DOT/FAA/AM-00/29

Russell J. Lewis

Travis L. Southern

Dennis V. Canfield

Civil Aeromedical Institute

Federal Aviation Administration

Oklahoma City, Oklahoma 73125

August 2000

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, Virginia 22161.

Distribution of

Butalbital in Biological

Fluids and Tissues Office of Aviation Medicine

Washington, D.C. 20591

U.S. Department

of Transportation

Federal Aviation

Administration
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-00/29

4. Title and Subtitle 5. Report Date

Distribution of Butalbital in Biological Fluids and Tissues August 2000

6. Performing Organization Code

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

Lewis, R.J., Southern, T.L., and Canfield, D.V.

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

FAA Civil Aeromedical Institute

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

Oklahoma City, Oklahoma 73125

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

Office of Aviation Medicine

Federal Aviation Administration

800 Independence Ave., S.W.

Washington, D.C. 20591 14. Sponsoring Agency Code

15. Supplemental Notes

This work was accomplished under the approved task AM-B-00-TOX-202.

16. Abstract

During the investigation of fatal aviation accidents, postmortem samples from the pilot/copilot are

submitted to the Federal Aviation Administration’s (FAA’s) Civil Aeromedical Institute for

toxicological analysis. Blood specimens are received in approximately 70% of the fatal aviation

accidents analyzed by the FAA’s Toxicology and Accident Research Laboratory. The lack of blood

available is usually due to the severe damage to a pilot’s body during an aviation accident and/or to

the length of time taken to recover the body following an accident. Therapeutic and toxic levels for

most drugs are reported in the scientific literature for blood and plasma only. Therefore, it is

imperative for an accident investigator and forensic toxicologist to be able to estimate drug

concentrations in a fatal aviation accident victim’s blood from the available concentrations in the

tissue. This is exemplified by a recent aviation fatality where butalbital was identified in muscle

tissue of a pilot, and the investigators wanted to know the approximate butalbital concentration

expected in the victim’s blood. Butalbital, a short-acting barbiturate found in combination with

other drugs such as acetaminophen, aspirin, codeine, and caffeine, is commonly prescribed for the

treatment of tension headaches. Certain side effects of butalbital, such as drowsiness, sedation,

dizziness, and a feeling of intoxication, could affect pilot performance and become a significant

factor in an aviation accident. Thus, our laboratory determined the distribution of butalbital in

various postmortem tissues and fluids. The distribution coefficients established for butalbital,

expressed as specimen/blood ratios, were found to be as follows: muscle (0.66 ± 0.09), kidney (0.98

± 0.09), lung (0.87 ± 0.06), spleen (0.75, ± 0.03), brain (0.96 ± 0.07), liver (2.22 ± 0.04), liver

fluid (0.89 ± 0.23), heart (0.91 ± 0.17), bile (0.94 ± 0.22), and urine (0.73 ± 0.16). The results

demonstrate that muscle, kidney, lung, spleen, brain, liver, and heart can be used reliably to estimate

butalbital blood concentrations.

17. Key Words 18. Distribution Statement

Forensic Science, Toxicology, Butalbital, Distribution, Document is available to the public through the

Sedative, Postmortem Drug Analysis, Aircraft National Technical Information Service

Accident Investigation Springfield, Virginia 22161

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

Unclassified Unclassified 7

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

1

DISTRIBUTION OF BUTALBITAL IN BIOLOGICAL FLUIDS AND TISSUES

INTRODUCTION

The Federal Aviation Administration’s (FAA’s)

Civil Aeromedical Institute (CAMI) is responsible

under the Department of Transportation (DOT)

Order 8020.11A, Chap 4, Par 170, to “conduct

toxicologic analysis on specimens from ... aircraft

accident fatalities.” Additionally, DOT Order

1100.2C, Chap 53, Par 53-15 requires that CAMI

“investigate(s) selected general aviation and air carrier accidents and searches 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

the CAMI’s Forensic Toxicology and Accident Research Laboratory where toxicological analysis is conducted on various postmortem fluids and tissues.

Due to the violent collisions, explosions, and fires

often associated with aircraft accidents, crew members’ bodies may be fragmented, incinerated, disintegrated, or scattered over large areas of rough terrain or

bodies of water. In many cases, the search for remains

results in only small fragments of tissues being submitted for toxicological analysis. In fact, the FAA’s

Forensic Toxicology and Accident Research Laboratory receives blood specimens in only 70% of fatal

aviation accidents. Therapeutic and toxic levels of a

drug are typically reported in the scientific literature

only for blood or plasma. Thus, accident investigators and forensic toxicologists are interested in estimating drug concentrations in a fatal aviation accident

victim’s blood from the available tissue drug concentrations. This is exemplified by a recent aviation

fatality where butalbital was found in the muscle of

a pilot and the investigators wanted to know the

approximate butalbital concentration expected in the

victim’s blood.

Butalbital, a short-acting barbiturate found in

combination with other drugs such as acetaminophen,

aspirin, codeine, and caffeine, is commonly prescribed for the treatment of tension headaches (1,2).

There are specific side effects, however, that could

affect a pilot’s performance and become a significant

contributory factor in an aviation accident. The most

serious of these side effects include drowsiness,

sedation, dizziness, and a feeling of intoxication

(1,2). Additional side effects of barbiturate exposure

include a withdrawal syndrome, characterized by

psychosis, personality changes, or seizures, and a

rebound syndrome, characterized by chronic and/or

daily pain or headaches (3,4).

Since scientific information concerning the distribution of butalbital at therapeutic and toxic levels is

not available, our laboratory determined its distribution in various postmortem tissues and fluids. A

search of the Forensic Toxicology and Accident Research Laboratory database identified four fatal aviation cases reported positive for butalbital where a full

complement of biological fluids and tissues, including blood, muscle, liver, kidney, bile, lung, spleen,

brain, urine, and heart was available. This report

details the quantitation and distribution of butalbital

in these postmortem specimens.

MATERIALS AND METHODS

Chemicals and Reagents

Butalbital and butalbital-d5 standards were purchased from Radian (Austin, TX) at 1.00 mg/mL

concentrations in methanol. The derivitization reagent, MethElute, was obtained from Pierce Chemical

Co.(Rockford, IL). All other necessary chemicals and

reagents were obtained from commercial sources in

high purity and were used with no further purification.

Gas Chromatographic-Mass Spectroscopic

(GC-MS) Conditions

All analyses were performed using a benchtop gas

chromatograph/mass spectrometer (GC/MS), which

consisted of a Hewlett Packard (HP) 6890 series GC

connected to a HP 5973 quadrupole MS operating

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 the autotune

voltage with no offset. All chromatographic separations
2

were achieved using an HP-ULTRA-1 crosslinked

100% methyl siloxane capillary column, 12 m X 0.2

mm i.d., 0.33 μm film thickness. Helium was the

carrier gas with a flow of 1.0 mL/min. An HP 6890

autosampler was used to inject 1 μL of sample extract

into the GC. The GC was equipped with a split/splitless

injection port operated in the splitless mode with a

purge time of 0.5 min and a temperature of 250 °C. The

oven temperature profile was 70 °C to 170 °C at 30 °C/

min with an initial hold of 0.5 min, then ramped at 40 °C/

min to a final temperature of 290 °C, which was held

for 1 min, yielding a total run time of 7.83 min.

Butalbital had a retention time of 3.5 min. Standard

solutions of butalbital and butalbital-d5

 were separately analyzed using the full scan mode from 50 to

500 amu in order to select unique quantitation and

qualifier ions. The ions chosen were 196, 138, 181

for butalbital, and 201, 200, and 184 for butalbitald5

. Using these ions, the mass spectrometer was

operated in selected ion monitoring mode (SIM)

with a Dwell time of 40 msec.

Butalbital concentrations were determined using

an internal standard calibration procedure. Calibration curves were prepared by plotting the linear

regression of the analyte/internal standard response

factor versus the analyte concentration. Response

factors were determined for each specimen, and the

various analyte concentrations were then obtained

from the respective calibration curves.

Preparation of Standards

Calibration curves were prepared by diluting a

butalbital standard in whole blood yielding concentrations ranging from 25 to 800 ng/mL. Controls

were prepared in whole blood at 80, 160, and 320 ng/

mL using separate drug standards than were used for

the calibration curve. The internal standard solution

was prepared by diluting a standard of butalbital-d5

 with

H2

O to yield a final concentration of 400 ng/mL.

Sample Preparation and Extraction Procedure

Tissues were homogenized with a Brinkmann Tissue Homogenizer (Brinkmann Instruments,

Westbury, NY) following a 1:1 dilution with water.

Three mL aliquots of specimen fluids, calibrators,

and controls, and 2 g samples of tissue homogenates

were transferred to individual 15 mL screwtop vials.

Smaller sample sizes were used if the extracted specimen concentrations were expected to be above the

calibration curve. To each specimen, calibrator, and

control, 1 mL of the internal standard solution (400

ng/mL) was added. The samples were vortexed and

allowed to stand for 10 min. The cellular debris and

proteins were precipitated and removed from the

samples by adding 9 mL of cold acetonitrile, mixing

on a rotory extractor for 15 min, and finally centrifuging at 820 × g for 5 min. The supernatant was

transferred to 15 mL vials and evaporated in a water

bath at 40 °C under a stream of dry nitrogen to a

volume less than 1 mL. To this 3 mL of 0.1 M sodium

acetate buffer, pH 7.0 was added. The extracts were

transferred to solid-phase extraction (SPE) columns

that were pre-conditioned with 3 mL methanol followed by 2 mL sodium acetate buffer, pH 7.0. The

SPE columns were Bond Elute Certify II columns

procured from Varian (Harbor City, CA). Care was

taken not to dry the column prior to sample addition.

A column flow rate of 1-2 mL/min was maintained in

each step using a Varian 24 port pressure manifold

with a nitrogen pressure of approximately 3 psi. Once

the samples had passed through the columns, the

columns were washed with 1 mL sodium acetate

buffer, pH 7.0, dried with 25 psi nitrogen for 5 min,

and then washed with 2 mL hexane:ethyl acetate

(95:5). Butalbital was eluted from the columns with

4 mL hexane:ethyl acetate (75:25). Eluents were

evaporated to dryness in a water bath at 40 °C under

a stream of dry nitrogen. The extracts were reconstituted in 75 μL MethElute and transferred to GC

autosampler vials for GC/MS analysis.

RESULTS AND DISCUSSION

The mass spectrum of butalbital and butalbital-d5

provided numerous ions, from which 196 and 201,

were selected as quantitation ions, respectively. Ions

138 and 181 were used as qualifier ions for 196, and

ions 200 and 184 were used as qualifier ions for 201.

A calibration curve (correlation coefficient = 0.998)

of six points was constructed from 25 to 800 ng/mL

and employed for butalbital quantitation for all specimens. With appropriate dilutions, all results fell

within the calibration curve. The extraction procedure provided a clean extract in a relatively short

period of time.

Therapeutic levels of butalbital in plasma range from

1 to 10 μg/mL (5). The blood concentrations found in

the four cases ranged from 0.221 to 11 μg/mL. With an

average blood/plasma ratio of 1.0, the blood concentrations ranged from slightly above therapeutic to slightly
3

below it (6). The distribution and concentration of

butalbital found in the various tissues and fluids are

presented in Table 1. The resulting butalbital blood

concentrations suggest that the individual victims either

took different amounts of drug or took the dose at

different times prior to the accidents.

The distribution coefficients for butalbital, expressed as specimen/blood ratios, are summarized in

Table 2. The distribution coefficients found for

butalbital are: muscle (0.66 ± 0.09), kidney (0.98

± 0.09), lung (0.87 ± 0.06), spleen (0.75, ± 0.03),

brain (0.96 ± 0.07), liver (2.22 ± 0.04), liver fluid

Table 1. Distribution of Butalbital in Postmortem Tissues and Fluids.

Postmortem Tissue and Fluid Concentrations

Represented as μg/g or μg/mL

Specimen Case 1 Case 2 Case 3 Case 4

Blood 11.000 0.638 0.368 0.221

Muscle 7.078 0.392 0.292 0.130

Kidney 11.162 0.541 0.394 0.214

Lung 9.838 0.528 0.343 0.180

Brain NA 0.564 0.375 0.216

Spleen 8.485 0.502 0.269 0.160

Heart 8.723 NA NA 0.227

Liver 24.453 1.415 0.839 0.481

Liver Fluid 8.000 NA NA 0.234

Bile 8.608 0.699 NA NA

Urine NA 0.394 0.308 NA

Table 2. Postmortem Tissue and Fluid Distribution Coefficients

for Butalbital.

Distribution Coefficients for Butalbital

Specimen Specimen/Blood ± SD % of blood ± CV

Muscle 0.66 ± 0.09 66 ± 14

Kidney 0.98 ± 0.09 98 ± 9

Lung 0.87 ± 0.06 87 ± 7

Brain 0.96 ± 0.07 96 ± 7

Spleen 0.75 ± 0.03 75 ± 4

Heart 0.91 ± 0.17 91 ± 19

Liver 2.22 ± 0.04 222 ± 2

Liver Fluid 0.89 ± 0.23 89 ± 26

Bile 0.94 ± 0.22 94 ± 23

Urine 0.73 ± 0.16 73 ± 22
4

(0.89 ± 0.23), heart (0.91 ± 0.17), bile (0.94 ± 0.22),

and urine (0.73 ± 0.16). The relatively small standard

deviations (SD) associated with these distribution

coefficients suggest that butalbital was in the postabsorption phase. Muscle, kidney, lung, spleen, brain,

liver, and heart ratios all have a coefficient of variation (CV) of less than 20%. Bile and urine had CVs

greater than 20%. This was not unexpected due to the

excretory nature of bile and urine. Liver fluid also

showed inconsistent ratios, having a CV of 26%.

Interestingly, liver fluid contained less then half of

the butalbital concentration found in liver tissue.

These results suggest that butalbital distribution in

select tissues, i.e., muscle, kidney, lung, spleen, brain,

liver, and heart, can be used to closely estimate blood

values over a broad concentration range and a wide

window of time since the last dosage.

CONCLUSION

Ideally, all toxicological analyses would be conducted using blood samples. Due to the nature of

forensic toxicology, however, the laboratory must

rely upon the specimens that are available. The need

is obvious for an ability to estimate drug concentrations in blood from values determined in various

other tissues or fluids. In this study, our laboratory

established the distribution of butalbital in various

postmortem tissues and fluids. The data from this

study suggest which postmortem tissues and fluids

are reliable for estimating butalbital concentrations

in blood. As expected, due to the excretory function

of bile and urine, our results indicate that these

specimens would not be desirable for estimating

butalbital blood levels. Surprisingly, liver fluid had a

relatively large CV for its distribution coefficient,

and also had substantially lower butalbital concentrations than did liver tissue. This suggests that liver

tissue would be a better choice than liver fluid for

estimating butalbital blood levels. The results demonstrate that muscle, kidney, lung, spleen, brain,

liver and heart can be reliably used to estimate

butalbital blood concentrations. The findings from

this study clearly emphasize the utility of various

postmortem tissues in approximating butalbital blood

concentrations.

REFERENCES

1. Hardman, J.G., Limbird, L.E., Molinoff, P.B.,

Ruddon, R.W., and Gilman, A.G., Eds. Goodman

and Gilman’s The Pharmacological Basis of Therapeutics, 9th ed., New York: McGraw-Hill, 1996.

2. Physicians’ Desk Reference. 54th ed., Montvale,

NJ: Medical Economics Co., 2000.

3. Preskorn, S.H., Schwin, R.L., McKnelly, W.V.

Analgesic abuse and the barbiturate abstinence

syndrome. JAMA, 1980; 244(4): 369-70.

4. Raja, M., Altavista, M.C., Azzoni, A., Albanese,

A. Severe barbiturate withdrawal syndrome in

migrainous patients. Headache, 1996; 36(2):

119-21.

5. Moffat, A.C., Ed. Clarke’s Isolation and Identification of Drugs, 2nd ed., London: The Pharmaceutical Press, 1986.

6. Baselt, R.C. Disposition of Toxic Drugs and Chemicals in Man, 5th ed., Foster City, CA: Chemical

Toxicology Institute, 2000.