110 Forsdahl, Gmeiner

Guro Forsdahl Quantification and stability of Salbutamol in human

G
nter Gmeiner
Doping Control Laboratory, ARC
Seibersdorf research GmbH,
A-2224 Seibersdorf, Austria
urine
A sensitive method for the quantification of free salbutamol in human urine is describ-
ed. Sample clean up is performed using SPE on a mixed phase extraction column.
Derivatisation is performed with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)
and the extract is analysed by GC-MS. The method was found to be suitable for use
in the doping field, where a cut-off limit of 1 lg salbutamol/mL urine is set by the Inter-
national Olympic Committee (IOC) and approved by the World Anti-Doping Agency
(WADA). Above that value a doping violation occurs. In addition, the stability of salbu-
tamol in human urine has been evaluated.
Key Words: Salbutamol; Gas chromatography-mass spectrometry; Solid phase extraction; Urine;
Doping;
Received: July 31, 2003; revised: October 16, 2003; accepted: October 20, 2003
DOI 10.1002/jssc.200301655

1 Introduction the presence of a prohibited substance in a bodily speci-

Salbutamol is a selective beta-2-agonist and is one of the
most commonly used bronchodilators for treating bron-
chial asthma [1]. Beta-2-agonists are included in the IOC
list of prohibited substances in sports due to their stimulat-
ing and anabolic effect. However, the IOC allows athletes
with asthma to be treated with some beta-2-agonists,
including salbutamol. But the administration is permitted
only by inhalation after prescription by a physician, and
must be declared in advance [2, 3]. A concentration of
non-sulfated salbutamol that exceeds 1 lg/mL urine con-
stitutes a doping violation [2]. The chemical structure of
salbutamol is shown in Figure 1 [4].
Figure 1. Chemical structure of salbutamol.
Salbutamol as a doping agent in urine is usually deter-
mined by GC-MS [5 – 7]. Other procedures described for
the quantitative determination of urinary salbutamol are
LC-MS [8, 9], nonchiral HPLC [10], chiral HPLC [11, 12],
and ELISA [5, 13]. In doping control, bioanalysis based on
MS detection is preferred, due to its better legal defensibil-
ity [14] and a variety of methods to unequivocally confirm
Correspondence: G
nter Gmeiner, Chemical Analysis, ARC
Seibersdorf research GmbH, A-2224 Seibersdorf, Austria.
Fax: +43 50550 3590. E-mail: guenter.gmeiner@arcs.ac.at.
men is advantageous.
Salbutamol is predominantly excreted in urine as a mix-
ture of free salbutamol and its conjugated metabolites
(e. g. sulphated salbutamol) [1]. The IOC cut-off limit, how-
ever, is based on the concentration of non-sulfated salbu-
tamol. The gas chromatographic analysis of the sub-
stance requires extraction and derivatisation. Both liquid-
liquid extraction (LLE) [4, 6, 7, 12, 15] and SPE [4, 8, 9, 12,
13, 15, 16] of salbutamol have been described. LLE has
disadvantages due to the large amount of solvents
needed, closely related to work safety and environmental
contamination issues. SPE, on the other hand, uses small
amounts of solvents, and in addition the automation of
SPE is readily possible.
The object of the present study was to develop and vali-
date a semi-automated method for quantification of salbu-
tamol in urine. The clean-up procedure involves solid
phase extraction. Derivatisation is done with N-methyl-N-
trimethylsilyltrifluoroacetamide (MSTFA). The analysis
was done by GC-MS. In addition, experiments were car-
ried out to evaluate the stability of salbutamol in urine sam-
ples.
2 Experimental
2.1 Materials and equipment
Salbutamol (free base) and terbutaline (hydrochloride)
were obtained from Sigma (St. Louis, MO, USA). Chloro-
form, 2-propanol, hydrochloric acid, sodium acetate,
anhydrous sodium acetate, potassium dihydrogen phos-
phate, disodium hydrogen phosphate (all analytical
grade), 25% ammonia (extra pure) and diphosphorus
pentoxide (P2O5) (extra pure) were purchased from Merck

J. Sep. Sci. 2004, 27, 110 – 114 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
 Quantification and stability of Salbutamol in human urine
(Darmstadt, Germany). Methanol (analytical grade) was
obtained from Scharlau (Barcelona, Spain), and MSTFA
was supplied by Machery and Nagel (D
ren, Germany).
b-glucuronidase/arylsulfatase and b-glucuronidase were
supplied by Roche (Mannheim, Germany). Oasis MCX
1cc columns were provided by Waters (Milford, MA, USA)
and purified water was obtained by a milli-Q reagent-
grade water system (Millipore, Bedford, MA, USA). Sulta-
nol tablets, containing 2 mg of salbutamol, were provided
by Glaxo Welcome Pharma GmbH (Vienna, Austria).
2.2 Reagents and solutions
Stock standard solutions of salbutamol and terbutaline
(1 mg/mL) were prepared in methanol. Working solutions
of 100 lg/mL and 200 lg/mL were prepared by diluting
the stock solutions. All solutions were stored at – 208C.
2.3 Sample preparation procedure
1 mL 1 M sodium acetat buffer pH 4.8 was added to 1 mL
human urine. Then 50 lL of internal standard (terbutaline;
20 lg free base/mL) was added, and the following auto-
mated SPE extraction was performed on an ASPEC XL
(Gilson France), using Oasis MCX extraction columns:
– Preconditioning: washing with 2 mL methanol fol-
lowed by 2 mL 0.1 M HCl
– Extraction: The samples were passed through the
cartridges (rate: 8 mL/min)
– Wash step: Thereafter the cartridges were rinsed with
2 mL 0.1 M HCl and 2 mL methanol
– Elution: 2 mL chloroform/2-propanol/ammonia 32%
(80:20:2 (v/v/v)) were added (rate 6 mL/min)
After evaporation, the samples were dried for 15 min over
P2O5. Derivatisation was performed by adding 100 lL
MSTFA to the samples, and subsequent heating at 608C
for 30 min.
2.4 Instrumental analysis
Chromatographic analysis was carried out using a Trace
GC 2000 gas chromatograph coupled to a Voyager mass
spectrometer (Thermo Quest, Austin, USA). Separation
was performed on a DB1 fused-silica capillary column,
17 m60.2 mm ID, 0.11 lm film thickness (J & W Scienti-
fic, Folsom, CA, USA). Helium was used as a carrier gas,
and the inlet pressure was set to 90 kPa. Oven tempera-
tures were programmed as follows: 908C initial column
temperature, 20 K/min to 1808C, 45 K/min to 3008C, held
for 3 min. The injector port temperature was set to 2508C,
and all the injections were performed in the split mode
(20 : 1 split ratio).
The mass spectrometer was operated in electron impact
(EI) mode at an ionisation energy of 70 eV. The MS acqui-
sition was performed in the selected ion monitoring (SIM)
J. Sep. Sci. 2004, 27, 110 – 114 www.jss-journal.de
111
mode by monitoring the following ions: 86 m/z, 294 m/z,
350 m/z, 356 m/z, 369 m/z, and 440 m/z.
2.5 Hydrolysis
Different hydrolysis procedures were tested in order to
evaluate their effect on the stability of salbutamol under
the respective conditions. The procedures were tested on
urine samples spiked with 1 lg free salbutamol/mL. In
addition, urine from the excretion study (see Section 2.7)
collected 6 h after administration of salbutamol was used
in these experiments. Terbutaline (1 lg/mL) was used as
an internal standard, and was added after hydrolysis, but
before the extraction.
The following procedures were compared to each other:
A Acidic hydrolysis: 0.5 mL of 6 M HCl was added to
1 mL of urine. After 1 h at 608C, 0.5 mL of 6 M sodium
hydroxide and 0.5 mL of 1 M sodium acetate buffer
pH 4.8 were added. The extraction, drying, and deri-
vatisation were performed as described in Sec-
tion 2.3.
B Enzymatic hydrolysis: 50 lL of b-glucuronidase/aryl-
sulfatase and 1 mL of 1 M sodium acetate buffer
pH 4.8 were added to 1 mL of urine. The samples
were heated for 1 h at 608C. The extraction, drying,
and derivatisation were performed as described in
Section 2.3
C Enzymatic hydrolysis: 50 lL of b-glucuronidase and
0.5 mL of phosphate buffer pH 7 were added to 1 mL
of urine. The samples were heated for 1 h at 608C,
and thereafter 50 lL of 6 M HCl and 1 mL of 1 M
sodium acetate buffer pH 4.8 were added. The extrac-
tion, drying, and derivatisation were performed as
described in Section 2.3.
Original Paper
D No hydrolysis: The samples were prepared as
described in Section 2.3.
All samples were quantified with calibration solutions con-
taining 1 lg of salbutamol and 1 lg of terbutaline from a
stock solution of 0.01 mg substance/mL methanol. The
standards did not undergo sample preparation. They were
just dried and derivatised as described in Section 2.3.
All analyses were performed under the instrumental con-
ditions described in Section 2.4.
2.6 Assay validation
The final method was validated by determining its specifi-
city, limit of detection (LOD), limit of quantification (LOQ),
recovery, linearity, intra-assay precision, and inter-assay
precision.
The linearity was studied by preparing a calibration graph
covering the expected concentration range. Urine sam-
ples were spiked with different concentrations of salbuta-
mol in the range 0.05 lg/mL – 3 lg/mL. LOQ and LOD
i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
112 Forsdahl, Gmeiner
were calculated according to the standard DIN 38 402,
Part 51 [17].
Specificity was evaluated by analysing 3 female and 4
male blank urine samples to exclude any influence of
interfering substances.
Intra-assay precision was evaluated after extraction and
analysis on the same day of six independent spiked urine
samples. This was done with 3 different salbutamol con-
centrations (0.05 lg/mL, 0.5 lg/mL, and 1 lg/mL).
Inter-assay precision was evaluated using calibration
points from the stability experiments with a salbutamol
concentration of 1 lg/mL urine. The samples were ana-
lysed in triplicate at least once a week for a time period of
60 days. Subsequently, the relative standard deviation
was calculated from the responses of all samples.
The extraction recovery of salbutamol was calculated by
analysis of spiked urine samples at 3 different concentra-
tions (0.5 lg/mL, 1 lg/mL, and 3 lg/mL). The internal
standard was added after the extraction, and the peak
area ratios were compared to peak area ratios obtained
when pure standards were derivatised. Consequently, an
arithmetic mean was calculated from all recoveries.
2.7 Excretion study
An excretion study was performed by using a healthy
male volunteer. Salbutamol (2 mg) was orally adminis-
tered, and urine was collected for 72 hours.
2.8 Stability experiments
Experiments were performed to evaluate the stability of
salbutamol in human urine. Urine samples from the excre-
tion study containing 2 different concentrations of salbuta-
mol (urine A and urine B) were stored for 60 days at 2
different temperatures, – 208C and 48C. Urine A had been
collected 4 h, and urine B 6 h after administration of the
drug. The samples were analysed in duplicate for the free
salbutamol content, using the method described in Sec-
tion 2.3 and Section 2.4.
3 Results and discussion
3.1 Sample preparation
The effect of acidic and enzymatic hydrolysis on salbuta-
mol was investigated and compared to sample prepara-
tion without any hydrolysis. The results are summarised in
Table 1. The acidic hydrolysis led to the cleavage of con-
jugates, but in return salbutamol was decomposed. The
enzymatic hydrolysis experiments showed that b-glucuro-
nidase/aryl-sulphatase from Helix pomatia had a limited
effect on the hydrolysis. b-glucuronidase from E. coli
showed no effect on the hydrolysis. Consequently, to get
an appropriate indication of the concentration of salbuta-
mol in urine it is recommended to solely quantify the con-
J. Sep. Sci. 2004, 27, 110 – 114 www.jss-journal.de
Table 1. Recovery of salbutamol in blank urine samples,
spiked with salbutamol, and urine samples from an excretion
study after different hydrolysis experiments. In both cases
the recoveries are expressed as a percentage of the highest
one (defined as 100%). All experiments are performed in tri-
plicate.
Spiked urine Urine samples from
samples excretion study
No hydrolysis 100% l 0.5% 33% l 1.9%
Acidic hydrolysis 63% l 1.1% 100% l 2.4%
b-Glucuronidase/ 99% l 2.5% 38% l 2.4%
arylsulphatase
b-Glucuronidase 96% l 3.0% 31% l 0.9%
centration of free salbutamol. A possible decomposition of
salbutamol by the conditions of hydrolysis is avoided. To
monitor a cut off limit as outlined by the IOC for non-sul-
fated salbutamol, this procedure is justified by the fact that
the amount of non-sulfated conjugates in urine is negligi-
ble. A change in the regulation for the cut off limit of salbu-
tamol should be considered for the future.
Our results are supported by earlier published literature
describing the decomposition of salbutamol during acidic
hydrolysis at a higher temperature (808C) [7]. A limited
effect of enzymatic hydrolysis is described, too, but the
hydrolysis conditions are not stated. Another study
describes higher recoveries of salbutamol during acidic
hydolysis (808C) compared to no hydrolysis and hydroly-
sis with glucuronidase/arylsulfarase and b-glucuronidase
under different conditions [18].
For extraction purposes, a mixed phase SPE column was
used, having both lipophilic and ion-exchange properties
to provide a simple and selective extraction of basic
drugs.
Derivatisation is used to improve the poor gas chromato-
graphic performance of beta-2-agonists [15]. In this case,
derivatisation was carried out only with MSTFA without
any catalyst to yield the tris-TMS-derivative, silylated at all
hydroxyl groups. The secondary amine is not silylated,
because of steric hindrance [19].
3.2 Method validation
The specificity of the method was investigated by analys-
ing 7 different blank urines, and no interference was found
at the retention times of salbutamol and terbutaline. How-
ever, a peak containing the fragment ion 356, was
observed close to the terbutaline retention time in some of
the blank urine samples. This peak, described in earlier
publications [16], turned out to be histidine-bis(trimethylsi-
lyl).
The IOC has determined minimum criteria necessary for
the unequivocal identification of compounds by MS tech-
i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
 Quantification and stability of Salbutamol in human urine 113

Table 2. Relative abundances of 3 ions (86 m/z, 369 m/z, and 440 m/z) in a salbutamol standard are compared to the relative
abundances of the same 3 ions in a urine sample from an excretion study.

Salbutamol standard Urine A Deviation

m/z Absolute area Relative area Absolute area Relative area Relative Absolute

86 467472 100% 718488 100%

369 461125 98.6% 697350 97.1% 1.6% 1.6%
440 10531 2.3% 15813 2.2% 2.3% 0.1%

Table 3. LOD, LOQ, method standard deviation, correlation

coefficient, recovery, intra-assay precision, and inter-assay
precision obtained for salbutamol according to the described
procedure.

LOD 0.03 lg/mL

LOQ 0.11 lg/mL
Method standard deviation 1.5%
Correlation coefficient 0.9999
Recovery 70%
Intra-assay precision
0.05 lg/mL urine 2.0%
0.1 lg/mL urine 1.7%
1 lg/mL urine 1.7%
Inter-assay precision 3.0%
Figure 2. Excretion of free salbutamol in urine after a single
dose of 2 mg salbutamol.
niques [20]. These criteria can be used as a measure of
the specificity of the analytical method. They imply that absorbed from the gastrointestinal tract and is excreted in
the retention time of the analyte should not differ by more urine as a mixture of free salbutamol and its conjugated
than 1% from the retention time of the standard solution, metabolites. About 24 – 33% of an oral dose of salbutamol
and at least 3 diagnostic ions must be evaluated. The rela- is excreted unchanged in urine, and due to first pass
tive abundances of the ions should not differ by more than metabolism about 48% of the dose is excreted as conju-
5% (absolute) or 20% (relative) from the relative intensi- gated sulfate. Salbutamol is not extensively metabolised
ties of the same ions acquired in a standard. In Table 2, in the lungs, and consequently the proportion of metabo-
the relative abundances of 3 ions in an extracted urine lite in urine after inhalation depends mainly on the dose
sample (0.22 lg salbutamol/mL urine) and a salbutamol swallowed [1]. The plasma half life is relatively short for
standard (containing 0.15 lg salbutamol from a stock salbutamol, about 2 – 6 h [18]. The excretion profile of free
solution of 0.01 mg substance/mL methanol) are com- salbutamol after oral administration of 2 mg salbutamol is
pared. As can be seen, both the relative and the absolute shown in Figure 2. Free salbutamol could be detected by
deviation are far below the limits determined by the IOC. the described method for more then 50 h after administra-
Results obtained for LOD, LOQ, correlation coefficient, tion.
recovery, relative standard deviation, intra-assay preci-
sion and inter-assay precision are given in Table 3.
3.4 Stability experiments
The LOQ of 0.11 lg/mL is well beyond the IOC cut-off limit
Experiments were performed to evaluate the stability of
set at 1 lg/mL, and a method standard deviation of 1.5%
salbutamol in urine. In a previously published study it has
is considered appropriate. The method was found to be
been demonstrated that the concentration of salbutamol
linear (r >0.99) over the standard curve range studied
decreases as much as about 80 – 90% within 25 days after
from 0.05 lg/mL to 3 lg/mL. The intra-assay precision
cold storage [18]. Other investigations, however, show
and the inter-assay precision proved to be satisfactory,
that the concentration of salbutamol is rather constant
and therefore an overall recovery of 70% is acceptable.
after a 45 day storage period at different temperatures [1].
Our investigations show, that the free salbutamol concen-
3.3 Excretion study tration decreased slightly after a 60 day storage period in
Pharmacokinetic parameters are main contributors to the the samples stored at – 188C (2 – 4%). The samples
detection window of the drug in urine. Salbutamol is well stored at 48C show a greater decrease of the salbutamol

J. Sep. Sci. 2004, 27, 110 – 114 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
114 Forsdahl, Gmeiner
Figure 3. Concentration of free salbutamol in urine A and B
over a storage period of 60 days at – 188C and 48C. The
lines shown are regression lines.
Urine A, stored at – 188C: g and 11. Urine A, stored at 48C:
f and – – – . Urine B, stored at – 188C: h and N N N N Urine
B, stored at 48C: 6and – - – .
Table 4. Decrease in salbutamol concentration in urine A
and urine B after a storage period of 60 days at – 188C and
+48C.
– 188C +48C
Urine A 2% 8%
Urine B 4% 12%
concentration in the range of 8 – 12%. The results are
summarised in Figure 3 and Table 4.
4 Conclusion
In conclusion, the presented method appears to be suita-
ble for quantification of free salbutamol in doping control.
The limit of quantification is well below the cut-off limit
determined by the IOC medical commission. The concen-
tration of free salbutamol in urine decreased only slightly
after long term cold storage. The quantification of free sal-
butamol without any hydrolysis step for the marginal
check of the IOC cut-off of 1 lg non-sulfated salbutamol/
mL urine is recommended.
Acknowledgement
The authors wish to thank Waters for the supply of Oasis
MCX columns.
J. Sep. Sci. 2004, 27, 110 – 114 www.jss-journal.de
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