Poppy Seed Consumption 

or Opiate Use: The 

Determination of 

Thebaine and Opiates 

of Abuse in Postmortem 

Fluids and Tissues

Robert D. Johnson

Russell J. Lewis

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

Rachael A. Hattrup

University of Central Oklahoma

Edmond, OK 73034

June 2005

Final Report

DOT/FAA/AM-05/11

Office of Aerospace Medicine

Washington, DC 20591
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.
Technical Report Documentation Page 

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

DOT/FAA/AM-05/11 

4. Title and Subtitle 5. Report Date 

Poppy Seed Consumption or Opiate Use: The Determination of Thebaine June 2005 

and Opiates of Abuse in Postmortem Fluids and Tissues 6. Performing Organization Code 

 

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

Johnson RD,1 Lewis RJ,1 Hattrup RA2 

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

 11. Contract or Grant No. 

1

Civil Aerospace Medical Institute 

P.O. Box 25082 

Oklahoma City, OK 73125 

2

University of Central Oklahoma 

Edmond, OK 73034 

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 

Work was accomplished under approved task AM-B-04-TOX-204. 

16. Abstract 

Opiates are some of the most widely prescribed drugs in America. Some opiate compounds are highly 

addictive and are often abused. Opiate abuse transcends all social, racial, and economic boundaries. 

Demonstrating the presence or absence of opiate compounds in postmortem fluids and/or tissues derived 

from fatal civil aviation accidents can have serious legal consequences and may help determine the cause of 

impairment and/or death. However, the consumption of poppy seed products can result in a positive opiate 

drug test. Therefore, the interpretation of positive opiate results must be viewed with caution. We have 

developed a simple method for the simultaneous determination of 8 opiate compounds from one extraction. 

These compounds are hydrocodone, dihydrocodeine, codeine, oxycodone, hydromorphone, 6-

monoacetylmorphine, morphine, and thebaine. The inclusion of thebaine is notable as it is an indicator of 

poppy seed consumption and may help explain morphine/codiene positives in cases where no opiate use was 

indicated. This method incorporates a Zymark RapidTraceTM automated solid-phase extraction system, gas 

chromatography/mass spectrometry, and trimethyl silane (TMS) and oxime-TMS derivitives. The limits of 

detection ranged from 0.78 – 12.5 ng/mL. The linear dynamic range for most analytes was 6.25 – 1600 

ng/mL. The extraction efficiencies ranged from 70 – 103%. We applied this method to 8 separate aviation 

fatalities where opiate compounds had previously been detected. The specimens analyzed for the 

determination of these 8 opiate compounds were blood, urine, liver, kidney, and skeletal muscle. This 

method has proven to be simple, robust, and accurate for the simultaneous determination of 8 opiate 

compounds in postmortem fluids and tissues. 

 

17. Key Words 18. Distribution Statement 

 

 

Forensic Toxicology, Opiates, Thebaine, GC/MS 

Aircraft Accident Investigation

Document is available to the public through the 

Defense Technical Information Center, Ft. Belvior, 

VA 22060; and the National Technical Information 

Service, Springfield, VA 22161 

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

Unclassified Unclassified 14 

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



Poppy Seed Consumption or Opiate Use: 

The Determination of Thebaine and Opiates

of Abuse in Postmortem Fluids and Tissues

Introduction

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

Aerospace Medical Institute (CAMI) is responsible under Department of Transportation Orders 8020.11B 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 opiate-related compounds are 

detected in postmortem specimens from general aviation 

accident victims. 

Opiates constitute a highly addictive group of 

commonly prescribed drugs.1, 2 The most commonly 

prescribed opiate-type compounds are hydrocodone, 

dihydrocodeine, codeine, hydromorphone, oxycodone, 

and morphine.3 These compounds may have severe side 

effects, including, but not limited to, drowsiness, dizziness, hypotension, unconsciousness, or mental clouding, 

which can lead to significant impairment.1 Due to these 

severe side effects, opiates constitute a potential causative 

factor in civil aviation accidents. Moreover, according 

to the Drug Enforcement Agency, these compounds are 

some of the most highly abused prescription drugs in 

America. Therefore, a procedure that allows for the rapid 

and accurate determination of opiate compounds is a 

necessity for the field of forensic toxicology.

Thebaine is a naturally occurring opiate that is introduced into the body, along with morphine and codeine, 

following the consumption of poppy seeds.4-8 Scientific 

literature has clearly demonstrated the distinct possibility of a morphine and/or codeine positive due to poppy 

seed consumption.4-8 The potential legal consequences 

of an opiate positive necessitates that laboratories, both 

drug testing and forensic, differentiate between an opiate 

positive due to morphine or codeine use and an opiate positive due to poppy seed consumption. In a case 

where morphine and/or codeine have been detected, a 

subsequent thebaine positive would indicate poppy seed 

consumption and could possibly rule out opiate use.

Heroin (diacetylmorphine) abuse is common in the 

United States. Heroin is quickly metabolized to 6-monoacetylmorphine (6-MAM), which is then metabolized 

to morphine. 6-MAM has not been detected following 

the use of morphine or other opiate compounds or the 

ingestion of poppy seeds and is only present following 

heroin use.5 Therefore, 6-MAM is considered a heroinspecific catabolite and is legally defensible evidence of 

heroin use.

Identification and quantitation of opiates 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. This 

report describes a rapid, automated procedure for the 

single-step extraction and simultaneous determination 

of hydrocodone, dihydrocodeine, codeine, oxycodone, 

hydromorphone, 6-MAM, morphine, and thebaine 

in postmortem fluids and tissues using a Zymark

RapidTraceTM automated solid-phase extraction (SPE) 

system and gas chromatography with mass spectrometry (GC/MS). Chemical structure for these 8 opiates 

can be seen in Figure 1. While published methods exist 

for the identification and quantitation of various opiate 

compounds, including thebaine,2, 5-7, 9-15none offer the 

sensitivity of this method, combined with the simultaneous extraction and analysis of the 8 opiate compounds 

in postmortem fluids and tissues.

Materials and Methods

Chemicals and Reagents

All aqueous solutions were prepared using double 

deionized water (DDW), which was obtained using a 

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

chemicals described below were purchased in the highest 

possible purity and used without any further purification. 

Hydrocodone, dihydrocodeine, codeine, morphine, hydromorphone, oxycodone, and 6-MAM were purchased 

from Cerilliant (Cerilliant Corp., Round Rock, TX) as 

methanolic standards at a concentration of 1.00 mg/mL 

in sealed glass ampules. Thebaine was purchased from 

RBI (Research Biomedical International Inc., Natick, 
MA). Hydrocodone-d3

, dihydrocodeine-d6, codeined3

, morphine-d6, hydromorphone-d3, oxycodone-d3, 

and 6-monoacetylmorphine-d6 were purchased from 

Cerilliant as methanolic standards at a concentration of 

0.100 mg/mL in sealed glass ampules. The derivatization 

reagent, BSTFA with 1% TMCS, was obtained from 

Pierce (Pierce Inc., Rockford, IL). Sodium acetate, hydroxylamine, potassium phosphate, glacial acetic acid, 

and β-glucuronidase were purchased from Sigma (Sigma 

Chemical CO., St. Louis, MO). Methanol, acetonitrile, 

and ammonium hydroxide were purchased from Fisher 

(Fisher Scientific, Pittsburgh, PA). Ethyl acetate was 

purchased from Varian (Varian Inc., Palo Alto, CA).

Gas Chromatographic/Mass Spectroscopic Conditions

All analyses were performed using a bench-top 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 tuned on a daily basis using perfluorotributylamine. The electron multiplier voltage was set at 106 

eV above the tune 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 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 

the purge time of 0.5 min. The oven temperature profile 

was established as follows: 160 – 195°C at 35°C/min, 

195 – 230°C at 5°C/min, 230 – 290°C at 40°C/min and 

a final hold time of 2.5 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 for each analyte were then selected based 

on both abundance and mass-to-charge ratio (m/z). To 

increase reproducibility and reduce 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 30 msec 

for each recorded ion.

Sample Selection and Storage

A search of the CAMI database identified 8 fatalities 

from separate civil aviation accidents from the previous 3 years that were reported positive for opiates 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 1.00% (w/v) sodium fluoride/potassium 

oxalate until analysis. All other specimens were stored 

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

concentrations determined in this study agreed well with 

those previously determined by our laboratory via another 

analytical method. All opiate concentrations found were 

within 10% of the value originally determined, verifying that no deterioration in opiate concentration had 

occurred.

Calibrator and Control Preparation

Calibration curves for all opiates except thebaine 

were prepared by serial dilution utilizing bovine whole 

blood as the diluent. Calibrators were prepared from one 

set of original stock standard solutions, while controls 

were prepared in a similar manner as calibrators, using 

bovine whole blood as the diluent, but from a second 

set of original stock solutions. Thebaine calibrators and 

controls were prepared using certified-negative human 

urine obtained from UTAK Laboratories Inc. (Valencia, 

CA) as the diluent. Calibration curves were prepared 

at concentrations ranging from 0.78 – 3200 ng/mL. A 

minimum of 7 calibrators were used to construct each 

calibration curve. Controls were prepared at concentrations of 100 and 400 ng/mL in pools large enough to 

provide replicate samples for the entire study. These 

concentrations were chosen to reflect typical values seen 

in our laboratory. The internal standard solution, containing d3

-hydrocodone, d6-dihydrocodeine, d3-codeine, 

d6-morphine, d3-hydromorphone, d3-oxycodone, and d6-

6-monoacetylmorphine, was prepared at a concentration of 400 ng/mL in DDW by dilution from the stock 

standard of each compound.

In human-derived specimens, codeine and morphine 

are predominately found as glucuronide derivatives. 

Therefore, we initially hydrolyzed each postmortem urine 

specimen using the enzyme β-glucuronidase. A solution 

of β-glucuronidase was prepared by adding 2.5 mL of pH 

5.00, 0.10 mM sodium acetate buffer to 250,000 units 

of the solid enzyme, followed by mixing. This yielded a 

final concentration of 100,000 units/mL. This solution 

was stored in a freezer at -20°C and discarded after storage 

of a maximum of 30 days. However, it typically was used 

entirely within 7 days following preparation.

Quantitation was achieved via an internal standard calibration procedure. Response factors for each compound 

were determined for every sample analyzed. The response 

factor was calculated by dividing the area of the analyte 

peak by the area of the internal standard peak. Calibration curves were derived by plotting a linear regression 

of the analyte/internal standard response factor versus 
the analyte concentration for each respective calibrator. 

These calibration curves were then used to determine the 

concentrations of each opiate compound in the prepared 

controls and biological specimens.

Sample Preparation and Extraction Procedure

Postmortem fluid and tissue specimens, calibrators, 

and controls were 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 30 mm 

in diameter and set to rotate at 22,000 rpm. Tissues were 

homogenized following a 1:2 dilution with 1.00% NaF in 

DDW. Three mL aliquots of specimen fluids, calibrators 

and controls, and 3.00 g aliquots of tissue homogenate 

were transferred to individual 16 x 150 mm screw-top 

tubes. To each specimen, calibrator, and control, 1.00 

mL of the internal standard mixture (400 ng) was added. 

Samples were vortexed briefly and allowed to stand at 

room temperature for 10 min. One hundred μL of stock

β-Glucuronidase solution (10,000 units), followed by 

2.00 mL of 0.10 M pH 5.00 sodium acetate buffer, was 

added to each urine sample. The urine samples were 

then vortexed briefly and incubated at 70°C for 3 h to 

facilitate hydrolysis of all glucuronide conjugates. In our 

initial investigations, complete hydrolysis of both codeine 

and morphine-glucuronide conjugates was achieved after incubation with β-Glucuronidase for 2.5 h at 70°C. 

However, an incubation time of 3 h was chosen to ensure 

that specimens with elevated opiate concentrations were 

also completely hydrolyzed. Following hydrolysis, samples 

were allowed to cool to room temperature. Nine mL of 

acetonitrile was added to all samples except urine. 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 820 x g for 5 min. The 

supernatant was then transferred to clean 16 x 100 mm 

culture tubes. To each sample, including urine, was added 

2.00 mL of pH 6.00 phosphate buffer and 0.50 mL of 

10% hydroxylamine, which was prepared fresh daily. The 

samples were covered with parafilm and incubated for 1

h at 60°C in a water bath.

Samples were extracted using a Zymark RapidTraceTM 

automated solid phase extraction (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 of methanol, 

followed by 2.0 mL of 0.10 M phosphate buffer pH 6.00, 

both at a flow rate of 8 mL/min. Following conditioning, 6.0 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 2.0 mL of 1.0 M acetic acid at a flow rate of 

8 mL/min, dried for 30 sec with nitrogen at a pressure of 

30 p.s.i., rinsed with 6.0 mL of methanol at a flow rate of 

8 mL/min and dried a final time for 30 sec at a pressure 

of 30 p.s.i.. The analytes were then eluted with 3.0 mL 

of 2% ammonium hydroxide in ethyl acetate, which was 

prepared fresh daily, into 15 mL round-bottom, screw 

top tubes. To avoid carryover, the RapidTraceTM column 

plunger was washed with 3.0 mL of elution solvent, and 

the RapidTraceTM cannula was washed by sequentially 

passing 6.0 mL of methanol and 6.0 mL of 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 

dry, 25.0 μL ethyl acetate and 25.0 μL BSTFA with 1% 

TMCS were added to each. The tubes were then capped 

tightly, vortexed briefly, and incubated in a heating block 

set to 90°C for 20 min. Samples were removed from the 

heating block and allowed to cool to room temperature. 

The samples were 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 linear dynamic range of 

the calibration curve.

Extraction Efficiency/Recovery

The method used for the determination of analyte 

recovery has previously been reported by Johnson et 

al.16 Briefly described, two groups of controls, X and 

Y, prepared using certified-negative whole blood (urine 

used for thebaine), 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.

Results and Discussion

Method Validation

The procedure described herein, which utilizes a Zymark RapidTraceTM automated SPE system and GC/MS 

for the detection of opiates and TMS and oxime-TMS 

opiate derivatives, provides a rapid, reproducible, and 

sensitive method for the determination of hydrocodone, 

dihydrocodeine, codeine, oxycodone, hydromorphone, 

6-monoacetylmorphine, morphine, and thebaine. Most 

analyte peaks were completely resolved, with the excep-
tion of thebaine and codeine, which had similar retention 

times. However, each provided ions with unique m/z, so 

no interference was observed. Deuterated hydrocodone, 

dihydrocodeine, codeine, oxycodone, hydromorphone, 

6-monoacetylmorphine, and morphine were used as 

internal standards for this study. Thebaine had no deuterated analog available, so codeine-d3

 was employed as 

the internal standard for this analyte. A representative 

chromatogram demonstrating the separation of the 8 

analytes is shown in Figure 2.

Numerous derivatizing agents can be employed for 

the GC/MS analysis of opiates. Propionic anhydride, one 

such derivatizing reagent, can be used to form propylopiate derivatives; however, propionic anhydride is often 

contaminated with trace amounts of acetaldehyde.17 Any 

acetaldehyde present can react with morphine to form 

6-MAM, thus falsely suggesting heroin use. Therefore, 

a trimethyl silane (TMS) derivative is most commonly 

used for the quantitation of opiate compounds.12, 18-21

However, when forming TMS derivatives of ketone-containing opiates, such as hydrocodone, hydromorphone, 

and oxycodone, numerous derivatives for each compound 

can be formed due to keto-enol tautamerization.1, 22 Tautamerization can be avoided by an initial pre-derivatization 

reaction with hydroxylamine. Hydroxylamine reacts with 

the ketone moiety of these opiates to form an oxime, 

which then forms a TMS derivative.22, 23

After evaluating numerous derivatizing agents, we 

chose to employ TMS and oxime-TMS derivatives 

due to the efficiency of the derivatizing reaction. We 

initially investigated the use of a 1% hydroxylamine 

solution for oxime formation, but this concentration 

did not produce consistent results across a broad opiate 

concentration range. It appeared that the 1% solution 

was not concentrated enough to ensure complete reaction 

between hydroxylamine and all available ketone moieties. 

Following further investigation, we found that a 10% 

hydroxylamine solution produced complete oxime formation for each of the ketone-containing compounds. 

TMS derivatization following oxime formation makes 

it possible to simultaneously quantitate both keto and 

non-keto opiate compounds in one extraction with reproducible results.

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. Analytes not meeting these criteria 

were reported as either negative or inconclusive.

The linear dynamic range (LDR), limit of detection 

(LOD), limit of quantitation (LOQ), and extraction 

efficiency (recovery) for each opiate compound, except 

thebaine, were determined using whole blood as the matrix. Analytical parameters for thebaine were determined 

in a urine matrix, since we would expect to see thebaine 

in urine specimens only. The LDR for each analyte is 

presented in Table 2. In general, LDRs were 6.25 – 1600 

ng/mL. Correlation coefficients for calibration curves 

used to ascertain that LDRs were all greater than 0.995 

when a weighting factor of 1/X was employed. Additionally, Table 2 shows the LOD and LOQ determined for 

each analyte. The LOD was defined as the lowest analyte 

concentration detectable that met 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 opiate related 

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

for these opiate-related compounds ranged from 0.78 

– 25 ng/mL. The LOD and LOQ values determined in 

this experiment were superior to what had been previously reported.22

Recovery of these opiate compounds at both 100 

ng/mL and 400 ng/mL ranged from 70 – 103% (Table 

2). Recovery values above 70% are exceptional when 

considering the simplicity of the extraction and the use 

of whole blood (urine for thebaine) as the matrix for 

these experiments. 

Carryover from one sample to the next did not occur 

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

the Zymark was investigated by extracting an opiatenegative 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. The injection of an ethyl acetate blank following the 3200 ng/mL calibrator showed no carryover 

contamination. Subsequently, ethyl acetate blanks were 

utilized between each postmortem specimen throughout 

the sample sequence to verify that no sample-to-sample 

contamination occurred.

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

accuracy and precision were examined for this extraction 

procedure. Accuracy was measured as the relative error 

between the experimentally determined and prepared 

concentrations of a whole blood control. Precision was 

measured as the relative standard deviation (RSD) of 

the experimentally determined concentrations of a 

group of whole blood controls. Pools of controls were 
created at 100 ng/mL and 400 ng/mL in volumes large 

enough to be used for the entire accuracy and precision 

investigation. 6-MAM was analyzed at concentrations 

of 10 ng/mL and 40 ng/mL, as opposed to 100 and 400 

ng/mL, due to the expected low concentrations of this 

analyte in vivo.

24 These controls were stored at 4°C for 

the duration of this study. For the intra-day accuracy and 

precision experiment, a calibration curve was extracted 

along with 5 replicates of each control concentration. 

As shown in Table 3, all analytes at both concentrations 

yielded relative errors within 15% of the target concentration. Furthermore, all analytes had RSDs within 5%. 

These results demonstrate the exceptional accuracy and 

precision of this method.

Inter-day accuracy and precision were evaluated by 

extracting 5 replicates of each of 2 control concentrations 

on days 4 and 7. The quantitative values determined on 

each of these days were derived from the calibration curves 

originally prepared on day 1. The results obtained after 

storage of each control lot at 4°C for 4 and 7 days can be 

seen in Table 3. For a majority of these opiate analytes, 

the concentrations determined on days 4 and 7 showed 

no significant difference from those obtained on day 1. 

This agrees well with other published findings.22 The 

RSD for each of the opiate compounds was less than 

10% on days 4 and 7. Even though no apparent decrease 

in concentration was observed over the 7-day storage 

period at 4°C, as a good laboratory practice and in an 

effort to maintain a high degree of accuracy, we would 

recommend (1) preparing new calibration curves at the 

beginning of each new opiate analysis and (2) prompt 

toxicological analysis once a postmortem specimen has 

been thawed.

Method Application: Postmortem Specimen Analysis

In the event of a fatal civil aviation accident, specimens 

from accident victim(s) are routinely sent to the FAA’s 

Forensic Toxicology Research Laboratory for toxicological 

analysis. Postmortem fluid and tissue samples obtained 

from 8 separate civil aviation fatalities that occurred over 

the past 3 years and had previously been screened positive 

for opiates by GC/MS were re-examined using this novel 

method. The fluid and tissue samples selected for analysis 

were blood, urine, liver, kidney, and skeletal muscle. The 

8 aviation fatalities chosen for this investigation had a 

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

analysis. The results obtained from this analysis can be 

seen in Table 4. 

The psychoactive concentrations of these opiate compounds, as well as their pharmacokinetics, are pharmacodynamics, are beyond the scope of this paper. These 

topics are, however, covered extensively elsewhere.25 As 

previously stated, deuterated analogs of hydrocodone, 

dihydrocodeine, codeine, morphine, hydromorphone, 

oxycodone, and 6-MAM were used as internal standards 

in this study. A deuterated analog of thebaine was not 

available. Therefore, to obtain reliable quantitative results, 

urine was used as the calibration matrix for thebaine, 

since the detection of thebaine was expected in urine 

specimens only. The interpretation of quantitative data 

resulting from the analysis of specimen types other than 

that used to create the calibration curve should be closely 

scrutinized due to variations in extraction efficiencies 

when deuterated analogs are not available.

Morphine was found in 3 of the 8 cases examined 

and was the only opiate detected in 2 of these cases. 

Morphine concentrations in the various fluids and tissues ranged from 0 – 260 ng/mL, 122 – 526, 0 – 870 

ng/g, 121 – 1786 ng/g, and 21 – 120 ng/g in blood, 

urine, liver, kidney, and muscle, respectively. The general 

trend observed for the highest to lowest concentration of 

morphine between specimen types analyzed was urine > 

kidney > liver > blood > muscle.

Codeine was identified in 2 of the 8 cases investigated 

and had a concentration of 13 ng/mL, 78 ng/g, 68 ng/g, 

and 19 ng/g in blood, liver, kidney, and muscle, respectively; urine levels ranged from 47 – 154 ng/mL. One of 

the codeine-positive cases tested positive for morphine 

and thebaine, while the other case tested positive for 

hydrocodone and dihydrocodeine.

Thebaine was identified in 1 of the cases examined at 

a concentration of 22 ng/mL in urine. For comparative 

purposes, morphine and codeine were found in the urine 

of this case at 122 ng/mL and 47 ng/mL, respectively. We 

did not identify any of these analytes in the corresponding blood or liver specimens. No additional specimens 

were analyzed in this case. An in-depth review of the 

case history revealed no medically legitimate opiate use. 

Furthermore, finding a urinary morphine concentration greater than that of codeine and both opiates at 

low concentration, along with the presence of thebaine, 

suggests that the morphine and codeine present in this 

case is a result of poppy seed consumption. It must be 

stressed, however, that it is possible following poppy seed 

ingestion to find morphine and codeine in urine without 

detecting thebaine. Therefore, the absence of thebaine 

cannot preclude poppy seed consumption as the source 

of morphine and codeine present in a case.

Hydrocodone and dihydrocodeine were found in 4 of 

the 8 cases examined. Blood concentrations for hydrocodone and dihydrocodeine ranged from 18 – 102 ng/mL 

and 13 – 270 ng/mL, respectively. Urine concentrations 

for hydrocodone and dihydrocodeine ranged from 35 

– 1447 ng/mL and 78 – 2023 ng/mL, respectively. Liver 

concentrations for hydrocodone and dihydrocodeine 

ranged from 20 – 447 ng/g and <1.5 – 18,000 ng/g, 
respectively. Kidney concentrations for hydrocodone and 

dihydrocodeine ranged from 36 – 210 ng/g and <1.5 

– 15,837 ng/g, respectively. Muscle concentrations for 

hydrocodone and dihydrocodeine ranged from <3 – 99 

ng/g and <1.5 – 3264 ng/g, respectively. As can be seen 

from these data, there was no apparent correlation between 

hydrocodone and dihydrocodeine concentrations within 

any of the specimen types analyzed. Additionally, no apparent trend was observed concerning which specimen 

type contained the highest concentrations of hydrocodone 

and dihydrocodeine. 

Hydromorphone was found in one of the hydrocodone/

dihydrocodeine positive cases. Hydromorphone was below 

the LOQ in all specimens except urine, which was found 

to be 178 ng/mL. Oxycodone was found in 1 of the 8 

cases examined. Oxycodone concentrations in the fluids 

and tissues investigated were 232 ng/mL, 2534 ng/mL, 

755 ng/g, and 298 ng/g in blood, urine, liver, and kidney, 

respectively. The trend observed for the highest to lowest concentration of oxycodone between specimen types 

analyzed was urine > liver > kidney > blood.

Each case was analyzed for the presence of 6-MAM; 

however, none was detected through the course of these 

experiments. Thus, the lack of 6-MAM was not surprising when considering the incapacitating consequences 

of heroin use and the resulting inability to operate an 

aircraft. To further validate this new procedure with regards to 6-MAM, we analyzed past College of American 

Pathologist Forensic Urine Drug Testing samples known 

to contain 6-MAM. Our findings were all within 10% 

of those previously reported.

Conclusion

An automated method that is rapid, robust, and 

sensitive has been developed for the identification and 

subsequent quantitation of 8 opiate compounds in 

postmortem fluids and tissues. This method offers the 

ability to simultaneously analyze numerous commonly 

encountered opiates and thebaine in one simple extraction. Application of this procedure demonstrates the effectiveness of GC/MS for the separation and subsequent 

detection of opiate and TMS/oxime-TMS derivatized 

opiate compounds. The relative simplicity of this procedure and its proven applicability to both postmortem 

fluids and tissues should make the quantitation of these 

opiate compounds more readily attainable for the field 

of forensic toxicology.

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2148.

FIGURES AND TABLES

6-MAM

Hydrocodone Hydromorphone

Dihydrocodeine

Thebaine

Oxycodone

Codeine

O

OCH3

NCH3

HO

Morphine

O

OH

NCH3

HO

O

OCH3

NCH3

HO

O

OH

NCH3

O

O

OCH3

NCH3

O

O

OCH3

NCH3

CH3O

O

OCH3

NCH3

HO

OH

O

OH

NCH3

CH3COO

Figure 1. Chemical structures of 8 opiate compounds. 
10

Figure 2. Representative chromatogram of eight opiate compounds in an extracted whole blood 

control. Peaks were obtained from a 1 μL injection of a 200 ng/mL whole blood, extracted control. 

Chromatographic peaks represent ions monitored in SIM mode for each compound as follows: 

1. dihydrocodeine; 2. thebaine; 3. codeine; 4. morphine; 5. hydrocodone; 6. 6-MAM; 

7. hydromorphone; 8. oxycodone. 

 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

 2

 4

 8

 1 3 5

6

 7

Table 1. Ions employed and retention times expected for the 

GC/MS-SIM analysis of 8 opiate compounds and their internal 

standards.

Compound Ions (m/z)* Retention Time 

d6-Dihydrocodeine 379, 318, 364 5.99

Dihydrocodeine 373, 315, 358 6.01

Thebaine 383, 353, 368 6.68

d3-Codeine 374, 375, 346 6.78

Codeine 371, 372, 343 6.79

d6-Morphine 432, 417, 404 7.65

Morphine 429, 430, 401 7.67

d3-Hydrocodone 389, 300, 374 7.93

Hydrocodone 386, 297, 371 7.94

d6-6-MAM 405, 406, 390 8.06

6-MAM 399, 400, 340 8.08

d3-Hydromorphone 358, 477, 432 8.31

Hydromorphone 355, 444, 429 8.32

d3-Oxycodone 477, 388, 462 8.74

Oxycodone 474, 385, 459 8.75

* Ions in bold used for quantitation. 
11

Table 2. LOD, LOQ, LDR and recovery data for 8 opiate compounds*. 

Recovery (%) ± sd¥

Compound LOD**

(ng/mL) 

LOQ

(ng/mL) 

LDR

(ng/mL) r^2

100 ng/mL 400 ng/mL 

Morphine 3.12 12.5 12.5-1600 0.998 85 ± 1 76 ± 3 

Codeine 1.56 6.25 6.25-1600 0.998 103 ± 9 97 ± 7 

Thebaine 6.25 12.5 12.5-1600 0.999 80 ± 6 70 ± 9 

Hydrocodone 3.13 6.25 6.25-1600 0.997 94 ± 5 90 ± 1 

Dihydrocodeine 1.56 6.25 6.25-3200 0.999 101 ± 5 94 ± 1 

Hydromorphone 0.78 1.56 1.56-1600 0.996 97 ± 4 89 ± 5 

Oxycodone 6.25 12.5 12.5-800 0.996 92 ± 13 94 ± 9 

6-MAM 0.78 1.56 1.56-3200 0.997 97 ± 9 100 ± 2 

* A blood matrix was used for all opiates in the determination of each analytical parameter above except for 

thebaine, for which a urine matrix was employed.

** Concentrations below 0.78 ng/mL were not examined. 

¥ n = 5 for each recovery group. 

Table 3. Intra-day and inter-day accuracy and precision data.* 

Day 1 Day 4 Day 7 

Target

(ng/mL) 

Mean

(ng/mL) RSD %E Mean

(ng/mL) RSD %E Mean

(ng/mL) RSD %E 

Morphine 100

400

99 ± 2 

424 ± 1 

2

1

-1

+6

94 ± 3 

410 ± 14 

3

3

-6

+3

98 ± 1 

432 ± 3 

1

1

-2

+8

Codeine 100

400

102 ± 7 

370 ± 2 

7

2

+2

-8

97 ± 2 

379 ± 5 

2

1

-3

-5

98 ± 1 

383 ± 2 

1

0.4

-2

-4

Thebaine 100

400

87 ± 3 

345 ± 7 

3

2

-13

-14

90 ± 1 

354 ± 4 

1

1

-10

-12

92 ± 2 

355 ± 7 

2

2

-8

-11

Hydrocodone 100

400

99 ± 4 

425 ± 12 

4

3

-1

+6

97 ± 4 

405 ± 11 

4

3

-3

+1

96 ± 7 

421 ± 11 

8

3

-4

+5

Dihydrocodeine 100

400

100 ± 5 

427 ± 2 

5

1

0

+7

92 ± 1 

415 ± 6 

1

1

-8

+4

96 ± 1 

430 ± 3 

1

1

-4

+8

Hydromorphone 100

400

90 ± 1 

369 ± 4 

1

0.4

-10

-8

89 ± 1 

362 ± 5 

1

1

-11

-10

93 ± 3 

360 ± 2 

1

3

-7

-10

Oxycodone 100

400

89 ± 3 

358 ± 1 

3

0.3

-11

-11

90 ± 4 

360 ± 15 

4

4

-10

-10

92 ± 5 

357 ± 2 

5

0.1

-8

-11

6-MAM 10

40

9.9 ± 0.6 

38 ± 1 

6

3

-1

-5

9.3 ± 0.8 

36 ± 1 

9

3

-7

-10

9.2 ± 0.8 

37 ± 2 

9

5

-8

-8

* n = 5 for all measurements. Accuracy was measured as the relative error (%E) from the target concentration. Precision was 

measured as the relative standard deviation (RSD) obtained from five replicate measurements. Blood was used as the matrix for 

all opiates except thebaine, for which urine was employed.
12

Table 4. Opiate concentrations (ng/mL or ng/g) found in victims of fatal civil aviation accidents. 

 Morphine Codeine Thebaine* Hydrocodone Dihydrocodeine Hydromorphone Oxycodone 

Blood - - - - - - - 

Urine 122 47 22 - - - - Case

#1

Liver - - - - - - - 

Blood 260 - - - - - - 

Liver 870 - - - - - - 

Kidney 1786 - - - - - - 

Case

#2

Muscle 120 - - - - - - 

Blood 23 - - - - - - 

Urine 526 - - - - - - 

Liver 83 - - - - - - 

Kidney 121 - - - - - - 

Case

#3

Muscle 21 - - - - - - 

Blood - 13 - 22 270 - - 

Urine - 154 - 1447 2023 - - 

Liver - 78 - 142 18,000 - - 

Kidney - 68 - 122 15,837 - - 

Case

#4

Muscle - 19 - 42 3264 - - 

Blood - - - 18 13 D - 

Urine - - - 262 78 178 - 

Liver - - - 20 D D -

Kidney - - - 36 D D -

Case

#5

Muscle - - - D D D -

Blood - - - 102 24 - - 

Liver - - - 447 106 - - 

Kidney - - - 210 39 - - 

Case

#6

Muscle - - - 99 13 - - 

Blood - - - 36 14 - - 

Urine - - - 35 376 - - 

Liver - - - 82 73 - - 

Kidney - - - 125 23 - - 

Case

#7

Muscle - - - 39 7 - - 

Blood - - - - - - 232 

Urine - - - - - - 2534 

Liver - - - - - - 755 

Case

#8

Kidney - - - - - - 298 

D = detected but below LOQ, (-) = not detected. 

* Urine was employed as the matrix in the preparation of the thebaine calibration curve.