Download Low Concentrations of Methamphetamine Detectable in Urine in the

Journal of Analytical Toxicology, Vol. 33, April 2009
Short Communication
Low Concentrations of Methamphetamine
Detectable in Urine in the Presence of
High Concentrations of Amphetamine*
John F. Jemionek†, Joseph Addison, and Marilyn R. Past
Armed Forces Institute of Pathology, Forensic Toxicology Division, Armed Forces Medical Examiner System,
1413 Research Blvd., Bldg. 102, Rockville, Maryland 20850
Abstract
Twenty-two urine specimens reported by military drug-testing
laboratories for the presence of high concentrations of
amphetamine only were subject to further analysis for the
presence of methamphetamine. The 22 urine specimens had
concentrations of amphetamine in the range of 28,028 to 241,142
ng/mL. The specimens were also assayed for the respective
isomeric ratio of d (S ) and l (R) amphetamine and
methamphetamine. The results suggest that urine specimens
containing high concentrations of amphetamine in which the urine
concentration ratio of methamphetamine to amphetamine is less
than 0.5% with similar isomeric distribution of d-(S ) and l-(R)
amphetamine and methamphetamine, respectively, may not
necessarily indicate polydrug use.
as-needed basis, for back pain. Each day, over the course of the
weekend prior to the urine analysis, the individual took multiple doses of what she believed to be Flexeril for acute back
pain relief. There was documentation of phone messages to her
physician that the medication, which the accused believed to be
Flexeril, was providing no pain relief. The generic Adderall
and prescribed Flexeril had nearly similar shape and color
characteristics. Subsequent analysis of one of the Adderall
tablets indicated 80% d-amphetamine (S). If correct, the mistaken ingestion of Adderall would explain the high concentration of isomeric amphetamine encountered, but what was the
source of the low level isomeric methamphetamine? Is it possible to encounter low concentrations of methamphetamine in
the presence of high amphetamine concentrations of similar
isomeric ratios in urine analysis specimens to collaborate the
proposed scenario?
Introduction
An individual with no documented history of drug abuse
and self-reported abstinence of drug use had a positive urine
analysis by immunoassay and gas chromatography–mass spectrometry (GC–MS) for amphetamine at 98,000 ng/mL and
methamphetamine at 275 ng/mL. The isomeric ratio was 75%
d-amphetamine (S) and 83% d-methamphetamine (S). The
individual denied use of amphetamine and methamphetamine.
A background investigation supported no history of drug abuse.
During administrative and legal review, the possibility of a
prescription medication substitution arose as the basis for the
positive urine analysis. A sibling with a documented history of
prescribed Adderall® had recently moved in with the accused
on a temporary basis pending relocation of her family. Believing that the accused was no longer using Flexeril®, the
sibling reportedly discarded the few tablets of Flexeril in the accused prescription bottle and replaced them with her generic
20-mg Adderall tablets that she had transported in a cigarette
case. The accused had a valid prescription for Flexeril, on an
* Disclaimer: The opinion or assertions herein are those of the authors and do not necessarily
reflect the view of the Departments of the Army, Navy, or the Department of Defense.
† Author to whom correspondence should be addressed. E-mail: [email protected].
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Materials and Methods
Specimen collection
Urine specimens (slated for discard post one year frozen
storage) were solicited from the military drug testing laboratories. Specimens solicited were those urine specimens reported as positive for the presence of amphetamine only and at
approximate concentrations equal to or greater than 25,000
ng/mL. No personal information was associated with the specimens obtained.
Standards and reagents
Amphetamine, methamphetamine, amphetamine-d11, methamphetamine-d14, d-(S)-amphetamine, l-(R)-amphetamine, d(S)-methamphetamine, l-(R)-methamphetamine, d/l-(S/R)amphetamine-d11, and d/l-(S/R)-methamphetamine-d14 were
purchased from Cerilliant (Round Rock, TX) in preparation of
calibrators and controls. (R)-(–)-α-Methoxy-α-(trifluoromethyl)phenylacetyl chloride (R-MTPAC) was purchased from
Fluka Chemical (Milwaukee, WI) for use in the isomeric analysis of amphetamine and methamphetamine. Reagents were
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Journal of Analytical Toxicology, Vol. 33, April 2009
purchased from Fisher Scientific or Aldrich Chemical and of
high-performance liquid chromatography (HPLC) grade.
Specimen preparation and analysis
For amphetamine-methamphetamine analysis, 2 mL each of
urine specimens, calibrators, controls, and negative specimens
were used in the extraction. A calibration curve was prepared
at 2, 5, 20, 50, 200, 500, and 1000 ng/mL. Dilutions, as required
using negative urine, were made to bring amphetamine concentrations within range. Methamphetamine concentrations
were determined from undiluted specimens.
To eliminate the possibility of methamphetamine detection
due to the presence of nondeuterated methamphetamine in the
methamphetamine-d14 internal standard, the initial amphetamine-methamphetamine analysis was conducted using
only amphetamine-d11 as the internal standard. Once the presence of methamphetamine was confirmed by GC–MS, quantitative analysis of methamphetamine was conducted using
methamphetamine-d14.
Deuterated internal standard (1000 ng/mL) was added to
each 2-mL aliquot of specimen. Specimens were made alkaline
with the addition of 200 µL of concentrated KOH. Following
the addition of 5 mL of chlorobutane, the specimens were
gently rotated for 15 min, then centrifuged for 10 min at 1920
× g, and the upper organic layer transferred to clean tubes. Following the addition of 100 µL of 1% HCl, the specimens were
evaporated to dryness under nitrogen at 50°C. The dried extracts were derivatized with 25 µL of chorodifluoroacetic anhydride and 100 µL of ethyl acetate. Specimens were vortex
mixed and incubated for 15 min at 70°C. Following incubation,
the extracts were evaporated to dryness, reconstituted with 50
µL of ethyl acetate, vortex mixed, and transferred to injection
vials for GC–MS analysis.
For isomeric analysis, 2 mL each of urine specimen, calibrator, controls, and negative sample were used in the extraction. A one-point calibration at 500 ng/mL was used against a
50% d/l (S/R) isomer ratios of amphetamine and methamphetamine. Internal standard (100 ng/mL) of d/l-(S/R)amphetamine-d11 and d/l-(S/R)-methamphetamine-d14 was
added to each tube. Specimens were made alkaline with the addition of 200 µL of concentrated KOH. Following the addition
of 5 mL of chlorobutane, the specimens were gently rotated for
15 min, then centrifuged for 10 min at 3000 rpm, and the
upper organic layer transferred to clean tubes. Following the
addition of 100 µL of 1% HCl, the specimens were evaporated
to dryness under nitrogen at 50°C. A previously published
method (1) using the chiral derivitization reagent R-MTPAC
was used to separate the isomers of methamphetamine and amphetamine into chromatographically distinguishable diasteromers.
GC–MS analysis
Amphetamine and methamphetamine analysis. An Agilent
6890 GC coupled to a 5973 MSD was used for the detection and
confirmation of amphetamine and methamphetamine. An Agilent DB-5MS (20 m × 0.18-mm i.d., 0.18-µm film thickness)
column was used for separation. Injections were analyzed with
a 4-mm straight inlet liner with deactivated glass wool in the
pulse split mode (10:1) and a pulse pressure of 35 psi for 0.9
min. Helium flow was constant at 1.0 mL/min. The injection
port temperature was 175°C with an initial GC oven program
of 70°C with a 1.0-min hold. Temperature was ramped at
20°C/min to 230°C with a final ramp of 50°C/min to 300°C followed by a 1.6-min hold. The MS acquisition was operated in
the selected ion monitoring mode (SIM) with the following
confirmation ions: m/z 170, 118, 91 for methamphetamine
and m/z 156, 118, 91 for amphetamine. For methamphetamine
analysis, the m/z 170 ion was the quanifying ion, and the m/z
118 and 91 ions were the qualifying ions. The methamphetamine ion ratios monitored were the m/z 118/170 and the
91/170 ions. For the methamphetamine-d11 internal standard,
the m/z 177 and 179 ions were the quantifying and qualifying
ions, respectively. The methamphetamine-d11 internal standard ion ratio monitored were the m/z 179/177 ions. For amphetamine analysis, the 156 m/z ion was the quantifying ion
and the m/z 118 and 91 ions were the qualifying ions. The
amphetamine ion ratios monitored were the m/z 118/156 and
91/156 ions. For the amphetamine-d11 internal standard, the
m/z 160 and 128 ions were the quantifying ion and qualifying
ions, respectively. The amphetamine-d11 internal standard ion
ratio monitored were the m/z 128/160 ions .
Isometric analysis of amphetamine and methamphetamine.
An Agilent 6890 GC coupled to a 5973 MSD was used for the
detection and confirmation of d/l-(S/R)-amphetamine and
methamphetamine in urine. An Agilent DB-1MS (30 m × 0.25mm i.d., 0.25-µm film thickness) column was used for separation. The GC injector was operated with a 4-mm straight inlet
liner with deactivated glass wool in the splitless mode. Helium flow was 1.0 mL/min. The oven had the following temperature program: initial temperature of 140°C with 0.50 min
holding time, followed by a ramp of 15°C/min to 215°C with a
1.5-min hold, and a final ramp of 35°C/min to 285°C with a 1min hold. The MS was operated in the selected ion monitoring
mode. The methamphetamine confirmation ions monitored
were m/z 274, 275, 176 for l-(R)-methamphetamine; m/z 274,
275, 176 for d-(S)-methamphetamine; m/z 281, 98 for l-(R)methamphetamine-d 14 and m/z 281, 98 for d-(S)methamphetamine-d14. For d/l-(S/R) methamphetamine analysis, the m/z 274 ion was the quantifying ion and the m/z 275
and 176 ions were the qualifying ions. The ion ratios monitored
were the m/z 275/274 and m/z 176/274 ions. For the d/l-(S/R)
methamphetamine-d14 internal standard, the m/z 281 ion and
the m/z 98 ion were the quantifying ion and qualifying ions, respectively. The m/z 98/281 ions were used in monitoring the
ion ratio The amphetamine confirmation ions monitored were
m/z 260, 118, 162 for l-(R)-amphetamine; m/z 260, 118, 162 for
d-(S)-amphetamine; m/z 264, 130 for l-(R)-amphetamine-d11
and, m/z 264, 130 for d-(S)-amphetamine-d11. For d/l-(S/R)
amphetamine analysis, the 260 m/z ion was the quantifying ion
and the m/z 118 and 162 ions were the qualifying ions. The ion
ratios monitored were the m/z 118/260 and m/z 162/260 ions.
For the d/l-(S/R) amphetamine-d11 internal standard, the m/z
264 ion and the m/z 130 ion were the quantifying ion and
qualifying ions, respectively. The m/z 130/264 ions were used
in monitoring the ion ratio.
Criteria for GC–MS acceptance. The retention time (tR) of
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Journal of Analytical Toxicology, Vol. 33, April 2009
the analyte in the specimens and controls must be within (±)
2% of the tR of the analyte in the calibrators. The ion ratios for
the analyte in the specimens and controls must be within (±)
20% of the ion ratios for the calibrator analyte. The negative
control should be less than the concentration established as the
lower limit of quantitation, and the positive controls must be
within (±) 20% of the laboratory established target concentration. The GC–MS lower limit of detection for amphetamine and
methamphetamine was less than 2 ng/mL, and the lower limit
of quantitation was 2 ng/mL.
Results and Discussion
A total of 22 specimens were analyzed. The total amphetamine concentration ranged from 28,028 to 241,142
ng/mL. The total methamphetamine concentration detected
ranged from 3.8 to 275 ng/mL. Isomeric methamphetamine determinations were conducted only on specimens where the
total methamphetamine concentration approximated 19 ng/mL
or greater.
As noted in Table I, data from the 22 urine specimens with
an amphetamine concentration greater than 25,000 ng/mL
were compiled. The following correlates were noted: 1. There
does not appear to be a correlation between the amphetamine
concentration and the subsequent methamphetamine concentration detected. 2. The only consistent correlation is the
ratio of methamphetamine to amphetamine that is below 0.5%.
3. Seven urine specimens with a methamphetamine concentration approximately 19 ng/mL or greater were analyzed for
isomeric correlation between the amphetamine and methamphetamine isomers.
Of the seven specimens subject to isomeric analysis: within
the variance of the isomeric analysis, there was a correlation in
the ratio of d/l-(S/R)-methamphetamine to the d/l-(S/R)amphetamine ratio for six of the seven urine specimens analyzed, and one urine sample showed an amphetamine to
methamphetamine isomeric correlation outside the normal
range of variance namely a 70% d-amphetamine (S) and an
87% d-methamphetamine (S) correlate.
The current study is not an attempt to distinguish knowing
versus unknowing ingestion of Adderall or illicit amphetamine
use but rather to determine if urine specimens containing high
concentrations of amphetamine also demonstrate the presence
of low concentrations of methamphetamine. It does appear that
low urine levels of methamphetamine may be associated with
high urinary concentrations of amphetamine. The two distinctive correlates in this limited analysis are first, the ratio of
methamphetamine to amphetamine is less than 0.5%, and
second, there appears to be a similar isomeric ratio of d/l (S/R)
between the amphetamine and methamphetamine encountered.
Table I. Amphetamine and Methamphetamine Concentration and Isomeric Analysis*
Amphetamine
Concentration
(ng/mL)
Methamphetamine
Concentration
(ng/mL)
% Methamp/Amp
29,610
43,938
50,115
130,278
28,028
30,613
36,209
38,044
48,228
60,221
64,641
65,392
66,237
79,852
98,227
4.2
4.3
3.8
4.7
10.6
7.0
6.2
6.6
9.7
7.5
9.4
14.3
7.5
15.4
12.0
0.014
0.010
0.008
0.004
0.038
0.023
0.017
0.017
0.020
0.012
0.015
0.022
0.011
0.019
0.012
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
No Isomeric analysis conducted when methamp < 19 ng/mL
Isomeric Analysis Results
59,451
81,391
67,409
98,000
181,079
241,142
19
230
21
275
63
24
0.032
0.283
0.031
0.281
0.035
0.010
Urine 60% d-amp and 68% d-methamp
Urine 44% d-amp and 44% d-methamp
Urine 71% d-amp and 74% d-methamp
Urine 75% d-amp and 83% d-methamp
Urine 68% d-amp and 68% d-methamp
Urine 72% d-amp and 70% d-methamp
148,239
55
0.037
Urine 70% d-amp and 87% d-methamp
* The isomeric composition of amphetamine and methamphetamine in 22 urine specimens containing a high concentration of amphetamine during random urine analysis testing.
Limitations in quantitative and isomeric analysis were based upon the analyte recovery with acceptable chromatography, peak symmetry, and mass ion ratios.
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Journal of Analytical Toxicology, Vol. 33, April 2009
The source of the urine methamphetamine cannot be determined in this limited study. Two considerations are offered.
First, is the small amount of methamphetamine found in the
urine a minor methamphetamine by-product component from
the amphetamine manufacture process? Second, does a minor
pathway of methylation exist in the metabolism and elimination of high blood concentrations of amphetamine? In a publication by Cone et al. (2), individuals receiving codeine-based
medications are known to excrete hydrocodone via a minor
metabolic pathway. Cone and co-workers (2) presented data
suggesting that hydromorphone may also be excreted in the
urine through a minor metabolic pathway of conversion of
morphine to hydromorphone. This observation has been subsequently validated in two other publications (3,4). Cone et al.
(2) suggest that in the interpretation of low urinary concentrations of hydromorphone in morphine-treated pain patients,
the presence of hydromorphone should not be considered as
conclusive evidence of hydromorphone misuse. Perhaps a similar degree of caution should be considered in urine specimens where low concentrations of methamphetamine are detected in the presence of high concentrations of amphetamine.
This preliminary work perhaps suggests that the presence of
low urinary concentrations of methamphetamine should not be
considered as conclusive evidence of methamphetamine abuse
when elevated urine amphetamine concentrations are encountered if the ratio of methamphetamine to amphetamine is
below 0.5% and there is a similar isomeric ratio of methamphetamine and amphetamine analytes in the urine. Validation
of these observations is requested by other laboratories encountering high concentrations of urinary amphetamine or
specimens from individuals under controlled amphetamine
administration. Confirmation of these observations would be
pertinent to the interpretation of toxicological analyses by
pathologists, toxicologists, medical review officers, and drug
counselors.
Acknowledgment
This work was funded in part by the Department of Defense
Counter Narcotics Program and by the American Registry of
Pathology, Washington, D.C. 20306-6000.
References
1. B.D. Paul, J.F. Jemionek, D. Lesser, A. Jacobs, and D.A. Searles.
Enantiomeric separation and quantitation of (±)-amphetamine, (±)methamphetamine, (±)-MDA, (±)-MDMA, and (±)-MDEA in urine
specimens by GC–EI-MS after derivitization with R-(–) or S-(+)-αmethoxy-α-(trifluoromethyl)phenyacetyl chloride (MTPA). J. Anal.
Toxicol. 28: 449–455 (2004).
2. E.J. Cone, H.A. Heit, Y.H. Caplan, and D. Gourlay. Evidence of
morphine metabolism to hydromorphone in pain patients chronically treated with morphine. J. Anal. Toxicol. 30: 1–5 (2006).
3. E.J. Cone, Y.H. Caplan, F. Moser, T. Robert, and D. Black. Evidence that morphine is metabolized to hydromorphone but not to
oxymorphone. J. Anal. Toxicol. 30: 319–323 (2008).
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The detection of hydromorphone in urine specimens with high
morphine concentrations. J. Forensic Sci. 53: 752–754 (2008).
Manuscript received August 25, 2008;
revision received October 21, 2008.
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