and biological properties of
J. Chromatogr. B 792: 241–247 (2003).
drugs due to deuterium labeling. In Advances in Pharmacology
14. P.R. Stout, C.K. Horn, and K. Klette. Rapid simultaneous deter-
and Therapeutics, Vol. 7. Biochemical-Clinical Pharmacology,
mination of amphetamine, methamphetamine, 3,4-methylene-
Pergamon, Oxford, U.K., 1979, pp 245–256.
dioxyamphetamine, 3,4 methylenedioxymethamphetamine and
3,4-methylenedioxyethylamphetamine in urine by solid-phase
extraction and GC–MS: a method optimized for high-volume
laboratories. J. Anal. Toxicol. 26: 253–261 (2002). Manuscript received June 1, 2010;
15. H. Kataoka, H.L. Lord, and J. Pawliszyn. Simple and rapid deter- revision received June 24, 2010.

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