Patrick S. Cardona

Arvind K. Chaturvedi

John W. Soper

Dennis V. Canfield

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

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-03/24 Simultaneous Determination of Cocaine, 

Cocaethylene, and Their Possible 

Pentafluoropropylated Metabolites 

and Pyrolysis Products by 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

Technical Report Documentation Page 

1. Report No. 2. Government Accession No. 3. Recipient's Catalog No. 

DOT/FAA/AM-03/24 

4. Title and Subtitle 5. Report Date

Simultaneous Determination of Cocaine, Cocaethylene, and Their December 2003 

Possible Pentafluoropropylated Metabolites and Pyrolysis Products by 

Gas Chromatography/Mass Spectrometry 

6. Performing Organization Code 

7. Author(s) 8. Performing Organization Report No. 

Cardona PS, Chaturvedi AK, Soper JW, Canfield DV

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS) 

FAA Civil Aerospace Medical Institute 

P.O. Box 25082 11. Contract or Grant No. 

Oklahoma City, OK 73125 

12. Sponsoring Agency Name and Address 13. Type of Report and Period Covered 

Office of Aerospace Medicine 

Federal Aviation Administration 

800 Independence Ave., S.W. 

Washington, DC 20591 14. Sponsoring Agency Code 

15. Supplemental Notes 

This work was accomplished under the approved tasks AM-B-98-TOX-202, AM-B-99-TOX-202, AM-B-00-

TOX-202, AM-B-01-TOX-202, AM-B-02-TOX-202, and AM-B-03-TOX-202.

16. Abstract 

During the investigations of fatal transportation accidents, samples from victims are also analyzed for drugs, 

including cocaine (COC). COC is abused by smoking, nasal insufflantion, and intravenous injection, and it is 

also taken with ethanol. Therefore, it is important to determine concentrations of COC and its metabolites, 

ethanol analogs, and pyrolysis products for establishing the degree of toxicity, the possible ingestion of ethanol, 

and the possible route of administration. In this study, a sensitive and selective procedure is developed for the 

simultaneous analyses of COC, benzoylecgonine, norbenzoylecgonine, norcocaine, ecgonine, ecgonine methyl 

ester, m-hydroxybenzoylecgonine, anhydroecgonine methyl ester (AEME), anhydroecgonine (AECG), 

cocaethylene, norcocaethylene, and ecgonine ethyl ester in blood, urine, and muscle homogenate. In the analysis, 

available deuterated analogs of these analytes were used as internal standards. Proteins from blood and muscle 

homogenate were precipitated with cold acetonitrile. After the removal of acetonitrile by evaporation, the 

supernatants and urine were extracted by solid-phase chromatography. The eluted analytes were converted to 

hydrochloride salts and derivatized with pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol.

The derivatized products were analyzed on a gas chromatograph (GC)/mass spectrometer system by selected ion 

monitoring. This method was successfully applied in analyzing 13 case specimens from aviation accident pilot 

fatalities and/or motor vehicle operators. AEME concentrations found in the 13 specimens were consistent with 

those produced solely by the GC inlet pyrolysis of COC controls, suggesting that COC was not abused in these 

cases by smoking. Although AEME remains a potential marker for establishing the abuse of COC by smoking, 

AECG was not a useful marker because of its low recovery and GC inlet production from COC metabolites. 

The developed procedure is unique because multiple analytes can be analyzed in urine, blood, and solid tissues 

by a single extraction with increased sensitivity through formation of hydrochloride salts and using a one-step 

derivatization. 

17. Key Words 18. Distribution Statement 

Document is available to the public through the 

National Technical Information Service 

Forensic Sciences, Toxicology, Cocaine, Metabolites, 

Pyrolysis Products, Analysis, Gas Chromatography/ 

Mass Spectrometry Springfield, Virginia 22161 

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price 

Unclassified Unclassified 19 

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized

iii

LIST OF ABBREVIATIONS

AECG _ _ _ _ _ _ _ anhydroecgonine

AEME _ _ _ _ _ _ _ anhydroecgonine methyl ester

BEG _ _ _ _ _ _ _ _ benzoylecgonine

BEG-D3 _ _ _ _ _ _ _ benzoylecgonine-D3

CAMI _ _ _ _ _ _ _ _ Civil Aerospace Medical Institute

COE _ _ _ _ _ _ _ _ cocaethylene

COE-D3 _ _ _ _ _ _ _ cocaethylene-D3

COC _ _ _ _ _ _ _ _ cocaine

COC-D3 _ _ _ _ _ _ _ cocaine-D3

CYP _ _ _ _ _ _ _ _ cytochrome P-450

ECG _ _ _ _ _ _ _ _ ecgonine

EEE _ _ _ _ _ _ _ _ ecgonine ethyl ester

EME _ _ _ _ _ _ _ _ ecgonine methyl ester

EME-D3 _ _ _ _ _ _ _ ecgonine methyl ester-D3

FAA _ _ _ _ _ _ _ _ Federal Aviation Administration

GC/MS _ _ _ _ _ _ _ gas chromatograph/mass spectrometer

HBEG _ _ _ _ _ _ _ m-hydroxybenzoylecgonine

LOD _ _ _ _ _ _ _ _ limit of detection

LOQ _ _ _ _ _ _ _ _ limit of quanititation

NBEG _ _ _ _ _ _ _ norbenzoylecgonine

NCOE _ _ _ _ _ _ _ norcocaethylene

NCOC _ _ _ _ _ _ _ norcocaine

NCOC-D3 _ _ _ _ _ _ norcocaine-D3

OSBI _ _ _ _ _ _ _ _ Oklahoma State Bureau of Investigation

PFP _ _ _ _ _ _ _ _ pentafluoropropylated

PFPA _ _ _ _ _ _ _ _ pentafluoropropionic anhydride

PFPOH _ _ _ _ _ _ _ 2,2,3,3,3-pentafluoro-1-propanol

SIM _ _ _ _ _ _ _ _ selected ion monitoring

SPE _ _ _ _ _ _ _ _ _ solid phase extraction.

1

SIMULTANEOUS DETERMINATION OF COCAINE, COCAETHYLENE, AND THEIR

POSSIBLE PENTAFLUOROPROPYLATED METABOLITES AND PYROLYSIS PRODUCTS BY

GAS CHROMATOGRAPHY/MASS SPECTROMETRY

INTRODUCTION

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

Aerospace Medical Institute (CAMI) is involved in the 

toxicological evaluation of samples collected from victims 

involved in fatal transportation accidents. During the 

investigations of such accidents, those biological samples 

are analyzed for the presence of combustion gases (carbon monoxide and hydrogen cyanide), alcohol and other 

volatiles, and drugs, including cocaine (COC). COC, a 

popular drug of abuse, is consumed by smoking, nasal 

insufflation, and intravenous injection (1). COC is also 

often taken in conjunction with ethanol, generating a 

transesterification product, cocaethylene (COE; 2). This 

COC analog has pharmacological activities similar to 

COC and may modestly prolong the COC-like effects 

(3). In recreational use, smoking may be the preferred 

route of ingestion of COC because its effects are immediate and intense. Furthermore, such effects are achieved 

without having to deal with needles and syringes, thereby 

minimizing the risk of contracting bloodborne diseases. 

Free-base COC (crack) is generally associated with smoking (1, 4, 5), while its salt form may be associated with 

other routes of administration.

Peak COC concentrations in blood usually occur 

30–60 min after nasal insufflation and within minutes 

after smoking or intravenous injection ingestion (6). 

COC is rapidly inactivated by the hydrolysis of its ester 

groups, producing benzoylecgonine (BEG), ecgonine 

methyl ester (EME), and possibly ecgonine (ECG; Fig. 

1). COC is further biotransformed by the cytochrome 

P-450 (CYP) enzyme system to norcocaine (NCOC), and 

BEG to norbenzoylecgonine (NBEG) and m-hydroxybenzoylecgonine (HBEG). Smoking produces the pyrolysis 

product anhydroecgonine methyl ester (AEME; 7, 8), 

which could be further hydrolyzed to anhydroecgonine 

(AECG). Furthermore, COE is metabolized into norcocaethylene (NCOE) and hydrolyzed into ecgonine ethyl 

ester (EEE), which could also be biosynthesized from EME 

in the presence of ethanol. COE could be converted into 

ECG and BEG, while NCOE and NCOC can generate NBEG. Additionally, NCOC can be converted into 

NCOE by transesterification (2).

Postmortem biochemical activity or improper specimen preservation and/or storage may also result in the 

conversion of COC into EME and BEG (Fig. 1). Even 

in water, at pH values greater than neutrality, COC is 

readily hydrolyzed to BEG. Cholinesterases in blood 

hydrolyze COC into EME, but this enzymatic reaction 

may be inhibited by freezing or by the addition of fluoride 

or cholinesterase inhibitors (9, 10, 11). A considerable 

amount of time may often pass before postmortem specimens are collected, chemically preserved, and refrigerated. Such a delay may possibly cause the conversion of 

all or some COC and COE into EME, BEG, and/or 

EEE. Therefore, concentrations of COC, COE, EME, 

BEG, other possible metabolites, and pyrolysis products 

in biological samples could be helpful in estimating the 

total amount of COC present in those samples at the 

time of their collection and/or at the time of an accident 

and/or death. Furthermore, the concentrations of COE, 

NCOE, and EEE may indicate concurrent use of COC 

and ethanol, and the concentration of AEME may assist 

in establishing whether COC was smoked or taken by 

other routes.

Numerous analytical methods for COC, COE, their 

metabolites, and other related products are reported in the 

literature (5, 7, 12, 13), but these methods involve multiple extraction and/or derivatization procedures and/or 

have not been shown to detect all the COC and possible 

related analytes in multiple specimen types. Although the 

pyrolytic product of COC, AEME, has been analyzed 

in blood, serum, and urine (5, 7, 12, 13), it has been 

demonstrated that COC can thermally degrade during 

gas chromatography, particularly at high injector port 

temperatures (8, 14).

In the present study, a selective and sensitive method, 

involving a single extraction and derivatization, was developed to simultaneously analyze COC, COE, their metabolites and pyrolysis products, and related compounds 

by gas chromatography/mass spectrometry in blood, urine, 

and solid tissues. Attempts were also made to establish 

a relationship between the concentration of COC and 

the production of AEME during gas chromatography 

and the suitability of AECG as an additional analytical 

marker for COC smoking. The developed method was 

successfully evaluated for analyzing blood, urine, and 

muscle specimens collected from 13 aviation accident 

pilot fatalities and/or motor vehicle operators. 
2 

EXPERIMENTAL

Materials

All solvents and reagents used during the analyses 

were of analytical grade and were of the highest available purity. These chemicals, analyte standards, and 

other reagents were obtained from commercial sources. 

Specifically, COC, BEG, NBEG, NCOC, ECG, EME, 

HBEG, AEME, AECG, COE, NCOE, and EEE were 

supplied by Cerilliant Corporation (Austin, TX) in certified 1.0 mg/mL solutions. Internal standards, cocaine-D3

(COC-D3), benzoylecgonine-D3 (BEG-D3), norcocaineD3

 (NCOC-D3), ecgonine methyl ester-D3 (EME-D3), 

and cocaethylene-D3 (COE-D3) were also supplied by the 

Cerilliant Corporation in certified 100 μg/mL solutions. 

Where a deuterated internal standard of an analyte was 

not available, the most structurally similar deuterated 

compound and/or the deuterated compound with the 

retention time nearest to the analyte in question was 

selected from the aforementioned internal standards for 

the quantitation of that particular analyte. For example, 

EME-D3 was used as an internal standard for the analysis 

of ECG, AEME, AECG, and EEE. Pentafluoropropionic 

anhydride (PFPA) and 2,2,3,3,3-pentafluoro-1-propanol 

(PFPOH) were purchased from Pierce Chemical Company, Rockford, IL. The 10-mL Certify solid phase extraction (SPE) columns, with a 130-mg sorbent bed, were 

obtained from Varian Corporation, Harbor City, CA.

Case Samples

A muscle specimen and a second paired blood and 

urine specimen were obtained from two aviation accident 

pilot fatalities—these specimens were submitted to the 

FAA’s Civil Aerospace Medical Institute by the National 

Transportation Safety Board for toxicological evaluation. 

Whole blood specimens from 10 motor vehicle operators suspected of being impaired by the abuse of COC 

were kindly provided by the Oklahoma State Bureau of 

Investigation (OSBI), Oklahoma City, OK. No historical 

information suggesting the abuse of COC by smoking 

was available for these 12 cases. The pilot fatality and 

motor vehicle operator specimens are referred herein as 

to “case samples” or “case specimens.”

Calibrators

A solution of 6400 ng/mL AECG and 800 ng/mL 

AEME was prepared in bovine whole blood, preserved 

with 2 mg/mL potassium oxalate and 10 mg/mL sodium 

fluoride. Serial dilutions of this two-analyte mixture resulted in calibrators of 50, 100, 200, 400, 800, 1600, and 

3200 ng/mL AECG and 6.25, 12.5, 25, 50, 100, 200, 

and 400 ng/mL AEME in the whole blood. To validate 

the precision of the calibrators, 80, 160, and 320 ng/mL 

controls of these two analytes were prepared together in 

human whole blood and preserved as above. 

A second solution containing 6400 ng/mL ECG and 

800 ng/mL each of COC, BEG, NBEG, NCOC, EME, 

HBEG, COE, NCOE, and EEE was prepared in bovine 

whole blood. This 10-analyte mixture was diluted in 

the same manner as the previously described AECG 

and AEME solution to obtain their respective calibrators. To validate the precision of the calibrators, 80, 160, 

and 320 ng/mL each of COC, BEG, NBEG, NCOC, 

EME, HBEG, COE, NCOE, and EEE controls were 

prepared together in human whole blood and preserved 

with potassium oxalate and sodium fluoride; these three 

controls also contained 800, 1600, and 3200 ng/mL 

ECG, respectively.

The generated AEME and AECG calibration curves 

were used to quantitate these analytes in the biological 

samples without any influence of their possible GC inlet 

pyrolytic production from COC and/or other possible 

derivatized/underivatized analytes. Similarly, the generated calibration curves of each of the 12 analytes were 

used for determining their concentrations in biological 

samples. Since AEME and AECG were not added to the 

10-analyte mixture, any amount of these analytes found 

in the 10-analyte calibrators or controls resulted from the 

artifactual pyrolytic production in the GC inlet.

Based on the bovine blood calibrator and human blood 

control analytical data, it was concluded that both biological matrices were comparable to each other for the analyses. 

Therefore, bovine blood was used for the preparation of 

calibrators and controls in analyzing blood case samples. 

Bovine blood calibrators and controls were also used during the analysis of urine and muscle samples.

Gas Chromatographic AEME Production

For establishing a relationship between the amounts 

of AEME pyrolytically produced on the instrument 

during analysis, several COC controls in bovine blood 

were analyzed. The COC controls ranged from 25 to 

6400 ng/mL. In addition, controls of other analytes 

were prepared and analyzed to determine the possible 

production of AEME. 

Extraction

Three mL of whole blood or urine and 3.0 g of muscle 

homogenate, prepared in 1.0% aqueous sodium fluoride 

in a 1:2-proportion (w/w), were separately transferred into 

150 mm × 16 mm screw-capped glass culture tubes. To each 

tube, 400 ng each of COC-D3, BEG-D3, NCOC-D3, EMED3

, and COE-D3 was added as an internal standard. With 

the exception of urine, 10.0 mL of acetonitrile (−20°C) was 

added to each tube to precipitate protein from blood and 

homogenate samples. The tubes were capped and secured 
3

Cocaine (COC)

Benzoylecgonine (BEG)

Norbenzoylecgonine (NBEG)

Ecgonine (ECG)

m-Hydroxybenzoylecgonine (HBEG)

Cocaethylene (COE)

Ecgonine Methyl Ester (EME)

Norcocaethylene (NCOE) Ecgonine Ethyl Ester (EEE)

Norcocaine (NCOC)

Anhydroecgonine Methyl Ester (AEME)

Anhydroecgonine (AECG)

 Smoking

(Pyrolysis)

Ethanol

CYP

HOH

CYP HOH?

HOH

HOH

HOH

CYP CYP

HOH?

Ethanol?

O

O

N

CH3

COOCH2CH3

H

H

OH

N

CH3

COOCH3

H

H

O

O

N

CH3

COOCH3

H

H

N

H

COOCH3

H

H

O

O

N

CH3

COOCH2CH3

H

H

OH O

O

N

H

COOCH2CH3

H

H

N

CH3

COOH

N

CH3

COOCH3

N

CH3

O

O

OH

COOH

H

N

CH3

O

O

COOH

H

N

H

O

O

COOH

H

N

CH3

OH

H

COOH

H

Figure 1: Possible biotransformation, pyrolysis, and ethanol products of COC (CYP = cytochrome P-450; 

HOH = hydrolysis).
4 

in a test tube rack. The entire rack was subsequently shaken 

manually for 2 min. The tubes were centrifuged at 770 × g

for 5 min, and the obtained supernatants were transferred 

into 16 mm × 125 mm glass culture tubes. The volumes of 

the supernatants were reduced to approximately 2 mL by 

placing the tubes in an evaporator with a water bath set at 

40°C, while purging with nitrogen. Two mL of phosphate 

buffer (0.1 M, pH 6.00) was added to each supernatant 

and to the urine sample. The solutions from each tube 

were then decanted into the SPE columns, which had 

previously been solvated with 2.0 mL of methanol, then 

with 2.0 mL of the buffer. Once the supernatants and 

urine had passed through the columns, they were washed 

with 6.0 mL of deionized water, then with 3.0 mL of 0.1 

N HCl. The columns were dried with 25 lb of pressure 

for 5 min. Subsequently, 9.0 mL of methanol was passed 

through the columns and the 25-lb pressure was applied 

to the columns for 5 sec. The analytes were eluted from 

the columns by using 4.0 mL of a mixture of ammonium 

hydroxide: isopropyl alcohol: methylene chloride (1:10:

40) into 10-ml screw-capped, glass conical tubes. 

Derivatization

To improve the analytical sensitivity, it was first necessary to convert all basic analytes into their hydrochloride 

salts prior to the PFPA/PFPOH derivatization. This was 

achieved by bubbling through the eluates hydrochloric acid 

vapors from a hydrochloric acid vapor generator (Fig. 2). 

An unused Pasteur pipette was attached to the generator 

for each eluate. The pipette tip was placed into the eluate, 

and the bulb of the generator was gently squeezed once. It 

is crucial to emphasize that only vapors from a generator 

with conditioned hydrochloric acid should be used during 

this step. Hydrochloric acid, which has not been previously 

conditioned, results in a large production of inorganic salts 

suspended in the eluates, leading to little or no recovery 

of the analytes of interest and possible damage to the GC 

column. The acid in the generator is conditioned by filling 

the generator approximately one-third with concentrated 

hydrochloric acid and bubbling air through the solution 

with the bulb 5 to 10 times daily for at least 10 days. 

Purging the acid solution for several hours with nitrogen 

or diluting the concentrated acid solution could not produce the same analytical result as the conditioning process 

produced. The conditioned acid solution is stable for at 

least two months. Therefore, by having two generators, 

one may be used for one month after conditioning while 

the other is being conditioned. Switching monthly to the 

generator with newly conditioned hydrochloric acid while 

the other is being freshly prepared and being conditioned 

with hydrochloric acid will ensure that a generator with the 

conditioned acid is always available for the hydrochloric salt 

formation prior to the PFPA/PFPOH derivatization. 

After bubbling acid vapors through the eluates, they 

were taken to dryness by using a water bath evaporator 

set at 40°C while purging with nitrogen. To the residues, 

50.0 μL each of PFPA and PFPOH was added. The tubes 

were capped and incubated in a heating block at 70°C for 

20 min. The reaction mixtures were cooled, then taken to 

dryness by using an evaporator set at 40°C while purging 

with nitrogen. Subsequently, the residues were reconstituted in 50.0 μL of ethyl acetate and analyzed.

Instrumentation

Using the electron impact mode, a gas chromatograph/

mass spectrometer (GC/MS; Model 6890/5973; Agilent 

Technologies, Wilmington, DE) was employed for the 

analyses of the reconstituted residues by selected ion 

monitoring (SIM; Table I). This system was configured 

with electronic pressure control. A bonded fused silica 

Figure 2: Hydrochloric acid vapor generator. 

This unit was assembled in the CAMI 

laboratory from a gas washing bottle (No. 

657750, Kimble Glass, Inc., Vineland, NJ), a 

vacuum/pressure bulb (No. 14-085, Fisher 

Scientific, Springfield, NJ), a small piece of 

polypropylene tubing, a bent piece of glass 

tubing, a Pasteur pipette polypropylene 

fitting (No. P0635-R, Organomation 

Associates, Inc., Berlin, MA), and disposable 

Pasteur pipettes. The hydrochloric acid 

vapors from this type of generator were used 

for converting various basic analytes into 

their hydrochloride salts prior to the PFP 

derivatization. Details are given in the 

Derivatization subsection of the 

Experimental section. 
5

Analyte Molecular Formula Molecular 

Weight 

Retention 

Index 

Retention 

Time (Min) 

Target 

Ion (m/z) 

Qualifying Ion I (m/z) 

Qualifyin

g Ion II 

(m/z) 

% Change in 

Analytical 

Response 

Due to HCl 

Salt 

%

Recover

y

LOD 

ng/mL 

LOQ 

ng/mL 

AECG-PFP C12H14F NO 5 2 299.24 1329 3.38 270 299 284 -31 0.2 †640 †640AEME C10H15NO2 181.23 1369 3.85 152 181 166 +233 40 13 13 

ECG-D3-2PFP C15H15D3F10NO4 466.26 1418 4.25 303 466 317 – 0.03 – – 

ECG-2PFP C15H15F10NO4 463.28 1418 4.26 300 463 314 – 1.5 640 800 

EME-D3-PFP C13H13D3F NO 5 4 348.25 1445 4.67 185 317 348 – – – – 

EME-PFP C13H16F NO 5 4 345.27 1446 4.68 182 314 345 +278 1.5 2 2 

EEE-PFP C14H18F NO 5 4 359.29 1505 5.32 196 314 359 +254 5 2 2 BEG-D3-PFP C19H17D3F NO 5 4 424.34 2080 8.21 303 424 319 – – – – 

BEG-PFP C19H20F NO 5 4 421.37 2081 8.22 300 421 316 +13 26 2 2 

NBEG-2PFP C21H17F10NO5 553.35 2105 8.45 214 312 431 0 – 25 25 

HBEG-2PFP C22H20F10NO6 584.38 2160 8.62 300 434 583 +57 25 50 50 COC-D3 C17H18D NO 3 4 306.33 2174 8.76 185 85 306 – – – – 

COC C17H21NO4 303.36 2174 8.77 182 82 303 +95 99 2 2 

COE-D3 C18H20D NO 3 4 320.36 2232 9.09 199 275 320 – – – – 

COE C18H23NO4 317.38 2235 9.10 196 212 272 0 77 2 2 

NCOC-D3-PFP C19H15D3F NO 5 5 438.32 2240 9.12 316 438 333 – – – – 

NCOC-PFP C19H18F NO 5 5 435.35 2242 9.12 313 435 330 +57 25 2 2 

NCOE-PFP C20H20F NO 5 5 449.38 2290 9.44 327 344 449 +48 22 2 2 

*

Analytes are listed in the order of their elution from the GC column. Cut-offs for AEME in the presence of COC and its metabolites were greater than the LOD due 

to its inlet pyrolytic production. The percent analytical response change due to HCl salt, percent recovery, LOD, and LOQ are based on extracted analytes from analyte fortified bovine blood samples. Details are given in the Experimental and Results sections. 

†AECG-PFP LOD and LOQ values in the absence of other analytes. AECG-PFP can also be artifactually produced from BEG-PFP, ECG-2PFP, and HBEG-2PFP (Fig. 6). 
6 

GC capillary column (12 m × 0.2 mm i.d., with a 0.33-

μm film thickness of 100% methyl siloxane liquid phase) 

was used for the analyses. The carrier gas was helium and 

was set at 1 mL/min constant flow. The inlet temperature was set at 210°C. The initial oven temperature was 

70°C. The temperature was then programmed to 135°C 

at 30°C/min, then to 140°C at 2°C/min, followed by 

230°C at 30°C/min, then to 234°C at 2°C/min, followed 

by 250°C at 50°C/min. There were no oven temperature 

hold times.

RESULTS

Analytical Sensitivity

As exhibited in Fig. 3, COC, COE, AEME, and the 

pentafluoropropylated (PFP) derivatives of other analytes 

chromatographed symmetrically with good response and 

analytically acceptable separation. Each of the derivatives 

produced significant molecular weight ions and characteristic mass fragmentation patterns, which allowed 

selection of unique target and qualifying ions during the 

SIM analyses (Table I; Fig. 4a-c). Although COE and 

NCOC-PFP overlapped chromatographically (Fig. 3), 

unique ions were selected for the identification of both 

analytes. Analyses of COC, BEG, EME, AEME, COE, 

NCOE, and EEE analyses produced calibration curves 

of good linearity in the range of 6.25 to 800 ng/mL. The 

calibration curves for the analyses of NBEG, NCOC, 

ECG, HBEG, and AECG were linear in the range of 25 

− 600 ng/mL, 6−100 ng/mL, 800−3200 ng/mL, 50−400 

ng/mL, and 800−6400 ng/mL, respectively. The calibration curves of all the analytes demonstrated coefficients 

of linearity ≥ 0.995. Limit of detection (LOD) and limit 

of quantitation (LOQ) values for these analytes are given 

in Table I. Some of the CAMI/OSBI case specimens 

contained analytes in concentrations greater than their 

respective assay’s highest calibrators. Those samples were 

diluted by a factor of 10 or 100 with 1.0% aqueous sodium 

fluoride. Three mL of the various diluted specimens were 

used to bring their concentrations within the linear range 

of their respective calibration curves prior to re-extraction. Where necessary, 1.0 mL aliquots of some of the 

case specimens were used for the analysis.

Pyrolytic Products

During the GC/MS analysis with the GC inlet temperature of 210°C, COC thermally degraded and predictably 

generated AEME (Table II; Fig. 5). The AEME production 

was COC concentration-dependent, as the net amount of 

AEME increased as the COC concentration increased in 

blood controls—13 to 18 ng/mL AEME was produced 

from 25 to 400 ng/mL of COC. However, the percentage 

of the pyrolytic production of AEME decreased as COC 

concentration increased in blood controls. Analytes—such 

as BEG-PFP, NBEG-2PFP, NCOC-PFP, ECG-2PFP, 

EME-PFP, HBEG-2PFP, COE, NCOE-PFP, and EEEPFP—did not increase the production of AEME over 

that observed with COC alone. Based on the chemical 

structures of AECG and AECG-PFP, it is chemically 

inconceivable that these compounds could pyrolytically 

produce AEME (Figs. 1 & 6).

The COC-AEME concentration relationship established under the current experimental conditions was 

used to predict artifactual production of AEME at a given 

blood COC concentration (Table II; Fig. 5). The predicted 

AEME value was subtracted from the AEME value in a 

specimen determined from the AEME calibration curve 

constructed from the AEME and AECG mixture. Therefore, the derived subtracted value would represent a true 

concentration of AEME in a biological sample.

GC inlet temperatures greater than 210°C produced an 

excessive amount of AEME, while the inlet temperatures 

of less than 210°C caused a decrease in the responses 

of all the analytes, negatively affecting the LODs and 

LOQs of the assay. Therefore, an injector temperature 

of 210°C was found to be most suitable for the analysis. 

Lower GC inlet temperatures sacrificed the sensitivity 

of the method for detecting analytes with no significant 

reduction in production of AEME. Higher GC inlet 

temperatures produced even more AEME but did not 

significantly increase sensitivity of the method enough 

to justify the higher inlet temperatures. The heated inlet 

artifactually produced large amounts of AECG-PFP from 

the pyrolysis of PFP derivatized BEG, ECG, and HBEG 

(Fig. 5). Authentic AECG-PFP can only be produced in 

vitro from the PFP derivatization of AECG since AECG 

is a possible hydrolytic product of AEME (Fig. 1).

Case Sample Analyses

As is given in Table III, COC was detected in seven 

samples and NCOC was found in one sample. The COC/

COE hydrolysis product, BEG, was present in considerable amounts in all the samples, including the muscle 

sample. Excluding the muscle and the blood samples of 

the CAMI cases, all samples were found to contain ECG 

in large amounts. Out of the 13 samples, seven samples 

were found to contain AEME ranging from 6−35 ng/mL. 

COE and NCOE were not detected in any of the case 

samples, but EEE was detected in small concentrations 

only in urine and muscle samples. Since AECG was artifactually produced in large amounts, concentrations of 

this analyte were not included in the table.
7

Figure 3: A total ion chromatogram of a mixture of COC, COE, AEME, and 

their PFP-derivatized metabolite and related products: 

1 = AECG-PFP; 2 = AEME; 3 = ECG-2PFP; 4 = EME-PFP; 5 = EEE-PFP; 

6 = BEG-PFP; 7 = NBEG-2PFP; 8 = HBEG-2PFP; 9 = COC, 10 = COE; 

and NCOC-PFP, 11 = NCOE-PFP. This chromatogram depicts resolution, 

sensitivity, and peak shape of 50 ng of each of these unextracted analytes on 

the GC column. Other unlabeled peaks are artifacts from some or all of the 12 

analytes. Although COE and NCOC-PFP overlapped chromatographically, their 

distinctive mass spectra allowed clear-cut identification of both analytes.
8 

 

/

300

82

42

463

119

150 444 64

Ecgonine-2PFP

C15H15F10NO4

M.W. 463.28

N

CH3

COOC3H2F5

H

OOC3H2F5

H

A

 

/

152

42

181

82

122

57 166 94 138

68 106

Anhydroecgonine Methyl Ester

C10H15NO2 M.W. 181.23

N

CH3

COOCH3

B

 

270

42

299

82

122

57 69 150 94 138 284

106 166

Anhydroecgonine-PFP

C12H14F5NO2

M.W. 299.24

N

CH3

COOC3H2F5

C

 

/

300

82

421

42 105

272

122

Benzoylecgonine-PFP

C19H20F5NO4 M.W. 421.37

N

CH3

COOC3H2F5

H

H O

O

D

Figure 4a: Mass spectra of (A) ECG-PFP, (B) AEME, (C) AECG-PFP, and (D) BEG-PFP.
9

 

/

82

196

94 42

317

272 212 55 122 67 108 166

Cocaethylene

C18H23NO4

M.W. 317.38

N

CH3

COOCH2CH3

H

H O

O

E

 

/

82

182

42 94

303

198 55 122 272 68 108

Cocaine

C17H21NO4

M.W. 303.36

N

CH3

COOCH3

H

H O

O

F

 

/

82

182

42

345

96

119 314

69

150

Ecgonine Methyl Ester-PFP

C13H16F5NO4

M.W. 345.27

N

CH3

COOCH3

H

OOC3H2F5

H

G

 

/

105

77 313

214

194

51

166 282 435 124 330

Norcocaine-PFP

C19H18F5NO5

M.W. 435.35

N

C3F5O

COOCH3

H

H O

O

H

Figure 4b: Mass spectra of (E) COE, (F) COK, (G) EME-PFP, and (H) NCOK-PFP. Figure 4b: Mass spectra of (E) COE, (F) COC, (G) EME-PFP, and (H) NCOC-PFP.
10 

82

196

42

359

314

96

119

67

150

168 256 286

Ecgonine Ethyl Ester-PFP

C14H18NO4F5

M.W. 359.30

N

CH3

COOCH2CH3

H

H

OOC3H2F5

I

300

82

583

267

434

m-Hydroxybenzoylecgonine-2PFP

C22H20F10NO6 M.W. 584.38

N

CH3

O

O

O

COOC3H2F5

C2H5

O

J

105

214

77

312 431

284

51

402 553

Norbenzoylecgonine-2PFP

C21H18F10NO5

M.W. 554.36

N

O

O

COOC3H2F5

C3F5O K

105

327

77

214

298

404 449

Norcocaethylene-PFP

C20H20NO5F5

M.W. 449.38

N

C

COOCH2CH3

H O

O

H

C3F5O

L

Figure 4c: Mass spectra of (I) EEE-PFP, (J) HBEG-2PFP, (K) NBEG-2PFP, and (L) NCOE-PFP. 
11

DISCUSSION

This study describes a sensitive and specific method for 

simultaneously analyzing COC, COE, their metabolites 

and pyrolysis products, and other related compounds. 

In this method, PFPA and PFPOH were used for the 

derivatization of the analytes. PFPA reacts with the 

hydroxyl and primary/secondary amino groups to form 

pentafluoropropionyl ester and amide derivatives, respectively, while PFPOH reacts with the carboxyl groups to 

produce pentafluoropropionyl ester derivatives. Therefore, the combined use of PFPA and PFPOH becomes an 

efficient reaction mixture for simultaneously derivatizing 

hydroxyl, amino, and carboxyl functional groups. Such 

derivatization was effective in selecting unique analyte ions 

for the SIM analysis of various analytes, particularly of 

those having more than one of these functional groups.

Findings of this study suggest that one must be 

cognizant of the potential misinterpretations of the 

presence of AEME when high concentrations of COC 

are encountered in biological samples. However, the 

relationship established between the AEME production 

and the COC concentration could be effectively utilized 

for calculating the amount of true AEME present in a 

biological sample. Additionally, AECG was not found 

to be a viable analyte for establishing whether COC had 

been smoked, because the inlet pyrolytic production of 

AECG-PFP from the PFP derivatives of BEG, ECG, 

and HBEG would completely mask the AECG originally 

present in a biological specimen. The artifactual GC inlet 

production of AECG-PFP is most likely mediated via a 

pyrolytic cleavage of one of the ester groups of the PFP 

derivatives of BEG, ECG, and HBEG, leading to the 

formation of the C=C containing AECG-PFP.

Neither the specimens from the pilot fatalities nor 

the blood specimens from the OSBI were found to have 

concentrations of AEME greater than those pyrolytically 

produced from the COC-spiked bovine blood samples 

(Table II). Although the AEME concentration in urine 

was higher than that in any other CAMI/OSBI case specimens, the urine AEME concentration of 35 ng/mL in 

the presence of 846 ng/mL of COC was consistent with 

the amount of AEME produced from the bovine blood 

sample fortified with 800 ng/mL COC (Table II; Fig. 5). 

These observations suggested that the individuals probably 

consumed COC by a route other than smoking. In addition, the absence of COE, NCOE, and EEE in the blood 

case specimens indicated that ethanol was not consumed 

during the ingestion of COC, since these compounds are 

synthesized in vivo in the presence of COC and ethanol 

(10). However, the presence of EEE—a possible hydrolytic 

product of COE and a biosynthetic product of EME—in 

the absence of COE and NCOE in the urine and muscle 

samples may not necessarily indicate that ethanol was 

consumed with COC in these two cases, but ethanol might 

have been consumed much later after the metabolism of 

COC and/or produced postmortem. This conclusion is 

based on the facts that EME was present in both samples, 

particularly in the urine wherein the EME concentration 

was very high (5203 ng/mL), and ethanol was found in 

the muscle and in the companion blood sample of the 

case with urine. Therefore, the production of EEE could 

have been attributed to the transesterification reaction 

of EME and ethanol. 

COC, COE, and PFP derivatives of EME and BEG 

have been analyzed previously in meconium, whole blood, 

and plasma (15), but the applicability of this method for 

the analysis of other COC and COE metabolites was not 

Table II: GC Inlet Pyrolytic Production of AEME at 210°C From Various Concentrations of COC in 

Bovine Blood*

Cocaine (ng/mL) Pyrolytically Produced 

AEME (ng/mL) 

Pyrolytically Produced AEME 

(%, COC Concentration) 

25 13 52.0 

50 13 26.0 

100 13 13.0 

200 15 7.5 

400 18 4.5 

800 30 3.8 

1600 40 2.5 

3200 75 2.3 

6400 132 2.1 

*

Details are given in the Experimental and Results sections. 
12 

0

10

20

30

40

50

60

0 1000 2000 3000 4000 5000 6000 7000

Cocaine in Blood (ng/mL)

AEME (%, COC Concentration)

0

20

40

60

80

100

120

140

AEME (ng/mL)

Figure 5: A graphic representation of the relationship between the amount of AEME produced pyrolytically from various concentrations of COC in bovine blood at the 210°C GC inlet temperatue. 

Table III: Analysis of CAMI and OSBI Case Specimens* 

Specimens Analytes (ng/mL)

Source Type COC BEG NBEG NCOC ECG EME HBEG AEME EEE

OSBI 1 Blood 24 1205 <1 †

ND 5497 18 25 11 ND

OSBI 2 Blood 25 1318 23 ND 15826 10 86 ND ND

OSBI 3 Blood ND 69 ND ND 1119 ND 5 6 ND

OSBI 4 Blood ND 1130 ND ND 8497 <1 5 ND ND

OSBI 5 Blood 10 1949 49 ND 21231 43 46 ND ND

OSBI 6 Blood 2 1270 ND ND 10148 2 23 13 ND

OSBI 7 Blood 2 696 ND ND 3344 9 ND 12 ND

OSBI 8 Blood ND 349 ND ND 4311 ND ND ND ND

OSBI 9 Blood ND 505 ND ND 3727 ND ND 10 ND

OSBI 10 Blood ND 218 ND ND 1215 ND ND ND ND

CAMI-1A Blood 62 159 ND ND ND 78 ND 12 ND

CAMI-1B Urine 846 7760 511 20 15126 5203 19 35 44

CAMI-2 Muscle ND 48 ND ND ND 8 ND ND 7

*All samples were negative for COE and NCOE. AEME concentrations are uncorrected with respect to the 

COC concentrations. AECG values are not included in this table because large amounts of AECG-PFP can 

also be artifactually produced from BEG-PFP, ECG-2PFP, and HBEG-2PFP (Fig. 6). 

†

ND = Not detected. 
13

Figure 6: PFPOH derivatization of AECG into AECG-PFP and predicted artifactual 

production of AECG-PFP from the GC inlet pyrolysis of three PFP metabolite 

derivatives. 

Pyrolysis

Benzoylecgonine-PFP (BEG-PFP)

Anhydroecgonine-PFP (AECG-PFP)

Pyrolysis

m-Hydroxybenzoylecgonine-2PFP (HBEG-2PFP)

Pyrolysis

Ecgonine-2PFP (ECG-2PFP)

PFPOH

N

CH3

COOC3H2F5

N

CH3

COOC3H2F5

H

H

O

O

Anhydroecgonine (ECG)

N

CH3

COOH

N

CH3

COOC3H2F5

H

H

O

O

OOC3H2F5

N

CH3

COOC3H2F5

H

H

OOC3H2F5
14 

demonstrated in the study. In another method, 11 COC 

metabolites in urine were analyzed by performing a single 

extraction and by converting them into their PFPA and 

hexafluoropropyl derivatives (13). However, the effectiveness of this method in other types of biological samples 

was not demonstrated. An analytical procedure for the 

pyrolysis product AEME was reported using N,O-bis-

(trimethylsilyl)trifluoroacetamide as a derivatizing reagent 

(7), but derivatives of this reagent are considered less 

stable than the PFP derivatives. Additionally, the methods 

reported in the literature lack sensitivity for analyzing 

the 11 analytes (not including AECG) in whole blood 

and other tissue types, as reported in the present study. 

In several methods (7, 8, 12, 14), the GC inlet temperature was ≥ 250°C, and concerns have been raised for 

the artifactual pyrolytic production of AEME during gas 

chromatography/mass spectrometry (4, 8, 14). Therefore, 

the AEME concentration reported in the literature may 

not represent the true concentrations of the analyte in 

the samples (7, 12). Although at 140°C no pyrolytic 

production of AEME was observed from COC (8), the 

sensitivity of the method at this temperature might not 

be analytically acceptable for detecting other analytes. The 

210°C-inlet temperature adopted in the present study 

was suitable for minimizing the production of AEME 

without compromising the sensitivity of the method for 

other analytes.

Overall, the present study describes a selective and 

sensitive method for the simultaneous analysis of COC, 

COE, and their metabolite and pyrolysis products, involving solid-phase column chromatographic isolation, 

hydrochloride salt formation, PFP derivatization, and 

GC/MS analyzation. Converting the analytes into their 

hydrochloride salts by bubbling hydrochloric acid vapors 

through the column eluates, prior to the derivatization, 

significantly increased the sensitivity for detecting COC, 

BEG, NCOC, EME, HBEG, AEME, NCOE, and EEE. 

Additionally, the method could be used to analyze NBEG, 

AECG, COE, and, in particular, ECG in biological 

samples. ECG found in large amounts in case samples 

further suggests that this metabolite is a quantitatively 

prominent analyte that can be used as a marker for COC 

abuse. The developed method allows the analysis of specimen types—such as whole blood and muscle—wherein 

the concentration of these analytes are relatively less in 

comparison to such analytes present in urine.

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