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Study on the Sampling of Methadone from Exhaled Breath

Article in Journal of Analytical Toxicology · June 2011

DOI: 10.1093/anatox/35.5.257 · Source: PubMed

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Beck.qxd:JATLynneTemplate 5/3/11 8:58 AM Page 1
Journal of Analytical Toxicology, Vol. 35, June 2011
Study on the Sampling of Methadone
from Exhaled Breath
Olof Beck1,*, Sören Sandqvist1, Michael Böttcher2, Paul Eriksen1, Johan Franck3, and Göran Palmskog1
1Department of Medicine, Section of Clinical Pharmacology, Karolinska Institutet, Stockholm, Sweden; 2Labordiagnostik
und Hygien Dessau GmbH, Dessau, Germany; and 3Department of Clinical Neuroscience, Division of Psychiatry,
Karolinska Institutet, Stockholm, Sweden
Abstract
This study aimed at develop and validate the procedure for
collecting exhaled breath for drug testing. Patients receiving
methadone maintenance treatment were recruited for the study.
Methadone levels were measured using liquid chromatography–
electrospray-tandem mass spectrometry. The sampling device was
based on a 47-mm C18 filter and used under pressure to aid flow
through the filter. The mouth was rinsed before sampling, and the
device was constructed to protect against any saliva
contamination. Methadone was present in breath samples before
and after the daily intake of methadone. The mean (± SD) pre-dose
level was found to be 135 ± 109 pg/min (n = 48, median 121). The
exhaled methadone increased after dose intake. Saliva levels of
methadone were high in comparison with exhaled breath levels.
Saliva contamination was suspected in about 10% of the collected
samples. Similar results were obtained using 1, 3, and 10 min
sampling times. The inter- and intraindividual variability were
found to be similar and in the order of 50%. Alternative sampling
using XAD-2 beads and solid-phase microextraction fiber was
found to be possible and enables sampling with low back pressure
and with no need for pump assistance. The presented results
confirm that breath testing is a new possibility for the detection of
drugs of abuse.
Introduction
It has recently been proposed that testing for drugs of abuse
can be performed by using sampling of exhaled breath (1–3).
Testing for drugs of abuse traditionally requires other speci-
mens such as urine, blood, saliva, sweat, and hair. These spec-
imens each have their specific features, advantages and disad-
vantages and are in many ways complementary to each other
(4,5). In many instances, however, it is important to be able to
* Author to whom correspondence and requests should be addressed.
Email: olof.beck@karolinska.se.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
collect a sample without need for specially trained personal
(blood, hair) or facilities (toilet). Breath testing offer a new pos-
sibility in the testing for drugs of abuse since it might be de-
veloped to be as convenient as alcohol breath testing.
Very little is known at the moment about how drugs of abuse
substances become carried in the exhaled breath. Human ex-
haled breath is known to contain a large number of substances,
both volatile and non-volatile (6–8). In our first study, am-
phetamine was selected as the main analyte because it is a
substance known to be volatile as free base in laboratory work
(1). The demonstration that also the less volatile compound
methadone is present in exhaled breath collected from pa-
tients receiving methadone maintenance treatment suggests
that it is carried in the aerosol fraction of human breath. One
way to sample this fraction has been by collecting exhaled
breath condensate. This was shown also to work for methadone
(3). However, collection of exhaled breath condensate requires
an instrument that cannot easily be transported and may not
be an optimal solution for drugs of abuse testing.
The present study was aimed to further validate the sampling
procedure used for methadone that is based on using a C18
solid-phase extraction (SPE) filter. Furthermore, two alterna-
tive sampling techniques are being proposed and evaluated
for the first time.
Materials and Methods
Chemicals and materials
Methadone and methadone-d3 were obtained as ampouled
methanol solutions from Cerilliant (Round Rock, TX).
Methanol, acetonitrile, and ethyl acetate of high-performance
liquid chromatography (HPLC) grade were from JT Baker
(Mallinckrodt Baker BV, Deventer, Holland). 2-Propanol of
“normapur” grade was from VWR International (West Chester,
PA). Formic acid of analytical grade was from Merck KGaA
(Darmstadt, Germany). Ammonium formate (> 99.995% trace
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Journal of Analytical Toxicology, Vol. 35, June 2011

metals basis) was from Sigma-Aldrich (St.

Louis, MO). The Milli-Q water was of
ultra-pure quality (> 18MΩ/cm) and pre-
pared in-house. The 47-mm C18 Empore
disc and the 30-mg SPEC DAS SPE car-
tridge were from Varian (Palo Alto, CA).
XAD-2 beads, with 20–60 mesh (Su-
pelpak-2) and solid-phase microextrac-
tion (SPME) 85-μm polyacrylate fiber and
fiber holder, were from Supelco Analytical
(Bellefonte, PA).

Preparation of methadone solutions

The ampouled methadone and
methadone-d3 solutions were diluted to
100 µg/mL using methanol. These solu-
tions were further diluted to suitable con-
centrations in 0.1% formic acid and
Figure 1. Outline of the sampling device used to collect exhaled breath samples on an Empore C18 disc. stored at –18°C for a maximum of 1 year.
The subjects were asked to breathe more deeply than normal during the 10 min sampling time. The mouth
was washed with water prior to sampling. A metal ring was inserted to separate incoming saliva from con- Study subjects
taminating the filter.
Patients undergoing methadone main-
tenance treatment (6 males, 1 female,
aged 44–58 years) were recruited from

A
the Methadone program in Stockholm
(Beroendecentrum Stockholm). The pa-
tients were in steady-state and received
supervised daily doses of methadone be-
tween 90 and 140 mg. The patients were
subjected to regular control of compli-
ance to treatment and any use of illicit
drugs by urine drug testing. Ethical ap-
proval was obtained from the Stockholm
Regional Ethics Committee (No.
2008/1347-31).

Sampling of exhaled breath on filter

Compounds present in the exhaled

B
breath were collected for 1–10 min by
suction through a 47-mm Empore C18
disc using a membrane pump to assist
the flow (about 300 mL/min). The sub-
jects were asked to breathe more deeply
than normal into an alcometer mouth
piece (Palmenco AB, Stockholm, Sweden)
mounted in the sampling device holding
the Empore disc (Figure 1). The mouth
was always washed with water prior to
the sampling. It was estimated that all
the exhaled breath was collected through
the filter during the sampling period. Fol-
lowing sampling, the Empore disc was
dismantled using a tweezers and stored at
–20°C. The sampling device was carefully
Figure 2. Outline of the sampling devices used to collect exhaled breath samples on XAD-2 beads (A)
and SPME fiber (B). The subjects were asked to breathe more deeply than normal during the 5- or 10-min
cleaned between uses with 70% ethanol.
sampling time. The mouth was washed with water prior to sampling. The XAD-2 beads were packed be- Following storage, the Empore disc was
tween two pieces of plastic foam. cut into 5 × 5-mm pieces using a scalpel
and transferred to a 10-mL glass test-

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Journal of Analytical Toxicology, Vol. 35, June 2011
tube. A 25-µL volume of 100 ng/mL methadone-d3 was added
and mixed using a vortex mixer, 300 µL of 2-propanol was
added (to wet the surface), mixed, and finally 5 mL of 20%
methanol in ethyl acetate was added. This mixture was shaken
for 1 h in a thermostatic bath at 37°C. Thereafter, the test-tube
was centrifuged for 15 min at 3000 × g at 10°C, the super-
natant transferred to a new 10 mL glass test-tube, and the ex-
traction procedure repeated using 1 mL of 20% methanol in
ethyl acetate. Finally, the two supernatants were combined, and
10 µL of 10% aqueous formic acid was added and evaporated to
dryness under a stream of nitrogen at a temperature of 40°C.
The dry residue was dissolved in 100 µL of 50% methanol in
ethyl acetate.
Standards for quantification were prepared from fortified
blank Empore discs. These were prepared by using solutions
containing 20 or 300 ng/mL of methadone corresponding to 6–
900 pg/disc. After drying, the discs were prepared for analysis
as described. Calibration curves were constructed using linear
regression analysis with weighting factor 1/x.
Sampling of exhaled breath on XAD-2 beads
Before use the XAD-2 beads was washed and activated by
shaking in methanol for 3 h and drying at 180°C under ni-
trogen flow. The activated XAD-2 was stored under dryness.
The exhaled breath was passed through a bed of XAD-2 beads
using a device shown in Figure 2A. The diameter was 10 mm,
three different resin bed lengths were used (5, 10, and 15 mm),
and the sampling time was 3 min. The sampling was per-
formed without support of pumping. Following sampling the
XAD-2 beads was collected and stored in plastic test-tubes at
–20°C.
Following storage, the XAD-2 beads were extracted by adding
0.3 mL 2-propanol, 4 mL methanol, and 20 µL of 20 ng/mL
methadone-d3 in methanol. The slurry was transferred to an ac-
tivated (by 1 mL methanol and 1 mL water) 30-mg SPEC DAS
cartridge, and the eluate was collected in a glass test-tube.
Four subsequent 0.5-mL aliquots of methanol were added to
the XAD-2 bed while avoiding drying of the bed. All fractions
were collected in the glass test-tube. Following addition of 10
µL of 10% aqueous formic acid, the eluate was evaporated to
dryness under a stream of nitrogen at a temperature of 40°C.
The dry residue was dissolved in 100 µL of methanol.
Standards for quantification were prepared by fortifying
XAD-2 beads (0.6 g) with methadone in methanol solution
(100–2000 pg/sample) followed by drying.
Influence from matrix was studied in an infusion experi-
ment. A prepared blank extract from XAD-2 beads was injected
while infusing methadone post-column. The infusion rate was
10 µL/min, and the solution was 0.5 µg/mL of methadone in
50% methanol in 0.1% formic acid.
Sampling of exhaled breath on SPME fiber
The SPME fiber was activated by holding it in a gas chro-
matograph injector at 200°C for 60 min. The activated fiber was
then kept in a capped glass test tube. The exhaled breath was
passed by an SPME 85-μm polyacrylate fiber mounted opposite
the air flow using a device shown in Figure 2B. The air stream
was focused by a conical design with a 5-mm exit hole towards
the SPME fiber situated 10 mm from the hole. The sampling
time was 10 min. Following sampling, the fiber was dismantled
and placed in a conical 0.3-mL autosampler vial containing 0.3
mL of methanol. The fiber tip was kept in the methanol for 5
min while ultrasound was applied, and then the methanol was
stored at –20°C. Following storage, the methanol was dried
under nitrogen after adding 10 µL of 10% formic acid in
methanol and 20 µL of 20 ng/mL methadone-d3 in methanol.
The residue was redissolved in 100 µL of 50% (v/v) methanol
in ethyl acetate.
Standards for quantification were prepared directly in
methanol and ranged between 5 and 50 pg/sample.
Mass spectrometry (MS) analysis system 1
A 3-μL aliquot was subjected to analysis by selected reaction
monitoring (SRM) ultra-performance liquid chromatography–
(UPLC)–MS–MS (Waters Quattro Premier XE, Waters, Milford,
MA). Details were previously published (2).
MS analysis system 2
A 10-μL aliquot was subjected to analysis by SRM LC–MS–
MS (Sciex API 2000). The chromatographic system was an
XTerra C18 column (50 mm × 2.1 mm, 3.5-μm particle size)
with an XTerra MS C18 guard column (10 mm × 2.1 mm, 3.5-
μm particle size, Waters), with mobile phase A = 0.1% formic
acid and B = acetonitrile with 0.1% formic acid. The mobile
phase was 85% A for 0.2 min, followed by a linear gradient from
15% B to 100% B to 2.5 min and kept at 100% B until 3.4 min.
The equilibration time between injections was about 2.5 min
(85% A). The flow rate was 0.425 mL/min.
Two product ions from the protonated molecules were mon-
itored for methadone (m/z 310 → 265; 310 → 105) and one for
methadone-d3 (m/z 313 → 268). This was done by SRM in the
positive electrospray mode with a 100 ms dwell time for each
channel. Other instrumental settings were as follows: declus-
tering potential, 14; curtain gas, 20 psig; collision gas (N2), 10
psig; and ion source temperature 300°C. The minimum de-
tectable amount (signal-to-noise ratio = 3) injected on column
was about ~1 pg.
Quantification of methadone in plasma
Venous blood was withdrawn in EDTA vacutainer tubes and
plasma was prepared by centrifugation. Plasma levels of
methadone were determined by an in-house routine method
based on LC–electrospray-MS operating in positive selected
ion monitoring mode. Plasma proteins were precipitated with
acetonitrile (4:1, v/v) containing methadone-d3. The chro-
matographic system was a Phenomenex Luna C18 column (50
mm × 2 mm, 2.5-μm particle size, Phenomenex, Torrance,
CA), with mobile phase A = 25 mM formic acid, and mobile
phase B = acetonitrile. The measuring range was 10–2000
ng/mL and the interassay coefficient of variation (CV) was
< 10% as estimated from quality control samples.
Quantification of methadone in saliva
Saliva was sampled using the liquid-based Saliva Collection
System (Greiner Bio-One GmbH, Kremsmünster, Austria) ac-
cording to the manufacturer’s instructions. Measured
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Journal of Analytical Toxicology, Vol. 35, June 2011

methadone concentrations in sampling solution were calcu- The length of the breath sampling time was studied in a
lated to neat saliva concentration. Saliva concentrations were separate experiment (Table III). Two values appeared to be out-
determined on an Olympus AU 680 using calibrators and con- liers and was omitted when calculating mean values that were
trols from Greiner Bio-One according to their standard pro- 142, 145, and 97 pg/min for sampling times of 1, 3, and 10 min,
tocol. respectively. A representative chromatogram obtained at the
A 100-μL aliquot of sampling solution was mixed with 200
μL methanol containing 80 ng racemic methadone-d3. After
centrifugation, 200 μL of supernatant was mixed with 200 μL Table I. Exhaled Methadone in Breath at Different Times
of 20 mM ammonium formate (pH 6.5), and 10 μL was injected in the Dose Interval: 10-Minute Sampling Time
into the UPLC–MS–MS system. Calibrators were prepared in
50% artificial saliva in Saliva Extraction Solution (SES, Greiner Methadone Pre-Dose Time after Post-Dose
Bio-One) in concentrations of 15–1000 ng/mL for R- Case Dose Methadone Dose Intake Methadone
methadone and S-methadone, respectively. The interassay co- No. (mg/d) (pg/min) (min) (pg/min)
efficient of variation (CV) was < 5% as calculated from quality
controls. 1 90 3840 27 2380
210 100
The chromatography system was a Chiral-AGP column (50
× 2 mm, 5-μm particle size) with a guard column (10 × 2 2 120 27 12 850
174 37
mm) obtained from Chiral Technologies Europe (Illkirch,
France) with mobile phase 30% (v/v) methanol in 20 mM am- 3 100 150 17 880
235 100
monium formate (pH 6.5).
4 110 120 35 320
216 140
5 100 90 29 280
Results 159 58

The sampling device using the Empore

C18 disc was modified to include separa- Table II. Exhaled Methadone in Breath and Saliva at Different Times in the Dose
tion of saliva fluid that could enter the Interval: 3-Minute Sampling Time
device during sampling (Figure 1). This
separation was designed to hinder any Methadone Methadone Pre-Dose Post-Dose Time after Post-Dose Post-Dose
saliva fluid to reach the filter surface. Case Dose Trough Breath Saliva Dose Intake Breath Saliva
Two experiments were performed to No. (mg/d) (ng/mL) (pg/min) (ng/mL) (min) (pg/min) (ng/mL)
study the amount of methadone in ex-
haled breath at three different times in 1 100 380 704 127 24 2238 6423
the dose interval (Tables I and II). In the MS* MS MS
first experiment using 10-min sampling 2 120 142 291 492 58 197 2054
time, there was a tendency for the excre- 229 210 571
tion rate of methadone (measured as 3 110 – 41 458 56 409 9746
pg/min) to be lower before and 2–4 h after 232 92 1244
dose intake than that measured 12–35 4 100 404 7188 564 52 2014 978
min after dose intake, but the high (out- 233 1734 665
lier) pre-dose value obtained for subject 1 5 120 566 138 1443 36 2434 88275
hindered a firm conclusion (Table I). A 219 failed 2299
second experiment was therefore per- * MS, missing samples.
formed using shorter sampling time of 3
min and measurement of plasma and
saliva levels (Table II). Also in this exper-
iment, one patient displayed outlier re- Table III. Sampling of Exhaled Breath for Different Lengths of Time
sults. In this case, it was observed that
the patient coughed during the sampling Case Methadone Dose Methadone Methadone Methadone
No. (mg/d) (pg/min) (pg/min) (pg/min)
periods. The excretion rate of methadone
at the pre-dose sampling time appeared
Sampling Time 1 min 3 min 10 min
not to correlate with plasma levels (n =
4). Neither did saliva levels appear to cor- 1 90 200 170 3840
relate with exhaled breath levels (r2 = 2 120 50 40 27
–0.18), apart from the fact that both 3 100 10900 233 150
breath and saliva levels were elevated di- 4 110 100 167 120
rectly after dose intake.

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Journal of Analytical Toxicology, Vol. 35, June 2011

shortest sampling time (1 min) is shown in Figure 3. The
amount of methadone collected during the 1-min sampling
time was enough for providing convincing analytical data. The
identification of methadone was based on a correct retention
time (± 0.5%) and correct relative ratio between the two
product ions (± 20%).
In these previous experiments it was noted that the repro-
ducibility of the sampling might be an important matter to
study. An experiment with triplicate consecutive sampling of
three patients was performed (Table IV). Mean, intra-, and in-
terindividual variability (CV) was calculated and were 54
pg/min ± 20 (SD), 62%, and 61%, respectively. Because of the
high intraindividual variability, the experiment was repeated
(Table V) with the following results: mean, 46 pg/min ± 15
(SD); intraindividual variability, 39%; and interindividual vari-
ability, 50%.
When taking all pre-dose determinations of exhaled meth-
adone together, a mean value of 681 pg/min was obtained (n =
53) with an SD value of 1920 and median of 133. Five of these
results were > 3800 and considered to be outliers (possible
saliva contamination), and calculation of the remaining 48
values gave mean (± SD) of 135 ± 109 pg/min (median 121).
Two alternative sampling techniques were evaluated in order
to find a sampling procedure not needing pumping assistance.
The two devices were constructed as shown in Figure 2. In the
first, a tube holding XAD-2 beads was mounted in the manifold.
Three different thicknesses of packing bed were tested (5, 10,
and 15 mm) using four patients. All three thicknesses made it
possible to blow breath air without any significant resistance.
Using a sampling time of 5 min, the results showed that
methadone was trapped on the XAD-2 beads. The mean excre-
tion rate was 11 ± 7 pg/min (n = 10) as estimated after deleting
a blank result and a result that was 454 pg/min. There was no
tendency for the trapped amount of methadone to increase
with a thicker bed, although the variability precludes any firm
conclusion. It was observed that extracts from XAD-2 beads
produced chromatograms with interfering peaks. A matrix ef-
fect experiment was therefore performed using a post-column
infusion of methadone. No suppression or enhancement could
be observed as compared with no matrix injection at or close
to the retention time of methadone. The overall extraction re-
covery of methadone from XAD-2 beads was 95% (n = 2). The
other alternative sampling procedure was using an SPME fiber.
This was done on three patients with a sampling time of 10
min. The chromatogram obtained for one of these samples is
shown in Figure 4. Methadone was identified using criteria of
retention time and product ion ratio. The amount of
methadone trapped on the SPME fiber was < 1 pg/min.

Table V. Reproducibility in Repetitive Sampling of

Methadone in Exhaled Breath, Second Experiment

Patient Patient Patient Patient Patient

Sample 1 2 3 4 5
No. (pg/min) (pg/min) (pg/min) (pg/min) (pg/min)

1 143 206 253 223 47

2 183 153 117 193 47
3 123 140 120 77 37

Figure 3. Chromatogram from the determination of methadone in ex-

haled breath by using the Empore C18 disc and a sampling time of 1 min.
Identification using LC–MS–MS was based on the presence of compound
with correct retention time and with correct relative abundance of the two
product ions.

Table IV. Reproducibility in Repetitive Sampling of

Methadone in Exhaled Breath, First Experiment

Patient Patient Patient Patient Patient

Sample 1 2 3 4 5
No. (pg/min) (pg/min) (pg/min) (pg/min) (pg/min)

1 170 87 80 143 260 Figure 4. Chromatogram from the determination of methadone in exhaled
2 37 133 80 40 87 breath by using the SPME fiber device. Identification using LC–MS–MS was
based on the presence of compound with correct retention time and with
3 27 70 137 40 133
correct relative abundance of the two product ions.

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Discussion
This study confirms that methadone is present in exhaled
breath from patients undergoing methadone maintenance
treatment. In addition, this was measured for the first time pre-
dose.
In sampling of exhaled breath contamination from the oral
cavity is of general concern (9). In the two previous studies of
methadone (2,3), exhaled breath was sampled closely after the
daily dose intake making contamination from the oral cavity
possible. This is also obvious from the relatively high levels of
methadone present in saliva after dose intake (Table II). In
our first study of methadone, sampling was performed imme-
diately (8–60 min) after dose intake, and the sampling device
was not constructed to protect the sampling filter from saliva
contamination. The observed levels were 10-fold higher than
when sampling of exhaled breath condensate was performed
with an Eco-Screen device (3). This device has a built-in sep-
aration mechanism to prevent any saliva from entering the
system. Because of this observation, the device used in our pre-
sent work was constructed to include saliva protection (Figure
1). However, the results at the first post-dose sampling time
point (Tables I and II) from this and the first study (2) are
comparable, which supports the idea that no significant saliva
contamination occurred. In addition, it has been documented
by measuring amylase activity in exhaled breath condensate
that saliva contamination is, at most, trivial (10).
Other observations also support the conclusion that
methadone is being carried in the exhaled breath. Washing of
the oral cavity with water was always performed before sam-
pling as a precaution. In the first study this was not done in a
few cases, but that did not make any difference. In one case
(number 4 in Table II) it was noted that the patient coughed
through the sampling procedure and this resulted in higher
measured levels. It should also be noted that in no case was a
methadone patient sampled without methadone being mea-
surable.
A sampling time of 10 min was originally used in this work.
The results from the present experiment showed that even a
sampling time of 1 min is sufficient. Based on this information,
a sampling time of 3 min was hence used which made sampling
from patients much easier. To make the sampling even more
convenient, it would be of great value to have a filter with less
back-pressure so that the need for pumping might be elimi-
nated.
Throughout this work a significant variability in measured
concentrations was noted. It was therefore important to per-
form the study of reproducibility (Tables IV and V). The in-
terindividual variability of 50–61% is not surprising consid-
ering that plasma trough values of methadone are likely to
range between 200 and 600 ng/mL in maintenance patients.
The intraindividual variability component 39–62% indicates
that the sampling procedure needs to be better understood
and standardized. Further study into this is warranted. For
example, it may not be the time of the sampling that should be
used for calculating excretion rates. In the field of breath anal-
ysis, this is a universal problem (6,8).
Other sampling techniques exist for sampling of volatile an-
262
Journal of Analytical Toxicology, Vol. 35, June 2011
alytes from air. The use of porous polymer adsorbents tubes is
common in environmental air analysis. The dimensions of
such products produce high back-pressure and were therefore
modified in our device in order to eliminate the need for
pumping. Although promising results were obtained, the
amount of methadone trapped was lower than with the Empore
C18 disc. The XAD-2 beads were difficult to handle from a prac-
tical perspective. The Empore disc extracts were previously
studied for matrix effects and found not to produce any influ-
ence (2). Because chromatographic peaks were observed from
extracted blank XAD-2, a possible matrix influence on
methadone ionization was again studied, but was not found to
occur. The experiment with the SPME fiber was based on the
publication that volatile analytes can be trapped from air by
this means, and the construction of the sampling device mim-
icked the published procedure (11). Although the results were
promising, the trapped amount was very low.
Although it is still unknown how methadone is carried in ex-
haled breath, it may be assumed that aerosol particles in mi-
crometer size will carry non-volatile substances (7,12). It is
likely that this is the case for methadone, but further study is
needed. A recent study on breath aerosol physiology has
demonstrated that the airway reopening is important for the
formation of exhaled particles (13). This may be of signifi-
cance for the previously mentioned intraindividual variability
between samplings.
So far, methadone has been utilized as a study compound be-
cause samples are readily available from patients receiving
maintenance treatment. It is critical at this point to investigate
if drugs of abuse other than amphetamine, methamphetamine,
and methadone are also present in exhaled breath. If this is the
case, breath testing for drugs of abuse may develop into a clin-
ically useful alternative to urine or other fluids, as patients par-
ticipating in these studies reported that breath sampling was
more convenient than urine collection.
Acknowledgments
We thank Inger Engman-Borg for assistance in the clinical
part if this work. This work was supported by grants from The
Swedish Governmental Agency for Innovation Systems—
Vinnova, the Swedish Research Council, and the Stockholm
County Council.
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Manuscript received October 22, 2010;
revision received January 11, 2011.
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