Russell J. Lewis

Robert D. Johnson

Michael K. Angier

Roxane M. Ritter

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

Holly S. Drilling

Shyla D. Williams

University of Central Oklahoma

Edmond, OK 73034

December 2003

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, VA 22161.

Office of Aerospace Medicine

Washington, DC 20591

DOT/FAA/AM-03/23 Analysis of Cocaine, Its Metabolites, 

Pyrolysis Products, and Ethanol 

Adducts in Postmortem Fluids and 

Tissues Using Zymark Automated 

Solid-Phase Extraction and Gas 

Chromatography-Mass Spectrometry
NOTICE

This document is disseminated under the sponsorship of

the U.S. Department of Transportation in the interest of

information exchange. The United States Government

assumes no liability for the contents thereof.
i

������������������������������������

�������������� ���������������������������� ��������������������������� � � �

����������������� � � �

��������������������� �������������� � � �

���������������������������������������������������������������������� ��������������

�����������������������������������������������������������������

����������������������������������������������������������������

�������������������������������� � � �

������������� �������������������������������������� � � �

�������������������������������������������������������������

������������

� � �

�������������������������������������������� �������������������������� � � �

� � � �������������������������� � � �

�

��������������������������������������

����������������

������������������������

�

����������������������

��������

����������������� � � � ��������������������������������������� �������������������������������������� � � �

����������������������������� � � �

�������������������������������� � � �

���������������������������� � � �

��������������������� ��������������������������� � � � ����������������������� � � �

�������������������������������������������������������������������� � � � ������������� � � �

����������������������������������������������������������������������������������������������������������

������������������������������������������� �������������������� ���������������������������������������

���������������������������������������� ���� ������������������������������������������� �������������������

����� ��������� �������� ���� ������������ ������ �������� ��� �� ������������� ���������� ����� ��� ���� �����

������������������ ���� �������� ��������������� ����� ���������� ������������� ����������������� �� �������������

������� ��������� ����� ��� ������� ��� ������������ ��� ���� ���� ��������� �� ������� �������� ���� ������ ��������

���������������������������������������������������������������������������������������������������

���������������������������������������������������������������������������������������������������������

��� ����� ���������� �� ������� ������� ���� ���� ������������� �������������� ��� ���� ���� ���� ����

����������������������������������������������������������������������������������������������������������

�����������������������������������������������������������������������������������������������������

��� ���� ����������� ���� ��� ���� ���� ��������� ���� ������ �������� ������������� �����������������

����������������������������������������������������������������������������������������������������

������������ ����������� �������� ���� �������������������� �������������� ���� ��������� ������������� ����

��������������������������������������������������������������������������������������������������������

���� ������������ ������� ���� ����������� ������������� ����� ����� ��� �� ����� ����� ���� ���������� ���

�������������������������������������������������������������������������������������������������������

��������������������������������������������������������������������������������������������������������

�����������������������������������������������������

� � �

�������������� ��������������������������� � � �

� � �

� � �

���������������������������������������������������

���������������������������������������������������

��������������

��������������������������������������������

�������������������������������������������

���������������������� � � � ��������������������������������������� ������������������������������������� ����������������� ���������� �

������������� ������������� ���

����������������� ������� ������������������������������������������

1

ANALYSIS OF COCAINE, ITS METABOLITES, PYROLYSIS PRODUCTS, AND ETHANOL

ADDUCTS IN POSTMORTEM FLUIDS AND TISSUES USING ZYMARK AUTOMATED

SOLID-PHASE EXTRACTION AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY

INTRODUCTION

The Federal Aviation Administration’s (FAA’s) Civil 

Aerospace Medical Institute (CAMI) is responsible, under Department of Transportation Orders 8020.11A and 

1100.2C, to “conduct toxicologic analysis on specimens 

from ... aircraft accident fatalities” and “investigate ... 

general aviation and air carrier accidents and search for 

biomedical and clinical causes of the accidents, including 

evidence of ... chemical (use).” Therefore, following an 

aviation accident, samples are collected at autopsy and sent 

to CAMI’s Forensic Toxicology Research Laboratory where 

toxicological analysis is conducted on various postmortem 

fluids and tissues. Occasionally during a toxicological 

evaluation, cocaine is detected in postmortem specimens 

from civil aviation accident victims. 

Cocaine (COC) is one of the most widely abused illicit drugs in America. COC abuse transcends all social, 

racial, and economic boundaries. It is available in two 

primary forms: COC hydrochloride, a white crystalline 

powder that can be snorted, swallowed, or injected, and 

“crack,” COC hydrochloride that has been processed into 

its freebase form through a reaction with either ammonia 

or bicarbonate (1). The intensity of the euphoric high 

derived from COC use is strongly dependent upon the 

route of administration. COC administered intranasally 

results in euphoria more slowly than when injecting COC 

or smoking crack (2). Crack use produces a “high” much 

more rapidly than other methods of COC administration 

(2, 3). The rapid rate by which crack affects the user has 

resulted in a dramatic increase in its abuse (2). Despite 

its popularity, smoking crack is an extremely dangerous 

method for the introduction of COC into the body. 

Reports of sudden death from smoking crack are not 

uncommon (2). This is primarily due to the unpredictable, sometimes fatal, quantities of COC delivered to the 

bloodstream via inhalation of COC-saturated smoke. 

The presence of COC, its metabolites, pyrolysis products and/or ethanol adducts in biological fluids and tissues 

provide markers of COC use and its possible route of administration (4-6). Numerous COC-related compounds 

and their unique transformation pathways are shown in 

Figure 1. The catabolism of COC results primarily in the 

ester hydrolysis products benzoylecgonine (BE) and, to a 

lesser extent, ecgonine methyl ester (EME) (7). In vivo, 

BE is predominantly produced from chemical hydrolysis 

of COC while EME results from enzymatic hydrolysis 

(8, 9). Other COC metabolites, which appear at lower 

levels than BE or EME, include norcocaine (NCOC), 

norbenzoylecgonine (NBE), m-hydroxybenzoylecgonine 

(HBE), and ecgonine (E). When COC is smoked, a unique 

pyrolysis product, anhydroecgonine methyl ester (AEME), 

is formed. AEME and its metabolite, anhydroecgonine 

(AE), have been used as indicators of crack use (4, 5, 10-

13). The concurrent use of COC and ethanol results in a 

biologically active molecule, cocaethylene (CE), and two 

non-active metabolites of CE, norcocaethylene (NCE), 

and ecgonine ethyl ester (EEE). COC and ethanol are 

frequently taken together, due to the effects of CE, which 

is nearly as psychoactive as COC but produces a much 

longer lasting high (14-16). CE is even more toxic than 

COC, and its potency results in an increased risk of death 

due to overdose (17). 

Identification and quantitation of COC, CE, AEME, 

and their related metabolites in postmortem fluids and 

tissues are important aspects of forensic toxicology and 

may provide crucial information in determining the 

cause of impairment and/or death. Described herein is 

a rapid, automated procedure for the single-step extraction and simultaneous determination of COC and its 

metabolites BE, NCOC, NBE, HBE, EME, and E, as 

well as the pyrolysis products AEME and AE and the 

ethanol adducts CE, NCE, and EEE in postmortem fluids 

and tissues using a Zymark RapidTraceTM solid-phase 

extraction (SPE) system and gas chromatography with 

mass spectrometry (GC/MS). While many methods exist for the identification and quantitation of COC and 

COC-related compounds (18-29), none offer the sensitivity of this method combined with the simultaneous 

extraction and analysis of all 12 related COC compounds 

discussed above.

MATERIALS AND METHODS

Chemicals and Reagents

All aqueous solutions were prepared using double 

deionized water (DDW), which was obtained from a 

Milli-QTplus Ultra-Pure Reagent Water System (Millipore®, Continental Water Systems, El Paso, TX). All 

chemicals were purchased in the highest possible purity 
2 

and used without any further purification. COC, BE, 

NBE, HBE, EME, E, NCOC, CE, NCE, EEE, AEME, 

and AE were purchased from Cerilliant (Cerilliant Corp., 

Round Rock, TX) as 1.00 mg/mL sealed glass ampules. 

COC-d3, BE-d3, EME-d3, and CE-d3 were purchased 

from Cerilliant as 0.100 mg/mL sealed glass ampules. 

The derivatization reagents, pentafluoropropionic anhydride (PFPA), 2,2,3,3,3-pentafluoro-1-propanol (PFP), 

and BSTFA with 1% TMCS (TMS) were obtained from 

Pierce (Pierce Inc., Rockford, IL). Sodium acetate was 

purchased from Sigma (Sigma Chemical Co., St. Louis, 

MO). Methanol, acetonitrile, ammonium hydroxide, 

hydrochloric acid, methylene chloride, and isopropanol 

were purchased from Fisher (Fisher Scientific, Pittsburgh, 

PA). Ethyl acetate was purchased from Varian (Varian 

Inc., Palo Alto, CA).

Gas Chromatographic/Mass Spectrometric 

Conditions

All analyses were performed using a benchtop gas 

chromatograph/mass spectrometer (GC/MS), which 

consisted of a Hewlett Packard (HP) 6890 series GC, 

interfaced with a HP 5973 quadrupole MS (Agilent, 

Palo Alto, CA). The GC/MS was operated with a transfer 

line temperature of 280°C and a source temperature of 

250°C. The MS was autotuned on a daily basis using 

perfluorotributylamine. The electron multiplier voltage 

was set at 106 eV above the autotune voltage. Chromatographic separation was achieved using a HP-ULTRA-1 

crosslinked 100% methyl siloxane capillary column (12 

m x 0.2 mm i.d., 0.33 μm film thickness). Helium was 

employed as the carrier gas and used at a flow rate of 1.0 

mL/min. A HP 6890 autosampler was used to inject 1 

�

���

� �

�

���

�������

�

���

� �

�

��

�������

�

���

� �

�

��

���������

�

�

� �

�

��

�������

�

�

� �

�

���

�������

�

���

� �

�

���

��

�

���

� ��

�

��

�

���

� �

�

���

�������

���

�

�

� �

�

���

�������

���

�

���

� �

�

���

��

���

�

���

� �

�

���

�

���

� �

�

��

��

���

��� ����

�

���� ���

��

���

���

��

���

��������������������������������������������������������������������
3

μL of extract into the GC/MS. The GC was equipped 

with a split/splitless injection port operated at 250°C 

in the splitless mode with a purge time of 0.5 min. The 

oven temperature profile was established as follows: 70 

- 130°C at 30°C/min, 130 - 140°C at 5°C/min, 140 - 

210°C at 35°C/min, 210 - 222°C at 4°C/min, and 222 

- 290°C at 45°C/min, with a final hold time of 0.49 

min resulting in a total run time of 11 min. Initially, 

neat standards of each compound (1 μL of a 100 ng/μL 

solution) were injected individually and analyzed using 

the full scan mode of the GC/MS, which scanned from 

50 to 600 AMU. Quantitation and qualifier ions used 

for each analyte were selected based on their abundance 

and mass-to-charge ratio (m/z). Because of their reproducibility and lack of interference, high mass ions were 

selected when possible. The ions chosen for each respective 

analyte can be seen in Table 1. Upon selection of unique 

ions, the MS was run in selected ion monitoring (SIM) 

mode with a dwell time of 20 msec.

Analyte concentrations were determined using an internal standard calibration procedure. Response factors 

were determined for each analyte present. The response 

factor was calculated by dividing the area of the analyte 

peak by the area of the internal standard peak. Calibration 

curves were then prepared by plotting a linear regression 

of the analyte/internal standard response factor versus the 

analyte concentration for all calibrators analyzed. These 

calibration curves were then employed to determine 

concentrations of the various analytes present in both 

controls and specimens. 

Sample Selection and Storage

A search of our toxicology laboratory’s database identified 5 fatalities from separate aviation accidents from the 

previous 3 years that were reported positive for COC or 

BE and also had a majority of the desired biological tissues 

and fluids (blood, urine, liver, kidney, and muscle) available for analysis. In all cases, blood was stored at -20°C in 

tubes containing 1.0% (w/v) sodium fluoride/potassium 

oxalate until analysis. All other specimens were stored 

without preservation at -20°C until analysis. Blood COC 

or COC-related metabolite values determined in this study 

were compared with those previously determined. The 

values found in this study agreed with those previously 

reported, verifying no deterioration had occurred.

�������������������������������������������������������������������

��������� ������������������ ������������ ���������������

��� ���� �������������� ����

����� ����� �������������� ����

�� ��������� �������������� ����

������� ����� �������������� ����

���� ����� �������������� ����

���� ����� �������������� ����

������ ����� �������������� ����

��� ���� �������������� ����

���� ��������� �������������� ����

���� ��������� �������������� ����

������� ����� �������������� ����

���� ����� �������������� ����

������ ����� �������������� ����

��� ����� �������������� ����

����� ����� �������������� ����

���� ����� �������������� ����

��������������������������������������
4 

Preparation of Standards

For each analysis, controls, and calibrators were prepared from the purchased 1.00 mg/mL drug standards 

using certified-negative whole blood as the diluent. 

Calibration curves were prepared by serial dilution at 

concentrations ranging from 0.78–3200 ng/mL for each 

analyte. Controls used for the determination of accuracy, 

precision, and analyte stability were prepared at 45 and 

450 ng/mL. Controls and calibrators were prepared 

from separate methanolic drug standards to ensure the 

accuracy of the prepared calibration curves. Controls 

separately containing COC or BE were prepared. These 

COC and BE controls were prepared at concentrations 

of both 250 ng/mL and 1000 ng/mL. All controls were 

prepared in pools large enough to provide replicates for 

the entire study. The internal standard solution containing 

d3-COC, d3-BE, d3-CE and d3-EME was prepared at a 

concentration of 400 ng/mL in DDW.

Sample Preparation and Extraction Procedure

Postmortem fluid and tissue specimens, calibrators, 

and controls were prepared and extracted in the following manner. Tissue specimens were homogenized using 

a PRO250 post-mounted homogenizer (Pro Scientific, 

Oxford, CT). The generator used with this homogenizer 

was 10 mm in diameter and set to rotate at 22,000 rpm. 

Tissues were homogenized following a 1:2 dilution with 

0.10 M acetate buffer pH 4.00. Three mL aliquots of 

specimen fluids, calibrators and controls, and 3.0 g aliquots 

of tissue homogenate were transferred to individual 16 x 

150 mm screw top tubes. To each specimen, calibrator, 

and control, 1.0 mL of the internal standard mixture 

(400 ng) was added. Samples were vortexed briefly and 

allowed to stand at room temperature for 10 min. Eight 

mL of 0.10 M acetate buffer, pH 4.00, were added to 

each sample. The samples were then mixed on a rotary 

extractor that was set to rotate at 15 rpm for 20 min. Following rotation the samples were centrifuged at 1230xg 

for 45 min. The supernatant was then transferred to clean 

16 x 100 mm culture tubes for extraction.

The samples were extracted using a Zymark

RapidTraceTM automated SPE system (Zymark Corp., 

Hokinton, MA). The SPE cartridges used were 3 mL 

Varian Bond Elute-Certify I with a 130 mg sorbent bed 

(Varian Inc., Palo Alto, CA). The RapidTraceTM was programmed with the following parameters: SPE cartridges 

were conditioned with 2.0 mL methanol, followed by 

3.0 mL 0.10 M acetate buffer pH 4.00, both at a flow 

rate of 3 mL/min. Following conditioning, 8.0 mL of 

sample was loaded on to each column at a flow rate of 

1.5 mL/min. The SPE columns were then rinsed with 

3.0 mL 0.10 M HCl at a flow rate of 3 mL/min, dried 

for 2 min with nitrogen at a pressure of 30 p.s.i., 

rinsed with 6.0 mL methanol at a flow rate of 3 mL/

min and dried a final time for 2 min at a pressure of 

30 p.s.i. The analytes were then eluted with 4.0 mL 

dichloromethane-isopropanol-ammonium hydroxide 

(80:20:2 v/v/v), which was prepared fresh daily, into 15 

mL round-bottom, screw top tubes. To avoid carry over, 

the RapidTraceTM cannula was washed with 6.0 mL of 

methanol, and the RapidTraceTM column plunger was 

washed by sequentially passing 3.0 mL elution solvent 

and 6.0 mL water to waste after completion of each 

sample extraction.

Each sample eluent was evaporated to dryness in a water 

bath at 40°C under a stream of dry nitrogen. Once dryness 

was achieved, PFPA (50 μL) and PFP (50 μL) were added 

to each. The tubes were then capped tightly, vortexed 

briefly, and incubated in a heating block set to 70°C for 

20 min. Samples were removed from the heating block 

and allowed to cool to room temperature. Two hundred 

μL of ethyl acetate was added to each sample to aid in 

the removal of the derivatizing reagent. The derivatizing 

reagent/ethyl acetate mixture was evaporated to dryness 

in a water bath at 40°C under a stream of dry nitrogen. 

Once samples were dry, they were promptly removed from 

the water bath to avoid unnecessary loss of any volatile 

metabolites. The samples were reconstituted in 50 μL 

ethyl acetate and transferred to GC autosampler vials for 

GC/MS analysis. All specimens were analyzed at one time 

to avoid inter-assay variations. Specimens with analyte 

concentrations above the associated calibration curves 

were diluted by an appropriate factor and re-extracted, 

so that the result fell within the calibration curve.

Extraction Efficiency

The method used for the determination of analyte recovery has previously been reported by Lewis et al. (30). Briefly 

described, two groups of controls, X and Y, prepared using 

certified-negative blood were extracted in the same manner 

as discussed above. Group X was spiked with a precisely 

known concentration of each analyte prior to extraction, 

while group Y was spiked with the same precisely known 

concentration of each analyte following extraction. Upon 

analysis, the average response factor obtained from group 

X was divided by the average response factor obtained from 

group Y to yield the percent recovery value (100 * (X/Y) 

= % recovery) for each of the compounds.
5

RESULTS AND DISCUSSION 

Method Validation

The procedure described herein, which utilizes a Zymark RapidTraceTM automated SPE system, PFP/PFPA 

derivatives, and GC/MS, provides a rapid, reproducible, 

and sensitive method for the determination of COC, BE, 

NBE, HBE, NCOC, EME, and E, as well as the pyrolysis 

products AEME and AE and the ethanol adducts CE, 

NCE, and EEE. All analyte peaks were completely resolved, with the exception of CE and NCOC. However, 

CE and NCOC each provide ions with unique m/z, so 

no interference was observed. Deuterated COC, BE, CE, 

and EME were used as internal standards for the quantitations performed in this study. For analytes with no 

deuterated analog available, the closest structurally related 

internal standard was employed. Surprisingly, even with 

such a simple extraction procedure, no analyte suffered 

interference from endogenous matrix components. A representative chromatogram demonstrating the separation 

of each of the 12 analytes is shown in Figure 2.

Initially, we investigated the use of BSTFA with 1% 

TMCS (TMS) as a derivatizing agent but found that 

it resulted in incomplete derivatization of secondary 

amines in the “nor” analytes. TMS was also found to 

be an unstable derivative for the analysis of the COCrelated analytes. The use of PFP/PFPA as a derivatizing 

agent provided a more stable derivative (20, 31) with a 

significantly higher molecular weight and less background 

noise than TMS, which is generally used for COC-related 

analyses. PFPA derivatizes hydroxyl and secondary amine 

functional groups contained within many of the COCrelated compounds. PFP derivatizes carboxylic acids 

contained within various COC-related compounds. 

The derivatives formed by the COC-related compounds 

can be seen in Table 1. Stability of these derivatives was 

determined by comparing the peak area of COC, which 

does not contain a functional group that will derivatize, 

to the peak areas of the derivatized analytes immediately 

following derivatization and periodically up to one week 

after derivatization. We found an insignificant amount of 

degradation for the derivatized analytes when stored at 

4°C, with the exception of HBE. Derivatized HBE was 

not as stable as the other analytes when stored at 4°C, 

giving a response that was 25% of its original response 

within 48 h.

AEME is a pyrolysis product of COC and is, therefore, 

an excellent marker for the smoking of crack COC. It is, 

however, theoretically possible to convert COC to AEME 

under high temperatures encountered in the GC injection port (4, 32-34). The injector port of the GC/MS 

used in this study was maintained at 250°C. The high 

temperature in the injector port necessitated the evaluation of the production of AEME from the heated injector 

port thermal decomposition of COC. This evaluation 

was accomplished by injecting 1 μL of a 100 ng/μL neat 

COC standard and monitoring the formation of AEME. 

The production of AEME, monitored by collection of its 

base peak at m/z 152, was found to be 0.5 ± 0.1% (n=4) 

of the peak area of the COC base peak at m/z 182. This 

�����������������������������������������������������������������������������������������������������������

�������������������������������������������������������������������������������������������������
6 

result agrees with other published findings (10, 19, 35). 

As a precautionary measure, we took steps to continually monitor the possible artifactual formation of AEME 

from COC throughout this study. All analyses contained 

numerous 1000 ng/mL and 250 ng/mL COC controls, 

which were monitored for the formation of AEME. The 

average conversion of COC to AEME for the 1000 ng/mL 

control was 0.7 ± 0.1% (n=8) and for the 250 ng/mL 

was 0.8 ± 0.2% (n=4). As can be seen in these data, there 

was no observed change in the percentage of conversion 

of COC to AEME over a broad COC concentration 

range. This lack of concentration dependence supports 

other published findings (19). Another route of artifactual 

AEME formation has also been suggested. The dehydration of EME in the heated injector port may form AEME 

(19). However, no AEME, as monitored by collection 

of its base peak at m/z 152, was detected following the 

injection of 1 μL of a 100 ng/μL EME neat standard. 

AE, a metabolite of AEME and marker for crack use, 

could potentially be formed from the thermal decomposition of BE (36, 37). Following the injection of 1 μL of a 

100 ng/μL neat standard of BE, the production of AE, 

monitored by collection of its base peak at m/z 270, was 

found to be 0.5 ± 0.3% (n=4) of the area of the BE base 

peak at m/z 300. This result also agrees well with other 

published findings (37). As a precautionary measure, we 

took steps to continually monitor the possible artifactual 

formation of AE from BE throughout this study. All 

analyses contained numerous1000 ng/mL and 250 ng/mL 

BE controls, which were monitored for the formation of 

AE. The average conversion of BE to AE for the 1000 

ng/mL control was 0.5 ± 0.2% (n=8) and for the 250 

ng/mL was 0.5 ± 0.1% (n=4). As our data indicate, there 

was no observed change in the percentage of conversion 

of BE to AE at various BE concentrations. 

While numerous studies investigating AEME and/or 

AE use an injector temperature of 250°C or greater 

(10, 19, 35, 37), a few reports recommend the use of 

cooler injector temperatures (12, 34, 37). To determine 

if there was a significant difference between 250°C and 

lower recommended temperatures, we simultaneously 

conducted the above conversion investigations at an 

injector port temperature of 210°C. We found no statistical difference between conversions found at 250°C 

and 210°C. Additionally, lower GC inlet temperatures 

have been reported to sacrifice GC/MS sensitivity (38). 

Therefore, we kept our injector temperature at 250°C 

for the entire study.

E is a di-ester cleavage metabolite of COC and has been 

proposed as a potential marker in postmortem specimens 

for the determination of COC use (39). Extreme caution 

must be used, however, when interpreting quantitative E 

results. Following derivatization and subsequent analysis 

of a 100 ng/μL EME neat standard, approximately 2% of 

the EME originally present was converted to E. While the 

hydroxyl group present on both EME and E derivatize 

with PFPA, only E should react with PFP since EME 

lacks the necessary carboxylic acid moiety. However, as 

is indicated by our results, EME appears to undergo a 

transesterification reaction with PFP during the derivatization process to form E. To discern other possible sources 

of E, we also investigated the formation of E from EEE 

and BE. EEE was found to consistently produce E at less 

than 0.6% of the original EEE concentration. Following 

derivatization, BE was also found to produce E but at 

levels of approximately 0.4% of the original BE concentration. While it appears that many COC metabolites can 

produce small amounts of E during the derivatization 

process, the presence of E, in the absence of EME and 

BE, or at concentrations substantially greater than that 

possible from artifactual formation, further supports the 

past use of COC.

The quantitation and qualifier ions for each analyte 

and internal standard examined in this study are listed 

in Table 1. Acceptability criteria employed for analyte 

identification and quantitation were as follows: 1) ion 

ratios for a given analyte, measured as the peak area of 

a qualifier ion divided by the peak area of the quantitation ion, were required to be within ± 20% of the average of the ion ratios for each respective calibrator used 

to construct the calibration curve for that analyte; 2) 

each ion monitored was required to have a minimum 

signal-to-noise ratio (S/N) of 5; and 3) the analyte was 

required to have a retention time within ± 0.20 min of 

the average retention time for each respective calibrator 

used to construct the calibration curve for that analyte. 

Any analyte not meeting these criteria was reported as 

either being negative or inconclusive.

The linear dynamic range (LDR) for each analyte is 

presented in Table 2. In general, the LDRs were approximately 0.78 - 3200 ng/mL. At concentrations greater than 

1600 ng/mL, the responses for NCOC, NCE, NBE, and 

E were not consistently linear. The correlation coefficients 

for the calibration curves used to ascertain LDR were all 

greater than 0.99 as demonstrated in Table 2. Additionally, Table 2 shows the lower limit of detection (LOD) 

and lower limit of quantitation (LOQ) determined for 

each analyte. The LOD was defined as the lowest analyte 

concentration detectable that meets the above-discussed 

identification criteria. The LOQ was defined as the lowest analyte concentration detectable that not only met 

all identification criteria discussed above but also had an 

experimentally determined concentration within ± 20% 

of its prepared value. The LOD for these COC-related 
7

compounds ranged from 0.78 - 12.5 ng/mL. The LOQ 

for these COC-related compounds ranged from 0.78 

- 25 ng/mL.

The average recoveries of COC and its metabolites at 

45 ng/mL and 450 ng/mL ranged from 26 - 84%, with the 

exception of AE and E. These values are listed in Table 2. 

At both control concentrations, the recoveries for COC, 

BE, CE, NCOC, NCE, and AEME were above approximately 60%. The experimentally determined extraction 

efficiency values for these compounds agreed well with 

previously reported recoveries in whole blood (39-41). 

At both concentrations, AE and E exhibited poor recovery from whole blood. The experimentally determined 

extraction efficiencies for these compounds ranged from 

approximately 1 - 4%. Given the high polarity of AE 

and E, the poor recoveries are likely due to compound 

loss during the sample loading and HCl wash steps of 

the extraction.

Carryover from one sample to the next did not occur 

with either the Zymark or the GC/MS. Carryover on 

the Zymark was investigated by extracting a negative 

control following the 3200 ng/mL calibrator. Carryover 

on the GC/MS was initially investigated and subsequently 

monitored by the use of ethyl acetate solvent injections. 

An ethyl acetate blank injected following the 3200 ng/mL 

calibrator injection showed no carryover contamination. 

Subsequently, ethyl acetate blanks were analyzed between 

each sample throughout the sample sequence to verify that 

no sample-to-sample contamination had occurred.

Intra-day (within day) and inter-day (between days) 

accuracy and precision were examined for this extraction. 

The accuracy was measured as the relative error between 

the experimentally determined and prepared concentrations of a sample. The precision was measured as the 

relative standard deviation (RSD) for the experimentally 

determined concentrations. Pools of controls, prepared 

using whole blood as the diluent, were created at 45 ng/

mL and 450 ng/mL in volumes large enough to be used 

for the entire precision and accuracy investigation. These 

controls were stored at 4°C until analyzed. For intra-day 

analyses, a calibration curve was extracted, along with 5 

replicates of each control concentration on Day 1 of the 

experiment. For the intra-day assay, all analytes at both 

concentrations yielded relative errors within 9% of the 

target concentration. Furthermore, all analytes had RSDs 

within 10%, with the exception of AEME and AE. The 

RSD for these COC pyrolysis products was as high as 

12%. This is likely a result of compound loss during the 

various dry-down steps in the extraction process, since these 

two compounds are highly volatile. The intra-day assay 

results are shown in Table 3. These results demonstrate the 

exceptional precision and accuracy of this method.

�����������������������������������������������������������������

��������� ������ ���� ���� ���� �������������

� �������� �������� �������� � ��������� ���������

���� ����� ����� ���������� ������ �� ���� �������

����� ����� ����� ���������� ������ �� ���� �������

��� ����� ����� ���������� ������ �� ���� �������

���� ����� ����� ��������� ������ �� ���� �������

���� ����� ����� ���������� ������ �� ����� �������

���� ����� ����� ���������� ������ �� ���� �������

�� ����� ��� �������� ������ ��� ������ ����������

��� ����� ����� ���������� ������ �� ���� �������

���� ����� ����� ���������� ������ �� ���� �������

���� ����� ����� ���������� ������ �� ���� �������

����� ����� ����� ���������� ������ �� ���� �������

��� ����� ����� ���������� ������ ��� ������ ����������

�������������������������������������������������������������������������

������������������������������������������������������

�

�����������������������������
8 

Inter-day accuracy and precision were evaluated by extracting 5 replicates of each of two control concentrations 

on Days 3 and 5. The quantitative values determined on 

these days were based on the calibration curves originally 

prepared on Day 1. The results obtained after storage 

of each control lot at 4°C for 3 and 5 days can be seen 

in Table 3. For a majority of the COC-related analytes, 

the concentrations determined on Days 3 and 5 showed 

no significant difference from those obtained on Day 

1. This agrees well with other published findings (42, 

43). The RSDs for compounds with deuterated internal 

standards were all within 3% on Days 3 and 5. For all 

other compounds, the RSDs were within 10% on Days 3 

and 5, with the exception of AEME and AE. Their RSDs 

ranged from 12 - 36%, but, again, this may be due to 

their extreme volatility as discussed above. Additionally, 

there was a substantial decrease in concentration observed 

over the course of 5 days for both AE and AEME. AE 

demonstrated an absolute decrease in concentration from 

Day 1 to Day 5 of 31% and 14% in the 45 ng/mL and 

450 ng/mL whole blood controls, respectively. AEME 

showed a similar decrease of 34% and 26%, respectively. 

The apparent decrease in AE and AEME prepared in whole 

blood and stored at 4°C emphasizes the need for prompt 

analysis once forensic samples have been thawed (44). 

�����������������������������������������������������������

� � ������ ������ ������

�������

��������

����

�������� ���� �� ����

�������� ���� ��� ����

�������� ���� ��

��� ��

���

���� ������

��� ����

���

���

����

��

���� �����

��� ����

���

���

��

��

�� ����

��� ����

�

���

��

����

���� ��

���

�� ����

��� �����

�

�

��

��

�� ����

��� �����

�

�

��

��

�� ����

��� ����

�

�

��

��

��� ��

���

���� ������

��� ����

���

���

��

��

���� �����

��� ����

���

�

��

��

���� ������

��� ����

���

���

��

��

��� ��

���

�� ����

��� �����

�

�

��

��

�� ����

��� �����

�

�

��

��

�� ����

��� �����

�

�

��

����

��� ��

���

�� ����

��� �����

�

�

��

��

�� ����

��� �����

�

��

�

��

�� ����

��� �����

�

��

���

��

��� ��

���

���� ������

��� ����

���

���

��

��

���� �����

��� ����

���

�

���

��

���� ������

��� ����

���

�

���

��

� ��

���

�� ����

��� �����

�

�

��

��

�� ����

��� �����

�

��

��

��

�� ����

��� �����

��

�

��

���

�� ��

���

���� ������

��� ����

���

�

����

��

���� �����

��� �����

�

�

��

��

���� ������

��� �����

�

�

��

��

��� ��

���

�� ����

��� �����

�

�

��

��

�� ����

��� �����

�

�

��

��

�� ����

��� ����

�

�

��

��

��� ��

���

�� ����

��� ����

�

���

��

��

���� �����

��� ����

�

�

���

��

�� ����

��� �����

�

�

���

��

���� ��

���

�� ����

��� �����

�

��

��

��

�� ����

��� �����

�

�

���

��

�� ����

��� �����

�

�

���

���

�� ��

���

�� ����

��� �����

��

��

��

��

�� ����

��� �����

��

��

���

���

�� ����

��� �����

��

��

���

���

���������������������������

��������������������������������������������������������������������

�����������������������������������������������������������������������������������
9

Method Application: Postmortem Specimen Analysis

In fatal aviation accidents, specimens from accident 

victims are routinely sent to the Federal Aviation Administration’s Forensic Toxicology Research Laboratory for 

toxicological analysis. Postmortem fluid and tissue samples 

obtained from 5 fatalities involved in separate aviation accidents over the past 3 years that had previously been screened 

positive for COC and/or BE by GC/MS were re-examined 

using this new method to determine the presence of various COC analytes. The fluid and tissue samples selected 

for analysis were blood, urine, liver, kidney, and muscle. 

The 5 aviation fatalities chosen for this investigation had 

a majority, if not all, of the desired specimens available for 

analysis. The results of this analysis are shown in Table 4.

����������������������������������������������������������������������������������������������������

��������������������������

������� ���� ���� ��� ��� ��� ���� ��� ���� ���� ����� ���

������� ��� �� ���� �� � ��� �� �� �� ��� ���

������� ���� ��� ����� ���� � ����� ��� ��� ��� ���� �����

������� ��� �� ���� � � ���� � � � ���� ����

�������� ��� �� ���� � � ���� � � � ���� ���

�������� ��� �� ���� � � ��� � � � ��� ���

������� � � � � � � � � � � �

������� ��� �� ���� � � �� �� � � ���� ����

������� ����� �� ������ ���� � ����� �� � ���� ���� �����

������� ��� �� ���� ��� � ���� ��� ��� �� ����� �����

�������� ��� �� ����� ��� � ���� � � ��� ���� �����

�������� ��� �� ���� �� � ��� � � �� ��� �����

������� � � � � � � � � � � �

������� �� �� ���� �� � ��� �� � ��� �� ����

������� ���� �� ����� ���� � ����� ���� ��� ���� ��� �����

������� ��� � ���� � � ���� ���� ��� ���� ���� �����

�������� ��� � ����� � � ���� ��� � ���� ���� �����

�������� ��� � ���� � � ��� ��� � ��� ��� ����

������� � � � � � � � � � � �

������� ��� � ���� �� � ���� ��� �� ��� ��� ����

������� ��� � ���� �� � ���� �� � ���� ���� �����

�������� ��� � ����� ��� ��� ���� ��� � ���� ����� �����

�������� ��� � ����� � � ���� ��� � ��� ���� �����

������� � � � � � � � � � � �

������� ���� � ����� � � ���� � � � ���� ����

������� ��� � ���� � � ���� � � � ����� �����

������� ��� � ���� � � ��� � � � ���� �����

������������������������������������������������������������������������������������������������

��������������������������������������������������������������������������������������������������������������

������������������������������������������������������������

���������������������������������������������������������������������������������������������������

�����������������������
10 

COC, BE, and EME were found in all cases examined. 

Blood concentrations for COC, BE, and EME ranged 

from <0.78 - 52 ng/mL, 151 - 847 ng/mL and 0 - 122 

ng/mL, respectively. Urine concentrations for COC, 

BE, and EME ranged from 348 - 1031 ng/mL, 6025 

- 41655 ng/mL, and 2837 - 11177 ng/mL, respectively. 

Liver concentrations for COC, BE, and EME ranged 

from 13 - 136 ng/g, 308 - 1099 ng/g, and 158 - 450 

ng/g, respectively. Kidney concentrations for COC, BE, 

and EME ranged from 31 - 94 ng/g, 517 - 1942 ng/g, 

and 343 - 614 ng/g, respectively. Muscle concentrations 

for COC, BE, and EME ranged from 15 - 49 ng/g, 145 

- 1060 ng/g, and 46 - 116 ng/g, respectively. As can be 

seen from these data, there is no apparent correlation 

between COC, BE, and/or EME concentrations within 

any of the specimen types analyzed. The general trend 

observed for the highest to lowest concentration of 

COC, BE, and EME between specimen types analyzed 

was urine>kidney>liver>blood~muscle. 

The identification of either AEME or AE is a good 

marker of crack use. However, since we previously showed 

that COC produces small amounts of artifactual AEME, 

and BE produces both AE and E, the interpretation of 

AEME, AE or E found in an actual case specimen must 

be approached cautiously. As previously described, whole 

blood controls containing COC and BE separately were 

run with each analysis. Following each analysis, the percentage of COC converted to AEME and the percentage 

of BE converted to both AE and E was determined. The 

percent conversion from one analyte to another was then 

multiplied by 5 to obtain a cutoff value, which was used 

to aid in determining whether AEME, AE or E found 

in a case specimen was actually present in the specimen 

or formed during analysis due to thermal decomposition 

of COC or BE in the injection port. A multiplication 

factor of 5 was chosen to provide a conservative cutoff 

that would ensure that any AE, AEME and/or E reported 

was real and not a false positive. During case analysis, 

the percent conversion, as determined by dividing the 

area count of the quantitation ion for AEME, AE or E 

by the area count of the quantitation ion for COC or 

BE, was found to be: COC to AEME = 0.7 ± 0.1% (n = 

4); BE to AE = 0.5 ± 0.2% (n = 4); and BE to E = 0.2 ± 

0.2% (n = 4). Therefore, a cutoff for AEME was established for this study at 3.5% of COC. For example, an 

AEME result having a detector response less than 3.5% 

of the COC detector response was reported as negative. 

The cutoffs for AE and E were established as 2.5% and 

1.1%, respectively.

AEME and AE were detected in all 5 cases examined. 

More specifically, it should be noted that their responses 

were substantially above our established cutoffs. E was 

detected in most of the cases examined but at levels below 

our established cutoff. Thus, the E observed may have 

been artifactually produced from BE and was reported as 

negative. Blood concentrations for AEME and AE ranged 

from 6 - 32 ng/mL and 0 - 927 ng/mL, respectively. 

Urine concentrations for AEME and AE ranged from 

29 - 434 ng/mL and 3433 - 16632 ng/mL, respectively. 

Tissue concentrations for AEME and AE ranged from 

22 - 5595 ng/g and 0 - 37052 ng/mL, respectively. We 

were unable to identify any correlation between AEME 

or AE and COC, BE or EME concentrations within 

any of the specimen types analyzed. Additionally, there 

was also no apparent trend observed for which specimen 

type contained the highest concentrations of AEME and 

AE. Since the recovery for AE is poor and BE, which can 

produce AE, is generally quite concentrated in typical 

COC-positive specimens, values reported for AE may be 

erroneously high. This is especially true in the presence 

of very high concentrations of BE. However, the true 

importance of the identification of AE is its presence 

as an indicator of crack use, not its specific specimen 

concentration.

CE, NCE, and EEE are good markers for the coadministration of COC and ethanol. CE, NCE, and 

EEE were found in 4 of the 5 cases investigated. Blood 

concentrations for CE, NCE, and EEE ranged from 0 - 20 

ng/mL, 0 - <1.56 ng/mL, and 0 - 83 ng/mL, respectively. 

Urine concentrations for CE, NCE, and EEE ranged 

from <1.56 - 255 ng/mL, 0 - 34 ng/mL, and 35 - 641 

ng/mL, respectively. Tissue concentrations for CE, NCE, 

and EEE ranged from 0 - 111 ng/g, 0 - 8 ng/g, and 0 

- 377 ng/g, respectively. No apparent correlation was 

observed between CE, NCE or EEE, and COC, BE, or 

EME concentrations within or between specimen types 

tested.

Various other COC-related metabolites were also identified. NCOC was found at very low concentrations in 2 

urine specimens and 2 liver specimens. NBE was found 

in various specimens in 4 out of the 5 cases analyzed. 

HBE was found in the kidney specimen of only 1 case. 

Since these metabolites are random in their presence and 

concentration, they are of little help in interpreting COC 

results, other than reaffirming the use of COC.

CONCLUSION

An automated method that is rapid, reliable, robust, 

and sensitive has been developed for the identification 

and subsequent quantitation of COC and 11 COCrelated compounds in postmortem fluids and tissues. 

This method offers the ability to differentiate between 

smoking crack and intranasal/intravenous COC use and 
11

is able to elucidate whether ethanol and COC were used 

simultaneously. Application of this procedure shows the 

effectiveness of GC/MS for the separation and subsequent 

detection of both underivatized and PFP/PFPA derivatized 

COC metabolites. One of the most important aspects of 

this method is the simultaneous analysis of 12 compounds 

using a single extraction procedure. The relative simplicity 

of this method should make the quantitation of previously 

obscure COC-related compounds more readily attainable 

for the field of forensic toxicology.

REFERENCES

1. Nakahara Y, Ishigami A. Inhalation efficiency of 

free-base cocaine by pyrolysis of “crack” and cocaine 

hydrochloride. J Anal Toxicol 1991;15(3):105-9.

2. Cone EJ. Pharmacokinetics and pharmacodynamics 

of cocaine. J Anal Toxicol 1995;19(6):459-78.

3. Perez-Reyes M, Di Guiseppi S, Ondrusek G, Jeffcoat AR, Cook CE. Free-base cocaine smoking. 

Clin Pharmacol Ther 1982;32(4):459-65.

4. Jenkins AJ, Goldberger BA. Identification of unique 

cocaine metabolites and smoking by-products in 

postmortem blood and urine specimens. J Forensic 

Sci 1997;42(5):824-7.

5. Kintz P, Sengler C, Cirimele V, Mangin P. Evidence 

of crack use by anhydroecgonine methylester identification. Hum Exp Toxicol 1997;16(2):123-7.

6. Cone EJ, Sampson-Cone AH, Darwin WD, Huestis 

MA, Oyler JM. Urine testing for cocaine abuse: 

Metabolic and excretion patterns following different 

routes of administration and methods for detection 

of false-negative results. J Anal Toxicol 2003;27:

386-401.

7. Isenschmid DS, Fischman MW, Foltin RW, Caplan 

YH. Concentration of cocaine and metabolites in 

plasma of humans following intravenous administration and smoking of cocaine. J Anal Toxicol 

1992;16(5):311-4.

8. Stewart DJ, Inaba T, Tang BK, Kalow W. Hydrolysis 

of cocaine in human plasma by cholinesterase. Life 

Sci 1977;20(9):1557-63.

9. Toennes SW, Thiel M, Walther M, Kauert GF. 

Studies on metabolic pathways of cocaine and its 

metabolites using microsome preparations from rat 

organs. Chem Res Toxicol 2003;16(3):375-81.

10. Cone EJ, Hillsgrove M, Darwin WD. Simultaneous measurement of cocaine, cocaethylene, their 

metabolites, and “crack” pyrolysis products by gas 

chromatography-mass spectrometry. Clin Chem 

1994;40(7 Pt 1):1299-305.

11. Jacob P, 3rd, Lewis ER, Elias-Baker BA, Jones RT. 

A pyrolysis product, anhydroecgonine methyl ester (methylecgonidine), is in the urine of cocaine 

smokers. J Anal Toxicol 1990;14(6):353-7.

12. Shimomura ET, Hodge GD, Paul BD. 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 

2001;47(6):1040-7.

13. Jacob P, 3rd, Jones RT, Benowitz NL, Shulgin AT, 

Lewis ER, Elias-Baker BA. Cocaine smokers excrete 

a pyrolysis product, anhydroecgonine methyl ester. 

J Toxicol Clin Toxicol 1990;28(1):121-5.

14. Erzouki HK, Allen AC, Newman AH, Goldberg 

SR, Schindler CW. Effects of cocaine, cocaine 

metabolites and cocaine pyrolysis products on the 

hindbrain cardiac and respiratory centers of the 

rabbit. Life Sci 1995;57(20):1861-8.

15. Wu AH, Onigbinde TA, Johnson KG, Wimbish 

GH. Alcohol-specific cocaine metabolites in serum 

and urine of hospitalized patients. J Anal Toxicol 

1992;16(2):132-6.

16. de la Torre R, Ortuno J, Gonzalez ML, Farre M, 

Cami J, Segura J. Determination of cocaine and its 

metabolites in human urine by gas chromatography/

mass spectrometry after simultaneous use of cocaine 

and ethanol. J Pharm Biomed Anal 1995;13(3):

305-12.

17. Dean RA, Harper ET, Dumaual N, Stoeckel DA, 

Bosron WF. Effects of ethanol on cocaine metabolism: Formation of cocaethylene and norcocaethylene. Toxicol Appl Pharmacol 1992;117(1):1-8.

18. Phillips DL, Tebbett IR, Bertholf RL. Comparison 

of HPLC and GC-MS for measurement of cocaine 

and metabolites in human urine. J Anal Toxicol 

1996;20(5):305-8.

19. Toennes SW, Fandino AS, Kauert G. Gas chromatographic-mass spectrometric detection of anhydroecgonine methyl ester (methylecgonidine) in human 

serum as evidence of recent smoking of crack. J 

Chromatogr B Biomed Sci Appl 1999;735(1):

127-32.
12 

20. Abusada GM, Abukhalaf IK, Alford DD, VinzonBautista I, Pramanik AK, Ansari NA, et al. Solidphase extraction and GC/MS quantitation of 

cocaine, ecgonine methyl ester, benzoylecgonine, 

and cocaethylene from meconium, whole blood, 

and plasma. J Anal Toxicol 1993;17(6):353-8.

21. Browne SP, Tebbett IR, Moore CM, Dusick A, Covert R, Yee GT. Analysis of meconium for cocaine 

in neonates. J Chromatogr 1992;575(1):158-61.

22. Campora P, Bermejo AM, Tabernero MJ, Fernandez 

P. Quantitation of cocaine and its major metabolites 

in human saliva using gas chromatography-positive 

chemical ionization-mass spectrometry (GC-PCIMS). J Anal Toxicol 2003;27(5):270-4.

23. Fandino AS, Karas M, Toennes SW, Kauert G. 

Identification of anhydroecgonine methyl ester 

N-oxide, a new metabolite of anhydroecgonine 

methyl ester, using electrospray mass spectrometry. 

J Mass Spectrom 2002;37(5):525-32.

24. Hernandez A, Andollo W, Hearn WL. Analysis of 

cocaine and metabolites in brain using solid phase 

extraction and full-scanning gas chromatography/

ion trap mass spectrometry. Forensic Sci Int 

1994;65(3):149-56.

25. Karacic V, Skender L. Analysis of drugs of abuse in 

urine by gas chromatography/mass spectrometry: 

Experience and application. Arh Hig Rada Toksikol 

2000;51(4):389-400.

26. Klette KL, Poch GK, Czarny R, Lau CO. Simultaneous GC-MS analysis of meta- and parahydroxybenzoylecgonine and norbenzoylecgonine: 

a secondary method to corroborate cocaine ingestion using nonhydrolytic metabolites. J Anal Toxicol 

2000;24(7):482-8.

27. Lewis DE, Moore CM, Leikin JB, Koller A. Meconium analysis for cocaine: a validation study 

and comparison with paired urine analysis. J Anal 

Toxicol 1995;19(3):148-50.

28. Mackey-Bojack S, Kloss J, Apple F. Cocaine, cocaine metabolite, and ethanol concentrations in 

postmortem blood and vitreous humor. J Anal 

Toxicol 2000;24(1):59-65.

29. Oyler J, Darwin WD, Preston KL, Suess P, Cone EJ. 

Cocaine disposition in meconium from newborns 

of cocaine-abusing mothers and urine of adult drug 

users. J Anal Toxicol 1996;20(6):453-62.

30. Lewis RJ, Johnson RD, Blank CL. A novel method 

for the determination of sildenafil (Viagra) and its 

metabolite (UK-103,320) in postmortem specimens using LC/MS/MS and LC/MS/MS/MS/MS. 

Department of Transportation, Federal Aviation 

Administration, DOT/FAA/AM-00/20 2000.1

31. Stout PR, Gehlhausen JM, Horn CK, Klette KL. 

Evaluation of a solid-phase extraction method for 

benzoylecgonine urine analysis in a high-throughput forensic urine drug-testing laboratory. J Anal 

Toxicol 2002;26(7):401-5.

32. Fandino AS, Toennes SW, Kauert GF. Studies on 

in vitro degradation of anhydroecgonine methyl 

ester (methylecgonidine) in human plasma. J Anal 

Toxicol 2002;26(8):567-70.

33. Toennes SW, Fandino AS, Hesse FJ, Kauert GF. 

Artifact production in the assay of anhydroecgonine 

methyl ester in serum using gas chromatographymass spectrometry. J Chromatogr B Analyt Technol 

Biomed Life Sci 2003;792(2):345-51.

34. Zhang JY, Foltz RL. Cocaine metabolism in man: 

Identification of four previously unreported cocaine metabolites in human urine. J Anal Toxicol 

1990;14(4):201-5.

35. Kintz P, Cirimele V, Sengler C, Mangin P. Testing 

human hair and urine for anhydroecgonine methyl 

ester, a pyrolysis product of cocaine. J Anal Toxicol 

1995;19(6):479-82.

36. Paul BD, McWhorter LK, Smith ML. Electron ionization mass fragmentometric detection of urinary 

ecgonidine, a hydrolytic product of methylecgonidine, as an indicator of smoking cocaine. J Mass 

Spectrom 1999;34(6):651-60.

37. Gonzalez ML, Carnicero M, de la Torre R, Ortuno J, 

Segura J. Influence of the injection technique on the 

thermal degradation of cocaine and its metabolites 

in gas chromatography. J Chromatogr B Biomed 

Appl 1995;664(2):317-27.

38. Grob K, Biedermann M. Video-taped sample 

evaporation in hot chambers simulating gas chromatography split and splitless injectors. II. Injection 

with band formation. J Chromatogr A 2000;897(1-

2):247-58.

1

This publication and all Office of Aerospace Medicine technical 

reports are available in full-text from the Civil Aerospace Medical 

Institute’s publications Web site: http://www.cami.jccbi.gov/aam400A/index.html
13

39. Smirnow D, Logan BK. Analysis of ecgonine 

and other cocaine biotransformation products in 

postmortem whole blood by protein precipitationextractive alkylation and GC-MS. J Anal Toxicol 

1996;20(6):463-7.

40. Winecker RE, Goldberger BA, Tebbett IR, Behnke 

M, Eyler FD, Karlix JL, et al. Detection of cocaine 

and its metabolites in breast milk. J Forensic Sci 

2001;46(5):1221-3.

41. Crouch DJ, Alburges ME, Spanbauer AC, Rollins 

DE, Moody DE. Analysis of cocaine and its metabolites from biological specimens using solid-phase 

extraction and positive ion chemical ionization mass 

spectrometry. J Anal Toxicol 1995;19(6):352-8.

42. Levine B, Ramcharitar V, Smialek JE. Stability of 

ecgonine methyl ester in postmortem urine specimens. J Forensic Sci 1996;41(1):126-8.

43. Moody DE, Monti KM, Spanbauer AC. Longterm stability of abused drugs and antiabuse chemotherapeutical agents stored at -20 degrees C. J 

Anal Toxicol 1999;23(6):535-40.

44. Fandino AS, Toennes SW, Kauert GF. Studies on 

hydrolytic and oxidative metabolic pathways of anhydroecgonine methyl ester (methylecgonidine) using microsomal preparations from rat organs. Chem 

Res Toxicol 2002;15(12):1543-8.
14