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Vol. 28, Issue 3, 286-291, March 2000
)-Methamphetamine and
S(+)- and
R()-N-(n-butyl)-amphetamine
in Mouse Hair after Systemic Administration
University of Colorado Health Sciences Center, Department of Molecular Toxicology and Environmental Health Sciences, Denver, Colorado
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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We examined the incorporation of unlabeled and tritiated
enantiomers of methamphetamine (MA) and a more lipophilic analog N-(n-butyl)-amphetamine (BA) into the
hair of pigmented (C57) and nonpigmented (Balb/C) mice after systemic
administration. We also compared the ability of extraction methods to
remove unlabeled and tritiated MA and BA enantiomers from the hair.
R(
)-MA, S(+)-MA, [3H]R()-MA,
[3H]S(+)-MA, R()-BA,
S(+)-BA, [3H]R-()-BA, and
[3H]S-(+)-BA were each administered to C57
and Balb/C mice (23 days of age) by i.p. injection at 8.8 mg/kg daily
for 3 days. At 44 days of age, hair samples from the animals were
treated with a brief methanol wash, a 24-h extraction with pH 6 phosphate buffer, and a final digestion in 1 N NaOH to free residual
drugs from the hair. Labeled drugs in the extracts were quantitated by
liquid scintillation counting. Unlabeled drugs were quantitated by gas chromatography/mass spectrometry (GC/MS). GC/MS analysis demonstrated MA and BA to be the major (>90%) species present in the blood during
the 24 h after administration. Less than 10% of the MA was
N-demethylated. No p-hydroxylated
metabolites were found. Blood concentrations of tritiated MA and BA
enantiomers measured by liquid scintillation counting agreed well with
blood concentrations of unlabeled enantiomers measured by GC/MS. Hair
concentrations of S(+)-MA were greater than those of
R()-MA in both mouse strains, paralleling blood
concentrations. There were no enantiomeric differences seen with BA
hair accumulation in either strain of mouse. Significantly more MA and
BA enantiomers were deposited in pigmented than in nonpigmented hair.
With labeled and unlabeled compounds, approximately 30% of
S(+)-MA and 60% of R()-MA in pigmented
hair could be removed by a phosphate extraction. A significant amount
of drug could not be removed from the hair by extraction. Greater
amounts of drug could be extracted from nonpigmented hair than
pigmented. Extracted and residual MA and BA concentrations in pigmented
hair were significantly greater when labeled compounds were quantitated by liquid scintillation counting than when unlabeled compounds were
quantitated by GC/MS. However, radiotracer and unlabeled drug
concentrations were the same in nonpigmented hair. The results demonstrate that hair pigmentation is an important determinant in MA
and BA deposition, and that MA and BA deposition is not enantioselective. The data demonstrate a significant amount of MA and
BA accumulated is not easily amenable to exhaustive aqueous extraction
from the hair. The use of tritiated MA and BA enantiomers demonstrates
that a significant amount of MA and BA stored in pigmented hair is
structurally different from parent MA and BA, perhaps associated with
melanin components of hair.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The suitability of hair as a reliable, quantitative indicator of systemic exposure to drugs of abuse remains controversial due to a number of unresolved questions. In particular, the chemical mechanisms of systemic drug incorporation into hair are as yet undetermined. Ultimately, a solid understanding of the chemical mechanisms of drug incorporation into the hair matrix and the dynamics of drug partitioning within the hair matrix is necessary for establishing the validity of hair sampling for drug detection.
Potsch and Moeller (1996)
suggested that hair is a multicomponent fiber
with compartments consisting of melanin, lipids, and proteins. Stout et
al. (1998a) also suggested that incorporation into hair involved a
multicompartmental process with partitioning of drugs between various
compartments. We have also demonstrated that carboxylic acid groups
within the protein matrix participated in the partitioning of
nonionized compounds into the hair. Our data have suggested that
cationic rhodamine rapidly incorporated into forming protein fibrils of
the cortex and medulla of hair when administered systemically, but was
incorporated into the cuticle cell junctions when applied externally
(Stout and Ruth, 1998). The deposited dye was found to be resistant to
removal by either methanol or aqueous buffer treatments.
A large amount of work has been reported on the deposition and
detectability of methamphetamine
(MA)1 in hair
(Nakahara et al., 1991, 1992, 1993, 1997; Niwaguchi et al.,
1993; Nakahara, 1995; Nakahara and Kikura, 1996; Rohrich and
Kauert, 1997). Nakahara and Kikura (1996) examined the effects of structural modification on drug deposition in hair with 32 analogs
of amphetamine. They found that increasing length of carbon substituent
at the N position increased drug incorporation into hair. Stout et al.
(1998a) also found that nonionized fentanyl was incorporated into hair
to a greater extent than ionized fentanyl. These results suggest that
increased lipophilicity may increase the incorporation of some
compounds into the hair matrix.
The purpose of this study was to examine the incorporation of MA and the more lipophilic N-(n-butyl)-amphetamine (BA) into hair from the systemic circulation. We examined the enantioselectivity of this incorporation and the stability of incorporated compounds to removal by several extraction methods. C57 (eumelanin-pigmented) and Balb/C (nonpigmented) mice were used to examine the effect of hair pigmentation on drug deposition. The use of tritiated amphetamine enantiomers as well as unlabeled drugs afforded an opportunity to carefully compare the mass balance of drugs during extraction from hair, and to detect possible occlusion or irreversible binding of drugs in the hair matrix.
Radiotracers were synthesized with the tritium label on the N-alkyl substituent to optimize the extent to which tracer deposition would be indicative of parent compound incorporation. A study of the serum distribution of metabolites was conducted to determine the profile of compounds available for incorporation from the circulation into hair.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Synthesis of Radiolabeled and Deuterated Compounds. The enantiomers of BA and MA were synthesized as described and as outlined in the reaction scheme presented in Fig. 1.
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(+)-N-Formylamphetamine. (+)-Amphetamine freebase (135 mg, 1 mmol) was dissolved in methyl formate in a 3-ml vial. The vial was capped and sealed with a Teflon disk and heated to 60°C in a sand bath for 48 h. At this point, gas chromatography/mass spectrometry (GC/MS) analysis indicated that the reaction was complete. The methyl formate was allowed to evaporate and the resulting clear liquid further dried under reduced pressure at room temperature to give N-formylamphetamine (150 mg, 92% yield) as a clear liquid, which crystallized on standing, m.p. 48-49°C.
1H NMR (CDCl3): 1.27 (d, J = 6.8Hz, 3H), 2.79 to 3.01 (m, 2H), 4.47 (m, 1H), 5.88 (broad s, 1H), 7.26 to 7.46 (m, 5H), 8.18 (s, 1H).13C NMR (CDCl3) 20.0, 42.3, 45.0, 126.5, 128.3, 129.3, 137.6, 160.4.()-N-Formylamphetamine.
This was synthesized as described above using ()-amphetamine (135 mg,
1 mmol) as starting material (155 mg, 95% yield).
[3H](+)-Methamphetamine. A vial containing 1.5 ml of 1 M [3H]lithium aluminum hydride (LAH; 10 mCi) was cooled to 0°C and opened under a stream of argon. A dry stir-bar was placed in the solution and the vial was resealed with a rubber septum, duraseal, and parafilm. A solution of the N-formylamphetamine (8.2 mg, 0.05 mmol) in dried tetrahydrofuran (THF; 0.5 ml) was added dropwise with stirring. The mixture was allowed to stir in a sand bath at 80°C for 48 h. The reaction was cooled to 0°C and freshly dried and distilled n-hexanal (7.5 mmol, 750 mg, 900 µl) in THF (2 ml) was added dropwise with stirring. The solution was stirred at room temperature for 30 min, then at 40°C for 15 min. The septa were removed and the mixture was allowed to stir at 30°C for 18 h to remove the THF. Dried ethyl acetate (2 ml) was then added to the solution followed by 100 mM pH 6 phosphate buffer (2 ml). The mixture was allowed to stir for 5 min. The mixture was allowed to settle, then the aqueous layer was decanted and filtered through celite. The aqueous phase was washed with two more aliquots of ethyl acetate to remove any neutral contaminants.
The yield of tritiated MA (60%) was determined by carrying out two parallel reductions of N-formylamphetamine with labeled and unlabeled LAH. The concentrations of these final solutions were determined by GC/MS using d14-MA as internal standard (Radian International, Austin, TX). Phosphate buffer (pH 6) was added to the [3H]MA solution to give a final solution of 0.88 mg/ml. A 1-mg aliquot of the labeled compound was chromatographed on a thin-layer chromatography plate with 1% ammonium hydroxide in methanol. The compound spot was visualized by UV illumination and compared to a concurrently run unlabeled control. This spot was then scrapped from the plate and radioactivity measured by liquid scintillation counting. Results were corrected for quenching by silica using control samples with a tritiated toluene standard (Packard, Meridan, CT) added. The specific activity was determined to be 20 dpm/ng. This same method was used to determine the specific activity for each of the labeled compounds.[3H]()-Methamphetamine.
This was carried out in a similar manner to the
[3H](+)-MA; the yield was 58% with a specific
activity of 61 dpm/ng.
(+)-N-Butyrylamphetamine.
Butyric anhydride (0.65 mmol, 102 mg, 105 µl) and pyridine (1.18 mmol, 93 mg, 95 µl) were added to a solution of (+)- or
()-amphetamine (80 mg, 0.59 mmol) in dichloromethane (5 ml). The
mixture was stirred under nitrogen for 48 h at room temperature.
The progress of the reaction was monitored by GC/MS. The reaction
mixture was diluted with dichloromethane (10 ml), and washed with a
saturated sodium bicarbonate solution, followed by dilute hydrochloric
acid and water. The organic solution was dried over magnesium sulfate and evaporated to a clear syrup that crystallized on standing (116 mg,
96% yield).
()-N-Butyrylamphetamine.
The procedure was carried out as described above using ()-amphetamine
(80 mg, 0.59 mmol) to give ()-n-butyrylamphetamine (110 mg, 91% yield).
(+)-N-(n-Butyl)-amphetamine. 1 M LAH in THF (1.2 ml, 1.2 mmol) was added to a stirred, cooled (0°C) solution of N-butyrylamphetamine (25 mg, 0.12 mmol) in THF (2 ml). The mixture was stirred at reflux for 48 h then cooled in ice, and water (1 ml) added dropwise. The mixture was basified with sodium carbonate, and the THF evaporated. The mixture was washed with ethyl acetate (3 × 1 ml), dried over magnesium sulfate, and evaporated under reduced pressure to give BA as a clear syrup (19 mg, 82% yield).
1H NMR (CDCl3) 1.16 (t, 3H), 1.31 (d, J = 6.3Hz, 3H), 1.45 to 1.65 (m, 4H), 2.60 (t, 2H), 2.85 (dd, 1H), 2.99 (dd, 1H), 3.15 (dd, 1H), 7.42 to 7.58 (m, 5H). 13C NMR (CDCl3) 13.9, 20.2, 20.4, 29.7, 32.1, 43.7, 54.6, 126.1, 128.3, 129.2, 139.6.()-N-(n-butyl)-amphetamine.
The procedure was carried out as described above using
()-butyrylamphetamine (25 mg, 0.12 mmol) with an
()-N-(n-butyl)-amphetamine yield of 20 mg or
87%.
(+)- and ()- d2-N-(n-butyl)-amphetamine.
A procedure similar to the above was used, with LAH (1 M in THF) used
as reducing agent. (+)- and ()-
d2-N-(n-butyl)-amphetamine
were obtained in yields of 85 and 88%, respectively.
[3H](+)-N-(n-butyl)-amphetamine. The vial containing 1.5 ml of 1 M [3H]LAH (10 mCi) was cooled to 0°C and opened under argon. A dry stir-bar was placed in the solution and the vial resealed with a rubber septum, duraseal, and parafilm.
A solution of the n-butyrylamphetamine (14.0 mg, 0.07 mmol) in dried THF (0.5 ml) was added dropwise with stirring. The mixture was allowed to stir in a sand bath at 80°C for 48 h. The reaction was cooled to 0°C and freshly distilled n-hexanal (7.5 mmol, 750 mg, 900 µl) was added dropwise with stirring. The solution was stirred at room temperature for 30 min, then at 40°C for 15 min. The septa were removed and the mixture was allowed to stir at 30°C for 18 h to remove the THF. Dry ethyl acetate (2 ml) was then added to the solution followed by 100 mM acetate buffer pH 4 (2 ml). The mixture was stirred for 5 min after which the aqueous layer was decanted and filtered through celite. The aqueous layer was washed with two more aliquots of ethyl acetate to give a solution of [3H]N-(n-butyl)-amphetamine (43% yield, 33 dpm/ng). The yield of N-(n-butyl)-amphetamine was established by carrying out two parallel reductions of butyrylamphetamine with unlabeled LAH, and determining the final concentration of BA in the pH 4 buffer by GC/MS using d2-BA as an internal standard. The specific activity (33 dpm/ng) was determined by thin-layer chromatography with 1% ammonium hydroxide in methanol and liquid scintillation counting of the eluted BA spot.[3H]()-N-(n-butyl)-amphetamine.
The procedure was carried out as described above.
()-N-(n-butyl)-amphetamine was obtained in 45%
yield with an activity of 31 dpm/ng.
Identification of Serum Metabolites after MA Administration. Concentrations of MA, amphetamine, and p-OH-MA were measured in blood after MA dosage by GC/MS. The MA concentrations measured by GC/MS were compared with the concentrations measured by liquid scintillation counting.
Three Balb/C and three C57 mice for each time point and each compound [30 min and 24 h for S(+)-methamphetamine [(+)MA] and R()-methamphetamine [()MA] and
[3H](+)- and ()-MA; 12 total mice] were
dosed once i.p. with 8.8 mg/kg of either (+)- or ()-MA (unlabeled) or
[3H](+)- or ()-MA. Mice were anesthetized
with 70 mg/kg pentobarbital and 0.5 ml of blood draw from the
retro-orbital plexus using heparinized microcapillary tubes. For GC/MS
analysis, internal standard was then added to the blood
(d14-MA and d11-amphetamine
obtained from Radian International) and the blood was vortexed with 2 ml of 1.5% isoamyl alcohol in heptane. The organic layer was decanted
and dried under nitrogen at 37°C. Samples were derivatized in 10 µl
of propionic anhydride and 100 µl of pyridine at 70°C for 20 min.
The excess derivatizing agent was evaporated under nitrogen at 70°C
and the sample reconstituted in ethyl acetate. The samples were then
analyzed by GC/MS using the conditions described later in the text.
For liquid scintillation counting, blood was drawn in the same manner
as for the GC/MS analysis and combined with scintillation cocktail (5 ml of Scintisafe Plus 50%, Fisher Scientific Co., Fairlawn, NJ) and
counted on a Packard 1600 TR liquid scintillation counter. Samples were
corrected for quenching using blood from untreated animals with added
tritiated toluene standard (Packard).
MA, amphetamine, and p-OH-MA were quantitated in the samples
by comparison to matrix-matched control samples made using untreated mouse blood. Analytical standards of MA (Radian International), amphetamine (Radian International), and p-OH-MA (Sigma
Chemical Co., St. Louis, MO) were added to produce concentrations from 0 to 4000 ng/ml. Curves for each of the compounds were linear over this range.
(+)- and ()-MA concentrations were compared between C57 and Balb/C
mice using a Student's t test (significance assigned at P < .05) at the two time points. (+)-and ()-MA
concentrations measured by GC/MS (unlabeled) were also compared with
the concentrations measured by liquid scintillation counting (labeled
compound) using a Student's t test (significance assigned
at P < .05) at the two time points.
In Vivo Study.
Groups of five Balb/C and five C57 mice were used for each of the eight
treatment groups (total of 80 mice). Treatment groups were: tritiated
and nontritiated (+)MA, ()MA,
S(+)-N-(n-butyl)-amphetamine [(+)BA], and
R()-N-(n-butyl)-amphetamine
[()BA]. All were administered at 8.8 mg/kg i.p. (+)MA and ()MA
were administered in 100 mM phosphate buffer (pH 6). (+)BA and ()BA
were administered in 100 mM acetate buffer (pH 4.5). All mice received
three consecutive daily doses and were then allowed to grow until 44 days of age.
,b).
At age 44 days the mice were sacrificed by asphyxiation in
CO2 and hair was shaved from the back with
electric clippers. Hair samples (10 mg) were subjected to a 30-s vortex
in methanol (1 ml). The methanol was decanted and analyzed. This wash
served as an estimate of very loosely associated drug or surface
contamination (Stout et al., 1998a). The hair was then subjected to a
24-h extraction in 100 mM pH 6 phosphate buffer (1 ml) at 37°C. Again
the solution was decanted and analyzed. The hair was digested in 1 N
NaOH (1 ml) at 37°C for 18 h. This digest was neutralized with 1 N HCl for scintillation counting.
Concentrations of each radiolabeled compound were measured in each of
the treatment solutions (methanol, phosphate, and neutralized digest)
by liquid scintillation counting. Scintillation cocktail (4 ml) was
added to each of the solutions. All data were corrected for color
quenching by comparison to matrix-matched control samples made with a
tritiated toluene standard (Packard).
Concentrations of each nonlabeled compound were measured in each of the
treatment solutions (methanol, phosphate, and digest) by GC/MS.
Deuterated internal standard (d14-MA from Radian
International or d2-BA described in the text
above) was added to the methanol wash solutions. This was then dried
under nitrogen and derivatized with propionic anhydride (10 µl) in
pyridine (100 µl) at 70°C for 20 min. This was dried under nitrogen
and reconstituted in ethyl acetate for analysis by GC/MS.
Compounds were isolated from the phosphate extractions by solid-phase
extraction using World Wide Monitoring columns (ZSDAU020 columns;
United Chemical Technologies, Bristol, PA) after the d14-MA internal standard had been added to the
aqueous phase. Columns were preconditioned by manufacturer's
specifications, the sample applied, and the columns washed. Analytes
were eluted with dichloromethane/isopropanol/ammonium hydroxide
(78:20:1, v/v/v). This was then dried under nitrogen at 37°C
and derivatized as above.
Compounds were isolated from the NaOH digest of hair by liquid/liquid
extraction using 1-chlorobutane [vortexed then mixed for 5 min after
Niwaguchi et al. (1993)] after the d14-MA
internal standard was added to the aqueous phase. The organic phase was
evaporated under nitrogen and the sample was derivatized and analyzed
as described above.
GC/MS Analysis. All samples were analyzed using a Hewlett-Packard 5890 GC coupled to a 5972 MSD. GC separation used an HP-5 MS column, 30 m × 0.25 mm × 0.25 µm. Head pressure was maintained at 21 kPa. An injection aliquot of 2 µl was used. The temperature profile was maintained at 100°C initially and then ramped to 225°C at 15°C/min; the purge valve was turned on at 30 s. The retention time for MA proprionamide under these conditions was approximately 7.6 min. The retention time of BA, under the same conditions, was approximately 7.2 min. The retention time of p-OH-MA was 12.6 min, and that of amphetamine was 7.0 min.
Electron ionization MS was used in single ion monitoring mode for quantitation. d14-MA was used as an internal standard on all samples and the area ratio of m/z 58 to 65 was used to quantify all MA samples. d2-BA was used as the internal standard for butylamphetamine determination. The area ratio of 100 to 102 was used to quantify all butylamphetamine samples. For p-OH-MA, m/z 58 was the quantitation ion and m/z 100 was used for amphetamine. The Hewlett-Packard RTE integrator was used to determine all areas. Dwell time was set at 100 ms on each ion and the electron multiplier voltage offset was set to 600 eV above tune. Calibration curves for MA, amphetamine, p-OH-MA, and butylamphetamine were linear.Statistical Analysis.
Data sets were analyzed by a one-way ANOVA using Statgraphics 6.0 software (Manugistics, Rockville, MD). Data sets were also tested by a
least significant difference multiple range test. Significance was
assumed at the 
= 0.01 level for all analyses.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Distribution of Serum Metabolites after Drug Administration. GC/MS analysis of blood from unlabeled MA-dosed animals revealed no detectable p-OH-MA for either enantiomer at either time point in either strain. MA was present at the concentrations presented in Fig. 2. These concentrations were not significantly different from the concentrations measured by liquid scintillation counting for either enantiomer in either strain at either time point (P > .11 in all cases). Amphetamine was present at one-tenth the concentration of MA.
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)BA in C57 or Balb/C mice were 1.70 ± 0.04 and 1.60 ± 0.04 ng/µl of blood, respectively. These concentrations are
approximately the same as ()MA concentrations depicted in Fig. 2.
In Vivo Study Results.
With the radiolabeled compounds, significantly more (P < .01) of each was recovered from C57 hair than from Balb/C hair. The total [3H](+)-MA concentration was
significantly greater than [3H]()MA in both
C57 and Balb/C mice. No significant differences between
[3H](+)BA and
[3H]()BA were observed. Significantly less
(P < .01) [3H](+)BA and
[3H]()BA were found in C57 hair when compared
with [3H](+)MA and
[3H]()MA concentrations. Additionally,
significantly more (P < .01)
[3H](+)BA and
[3H]()BA remained in the hair than was
recovered in the extractions. Less than 5% of the total
[3H](+)BA and
[3H]()BA was recovered in the methanol wash
and less than 20% of the total [3H](+)BA and
[3H]()BA was recovered in the phosphate
buffer extraction (Table 1). A
significant portion of the total [3H](+)MA and
[3H]()MA were also not recovered in either
the methanol wash or the phosphate buffer extraction. For C57 mice,
less than 5% of the total [3H](+)MA and
[3H]()MA was recovered in the methanol wash
and approximately 30% of the total [3H](+)MA
and [3H]()MA was recovered in the phosphate
buffer extraction (Table 1). A similar recovery (approximately 30% of
the total [3H](+)MA and 15% of the total
[3H]()MA) was observed in phosphate buffer
extraction of Balb/C hair (Table 1). Significantly less
(P < .01) [3H](+)BA and
[3H]()BA were recovered by phosphate buffer
extraction from C57 hair than from Balb/C hair. Approximately 5% of
the total [3H](+)BA and
[3H]()BA were recovered from C57 hair whereas
approximately 15% of the total [3H](+)BA and
[3H]()BA were recovered from Balb/C hair
(Table 1).
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)MA were
detected in C57 hair by GC/MS than by radiotracer quantitation (Table
1). The concentration of MA remaining in the hair as measured by
GC/MS was only 50% of that determined by liquid scintillation
counting. No significant differences were observed between radiotracer
measurements and GC/MS measurements of Balb/C hair concentrations of
(+)MA and ()MA (Table 1). No amphetamine was detected in the hair of
either C57 or Balb/C mice.
Significantly less (P < .01) (+)BA and ()BA were
measured in hair by GC/MS than by radiotracer (Table 1). Less than 1%
of the (+)BA and ()BA detected in digested hair after the methanol wash and phosphate buffer extraction by radiotracer were measured by
GC/MS of the organic extract of digested hair (Table 1). Significantly less (P < .01) (+)BA and ()BA were measured in the
phosphate buffer extraction of C57 hair by GC/MS than by radiotracer
(Table 1).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The data demonstrate little overall metabolism of MA and BA during
the 24-h period after drug administration. Of the reported metabolites
of MA (Caldwell et al., 1972), only MA and p-OH-MA would
retain the label in the tritiated analogs. The metabolism of BA would
result in the same pattern of BA and p-OH-BA retaining the
label. The data demonstrate that no p-OH-MA was detected in the blood and that the concentration of MA as measured by liquid scintillation counting of tritiated species matched the MA
concentration as measured by GC/MS. These results were observed in both
strains for both enantiomers at two time points. Approximately 10% of the MA was N-demethylated, which would result in the loss of
the tritium label. Thus it is likely that the circulating tracer does reflect parent MA. The same situation was obtained with BA enantiomers.
As with all radiotracer studies, isotope effects may alter the metabolism of the labeled analog relative to the unlabeled drug. The consistently lower, although not significantly different, measurements of serum MA by GC/MS than by radiotracer suggest that N-dealkylation may be nominally faster for the unlabeled compound than for the labeled compound. However, this metabolic difference appears slight, and would not account for the substantially lower hair concentrations of each compound measured by GC/MS (unlabeled) when compared with radiotracer results in pigmented hair. Furthermore, this difference in MA concentrations determined from labeled and unlabeled drugs was not observed for deposition in nonpigmented hair.
The same metabolic situation exists for BA. Coutts et al. (1976)
reported that less than 15% of BA administered to rats was excreted in
urine as p-OH-BA and of that, only 2% was free,
nonconjugated drug. This coupled with the nondetectable ring
hydroxylation of MA in mice suggests that ring hydroxylation was not a
significant metabolic route for the purposes of this study.
Metabolic N-dealkylation would result in the loss of the label as either formaldehyde, from MA, or butyraldehyde, from BA. It is possible that these could contribute some radioactivity and account for some of the higher drug concentrations in pigmented hair when determined by scintillation counting of tritiated analogs.
From the results of this study, both MA and BA were present in greater
concentrations in pigmented hair than in nonpigmented hair, but no
significant enantiomeric preferences were observed. The greater
concentrations of (+)-MA than ()-MA deposited in the hair of both
strains of mouse reflect the higher blood concentrations of (+)-MA
achieved after systemic administration. MA levels in hair were
consistent with levels reported by Nakahara et al. (1993) for
rats administered repeated doses of MA. For the butylamphetamines, the
concentrations of (+)BA and ()BA were not significantly different. The significant strain difference suggests that pigmented hair can
store more MA and BA than can nonpigmented hair. This is consistent with numerous authors who have found significantly greater
concentrations of various compounds in hair containing more pigment
(Cone and Joseph, 1996). This is also consistent with previous results
from our laboratory in which we found significantly higher
concentrations of systemically administered, radiolabeled serum
constituents Ca2+, cysteine, and
Mg2+ in pigmented hair (Stout et al., 1998b).
The presence of greater MA than BA concentrations in pigmented hair is
inconsistent with the report of Nakahara and Kikura (1996), who
concluded that increased length of carbon substituents at the nitrogen
position on amphetamine resulted in increased incorporation into hair.
However, at the equivalent doses used in this study, significantly
lower serum concentrations of BA than MA were obtained, and the
hair concentrations reflect that difference. This may also reflect a
species difference in drug distribution by the mouse relative to the
rat in the Nakahara and Kikura study.
A comparison of the results of the analysis of MA concentrations in
digests of pigmented hair by GC/MS with scintillation counting also
suggests that in pigmented hair MA may be present in a chemically
associated or modified form. Analysis of pigmented hair extracts and
total hair digests by GC/MS resulted in much lower concentrations of MA
than did measurement of MA by counting of the radiotracer. A similar
pronounced difference was seen with BA enantiomers. The necessity of
sodium hydroxide digestion to solubilize hair-associated radioactivity
after MA and BA accumulation suggests that the drugs may be very
tightly associated with other hair components or structurally modified.
Modifications of the amphetamines such as covalent association with
protein or pigment components of the hair could produce structural
derivatives quite different from the parent amphetamine. Extremely
tight binding or association of MA or BA enantiomers with the melanin
components of pigmented hair could also result in the inability of
extraction to completely remove the drugs (Stout and Ruth, 1999). It is
also possible, yet less consistent with the present data, that a minor circulating metabolite of MA or BA could selectively be sequestered in
the hair follicle of pigmented mice. This would also give rise to a
disparity between MA or BA content as measured by liquid scintillation
counting and GC/MS quantitation of unlabeled compounds.
The small quantities of MA and BA detected in the methanol wash are
indicative that little urine contamination occurred. This is consistent
with previous work from our laboratory showing the Na+ to K+ ratio in the hair
is the reverse of that in urine, indicating a lack of urine
contamination under the study conditions (Stout et al., 1998b).
The presence of small amounts of MA and BA in the phosphate extraction of hair suggests incorporation of BA and MA into a water-accessible compartment. The lower recovery of BA compared with MA is consistent with BA being less polar and more lipophilic, thus less likely to extract into aqueous media. However, the generally low aqueous buffer recoveries of BA an MA indicate that the accumulated MA and BA in hair have limited water accessibility or is of a limited compartmental size.
Several authors have reported parent drug to be the dominant species
deposited into the hair even when drug metabolites predominate in the
serum (Nakahara, 1995; Cone and Joseph, 1996). Nakahara et al. (1997)
also reported the appearance within 5 min of high concentrations of MA
in the hair bulbs of rats administered lethal doses of MA. This is also
consistent with the appearance less than 5 min after systemic
administration of fluorescent tracers, rhodamine, and fluorescein, in
the forming hair matrix of the hair bulb (Stout and Ruth, 1998). Like
rhodamine, parent drugs such as cocaine may rapidly accumulate in the
forming protein or melanin matrices and become trapped as the matrix is
progressively cross-linked and dehydrated during hair formation (Stout
and Ruth, 1999) Although in the current study the data strongly
indicate that the radiolabeled drugs accumulated in the hair are
unmetabolized MA and BA enantiomers, the chemical identities of the
radioactive species is not known and the deposition of metabolites
cannot be excluded.
The current data certainly suggest that the frequently used procedure
of determining extraction efficiencies of drugs from hair samples by
using externally loaded hair samples is not physiologically valid. The
external exposure of hair to drugs does not produce incorporation
analogous to in vivo incorporation (Stout and Ruth, 1998; Stout et al.,
1998a). In addition, it appears essential that methods used for the
extraction and quantitation of drugs in hair must be very thoroughly validated.
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Footnotes |
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Received February 26, 1999; accepted November 10, 1999.
This work was supported by National Institutes of Health Grant DA09545.
Send reprint requests to: James A. Ruth, University of Colorado Health Sciences Center, Department of Molecular Toxicology and Environmental Health Sciences, 4200 E. Ninth Ave., Box C238, Denver, CO 80262. E-mail: James.Ruth{at}uchsc.edu
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Abbreviations |
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Abbreviations used are:
MA, methamphetamine;
()MA, R()-methamphetamine;
(+)MA, S(+)-methamphetamine;
BA, N-(n-butyl)-amphetamine;
()BA, R()-N-(n-butyl)-amphetamine;
(+)BA, S(+)-N-(n-butyl)-amphetamine;
LAH, lithium aluminum hydride;
GC/MS, gas chromatography/mass
spectrometry;
THF, tetrahydrofuran.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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