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Maha Alghamdi
Chem 624-Chemical Separation
Journal Article

Analysis of Cocaine and its Major Metabolites in Human Urine by
Ion Trap Tandem Mass Spectrometry



Keywords: Cocaine; GCMS/MS; Saliva; Ion-trap; Dissociation pathways

Introduction

Cocaine or benzoylmethylecgonine is an alkaloid that is found either naturally in the
leaves of the coca bush (Erythroxylum coca) or can be synthesized from ecgonine. In the early
1900s, purified cocaine was used medicinally to treat a variety of maladies. However, due to the
addictive nature of the drug, coca leaf and cocaine was classified as Schedule I of the Single
Convention on Narcotics Drugs in 1961(United Nation Office on Drugs and Crimes, 2012).
Cocaine is a powerful nervous system stimulant, providing the user with increased energy
and feelings of euphoria. The initial side effects of cocaine include paranoia, agitation,
hallucinations, anxiety, nausea, seizures and strokes. However, continued and prolonged use of
cocaine could lead to more detrimental side effects such as cocaine psychosis such as paranoia,
aggressive behavior, hallucinations, gastrointestinal problems, respiratory problems, kidney
failure, sexual inadequacy, and damage to the nasal cavity or septum depending on nasal
administration (Goldstein et al., 2009). Cocaine is an extremely addictive drug and continued use
creates a powerful dependence on the drug as the body quickly builds up a tolerance and
subsequently greater doses are needed to achieve the high. Inhalation of cocaine as a fine
powder through the nasal passageways, otherwise known as snorting, is the most common
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route of administration. There are also several other routes of administration that include
through the eyes, ears and throat and smoke inhalation when using crack cocaine.
Cocaine is quickly metabolized to benzoylecgonine (BZE) and ecgonine methylester
(EME) via chemical hydrolysis (Jatlow, 1987). Esterase hydrolysis forms primarily ecgonine
and minute amounts of the active norcocaine. Crack cocaine is formed via a reaction with
ammonia or bicarbonate and when smoked, a pyrolysis product, anhydroecgonine methylester
(AEME), is formed (Cognard et al., 2005). Frequently, users of cocaine consume alcohol and
cocaine together, creating an active homologue cocaethylene which forms through
transesterification following consumption of the COC and ethanol (Cognard et al., 2006). The
following figure depicts the chemical structures of cocaine (COC), and its metabolites
cocaethylene (COET), ecgonine methylester (EME), and anhydroecgonine methylester (AEME).

Figure 1: Chemical structures of cocaine (COC), cocaethylene (COET), ecgonine methylester (EME)
and anhydroecgonine methylester (AEME) (Cognard et al., 2006).
Since cocaine has been internationally claimed as a Schedule 1 drug, it is of prime
importance to have a sensitive and easy method of detection for the presence of cocaine and its
metabolites in suspected users. The detection of psychotropic drug substances has traditionally
been in either blood or urine matrices. However, in the past three decades, testing for
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psychotropic drugs via saliva has been introduced as a new matrix. The use of saliva instead of
urine and blood provide an advantage because it is non-invasive, simple sample collection, and
the chances of adulterating the sample are minimized (Campiglia et al., 1998).
This study is the first of its kind to selectively test for cocaine, cocaethylene, ecgonine
methylester , and anhydroecgonine methylester in the saliva of cocaine drug addicts via gas
chromatography/ mass spectrometry (GC/MS). More importantly, the results of this research are
significant in that the structure, identity, quantitation, and detection of cocaine and its
metabolites are simplified by analyzing the mass spectra data for COC, COET, EME, and
AEME.
Experimental
Chemicals
Acetonitrile solutions of COC, COET, EME, and AEME (1000g/ml) and acetonitrile
solutions of deuterated cocaine (COC-d3) and deuterated ecgonine methylester (EME-d3),
100g/ml were bought from Cambridge Isotope Laboratories Inc. Solutions of methanol,
toluene, acetic acid (100%), hydrochloric acid (37%), ammonium hydroxide (25%), sodium
hydroxide, potassium hydroxide, sodium hydrogenophosphate, and potassium
dihydrogenophosphate were purchased through Merck. Methylene chloride and isopropyl
alcohol were purchased from Fluka.
Instrumentation
Gas chromatographic analysis was done via a Varian 3400 CX gas chromatograph which
included a Varian Saturn 2000 ion-trap detector. The carrier gas used was high purity helium and
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at a pressure of 0.069 MPa (10 PSI). A DB5-MS J&W Scientifics fused silica capillary column
with a composition of 5% phenyl-95% methyl-polysiloxane stationary phase was utilized with a
capillary column of dimensions 15m0.25mm (0.25m film thickness). The column
temperature was initially at 75 C for a duration of 1 min, increased to 170 C at a rate of 15
C/min, then increased to 210 C at a rate of 5 C/min, and then finally to 310 C at a rate of 30
C/min. The ion-trap was functioning in positive chemical ionization (CI) with isobutane as the
gas. Tandem mass spectrometry was operated in the non-resonant mode; and the collision
induced dissociation (CID) values are displayed in Table 1. The ions that were left to quantify
were COC m/z 182, AEME m/z 122, EME m/z 182, COET m/z 196, COC-d3 m/z 185 and EME-
d3 m/z 185 (Cognard, 2006).
Table 1: CID parameters and primary product ions for each compound (Cognard, 2006).


Sample Preparation
Saliva samples were simply collected with a cotton swab. Blank saliva was collected
from non-cocaine users and no traces of COC and its metabolites were detected. Saliva samples
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were homogenized and 500 l were obtained for studies. The 500 l was diluted with 1ml of
deionized water and buffered with 1ml of phosphate buffer pH 7. A concentration of 1.6 g/ml
of COC-d3 and EME-d3 was added. Samples were mixed well and an ASPEC system was
operated to extract samples. Calibration samples were prepared by spiking blank saliva with
specific volumes of standard COC, AEME, EME and COET solutions to obtain concentrations
of 2, 5, 10, 50, 200 and 500 ng/ml for each analyte. Quality control (QC) samples were similarly
done by spiking blank saliva with COC, AEME, EME and COET solutions to obtain 2, 5, 10,
100 and 800 ng/ml.
Results
The linearity of any analytical experiment is when results are directly proportional to the
concentration of the analytes in the sample (Chapuzet, 1997). The linearity in this study was
calculated by fitting the back-calculated concentrations of the QC versus theoretical
concentrations by utilizing the linear regression based on the least square method (Chiap, 2001).
Good linearity (slopes close to 1.000.05) and good closeness R
2
above 0.999 for all analytes
were observed.
Since tandem mass spectrometry analysis is very selective, a small portion of the
precursor ion has been kept in CID spectra so that the precursor ion intensity accounts for
approximately 10% of the base peak. Since this is the first study in which the mass spectra of
cocaine and its metabolites have been reported, it was found that it was impossible to fragment
all the precursor ions so that their intensity related to about 10% of the base peak. The most
obvious example is the protonated COET (Fig. 2C), in which the corresponding abundance of the
precursor ion remained above 50% whatever the excitation voltage between 46V and 100V.
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Similarly, the precursor ion peak of protonated COC (Fig. 2B) stayed above 25%. The
explanation for this can understood in terms of precursor ion stability. Figure 2 depicts the first
mass spectra of cocaine and its metabolites.

Figure 2: CID spectra of (A) anhydroecgonine methylester, (B) cocaine, (C) cocaethylene, and (D)
ecgonine methylester obtained in the positive CI mode with an ion trap mass spectrometer (Cognard,
2006).

The major product ions of the CID spectra result from dissociation mechanisms that
suggest protonation on an ester or hydroxy group. Apparently, the protonation on the tertiary
amino function is thermodynamically favored. The presence of product ions from molecules
protonated on the amino group suggests the breaking of at least two bonds since the nitrogen
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atom is involved in two rings. This probably explains why the fragmentation yields of COET and
COC are low, despite having high non-resonant CID voltages.
The applicability of the experimental procedure was applied by testing several real
human saliva samples for the presence of cocaine and its metabolites. The saliva samples were
processed by solid phase extraction and studied via GC-MS/MS as explained above in the
experimental section. Figure 3 presents the chromatogram for a cocaine user while Figure 4
presents a chromatogram that allows for the comparison between a cocaine user, a quality
control at 100 ng/ml and a blank saliva sample. These results indicate the effectiveness of the
experimental method in the detection of COC, EME, AEME, and COET.

Figure 3: Chromatograms of a saliva sample from a cocaine abuser conveying peaks of anhydroecgonine
methylester (~4 ng/ml), ecgonine methylester (140 ng/ml) and
cocaine (302 ng/ml) (Cognard, 2006).
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Figure 4: Chromatographs of a cocaine abuser, QC at 100 ng/ml, and a blank saliva sample (Cognard,
2006).

Table 2 shows the ranges determined for concentration: 5-500 ng/ml for EME, 2-500
ng/ml for COC and COET, and AEME could only be determined approximately between 2 and
200 ng/ml based on selected acceptance limits. The selected acceptance limits used in this study
are as follows: trueness 10020%, repeatability 15% and intermediate precision20%.
Table 2: Concentrations, Trueness, Repeatability, and Precision for QC samples (Cognard, 2006).

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It is important to elucidate the dissociation pathways in order to understand the CID
spectra of the compounds that are analyzed. Figure 5 shows the dissociation of the protonated
cocaine goes through a loss of benzoic acid followed by a loss of methanol. The transitions m/z
307m/z 185 and m/z 307m/z153 seen from COC-d3 agree with the dissociation mechanism
just proposed. The dissociation pathway of COET is similar to COC, with the exception that the
second step involves the loss of ethanol rather than methanol.
Figure 6 shows the dissociation of the ecgonine methyl ester initiates by depletion of a
water molecule to create the m/z 182 ion. Water elimination results either from protonation on
the hydroxyl group, as shown in Fig. 6; or it can happened from protonation of the ester group
followed by transferring H
+
to the hydroxy group. The subsequent loss of methanol from m/z
182 produces m/z 150, which goes through an intermediate structure to finally yield the m/z 82.
The dissociation pathway for EME is in agreement with the CID spectrum of EME-d3 which
displays m/z 203,m/z 185,m/z 153 and m/z 85 ions.
Figure 7 shows the dissociation of protonated anhydroecgonine methyl ester initiates by a
loss of methanol after protonation on the carbonyl group. The ion that forms is the m/z 150 ion
and that dissociates by two pathways. The first pathway creates the m/z 122 by CO elimination,
the second pathway produces the m/z 118 ion through elimination of a methanol. The
dissociation mechanism suggested in Fig. 7 to elucidate the production of the m/z 118 ion may
appear complicated but a much simpler one has not yet been found since this is the first study in
which the mass spectra of these compounds have been established.

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Figure 5: Predicted dissociation pathways of protonated cocaine under collisional activation (Cognard,
2006).







Figure 6: Predicted dissociation pathways of protonated ecgonine methylester under collisional activation
(Cognard, 2006).
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Figure 7: Predicted dissociation pathways of protonated anhydroecgonine methylester under collisional
activation (Cognard, 2006).

Conclusion
This study is significant in that it has accomplished reporting for the first time the mass
spectra of cocaine and its major metabolites via GC-MS/MS. This novel method allows a semi-
automated and very sensitive detection for the simultaneous determination of cocaine and its
metabolites in saliva media. The effectiveness of this method has been proven in terms of
linearity, trueness, precision and limits of quantification for COC, EME and COET. Despite
being unable to precisely quantify the metabolite AEME due to our specific criteria for
repeatability and intermediate precision, the trueness was fully established. Therefore, this
method only allows a close approximation to AEME concentrations in saliva. The identification
and detection of metabolites is enough to confirm the usage of cocaine, but more importantly, the
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primary concern of forensic toxicology is detection and quantitation of the main drug substance
cocaine. The mass spectra reported allows for detection, quantitation, and validation for routine
analysis of cocaine and its metabolites in forensic toxicology.














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