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1 Division of Forensic Toxicology, Office of the Armed Forces Medical Examiner, Armed Forces Institute of Pathology, Rockville, MD 20850.
aAddress correspondence to this author at: Division of Forensic Toxicology, AFIP Annex, 1413 Research Blvd., Rockville, MD 20850. Fax 301-319-0628; e-mail paul{at}afip.osd.mil.
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Abstract |
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Methods: Liver, brain, blood, and urine specimens obtained from 15 postmortem cases were extracted using solid-phase extraction, derivatized, and analyzed using gas chromatography–mass spectrometry with selective-ion monitoring.
Results: Median concentrations (range) of drugs observed in postmortem liver, brain, blood, and urine were 0 (0–10) ng/g, 7 (0–92) ng/g, 0 (0–42) µg/L, and 62 (0–2030) µg/L, respectively, for MED; 655 (90–3274) ng/g, 22 (0–52) ng/g, 119 (13–773) µg/L, and 456 (109–7452) µg/L, respectively, for ED; 57 (0–503) ng/g, 187 (0–1403) ng/g, 12 (0–88) µg/L, and 1208 (37–28 062) µg/L, respectively, for COC; and 821 (45–4980) ng/g, 524 (46–5153) ng/g, 458 (30–2071) µg/L, and 6768 (917–116 430) µg/L, respectively, for BZ. MED was detected in 12 of 15 postmortem cases. The concentrations were highest in urine compared with liver, brain, and blood. The hydrolysis product ED was detected in all postmortem cases, and the concentrations were substantially higher than MED in all liver, blood, and urine specimens.
Conclusion: ED may be a more useful indicator of crack COC smoking.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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800 °C. On the basis of this information, it would be reasonable to expect individuals smoking crack COC to inhale significant amounts of MED. As a result, the detection of MED would be a valuable marker to aid in identifying smoking as a route of administration and in estimating COC concentrations at the time of death.
There are additional considerations in the analysis of MED. Once in the body, the concentration of MED might be expected to be lower because of conversion to ecgonidine (ED). The hydrolysis of MED to ED has been reported, and ED has been detected in human urine (7)(8). Urine specimens that had tested positive for benzoylecgonine (BZ) in the military drug-testing program were tested for MED and ED. In 22 of the 23 specimens tested, ED was detected, and the concentrations were at least an order of magnitude greater than those of MED. These results suggest the value of quantifying both MED and ED to identify COC smoking as the route of administration.
Although MED and/or ED have been examined in various tissues and fluids (2)(3)(7)(8)(9)(10)(11)(12)(13), MED has not been examined in liver and brain tissue, and ED has not been examined in liver, brain, and blood specimens. We examined the available specimens from 15 postmortem cases for the presence of MED, ED, COC, and BZ.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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specimens
Specimens from 15 postmortem cases were obtained from the
Postmortem/Human Performance Laboratory, Division of Forensic
Toxicology, Armed Forces Medical Examiners’ Office, Armed Forces
Institute of Pathology. The research protocol was approved by the Armed
Forces Institute of Pathology Institutional Review Board. Specimens
reported to be positive for COC/BZ had been screened positive by
immunoassay, confirmed positive by gas chromatography–mass
spectrometry (GC-MS), and stored frozen at -18 °C for 6 months
after testing was completed. Before use in this study, specimen
identity was removed in preparation for disposal. Complete information
concerning the specific source for each fluid and tissue was not
available for any case. A significant impact on results was unlikely
for the liver specimens because drug distribution in liver tissue would
be expected to be uniform as a result of normal high hepatic profusion
and metabolic activity. Brain tissue may have been obtained from
various portions of the brain because there is no standardized
procedure concerning the source of brain tissue. This most likely did
not have a significant impact on our results, based on the reported
uniform distribution of COC in postmortem brain tissue (14).
Urine was obtained directly from the bladder. In a few cases, blood
specimens were obtained directly from the heart or from an intravenous
catheter.
instrument
The Hewlett-Packard GC-MS system consisted of an HP 5890 Series II
Plus gas chromatograph and a Model 5972 quadrupole mass-selective
detector. An HP 18593B autoinjector was used to inject the samples into
the GC-MS instrument.
synthesis of methyl
N-desmethyl-N-[2H3]methylecgonidine
(d3-MED)
The starting compound,
N-desmethyl-N-[2H3]methylecgonidine
(d3-ED) was prepared from 200 µg of
d3-EC by the procedure published
previously (7). DMF dimethyl acetal (50 µL) was added to
the compound in a Reacti-vial. The vial was tightly capped,
vortex-mixed, and heated at 50 °C for 5 h. The solution was
cooled to room temperature. The
d3-MED, without further purification,
was quantitatively transferred to a 50-mL volumetric flask and diluted
to the mark with acetonitrile. When compared against a known amount of
nondeuterated MED, the concentration of
d3-MED was found to be 2.08 mg/L. On
the basis of the molar concentration compared to the starting material,
d3-ED, the yield was 84%.
preparation of stock solutions
Solutions of COC, d3-COC, MED,
and d3-MED at appropriate
concentrations were prepared in acetonitrile. Hydroxy solvents were not
used to avoid hydrolysis of methyl esters. Solutions of BZ,
d3-BZ, ED, and
d3-ED were prepared in methanol.
sample preparation for COC, BZ, MED, and ED determination
Liver and brain tissue samples.
The flowchart for the
extraction is shown in Fig. 1
. For each specimen and control, 1 g of tissue (negative
tissue for control) was weighed into flat-bottomed plastic tubes and
stored frozen. Controls were prepared at concentrations of 100, 50, and
20 ng/g of tissue by adding COC (100 µL of 1.0, 0.5, or 0.2 mg/L), BZ
(100 µL of 1.0, 0.5, or 0.2 mg/L), MED (100 µL of 1.0, 0.5, or 0.2
mg/L), and ED (100 µL of 1.0, 0.5, or 0.2 mg/L) into three empty
glass centrifuge tubes. Internal standards (100 µL of 0.5 mg/L
d3-COC, 100 µL of 0.5 mg/L
d3-BZ, 50 µL of 0.68 mg/L
d3-MED, and 100 µL of 0.5 mg/L
d3-ED) were added to the control tubes
and to empty glass centrifuge tubes that were later used for specimen
analysis.
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Freshly prepared NaF (200 µL; 10 g/L) was added to the tissues in the plastic tubes. The tissues were thawed, homogenized with 1 mL of 0.1 mol/L phosphate buffer (pH 6.0), and poured into the corresponding glass tubes containing drugs and internal standards for controls and internal standards only for specimens. Two milliliters of 0.1 mol/L phosphate buffer (pH 6.0) was then added to the residual tissue and homogenized again. The resulting homogenate was combined with the initial homogenate.
After homogenization, the tubes were capped and placed in an ice bath. The homogenates were vortex-mixed for 10–15 s and centrifuged for 60 min at 3834g at 7–10 °C. A set of SPE columns was placed in the extraction chamber and conditioned with 3 mL each of methanol, water, and 0.1 mol/L phosphate buffer (pH 6.0), in that sequence, under slightly reduced pressure.
A set of glass centrifuge tubes was placed in the chamber to collect
the next elution fraction (containing ED). The supernatants from the
tissue homogenates were poured onto the columns and allowed to elute by
gravity or under slightly reduced pressure. Deionized water (1 mL) was
added to the columns, and that fraction was also collected. The tubes
from the chamber were then removed and set aside for extraction of ED
(Fig. 1
). The SPE columns were washed with 2 mL of deionized water, 3
mL of 0.1 mol/L HCl, and 3 mL of isopropanol, and then dried for 5 min
under reduced pressure. The MED, COC, and BZ were then eluted with 3 mL
of a mixture (9:1:0.2 by volume) of dichloromethane–methanol–aqueous
NH3 (14.8 mol/L).
The solutions were evaporated to dryness under nitrogen at room temperature to minimize loss of MED. The extracts were dissolved in 50 µL of dry acetone, transferred into autosampler vials, and tested for MED and COC by two separate GC-MS procedures. After the MED and COC tests, the samples were tested for BZ as the propyl derivative, using another GC-MS procedure.
To extract ED from the eluates, the pH of the solutions was adjusted to 2.0 ± 0.2 with 0.5 mol/L HCl. Methylene chloride (1 mL) was added to each tube and vortex-mixed. The solutions were centrifuged at 2177g for 3 min. The clear upper aqueous layers were then poured onto a second set of SPE columns preconditioned with 3 mL of methanol, 3 mL of deionized water, and 1 mL of 0.01 mol/L HCl. The solutions were allowed to elute by gravity flow or under slightly reduced pressure. The columns were washed with 1 mL of 0.1 mol/L HCl and 3 mL of methanol, and dried under reduced pressure for 5 min. The ED was eluted with 3 mL of a mixture (4:6:0.25 by volume) of methanol–isopropanol–aqueous NH3 (14.8 mol/L). The solutions were evaporated under nitrogen at 50 °C. To remove the white residue from ED, 2 mL of dry acetone was added. The solutions were vortex-mixed and centrifuged. The acetone solutions were separated and evaporated to dryness. The liver specimens were tested for ED as the tert-butyldimethylsilyl derivative by a GC-MS method, and the brain specimens were tested for ED as the trimethylsilyl derivative by GC-MS.
Urine and blood samples.
An initial 3-mL aliquot of each urine
specimen was prepared as published previously (7). The
procedure used for preparing blood specimens was the same as for the
liver and brain tissues with the following exceptions. The initial 1-mL
aliquots of blood were diluted with 4 mL of 0.1 mol/L phosphate buffer
(pH 6.0). No methylene chloride wash was used in preparing the
ED-containing fractions for the SPE steps.
derivatization
Propylation of BZ at the GC injection port for specimen
analysis.
After COC and MED analysis in acetone solution, DMF-DPA
(20 µL) was added to the autosampler vials. The contents were mixed,
and the vials were recapped. Analysis was performed for propyl-BZ as
reported previously (7).
Silylation of ED by MTBSTFA for specimen analysis.
The
extracts from liver and urine containing ED were dissolved in MTBSTFA
containing 1% TBDMCS and prepared as described previously
(7).
Silylation of ED by BSTFA for specimen analysis.
The extracts
from brain and blood containing ED were dissolved in 50 µL of BSTFA
and heated in closed tubes at 70 °C for 15 min. The samples were
centrifuged immediately at 7–10 °C for 2–3 min at 1700g
and transferred to autosampler vials for GC-MS analysis.
GC-MS analysis
Samples were introduced in 1- to 4-µL volumes using an
autoinjector. The GC analysis was performed using a DB-5MS capillary
column [5:95 phenyl-methylsiloxane; 15 m x 0.25 mm (i.d.); J & W
Scientific] or a ZB-5 [5% phenyl polysiloxane; 15 m x 0.25 mm
(i.d.); Phenomenex] at 10 psi constant pressure helium flow. The
mass-selective detector was operated in the electron ionization mode at
70 eV with a source temperature of 200–250 °C. The electron
multiplier voltage of the detector was set at 200–700 V above
autotune, and the dwell time for each ion monitored was 50 ms.
GC-MS conditions for MED.
The analysis was performed at
injector and transfer line temperatures of 140 and 280 °C,
respectively. The oven temperature started at 90 °C (held for 1 min)
and increased to 140 °C at 20 °C/min (held for 2 min). The GC was
started in splitless mode, and injector port purge was turned on after
0.3 min. The monitored ions were m/z 181, 166, and 152 for
MED and m/z 184 and 155 for the
d3-MED. Ions m/z 152 and
155 were used for quantification. Because COC, BZ, and MED were
extracted together, injector temperatures were kept 
140 °C to
avoid possible formation of MED from any COC present in the sample.
After each injection, 2–3 µL of acetone was injected as a solvent
blank.
GC-MS conditions for COC.
The analysis was performed at
injector and transfer line temperatures of 280 and 270 °C,
respectively. The oven temperature started at 170 °C (held for 0.5
min) and increased to 270 °C at 30 °C/min (held for 2.7 min). The
GC was started in splitless mode, and injector port purge was turned on
after 0.3 min. The monitored ions were m/z 303, 272, and 182
for COC and m/z 306 and 185 for the
d3-COC. Ions m/z 303 and
306 were used for quantification. After each injection, 3 µL of
acetone was injected as a solvent blank.
GC-MS conditions for propyl BZ.
The analysis was performed at
injector and transfer line temperatures of 280 and 270 °C,
respectively. The oven temperature started at 170 °C (held for 0.5
min) and increased to 270 °C at 30 °C/min (held for 2.7 min). The
GC was started in splitless mode, and injector port purge was turned on
after 0.3 min. The monitored ions were m/z 331, 272, and 210
for BZ and m/z 334 and 213 for
d3-BZ. Ions m/z 331 and 334
were used for quantification. After each injection, 3 µL of MTBSTFA
containing 1% TBDMCS was injected as a solvent blank.
GC-MS conditions for tert-butyldimethylsilyl ED.
The analysis
was performed at injector and transfer line temperatures of 250 and
270 °C, respectively. The oven temperature started at 135 °C
(held for 0.5 min), increased to 175 °C at 10 °C/min (held for
0.5 min), and increased to 275 °C at 30 °C/min (held for 1 min).
The GC was started in splitless mode, and injector port purge was
turned on after 0.2 min. The monitored ions were m/z 281,
252, and 224 for ED and m/z 284 and 227 for the
d3-ED. Ions m/z 224 and 227
were used for quantification. After each injection, 3 µL of MTBSTFA
containing 1% TBDMCS was injected as a solvent blank.
GC-MS conditions for trimethylsilyl ED.
The analysis was
performed at injector and transfer line temperatures of 200 and
220 °C, respectively. The oven temperature started at 111 °C
(held for 1 min) and increased to 210 °C at 25 °C/min (held for 2
min). The GC was started in splitless mode, and injector port purge was
turned on after 0.2 min. The monitored ions were m/z 239,
224, and 210 for ED and m/z 242 and 213 for the
d3-ED. Ions m/z 210 and 213
were used for quantification. After each injection, 3 µL of BSTFA was
injected as a solvent blank.
statistical analysis
The statistical analysis of the data sets was performed using
ANOVA. To be a significant difference, both sets had to show a
difference at P <0.05.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Separation of ED from COC and COC metabolites minimized the formation
of artifact ED. MED in the extract of COC and BZ was analyzed
separately at a GC injection port temperature of 140 °C. Higher
temperatures were avoided to minimize formation of artifact MED. To
ensure that artifacts were not formed during analysis, a control
containing COC, BZ, EC, and MEC at 1, 5, 5, and 5 mg/L, respectively,
was tested. No ED or MED was detected under the extraction and GC-MS
conditions described. COC was also injected separately before
derivatization of BZ because the alkylating agents contained a small
amount of methylating agent as impurity. In our experiment, DMF-DPA,
DMF-diisopropylamide, or DMF-diethylamide with only BZ produced a small
amount of COC as byproduct (<1%). Although MED and COC were injected
under two different conditions (injection temperatures 140 and
280 °C, respectively), the samples could be analyzed in one
injection batch by use of an autoinjector with two GC-MS settings. ED
extracted from blood and brain and tested as the
tert-butyldimethylsilyl derivative showed chromatographic
background. Analyzing the compound as the trimethylsilyl derivative
minimized the background.
The linearity, correlation coefficient squared
(r2), limit of quantification (LOQ),
and extraction efficiency are summarized in Table 1
. In the range of linearity, all compounds showed ion ratios
within ± 20% of the mean values. At least eight concentrations
were used in the range of linearity. Only one sample was tested at each
concentration. Good correlation (r2)
was observed in the linear range mentioned, where acceptable values
were defined as 
0.9900. Signal-to-noise ratios in all analyses were
>4:1. In this study, LOQ was defined as the concentration at which one
of the two ion ratios was outside ± 20% but within ± 30%
and the concentration was within ± 20% of expected value.
Quantification ions in some compounds showed better chromatographic
background, allowing a LOQ below the limit of linearity. This
observation was most likely attributable to the variability of the
biological matrices among the various tissues and fluids. Specimens
were analyzed in several batches, and controls at concentrations of 0,
20, 50, and 100 µg/L were used in each batch analysis to validate the
results. The criteria for batch validation were the same as those used
for linearity studies.
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Case histories for the specimens were extremely limited, although
preliminary causes of death were not attributed to drug overdose. The
results from the analyses by drug are summarized in Tables 2
and 3
. Fig. 2
shows chromatograms for ED extracted from blood specimen 4
(Fig. 2a
) and MED extracted from brain specimen 11 (Fig. 2b
), where the
concentrations were 25 µg/L and 22 ng/g, respectively. The ranges for
MED concentrations in liver, brain, blood, and urine were 0–10 ng/g,
0–92 ng/g, 0–42 µg/L, and 0–2030 µg/L, respectively. The number
of samples in which MED was detected was 5 of 15, 7 of 14, 3 of 11, and
10 of 13 in liver, brain, blood, and urine, respectively. Although MED
was detected in 5 of 15 liver specimens, the observed concentrations
did not exceed 10 ng/g in any specimen. In contrast, urine
concentrations as high as 2030 µg/L were observed, and 6 of the 13
urine specimens reached concentrations in excess of 100 µg/L. In
general, MED concentrations were much lower in brain than in urine. The
range of MED concentrations in the liver specimens was slightly larger
than in the brain specimens. Although examining brain for estimating
COC concentrations at the time of death may have some advantage because
of the free passage of COC across the blood-brain barrier, the
characteristics of MED relative to the blood-brain barrier have not
been established. The high number of blood samples negative for MED or
with low MED concentrations may indicate that detection of MED may be
difficult in blood and be of limited usefulness in the estimation of
COC concentrations at the time of death. On the basis of these
observations along with other concerns for the use of blood in
evaluating COC use in postmortem cases (15), other tissues
and/or fluids may be more useful in postmortem examinations.
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ED concentrations were noticeably higher than MED in liver, blood, and urine. In liver, the median ED concentration was 655 ng/g (range, 90–3274 ng/g) compared with 0 ng/g (range, 0–10 ng/g) for MED. Similar results were observed in blood. The amount of ED was relatively low in brain, and there was no statistical difference compared with MED concentrations in brain tissue. This indicates that ED does not easily pass through the blood-brain barrier, which might be expected based on the hydrophilic nature of ED, and may also be accompanied by extensive first- or second-pass metabolism of MED in the liver and efficient excretion of ED by the body via the kidneys. This may also indicate that any MED that may enter the brain is converted to ED via enzymatic hydrolysis at a very low rate, which may parallel the observation that cocaine methyl esterase activity in brain is only 1–6% compared with liver in rats (16). Although MED was detected in only 3 of 11 blood specimens, ED was detected in all blood specimens. This suggests that MED is extensively converted to ED in the blood as well as in the liver, which is supported by the previously reported hydrolysis of MED to ED in liver homogenates (7) and the enzymatic hydrolysis observed in unpreserved sheep plasma (17).
When the ED concentrations in liver and urine were compared, 6 of 13 specimens contained more ED in liver than in urine. In 8 of 15 liver specimens, the amount of ED actually exceeded the amount of BZ. ED was detected in all 15 cases (50 of 53 total specimens), indicating that smoking was a route of COC administration. Compared with the detection of MED in 12 of 15 cases (25 of 53 total specimens), ED appears to be more easily detected than MED and may be a more useful marker than MED for determination of COC smoking in postmortem cases.
In conclusion, a review of the data reveals that ED concentrations were significantly higher than MED concentrations in liver, blood, and urine. In brain the difference was not significant. Of 53 tissue and fluid specimens collected from 15 postmortem cases, 50 specimens were positive for ED compared with only 25 specimens positive for MED. Only three brain specimens were negative for ED. The presence of ED and MED suggests that smoking was the route of COC administration in all 15 postmortem cases.
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Footnotes |
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2 The opinions expressed herein are those of the authors and are not to
be construed as official or as reflecting the views of the Department
of the Army, the Department of the Navy, the Department of the Air
Force, or the Department of Defense.
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