| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
University of Rochester School of Medicine, Departments of
1
Pathology and Laboratory Medicine and
2
Obstetrics and Gynecology, Rochester, NY 14642.
a Address correspondence to this author at: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Ave., Box 608, Rochester, NY 14642. Fax 716-273-3003; e-mail Tai_Kwong{at}urmc.rochester.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: We developed gas chromatography–mass spectrometry (GC-MS) assays for MEG and EC. Plasma was collected from sheep blood containing 0, 0.06, or 0.24 mol/L (0%, 0.25%, or 1%) NaF. MEG was added to these plasmas, and they were incubated at -80, 1, 21, or 37 °C to determine whether there were temporal, temperature, or storage effects on MEG stability over 48 h.
Results: Decreased temperature and increased NaF concentrations limited MEG degradation and EC formation. MEG stored in plasma at -80 °C was stable up to 1 month, even in the absence of NaF.
Conclusions: MEG is stable in sheep plasma collected in commercially available, evacuated blood-collection tubes containing NaF and stored at -80 °C. In vitro formation of EC can be minimized with appropriate sample handling, and its in vivo formation may provide a better marker of crack smoking than its parent pyrolysis product.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We hypothesized that the scarcity of reports detecting MEG in blood
could be attributed to biotransformation or loss during sample
processing and storage. Our research paradigm uses a sheep model to
study physiologic responses to and pharmacokinetics of MEG. Thus, we
were concerned about proper sample handling techniques and MEG
degradation in stored study samples. Our objective was to investigate
the effects of different temporal and temperature storage conditions on
MEG concentrations and conversion to its hydrolytic product, ecgonidine
(EC). To accomplish this, an assay capable of measuring MEG plasma
concentrations was developed. Because EC is more polar than MEG, MEG
extraction conditions were incapable of recovering EC, and a separate
assay was developed. NaF is routinely added to samples
collected for cocaine analysis to inhibit esterases responsible for
biotransformation of cocaine (6)(7). It has been
hypothesized that EC is a hydrolytic product of MEG (Fig. 1
), and EC has been reported in urine of crack smokers
(1)(8). Conversion of MEG to EC presumably
results from the same metabolic mechanism as hydrolysis of cocaine to
benzoylecgonine (BE). Thus, we investigated whether NaF or temperature
prevents degradation of MEG to EC in sheep plasma under different
storage and pH conditions. These experiments established appropriate
collection and storage conditions, which assure accurate determination
of MEG and EC concentrations in blood/plasma.
|
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
synthetic procedures
MEG and EC.
MEG and EC hydrochloride were synthesized
according to a method published by Zirkle et al. (9). MEG
was purified by conversion to fumarate salt. Impurities were removed by
washing the solid residue with acetone. The salt, recrystallized from
methanol-ether, had a melting point of 178–180 °C (melting point in
the literature, 178–179 °C). MEG was dissolved in methanol and
analyzed by gas chromatography–mass spectrometry (GC-MS); major peaks
were at m/z 152 and 166, with the molecular ion at
m/z 181. EC hydrochloride was derivatized by treatment with
BSTFA-TMS at 65 °C for 10 min and analyzed by GC-MS; major peaks
were at m/z 210 and 122, with the molecular ion at
m/z 239.
Ethylecgonidine (EEG).
A mixture of 980 mg of EC hydrochloride
and 50 mL of absolute ethanol, saturated with hydrogen chloride, was
refluxed for 24 h. After removal of solvent, the residue was
dissolved in water, and the aqueous layer was adjusted to a pH of 8.5
with saturated potassium carbonate and extracted four times with 25-mL
volumes of dichloromethane. The combined organic layers were
evaporated, and 850 mg of the ethyl ester was obtained. GC-MS analysis
of the product showed major peaks at m/z 138 and 166, with
the molecular ion at m/z 195. The fumarate salt of the ester
was prepared and recrystallized from ethanol-ether; its melting point
was 144–146 °C. The fumarate salt was subjected to thin-layer
chromatography on silica gel developed with
chloroform:methanol:ammonium hydroxide (80:18:2, by volume) and was
visualized under ultraviolet light. The
Rf was 0.78.
N-Ethyl-N-norcocaine.
N-Ethyl-N-norcocaine was synthesized via
N-norcocaine by mixing 1.56 g of
N-norcocaine, 0.75 g of potassium carbonate, and
0.89 g of ethyl iodide in 50 mL of acetone and stirring overnight
at room temperature (10). The solid was removed by
filtration and washed with acetone. Removal of solvent from the
combined filtrate and washings yielded a solid residue that was
purified by chromatography on a silica gel column with
chloroform:methanol:ammonium hydroxide (190:9:1, by volume) as
eluant. GC-MS revealed major peaks at m/z 196 and 286, with
the molecular ion at m/z 317.
N-Ethyl-N-norecgonidine (NENE).
N-Ethyl-N-norcocaine hydrochloride (1.36 g in 15
mL of 370 g/L hydrochloric acid) was refluxed for 17 h.
The mixture was cooled in a refrigerator, allowing benzoic acid to
precipitate, and was removed by filtration. The filtrate was evaporated
to dryness, and the white solid residue was triturated with several
portions of ether to remove traces of benzoic acid. The solid residue
was dried in a vacuum oven at 100 °C and recrystallized from
ethanol-ether; the melting point of the solid was 239–240 °C. The
product was derivatized by treatment with BSTFA-TMS and analyzed by
GC-MS. The spectrum showed peaks at m/z 136 and 224, with
the molecular ion at m/z 253.
sample preparation
MEG and EC calibrators (30, 50, 100, 250, and 500 µg/L of MEG or
EC) were prepared daily in sheep plasma using stock 1000 µg/L MEG or
EC in sheep plasma solution. Three interassay controls used for MEG and
EC (55, 300, and 2500 µg/L for MEG and 55, 300, and 3000 µg/L for
EC) were prepared by adding MEG and EC dissolved in methanol to the
plasma and were stored in aliquots at -80 °C. A 500-µL aliquot of
calibrator, control, or sample sheep plasma was pipetted into a clean
test tube, and internal standard (EEG for MEG analysis and NENE for EC)
was added, creating a final concentration of 250 µg/L for each
internal standard. After the internal standard was added, each separate
sample was incubated for 15 min in an ice bath. Chilled methanol (1 mL)
was added dropwise to each sample, while vortex-mixing, to precipitate
plasma proteins. Samples were then centrifuged at 1000g for
20 min in a 10 °C refrigerated centrifuge.
For analysis of samples collected from MEG incubated in buffer, MEG and EC calibrators were prepared in phosphate-buffered saline (PBS, pH 7.4) by the addition of MEG dissolved in methanol. Methanol precipitation was not performed; however, sample and calibrator preparation, extraction, and GC-MS analysis was conducted according to the protocols for plasma.
meg extraction
MEG was extracted according to the method of Cone et al.
(2) on solid-phase columns, with a modification using
methanol to precipitate plasma proteins. The total supernatant
collected at the end of the sample preparation step was diluted with 5
mL of sodium acetate buffer (2 mol/L, pH 4.0 ± 0.1). With the
methanol protein precipitation procedure, 
10% more MEG was
recovered than with filtration. In brief, extraction columns were
conditioned sequentially with elution solvent
(dichloromethane:2-propanol:ammonium hydroxide; 78:20:2, by volume),
methanol, distilled water, and 2 mol/L acetate buffer. Diluted samples,
calibrators, or controls were added to preconditioned columns and
eluted by gravity. Columns were washed sequentially with distilled
water, 0.2 mol/L hydrochloric acid, and methanol. After methanol
washing, columns were dried under reduced pressure for 2 min.
Analytes were collected in tubes, using 5 mL of elution solvent, and
dried under nitrogen at 37 °C. The analytes were resuspended in 50
µL of ethyl acetate, transferred to autosampler vials, and analyzed
by GC-MS.
ec extraction
The EC extraction procedure was based on the method of Peterson et
al. (11). The total supernatant collected at the end of the
sample preparation step was diluted with 5 mL of distilled water and
adjusted pH to 2.0–2.5 by the addition of 0.2 mol/L phosphoric acid.
Extraction columns were sequentially preconditioned with methanol,
distilled water, and 0.2 mol/L phosphoric acid. Diluted sample
supernatants were added to the columns. Columns were then washed with
0.2 mol/L phosphoric acid and methanol. After the methanol wash,
columns were air-dried for 1 min. Five milliliters of 3% ammoniacal
methanol (30 mL/L ammonium hydroxide in methanol) was used to
elute analytes into clean tubes. Eluates were transferred to
Reacti-vials (Pierce Chemical) and dried at 50 °C under nitrogen.
BSTFA-TMS (50 µL) was added under nitrogen, and the vials were
immediately capped, vortex-mixed, and incubated at 65 °C for 15–20
min. Derivatized mixtures were transferred to autosampler vials and
analyzed by GC-MS.
instrumentation
A Hewlett Packard model 5890 series II gas chromatograph equipped
with a HP 6890 series autosampler and a HP 5972 mass selective detector
was used for MEG and EC analysis. Sample (2 µL) was injected into a
250 °C injector operated in the splitless mode. A Hewlett Packard
Ultra II column [5% phenyl methyl siloxane; 12 m x 0.2 mm
(i.d.); 0.33 µm film thickness] was used for both MEG and EC. The
oven for MEG analysis was temperature programmed from 70 to 250 °C
at 30 °C/min with no initial hold time and a final hold of 4 min.
The oven program for EC had an initial 1-min hold at 70 °C, followed
by a ramp from 70 to 200 °C at 20 °C/min, another 1-min hold, and
then a second ramp from 200 to 250 °C at 30 °C/min and a final
hold of 2 min. Ultrapure helium carrier gas was used with a flow rate
through the column of 1 mL/min. The transfer line temperature was
280 °C, and the mass spectrometer was set to use electronic
ionization of 400 V relative to the daily tune voltage. For MEG
analysis, the following ions were monitored: m/z
(152), 181, and 166 for MEG and (166), 138, and 195 for EEG. For EC
analysis, the ions monitored were m/z (210), 239, and 122
for EC and (224), 253, and 136 for NENE. Ions in parentheses were base
ions and were used for quantification. The dwell for all ions was 80
ms. The retention times for MEG and EEG were 3.366 and 3.638 min,
respectively. The retention times for EC and NENE were 6.027 and 6.304
min, respectively.
assay validation
MEG and EC assay linearity was investigated by adding MEG/EC to
sheep plasma from duplicate stock solutions diluted from
calibrators weighed out separately. The expected MEG
concentrations of the linearity samples were 100, 500, 1000, 2500, and
5000 µg/L. The expected EC concentrations of the linearity samples
were 50, 250, 500, 1000, 2500, 3000, and 5000 µg/L. Internal standard
was added to a final concentration of 250 µg/L, and the linearity
samples were extracted along with fresh calibrators. The linearity
samples were quantified using the calibration curve. Recovery was
determined for each assay’s lowest calibrator (30 µg/L). Interassay
precision was determined using MEG (55, 300, and 2500 µg/L) and EC
(55, 300, and 3000 µg/L) controls that had been prepared in sheep
plasma and stored at -80 °C.
experimental design
Solutions of sheep blood were prepared to contain 0, 0.06
(0.25%), and 0.24 mol/L (1%) NaF (Fig. 2
). Fresh blood was centrifuged at 1000g for 7
min, and plasma was collected. Concentrated MEG stock (70 mg/L) was
prepared in NaF-free plasma by adding MEG dissolved in methanol. MEG
solutions (2000 µg/L) were prepared by adding 1 mL of 70 mg/L MEG in
plasma to 34 mL of plasma containing 0, 0.06, and 0.24 mol/L (0%,
0.25%, and 1%) NaF and mixed thoroughly. A 10-mL aliquot of each 2000
µg/L solution was stored in an ice-water bath (1 °C), at room
temperature (21 °C), and in a 37 °C incubator. From each
solution, 500-µL aliquots were collected for separate MEG and EC
analysis at 0, 1, 3, 6, 13, 18, 24, and 48 h and immediately
placed in a -80 °C freezer until analysis. Additional aliquots of
each 2000 µg/L MEG plasma solution were placed in a -80 °C
freezer immediately after solutions were prepared.
|
To investigate the effect of pH on MEG stability, 4 mL of each MEG plasma solution (0, 0.06, and 0.24 mol/L NaF) prepared above was aliquoted, and the pH was adjusted to pH 6.0 ± 0.1 with acetate buffer (0.2 mol/L, pH 3.0). Initial samples were collected for MEG and EC analysis as soon as they were buffered, and the remaining solutions were placed in a 37 °C incubator. Samples were collected for MEG and EC analysis at 13 and 24 h and stored at -80 °C.
To investigate MEG degradation in the absence of enzymes, 50 mg/L MEG
dissolved in methanol was added to 25 mL of PBS, pH 7.4 (final MEG
concentration, 2000 µg/L). Aliquots were split into tubes, stored on
ice (1 °C), at room temperature (21 °C), and in a 37 °C
incubator. From each condition for MEG and EC analysis, 500
µL was sampled immediately after solution preparation and at
6, 9, 24, and 48 h. The samples were stored at -80 °C until
analysis (3 weeks).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. The limit of detection was defined as the lowest expected
analyte concentration that produced ion ratios within 20% of the
averaged calibrator ratios. The lowest value of the linear range was
the limit of quantification, which was defined as the measured
concentrations within 20% of the expected value. The recovery for the
MEG extraction method, after methanol precipitation of plasma proteins,
was >90%. A linearity plot was constructed of the measured vs
expected concentrations of analyte (Fig. 3
). The 5000 µg/L samples were not included in the fits and are
not shown because of increased variability for MEG and loss of
linearity for EC. Thus, working linear ranges were established as
20–2500 and 30–3000 µg/L for MEG and EC, respectively. The standard
deviations around the regression lines were 87.2 and 51.5 µg/L for
the fitted MEG and EC linearity curves, respectively. Table 2
lists the interassay characteristics for the MEG and EC assays.
Interassay CVs were <10% for both assays.
|
|
|
There was a greater rate of loss of MEG with increasing temperature in
all NaF conditions (Fig. 4
). The 0.24 mol/L (1%) NaF data are not shown for sake of
clarity, but they did not differ by more than the assay’s precision at
2500 µg/L (8.6%) from the 0.06 mol/L (0.25%) NaF data. The
disappearance of MEG in non-NaF-treated plasma incubated at 37 °C
was striking: MEG concentrations after 24 h were 50% of
initial concentrations at 37 °C. EC formation increased with
increasing temperature and correlated directly with the loss of MEG.
Summation of the total moles of MEG and EC present at each time point
of the 37 °C incubation revealed that total moles differed by <10%
of the moles of MEG initially present. This mass balancing indicates
that EC is a major product of MEG. MEG concentrations in all ice-bath
incubation samples were stable, with concentrations varying by <10%
of the starting concentration for all NaF conditions. A key observation
of this study was finding that EC concentrations in 0.06 mol/L (0.25%)
NaF-plasma incubated on ice were nonquantifiable even after 48 h,
whereas the EC concentration in the equivalent sample in
non-NaF-treated plasma was 158.20 µg/L.
|
The top panels of Fig. 4
illustrate that decreased temperature enhanced
the stability of MEG within both non-NaF- and NaF-treated [0.06 mol/L
(0.25%)] samples. The ice-bath incubation consistently provided the
most stable MEG concentrations by limiting EC formation. The bottom
panels of Fig. 4
show that 37 °C incubation possessed the highest
concentrations of EC and that 0.06 mol/L (0.25%) NaF reduced formation
of EC. The EC concentrations were low in the 0.06 and 0.24 mol/L
(0.25% and 1%) NaF samples (153.82 and 145.04 µg/L) compared with
781.71 µg/L in the non-NaF-treated sample after 48 h at room
temperature.
Additional studies revealed that MEG was stable when stored at -80 °C under any of the NaF conditions for up to 3 months of storage, showing no significant loss of MEG. MEG concentrations under these conditions did not vary by more than 14% from 2000 µg/L. Additionally, EC conversion from MEG under the same storage conditions was negligible, never being >40 µg/L. This observation suggests that measured concentrations of EC in the incubation study samples are a result of MEG conversion during incubation and not from conversion during sample storage.
The addition of NaF improved the stability of MEG (limiting formation of EC). However, MEG concentrations in both NaF groups for all temperature conditions never varied from each other by more than the assay’s precision. This suggests that increasing the NaF concentration from 0.06 to 0.024 mol/L (0.25% to 1%) had no effect.
MEG concentrations decreased by more than the assay’s precision after 3 h of 37 °C incubation in plasma lacking NaF, and plasma adjusted to 0.06 mol/L (0.25%) NaF varied by more than the assay’s precision after 24 h under the same incubation conditions. MEG concentrations decreased by more than the assay’s precision after 13 h of room temperature incubation in plasma without NaF and after 48 h in plasma adjusted to 0.06 mol/L NaF.
Reducing the pH to 6.0 produced a greater effect than NaF: MEG
concentrations did not vary by more than the assay’s precision after
24 h in all NaF conditions when incubated at 37 °C (Fig. 5
).pH measurements immediately after solution preparation and at the
conclusion of the experiment are listed in Table 3
for endogenous and pH 6.0 samples.
|
|
When MEG was dissolved in PBS at pH 7.4, it demonstrated little variation of MEG concentrations in all temperatures tested (data not shown). The MEG concentrations did not vary by more than the assay precision even after 48 h of 37 °C incubation.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This study demonstrates that samples collected and processed according to procedures similar to those for cocaine samples should produce MEG concentrations that reflect in vivo metabolism. Standard cocaine collection and handling procedures include samples collected in commercially available, evacuated blood-collection tubes containing NaF [0.06 mol/L (0.25%) NaF] and storage at 0 °C (or lower) as soon as possible. These conditions are adequate for cocaine if analysis is performed within 24 h. However, for longer-term storage it is necessary to increase NaF concentrations to 0.24 mol/L (1%), decrease the pH to 5–6, and store samples at -20 °C. Unlike cocaine, NaF concentrations found in commercially available evacuated blood-collection tubes that contain NaF adequately maintain MEG stability during storage and sample processing.
This study suggests that MEG concentrations are stable if specimens are refrigerated after collection for short-term storage. Because previous studies determining MEG in blood and plasma followed standard cocaine handling and storage procedures, it is unlikely that the absence of MEG in blood samples reported in these studies is attributable to the in vitro disappearance of MEG. This leaves open the possibility that MEG has a short plasma half-life, is rapidly cleared from the central compartment, distributes to other compartments in vivo, and thus can be detected in matrices other than blood or plasma. This study demonstrated that EC concentrations measured in specimens collected with commercially available, evacuated blood-collection tubes containing NaF and stored at -80 °C for less than 1 month accurately reflect in vivo metabolism that occurred before collection and storage. Thus, if EC does form by a mechanism similar to BE from cocaine, EC might provide a stable and more persistent marker for MEG and, thus, crack cocaine smoking.
Paul et al. (8) examined MEG incubation in human liver homogenates and whether NaF had any effect on EC formation. These authors demonstrated that NaF at a concentration of 0.04 mol/L inhibited formation of EC from MEG in liver homogenates (2000 µg/L) when incubated for 3 h at 37 °C. Despite species and matrix differences, our sheep plasma data under similar temperature and NaF conditions at 3 h are strikingly similar. Initial MEG plasma concentrations of 2000 µg/L when stored at 37 °C for 3 h without NaF decreased to 1791.55 µg/L; the corresponding EC concentration was 138.60 µg/L. The MEG and EC concentrations were 1853.54 µg/L and undetectable, respectively, when the sample was adjusted to 0.06 mol/L (0.25%) NaF. These data, like those for MEG in liver homogenates, suggest that metabolic hydrolysis of MEG to EC occurs.
Reducing the plasma pH to 6.0 maintains MEG concentrations, even at 37 °C, without any added NaF. Acidic pH previously was shown to be better than NaF for maintaining cocaine stability, probably by inactivating enzymatic hydrolysis (13). The enhancement of MEG stability by reduced plasma pH does not in itself differentiate whether in vitro MEG conversion to EC occurs via chemical or enzymatic hydrolysis. Reduced pH might impair the enzymatic activity responsible for MEG degradation as well as provide suboptimal conditions for chemical hydrolysis. However, our data demonstrating that MEG incubated in PBS (pH 7.4 at 37 °C) disappears at a slower rate than plasma lacking NaF (incubated at 37 °C) suggest that MEG conversion to EC in sheep plasma does not occur predominantly via chemical hydrolysis. Because fluoride is a general enzyme inhibitor and because similar results were obtained with PBS and NaF-containing plasma at 37 °C, conversion of MEG to EC appears to occur enzymatically. A third possibility is that MEG undergoes both enzymatic and chemical hydrolysis to EC.
Studies of MEG and EC concentrations after incubation with isolated esterases could help to clarify the mechanism of MEG conversion to EC. Reports of cocaine hydrolysis to BE are conflicting, with hydrolysis proceeding chemically or enzymatically. Stewart and co-workers (14)(15) reported that the conversion of cocaine to BE occurred via chemical hydrolysis because they observed similar formation of BE in plasma and buffer. More recently, a carboxylesterase has been isolated and purified from human liver with specificity for transforming cocaine to BE; this likely accounts for in vivo BE formation from cocaine (16)(17). A recent review of reports describing cocaine conversion to BE concludes that BE is formed predominantly via enzymatic processing in vivo and predominantly via chemical hydrolysis in vitro (17). Our findings that in vitro conversion of MEG to EC occurs predominantly via enzymatic hydrolysis contrasts with the finding that in vitro conversion of cocaine to BE proceeds via chemical hydrolysis. It is important to note that species variation in regard to sheep and human metabolism of cocaine exist, with sheep clearing cocaine more rapidly than humans (18)(19). Therefore, in vitro conversion of MEG to EC in human plasma might occur at a different rate than conversion in sheep plasma.
In conclusion, based on the current study, collection and handling of blood/plasma samples should be performed according to cocaine procedures to assure accurate measurements of both MEG and EC. This study demonstrates that NaF, reduced temperature, and reduced pH prevented MEG conversion to EC in sheep plasma. The lack of MEG degradation in buffer suggests that MEG conversion to EC results predominantly from enzymatic processing. EC could provide a useful marker of crack smoking.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  K. B. Scheidweiler, M. A. Plessinger, J. Shojaie, R. W. Wood, and T. C. Kwong Pharmacokinetics and Pharmacodynamics of Methylecgonidine, a Crack Cocaine Pyrolyzate J. Pharmacol. Exp. Ther., December 1, 2003; 307(3): 1179 - 1187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. T. Shimomura, G. D. Hodge, and B. D. Paul Examination of Postmortem Fluids and Tissues for the Presence of Methylecgonidine, Ecgonidine, Cocaine, and Benzoylecgonine Using Solid-Phase Extraction and Gas Chromatography-Mass Spectrometry Clin. Chem., June 1, 2001; 47(6): 1040 - 1047. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, sample adjusted
to 0.06 mol/L (0.25%) NaF; 
, sample adjusted to 0.24 mol/L (1%)
NaF. EC concentrations: 
, non-NaF-treated sample; 
, sample
adjusted to 0.06 mol/L (0.25%) NaF; 
, sample adjusted to 0.24 mol/L
(1%) NaF.