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Published:
Journal of Analytical Toxicology,
ISSN 0146-4760,
Volume 24,
Number 6, September 2000, pp. 453-455
Here is where the title stuff goes
Letter to the Editor—A Gas Chromatographic–Positive Ion Chemical Ionization-Mass
Spectrometric Method for Determination of Cocaine, Benzoylecgonine, Ecgonine
Methyl Ester, and Norcocaine in Plasma: Detection of Norcocaine in Plasma After
Oral Administration of Cocaine
Alan C. Spanbauer[1], David E. Moody[1], Rodger L. Foltz[1], and Sharon
L. Walsh[2]
[1]University of Utah Center for Human Toxicology, 20 S 2030 E, Room 490, Salt
Lake City, Utah 84112-9457 and
[2]Behavioral Pharmacology Research Unit, Department of Psychiatry and Behavioral
Sciences, Johns Hopkins University School of Medicine, 5510 Nathan Shock Drive,
Baltimore, Maryland 21224
To the Editor:
In a previous publication (1), we presented a gas chromatographic–positive
ion chemical ionization-mass spectrometric (GC–PICI-MS) method that employed
solid-phase extraction (SPE) and derivatization with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide
that allowed detection and accurate and precise quantitation of cocaine, benzoylecgonine
(BE), and ecgonine methyl ester (EME) in human plasma. This method allows detection
of the parent compound and the primary metabolites of two hydrolytic pathways.
While the underivatized primary metabolite of oxidative metabolism, norcocaine,
was detectable, the chromatography was not sufficient for accurate and precise
quantitation. A pharmacologically active metabolite of cocaine (2), norcocaine
is readily identifiable in urine and tissue samples (3,4), but its detection
in plasma has been limited until recently to postmortem cases where overdose
and postmortem redistribution are possibilities (5,6). Jufer and colleagues
(7) recently reported detection of norcocaine in human plasma following repeated
administration of oral doses of cocaine, demonstrating that norcocaine is a
plasma metabolite of cocaine worth monitoring.
To include norcocaine in our method, we hypothesized that the use of hexafluoroisopropanol
(HFIP) in combination with pentafluoropropionic anhydride (PFPA) as the derivatizing
agent would permit derivatization of norcocaine at the amine, as well as derivatization
of BE and EME. To test this hypothesis, we performed validation experiments
in human plasma fortified with cocaine, BE, EME, and norcocaine and analyzed
plasma samples from another human oral dosing study to confirm the ability of
the method to detect norcocaine. As a change in derivatizing reagent is a major
change to the method, it was necessary that we show accurate and precise quantitation
of not only norcocaine, but also cocaine, BE, and EME.
The reagents unique to this modification were norcocaine (Radian Corp., Austin,
TX), norcocaine-d5 (Research Triangle Institute, Research Triangle Park, NC),
HFIP (Aldrich Chemical Co., Milwaukee, WI), PFPA (Regis Technology, Morton Grove,
IL), glacial acetic acid (Sigma Chemical Co., St. Louis, MO), and sodium hydroxide
(Malinckrodt Inc., St Louis, MO). The GC column was a DB-5 (15 ¥ 0.32 mm,
1-µm film thickness) fused-silica capillary column, obtained from J&W
Scientific (Folsom, CA). Other reference standards and materials, including
the SPE columns, were obtained as previously described (1).
Cocaine-d3, BE-d3, EME-d3, and norcocaine-d5 were added as the internal standards
(100 ng/mL) to 1-mL aliquots of calibrators in blank plasma (1.0–1000 ng/mL),
quality-control samples, and study specimens. Samples were allowed to equilibrate
1 h and the plasma was made acidic by the addition of 0.1M acetate buffer (pH
4.0). The tubes were vortex mixed briefly and centrifuged for approximately
10 min at 2000 rpm. The supernatant from the samples was extracted by SPE and
dried as previously described (1) with the following modifications: the final
conditioning step used 2 mL of 0.1M acetate buffer (pH 4.0) rather than phosphate
buffer and water was omitted from the wash. The dried residues were reconstituted
with 100 µL HFIP and 100 µL PFPA, and placed in a heating block
at 80°C for 45 min. After derivatization, the tubes were removed from the
heating block, allowed to cool to room temperature, and evaporated to dryness
in a 40°C water bath under a stream of air. The residues were reconstituted
with 50 µL ethyl acetate and transferred to autosampler vials for analysis.
The GC–MS conditions were essentially as previously described (1) with
the following modifications: a DB5 capillary column was used; the temperature
program was 100°C for 1 min, then increased to 310°C at a rate of 20°C/min
and held at 310°C for
1 min. The transfer line was maintained at 265°C. The ions monitored for
the analytes and respective internal standards were as follows: cocaine (d0/d3),
304/307; BE (d0/d3), 440/443; EME (d0/d3), 346/349; and norcocaine (d0/d5),
453/458.
Analysis of extracted cocaine, BE, EME, and norcocaine by GC–PICI-MS produced
major ions at m/z 304, 440, 346, and 453, respectively. These ions correspond
to the protonated molecules of underivatized cocaine, the hexafluoroisopropyl
ester of BE, the pentafluoropropionate ester of EME, and the ammonium adduct
of the pentafluoropropionate amide of norcocaine. Selected ion monitoring of
cocaine and the derivatized metabolites under the established chromatographic
conditions resulted in good separation of the compounds, the signal-to-noise
ratios suggested that the compounds could be quantitated to a lower limit of
quantitation (LLOQ) of 1 ng/mL.
Validation experiments were carried out at the specified concentrations using
previously described experiments (8), and the results are presented in Table
I. The method had sufficient recovery, specificity (lack of interference in
blank plasma), and linearity to accurately and precisely quantitate all analytes
from a LLOQ of 1 ng/mL to an upper limit of 1000 ng/mL. All four analytes had
acceptable stability in quality-control samples subjected to storage at room
temperature for 24 h and 3 freeze-thaw cycles.
The ability of this assay to measure cocaine, BE, EME, and norcocaine in human
plasma was assessed in samples collected from human volunteers receiving oral
doses of cocaine. Plasma samples were prepared from blood collected from five
male participants of a larger study that will be described in detail elsewhere
(9). On the day the samples were collected, each subject received oral doses
of 100 mg cocaine at 0800, 1200, 1600, and 2000 h, and three successive intravenous
doses of 0, 25, and 50 mg cocaine at 1300, 1400, and 1500 h. Samples were collected
5 min before and 55 and 475 min after the last dose of the day. In agreement
with the results of Jufer et al. (7), BE concentrations in the plasma samples
were highest among the analytes measured and EME concentrations were slightly
higher than cocaine. Norcocaine did not exceed a mean of 6 ng/mL, but was detectable
at all three time points before and after the last dose of the day (Figure 1).
The concentrations of norcocaine in plasma reported here are lower than those
observed in postmortem blood of 10–20 ng/mL in one study where norcoaine
was detected in 2 of 9 cases (5) and 23–264 ng/mL in another study where
norcocaine was detected in 3 of 13 cases (6).
This study demonstrates that the GC–PICI-MS method described can simultaneously
quantitate cocaine, BE, EME, and norcocaine in human plasma at concentrations
ranging from 1 to 1000 ng/mL. The current method offers some advantages over
previously described GC–PICI-MS methods developed in our laboratory. SPE
provides for a more time-efficient extraction than multiple liquid–liquid
steps (5), and using HFIP and PFPA has added the ability to detect norcocaine
to our previously described SPE method (1). In comparison to electron-impact
methods (3,4), the lower LLOQ achievable with this method should allow detection
of norcocaine for longer periods after ingestion of cocaine. Our data establish
that the assay is accurate and precise. This study also confirms detection of
norcocaine in plasma following oral administration of cocaine to humans.
References
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Moody, and A.A. Chasin. Analysis of cocaine and its metabolites from biological
specimens using solid-phase extraction and positive ion chemical ionization
mass spectrometry. J. Anal. Toxicol. 19: 352–358 (1995).
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6. A.J. Jenkins and B.A. Goldberger. Identification of unique cocaine
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7. R.A. Jufer, S.L. Walsh, and E.J. Cone. Cocaine and metabolite concentrations
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8. D.E. Moody, J.D. Laycock, A.C. Spanbauer, D.J. Crouch, R.L. Foltz,
J.L. Josephs, L. Amass, and W.K. Bickel. Determination of buprenorphine in human
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9. S.L. Walsh, K.A. Haberny, and G.E. Bigelow. Modulation of the effects
of intravenous cocaine following chronic oral cocaine in humans. Psychopharmacology,
in press. Reproduction
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