Social use of ethanol and its abuse continues to be important
from the viewpoint of clinical and forensic interest (1). Accord-
ingly, almost every state in the United States and many foreign
countries around the world have per se laws governing the mea-
surement of ethanol concentrations for forensic purposes (1). The
goal of measuring ethanol concentrations is to establish the degree
of intoxication either at the time of sample collection or another
time. For this purpose, one of three principal media, viz., blood,
breath and/or urine, are used to measure ethanol concentrations.
Often the sample choice is single and ethanol concentrations in the
other samples may need to be derived. If the sample of choice were
urine, then the interpretation of the degree of intoxication would be
difficult but not impossible, because urine is not a dynamic body
fluid like blood. Accordingly, the accuracy of measurement of
ethanol concentrations in evidentiary samples and their interpreta-
tion, especially in the case of urine, are extremely important.
The accuracy of measurement and interpretation of ethanol re-
sults may be compromised for many reasons. One such scenario is
the presence of high concentration of sugar, contamination of such
samples by ethanol producing/metabolizing bacteria and/or yeast,
e.g., E. coli and C. albicans, and storage of contaminated samples
at room temperature that promotes in vitro ethanol production/con-
sumption (2–8). To correct such a situation NaF (10 mg/mL) is
added to samples at the time of collection and the samples are
stored at 0–4°C prior to analysis (9–11). The latter corrective step
is made possible by collection of samples in containers having a
fixed amount of sodium fluoride. This in turn will lead to the pres-
ence of elevated levels of NaF in samples because small volumes
of samples are collected in bottles containing a fixed concentration
of NaF, which has been shown to result in salting out of ethanol
during storage and analysis (9,10,13).
Improper storage methods and procedures may also result in
compromising the accuracy of measurement and interpretation of
ethanol results. Described herein is one such scenario, which we
have discovered and corrected. We, like many other laboratories,
routinely treat urine samples with NaF (10 mg/mL) and store them
at 4°C prior to any toxicological analysis. After the completion of
ethanol analysis, the urine samples are treated in one or more of the
following ways. First, urine samples are stored frozen at ?20°C
until they are tested for drugs of abuse (a preventive measure to
preserve drugs of abuse and their metabolites). Second, urine sam-
ples are stored frozen at ?20°C until they are returned to the agen-
cies that collected them for safekeeping and/or to use them as items
of physical evidence in court proceedings. Third, urine samples are
stored frozen at ?20°C until they are transferred to defense coun-
sel for re-testing of ethanol and drugs of abuse when the state lab-
oratory results are disputed. Finally, urine samples are stored
frozen at ?20°C until they are destroyed when they are no longer
required. Although this practice has been discontinued lately, the
Copyright © 2003 by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959.
Lakshmaiah Sreerama,1,2Ph.D. and Glenn G. Hardin,2M.P.H.
Improper Sealing Caused by the Styrofoam
Integrity Seals in Leakproof Plastic Bottles
Lead to Significant Loss of Ethanol in Frozen
Evidentiary Urine Samples*
ABSTRACT: Evidentiary urine samples (n ? 345) stored frozen at ?20°C in their original containers (leakproof 100 mL plastic bottles) upon re-
testing for ethanol resulted in concentrations that were significantly lower (average loss ? ~30%) than those prior to their storage at ?20°C (p ?
0.0001). The observed loss of ethanol was independent of the method of thawing or the concentration of ethanol in the samples, but was dependent
on the sample volume in the container, i.e., the larger the volume of sample the larger the magnitude of ethanol loss. The loss of ethanol was deter-
mined to be due to improper sealing by a Styrofoam integrity seal attached to the mouth of the container. Accordingly, adopting leakproof plastic
containers that do not contain Styrofoam integrity seals, but rather an outside and across the cap tape integrity seal for evidence collection and long-
term storage, will prevent loss of ethanol due to evaporation.
KEYWORDS: forensic science, ethanol, urine, head-space gas chromatography
J Forensic Sci, May 2003, Vol. 48, No. 3
Paper ID JFS2002284_483
Available online at: www.astm.org
1Department of Chemistry, St. Cloud State University, St. Cloud, MN.
2Forensic Science Laboratory, Minnesota Bureau of Criminal Apprehen-
sion, St. Paul, MN.
*Parts of this study were presented at the 54th Annual Meeting of the
Academy of Forensic Sciences in Atlanta, GA in Feb. 2002.
Received 17 Aug. 2002; and in revised form 20 Dec. 2002; accepted 20 Jan.
2003; published 1 April 2003.